ACKNOIILÐGEMENTS

Tt¡e aubhor wishes to express her appreciatlon to the following people for

their assistance durlng the course of this proJect and preparation of this

the si s.

Dr. Ran Tr:1si for his supporü, encouragenent, friendshlp and exlensivenorphological expertise particularly in relatlon to the nentous systen.

Dr. Ivor Dreosti fon his biochenÍcal expertlse, excellent sclentÍficguidance, and continued encouragenent and advice.

Dr. Bob Barbour for his continued interest and for pernission toundertake this project ln the Anato4r and Histologr Department.

Mr. Chris Lelgh fon hÍs experü technlcal advlce.

Dr. Ian Record for assÍstance in the early part of ùhe project wÍthexecutlon of the abryo culture technique.

Dn Peter Baghurst for statistical advice, and developnent of a progranfor analysfs of the proJect data.

Mr. Reg Buckley for trace elenent analysls.

Mr. Rob Murphy and Mr Jeff Tonlfnson for pneparation of the photographicplates.

The sbaff of the Departnent of Anatony and HÍstology' the CSIRO Divlsionof Human Nutrition, and the Electron Optical Centre for their assistanceand advice.

Mr. Al1an Partridge for his excellent t,ypingr and continued support,frfendship and tolerance.

l,frs. HiIda Joschko for her unceasÍng noral support and encouragemenf .

ABSTRACT

Recently there has been a heighlening of interest in the underlying

causes of birth defects, and the vulnerability of mother and developing

embryo and fetus to nutritional and environnental factors, which are now

recognised as major areas requiring attention.

This thesis details Lhe results of in vivo and in vitro studies by the

author which provide new insight,s in relation to the effecLs of zinc

deficiency, hypervitaminosis A, alcohol, nicotine and sal-icylic acid, at

the morphological and ul-trastructural leveIs in the developing enbryo. AJ-I

these agents have been previously suspected or proven to exert teratogenic

effects on the offspring of animals and humans.

The major aÍms of the thesis were first to examine mainly Lhe

morphological abnormalities and associated cytological and ultrastructural

changes particularly of the neural tube about the period of neurulation'

followÍng exposure to the aforementioned agents. Secondly, attention was

aLso focused on the contribution that concomitant exposure to several of

these teratogens nakes to the overalL aetiolory of malformation at both

the morphological and ultrastructural 1evels, whÍch to daùe only limit'ed

studies have explored.

Studies were perforned on rat embryos i¡ vivo, as well as -i¡ vitro using

the recently deveJ.oped enbryo culture technique. Growth and norphological

features of the embryo Ìüere examÍned using a dissecting microscope and the

results were statistical.ly analysed. Exanination of the enbryonic neural

tube was perforned principally with scanning and transnission electron

nicroscopy, and light microscopy vras also utilised to exanine neural cell

densi ti es.

Because of difficutties with inducing a teralogenic zinc deficiency in

culture, a 1ow dietary zinc status and an excess of vitanin A were induced

;ln vivo prior to the critical period of neurogenesis which Ín the rat

occurs between days 9.5 and 11.5 of gestatÍon. The results revealed that

when adninistered alone, zinc deficiency and hypervitaminosis A induced

severe neural dysnorpholory which was accompanied by extensive cell death

within the neuroepithelium and mesenchyrne respectively.

A furLher study was performed in w hich zinc deficiency and

hypervitarninosis A were adninistered concurrently which Ied to increased

numbers of malformed enbryos with neural tube defect,s including

exencephaly, as well as severe neural cell death, whlch were far greater

than the effect of the individual agents. Statistical analysis indicated

that concurrent exposure to these agents exerted an interactive effect on

neural tube defects which was reflected in the extensive ultraslructural

danage observed in the cranj.al neural tube.

J¡ vÍtro culture of 9.5 day rat enbryos was used to study the effects of

nicotine, alcohol and salicylic acid on embryonic growth and norpholory

over a range of concentrations. Examination of day 11.5 embryos revealed

that all three conpounds led to growth retardation and dysnorphology of a

number of structures including the neural tube, demonstrated by the

presence of hypoplastic forebrains, nicnocephaly, and/or open neural

tubes. These abnormalities were often acconpanied by a dose-dependent

cetlular disruptÍon and necrosis predominantly within the neuroepithelium

which consisted of the disruptÍon of t,he cytoplasmic contents, and in

addition, alterations ín nuclear structure in nicotine-treated enbryos.

The present study also found that ceIl death in the neural tube was not

always Iinked with neural dysnorpholory as exencephalfc enbryos exposed to

saticylic acid did not exhibit neuroepÍlhelial necrosis at Iower doses.

The author then investigated whether concurrent administration of alcohol

and nicotine would lead to an exacerbation of teratogenesis The results

showed that growth retardation and the frequency of dysmorpholory in the

combined treatment group was additive for a number of structures incLudlng

neural tubes, which suggests that the two agents exerted their teratogenic

effects independently. This result was reflected in the level of severity

of cellular disruption and necrosis in the neuroepit,helium whÍch also

appeared additive.

In the final study, the author investigated the possibÍIity of an

alcohol--saticylic acid inberaction The frequency of abnornalities when

the two treatnents were combined indicated that these two agents exerted

their teratogenic effects independently for a number of structures

examined,' including the neural tube. Many of the embryos denonstrated a

collapsed bub closed anterior neural tube particularly in the forebrain-

midbrain region. For embryos that failed to complete neuruLation when the

treatnents were combined, the frequency of this defect demonstrated an

interactÍon, which vJas aneliorative, producing fewer and possibly less

Iife threatening abnormalitÍes. These observations suggest that alcohol

and salicylic acid nay interfere with neurulation and thus may exerb their

neurotoxlc effects through similar nechanisms. Cell death and ceII Loss

which occurred mainly in the neuroepithel ium accom panied t'his

dysnorphology and was preceded by a breakdown of mitochondria and a

reduction in the intensity of polyribosomes while the nucLei generally

renained intact. The level of necrosis induced by concomitant exposure to

aLcohol and salicylic acid appeared to be far in excess of that observed

with the individual teratogens, suggesting an intenactlon at t'he

uI trastructural 1evel.

From these studies it can be concluded that al-I five agents investigated

act as nervous systen teratogens alone or in conbinatÍon, and depending on

the agents they exert eÍther an interactive or addÍtive effect on neural

tube dysmorphology and cell necrosis. The presence of extensive neural

necrosis in sone experimental enbryos appears to be an additional

teratogenic effect of the agent, rather than an underlying cause of failed

neurulation, as it was not consistently linked wÍth exencephaly and open

neural tubes. These neural abnornal-ities may arise as a result of severe

blebbing of the neuroepitheliun at the sites of neural fold fusion, and/or

by other mechanisns yet to be elucÍdated. The present observatÍons

strongly implicate neural tube necrosis as an underlying cause of

nicrocephaly, and suggests that it may also be linked with long term

intellectual and behavioural deficÍts reported Ín aninaÌs and hu¡nans.

CONTENTS

CHAPTER 1 : INTRODUCTION

INTRODUCTIONB ]BL IOGRA PIIY

CHAPTER 2: MATER]ALS AND METHODS

ANIMAL SDIETSTISSUE REI'OVAL AND EI'ßRYONIC ASSESSMENT

IN VITRO CULruRE TECHNIQUES

.1 Explantation of embrYos

.2 Preparation of culture medium

.3 Culture chanbers and gas mixturesBIOCHEMICAL ANALYSIS

.1 Protein estination

.2 Trace elementsMICROSCOPY

.1 LÍght and transmission electron microscopy

.2 Scanning electron nicroscoPYSTATISTICAL CONSIDERAT IONSBIBLIOG RAPHY

CHAPTER 3: IN UTERo GRoI'{TH AND DEt/EtoPMu\IT 0F ZINc DEFICIENIEMBRYOS DURING NH]ROGn{ESIS.

INTRODUCT ION.1 Zinc metabolism.2 Zinc requirenents.3 Absorplion and secretion of zinc.4 Biochenical roles of zinc.5 Aetiolory and clÍnical nanifestations

of zinc deficiencY.6 ZLnc and hunan develoPment.T Zínc and develoPment in animals

MATERIALS AND METT{ODS

.1 Ani.mals and dietsNESULTS

.1 Growth and norphological develo@ent

.2 CeIIuIar develoPrnenl

.3 UI trastructural observationsDISCUSSIONCONCLUSIONBIBLIOGRAPHÏ

4.1 INTRODUCTION4 .1 .1 Hisbory4.1 .2 Metabol isn

1

1

1

2

1-11-15

2-12-22-32-52-52-72-T2-82-82-92-92-g2-102-102-14

2.12.22.32.42.42.42.42.52.52.52.62.62.62.72.8

3.13.13.13.13.13.1

3-13-13-23-23-3

3.13.13.23.23.33.33.33.33.43.53.6

3-53-73-103-1 43-143-153-153-253-303-363-4 5

3-47

CHAPTEN 4: GROV{TH AND MORPHOLOGICAL EFFECTS OF HYPEMIITAMINOSIS A'

IN UTERO.

4-14-14-2

4.1 .34.1.44.1.54.1.64.1 .7

4.1.8

Biochenical roles of retinoidsRetinoid toxicity and cell functionDÍetary all-owances of vitamin A

Clinical assessment of hypervitaninosis A

Vitanin A and retinoid toxicity in enbryonicdevelo¡ment in animal sVÍtanin A and retinoid t,oxÍcity in enbryonic

4-54-74-84-9

4-11

4- 184-214-234-244-244-254-264-264-264-324-344-424- 51

5-15-35-35-35-45-45-85-145-1I5-27

6-16-16-36-1 06-1 6

6-196-206-226-226-276-3 1

6-3 46-426-49

development in humans.9 Mechanims of vitanin A action.10 Justification for vilanin A study

MATERIALS AND METHODS

.1 Aninals and diets

.2 Tissue preparation and analytical nethods

.J Statistical analysisRESULTS

,1 Gnov¡th and norphological development.2 CelluLar abnormal ities.3 Ultrastructural observafÍons

DTSCI'SSIONB TBL TOGRA PHY

CHAPTER 5: ZINC-VITAMIN A INTERACTIONS AND TERATOGMJBSIS.

INTRODU CT IONMATERIALS AND METHODS

. 1 Anina1 s and diet s

.2 Tissue preparation and analytical- net,hodsREST]LTS

.1 Growth and morphological developrnent

.2 Microscopic observations

.J Ultrastructural observationsDTSCUSSTONBIBLIOGNAPHY

INTRODUCTION.1 Ethanol netabolisn.2 Human studies.3 Ani:¡aI studies.4 Mechanisms of aÌcohol teratogenesÍs.5 Ains of the study in relationship t,o alcohol

t,eratoge ne si sMATffi IALS AND I'ÍEÍTIODSNESULTS

.1 Growth and norphological development

.2 Scanning electron nicroscopy

.3 Cellul-ar observations

.4 Ultrastructural observati.onsDTSCUSSTONBIBLIOGRAPHY

CHAPTER 6:THE EFFECTS OF ETHANOL IN VTTRO ON THE DEVELOPTNG NERVOUSSYSTEM.

4.14.14.24.24.24.24.34.34.34.34.44.5

5.15.25.25.25.35.35.35.35.45.5

6.16.16.16.16.16.1

6.26.36.36.36.36.36.46.5

CHAPTER ?:THE EFFECTS OF NICOTINE IN VITRO ON THE DEVELOPING NERVOUS

SYSTEM AND SOI.fE OTHER STRUCruRES.

INTRODU CT ION.1 History.2 Conponents of cigarette smoke.3 Pharmacological actions of nicotine.4 Metabolism of nicotine.5 Clinical characteristics of nicotine.6 fuoking and embryonic development in humans.7 Nicotine and embryonic development in animals.8 Aims of the PresenL studY

MATERIALS AND METHODS

.1 Aninal s

.2 ftrbryo culture and teratological screening

.3 StatisticsRESTJL TS

.1 Growth and norphological deveLopmenf

.2 Scanning electron microscope observations

.3 CeIluIar observatÍons

.4 UI trastructural obser¡¡ationsDISCUSSIONB ]BL IOGRA PHY

INTR ODU CT IONMATENIALS AND METT{ODS

RESULTS

.1 Growth and norphological observations

.2 Ul-trastructural obsenvationsDISCUSS IONBIBLTOGRAPHY

CHAPTER 9: ITIE IN VITRO ACTION 0F SALICYLIC ACID 0N RAT EÌ"ßRYOS.

7.17.17.17.17.17.17.17.17.17.27.27.27.27.37.37.37.37.37.47.5

T-17-17-2T-3T-3T-67-87 -187 -227 -237 -237 -237 -237 -247 -247 -zl7-307 -327-387 -46

CHAPTER 8: THE EFFECTS OF CONCURRENT ADMINISTRATION OF ALCOHOL AND

NICOTINE ON GRO.¡TTI AND DEI/E.OPMB¡T OF RAT EMBRYOS.

8.18.28.38.38.38.48.5

9999

8-18-48-58-58-148-218-30

1

1

1

1

.1

.2

.3

.1

.2

.3

9-19-19-3

INTRODU CT TON

Hi storyMetabol Ísm of sa1 icylate conpoundsPhamacological and biochenical actionsof the salicylatesThe effects of aspirin on embryonic developrnentin aninalsSalicylate compounds and embryonic developmentin hunansMATERIALS AND MEIT{ODSAnimaL sfubryo cuLture and teratological screeningSta t,i st i csRESIJL TS

Grow thMorphological dev eloprnentCeltular observationsUl tra structural observations

9.1.4

9.1 .5

9-6

9- 10

9-169-20g-209-20g-209-219-219-219-259-zl

9.29.29.29.29.39.39.39.39.3

.1

.2

.3

.4

DTSCU SSIONB IBL IOGRA PHY

1 0.1 ]NTRODUCTTON

10.2 MATERIALS AND METHODS

1 0.3 RESULTS

10.3.1 Growth and norphological observatÍons10.4 DISCUSSION10.5 BIBL]OGRAI'I{Y

CHAFTER 1 1: GHIIERAL DISCUSSION.

9.49.5

9-339-42

10-11 0-31 0-41 0-410-231 0-33

11-111-511-281 1-4011-481 1-50

CHAPIER 10: THE TERATOGÐ{IC EFFECTS 0N CULIIJRED RAT EMBRYOS 0F CONOIIRRENT

EXPOS'RE TO ALCOHOL AND SALICII,IC ACID.

11 .111 .211 .311.411 .511 .6

INTRODU CT TON

General morphologicaL observationsCeII death and neural teratogensTeratogenesis and the hunan conditionConcl usionsB TBL IOGRA PHY

APPENDIX 1: PUBLICATTONS AND ABSTRACTS

DECT.ANATION

This thesis conbains no naberial which has been accepted for the a$¡ard of

ar¡y institution, and to bhe best of the candidatets knowledge contains no

naterial prevlously published or written by any other personr except where

due reference is given

ldarlon A. Joschko.

Novenber 1991.

ilArÍß

lllGl{EDr

*¿t*ßÆil/*-þfrss++o courr' **f/ô--D --.{Heeæ*gÀ.r $n md:ai þ str eny ct þ trr, nta cqqff ln ü! I¡Úrruftr lfu¡I. r*qs r¡dHnË h tornatdpüump¡hg.

1-1

CHAPTER 1

INTTODUCTION

In recent years, there has been an upsurge of interest in t,he aetlology

and prevention of birth defects, probably partly due to t,he thalidonide

dÍsaster and partly because of the decllne 1n lnfant mortality fron other

more preventable causes such as infectious and nutnitional diseases. Fron

the analysis of many epidemiological studies the incidence of obvÍous

gross malfornations in tr'Iestern countries has been estÍmated at 3í aL

birth, (Shepard 1979t Kalter & Ïlarkany 1983), with one third of these

being Iife threaLening (Shepard 1979). The percentage of congenital

defeets is doubled aE2years of age through the further recognftion of

cardiovascular, renal and central nervous system anomal-ies (TuchDântr-

Duplessis 1977). Mental retardation which occurs in approximately 3l of

school age children is connorùy of congenital onigin and frequently is not

associated wlth a defÍnable structural change in the nervous systen

( Shepard 1986).

It has also been recognised that there ane a number of correlations

between pre- and peri-natal death and malfunctlon in hunans. Nornal1y,

embryonic and fetal loss occur in approxÍmately one of every two

pregnancles, whiLe around 75Íl oî structurally abnornal enbryos and fetuses

never reach the vfable stage (Shepard & Fantel 19TÐ. Anong sponüaneously

aborted ernbryos and fetuses, the rate of structural abnornalÍties varfes

fron 7-24f (Fantel et al 1980), whÍle maJor nalformatlons l¡ere reported fn

141 of stillblrths and 3Tí of neonatal deaths (Drew et al 1978). In terns

of wasted pregnancies due to defective fetal developnent nore than 5601000

Iives a year are clalned Ín the Unlted Süates t,hrough lnfant death,

spontaneous abortÍon, st,illbirth, and nlscarriage (Natlonal Foundation,

1-2

March of Dines 1g7Ð. In the aetiolory of congenitat malfornations (Table

1.1) around 251, of abnormalities are caused by genetic díseases or

chroroosonal abnormalitÍes, 10í can be attributed to exogenous factors such

as drugs and environnental agents, while the rnaJorlty (65%) have no known

cause (BeeIey 1981). It islikely bhat the aetiology of sone of these is

nultifactorial in origfn and nay be induced by an interplay of genetÍc and

environmental components (Fraser 1977).

Table 1.1 Aetiolory of hurnan nalformations.a

Suspected causedl0

Autosomal genetic dÍsease

Chronosomal abnormalltle s

Maternal conditÍonsdÍabetesendocrÍnoPathiesnutrltion def icienciesdrug addictions

Miaternal Ínfecfions

Mechanlcal def ormities

Chenicals, drugs, radiatÍon, hyperthernia

Unknown? polYgenic? Multifactorial

( gene- env Íronnent interactlons)? spontaneous? synergistic action of tenatogens

))

))

4

15-20

5

3

1-2

65

)))))

(a) Aften Beckman & Brent (1984).

So far only a handful of drugs and envÍronnental chemlcals have been

established as known hunan teratogens (TabIe 1,2), although the nunbers

lncrease when agents under suspiclon are also considered (Table 1.3). It

should be noted that nof all workers classify these agents in hunans in

the sane $¡ay as Shepard (1986). The nunber of agents demonstrated in

1_3

humans as having teratogenic activlty is surprisingly small, 1n contrast

to the hundreds of dysnorphogenic agents which have been recognised in

animals (Shepand 1989). ltre potential hunan developmental toxicants and

theÍr experÍmental effects in a nunber of species have recently been

reviewed by Schardein and KelIer (1989).

Table 1.2. Known Teratogens in Humans.b

RadÍationIhera peuticRadioiodineAtonic weapons

InfectionsRubella virusClt,onegal ov lrusHerpes simplex virus I and IIToxo plasmo sisVenezuelan equine encephal itis virusSy phttis

l,laternal metabolic ÍnbalanceEndemic cretÍnfsnDi abe tesPhenylke t,onuriaVirilízing tunors and netabollc conditfonsAI cohol I snHypertherniaRheunatic disease and congenital heart block

Drugs and enviromental cheuicalsAndrogenic hornonesAninoptenin and nethyl aninopterinCycl ophospharnideBusulfanThal idonideMercury, organicChl orobÍ ph enyl sDle tlyl s t Í1 be s tr oIDiph erryIhyda ntof nTrinethadione and ParanethadioneCounarf n anticoagul antsPer¡lcillanine ( possibly)Valproic acldGoftrogens and antÍthyrofd drugsTetracyclfnes13-cls- retinoic acid (fsotretirnin, Accutane)Li thfumMethfnazole and scalp defects

(b) Adapted fron Shepard (19S6).

1-4

Table 1.3 Possible and unllkely Teratogens in Humans.c

Possible teratogensCigarette srnokingDlazepan (Valtun)Zinc deficiencyHigh vitanin A intakeVaricella infectionBÍnge drinkÍngOrganic solvents (laboratory workers)

Unlike1y tenatogensAspi rl nBirth control pillsUl tra soundSpernicidesBendect,in (antf nauseant conbinatlon producb)Illiclb drugs (Marijuana, LSD, cocaine)Vldeo display teminalsAspartaneAnestheticsRubella vaccineMetronldazol eAgent Orange

(c) Adapted from Shepard (1986).

The apparent discrepancy between experinental and clinical observations

is probably due to the complexÍty and multiplicfty of factors Ínvolved in

the induction of congenital abnornalitfes. These include factors such as

appropriate anounts of agent, adninistered at the preclse monent in

morphogenesis, toget,her wilh the appropriate genetic susceptibility to

react. The tenporal coÌnponent in the inductfon of teratogenesis is of

extreme inportance. In order for an agent to induce dysnorphology ft nust

be adnlnistered during enbryological differentation or rthe crlbicalperlod of organogenesisn which varles between specles depending on the

length of gestation In rats Ít extends fron days 9-17, and fron days 20-

55 fn humans (Schardein 1985). The conparative tine periods for the majon

developnental events in hunans and rats are given in TabIe 1.4.

Mosf organs are usually highly susceptible to the effects of teratogens

for short tines within the perlod of organogenesJ.s (Ffg 1.1¡. For exanple,

1-5

the critical perlod for neurogenesls occurs between days !-11 in rats. An

acute dose of a teratogen on day 10 of gestation j.s 1lkely to have an

optinun effect on brain development, ¡.¡it,h lesser effects on other organs.

I{hi1e these conditlons may be readily net in laboratory aninals, it nay be

assumed that they are present only exceptionally durÍng the development of

the human concepfus.

Table 1 .4 Developnental events in ht¡mans and rats. d

Species age ( in days)

Hrman Rat

Neural plateFirst somiteBranchial arch, firstHeart-first beatsPronephros0ra1 plate perforationAnterior neuropore closedMesonephnosOtocyst closed10 sonÍtesThree branchial arehesMesonephric duct to cloacaThyroid appearsUpper lí.mb budPosterior neuropore closed20 somitesMetanephric bud appearsLung bud appearsCrovlr¡runp length, 5nnLower linb bud appearsSplral septun beginsHerniatfon of gubEle pigmenlDigÍtal rays-upper extrenityCrq¡r¡-rt¡mp length, 1Onr¡0ssification beginslfuLlerian duct appearsCIoaca dfvided by urorectal septunTestes, histological differentiationDlgit separatfonHeart septatÍon conpleteEle 11ds closedPalate closed conpletely

1 8-2020-212022222424-25252525-2626262727-2826-27zt-28282829-3029-30343434-35353T40-4340434343-4746-4756-5856-58

9.5101010.2101010.511-1211 .310 .511 .5121010.511 .311 .312.312 .11311 .2?11.512.5

13 .41517 -1813.517

14.5

15.51816-17

d Aoapted fron Shepard (1989).

1-6

40 eE

A Enef Pulse of Terotogenlc Treclmenl on

lhe lOlh Ooy of Geslol¡on Would Resullin lhe Followlng lncrdence of Molformo'ltons

35"/" Brorn Def ec ts33% Eye 0ef ecls24Y" Heorl 0ef eclsI B% Skelelol Oefecls

6 "/. Urogenito I OeleclsO"/o Po lole Defe cl s

Po lo leUro enllol

.9(!

Eoo

=èe

30

20 .tst

Broin

Heorl on ó

A¡r olelelon

Aor I i--..Arches

ro

ltii

89 ro ll t2 13 t4

Days of Ge¡tation in Rat

15 16

Fig. 1.1. Group of curves representing the susceptibÍI1ty of particularorgan systems Ín rabs to a hypothetical teratogenlc agent given ondifferent days of gestatlon (I,tllson 1973).

In order to consider the pathogenesis of neural tube defects, it fs

necessarV to consider the nornal process of neuroernbryological developnent

or prfmary neurulatÍon which beglns in the rat on day 9.5 and ln hunans on

days 16-18 of gestatlon tJhile there are sone varlalfons in the stages of

neural tube formation anongst species, generally primary neurulation or

closure of the neural tube can be considered to begin with the appearance

of the neural plale as a thickening of the dorsal ectoderm (0tRahitly &

Gardner 1979), followed by elevatlon of the lateral narglns of the neural

plate to forn the neural folds, neural cnest cell nigrati.onr and fusion of

the neural folds to forn a neuroepithelium discrete fron the surface

ectodern (Canpbell et aI 1986). In nannalfan enbryos lthas been shown

(Morris & Solursh 1977, Morriss & New 1979, Copp et aI 1990) bhat neural

tube fusÍon occurs initially ln the cervical reglon conüÍnulng in rostral

and caudal dÍrections. Closure contlnues to the upper hindlinb (otic

1_7

Ievel) whÍIe cephalic neural tube closune occurs by fusion of the rostral

tip of the neural folds and an fnterrnedfate point in the region of the

posberior forebrain forming the large prlnary forebrain vesiclq This is

followed by complete closure of the cephalic neural tube and then

progressive fusion caudally from bhe cervical region. Fusion of the

anterior and posberior neuropores is conpleted by gestation days 26-30 Ín

hunans (LemÍre 1982) and 11.5 in rats (Shepard 1989). Following cl-osure of

the posterj.or neuropore, secondary neurulation occurs in the caudal region

which fs nore relevant in species such as rodents where nearly half of the

vertebrae are caudal (Copp et al 1990). The events comprising secondary

neurulation, which has been associ.ated with spinal malformatlon (Hughes

and Freeman 1974), occurs sonetime following the period during which

embryos were exa¡nined in this thesis.

While the aetiology of nost teratology Ís unknown, the vulnerability of

mother and developing fetus to nutrftlonal and environmental exogenous

factors has been recognÍsed. Marry, if not nost of these substances readÍly

cross the placenta and/or are excreted into the breasü nilk, and nay even

accumulate Ín the fetus, which does not yet have the well developed

netabolic and excretory nechanÍsns of the adulb (Mirniran & DeBoer 1988).

The author has chosen to exanÍne the teratogeni.c effects of five of these

exogenous factors to whÍch young pregnanf vJornen are frequently exposed as

part of their diet and/or Llfestyle. I'Ihile they all fall outside the

category of known hurnan teratogens (see Tabte 1.3)r alt fÍve agents have

been lnpllcated in sone studies in both humans and aninals as havÍng a

detrímental effect on the developlng brain, anong other slructures. Hlgh

levels of these agents n¡ay accunulate fn the brain which is not adequately

protected by a blood - brain banrler early in developnent. This study will

be concerned nalrùy with an assessnent of the risk to brain developnent by

1-8

detailed exanÍnabion and evaluation of neural tube dysnorphology at gross,

histological and ultrastrucbural 1evels.

Although it has been recognised that wonen are exposed to a whole range

of exogenous agents whether voluntarily or involuntarily during pregnancy,

mosl experinental studies so far have exanined bhe dysmonphogenic effects

of a particular toxicant in Ísolatlon rather than conbined with ot,her

agents. Hence the contribution that conconitanf exposure to two or more

teratogens nakes to the overall aetíolory of hunan malfornation Ís still

unknown. It, is likely that studles of this kind wÍIl lead to the

recognition of nore human teratogens since sone compounds nay only er¡er be

ingested/admlnisbered at sub- or low teratogenlc IeveÌs alone, and Ít ls

orùy when several agents are present sinuLtaneously thaü potentiat,ion nay

occur, thus raising the threshold such that an obvious teratogenic

response ensues or is enhanced, resulting in a change in the frequency or

severity of a defect. This may occur eÍther as an interactive or addltive

(independenü) process possibly depending on whether the two teratogens

exert their effect on the enbryo lhrough the same or different nechanlsms

respectively. For example, si.nce compounds 11ke alcohol and salicylic aeid

cause simÍIar dysnorpholog¡r such as neural tube defects in rats, it is

likely that administration of both concurrently may lead to an lncrease in

frequency of dysnorphology which when assessed statistÍcalIy can establish

¡¡hether the two teratogens exert t,heir effect on the neural tube

independently or intenactively. Hence, Íf pregnant !¡omen expose

thenselves to several noxious agents I ike al cohol and tobacco

slnultaneously, the potential fs there for nore serÍous consequences to

develop fn theÍr unborn chÍldren than if an agenl is ingested alone.

Ihe dietary nanipulatlons irvestlgated for their teratogenÍc potential Ín

1_9

this study were a deficÍency of the trace el-ernent zinc and an excess of

the vitanin A. Optimal nutritional status requíres that at least 10 trace

elements including zinc, which are known to be essential for higher

animals (Moghissl 1981) are represented in the dlet. In the human setting,

diet' or lifestyle can lead to marginal supplies of trace elenents, which

Ís of particular relevance as Oberleas & Caldwell (1981) have poÍnted out

t'hat mild deficiency states nay be equally as criticaL Ín produclng

effects as severe malnutrition Recent studies from our laboratory (Record

et aI 1985a) have reported that nany prenenopausal women are nildly zinc

deficient, and nauseated pregnant r{omen may also suffer frorn zinc

deficiency (pfeiffer & Barnes 1981). Of particular interest Ís the

observat,ion that in areas of the world where zinc deficiency is prevalent,,

nalfornations of the central nen¡ous systen are especÍa1ly high (Sever &

Emnanuel 197Ð and include anencephaly (Cavdar et al 1982). It nay also be

no coincidence that children in these areas with a dietary insufficÍency

are slow learners (PfeÍffer & Barnes 1981). Furthermore, ÌJor¡en who suffer

fron acrodermaüitis enteropathica, a dtsease which 1nüerferes with

adequate dietary zfnc absorption, have also been delivered of anencephalic

lnfants (Hanbidge et al 197Ð.

Despite the unlversal use of various vltanln preparations by pregnant

wonen, only 18 cases have appeared over the past 25 years that rerate

excess vÍtanÍn A lnt'ake to adverse effects on the progeny (SchardeÍn &

Keller 1989). Ihe fads wlth rrnegavitaninn consumption however may change

this sltuaü1on as the so-called health giving benefÍts of large doses of

vltanins dÍseussed 1n sone popular health nagazines rnay glve way to less

than healthy consequences 1n wonen and thelr unborn chfldren Vftanin A

and analogues such as isotretÍnoin have been frequently ingested for the

treatnent of serious acne, often with serlous slde effects (Stange et al

1-10

1978, Stutt,gen 1975¡ l.lindhorst & Nigra 1984), and when used by young,

pregnant women have been shown to Iead to central nervous systern (CNS)

abnornalities, craniofacial, cardiovascufan and ear defect,s in their

unborn children (Rosa et, al 1986).

In contrast to these nutritionally derÍved teratogens, a nunber of agents

that are used voluntarily for personal or social reasons, or for

therapeutic benefit will also be considered for their potentÍal as neural-

tube teratogens. these Íncl-ude tobacco use through smoking, alcohol

consunption and the ingestionof analgesics. Since ¡nost or aIl- of these

factons enlail sone risk whether to pregnancy or to other aspects of

health, there is concern over their use, and nany reports have suggested

that these controllable factors in t.he envinonnent nay account for a

significant nunber of reproductÍve failures.

It has been estimated that fron 20 Lo 501l of women smoke during pregnancy

(AbeI 1980) which is an alarming proportion as smoking presents particular

dangers to the offspring, particularly in relabion to substantial

diminution of birthweight (l'feredÍth 197Ð. Smoking nothers were also BOf

nore likely to have a spontaneous abortion than were non-smokers, while

of 870,000 perinatal deafhs in the U.S, 4600 or over 5Í were attributed

directly to this cause (Epstein 1 978). Vlhether smoking has any association

with congenital abnormalities 1s stilL uncertain, although an increased

risk of congenital nalfornations has been reported (Kelsey et al 1978,

Christianson 1p80, Van den Eeden et aI 1990) anong offspring of wonen who

snoked nore than one pack of cigareütes per day. Neural üube defects such

as anencephaly (l'laeye 1978), along w ith impaired learning ability (Butler

& Goldsteln 1973, Streissguth 1978), have been found to be markedly

increased ln fhe offspring of women who are heavy snokers .

1-11

Alcohol, whÍch has been referred to as the major teratogen of the decade

(Schardein 1985) has been reported by one group (HiII eb al 1977) to be

fngested by 68f of wonen during pregnancy, wifhTl of them consunÍng it

daily. In another studyr gl of pregnant wo¡nenwerereported to be heavy

drlnkers (Rosett et aI 1978). Jones & Smith (.l973) descrÍbed a distinct

dysnorphic conditlon associated w ith maternal, gestational aI cohol ism

which they terned the fetaL alehol syndrone (FA,S) and whieh included CNS

defects such as nicrocephaly and hydrocephaly, and facial anonalÍes.

MenbaI retardation is one of the nost conrnon and serÍous problens

assoclated with the FAS, and alcohol has been reported to be its most

frequent cause in the western world (Clarren & Smith 1978) with between

3000 and 6000 babies born mentally retarded 1n the U.S. annually. 0n1y

linited neuropathological studies have been performed to date in humans

and these have indfcated cerebellar dysplasla and helerobopÍc cel1

clusters as consistent anonalies. Aninal studles have denonstrated all

aspects of bhe FAS reporfed in humans, and several have reported cel1

death in the developing neural tube (flebster et al t983; Bannigan &

Cottell 1984; Sullk et al 1988) which may be llnked with the gross neural

dysnorphologr frequently reported fn these studÍes.

SalÍcylic acid, or aspirin, the final agent exanined Ín this süudy, 1s

the nost widely used drug ln the world and anong its nar:y uses it acts at

hfgh doses as an anti-inflannatory agent in condÍtions such as arthrÍtis,

or as an analgesic, where in sone cases when taken in excessive quantÍtfes

it can be considered as a substance of abuse. The knowledge that two-

ühinds of pregnant wonen are reported to have taken aspirin durlng

pregnancy (H111 1973, Forfar & Nelson 1973) is sonewhat alarnlng as a

nunber of reports have associated salicylates wlth the fnduction of

congenltal abnornalltfes ln the offspring of sone pregnant women, and the

1- 12

observation that these drugs are active teratogens in animals has also

heighbened concern over this potentíal. As is the case for so nany

potentially teratogenic agents, there are some reports which demonstrate

thaf a high analgesic intake can lead to the birth of infants with a

raised incidence of CNS defects and other abnornalities (Ne1son & Forfar

1971), while other studies have found no assocÍation between the use of

these drugs in pregnancy and congenital malfornations (Slone et aI 1976),

stillbirth, neonatal death and reduced birthweight (Shapiro et al 1976).

For many of the agents which induce dysmorphogenesis in humans and

animals, the mechanisns whtch underlÍe these abnormalitÍes are yet to be

elucidated, and will probably require a multidisciplinary approach

involving electron microscopists, molecular biologists, geneticists and

others. Some clues towards an eventual solution may be found in

hisbological and ultrastructural anaLysis, since these techniques can

reveal pathologicat information such as the sibe and severity of cell

death and the types of organelles affected, which may provide some insight

inbo bhe kinds of cell functions that the terafogenlc agent may inberfere

w 1th.

Although zínc deficiency, hypervitaminosis A, nicotine, alcohol and

salicylic acid have alL been previously examÍned for their teratogenic

potent,ial in humans and animal studies, few have assessed the pathological

effects of the agents on the developing conceptus and especially on the

neural tube, hence nany questions renaln unanswered. Most studies have

examined drug and environmentally-induced dysnorphology at the end of

gestation, far fron the critical period of organogenesis when most

dysnorphogenic compounds exert theÍr effect. Furthernore, nost

observations have been u¡ade predoninantly on fetuses extracted from the

naternal environmentr rather than

direc! effecb of the agent on the

fron embrYo cu1ture

1_13

whÍch a1lows bhe

conceptus to be determined.

The first aÍn of this thesis is bo examine the effects j-¡0 vivo or in

vitro on the development of rat embryos exposed to each of fiveagentst

during bhe criticar period of neurogenesls (from days 9'5 to 11'5) in

order to gain furüher insight into the norphologícal abnormalities and

associated cytological and ultraslructural changes, with particular

emphasis on the neural tube. A conparisonwill then be made between the

neural abnornalitÍes induced by these agents at alI these Ievels' From

these observalions the author hopes to assess whether a link could exist

between any overt neural tube dysnorphology and pathologÍcal changes

observed.

The second aim of this thesis is to study the effects of several

combÍnations of the agents on enbryonic growth and development on day 11.5

of gestatÍon following t,he closure of the neural tube, and to assess both

quantitatively and qualitatively whether simultaneous exposure to two

agents witl bring about an additive or synergistic mode of action'

parbÍcularly in relatÍon to morphological and ultrastructural changes in

the developing neural tube.

Throughout this study, an animal model was used to exanine the enbryos

nidway in pregnancy Ímmediately followíng the crÍtical peniod for

neurogenesis. This is of particular relevance for studies of neunal bube

dysmorphology as it enables assessrnent of early ulbraslructural and

cytological aberratÍons often not evident later in pregnancy, but which

nay be relevant to the presence of nental retardatlon and intell-ectual and

behavioural dysfunction postnatally. It also provides a more accurate

determination of the frequency of nalfornabi.ons as abnormalities in ter¡n

1-14

studies may be nasked by early resorptions.

In order thab zinc deficiency and hypervitamÍnosis A could be studied

individually and in conbination these studies were performed ;Ln vÍvo since

embryos grown j.n zinc deficient nedium do nob demonstrate comparable

dysnorphologr wlth those developed j.n vivo (Record et aI 1985c). Hence in

order to nake the condltÍons of treatment of zinc defÍciency and

hypervitaninosis A equivalent for the combined study, the viüamÍn A study

was also perforned in vlvo. The remaining agents were administered in

vitro which ensures that ar¡y enbryonic dysnorpholory was a direct

consequence of the teratogenic properties of the agent rather than a

secondary effect of some disturbance to the naternal organism. The

technÍque also allowed developnent to be followed throughout the period of

neurogene si s.

Because lhe author wished to provide a conprehenslve background on the

llteratwe pertainlng to each agent examlned, which was often extensive,

Iiterature reviews for each agent w111 be contained in separate

introductions at the beglnning of the appropriate chaptens, with

correspondlng bibllographies at the end. Because of the number of agents

exa¡nined and the diversity and conplexity of the Íssues involved the

author found 1t necessary t,o provide exbensÍve dÍscussions of results

within the respectlve chapters of this thesis. Therefore the general

discusslon which constitutes chapter 11 will prwlde an overview of alt

discussions brÍngÍng together the najor thenes presented 1n the indivÍdual

chapters. FuI1 papers, and abstracts of conmunfcat,lons containing work

fron thls thesis which have been presented at cor¡ferences Ís docunented 1n

Appendix 1.

1-15

1.2 BIBLIOGRAPITY.

Abel, EL on growth

and develBanniga mice: an

electron George, RToxicol. 24, 483-500.

of drugs in the first trinester of261-214.moking in pregnancy and subsequent57 5.

canpbelI, LR., Dayton, DH. & SohaI, GS. (1986) NeuraI tube defects:a review ofhunan ãna aninal studies on the ebiology of neural tube

defects. Teratolory 34, 171-187 'Cavdar, AO., Bala;;, 8., Arcasoy, A. & Erten, U. ( 1982). Ef fect of

nubrÍtion on serun zÍnc concentratio¡ during pregnancy in lurkish wonen'

An. J. cltn. Nutr. 33, 542-544'Christtanson, n. iiggo). The relationship betw een naternal smoklng and

the incidence of congenital anonalies. An. J. EpldenÍol' 112' 684-695'

crarren, sK. ¿ J;ät, D!ü. (19?8). Fetal alcohor svndrome, N' Engl' J'

Med. 298, 1063-1067.Copp, AJ., Brook, FA., Estibeiro, JP', Shun, ASIJ' & Cockroft' DL' (1990)

The enbryonic dévelopnent of mannalian neural tube defects' Progr'Neurobiol . 35, 3$3-403.Drew, JIL, lueír, DA: & Beischer, NA. ( 197 8). Congeni tal mal formatj'ons of

abnornal glucose tolerance, and estriot excretÍon in pregnancy' 0bstet'Gynecol. 51, 129-132.Epstein, ss. írgiAl. The polilics of Cancer. sferra Club Books' san

Francisco.Fantet, AG., Shepard, TH., Vadhei¡n-Roth, C., Stephens, TD. & Coleman, C.

(1g80). Embryoniqand fetal phenotypes: Prevalence and other assoclatedfactors in a large study of sponta eous abortions. In: 9th BÍrth Defecbs

Synposfum of theNew york State Birth Defects Instítute on Hurnan Embryonic

and Fetal Death. Porter, IH. & Hook, m. (eds)' Acadenic Press' New York'

Forfar, JO. & Nelson, I.'íU. (1973) Epideniology of drugs taken by pregnanb

lronen: Drugs that nay affect the fetus adversely. cIÍn Pharnacol' Ther'

14,632-635.fi'""ã", Éõ. t 1g77). Relationshíp of aninal studies to naru In: Handbook

of Teratology, VoI 1. Ïlilson, JG. & Fraser, ¡'C. (eds). Plenum Press, New

York.Hanbfdge, KM., Neldner, KII. & ÏIalravens, PA. (19?5). Zinc, acrodernatitis

enteropathfca and conger¡ttal malfornations. Lancet 1, 5TT-5'18'-iift,'nn (1973) orugs ingested by pregnant women CI1n Pharnacol. Ther'

14' 654-6fl' ¡{ c .Ip-- ehânev, LM. & Mccurrey, LB. ( 19??)'Hitf, RM., Craig, JP., Chaney, MD', Tennysont

utir izatf on of ñ""- lhe-counter drugs during pregnancy. crln. obstet'Gynecol. 20, 381-394

Hughes, AF. a ï"ãér"n, RB. ( 19?4) Conparative renarks on the development

of the tailcord amon8 higher vertebrates. J. Enbryol. Exp' Morphol' 32'

3 55-363 .

Jones, KL., Snith, Dl,l., UIleIand, CN., et al (1973). Pattern ofnalformabtons in offspring of chronic aIcohoIlc nothers' Lancet 2'

1267 -1Zl 1 .

KaIter, H. & I{arkany, J. (19s3). congenftal Malfornations' New Engl' J'

1-16

Med. 308, 424-431.Kelsey, JL., Dwyer, T.' Holford, TR. & Bracken, MB. (1978). Maternal

smoking and congenital nalformations: an epideniological study. J.Epidemiol. Commun. HIth. 32, 102-1OTLenÍre, RJ. (1982) Neural tube defects: CIinicaI correlations. In:

Proceedings of the Congress of NeurologÍcaI Surgeons, Toronto, Canada.t{Ítlians & }Iilkins, Baltimore. pp 165-177.

Meredibh, HV. (197Ð, Relation between tobacco smoking of pregnant wonenand body sÍze of their progeny: a compilation and synthesÍs of publishedstudies. Human Biol. 47, 451-472.MÍrrniran, M. & De Boer, S. (1988). Long term effects of chemicals on

developing brain and behaviour. fn: Teratogens - Chemicals which causeBirth Defects. Meyers, VK. (ed). ELsevier, New York. pp27 1-314.Moghissi, KS. (1981). Risks and benefits of nutritional supplements

during pregnancy. Obstet. Gynecol. 58, 68-78.Morriss, GM. & New, DAT. (1979) Effect of oxygen concentration on

morphogenesj.s of cranial neural folds and neural crest in cultured ratembryos. J. Embryol. Exp. Morph. 54, 17-35.Morrlss, GM. & SoIursh, M. (1978) The role of primary nesenchyne in

normal and abnorrnal morphogenesis of namnalian neuraL folds. Zoon(Formshaping Movements in Neurogenesis) 6' 33-38.Naeye, RL. (1978). Relationships of cÍgarette smoking to congenital

anomalÍes and perinatal death. Am. J. Path. 90, 289-293.National Foundation (1975). Natlonal Foundation. March of Dirnes: Facts.

New YorkNelson, MM. & Forfar, J0. (1971). Associations between drugs adnÍnistered

during pregnancy and congenital anomalies of the fetus. Br. Med. J. 1t523-527 .

Ober1eas, D. & Caldwell, DF. (1981). Trace Minerals in pregnancy. fnt. J.Envlron. Stud. 17, 85-98.0'Rahilly, R. & Gardner, E. (1979) The initial developnent of the human

brain. Acta Anat. 104, 123-133.Pfeiffer, CC. & Barnes, B. (1981). RoIe of zinc, nanganese, chromium,

and vitanin deficiencÍes in blrth defects. Int. J. Environ. Stud. 17, 43-56.Record, IR., Record, SJ., Dreostí, IE. & Rohan, TE. (t985a). Dietary zinc

Íntake of pre-nenopausal wonen Hun. Nutr. Appl. Nutr. 394, 363-369.Record, fR., Dreosti, IE. & Tulsf, RS. (t985b). J.n vitro development of

zinc deficient and reptete rat embryos. Aust. J. Exp. BÍo1. Med. Sci.63,65-7 1 .Rosa, FtJ., Wilk, AL. & Kelsey, F0. (1986).Teratogen update: vitamin A

congeners. Teratotogy 33, 355-364.Rosett, HL., OuelIetLe, EM., I{einer, L. & 0wens, E. (t978). Therapy of

heavy drÍnkÍng during pregnancy. Obstet. Gynecol. 51, 41-46.Schardein, JL. (1985). ChenicalJ.y Induced Birth Defects. Marcel Dekker,

New York.Schardein, JL. & Kel1er, KA (1989). Potential human developnental

toxicants and the role of aninal testÍng Ín t,heir ldentificalÍon andcharacterisation Crit. Rev. Toxicol. 19, 251-339.Sever, LE & Ennanuel, I. (1973). Is there a connectÍon between naternaL

zinc deficlency and congenftal rnalfornations of lhe central nervoussysten in man? TeratoLogy 7, 117-119.Shapiro, S., Monson, RR., Kaufnan, DW., SiskÍnd, V., Heinonenr 0P. &

Slone, D. (1976). Perinat,al nortality and birth-wefght in relation toaspÍrin taken during pregnancy. Lancet. 1, 1375-1376.Shepard, TH. (1979). Teratogenicity of therapeutic agents. Current

1-17

problens in pediatrics. year Book Medical publishers. chicago.shepard, TH. (1986). Human Terat,ogenicity. Adv. pediatr. 3t, 225-26g,shepard, TH. (1989). A catalog of reratogenic Agents. 6tr¡1on The JohnsHopkins University press, Balti¡nore.shepard' TH. & Fantel, AG. (1979). Embryonic and early fetal Ioss. Clin.Perinatot. 6, 219-243.Slone, D., Siskind, V., Helnonen, Op, et aI. (1976). Aspirin andcongenital malformations. Lancet 1, 1373-1375.st'ange, L., Carlstron, K. & Erikkson, M. (197g). Hypervitaminosis inearly human pregnancy and malfornations of the centraL nervous system.Acta Obstet. Gynecol. Scand. 57, Zgg-291.streissguth, Ap. (1986) smoking and drinking during pregnancy andoffspring learnÍng disabilities: A review of the literature anddeveloprnent of a research stralegy. rn: Learning disabilities and prenatalrisk. Lewis, M. (eo). university of rllinois Press, rltinois. pp e-g-62.stuttgen,G.(1 gT s).oral vitanin A acid therapy.Act,a Dernatoi. venerol.(Suppl). 24, 174-179.surik, KK., cook, cs. & Ï'lebster, vls. ( 19gg). Teratogens and craniof acialnaLformations: Relationships to cell death. Developnent, 103 (suppl).

213-232.Tuchnann-DupIessis, H. (19TT). Drug Effects on bhe Fetus. Adis press,

New York.Van den Eeden, SK., Karagas, MR., Daling, JR., Vaughan, TL. (1990) Acase-control study of naternal snoking and congenital nalfornations.Paediatr. Perinat. Epideniot. 4, 147-155.ïlarkany, J. (1928). Terathanasia. Teratology 17, 1g7-192.Íüebster, WS., Ïf a1sh, DA., McEwan, SE. & Lipson, AH. ( 19S3). So¡oeteratogenic properties of ethanol and acetaLdehyde in c57B1/6J mice:rnplications for the study of t'he fetaL alcohol syndrone. Teratologr 2f,231-243.ÏJiIson, JG. ( l9T3). Envi.ronnent and Birth Def ects. Acadenic press, New

York.Windhorst, DB. & Nigra, T. (1982). General clinical toxlcology of onalretinoids. J. A¡n. Acad. DermatoL. 6, 675_692.

2-1

CHAPTER 2

MATERIALS AND METTTODS

2.1 . Animal s

The Sprague-Dawley straín of rat was used throughout these studÍes. They

w ere housed and naintained within the aninal facilfty of CSIR0

DivÍsion of Hunan Nutrit,ion, Adelaide, South Australla. AII animal

experinents were perforned according to the requÍnenenls of the

CSIRO/NHMRC guidellnes on the use of aninals for scientific purposes, and

$tere approved by the appnoprÍate ethics commÍttee prÍor to co¡nnencement.

Aninals were naintained on a 12 hour Iighf-dark cycle and were glven free

access to both colony diet and water untÍI the tine of nating. !{hen

enbryos $¡ere required, vlrgfn females of the appropriate age and weighf

wene placed overnight with suitable nales of the same strain kegnancy

was always established by the detectlon of spern in the vaginal snear the

following norning. CopulatÍon vras assumed to have occurred at mÍdnight,

nidway through the dark cycle. Hence the time of det,ection of vaginal

sperm vJas designated as day 0.5 of gestafion At the tfme of spern

detectÍon dams used in the i¡ vivo studies were randomly assigned to

appropriate experi.nental and control groups and were housed Índividually

f n plastlc and sbairùess steel cages wÍth staÍrùess steel mesh floors to

ensure that, faecal ingestion dld not occun The aninals were provlded with

glass disfÍlled water ad 1Íbltun, and food 1n accordance with the

requirenents of the lndlvidual experinent. Pregnant fenales used ln the ;i¡

vÍtro studies were housed comnunally in chronium plated wire cages with

free access to glass distilled $¡ater and colony diel unüil they $¡ere

sacrfficed on day 9.5 of gestatÍon

2-2

2 .2 Diet s

The colony diet that all animals were maintained on prior to matingrand

until the time of sacrifice for rats used in the in viÈro studies

consisted of a commercially available pelleted food (pno-nAt Milling

Industries, Adelaide, S.A.). During the experinental period, the rats used

in the in vivo studies received semi-synthetic soybean based diets which

were either zinc deficient or zinc replete according to the group to which

the animals were assigned.

Preparation of the semi-synthetic diet consisted of initially heating the

soy flour (SoYlArn, H.J. Langdon & Co, Melbourne, Victoria) in an oven for

one hour aË 105oC in order to inactivate the trypsin inhibitory factor. An

appropriate atrount (approximately f.5 Kg) of the soyflour was nixed with

hot 95i,, ethanol for 30 nins and filtered through a Buchner funnel to

remove the fat componenÈ of the flour. The trace elements including zinc

$rere removed by extracting divalent cations fron the flour by suspending it

in approximately 8 liÈres of water and 14g of Na EDTA and adjusting the pll

to the isoelectric point (pII 4.0) to prevent protein fron becoming ionised

and lost Ëhrough filtration. The mixture was stirred for 2 hours and

filtered. The EDTA step was repeated and Èhe flour was washed Èhree times

with metal-free distilled water to remove any Ëraces of EDTA. After a

further extraction with re-distilled hot ethanol, the flour was allowed to

air-dry and was stored at -20oC until required. The zinc content of the

soy flour was reduced fron 40-50 ug/g to less than 0.5 ug/g, which nas

deËermined by flame atomic absorption spectrophotonetry. Íthen required,

levels of zinc qrere adjusted by adding it in the forro of zinc sulphate

to a final level of 100 vglg. Each batch of the soy-based diet was

assayed for zinc and copper prior to use. Together with soy flour which

formed the basis for the semi-synthetic zinc-deficient diet, the

2-3

percentages of the najor components are listed Ín Table 2.1.

Table 2.1 ConposÍtion of t'he zinc-def icÍent dieb.

Conponent dþ

Soya-bean flour

Sucro se

Sunflol,¡er oil

SaIt nix

DL-methionine

Vitanin nix

43

44.2

8

4

0.7

0.1

The constituents of the seni-synthetic and comnercial diets are detailed

in Tabl e 2.2, where they are conpared with the nubrient requirements of

laboratory aninals of the Anerican InstÍtute of NutrÍtion (Natlonal

Academy of Sciences 1978).

Prevlously deternined growth rates (Record 1986, unpublished PhD thesÍs)

had demonstrated that animals fed the soy flour based dlet received the

essential nutrients provided by the conmercial diet when supplenented with

zinc, and that the onisslon of zinc Led to a deleterious effect' on growttt"

2.3 Tissue removal and embryonic assessmenf.

For the j¡ vivo experiments dans were sacrificed on day 11.5 of

gestation under dÍethyl ether anaesthesÍa and blood sanples were removed

by cardiac puncture and spun inmedialely to recover serum. The peritoneal

cavity ï¡as exposed and the uterus waa renoved. Each uberine horn was cut

longttudinally to the Ievel of the ovarÍes and the inplanted enbryos

encased ln decLdual tissue were excised and transferred to a petrl dish

containing Hanks Balanced SaIt solution (HBSS) pH 7.3.

2-4

Table 2.2 Conparison of commercial and semi-synthetic diets with

recommendations of the National Academy of Science'

ConstituenË Conmercial Semi-synthetic N. A. S.

ProteinCrude fatDigestible energyCrude fibreS ucrose

Cal ciurnPho s ph or ousZincCopperIronMangane seMolybdenumIodineSodiun chlorideMagne sium

23%(mixed protein) l9% (soy)3 .1"Á 87"

12800 rJ/Ke 16000 r.¡/Kg4.8i4 N.D.N. S. 44i(

121Á(ídeaL)57"

15900 x¡/Kg

.57.

.4/o

N. S.N.S.

I0gmg

mg

mg

00

T25

3550

ngmg

mgEg

I0

615

66L02

I20

42r

4000 ru4mg3rg

20 mg

8rg6rg

50 ugN. S.

1000 ru30 IU50 ugIrgN. S.N. S.N. S.

7 500 ru15 mg

5rg15 mg

25 mg15 mg

15 ug50 mg

750 IU60 IU12 .5 rog

0.3 mg

125 ug250 mg

.rÁ

.967"mg

r0g

.867.mg

0 .57"r.07"

100 mg12 og

190 mg

28 mg

N. D.7rg0.67"

470 ng

5mg

b

N. S.0 .15 mg

0.05"/"400 mg

Choline chlorideMeth ionine

Vitanin A 22500 rtJThiamine 58 ngRiboflavin 5 mg

Niacin 18 ngCalcium Pantothenate 20 ngPyridoxine 5 mg

Vitamin Bl2 120 ugAscorbic Acid N.D.Vitamin D3 4000 IUVitanin E 36 IUVitamin K 8.3 mg

Folic acid 2 ,gBiorin 110 ugInositol N.D.Para-amino benzoic acid N.D.

7004

500 mg

3.5 e

mg

.2g1000 mg

6s

N. D.N. S.

ab

: not deternined: not specified: sunflower oil: zinc was onitted frorn the zinc-deficient diets

2_5

Under the dissecting microscope, each embryo was then dissected free

of decidual Èissue using fine stainless steel forceps. The visceral

yolk sac was examined for the presence of viable blood vessels, opened

and the embryo exanined for the occurrence of fusion of the allantois

with the chorion Eo f orm the chorio-allantoic placenta which was then

renoved together with the amnion.

Several growth parameEers were assessed for each embryo using a

dissecting nicroscope. The crown-rump lengEh ÞJas measured with the aid of

a graticule inserted inËo the eyepiece and Èhe number of sonites was also

counted. In some cases protein estimations of t.he whole embryo were

determined by a modification of the method by Lowry et al (1951) after

storing the tissue al -20o C. Erobryos were also examined under the

dissecËing microscope for evidence of gross dysmorphology, which in order

to ensure uniformity in each litter rüas recorded on standard scoring

sheets based on those of Brown and Fabro (f9Bl). Some embryos were

photographed when required using Kodak Tri-X pan film, 400 ASA, while a

number from each dam were selected and prepared for scanning electron

microscopy (SnU). The anterior neural tubes of other embryos were prepared

for exanination at the cel1ular and ultrastructural levels using the

light and transmission electron nicroscopes (rnu) respectively.

2.4 In vitro culture techniques.

2.4.L Explantation of embryos.

The methods of explanting and culturing rat embryos in vitro in this study

is baged on those described by New (1971,1978), and were performed under

aseptic conditions. The uterus froro each 9.5 day pregnant animal was

removed and the uterine horns were opened longitudinally with fine

2-6

scissors, exposing the pearshaped rnasses of decidual tissue w hich

surrounded each embryo and its nenbranes . These decidua r¡ere carefully

renoved from their implantation sites with curved forceps, and

transferred to a petri dish containing HBSS. The embryo and its nembranes

(fig 2.1) vrere dissected free of the decidua by carefully teasing it

apart at the broad end of the decidual pear with fine watchmakers forceps

until it separated into trso halves. The headfold stage rat embryos and

surrounding membranes lay inEact. in one of these halves and could be

dissected free with relative ease. The delicate Reicherts menbrane with

attached trophoblast and parietal endoderm sras torn apart using fine

forceps, leaving intact the embryo, with ectoplacental cone and yolk sac,

Ectoplacenta I

Utcrine cone

Chorion

Yolk sac

rval I

Decidua

Amnion

Head fold

Reichert Allantoismembrane

Fig. 2.I. Section through head-fold stage rat embryo and embryonicmembranes of 9.5 days gestation. In the uterus (tett) and as explanÈed forculturing (rigbt). The total length of the conceptus at this stage, fronthe ectoplacenËal cone at one end to the developing enbryo at the other,is about I.5-2.0 nm. Later the embryo becomes surrounded by the amnion andthe yolk sac, and the allantois extends and fuses with the chorion andectoplacental cone to form Èbe placenta. After New (1978).

which was explanted with the conceptus' Removal

structure peculiar Èo rodents and insectivores,

can expand in cultu¡g (New 1973).

2-7

of Reicherts membrane, a

ensures ÈhaÈ Èhe yolk sac

2.4,2 Preparation of culture medium.

Blood vras collected by cardiac puncture from male and PregnanÈ female

raEs, and immediately centrifuged before clotting could occur so that

blood cells r{ere precipitated and a clear fibrin clot was formed

separa¡ely from the cells in the plasma layer above (New 1978). The clot

rdas squeezed using curved forceps releasing the serun and then discarded.

The blood was re-centrifuged and the serum was collecÈed using a glass

pipette and stored at -20"C until required. The serum was always

imnediately cenErifuged as this is considered to provide a betÈer growth

medium for headfold stage embryos than delayed centrifuged serum (Steele &

New 1974). Prior !o use, Ëhe serum sras heat inactivated by immersing it in

a rdater bath at 56"C for 30 minutes, a procedure which is also considered

to inprove its capacity to support embryonic growth and prevent

abnormalities in cultured embryos (Steele & New I97Ð., The heat treaÈnent

is a standard method for inactivating complement in serum when Reicherts

membrane which might act as a partial barrier to immune reactions between

rnother and embryo is no longer intacÈ (Steele & New L97Ð, although it has

also been suggested that the effects on embryonic growth of heat-

inactivated serum nay be concerned nore with nutrition than antigenicity

(New I 97 8).

2,4.3 Culture chanbers and gas mixtures

Three undanaged head fold sÈage enbryos were placed in sÈerile 60 ml

cylindrical glass botÈles together with 90% of imnediately centrifuged,

heat inactivat,ed, pooled rat serum made up to a volume of 3 nl with

2-B

sterile water, antibiotics, streptomycin-G-sulphate (00 ug/mI) and

penicillin-G-sulphate (6 ug/nl) (rlein et al 1980), and where appropriate,

Ehe Èreatment conpound. The culture bottles were placed in a water batb

aE 37 oC and equilibrated at the commencenent of the culture period with 5%

OZ, 57" CO2 and 902 N2 which was humidified by bubbling through water. The

bottles were Èhen laid horizontally on rollers in an incubator and rotated

at 30 r.p.m. f or 48 hours aE a tenperaLure of. 37"C. Rotating rnediurn

promotes oxygenation and assists respiration through the gentle swirling

action of the explants in the medium, and also ensures maximun and even

exposure of the embryo Eo the teratogenic agenÈ (New & Cockroft 1979).

The enbryos were regassed with progressively increasing concentrations

of. O, at 20 hours when the gas mixture contained 20i¿ 02, 57. CO2 and 757"

N2 and at 40 hours when it consisted of 407" 02, 57" Co2 atd 557" N2.

Rat embryos explanÈed at the headfold stage (9.5days) by this method and

cultured for 48 hours in 907" rat serun have been shown to be

indistinguishable from those in vivo (New et al L976). After 48 hours in

culture, the enbryos were removed from the incubator and examined for

the presence of a heartbeat and yolk sac circulation, and for growth and

morphological development under a dissecting microscope.

2.5 Biochemical analysis.

2,5,L Protein estination.

Determination of protein content of the whole embryo was based on the

nethod of Lorury et aI (1951). Embryos previously removed from the uterus

and frozen aÈ -20"C were thawed and digested in a solution containing

0.057" Triton X-100 in 0.1 M NaOH. After digestion, the protein content of

equal aliquots rùas determined exactly by this method including the

standard (crystalline bovine serun albunin, Sigma, St.Louis) w hich was

2-9

also dissolved in this solution.

2.5.2. Trace elements.

A Perkin-Elmer HG-500 Atomic Absorption Spectrophotometer (Perkin-

Elmer,Norwa1k, N.J.) was used Ëo determine zinc levels in sera, by the

direct aspiration flame-mode technique (Wilkins et al L972) following

dilution with glass-distilled water. Diet sanples were wet-ashed in a

mixture of nitric and perchloric acids. Residues were then diluted

appropriaËely w ith glass-distilled water prior to trace element

determination (Record eÈ a1 1982).

2.6 Microscopy.

2.6.L. Light and transmission electron microscopy.

Whole embryos were fixed for 24 hours at 4"C in 0.1M phosphate buffer

with 3% paraformaldehyde and 37" glttaraldehyde at pH 7.4. The tissue !ùas

then placed on a rotator, washed twice for 10 minutes in 0.lM phosphate

buffer (pll 7.4), and posÈ-fixed for I hour in 1% aqueous osmium tetroxide

to preserve unsaturaÈed lipids and phospholipids, after which it was

rewashed for a furtber 10 minutes in 0.lM phosphate buffer before being

dehydraÈed through a series of alcohols of increasing concentration from

307. to L00%. Specimens were then treated with two changes of. L007" alcohol

and copper sulphate in a 1:1 ratio, followed by inmersion in propylene

oxide (l12 epoxy propane) aloner and Èhen in conbination with increasing

proportions of resin (Spurrs resin, TAAB, Berks, U.K.), until iË eras

finally enbedded in pure resin aÈ ¡rhich stage it was allowed to

polymerise at 60oC for 24 hours.

Glass knives were used to cuE rtÈhickt'horizont.al sections of cranial

tissue (0.5 un-l.0 un) on a Porter-Blun MT2-B Ultramicrotome. The tissue

was collected on slides, stained with hot toluidine blue (0.025Ð - borax

2_ 10

(0.51), coverslipped, and exanined and phot,ographed using an orynpus BH

compound light mÍcroscope. Thin sections (60-90 nm) were cut using a

dianond knife ( Diatone, switzerland) and coLlected on 3nn, 2oo mesh

copper/rhodium grids and staÍned with uranyl acetate and Iead citrate.Uranyl acetate was dispensed through a nillipore filter into smaLl_ !ùax

nboatsr¡ in a petri-dish, surrounded by Zof alcohor. The grids were

floated in the nboatsn for 25 ninutes in the dark, then dipped in TO%

alcohol and washed in distilled water and dried with filter paper. Lead

citrate drops were pipetted into a partitioned petri dish contafnine 10-20

sodiun hydroxide pelrets. The grids were appried to the drops for 12

minutes, washed in glass dist,llled water and then dried on filter paper

before being examined wfth a JEOL 100S electron nicroscope at 60 KV, and

photographed as required.

2.6.2. Scanning electron microscopy.

Enbryonic tissue for scanning etectron microscopy Þras processed in a

similar nanner to that, used for transmission electron nlcroscopy except

that it was dehydrated through a graded series of acetones (30l,-1ool)

instead of a1cohols, and then inmediat,ery critÍcally point dried(Balzers-Union, LÍchtenstein) from iso-anyl acetate using carbon dÍoxfde

as the exchange fluid. SpecÍ.nens $¡ere nounted and secured on aluninlunstubs by double sided tape and sputber coated to a thickness of 10-20 nn

with carbon and gold-palladÍun using a Denton Vacuun Evaporator (DSM-1).

The tlssue ÞJas then examined wtth an ETEC scanning electron nicroscope at20 KV and photographed when required uslng Kodak 120 panatomfc X fitn.

2.7. Statisülcal consideratfons.

Continuous growth varj.ables such as proteÍn content, sonlte nunbers,

crosllFrunp lengthr âs well as naternal serun zlnc levels were analysed

2-11

using a standard anal-ysis of variance (ANOVA) technique which tested for

main effects of the teratogens, alone and in conbinat,ion w here

appropriate. Tukey tests for multiple comparÍsons between neans of Lhe

treatment groups (Zar 1984) were aLso used.

For discrete developnent data such as the number of embryos with

defects or $rith specific nalformations, a maxinun Iikelihood of estimation

of the nain effects of the teratogens alone or in conbination was used

which involved treating the number of affected embryos within each

treat¡nent group as being binomially distributed as X2 on the appropriate

degrees of freedom (Baker & NeIder 1978). An available progran was

modified to suit, the data by Dr. Peter Baghurst of the CSIRO, DivÍsÍon of

Human Nutrition, Adelaide.

In chapters 5r I and 10 where the combined effects of two treatments were

exanÍ.ned, in order to evaLuate the degree of independence which the two

teratogens exert when adminÍstered concurrently, a specific progran was

developed by Dr. Peter Baghurst. Five models were postulaled as likely

alternatives to fit the data describÍng the effect of the teratogens on

dysnorpholory of a parlicular enbryonic structure, when the treatnents

w ere adm inistered concurrently :

Model 1:p=Po (deviance 4) proposes that there is no effect of either

of the treatments.

Model 2:p=Po.rA (deviance 3) proposes that there is an effect of

treatment A only.

Model 3:p=Po.rg (deviance 2) proposes that there is an effect of

treatnent B only.

Model 4: P=Po.r¡.rg (deviance 1) proposes that there are independent

effects of both treatnents.

2- 12

Model 5z P= Po.rA. rB."RS (deviance 0) proposes that there is an

interaction between the two treatments'

The relationship of the model to probability theory is outlined below'

Po indicates probability of an evenb (eg. a defect) given no nexposuren'

t.¡r FBr rAB are relative risks associated wÍth nexposurert to A,B and AB

respectiv elY.

o (3df) Model 1, (dev. 4)

Po."B (ZOr¡ Model 3, (dev. 2)

P

Model l, (dev. 3)

.rA.rB (1df) Model 4-, (dev. 1)

.rA.rB."ts (0df) Model 5, (dev. 0)

Differences between deviances for the appropriabe degrees of freedom for

X2 indicate whether the model should be accepted or rejected'

Differences between devÍances of Model 1 (dev.4) and Model 4 (dev.1)

when signifieant indicate that Model 1 shouÌd be rejected.

Differences between Model 2 (dev. 3) and Model 4 (dev' 1)' and between

Model3(dev.2)andModel4(deu1)whensignÍficantindicateaneffect

of treatnent B and of treatment A respectively affer taking the

alternale treatmenb inbo account wÍthin the conbined treatnent Sroup'

Po'"A ( 2df)

Differences between ModeL 4 (0ev. 1) and ModeI 5 (dev. 0)

signifícant indicate an interactive effect of the two treatments. A

significant value for X2 indicates that the two treatnents

independently when conbined.

2- 13

w hen

non-

act

As the deviance value for Model 5 wiII always be nOtr since 1t contains

sufficÍent pararneters to describe any data set of this nature exactly, a

separate colunn for Model 5 has not been Íncl-uded in the appropriate

Tables in the text.

2-14

2.8 BIBLIOGRAPHY.

Baker, R.I. & Nelder, JA- (1978). The GLIM system. Release 3. GeneralísedLlnear Interactive ModellÍngs. Numerlcal Algorithns Groupr Oxford.Brown, NA. & Fabro, S. (1981). Quantitatlon of rat embryonic development

:Ln vitro: A norphological scoring system. Teratology. 24, 65'78.Lowry, OH., Rosebrough, NJ., Farr, AL. & Randallr nJ. (1951). Proteln

measurenent with the Folin-Pheno1 reagent. J. Biol. Chen. 193, 265-275.NatÍonaL Acadeny of Sciences. (1978) Nutrient requÍrements of donestÍc

anÍmals, No. 10. Nutrient requirenents of laboratory anfmals. llashingbonD. C. ' 7-37 .New, DAT. (1971). Methods for the culture of post-implantation rodents.

fn: Methods in Mamnalian Embryology. DanÍ.eI, JC. (ed). ÌJ.H. Freenan &

Co,. San Francisco. pp 305-319.New, DAT. (1973). Studies on nammalj.an fetuses in vÍtro during the period

of organogenesis. In: The Mammalian Fetus:h vitro. AustÍn, Cn. (ed).Chapnan & HaIl, London pp 15-65.New¡ DAT. (1978). llhole-enbryo culture and the study of mammalian

embryos duning organogenesis. Biol. Rev. 53, 81-122.New, DAT. & Cockcroft, DL. (1979). A rotating bot,tle culture nebhod with

continuous replacenent of the gas phase. Experlent'ia 35r 138-140.New, DAT., Coppola, Pf. & Cockroft, DL. (1976). Conparison of growth lI

vÍtro and in vivo of posl-inplantaion raü enbryos. J. Enbryol. Exp.Monphol. 36, 133-144.Record, IR., DreostÍ, IE., Manuel, S. &Buck1ey, BA. (1982). Interaction

of cadmlun and zÍnc 1n cultured rat enbryos. Life Sci. 31, Zl35-2143.Record, IX. (1986). Unpublished PhD thesis. Dept. Anatony and Histofogy'

Univ. of Adelaide.Steele, CE. & New, DAT. (1974). Serum variants causing lhe fornation of

double hearts and other abnornalitles in explanted rat embryos. J.Enbryol. Exp. Morph. 31 , 707 -7"19.Vlilkins, PJ., Grey, PC. & Dreosti, IE. (1972). P1asma zinc as an

indicator of zinc status in rats. Br. J. Nutr. 27, 113-120.Zar, Jlt (1984). Biostaülstical Analysls. Prentice-HalI Inc, New Jersey.

3- 1

CHAPTER 3

IN UTERO GRChITI aNd DEVELOPMENT OF ZINC DEFICIENT EMBNYOS DURING

NEUROGENESIS

3.1 INTRODUCTION.

3.1.1 . Zínc netabolim.

Recognit'ion of the trace eL em ent zinc as an essential- nutrient f or

tivÍng organisns began in 1 869 when RaulÍn deternÍned that, zlnc $¡as

required for the growth of the bacterlun Aspergillus niger (RauIin 1869).

Todd et aI (1934) further denonstnated its inportance Ín the growth and

naintenance of rats, and since then a dietary lnsufficiency has been shown

in nany species lncludÍng hunans, t,o lead to Ímpaired maintenance, growth,

reproduction and even death Ín the nost severe cases (Underwood 1977).

Because of the extensive literature in exfstence pertalning to the

netabollsm and blochenical roles of zÍnc the auühor will mairùy provÍde an

account of those aspects of zinc that are relevant to bhÍs thesis and

dlrects the reader to a number of comprehenslve reviews (Underwood 1977,

Nriagu 1980; Prasad 1976,1983; Sigel 1983; Cunnane 198S) dealfng wlththese topfcs.

The zinc content of the adult hunan body 1s approxinatery 1.5 to 2.oc

wfth the hfghest, concentrations (100 uelg - 200 uB/Ð occurring in the

rellnar male reproductÍve organs, halr and bone, with intermediate

concentratlons (50-ug/g) present tn kldney, nuscle, l1ver, and lesser

concentratlons of zJ,nc Ín nost other tissues of the body (Underwood

19TT). About 80f of the total zinc in blood occurs in the erythrocytes,

nainly assoclaüed wfth the enzyne carbonfc anhydrase (Gardiner 1984)

3-2

which is of inportance in the bransport of carbon dioxide from the tissue

to the lungs (Lindskov 1971), as well as wit,h the proteÍn superoxide

disnutase (Bannister eb a1 1971) which is involved in the protecbion of

tissue (and erythrocytes) from danage by superoxide ions (McCord et al

197ù. The nean plasma zinc level in hunans is around 90ug/100ml wÍth nost

bound to proteins especial-Iy albumin which loosely binds about half of the

zinc and represents zinc in transport, whiJ.e aldla 2-macroglobulins and

ganma-globulins tightly bind rnost of the remainder w ith orùy a smal-l

percentage (7Ð toosely bound to amino acids (Solomons 197Ð.

3 .1 .2 . Zinc r eq uÍ renent s.

Ib see¡ns that the ninÍnaI dietary requirement for zinc cannot be readily

deternined. Metabolic balance studies have suggested an approxinate zÍnc

requirenent value of 6 mglday based on observations in several dífferent

populations (Sandstead 1982), however experimental inductÍon of zinc

deficiency by Sandstead (1985) in adult nen on mÍxed diets set the mínimal

zinc requÍrement at 4ng/day. These values depend largely on the dietary

bioavailability of the trace elenent for absorption which is believed to

be reduced 1n plant foods by the intake of high levels of dietary fibre

and phytate (inositol hexaphosphate) (HaLsted et aI 19741, Reinhol-d eb aI

1976; Sandstead 1982). The possible public health significance of the

inhibibion of zinc retention is evident when one considers thal foods

derived fron cereal grains are the major food source for much of the

worlds population

3.1.3 Absorplfoh and excretion of zinc.

In humans onLy 2Ol Eo 30ll of avaflable ingested dÍetary zÍnc is absorbed

(Sandstron & Cederblad 1980)r but the site and nechar¡ism of absorpbfon

have not been fully resolved. It appears however that zÍnc absorption Ín

3-3

hunans occurs nainly in the duodenum (EVans 1976), while in rats (Cousins

197Ð and dogs (Naveh et al- 1988) the ileum and jejunum feature as well.

The nechanÍsm of absorption may invol-ve a zinc-binding ligand which

appears to facililate the uptake and active transport of zÍnc across the

Íntestinal border (Hurley et aI 1977). The zinc-binding Iigand has been

varÍously suggested to be of pancreatÍc origin Ín sone studies (Evans &

Johnson 1980a) bub not in others (Naveh et aI 1988), and to lnvolve

prostaglandins (Song et aI 1988), picolinic acid (Evans & Johnson 1980b)

and cÍtrate (Hurley et aI 19TT). Zínc absorption seems to be dependent

upon a nunber of factors (Underwood 1977) Íncluding thelevel of intake

and nutriüional status of the individual, with higher levels being

absorbed in persons with poor nutritional status (Spencer et al 1980).

The elimination of zinc fron the body occurs nainly by way of the faeces

(Miller et aI 1966; Robinson et aI 1973) with a minimal urinary zinc

loss frorn 0.1 to 0.7 ng./day (Underwood 1977), conpared with a dletary

intake of 10 to 15 ng (Robinson et aI 1973, Spencer et al 1978).

Menstrual losses are snall (approxinat,ely 65 ug/day) (Underwood 1977),

but nay be of signifÍcance in a zinc deflcient lndividual, whlle

sweat l-osses are also generally neglÍgibte but nay become Ínportant

unden climatic or other siüuations which induce excessive sweating (Prasad

et al 1963b).

3.1.4. Biochemical roles of zinc,

The bÍochenlstry of zÍnc 1s extensi.ve and w111 only be outllned in this

Introduction Several publfcations provfde very adequaüe accounts of the

biochenfcal roles of zÍnc (Prasad 1976, t983; Sigel 1983). Zinc plays an

important erzynlc role with over 24 metallo-enzynes 1n hunans known to

requfre zfnc for üheir functions (Parfsl & Va1lee 1970), and around 100

netallo-enzynes recognised across specles (Rlordan 1976). ZLnc enzy¡nes

3-4

play a role in Ímportant metabolic processes including carbohydrate'

tipÍd,probeinandnucleicacidsynthesisanddegradation,andarefound

as menbers of all major classes of enzymes (Kirchgessner & Roth 1980)'

Both DNA and RNA synthesis require the presence of zinc as a component of

enzymes and the macromolecules themselves' In nutritionally zinc deficient

animals, a reduced leveI of tritiabed thymidine is incorporated into DNA

(foasad & Oberleas 19?4). lhese workers have shown that decreased activity

of the netallo-enzyme, deoxythynidine kinase may have caused this early

reduction in DNA synthesis. zinc deprivation has also been shown to Iead

to reduced revers of the zinc dependent enzymes required for synthesis of

RNA, and increased leveIs required for RNA catabolis¡n (Terhune &

Sandstead 1972; Prasad & Oberleas 19?3)' Hence a primary effect of zinc

is on several zinc-dependent enzy¡nes which regulate the biosynthesis

and catabolic rate of RNA and DNA'

z'nc is also involved in the progression of the cerl cycre' Zinc

deprivation interferes with the biochenical processes essential for

cellstopassfrononepartofthecycletothenext.Aninvitro

study of the eukaryote Euglena gracilis which undergoes growth phases

similar to those Ín prokaryobes has shown that a deficiency in zinc

adversely affects all the stages of the cel1 cycle (Riordan 1976)

including nitosls (Eckhert & HurIey 1977i Chesters 1978' 1982' Record et

aI 1 985a).

other functions include a role in the structure, function and

stabÍlisation of biological menbranes (Chvapil 1976; Bettger & 0tDelI

1981), and zinc deficiency nay lead to free radical danage inthe fetus

(Dreosti

though t

1 9 87 ; Dreo sti & Par tick 1 9 87) '

to be the PrÍmarY initiator of

The superoxide radical is

cellular oxidabÍve injurY'

3-5

Ieading to Iipid peroxidation and changes in membrane structure and

functÍon (Slater et aI 1987), Zínc also appears to be involved in many

aspects of imnune function, especially T-Iymphocyte mediated actÍvity

(Prasad 1 983), as w eII as a number of other physiological

processes including hormone netabolism and neuroendocrine interactions

(Kirchgessner & Roth 1980), tissue growth and sexual developnenf

of males (Sandstead et aI 1967; Underwood 1977), taste acuity (Henkin

1984) and behaviour (HaIas 1983).

3.1.5. Aetiolory and clÍnical manifestations of zÍnc deficiency'

Inadequate zinc Levels in humans nay arise through a varÍety of

different causes described by Prasad (1979b), although dietary factors are

the najor determinanb of zínc status in the géneraI population A

comprehensive review of the likely mechanisns involved can be found in

Sandstead et aI (1976) and Prasad (1976). Inadequate dietary zinc has been

neported in infants and children receiving diets otherwise sufficient

(Sandstead 1923; Hanbidge 1977) as wel-l as in institubionalised or

hospitalised patients, people of poor econonic status, and those receiving

total parenteral nutrition not supplenented with zinc (Hauer & Kaninski

19?B). The likely outcome of this latter condibion is severe zj.nc

deficiency (Van Rij & McKerøie 1977).

As well as a nutritionally derived zinc insuffÍciencyr conditÍoned

zinc deficiencÍes are also of extreroe inportance and nay occur as a

consequence of factors other than a primary lack of the nutrient in the

dieb (Sandstead et aI 1976). Cereal protein based dlets such as those

regularly consumed in Egrpt and Iran have been shown Lo provide a

predispositlon to zinc deficiency even lhough dietary tntake of zinc

appeared to be quite adequate (Sarran et aL 1969, Prasad 1983). The

3-6

availability of zinc Ín these diets Ís poor however because of the high

IeveIs of phytate and fibre which bind zÍnc and prevent its adequate

absorpbion (Reinhold et aI 1976) and which probably contribute to

the development of a nubritional zinc deficiency. Adolescent patÍents

suffering from zinc nalabsorption in the Middle East were characterjzed

by dwarfÍsn, hypogonadism Ín maIes, poor appetite, mental lethargy and

severe skin diseases (hasad et aI 1961' 1 963a, b' Prasad 1966). All these

dysfuncbÍons were shown to be reversed with zinc supplementation

Zinc avaiLability 1s also reduced in infanb formula conpared with

breast nilk (Johnson & Evans 1978), while zinc deficlency has also been

reported in patÍents wibh malabsorption syndrome, leading to an intesüfnal

loss of zinc, and Ís often associated wlth other deficiencies (Prasad et

aI 1 963b). Genetic dÍsorders such as sickle cel1 anaenia and

acrodermatftfs enteropathica are also associated w ith zinc deficiency

probably caused by impaired intestinal- zinc absorption (NeIdner et al

1975). The cIinlca1 manÍfestations of the deficÍency, which are

reversible upon zÍnc supplenentation conslst of skin lesions and alopecia,

diarrhoea, nental letharryr and depression as well as eye lesionst

although untreated acrodermatitis enteropathÍca is usually fatal (Barnes &

Moynahan 197Ð.

Excessfve losses of zinc which occr¡r through hyperzlncuria can lead to

eventual zinc depletÍon Thfs ls synptomatlc of patients with cirrhosis of

the lfver, nephroüic syndrome and sickle cell disease (Prasad 1982).

IÌypem incuria has also been shown to occur fn condÍtions associafed wilh

burns, surger!¡, multfple ir\junles and major fracüures, as well as diabetes

nellitus , protein deprlvation, stan¡ation (Klingberg et aL 1976; Spencer

et aI 1976), alcoholisn (Prasad 1983) and the use of chelating

3-7

agents as therapeutic a8enls for disease (eg' penicilJ-amine for ÌJilsonfs

disease) (Kltngberg et al 1g76). Certain physiologicat conditions such as

pregnancy & lactation require an increased zinc intake (Prasad 1983) to

accornmodate the growing bissue nass. Plasna concentratiOns of zínc

decrease in hunan pregnancy (Henkin et aI 19?1). Presumably the decrease

reflects in part the uptake of zinc by the fetus and the other products of

conceptioru It has been estimated that the pregnant I'f olnan nust retain

approxÍmately 750 ug of zínc per day for the growth of producbs of

conceptÍon during the tast two thirds of pregnancy (sandstead 1973)' Thus

when zinc defÍciency occurs Ín pregnancy' a condibioning factor is the

denand of the fetus for zLnc. studies in the rab suggest that' the

placenta actively provides zinc to the fetus (sandstead et al 1970)'

zínc deficiency syndrone in pregnancy is characterised by íncreased

maternar morbidity, abnornar taste sensations, prolonged gestationt

inefficient labour, atonic bleeding and increased risk to the fetus'

Íncluding congenibal malfornations (Jameson 1980).

3.1.6. Zinc and hunan development'

Intheprevioussection,thecausesandclinÍcalmanifestationsofzlnc

deficiency were described, and specifical-Iy a naternally-mediated effect

on the fetus was alluded to. In bhis seclion the linited data available

which describes the likely implications of zinc deficiency in utero on

enbryonic developnenb in hunans wiIl be outlined' CIinically recognisable

zinc defÍciency in humans was nanifested as dwarfisn and hypogonadlsn in

adolescent youths, and was shown to be prevalent in Erypt (Prasad et aI

1gSla,b), rural lran (Ronagty et af 1968; Halsted 1973) and Turkey (Sever

and Ennanuel 19?3). These aræs have also been reported to have very high

rates of anencephaly (stevenson et aI 1966; Danyanov & Dutz 1971; Cavdar

eta].1980),whichtedtothesuggestionofallnkbetweenzinc

3-8

deficiencyandcentralnervoussystemanonalies(Prasad1979b).In

some of these studies serum zlnc leveIs were significantly lower in

poonlynourishedthaninwell-nourishedpregnantwonenandinmothers

of anencephalics compared with control-s (cavdar et al 1980)' In several

other studies (Janeson 1976; Janeson et aI 1 977)' increased maternal

morbidity and congenital malformations, including nyelomenÍngocoele could

be correlated with 1ow naternal serum zinc 1eveIs, which supported the

notion of a teratogenic effecb of zrnc deficÍency. Furthermore'

mothers who Írere zj¡c deficienb early in preSnancy and who $¡ere

supplementedwithzíncsulphatethroughout,deliverednorenormal

infants than those whose zÍnc leveIs renained low (Janeson 1982)'

Further evidence of the invoLvement of zinc in fetal devel0pment was

obbained from pregnant women with acrodernatitis enteropathica' which

is characterized by severe skin Iesions and diarrhoea (Moynahan

1973), synpt,ons which are exacerbated during pregnancy (Verbug et aI

19?4),andreversedbyzlncsupplementation(Barnes&Moynahanl973).In

three pregnant women with this disease who were not supplemented with

zinc, or who receÍved only diodoquinr âD earlier treatnent (Dillaha et

aI 1 953), seven pregnancies ended in abortion and congenibal

nalformations, nainly anencephaly and achondroplastic dwarfÍsm (Hambidge

et aI 1g75). However, Ín Wonen supplemented with zinc sulphate throughout

pregnancy nc congenital abnormal-ities were observed (Brenton et aI 1981)'

lfomen with acrodermatitls enleropathica which is known to produce

abberrant zinc metabolisn also had lower than normal plasna zinc levels

in the thlrd trlnester of pregnancy (Moynahan 19?4; Nerdner & Hanbfdge

1975).

stnilar abnormalitles were described earlier by Hurley and swenerton

3-9

(1966) in the offspring of zinc deficient rats, where reduced birthweÍght

andcongenitalabnormatitieswerelinkedwithmaternalserunzinclevels

at term. In wonen however, this association is not so cleart since a low

zj_nc sbatus at tern was correlated with gnowth retardation and fetal

dysnorpholory in some studies (Cavdar et at 1980; Soltan & Jenkins 1982)

bub not in others (Janeson 1976; Dreosti et aÌ 1982)' Raised maternal

zinc Ievels have been observed in the blood of mothers whose infants

were born with neural tube defects (McMichaet eb al 1982) and in the

hair of nothers of infants with spina bifida (Bergnann et aI 1980)' It

appears thaf in experimental studies and in poorly nourished

populations a correlation exists between inpaired fetal brain

development and low maternaf zinc status. However, adequately

nourished women nay also demonstrate low serum zinc Ievels if there

is above average t,ransfer of zinc to a large fetus' Alternatively'

nothers with growth retarded infants may show raised serun zinc IeveIs

as a smaller fetus has lower zinc requirenenbs (Dreosti et al 1982)'

Hair zinc content has been shown to be reduced in young healthy pregnant

women (Hanbidge & Droegemueller 19?4) and 1n children with mil-d or

moderate zinc deficiency (Hambidge et aI 1 9'12)' In contrast, raised

levels of hair zinc have been observed in cases of extrene zi-nc

deficiency (Neldner & Hambidge 1gTÐ, and in nothers of Ínfanbs with

spÍna bifida which was accourpanied by significantly lower birthweight

and infant lengfh 1n this group (Bergnann et aI 1980)' These authors

suggested that the dlfferenees Ín zínc and growth paraneters

lndicated an abnornality of zinc availability or metabolism in bhe

mothers of infants with spina btfida. Hence, haír zÍnc nay prove to be a

nore reliabLe marker for the detection of congenital abnornalities fhan

plasna zinc in the zínc deficient syndrone'

3- 10

Adequate zinc levels are also requÍ.red postnalally for continued brain

development. During the first I to 4 weeks in rat neonates when the

cerebellun and hippocampus are developing (Aftnann & Das 1965), zinc has

been shown to accumulate in intrahippocampal mossy flbre pathways

(Crawford & Connor 1975; Dreosti et aI 1981). The hippocampus has a role

in nenory, cognÍtion and enobion (Saghal 1980) and the involvement of zinc

in this region suggests that low levels of zinc at this tine ín neural

development nay lead to neuropsychiatrÍc dysfunction (Henkin eb al 1975i

llalravens et aI 1978), including irritability, lethargy and depression,

as we1l as rnental retardation (Pihl & Parkes 1977i Krischer 1978).

3.1 .7. Zínc and development in animals.

The inportance of zinc for intrauterine development has been fÍrm1y

established since Turk et aI (1959) found that naternal Íngestion of a

zinc deficient diet led to weakness and death in chicksr âs well as to

brain and skeletaL defects (Blamberg 1960; Kienholz et al 1961). SÍnce

ühen a reduced naternal dietary zinc intake has been shown to cause a

severe teratogenic response in several- other species including rodents

(Hurley & Swenerton 1966; HurIey & Shrader 1972, Hackman & HurIey

1983; Record eb al 1986), and nonkeys (Swenerton & Hurley 1980)'

Ieading to a range of undesirable consequences including impaired

intrauterine growth, severe congenital nalformations and enbryonic death.

Maternal dletany deficÍencies, even of a transilory nature (Hur1ey et aI

1971), have been demonstrated Lo cause congeniLal abnornalities over a

wide range of organ systens lncludÍng the brain¡ €Vêr palate and skeleton'

as well as cardiovascular, resplratory and urinogenital sysbens (HurIey &

Swenerton 1966; M111s et' al 1969i Hurley & Schrader 197 2; Warkany &

Petertng 1972; Record et al 1985b; Rogers & Hurley 1987; Harding et al

3-11

1988; da Cunha Ferreira et aI 1989; Joschko et aI 1989). While zinc

deficlent dysmorphology ranged over a number of ongan systens the

central nervous system (CNS) seened to be the nost seriously affected.

CNS abnormalities occurred when a dietary zinc insufficiency was

induced durÍng the period of peak neurogenesis (Record et aI 1985a;

Joschko et aI 1989), which in the rat occurs between days 9 and 11 of

gestatÍon (Beaudoin 197 9). Gross dysmorphology of the anterior brain is

thought to arise from cl-osure of the aqueduct of SylvÍus leading to

hydrocephalus (HurJ-ey & Shrader 1972; Adeloye & I'larkanV 197 6), and f rom

linited closure of several regions of the developing neural tube midway

through gestatÍon (Joschko et aI 1989). The inadequate neural tube closure

and acconpanyÍng celluLar necrosis observed in our laboratory (Record et

aI 1985b; Joschko el al 1989) nay underlie the exencephaly, anencephaly

and spina bifida reported in tern aninals (Warkany & Petering 1972; HurIey

& Schrader 1972; Dreosti 1983).

It has been proposed (Record et aI 1985b) that the nechanlsm underlying

the extensj.ve celluLar necrosis which occurred mainly in areas of norrnal

rapid proliferation in the neuroectoderm may be due to damage to

intracellular membranes, since zinc is known to play a role as a nenbrane

stabitiser (Chvapit 1976; Bett,ger & 0'Dell 1981). Zinc Ís also involved

in antioxidant and free radical defence ¡nechanisns (Dneosti 1986, l{illson

1987), hence a zLnc deficient status during fet,a1 Iife when free radical

prolective nechanisns are poorly developed (Dreosti & Partick 1987), lnây

lead to cellular oxidative damage (Dreosti 1986, 1987), and subsequent

ne¡nbrane dÍsnuption, as observed in the neural tube of zínc deficient

enbryos (Record et aI 1985b).

Biochenical sfudfes have suggested addÍtfonal or alternative nechanisns

3'12

of action of zinc deficiency in the developÍng embryo. A reduced uptake of

tritiated t,hymidÍne in developing neural tissue suggested that DNA

synthesis was reduced (Eckhert & HurI ey 1977; Record & Dreosti 1979i

DreostielaIlgso),whileanincreasedmitot'icindex(Hurley&

Schrader 1972i Eckhert & HurIey 1977) nay indícate ühat mitosis fs

blocked, or is compensatorily rsped upn when maternaÌ serun zinc Ievels

increased (Record et a1 1985b).

Post-natal effecbs of zinc deficiency in rats have also been

investigated and although tenatogenesis is nost evident in rats exposed

to zinc deficiency during organogenesis, zLnc depletion during the

perinatal perÍod has been shown to lead to changes in behaviouraL patterns

in rats (Lokken et al- 1973i Sandstead et al 1975) and in monkeys

(Sandstead 'l 9?7), as weII as reducing short term memory (Lokken et al

1973). A full description of these and other studies including the

possible aetiology, and biochemistry of the brain areas involved are

reported by Dreosti (1983). It should be noted that parallel

obserr¡ations have been made in humans as psychological disturbances

lncluding depression and mental Iethargy have been reporLed in

patients suffering fron dietary Índuced zinc deficiency in bhe MiddIe East

(prasad 1966; Sandstead et al 1967), as well as from the inherited zinc

deficiency dlsease acrodermatitis enteropathica (tlalravens et aI 1978).

InterpretatÍon of the adverse effects of zinc deficiency on the

developing enbryo may be confounded by several naternally-nediated

mechanisns such as the possible mobiLisation of naternal zinc stores or

hormonal influences, w hich suggests use could be made of the technique of

enbryo culture to dÍstÍnguish these effects. However, in the present

study, attention was focused on the j¡ vivo effects of zinc deficiency

3- 13

during neurogenesis in preference to the enbryo culture technique in the

J-ighb of observations rnade by Record et aI (1985c) who showed that normal

day 9.5 embryos were not affected adversely when grolrn on zinc defícíent

serum. Since the tíne when the experiments described in thÍs bhesis were

performed, Mieden et aI (1986) have reported successfu]-Iy growing

dysmorphic embryos in vitro, however while they demonstrated growth

retardatÍon, the Sroup did not report malformations of the CNS

characteristic of zínc deficiency in utero in rats. The uncertaÍnty

associated with Ín vitro studies on zinc deficient enbryos may arise

because of the ability of the yolk sac of enbryos in culture to endocytose

macroglobul-in-bound zinc and util ize it'. Such zinc stores are not

avaÍlable to the enbryo j¡ vivo, possibJ.y because of the presence of

Reicherts nembrane which may function as a filterr preventing

macroglobulins from reaching the enbryo, and which is removed prior to

embryo cultwe.

That abnormal cel1 death in developing neuraf tissue had not been

reported in previous histological studies until recenlly (Record et al

1985b; Harding et al 1987; Joschko et aL 1989), demonstrates the relevance

of performing tissue exaninations in developÍng enbryos close to the tine

of neurulation, so that the full extent of zinc deficíent neural tube

dysmorphology can be recognlzed. In this study, while gross and cellufar

dysnorphology were investigated, the ulbrastructural changes Índuced by

zinc defÍciency at the time of neurogenesls were a naion focus of

examination in an effort to gain further information as bo the site of

actíon and also the possible roechanisrû/s underlying lhe serious neural

tube dysnorphologr induced by this teratogen

3- 1 4

3.2 MATERTALS AND MEÏI]oDS.

3.2.1 Animals and DÍets.

Virgin fenale Sprague-Dawley rats (180-210e) were placed overnight wlth

nales of the sane strain, and pregnancy was established by the detectÍon

of spern 1n the vaginal snear the following norning (gestation day 0.5).

hegnant dans were housed Índividually in stainless steel and plastic

cages and were assÍgned lo an EDTA-extracted soybean based diet (tlilkins

et aI 1972) which was eiLher zlnc-deficient, containing less lhan 0.5 ug

Zrdg or was supplemented wÍth 100 ug oî Zn/g as zi.nc sulphate. Both zlnc-

deficfent and replete groups $rere fed dÍets in a cyclical pattern (Fig

3.1) nodified fron Record eü al (1985a), in order bo ensure high dietary

intakes on days 8 and $ of gestatÍon, with correspondingly low serum zfnc

levels fn bhe zinc-deflcient anÍnals whÍch was shown by thÍs group to

enhance neural tube teratogenesis.

Df etarylntake

(e)

25

20

15

10

5

II

II

IIIIIIIIIIIIII

IIII

0 8 10 12

Days of gestation

Fig. 3.1 Patt,ern of feeding of zinc replete and zlnc defÍcfenb dams toinduce peak consunption and hence lotr senum zinc levels on days I and 9of gesüat1on.

AnÍnals 9¡ere sacnificed on day 11.5 by ether anaesthesia and blood was

obtained by candiac puncture for serun zlnc analysls by flane atonic

absorption spectroscopy (Record et aI 1982). Enbryos l¡ere renoved fron

3- 15

the uterus into Hanks Balanced Salt Solution (PH 7.3), and examined under

the dissecting nicroscope for growth and norphological deveLopment.

SeveraL representative embryos fnom each dam were selected for

exanination by light and transmission electron microscopy (Record et al

1985b). Embryos prepared for scanning electnon microscopy were fixed

in o.1M sodiun cacodylate, 2% paraformaldehyde and 3f glutaraldehyde

(PH 7.3) al 4'C overnight, dehydrated through a serÍes of graded

aceLone solutions and crÍticaIly point dried (BALZERS UNION'

LÍchtenstein). The embryos were mounted on stubs, coated with carbon

and gold-palladium and examined with an ETEC scanning electron

mÍcroscope at 2o KV. The renaÍning enbryos within each litter were

stored at -20'C for proteÍn determination (Lowry et al 1951).

Continuous growth variabLes such as protein and somibe numbers were

analysed using standard analysís of variance. For discrete varÍables such

as number of dead enbryos or nunber of embryos with any kind of defect, a

naximum tikelihood nethod was used, assuning the data to be binomially

distributed as X2 on the approprÍate degrees of freedorn (Baker & Nelder

1978).

3.3 RESULTS.

3.3.1. Growth and morphological development.

The enbryos in the zinc-deficient group were recovered from dams fed Iess

than 0.5ug Zrúe in bhe diet, conpared r¡ith more than 100 ug Znlg îor

controLs and whose serun zinc levels were correspondingly Lower (0.70

uglml) than levels in the serum of zÍnc replete dams (1.24 ug/nl) fron

whon control enbryos were oblained (Table 3.1). The established

relardaüÍon of zinc deficiency on lnbrauterine growth (HwIey & Schrader

197Zi Record et aI 1985a, 1986; Harding et al 1988) was also observed

3- 16

in this study. A conparison of the control embryo Ín Fig. 3.1a and the

zínc deficient enbryos in FÍg. 3.1b photographed aü the same

magnification shows this cIearly. Ab 11.5 days of gesbation the zinc

def icent enbryos !ù ere generally up to 25l shorter, wit'h 5 f ew er

somÍtes and whole body proteÍn levels which were about 40Í less

bhan their zlnc replete counterparts (TabIe 3.1).

Table 3.1 The Effecl of Zinc Status on hbryonic Growth in Rats.1

Zinc replete : ZÍnc defÍcient

No. of embryos (Aans)

l,laternal serum zinc (ug/nt)

Crowr¡rump Ienglh (mn)

Nunber of somites

tubryonÍc protein (uB)

61 (5)

1 .25+0.01

3.68lo .08

22.58-0.33

245 .4011 8 .6

66 (5)

0.f010.08b

2.83!O .lZc

17.4611 .o1c

1)as.00g13 . Oìa

1v"lu"" are neans +SEM.

a'b'c Significantly ress than control values. (ANovA). ap(O.02, bp(O.01,

cp(O .005.

There was however no difference Ín lnplantation rates between zinc-

replete and zÍnc deffcfent enbryos (Table 3.2). It is possible t'hat a

dietary zinc insufficíency had eibher not been induced or was , orùy

narginal at the ü1ne of inplantation (day 6 of gestation) in the

present experÍnents, as self-lnduced cyclfcal feeding by the animals nay

have rafsed plasna zinc to more normal levels at this tlne. Studles by

Record (1986, unpublished PhD thesÍs) also fafled to find a speciffc

effect of zfnc deficlency on fhe implantatlon process. llhlle enbryonic

loss was also not signiffcantly affecfed by a dietary zLnc deficÍency,

the rate of dysnorphologr Ín zÍnc deflcÍent preplacental embryos Ìlas over

50Í for all organ systens as well as for several lndlcators of ovenal-l

3- 17

Dev elo pmentTabIe 3.2 The effect of Zinc Status on Morphological

in rats.1

Zinc Replete z ZLnc Deficient

No. of eobryos-dams.

fubryonic 1oss.

Total øbryos with defect.

Neural tube defects.

Ele defects.

Ear defects.

BranchiaL arch defects.

Heart defects.

LÍnb defects.

Incomplete flexÍon.

ImpaÍred chorlo- allantoic fusíon.

61-5

3(4.e)

3(4.e)

2(3.21)

0( 0)

0( 0)

0( 0)

0( 0)

0( 0)

2(3.2)

3(4.8)

66-5

6(e.1)

34( 51 .5)c

26(39.4)c

9( 13.6)c

7( 10.6)b

7( 10.6)c

4(6.1)a

18(2t.3)c

1 9( 28.8) c

31(47.0)c

1 Percentage of embryos in parentheses.

a p(0.05, b p(0.01 , c p(o.oo1 .

development such as dorso-lateral flexion and chorio-allantolc fusion

( TabI e 3.2).

By day 11.5 of gestation, control embryos exanlned under the dissecting

rnÍcroscope vJere observed to have rotated lnto the dorso-convex posiflon

(Fíe 3.1a), whÍle the allantois had fused with the chorlon üo form the

chorio-allantoic placenla which provides the rat with lts nutrlüÍon after

day 11 of gestation, at the tine when the enbryo 1s too large to depend

only on the yolk sac for this function (New 1 976). In the control enbryos

neurulablon was also conplete at thfs tfne, optlc and otic placodes had

developed togethen wÍth 3 sets of branchial arches, a well forned heart

tube and forelinb buds (FÍe 3.1a). Nearly 471 of the zinc deficient group

3-18

Fig. 3.1a. Zinc replefe 11.5 day enbryo. 0P, optÍc placode" 0T, oticplacode., 11213, branchial arches., H, heart', S, sonltes', F, forelinbbud.

Ffe. 3.1b. 11,5 day zinc-deficient embryos removed fron the same dan

wittr varying degrees of growth retardation and dysnorphology. Tkre twoenbryos on the rÍght hand side denonstrate Íncomplete or partial lack ofrotation into the dorsally convex positfon and severe neuraldysmorpholog. Note the presence of the unfused allantois (A) Ín three ofthese embryos.

Fie. 3.1 c. 11 .5tube anonalies.tubes fncluding(arrowheads).

day zÍnc-defÍcient enbryos showfng1,3, hydrocephalY (asterisks).' 2

exencephaLy (arrows) and 213,

a

,4,6,

range of neural516, open neural

cranial cysts

Fig. 3.1d. Higher magniffcatlon of enbryo 2 in Fig. 3.1.c shows fusedbut dimÍnished forebrafn vesLcles (asberisk), open midbrain and a largecranlal cys! on the rlghb side of the craniun (arrowhead) with a snallerone on the left side whlch can be nore easily dlslinguished in s

Fig. 3.1e. Dorsa1 and ventral aspects of early 10.5 day zinc repleteembryos showing lhe cranial neural fold (arrows) and caudal neural folds(arrow heads) as well as synneürical, unfused forebraln vesÍcles(asterlsks) and first branchial arches (1).

Fig. 3.1f. Lateral aspect of mfd to late 10.5 day zlnc-replete enbryosshowing nornal relationshÍp between growth and development. At ühis stageof normal developnent the neural tube f s orùy open in the nidbrafr¡-hindbrain region (arnow) and at the caudal end of the enbryo (arrowhead).

Bars= 1 mn.

ç

ï

3-19

demonstrated inconplete or tot,a1 lack of fusion of the chorlon and

allantois (TabIe 3.2). In Fig 3.1b, the unfused allantoÍs is shown in

these 4 littermates removed fron a zinc-deficÍent dan. The photograph also

denonstrates the wlthÍr¡-lifter variation frequently associated with zínc

deficiency. Although the two ernbryos on the lef t were much less affecbed

and larger than those on the righb, none had developed a viable placenta,

hence without Ít even the nore nornal ernbrryos were urù1kely to have

reached tern. Failure of the zinc deficient enbryos to rotate into fhe

dorsally convex positÍon occurred Ln 29l of the embryos and was often

associated with the absence of placental formation The two embryos on the

rÍght in Fig 3.'lb denonstrate incomplete or partial lack of rotation

These enbryos were also often snaller than enbryos vrhich had rotated. The

developing nervous systen of the zinc defÍcÍent rats demonstrated the

hlghesb rate of anonalies (39f) of all the organ systems exanined (Table

3.2). Fig 3.1c reveals a typical range of antenior neural tube anonalles

associated wfth zinc deficient enbryos. These six embryos were lftter

mates and abnornalÍ ties included trydrocephaly ( 1 ,3), open neural tubes

(exencephaly) (2r4r5,6) and cranlal cysts (216). The latter abnormality

cor.úd not be denonstrated by scanning electron mf crosco py as the cysts

collapsed during critfcal polnt drying. Higher nagniffcatlon of embryo 2

(Fig 3.1d) shows that the forebrain vesfcles have closed but are

diminlshed in sÍze. The nfdbrain region is open and a very large

cyst occuples the Lateral rfght side of the cranium. The snaller

menlngocele on the left side Ís Iess evidenl due to the angle on which the

enbryo was pholoþraphed. A conpanison of the abnornalities in Figs.

3.1b,c and d, with typical 11.5 day (ffg 3.1a) and late 10.5 day (Fies

3.1e,f) control enbryos shows that the abnornalities of the cranlal

neural üube are nob nerely reflectfons of a reduced rate of growth due to

3-20

zinc deprÍvation but rather teratogenically-induced anomalies.

Special süructures 11ke the developíng optic placodes and branchial

arches r.Iere also susceptible to zÍnc related teratogenesis (TabIe

J.2, Fig. 3.2), Figure3.2 presents some of these abnorrnalities, and

indÍcates the range of dysnorpholory induced by zínc deficlency both

between and within litters. In a typÍcal zinc replete day 11.5 enbryo

(Fig. 3.Za) rotation has been completed, the neural folds have fused,

the forelimb buds are clearly visible and the optic placodes, heart

and branchial arches are well forned. The otic vesicLes whieh lie

dorso-lateral to the second branchial on hyoÍd arches are not vfsible at

thÍs orlentation (see Fig. 3.1a). FÍg. 3.2b shows an enbryo at an

earller stage of developnent. At nid to late day 10.5 of geslatÍon

rotaüion Ís inconplete and the neural folds have not fused at the

cranial and caudal extremities. Nearly 14f of the zinc deficient

enbryos exanined exhfbited eye defe ct s w hi ch included

nÍcrophthalmia or anophthalnla, and 11f displayed dysnorpholory of the

nandibuLar arches (Figs. 3.2crdrerf) which were abnormally shaped and

ellher fused together at the nidline and/or to the perÍcardium (Fies.

J.2c,e,f), or s¡ere abnornally dimínÍstred in sÍze (Fig. 3.2d). Ttre hyofd

and thÍrd branchÍal arches were also absent in some enbryos (Figs. '3,2c-

f). Absence of forellmbs also occurred (Figs. 3.2c,d) and pericardia were

oflen enlarged when renoved fron the dan but frequently collapsed during

processÍng. In the enbryo shown ln Fig 3.2e the perÍcardlun had been

renoved from the heart, üo reveal a well developed heart tube.

Sone zinc deficient, enbryos denonstrated neural tube defects whereby the

forebrain and nldbraÍn were closed but the enbryos ÌJere frequently

nlcrocephalic wfth severely reduced forebrains (Flg. 3.Zc). Many of the

3-21

FÍg. 3.2a. In this normal 11.5 day zinc-replete embryo, the aninal has

rotated into the typical dorso-convex orientation The forebrain (Fb),

nÍdbraln (Mb) and hindbraÍn (gU) are clearly defined and the neural foldshave fused along the dorsal nldlÍne (arrows). At thÍs stage the enbryo has

developed optic placodes (OP), I sets of branchial arches (1rZr3'1 and oticvesicles which åre nornally located dorso-lateraI bo the second branchialarch, but are not visÍble at this orientation Tlre lÍne depicts theapproximate level at which the cranial neural tube was sectioned forhistologÍcal examÍnation H' heart., S' somites., F, forelinb bud.

FÍg. 3.2b. Dorsal view of a nÍd to late 10.5 day zinc'replete embryowiilianopen anterior (arrows) and posterior (P) neuropore. The embryohas not completed rotation S' somites.

Figs. l.2crd,e,f. Three severely affected 11.5 day zfnc defÍcÍentenbryos showing overall developnental retardation

e- The embryo is nicrocephalic wilh severely reduced forebrain vesicles(Fb). Ihe mandibular arches $¡ere fused in the n1dl1ne (1) and t'he second

and ühlrd branchÍal arches were underdeveloped. The optlc placodes had notdevetoped and the embryo had only partially rotated. H, heart.

d. In this zinc-deflcient enbryo bhe neural tube is open (arrowheads)inbhe cranial region and caudal to the hÍndbrain(Hb) where it is brieflyclosed. The nandibular arches (1) were underdeveloped, the pericardiunand heart (H) were enlarged, and the enbryo had not rotated into thedorso- convex posftion Fbr forebraÍn

e. A ventral vlew of this zinc-deficÍent embryo showfng severely reducedforebraln (Fb) wÍth mandibular arches fused together (1) and to theperfcardir:n (Pe), which had been partlally renoved to reveal the hearttube (H).

f. A different orientatlon revealed that the cranial neural folds(arrows) had not fused Ín thÍs enbryo and the forebrain (Fb) appeared tobe assynetrÍc. 1, fused mandibular arches, vsr ventrÍcular surface of theneuroepith eIiun.

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3-22

more affected zinc defÍcient enbryos showed fncomplete closure of the

cranial and spinal neural tubes (Figs.3.2d,f). The spinal neural tube was

often open in its entire tength (Fig. 3.2d) while the rostral end was open

in the forebraÍn and midbrain regions but the hindbrain was sometj.mes

closed. The headfolds, despite their lack of fuslon were often observed to

be quite rigid and vertlcal as seen fn Figs. 3.2d and 3.2fr whÍch suggests

that the abnornality may derive fron the neural epithelial or crest cells'

rather than fron the underlyÍng supporting mesenchyme. Clearly a

conpari.son between the 10.5 day control enbryo and the zLnc deficient

enbryos in this figure shows that the abnornalities are teratogenically-

Índuced rather than simply a consequence of the reduced growth rate.

Apart frorn the obvlous structural abnornalitÍes whÍch may be attrlbuted

to enbryonic zinc deficiency, scannfng electron microscopy revealed that

these were often accompanied by changes Ín the surface epithelium as well

as the neuroepithelium of the anterior neural tube abufting the

ventrlcular lumen in nore severely affected enbryos. In a typical section

fron the forebraj.n region of the neural tube in an 1 1.5 day zinc replete

ernbryo (Fig 3.3a) the cells of the surface ectodern were relatively

snooth, except for a line of microvilll nainly concentrated around the

perineters of the cel1s, wtth a few scattered over the remafning

surface. Examination of the nídbraÍn region of a number of 11.5 day

control enbryos showed a simÍlar dlstribution of nÍcrovilli except that

there were usually fewer dlstrlbuted over the cell surface (noü shown).

In FÍg 3.3b, the surf ace ecf o'dern ín the f orebrain- ¡nldbrain region of

another zinc replete enbryo had been carefully renoved to reveal the

underlylng layers. The apical surface of the pseudostratified colunnar

neuroepfüheliaI cells whfch abuts the venünicular lunen was covered

with nar\y cytoplasmic extensions proJectfng fnto the lumen to forn a

3-23

Fig. 3.3a. Forebraln region of a day 1 1.5 zinc-replete embryo' The cellsof the surface ectoderr aru relatively srnooth except for a dense rayer of

microvilli (arrowhead) around bhe perÍmeters, and a few scattered over the

renaining surface.

Fig. 3.3b. Layers of the anterlor neural tube of an 11.5 day zincreplete embryo. I?¡e apÍcal surface of the neuroeplthellun (N) abuts the

ventricul-ar Lumen (L) and consists of cytoplasnic extensÍons, or blebsproJecting into the lunen (arrowheads). Itre basal end of these oells reston the basal lanina (nob visible) which apposes the stellate'shapednesenct¡rma] cells (M) with their nyriads of f ilopodia (arrows) whích make

connection between the cells.

Fig. 3.3c. Section of an open neural lube in the midbrain region of an

11.j day zinc deff cient enbrgo showing lhe neural cresb area (llC¡. Et

surface ectodern., L, Iumen

FÍe. 3.3d. Hieher nagnlfÍcatÍon of c, showing neuroeplthelíaI cells (N)

fn the neural crest regton which appear shrunken and less tlgbtly packed

than normal cells. The neural folds have not fused and the surfaceectodern (E) has shrunk away fron the underlying neuroepithelial ceIIs.Dense nicrovfLll cover the ectodermal surface (arrowheads).

Ffg. 3.3s MÍdbraln surface of 10.5 day zinc replete embryo showingAense distribulion of microvilll (arrow) around the perfneters ofectodermal cells (E) and large nunbers over the renairdng surface area'

athe

FÍe 3.3 f.enbryo wiùh(arrowheads).

Surfacelange,

of the ventrlcular lumen of 10.5 day zinc repletedense ni cr ov 111 Í ( arrow s) and nun erous blebs

Fig. 3.3g. Surface of the ventricular lumen ofembryo wlth nar¡y variabl+sized blebs coveredand with extended fibrillar network (arrowheads)'

1 1.5 day zinc-defícienbin nicrovilli (arrows)

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3-24

series of snall nblebsn. The body of the neuroepithellal cells

appeared relatively snooth and slender and tíghtly packedr with the

basal ends lying on the basal lanfna, an extracellular supporting

layer whÍch abuts the nesenchymal ce1ls. These cells appeared stellate

shaped with nyrÍads of cytoplasmic projectlons or fj-lopodia running in

every direction giving then a tangled appearance. The nicrograph in Fig.

3.3d is fron the neural cresb area (see inset Fig. 3.3c) visible because

the neural folds in the midbrain of a zinc deflcienb enbryo have not

conplebely fused, and which in nornaL day 11.5 enbryos is the sÍte

where fusÍon of the neural folds occurs. The surface ectoderm is

covered in nicroviLll whlch appear dense and evenly distributed over

the entire cell surface, but are not heavlly concentrated around the

cell perimebers, ln contrast to sÍmfIar sites 1n both 11.5 day (Ftg

3.3a) and 10.5 day control enbryos (Fie 3.3e) which corroborates

observations ¡nade recently by Harding et aI (1988). The neuroepÍtheIiaI

cells of bhe zinc deficient embryo (Fig 3.3d) lrere also different from

control neuroepithelÍal cells as seen Ín Fig 3.3b. Tl¡ey appeared less

tÍghtly packed and possibly shrunken with more extensive cytoplas¡aic

projectlons compared wtth control neuroepitheliat cells. ExaminatÍon

of ühe ventricular surface of the neuroepithelfal cells revealed

that whiJ.e blebbing was a normal phenonenon in the neuroepÍtheliaI cells

of zinc replete day 11.5 enbryos, (Fig 3.36¡, and day 10.5 embryos (Fig

3.3f), exposure to zfnc deficiency 1n utero (Ffg 3.3t¡ appeared to

cause a vasl increase in t,he nunber and size of blebsr which were also

covered 1n microviffi. l?¡e nicrovilli however ¡¡ere considerably snaller

than ühose observed fn ühe neural tube of day 10.5 embryos (Flg. 3.3f).

Agatn lhe dÍfferences rer¡ealed at the scanning electron nlcroscope Ievel

suggests thaü the changes brought about by zinc insufff.ciency can be

3-25

attributed to nore than merely a reduction in growth.

3.3,2 Cellular develoPment.

Light nicroscopy of the crani.al region of grossly malforned zinc-

defÍcient embryos revealed thaf ühe dysmorpholory observed under the

dlssecting and scanning electron nicroscopes couLd be associated with

celluLar disorganisation In a typical 11.5 day zinc replet,e embtAo (fig

3.4a) sectioned horizontally through the cranium af the level of the optic

pits the neural folds have fused throughout the cranial reglon TL¡e

neuroepithelÍum is a thick layer of cells and the apical ends whÍch line

the venüricular lunen are essentÍally snoottt Outside the neuroepitheliun

lies the nesenchyne whÍch contains frregularly shaped ce1ls, and

developÍng blood vessels.

Higher nagnificablon (FÍg 3.4¡¡ of a sectÍon fron the nfdbrain of the

embryo in Fig 3.4a shows that, the neuroepitheliun consists of

pseudostratified columnar celIs (observed first under the scannlng

electron nicroscope, FiS 3.3b), which are arranged Ín a regular pattern

with mitotic cells abutting the ventrlcular lunen the columnar cells

contain oval straped nuclei with proninent nucleoli which occupry a

considerable portíon of the cells. Higher nagnification also revealed'that

the lumen end of these cells which appeared snooth In Fig 3.4a actually

for¡ned small cytoplasnic protrusions or nblebsn proJecüing into the lunen

of the cranial ventricles consistent with the irregular lumenal surface

observed under the scannÍng electron nfcroscope (Fig 3.lb)r while the

other end rested on a contÍnuous basal IanÍna adjacent to the nesenct¡tne

(not visfble at this nagnification).

Higher nagniflcation of the nesenchyne within the anterlor neural tube

(ffg 3.4b) shows thaü rnesenchynal cells are stellate-shaped and appose

3-26

Fig. 3.4a. Light nicrograph of the crar¡1aI region of an 11'5 day zinc-replete embryo sectÍoned horizontally at the leveI of the optic vesicles(Oi. The neuroepithelium (N) Ís bhick and snooth with dense mesenchyroe (M)

surrounding it. The dense regÍons in the nesenchyme are developing bloodvessels (tãrge arrowheads). The arrow poinüs to the site of fusion of bhe

neural folds. Snatl arrowheads nank the regfon seen ab highernagnlfication in b. Fbr forebraini Mb, nfdbrain; Hb, hindbrain, Et surfaceectodern.

Fig. 3.4b. Hlgher magnlfication of a section of the nidbrain neurallube of this 11.5 day zinc-replete enbryo. The pseudostratifled col-umnar

cells are arranged in a regular pattern with relatÍvely smooth apical ends

with only snall blebs (arrowbeads) projecting into the ventrÍcular lunen(L). Mltoflc figures (Mf) were also observed close to this sÍte. The basalends of lhe neuroepithelial cells are snooth and are located close to thenesenchyne (M) which contains stellate shaped cells with Ínüercellularcytoplasmic processes (arrows) and blood vessels (bv). N' newoepfthelium.

Fig. 3.4c. Section of forebrafn of 11.5 day zinc replete enbryoindicated by arrow 1n Ffg 3.4.a" kograrnned celt death (arrowheads) withinthe neuroepithelÍun occurs at the site of fusion of the neural folds. Note

the ceII debrls (arrow) Ín the lumen, whlch was probably extruded fron theneuroepfttretÍun (N); M, mesenchyne; surface ectoderm (E); blood vessel(bv).

Flgs. 3.4d,e. Conparable secüÍons through a 10.5 day zinc replete embryostrowtng siniLar organlsatfon The cranial neural folds (arrows) have notyeü fusid. The arrowheads fn d. nark the site seen at higher nagnificatlonln s. N, neuroepÍthelluni M, mesenctryne; bv, blood vesseli Mf' mitoticfigure; NC, ner¡¡al crest.

Bars=100un a, b, c, d; Bar=50un e.

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3-27

each other through a dense fibrirlar network of cytoplasmlc projections

orpseudopodia,andarelocatedlnarnatrixoforganlcnaterialnot

visiblebynornalstalningmethods.Interspersedanongstthemesenchymal

ce1ls are developfng endothelial-lined blood vessels which often contained

nucleated erythrocytes. The neural tube is bordered by a single layer of

surface cells (Fig. 3'4c) which enables efficient dÍffuslon of nutrÍents

directly bo the underlying tissue during the time prior to placental

fornation There was very IÍttIe evldence of ceII disruption in the zinc

repleteembryosexaminedinthissludy'exceptatthemostrostralsite

where the neural tube had recently closed (Fig' 3'4c), and whlch

represenbs the sibe where nornal prosralnmed celI death occurs most

frequently during neurulabfon in heatthy rat embryos' At this slfe many

dense dark intraceLlular and extracellular bodies were obser¡¡ed' In order

to denonstrate that the abnormaj- zinc deflcient enbryos were not nerely

growth retarded, normal zlnc replete 10.5 day enbryos were also sectÍoned

and photographed at a comparable Ievel (FÍgs. 3'4d,e)' Although the

anberior neural tube has not yet closed in the forebrain and hindbrain

regions,thecellularconponentsarehíghlyorganisedlunlikesfnilar

sftes fn zinc deficient enbryos. To avoid unnecessarv repetition of

control photographs, Fig. 3.4 wtIl be referred to where appropriate in

subsequent chaPters.

HÍstological examination of 11.5 day zinc deficlent embryos as seen in

Fig. 3.5a revealed that While the rosfnal part of bhe cranlal neural tube

had closed, neurulatlon was fnconplete wlth the hindbraln region renaining

open due to the lack of fuslon of the neural'folds' Thts enbryo

denonstrated thaü the norphological distortfons thoughb to be

neningocoeres under the dissectÍng microscope (Flgs. 3.1c,d) observed Ín

nany zinc deficlent enbryos 1¡ere 1n fact fluid fllIed cysts as they did

3-28

Fig. 3.5a. Light mÍcrogr-aph of on of an 1 1'5 day zinc

deficlent enuryJ showing a large m cyst (asterisk) in the

lateral region of the crãnfum leading and distortion of the

neural tube. The neural folds have e hindbrain region and

there are condensed bodies within the neuroepithelium and the mesenchyme'

sone of which appear to be located on the ectodermal surface (arrow) inthe hindbrain á"u". Arrowheads nark the region seen at highernagnification in b. Fb, forebrain; Hb, hindbrain' E, ectoderm; bv' blood

vessel s.

FÍg 3.5b. Higher nagnification of a region in bhe forebrain-midbrainwhÍch shows that the basal surface of the newoepÍthelium (N) 1: disrupted

(arrowhead) and that large nunbers of inclusions and extracellu'lar debris

(arrows) are present in the neuroepithelium and nesenchyne (M)' Note the

cell debris within the blood u""""i (Uu) and ín the ventricular lumen (L)'

Figs. 3.5c,d. Severely díslorted neural '5 day zÍnc-

def icÍent embryo with neural folds (NF) fused' Snall

arrowheads in c, nark the siþe seen at hi in d' The

neuroepithelium (N) in d. is disorganised intra - and

extracellular bodies (large arrowhãads), and Iarge blebs. (sna[ arrows)

protrude Ínto the ventricular lumen (L)' The mesenchyme (M) also contains

some cerr debri; ii;;;; "r"ow")' Mibotic fisures (Mf) are vislbre' bv'

btood vessel.

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3-29

noU involve the neural tube. Except for the presence of bhese cysts,

the abnornatities observed at the cellr:Lar IeveI ln thls anlnal generally

reflects the cranial dysmorphology of the embryo ln FÍ9. 3.2c viewed

under the scanr¡Ing electron nlcroscope. The cyst led to distortlon of the

neural tube on the sane slde of the drar¡1un causing conpressÍon of the

optÍc plt, neuroepitheliun and mesenchyne. Dense, dark bodies were also

noted at these sites.

HÍgher magnÍfÍcaüion of a sectlon of the neural tube (Fig. 3.5b) clearly

showed a considerable degree of disruption of the newoepithelÍun at

various sites ]n the forebrair¡-nidbrain regions which resulted Ín bhe

forrnation of large, extracellular spaces probably because of cell

shrinkage or death wlth cells appearing to be lost ínto the mesenchyne and

ventricglar lumen Many darkly stained bodies were observed 1n the

neuroepitheliat cells as well as in the Iu¡nen and the nesenchyme. These

bodies occurred either free in the üissue or were located wfthln cellS.

The nature of these dense bodles will be discussed later in this section

As w ell as dense, dark bodÍes wÍthin the mesenchyne, the ¡nesenchynal

celrs themselves were arso affected by zÍnc insufficíency (Fig' 3'5b)' The

cells appeared to be nore rounded than stellateshaped and with

few er inbercellular cytoplasnic connections. The crowded appearance

of the nesenchyne could probably be attrÍbuted to a nunber of features

including rounded cel1s, considerabLe numbers of intracellular and

extracellular dense, dark bodies sone of which probably originated in the

neuroeplthelium, and compression of mesenchynal contents due to the fluid

fil1ed cranlal cyst. The aggregation of condensed bodies observed in the

hindbrain region probably occurred as a resulb of nigration fron other

parts of lhe mesenchyme as well as the neuroepithellun. This site appeared

3-30

to provide an exit for the debris fron the neural tube as higher

magnlficaüion of this area (not shown) showed thaù naüerial was being

extruded from fhe neural tube through a break in the ectodern. Ïhis

procedure along wllh deposltion of naterial lnto the lumen tI¡ay provide the

enbryo with several mechanisms by whlch it nay rÍd lbself of pathological

materÍa1 durlng organogenesls.

The severely malforned anterior neural tube of another zinc deffclent

embryo shown fn Figs. 3.5c and d, probabl-y reflects the serlous neural

tube dysmorpholory seen Ín ühe enbryo 1n Fig 3.2d observed under the SEM.

Ihe neural folds of the forebraln and hlndbrain areas have nof fused and

are also severely dlstorted especlally in relation to the orlentation of

the forebrain neural folds. The lumenal- surface of bhe newoepithelÍal

cells showed severe blebblng and budding inbo the ventricul-ar space, whlle

the basal nargin of the neuroepitheliun was convoluted and 1n sone areas

the ner:¡oepithellal cells appeared disrupted at these sites. Despite these

abnormalities nitotic cetls vlere still visÍble at lhe lunen T?¡e ¡nesodern

showed sinilar abnormally rounded cells and an absence of cytoplasnic

connections as seen in Figs. 3.5a and b, however there was far less

evidence of intracellular and extracellular dense bodies. Hence the

disparities Ín dysmorphology and in the presence or absence of

condensed bodies may refLect a difference in efficiency between enbryos

to remove this debrfs, but is more probably related to the level of

zinc defÍciency and/or the stage of neurulatÍon when the deficiency is

nosf pronounced.

3.3.3 Ultraslructural observations

The transmission el-ectron microscope enables the ultrastructure of the

anterior neural tube of 11.5 day zinc replete embryos to be described

FiS. 3.6. Transmission electron micrographs of features ofcranlal neural tube cells.

3-31

normal 11.5 day

a. A region of the neuroepltheliurn (N) at, the apical end adjacent bo the

ventricularlumen(L)withcellsincloseproximitytoeachother.Thereare snall cell processes or nblebsn whÍch project, into the lumenal space

(arrow) with junótional complexes between apposing cells (ic). The nuclei(nu) are generalì.y oval in shape with dístincbive nucleoli (n) wibh

condensed heterochromatin (c) in a clockface arrangement around the

nuclear PeriPherY.

b. A rnitotic cellThe chronosomes (ch)

in late telophase adjacent to the ventricular lunenand nuclear envelope (arrow) are vÍsible. L, Iumen

c. Higher magnificatÍon of organelles in a normal neuroepithelial celIshowinj the cytoplasm fÍIled with free rÍbosones, nitochondria (m) withcharacteristic IàneIlar or plate-1ike cristae and rough endoplasmicreticutum (r) which were either straight or branched. The nucleus issurrounded by a nuclear envelope (arrow). Dr nucÌeolusi cr

heterochromatin; 8¡ Golgi apparatus.

d. The mesenchyme contains stellate-shaped celLs whose extensivecytoplasmic projections connecb the cel1s together (arrow),(confirned at much higher magnification than shown here), and aresuspended in an extracellular rnatrix (ECM). DUr nucleus; n, nucleoÌus'

e. Dorsal midline region of cranÍum at the most recent point of neuraltube closure showing norrnal programned cetL death. Dead cell components

were either tocateã within the extracellular space (open arrow) orphagocytosed by other cells (arrowheads), where in sone cases they

occupied most of the space within the host cell (asterisk)' Note t'he

vacuoles in severaL cells which appear to be phagocytosÍng extracellularnaterial (arrows). L' Iumen

f. Higher magnification of a sectÍon of the host cell marked by an

asterisk in e. cont,aining many phagocytosed, condensed cell remnants atvarious stages of digestion, contained within heterolysoso!¡es (arrows)

causÍng the ceLl components to be indistÍnguishable. A few membranes

probabiy of mÍtochondrial origin couLd stil1 be identified (largeàrrownea¿). The polyribosornes of the host cell were dispersed'mitochondria (m) were condensed and the cÍsternae of the rough endoplasnicretÍculum were vesiculated (open arrow). Note the abnormal shape of thenucleus (nu) and its outer envelope (small arrowheads).

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3-32

(Fie. 3.6) and to be conpared with similar sites in 11.5 day zinc-

deficient embryos. The anterÍor neural tube consj-sts of a thÍck'

pseudostratified columnar epithelium with most of the neuroepithelial

cells extending fron the ventricular lurnen to the basal lanina. At the

lunenal surface (Fig.3.6a) of nornaL 11.5 day enbryos, snall apical ceII

processes referred to as nblebsn at the scanning electron nicroscope level

are closely apposed with conspicuous junctional complexes or terminal

bars, and nicrotubules are conmon in this region. Mitotic cells were found

at or near the lunen 1n the lateral regions of the anterÍor neural tubet

in various stages of the cell cycJ.e. Fig. 3.6b shows a mitotic ceIl in

late teLophase. Fig 3.6c denonstraLes a nu¡nber of features chanacteristic

of neural cells at this stage of developnenb. Thnoughout the

neuroepitheliun, the cytoplasnic membranes of neighbourÍng cells are

closely apposed but bhere are rþ junctional compl-exes. The interphase cell

body characterÍstically contains a J.arge nucleus with dense chronatin'

which is either concentrated in patches (heterochronatin) on finely

dispersed (euchronatin), and a nucl-eolus which is either centrally or

peripherally located. The cytoplasm contains free rÍbosomes, which at

hÍgher nagnification (not shown ) can be seen to be arranged polysomally,

which is indicative of protein synthesis. Rough endoplasnic reticulun

located in the cytoplasm varies fro¡n short fragnents to long, sometines

branching cisternae, whÍch nay even be dllated.

The cells often contain a prominent GoIgi apparatus, and nunerous

nÍtochondria with Ianellar crlstae. Fig. 3.64 shows the underlylng

nesenchyme which is situated adjacent to the basal surface of the

neuroepitheliaL layer and is separated fron it by the basal lanina (not

shown), which in nornal 11.5 day enbryos is intact. The cells which are

suspended in an extracellular natrix (ECM) are stellate-shaped r¡ith

3-33

extensive cytoplasmlc proJecttons or npseudopodlan which closely appose

neighbouring ce1ls as well as the basal lanlna by neans of tight and close

Junctions (Morriss 19TÐ. rhe organerles appear slntlar in structure to

those in the neuroepit,hel ÍaI cel1s. NonnaI progranned celI death isconcentrat'ed along the dorsal aspects of the anterÍor neural tube where

closure has ooourred nost recently (Fig 3.6.¡. Dead cell conponents were

either located in the extraceLlular space or phagocytosed by other

apparently hearthy cells and in sone cases occupied nost of ühe

space of the host cell. Ttre oelr debris usually varied ln densitv (Fig.

3.6f) depending on t,he ceLl conponenüs and the leve1 of dfgestion whÍch

had occurred. The highly dense Ínclusions in this nÍcrograph were

probably nainly nuclear rennants, with sone cytoplasnlc organelles

around the less dense perÍpheny. Sone membnanous remalns probably of

nitochondrÍa1 origln were also evident. Ser¡eral features of the host

cel1 whlch suggested that it too night succunb to eventual celldeath, included dÍspersed polyrfbosomes, condensed nitochondria and an

abnornally shaped nucleus wÍth Íts outer envelope no longer in close

proxfmfty.

Trans¡nission electron nicroscope exaninatlon of a number of zinc

deficient' enbryos showed extensive ultrastructural disorganisatlon

throughout the anterior neural tube, some aspects of which are

lllustrated in Flg. 3.7. The first nicrograph (FÍe 3.Za) shows largecytoprasnic protrusions or blebs at the apical end of the

neuroepithelial cells whÍch project into the Iunen of the ventriclegiving Ít the severely disrupted appearance first observed under the

SEM. Sone of these blebs were filled with electron dense cellremnants, seen as dark Ínclusions at the 1lght nicroscope level. The

accumulatlon of celLular naterial and lysoso¡ne-l1ke structures at this

ebs) proiecting into the ventricularcel-I remnants including mitochondria

ar structures are probably lysosomes

one of these processes appears to be

aring to extrude ceII remnants intodebris in the lumen (ttr:'n arrows)'

L, Iumen; Nr neuroePÍthel iun'

Fie.3.?.Ultrastructuraldisorganisationwilhintheanteriorof ã aãy 11.5 zinc def icient enbryo'

b.Neuroepithelialdeadcetlrennantstocatedextracellularly(arrowheads) and intracellularly (arrows), of varying density ranging

i"ot light fLocculent to dense bodÍes'

co nde nsedand other

3-34

neural tube

n itochondriaceII nemnantsc. A neuroepithel ial ceII ( N) containing n-any

with broken cristae (arrows), large vacuoles (v),

probably of nuclear origin (arrowheads)'

d. A neuroepithelial ceIl (N1) showing the early signs of cell disruptíon

including breakdown of the ceIl mernbrane (: rrows), dispersal of

cytoplasnic ribosornes, damagednitochondria (asterisks), and membrane

fragnents surrounded by rough "nooplasnic reticulum (1arge arrowheads)'

cornpare the sparse disbribution of polyribosones (smaII arrowheads)in the

cyLoplasrroftheaffectedcellwiththoseoftheadjacenthealthynäurðepithelial cetl (N2)' cv, coated vesicles'

tual fabe of neuroepithelial ceIIscomponents (arrow s)' The condensed

nucleus of the host ceII clearlYs; N' neuroePitheliun'

f.AsectionofthenesenchymewÍthdeadcellrennantslocatedclear origin, and extracelluJ'ar1yonganelles sbill disfinguishable'is are also visible and the large

e in the Process of engr:tfing ceII

components (oPen arrow)'

g. condensed dead cells and cell rennants (arrows) wlthin the nesenchymal

extracellular matrix (ECM)' The basal lamina was disrupled at several

sites (open arrows). N' neuroepithelium'

Bars=2um.

\sf,ÞTft/t

t'.

{

w Iv;

ECM

1

c:

)-r

,Æs{ì

3-35

site, and the dlsruptlon of the cell nenbrane suggesfs that the neural

tube disposes of dead naterial by extrusÍon into the ventricular

Iunen. Ffg. 3.7b shows a range of electron dense naterial situated

both lntra- and extra-cellulan1y in the walI of the neuroepithellun. Tlre

dark bodies varied considerably in content and were either phagocytosed

by apparently healthy neuroepÍt,hellal cells or were locaüed in the

extracellular spaces. Tt¡e dead ceII rennants ranged fron light flocculent

material to dense bodies, and appeared to represent all conponents of

,celIs both nuclear and cytoplasmic (Fie. 3.7c).

It appears 1Íkely that ühe ultimate fate of the apparently healthy

neuroepifhelial cells which phagocytose dead cell remnants in the

absence of nacrophages at this tine Ín enbryonic developnent fs death, as

denonstrated by the irregularly shaped nucleus and condensed cytoplasm of

the scavenging rhostr celI 1nFig.3.7d. Sone of the early signsof cell

disruption prior to condensation of the celI conponents are shown in Fig.

3.7e. The cell menbranes are disrupted, cytoplasn is dlspersed, bhe

cytoplasnlc ribosones have lost theÍr polysonal confÍguratlon, and nany

of the mitochondria are dÍsrupted. Sections of rough endoplasmic

retÍculun contain nembrane fragmenbs within the ci.sternae which nay be

digested by ribosonal enz ymes (Ghadially 1988). Dispersal of the

polysomes has also been previousl-y observed to be the firsb norphological

sign of actÍon of the toxic agent fluorodeoxy-uridine on embryonic

neuroepithellum (Langman & Cardell 1978).

The nÍcrograph in Fig. 3.7 f show s extracellular and j.ntracellular

remnants of varying density in the mesenchyme sinilar to cell

debris observed Ín the neuroepithelÍum, with sinilar organelles being

affected. Small vacuoles were frequently observed in nesenchynal cell-s

3-36

often containlng ceII remnanbg I?re large nunbers of dead cell rennanfs

observed in the mesenchyne close lo the basal lanlna 1n sone zinc

deficlent embryos nay be due to the extractlon of dead

neuroepithelial cells and ceLl rennants lnto the extracellular natrlx. Tt¡e

presence of a disrupted basal Laroina (fig. 3.79) Ín sone zinc deficient

embryos nakes this a llkeIy neans by which the neuroepitheliun rids ltself

of sone ceII debrls.

3.4. DISCUSSION.

The data pnesented in this study confirm previous observations (Hurley &

Swenerton 1966; Eckhert & HurIey 1979i Rogers et aI t985; Record et at

1986; Harding et aI 1988) ühat low dietary zlnc levels in pregnant dans is

highly correlated with growth refardatÍon in enbryonic and fetal raüs. Ihe

observation that embryonic growth is severely retarded by fn utero zinc

Ínpoverishnent denonstrates that growth retardation occurs at, the tlne

of neurogenesis (days 9-1 1).

Although nary embryos exposed to a zinc deficient envÍronnent displayed

severe nalfornations of the central nervous systen and craniofaclal

defects as reported previously (Hurley & Shrader 1972i Record et al

1986) the hfgh incidence of intrauterine deaths and resorptÍons

reported eLsewhere (Duncan & HurIey 1978b) was not observed Ín this

study. Thls difference may have arisen at Ieasf in part fron the variation

1n tine of renoval of the enbryos, which Ín previous studies was close to

term, whereas in the present investigatÍon occurred at mid-gestation, a

tine when the rab enbryo is not yet dependent on a functioning placenta.

It is shortly aften this period however, that the fuIl consequences of

the lack of chorÍo-allantoic fusion and of subsequenb placental fornation

becomes apparent as the embryo is dependent on ühis structure for adequate

3-37

growth and nutrition as it increases in size after day 11 or the 17 somife

stage in rats (New et al 1g76). Hence by renoving the embryos from the

uterus on day 11.5 followÍng neurulabÍon, the incidence of enbryonic

deaths and resorptions was mininised and the number of |tusefuln observable

malf ornabions wa s maxinised.

!Ihile nost embryonic structures were affected to some extent by zinc

deficiency, norphological and nicroscopic evidence revealed that the

developing nervous systen appeared to be most frequently and severely

affected. Biochemical studies supporb this organ selectivÍty as Eckhert &

Hurley (1977) reported a greater reduction in nuclelc acid synthesis in

brain compared with body regions fotlowing zÍnc impoverishnent which was

accompanied by an enhanced recovery in neural tissue following zlnc

replacenent. Hence the nore severe dinÍnution in neural tissue

demonstrated by reduced nucleic acid synthesÍs could account for this

Íncreased nervous systen vulnerability'

Dysnorphology of the brain and associated structures which occur as a

resul-t of in utero zinc inpoverish¡nent durÍng organogenesÍs including

abnormalities of the braÍn (Hur1ey & Swenerton 1966; Dreosti et al 1972'

Hurley & Shrader 1972; Record eb aI 1986; Harding et aI 1988; Joschko et

al 1989), craniofacial- processes (Dreosti eb al 1972i ÏJarkany & Petering

1972) and eye (Hurley & shraaer 1972i Record et al 1986; Rogers & Hurley

1987i Joschko el al 1989) was confirmed in this study. The vast range and

variation in intensity whÍch was characteristicalty seen in the devel-oping

nervous system is thought to occur as a consequence of the large changes

in serun zinc levels during organogenesis brought about by the zinc

defÍcient dans regulabÍng theÍr food intake in a cyclÍcal nanner of about

4 days duratfon (Record et aI 1985a), and hence the specific nalformation

3-38

is Iinked intinately to the timíng of the feeding and fasting stages of

the cycle (Record et al 1986). By fasting the dams on days 6 and 7 of

gestation, the author was able to induce Ìow serum zÍnc leveIs during

neurogenesis which was reflected in the high incidence of brain and eye

anomalies, since these organs are developing maximalJ-y (and are therefore

nost affected by zinc deficiency, see Fig. 1.1) between days 8 and 10 of

gesfation (Hur1ey et at 1g71). These differences in timing and degree of

zinc deficiency are likely to explain in part belweerrlitter variations ín

malfornati.ons observed in the neural- tube in this study, and previously

(Record et aI 1985a, Harding et aI 1988) where the 11.5 day enbryos in

some 1itters were so severely affected that, the neural tube was open from

the anterior end to the caudal nost regions, while in others the anlerior

neuraL tubes !ùere cÌosed or were slightly open in one region of the

cranium only. The gross neural tube abnormalities observed are

unlÍkely to arise from a delay in developnent but rather as a result of

abnormal development. This was supported by the cellular and

ultrastructural data, which showed serious damage associated with the open

neural tube and refuted bhe consideration thal neural tube abnormalities

were induced by a slowing of neurulation

Ultrastructural observations demonstrated that the major site of action

of z¡nc deficiency in the neural tube is on the neuroepithelial cells.

This action of a teratogen specifically on one kind of cell type has been

noted previously (Morriss 1972,1973; Joschko et al 1989) when excess

vÍtanin A was observed to preferentÍaIIy affecb tissues derived fron the

nesodermal gern 1ayer, with a ninÍnal effect on the neuroepithel-ium. Hence

it appears that zinc deficiency may be tissue specific exerting its affect

on t,he neuroepithellun which dlfferentiates into various brain

structures Iater in pregnancy (ÏIendell-Snith & ÏJillians 1984).

3-39

Tt¡e neural crest cells are anolher popr:lation of cells that appear to be

affected by ztnc inpoverishnent. Sfnce neural crest ceIls could nol be

readlly dlstinguished hlstologically fron other cells in the reglon of

closure of the neural tube, ühis observation could only be nade

lndfrectly in ühis study by exanination of neural crest ceII derlved

structures, such as branchial arches (Noden 1986). It is now well

established that nost of the faclal nesenctyme Ís derfved from the neural

crest cells (Pratt & Christiansen 1980; Noden 1982) This population of

enbryonlc cells 1s usually first identified in the neural ectodern

Ímnedfately before fusion of the neural folds in mannals (Verwoerd &

Oostran 1yl9) and migrates lnto the fÍrst and second arches by the 23

sonite stage (day 11) Ín rats (Sul1k et aI 1988). An absence or dimÍnution

of branchlaL arches at this tlne suggests that zÍnc deficiency

lnterferes with neural crest cells possibly by impeding crest cell

nÍgration. Hal1 (1984) previously concluded bhat defects of arch

derlved structures occurred through an interference with crest ceLL

migratlon fron their position between presumptive neural and epidernal

ectodern, and that this cor¡ld occur by inhiblting the Ínteraction between

the cells and the extracellular producls such as hyaluronfc acid through

whÍch they nigrate (Thorogood et al 1982). However, not all branchial arch

defects observed 1n this study nay be attributed to reduced crest cell

migrationr âs ín sone enbryos the nandibular processes were present, but

abnorrnal, often beÍng fused at the ¡nÍdlÍne and/or to the pericandiun

suggestlng lnstead that, the defect nay J.Íe Ín the incorrect positioning of

neural crest cells in these süructures. In contrast to facial

processes, zinc deficiency may exert its effect on optic placodes by

reducing the number of neural crest cells which derive fron the neural

plate, since evidence reported by Johnsbon (197Ð suggested bhat eye

nalfornations nay be due to an actual def,fclency of newal

3- 40

crest ceIIs,

raüher than to defective cell nigration

Itre nechar¡isn through which zinc deficiency exerts ibs teratogenic

effect on preplacental enbryos 1s as yet uneeitaÍn, however several

proposals have been forthconing and a nunber of studles have suggested

that zinc deficient teratogenesis 1s biochemically derived. Sone workers

(Swenerton et aI 1969t Grey and Dreosli 1972: Hurley 1976) have ascribed

the teratogenlcity of natennal zinc deficÍency to lmpaÍred nucleic acÍd

synthesis during enbryonic developnent resulting ln reduced cellular

proliferatfon, leading to inpaired cell and organ development. Decreased

celluLar proliferation nay be nediated by reduced activity of the zínc

dependent enzyme ttymidÍne kinase whlch is thought to lead bo a dfnÍnution

fn nitotic ativity (Prasad & Oberleas 1974; Dreosti & Hurley 1975i

Dreosti et aI 1980) since the enzyme is wÍdely recognised to be a raüe

J.fmibing step in DNA biosynthesis. The Ínvolvenent of thynidÍne kinase

ln reduced nitosls is further supported by the observafions thaf

3tt-ttynidlne incorporation has been reported by several groups to be

reduced 1n whole body DNA in zinc deficient fetuses (Swenerton et al

1969i Ecklnert & Hurley 1977, Dreosti et aI 1980)r with an even great,er

reductlon in the head regions of day 12 embryos of zinc deficient dams

compared with pair-fed controls (Eckhert & Hurley 1977). Other workers

(HurIey & Shrader 1972; Record et al 1985b) have proposed that the

zinc defícÍent induction of inpaired nucleic acld synthesis is related

to an increase in nitotic index, which suggesls that zinc de,ficiency

exerts its effecf by blocking or delaylng mibosis at a crÍtÍcal

stage of organogenesis (Record et al 1985b). !Ihile it was conflrned

in this study that nitosis occurs in areas where necrosis is severe,

Record et aI (1985b) suggested prwiously that zinc deficiency nay affect

only a specific Phase

able to accumulate

neighbouring ceII s.

3-41

of the celt cycle, or that perhaps sone ceLls were

and utilise zinc released as a result of death of

Ílhile bhere is strong evÍdence which suggests that zinc deficiency-

related teralogenesis may Ínvolve an action on the ce]l cycIe, the

appearance of the dysnorpholory of the neural folds and of the

ultrastructural nembrane defects, suggests lhat zinc impovenishment may

Ínterfere with neural tube closure directly by an action on cell

nembranes, principally those of the neuroepithetÍum' This was ilÌustrated

by an effecb on the internal Iiniting nembrane which $tas particularly

dislorted and disrupted by zinc inpoverishment, as well as the nembranes

of organelles, especially nitochondrial cristae and endoplasnic reticulum'

A menbrane-raediated action of zinc deficiency Ís considered to be

particularly 1ikely since an absence of zinc has been shown to compronÍse

the integrity of rnembranes (Chvapil 1976). Furthermore' in view of the

rapid onset of cell death and disruption observed only hours after

adninistration of a nunber of teratogens lncluding excess vitanin A

(Morriss 1973) and ethanol (Sulik et al 1988), and since the

uftrastrucbural defects and gross dysnorpholory which occurred with these

tvlo teratogens $¡as quÍte sinilar to observations made in zinc deficient

enbryos Ín this study, 1t is likely that ceLl death and disruptÍon may

rapidly folLow the inductÍon of a zlnc deficient state, and may underlie

the severe gross abnormalities characberistic of t'he zinc deficiency

syndrome.

Hence the present author proposes that the frequently reported

observation of reduced nucleic acid synthesis and cellular proliferation

is a consequence of the zinc deficient, state and related teratogenesis

3-42

nather than the cause of 1t, and that zinc deficiency exerts lts rapld,

prfmary node of action directly on ceLl and orgêne1le nembranes leading to

thefr disruptfon and destabllisatfon, and to the rapid and ser¡ere onset of

cell death. The dfsruptfon of the entire cell cycle fncluding nitosis and

DNA synthesis nay then ensue. I,lhile fhe present results support nenbrane

destruction and subsequent cell death as being closely correlated with

neural tube dysmorphologr lnduced by zinc defíciency, this study was the

first of a nunber perforned by the author, and further result,s descnibed

in later chapters casb sone doubt on the valldf ty of t,hisr associatlon,

causlng the aufhor to consider that neuraL tube necrosis nay be related

to functÍonal deficits nanifested after birth, rather than the underlying

cause of failed or inconplete neurulation

Membrane IÍpid peroxÍdation couLd be considered a likely nechanisn

through whfch membrane breakdown observed in developing enbryonic tissue

nay occur, since the involvenent of zinc Ín the free radical and cellular

antÍ-oxidant defence mechanisms has now been establlst¡ed (Dreosti 1986).

fubryonic tlssue nay be partlcularly vr.rlnerable to free radÍcal danage

slnce many of t,he mechanisms protecting the enbryo fnon fnee radical

damage are scarcely actlve in utero (Dreost,i & Partick 1987). Lipid

peroxidation has been shown fo Lead to disruptÍon of lysosomal menbranes

and subsequent release of Ìrydrolytic enzynes (l{ iIIs & I'liLkinson 1966)

and Ín view of the presence of lysoson+'like structures associated

with regions of subcelluLar disruption this nay Ín turn lead to the

extensi.ve cel1 death observed in the neural tube of zinc-deficlent

enbryos. Since lipid peroxidatlon has been reported t,o lncrease fn fetal

liver buü not in brain on day 20 of gestaùion (Dreosti & Partick

1987), nembrane lipid peroxidation would seen not, to be a likely

mechanisn of neural menbrane danage. Ho!,lever, the author suggests

3-43

that the difference 1n llpid peroxidatfon between the two organs

night be explained by the activation of a nbrain sparlngn effect later

in pregnancy when Iiver functlon nay be sacrificed by direcbing nore of

the available zfnc to the braln This proposal Ís further supported

by the absence of abnornal ceII death Ín neural tissue in the nost

extensive histological studies of older ztnc deficient enbryos and

fetuses (HurIey & Shnader 1972) as well as the observation that' both

serun and braÍn zfnc levels ín zlnc deficfent fetuses were unchanged

fron controls, whlle in Iiver values vtere sharply lower. These

observations poÍnt to the operation of an effective homeostatLc

nechanism for zinc 1n ùhis vital organ, leading to the rbraÍn-sparingn

effect. Record et a] (1985b) suggested that exposure of enbryos to waves

of zinc deficiency later 1n gestation is less Ilkely to cause gross

cellrùar necrosis because of a functional placenta allowing access to a

large volune of circuLating rnaternal zinc, and also because of the

regressÍon of decidual üissue which has a large nass conpared wlth the

enbryo, which probably contrlbutes to nut,rient requÍrenents of the enbryo.

The growth rate of the embryo decllnes with age fn nid-gesfation and thus

the proportion of cells for which extra zinc is required dimÍnist¡es

(Record et al- 1985b). Although large numbers of cells are involved' it 1s

also posslble ühat these cells acquíre sufficient zinc to naintaln fheir

integrity fron other quiescent celLs wilhin the embryo. Hence the enbryo

nay be selective about the organs which receive additÍona1 zinc and this

might explain wty brain is spared but Iíver is not. If it is aecepted that

the fetus has better access to circuLating zínc, and requires less zinc

later in pregnancy when the growth rate 1s reduced, this would explain why

roenbrane lipid peroxidation is not indicated at term, but it does not

disniss the possibility that the extensive cell death observed

preplacentallY occurs

radical danage.

3-44

as a dÍrect nenbrane effect nediated through free

The severe cell death or necrosis which was far in excess of the nornally

occurrÍng physlological or progralnned cell death (eCO¡ (Schwelchel &

Merker l¡g13i Geelen & Langnan 1977i WyIIie 1981)¡ wâs located throughout

the neuroepitheliun of the anterior neural tube. In contrastt

physÍoJ.ogical celÌ deabh was observed in control enbryos üo be nairùy

confined to the sites of neural tube cl-osure located dorsally along fhe

rostro.caudal axls, while the nitotically active Iateral regions displayed

only fhe occasj.onal dead or dying ce]l. Sulik et aI (1988) have

reported sinÍIar findings of extensive areas of cell death in the

developing neural. tube following exposure to a nunber of teratogens,

fncluding ethanol which demonstrated sone quite similar neural tube and

craniofacÍal dysnorphologl to that observed in zinc deficÍent embryos.

Nile blue stain was seen to be taken up specifically by necrotic cells in

regfons of the neural tube and other prolJferatÍng areas of the enbryo Ín

an as yet unknown manner at, sÍtes which corresponded to areas of normal

pCD and which had expanded from there. Hence Sulik et aL (1988) proposed

that the expansion of regions of progranmed cell death 1¡as a suÍtable

pathogenic nechanism underlying craniofacial malformations. While there

was sone expansion of ceII death fn the regions of PCD in the neural tube

of zÍnc deficÍent enbryos in the present study, histologÍcaI and

ultrastrucbural exanination revealed an extensive area of necrosis in bhe

nltotically active lateral regions of the neural tube, which suggesfs

that, the nechar¡lsm underlying necrosis night not be the same as that

underlying PCD. Hence it can be concluded that the extensive cell death

observed in experi.mental aninals in bhis study was not part of the

progranmed cell death seen routinely in control aninals, which has its

3-45

major function in the shaping of enbryonÍc tlssue and rcullingn of

unnecessary or unvíab1e cells, bul rather that, it refLected the

teratogenic outcome of zinc impoverishnent in rapidly proliferatÍng

regions of the preplacental embryo. I,lhile these observations point

towards a distinction between the aetiology of PCD (or apoplosis) and

necrosis, in the present study however, the author was unabLe to

differentiate between them uLtrastructunally. Although separate models

of cetl degeneratíon for apoptosis and necrosis have been proposed

(!,1y111e 1981)r all stages of both models trere observed in the present

study in regions of the neural tube where celI death has been designated

as either predoninantly necrotic or apoptotíc. Hence bhe author

proposes that if any distinction is to be made between apopbotic and

necnotÍc pathology, it should be based on a difference in locations

withÍn the neural tube rather than on rigidly defined norphological

criteria at the ultrastructural Ievel. It should also be considered

however that a possibte biochenical distinction and biochenÍcaL pathways

nay separate apoptosls from necrosis, mediated through different

lysosonal enzymes or as yet unidentified compounds being released by the

dying or dead cells.

3.5 CoNCT.USroN.

The presence of severe nenbrane danage and cell death predominantly in

the neuroepitheliun during neurulation nay underlie the Sross

dysnorpholory which included open neural tubes and poor branchÍal arch and

optic placode developnent in zÍnc deficient enbryos aE the tine of

neurulatlon in this study and exencephaly, aleft palate and eye defects 1n

term fetuses obsen¡ed previously. The nechanism by which zinc deficiency

exerts its teratogenlc effect is 1Íkely to be through a rapid and direct

3-46

action on ceII and organelle nenbranes Ieadlng to their destab1.lfsatlon.

This tnnediate aotlon nay be nedlated through free radfcal danage or

lysosonal enzy¡ne activtty, and nay ln turn lead to a secondary effect

lnterferlng wlth blochenlcal nechar¡lsns of the cell cycle lnoluding those

lr¡volved tn DNA synthesls and nlfosis.

3-47

3.6 B]BLIOGRAPHY

AdeIoye, Á. & Ì,larkany, J. ( 1976) Experinental congenital hydrocephalus: a

review with special- consideration of hydrocephalus produced by zincdefÍciency. Childs Brain 2, 325-360.AItmann, J. & Das, ED. (196Ð. AutoradÍographic and histological evidence

of postnatal hippocampal neurogenesis Ín rats. J. Comp. Neurol. 124r 319-

326.Baker, RJ. & Nelder, JA. (19?8). The GLIM systern. Release l. Generalised

Linear Interactive Modellings. Nunerical algorÍthms Sroup' Oxford.Bannister, J., Bannister, Í1. & Ïlood, E (lgll) Bovine erythrocyte cupro-

zinc protein: I. fsolation and general characterisation Eur. J. Biochen.18, 178-186.Barnes, PM. & Moynahan, EJ. (19?3) Zinc deficiency Ín acrodermatitis

enteropathica: MuItiple dietary intolerance treated with synthetic diet.Proc. R. Soc. Med. 66' 327-329.Beaudoin, AR. (19?9). Embryology and Teratology. In: The laboratory rat'

1íoL2. Research Apptications. Baker, HJ., Lindsay, JR. & lleisbrobh' SH.

(eds). Acadenic Press, New York.Beise1, WR. (1976) Trace elenents in infectious processes. Med. CIin.

Nth. Am. 60, 831-849.Berglund, T. (19S4). Zinc-Biological Effects. University of Stockholn.

fnstitute of Physics Report No. 84-12.Bergnann, KE., Makosch, G. & Tews, KH. (1980). Abnornalities of haír zÍnc

concentratlon in nothers of newborn infants wÍth spina bifida. Am. J.

Cl ín. Nubr. 33, 2145-2150.BetLgen, WJ. & OtDelt, BL. (1981) A cribical physiological rofe of z|nc

in the strucLure and function of biomembranes. Life Sci. 28, 1425-1438.Blamberg, DL., Blackwood, tIB., Supplee, WC. & Conbs, cF. (1960). The

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Todd, Ï,|R., Elvehjem, CA, & Hart, B. (1934). ZÍne in the nubrition of therat. Arn. J. Physiol. 1O?, 146-1 56.Turk, DE., Sunde, ML. & Hoekstra, WG. (1959). ZÍnc deficiency experinents

with pou.ì.try. Poultry Sci.38' 1256,Underwood, nI. (1gi7). Trace Elenenbs in Human and Aninal NutrÍtion. 4th

Edn Acadenic Press, New York. pp 196-242.Van Rij, AM. & McKerøie, JM. (1977). Zinc in parenteral nutrition hoc.

Nutr. Soc. (NZ). 2, 87-94.Verbug, DJ., Burd, LI., Hoxtell, EO, & MerriIl, LK' (1974)'

Acrodernatitis enteropathica and pregnancy. Obstet. Gynecol. 44' 233-237.Verwoerd, CDA. & Van Oostran, CG. (1979) Cephalic neural crest and

pJ.acodes. Adv. Anat. Enbryol. CelI Biol. 58, 1-75.l{alravens, PA., van DoornÍnk, t'IJ. & Hanbidge, KM. ( 197 8). Meta1 s and

nental function J. Pediat. 93, 535-541.!larkany, J. & Petering, HG. (19T2) Congenital malfor¡ûations of the

central nervous system in rals produced by maternal zinc deficiency.Teratologr 5r 319-334.hIendell-Snith, CP. & ViliI1Íams, PL. ( 1984). Basic human embryology.

Pitnan, Londontlitkins, PJ., Grey, PC. & Dreostir IE. (1972) Plasma zinc as an indicator

of zinc status in rals' Br' J' Nutr' 27r 113-120'Ïllllson, R. (198?). Vitarnj.n, seleniun, zínc and copper interactions

in free radicat protection against iJ.1-placed iron. Proc. Nutr. Soc. 46,27 -34 .ïlyllie, AII. (1981) Cell death: a new classification separating apoptosls

from necrosis. fn: Cell Death in Biology and Pathology. Bowen, ID. &

LockshÍn, RA. (eds). Chapman and HaII, London. pp 9-34.

4- 1

CHAPTER 4

GRCI¡TH AND MORPHOLOGICAL EFFECTS OF HYPERVITAMINOSIS A IN UTERO'

4.1 TNTRODUCTTON.

4.1 .1 HÍstorY.

Fron the time of the ancient Egptians synptoms of vitamin A def iciency

such as night blindness have been recognised as a disease which was

nutritionalty derived. In the mid to Iate 19th century endemic night

blindness observed in poorly nourished slaves, as weII as in orthodox

Russian catholics and in infants of mothers who upheld the Ientern fasts

was also alt.ributed bo a dietary inadequacy. It was not until 1913 however

that tw o groups (osborne & Mendel 1g13i McCoIlum & Dav is 1 91 3)

simultaneously reported that an essentiat Iipid soluble substance in

certain foods such as butter, mi)-k fat, egg yolk and cod liver oil

pronoted growth in experi¡nental rats, and that wibhout it xerophthalmia

and night blindness were frequently acconpanying symptons. The compound

was ab that tine referred to as fat soluble A and later as vitamin A The

experimentally induced symptoms of vitamin A deficiency were shown to have

a clinÍcal l-ink during !Iorld fiar 1 when xerophthalnia was reported in

soldiers deprived of dietary butterfat. The term vitanin A or retinoid is

the generic description of any substance, or mixture of subsLances

possessing vitamin-A like biological activity which is expressed in

internationaL units (ru) where 1 ru is equivafent to 0.3 ug retinol. 0ne

IU of vitamin A 1s also equivalent to 0.6 ug of the provitamin, Ècarotene

(WoIf 1984).

4-2

Vlhjl-e hypovitaminosis A has been, and continues to be, a najor public

health concern particularly in underdeveloped countries (See Underwood

1984 f or a comprehensi.ve review), hypervitaminosis A is a relatively

recent and infrequent problen with only 579 reported cases from 1850 to

1978 (Bauernfeind 1980), and fewer than 10 cases per year frorn 1976 to

1987 (Bendich & Langseth 1989). There is growing concern however that in

developed countries the prevalence of chronic hypervitaminosis A nay

increase in future years as the use of vitamin A' available withouL

prescription becones more widespread, particularly among sufferers of acne

and skin diseases, and health food faddists, with a further 10,000 new

prescriptions for the synthetic retinoid isobetrinoin, ( 13-cis-retÍnoie

acid, Accutane) written each nonth in the United States for s,omen of

reproductÍve age (Lammer et al 1985). This group is especially at rÍsk of

the consequences of excess vitamin A which include birth defects' since

young !{onen of chjl-d bearing age often suffer fron severe acne and

continue to ingest megadoses of vitanÍn A during the first trimester of

pregnancy. A recent review by Underwood (1989) describes the current

teratogenic status of vitamin A. AÌso at risk fron exposure to high

levels of vitamin A are infants and young children of welL meaning

parents overly concerned about the health of their youngsters and of

thmselves.

4 .1 .2 Metabol im.

Due to the extensive Iiterature available on Vitamin A metabolism, thÍs

section will onty outLine sone aspects which nay be relevant to thÍs

thesis. For a nore conprehensive report the reader is referred to a

review by Goodnan and Blaner (1984). Dietary vltanin A is derived from the

bfosynthesis of plant carotenoid pignenbs of which B-carotene has the

nost provitamin A activity and whlch occurs in significant amounts in

4_3

foods generally eaten by vertebrates (Bauernfeind 1972). Preforned vitamin

A can only be found in significant quantities in aninal tissues in the

form of long chain retinyl esters like retinyl palmitat,e. Mammal, fish and

fowl livers provide the richest supplies of preformed vitamin A because of

the speciaL role of the liver in uptake, storage and nelease of dietary

vitamin A (Bauernfeind 1980). Several reviews deal in greater detail with

the strucLures and functions of provitanin A and preformed vitamin A

(Underwood 1984; l,lolf 1984; 0lson 1986).

The biosynthesis of vita¡nin A occurs nainly in the intestinal- mucosa and

invoLves the cleavage of the provitanin A, B-carotene, to form two

molecuLes of retinaldehyde which are then reduced to retinol (Huang &

Goodman 1965; Fidge et al 1968; Goodnan & 0tson 1969) by the enzyme

retinaldehyde reductase which can also be found in the liver (Zachman &

Ol-son 1963) and in the eye (B1aner & Churchich 1980). The retinol so

forned is esterifÍed in the mucosal cel1 principally by pa1 mitic

acid by a specific enzyne reaction, and the reLinyl esbers enter

the lymph chylomicrons to be transported to the liver.

Preformed vitamin A in the forn of dietary retinyl esters are hydnolysed

in the inlestinal lunen to form retinol which is absorbed into the mucosal

ceIl, re-esterified with long chain maínly fatty acids, and the retinyl

esters fcrmed are aLso transported to the liver, the major storage site

for vitamin A, by way of lymph chylonicrons (Goodnan 1984b; Helgerud et al

1983). After uptake of the retinyl esters in the liver, a further

hydrolysis and re-esterj.fication occurs. Hepatic vitanin A usually

represents approxinately 90f of tot,a1 body reserves of vÍtamin A, where

both the hepatÍc parenchymal cells and the fal storing stellate cells,

seen to play a role in its storage (01son & Gunning 1983). Their reLative

4-4

roles in retinol storage under different conditions are yet to be

elucidated, however it is known that the parenchymal cell is the major

cell type involved in the hepatic uptake and initial metabolism of newly

absorbed retinot which is then transferred to the nottsparenchymal fat

storing cells after initiat uptake as retinol esters into lipid droplefs

for storage (Blonhoff et al 1982).

The parenchymal cel1 has also been identified as the celI type

responsible for the mobilisation of retinol from the liver (BIomhoff et

al 1982), white it has been suggested (McLaren 1981) that' the fat

storing cells act as a sump for excess vitamin A up bo a point, beyond

which hypervitaminosis A occurs. EVidence has been obtained that the fat

storing cells rnay play an important role in vitamin storage in the Iiven

under normaL as well as hypervitaminotic conditions (Knook et aI 1982).

Vitamin A is mobilised from the liver as the free alcohol- (retinol)

bound to a specific plasma transport protein, retinol binding protein

(nBP), which is responsible for the delivery of retinol to the extra-

hepatic sites of action of the vitamin

Retinol mobjLÍsation and delivery are highly regulated events that are

particulanty controlled by processes that regulate the rates of synthesis

and secretion of RBP by the liver (reviewed in detail by Goodnan 1984a

and VloLf 1984), and it seens that an adequate dietary zinc is a

requirement for the naintenance of mobilisation of vitanÍn A from the

liver (Fan et aI 1973, Smith et aL 1973i Smith 1982) since zinc

defÍciency has been shown to depress the plasma and liver mP leve1s to

about 1/4 and 1/2 of normal values respectÍveIy (SmÍth et aI 1974). The

interrelationship between zinc and vitamin A will be discussed further in

the folIow ing chapter.

4-5

DeIivery of retinol to peripheral target tissues may involve specÍfic

cell swf ace receptors that recognise RBP. Chen & HeIIer (977) show ed

that bovÍne pigment epithelial cells were capable of taking up retinol and

retinoic acid when these retinoids were presented to the cells as

complexes with serum RBP, but vitamin A was not taken up when presented to

the ce]Ls as serun albumin conplexes. The uptake of the retinoids was

accompanied by dissociation of the retinoid-HBP complex, with the retinoÍd

being taken up by the epithelial cells, while the protein renained

out side.

The eIÍmination pathways of vitamin A, retinol and retinoic acid

involve cleavage at several different sÍtes on the molecul-es to form a

nunber of metabolites which are excreted in the faeces having been derived

from the Iiver via the bjLe (Haenni & Big1er 1977)' Another rnajor

excretory rouLe of vitamin A netabolites is via the urine (Haenni et aI

1976). Under experimentat conditions (Roberts & Del-uca 1967; Varma &

Beaton 1972, Sundbloon & Olson 19S4) the anount of metabolites excreted in

the faeces is roughly twice that, in the urine during the first few days,

but in longer term experiments urinary excretion tends to predoninate.

4.1.3 Biochemical roles of retinoids'

Ttre best defined role of rebinoids is in visÍon where it functions as the

prosthetic group of visual pigrnents. The role is adequateJ-y covered in a

review by Bridges (1984) and is clearly different fron its general role in

epithelial and other tissues of the body, since the actÍon of eye vitamin

A Ís a passive or subsidiary one. Vitamin A probably has a number of

biochenical roles which Ís suggested by the presence of two functionally

active forrns of netabolic vÍtamin A, retinol and retinoic acid, which

are associated with t,wo ceIIuIar binding proteins, cellular retinol

binding protein (cmP) and cell-uIar retinoic acid binding

4-6

protein (CRABP).

The functÍons ascribed to retinol or retinoic acid include an action

on the cell nucleus to alter the expression of genetic information within

epithelial cell-s in lhe course of controlling bheir differentiation

(Chytil and Ong 1983; l¡olf 1984). Bashor et aI (1973) proposed that the

nucleus Ís responsibte for the ¡nolecular mechanisms underlying vitamin A-

dependent ceIIu]ar differentiation Two celluIar bindÍng proteins for

retinol and retinoic acid, distinct fron the serun RBP were shown to

interact with the nucleus (Ong & Chyt'il 1975; Chytil & Ong 1979) and to

transfer retinol specifically into the nucleus where it becomes bound to

the nuclear chromatin (Liau et aI 1981).

In the absence of sufficient vitamin A in the whole anÍmal, mucous

epitheliun has been shown to be transforned into squamous keratinising

epiLheliun (!lotf 1984), whereas Fritton-Jackson & FeII (1963)

transforned 12 day otd chick enbryo skin cultured originally in a

vitamin A-deficient medium fron squamous keratinising epithelium to form a

mucous secreting (ciliated) epithelÍun when the mediun contained

vitamin A. Further experinents perforrned by Fuchs & Green ( 1 981 )

showed that vitamin A controls a process that' determines Lhe amount

of different specific keratin nessenger ribose nucleic acid (nRNA)

present. How vitamÍn A directs the nRNA is uncertain, however it

denonstrates for the first time a controlling action of vitamin A on the

IeveL of one specific pRNA molecule coding for one specific proteirL

The other biochenical role in which vitamin A plays a parb is an action

on the ceII surface (ie. plasna menbrane). Deluca (1977) has shown changes

in glycoprotein synthesis, frequently in celL surface nenbranes Ín

response to vÍtanin A treatnent or deficiency, hence vitamin A may be

4_7

controlling the synthesis of specific glycoproteins. Most ceLl surface

proteins are glycoproteins and in fact it is possible that the action of

retinol on glycosylation in the endoplasmÍc reticul-um ultimately results

in changes in the cell surface, because the plasma membrane is forned

fron this structure llrus it appears like1y that a cell surface function

of vitamin A may be nediated by the principal cell surface menbrane

conslituents, the glycoprotei.ns, and that regulation of the glycosylation

of specific glycoproteins may affect certain cell surface phenomena such

as adhesion and fibronectin attachnent. There is a nultiplicity of results

on the decline of glycoprotein synthesis in vitamÍn A deficiency and the

stimulation of it by vitamin A administration A possible nechanÍsm for

the role of retinol in the formation of specific glycoproteins was

proposed by De Luca (1977) and involves the formation of phosphorylated

mannose derivatives from retinol. The evidence available in support of

this nechanism is described by I'loIf (1984).

4.1.4 Retinoid toxicity and cell function.

Although vÍtarnin A is essential for normal growth and function, excess

vitamin A has been shown to produce many pathological changes. lthile a

nunber of the early studies descrÍbing the action of vitamin A on cells

were performed in isolated systens the results are particularly reLevant

to the observations nade in the whole ani.mal, as many sÍnilarities v{ere

observed between the ceIl studies and in vivo dala in this thesÍs. Ïhe

additlon of retinol to various cell-s has led to serious disturbances in

ceI1 rnenbranes (Dingle & Lucy 1962; Kinsþ 1963) including those of

erythrocytes, where retinol caused isolated erythrocytes to lyse and be

destroyed in marly species.

It has been suggested (Lucy & Dingle 1964) that one of ùhe physÍological

4-8

roles of vitamin A was to stabilise membranes by acting as a

crosslinking agent between the Iipid and protein constÍtuents, since the

vitanin can be strongly bound to both molecular speci-es' LocaI

distensions (smal1 invaginations) in plasma membranes which finally

disintegrated were observed in mamnalian fibroblasts following the

addition of retinol (Dingle et al 1962). Nuclear membranes as well as

mitochondria were also distorted, with the latten becoming swollen and

internally disrupted. Endoplasmic reticul"ar membranes also demonstrated

disinLegrated cisternae, and an increase in the numben of free

ri-bosomes. Retinol also has an effect on lysosornes, causing a release

of lysosomal enzymes in both Iiving cells as well as isolated organelles

(FelI & Dingle 1g63) with the release of different enzymes depending on

the tissue from which the lysosomes have been isolated.

4.1 .5 Dietary allowances of vitamin A.

Several regulatory bodies have recommended daily dietary al-lowances of

vitamin A, since an inadequale intake of vitamin A means that the body

does not have a sufficienb IeveI of the nutrient for oplimal ceII

fornation and function, and depending on the degree of deficiency'

biochenical and clinical signs will develop. If excessive anounts of

vitanin A are ingested , biochenical and clinical abnormalities arise in

body tissues' many of which cause disconfort and pain

Several different regulatory authorities (cited in Bauernfeind 1983) have

set recommended dietary allowances (RDA) with considerable vari.ation

between them (TabIe 4.1). The recommended daily dietary intake set by

FAO/WHq19T4)ranees fron 825 IU for young children up to 4000IU/day for

lactating woneru Ttre National Instltute of Science (NAS/NRC/FNB 1980)

has set daily allowances higher in all calegories except for lactation

4_9

ThesevaLues range from 1320 IU in chiLdren 1-3 years, up to 4000 IU for

Iactating women. The U.S. RDA issued by the Food and Drug Association

(FDA 1983) requires 2500 IU for children under 4 years, and 5000 IU for

persons over this age. Pregnant and lactating women have RDA|s of 8000 IU.

TabIe 4.1 The recommended dietary allowance (RDA)a of vitamin A in humans.

FA0/!tH0 NTS FDA

1-3 years :

7-9 years :

16 years + (na1es) :

16 years + (fenales):

hegnant women :

Lactating wonen :

825

1320

2500

2500

2500

4000

1320

2320

333 0

26 40

3300

4000

2500

5000

(up to 4 years)

( 4 years +)

8000

8000

a n¡A values are given in international units (IU).

4.1.6 Clinical assessnent of hypervitaminosis A.

Urùike vitanin A deficiency, hypervitaminosis A was not reported bo be

a problen of nagnitude in public health until recently. Now there is a

growing concern that vitamin A intake in excess of 10 to 20 times the

recomnended daily al-lowance is becoming a clinical problem of increasing

frequency and risk because of extensive self-medication since vitamin A Ís

available comnercially without prescription in nraegadosen concentrations

of 25,OOO ru to 50,ooo ru. Hypervitaminosis A syndroroe which is

comprehensively reviewed by Kamm et al (1984) and Biesalski (1989), can be

either acute or chronic dependÍng on the age of the patientr and the dose

and duratÍon of ingestion (Aegerter & Kirkpatrick 1975; Lichtenstein

1975).

Acuüe toxicity was first observed in EskÍnos and arctic travellers who

4- 1 0

ingested polar bear or seal liver (Rodahl & Moore 1943; Southcott et aI

1g71). The nervous systero is usually severe]-y affected aften excessive

doses of vitamin A with symptoms in adults inctuding increased

intracranial pressure, pseudotumour cerebri and headache, fol-lowed by

effects on the gastrointestinal tract including nausea and vomiting

(Koerner & Voellm 197Ð as well as severe skin irritation After a single

dose symptoms may occur in adul-ts and proportionately in chiJ-dren when the

levels exceed 5OO'OOO IU (Bendich & Langseth 1989).

Chronic hypervitaminosis A was first described by Josephs (1944) in a 3

year old boy given halibut liver oil , 72 mg retinol equivalents/day for 3

months. Tk¡e chjLd presented with erùarged Iiver and spleen and abnormal

skeletal developnent. Sone of the nany symptoms of chronic vitarnin A

intoxication include cutaneous abnornalities, such as desquamation and

skin flaking, dryness of mucous membranes, bone changes in chÍIdrent

increased cerebrospinal fluid pressure' disturbed blood clotbing'

headaches, papillary edena, anorexia, weÍght 1oss, increased serum calcium

and alkaline phosphatase (Koerner & Voellm 1975) ' as well as inpaÍred

Iiver function following large daily doses for periods up to 9 years

(Meunter et al 1971; Babb & Kieraldo 1978).

Since then nany other reports of retinoid toxicity in children and aduLts

have been docunented and are cited in Bush & Dahns (1984). Most synptoms

of retinol ingestion have been observed to abate fotlowing cessation of

ingesüion with bony changes in chitdren the mosL persistent (Pease 1962).

A fatal case of hypervitamÍnosis A was reported ( Bush and Dahms 1984) in

a neonabe who ingesLed 90,000 IU (60 tines the suggested dose) for 11

days Among the nunerous synptoms contributing to the childs declÍne was

accelerated bone resorption, Ieading lo hypercalcemia and extensive

4- 1 1

calcification of respiratory alveolar septa and bronchioles, with

metastatic calcifications of kidneys, stonach, soft tissues and skÍn

DerÍvatives of vitanin A including tretinoin and isotretinoin ( 13 -cis-

retinoic acid) have al-so been reported Lo lead to cl-inical abnormalities.

patients treated with topical tretinoin, frequently prescribed in the

19Tors fon a wide variety of dermatoses including acne, experienced severe

skin irritations (Peachy & Connor 1971), and erythema (Bottag & Ott

1g7Ð, while oral tretinoin showed similar side effects to vitamin A.

Headache and other CNS problens, gastrointestinal and mucocutaneous

abnormalities were common symptoms (Stuttgen 197Ð, bul hepatotoxic

synptons were not observed presumably because oral tretinoin is not

stored in the liver. Isotretinoin, a Iess toxic synthetic derivative of

retinoic acid used successfully for the treatroent of dermatological

dÍsorders, has side effects greatly reduced in severity and frequency

compared with hypervitaminosis A. A review by Windhorst and Nigra (1982)

provides a summary of clinical assessnents of hundreds of patients treated

with this analogue of vitamin A-

4.1 .7 Vita¡nin A and retinoid ùoxicity in ernbryonic developrnent in animals

4.1 ,7 .a Vitanin A and embrryonic development.

Like zinc, vitanin A also plays a role Ín adequate growth and

dÍfferentiation of the ernbryo (Moore 1957) but urùike zínc il is both an

excess of the nutrient (Fell 1962; Glauert et al 1963; Morriss and

Thonson 1974) and a defieÍency (llarkany and Roth 1948; Kalter & Ïlarkany

1g5g, Duncan & Hurley 19?8) that are teratogenic Ín aninals. The

teratogenicity of excess vitamin A was firsü reported nore than 30 years

ago by Cohlan (1953,1954) who descrÍbed a devastatlng effect of the

vitamin on rat fetuses, which included a marked increase in resorptions'

4-12

stil-Lbirths and a number of gross congenital abnormalitíes of cranial and

facial stuctures in treated pregnancies compared with controls. The most

frequently observed anonalies following daily doses of 35'000 IU of

vitamin A were exencephaly, cleft palate, harelip, and eye defects with

less frequentJ-y observed abnormalities such as spina bifida

(nryeloschisis).

Since these early studies in rats, extensive reports have confirmed the

teratogenicity of vitamin A in a large nunber of species and have

added to then. Mice, guinea Pigs, hansters, rabbits and monkeys $¡ere

found to be susceptible to suprapt¡ysÍologicaL levels of vitanin A and

its analogues (studies up to 1979 are reviewed extensively by Geelen).

Ïlhile exencephaly and cranÍofacial defects are frequently reported to

occur in the offspring of vitamin A treated fetuses (Cohlan 1953r 1954;

Giroud & Martinet 1956; Kalter & Ïlarkany 1959; Langman & VleIsh 1966;

Morriss 1973, Theodosis & Fraser 1978; Padmanabhan et al 1981; Kochhar

et aI 1988), alnost all other organ systems can be affected. These

include respiratory (Mathews eb al- 1981; Singh et al 1983), limbs and

skeLeton (Marir¡.PadiLla & Ferm 1965i Shenefelf 1972; Kochhar et aI 1988)

and cardiovascular (Kal ter & I'Iarkany 1971; Fantel et al 1977), of w hich

the frequency and severity depends on factors such as the dose and

forn of vitamin A administered, the time and route of administration

during gestation and species susceptibiLity.

Since this study Ís concerned mainly with the teratogenic effects of

hypervitaminosis A on the development of the nervous system, brain

maLformations and craniofacial abnormalities will be described in nore

detail, while the reader is referred to Geel-en (1979) and Underwood (1984)

for further details on vitamin A induced anonalies in other organ systens.

4-13

Exencephaly, the rnost prevalent brain anonaly associated wifh excess

vitamin A is characterised in the early stages (day 11 of gestatÍon Ín

rats) by lack of fusion of the cephalic neural- folds which instead expand

lat,era1Iy and renain continuous with the surface ectodern' Thus neural

tube closure does not take ptace and the nalformed brain remains exposed

to the anniotic cavity. The prosencephalon and rnesencephalon remain opent

while the caudal part of the rhombencephalon is closed' Rathkets pouch'

the otic vesicles and the Gasserian ganglion are nornar (Geeren 1979)'

A number of other cNS abnormalities associated with hypervÍtaninosis

A, observed in human term fetuses have also been observed in rodents'

These incrude encephalocoere, anencephary and nicrocephary (Giroud

1 g60; Murakami & Kameyama 1 96 5), hydrocephaLus (cohlan 1 954;

Benito-Arranzi1964),aswel-lasmeningoceoleandrneningoencephalocoele

(Morriss 1g7Ð, and spina bifida, of which two distinct forms have been

described depending on the species examined (Coh1an 1954; Langnan &

llelch 1966; Shenefel E 1g72). These abnormalities have been reported fo

occur followÍng doses ranging frorn 15rO0O IU to 100'0oo IU of vitanÍn A

retinoL or esters, over various periods of gestation, bub always

including the tine of neurulation (days 8-11 i.n rats, days 7-10 in mice)'

central nervous systen defects were also reported Ín day 18 rat

embryos (pillans et aI 1988), when doses of vitamin A between 10'000 and

3O,OOO IU were administered during the earlier pre-inplantation periodt

which is thought to be relatively resistant to toxic inJury (Austin 1973i

Tuchnann - Duplessis 1gT7i Adams el al 1961). Since there $Ias no

indicalion that vitanin A adversely affected 81 hour blastocyst

viabiliby, cell nunber, nitobic index or chronosone structure, it

l¡as suggested that a persistence of vitarnin A in the nother or enbryo

for a number of subsequent days is the nost Likely explanation for the

Iater expression of toxic ir¡jury' which is characteristic of the

thatarenotedasaresultofexposuretotheteratogenduringthe

of organogenesis (PiÌlans et aI 1988)'

4- 1 4

effe ct s

period

Craniofacia].anoma]ieshavealsobeenwidelyassociatedwith

administration of excess vitamin a. one of the most comnon abnormalities

reported in a nunber of studies was cleft patate which was induced in nice

by administration of vitamin A (Takekoshi 1 964; Sauer and Evans 1980;

NeweIl and Edwards 1981) later in gestation (days 10-13) than that

required to induce exencephaly. Administration of a single dose of

20,OOO IU of vitamin A earlier in gestat,ion (day ?) (Marin-Padilla &

Ferm 196Ð led to cleft lip and palate as vrell as anencephaly in

ha¡osters. A high incidence of cleft palate has also been reported in

rats (Kochhar & Johnson 1g65i Nanda 1971; Morriss 1972, 1973i Lorente &

Millertg?8a,b)with50fofembryosexposedto60'000IUvitaninAfor3

days from day 9 or 10 of gestation resorbed, and Bof of those remaining

with cleft palate and high incidences of eye defects and exencephaly'

Nanda (g71) reported that the palafal processes of rab embryos exposed

to 4o,ooo IU of vitamin A frorn day g-11 of gestation showed

significantly fewer labelled ceIIs and milotic figures when injected

with tritiated thymidine into the amniotic sac, and suggested that

vitamin A might cause cleft palate by disturbing the proliferative abiltty

of the cells of the palataÌ process by interfering with bhe DNA synthesis

phase and generation tine.

palatal abnormarities were often accompanied by optÍc and otic defects'

Ear anomalies which often accompanied palatat defects included absence of

external ear ( Morriss 1973 Singh et aÌ 1977), anotia, microtia'

nacrotia, double ears and malposÍtÍons of the pinna (singh et aI 19'.17)'

4-15

Histological sectionÍng of the cranium on day 20 (Padmanabahn &

Singh 1980) frequently revealed malformations of bhe middle and ínternal

ear, often without abnornalities of the exlernal earr when doses of

10o,o0o to 200,000 IU of vitanin A palnitate were administered on days 8-

14 of gestalion Most studies examined have aLso reported eye defects of

varíed frequency, severity and abnormafities, including exophthalmia and

anophthalmia (Marin-Paditla & Fern 1965; Padmanabhan et aI 1981) (See

GeeIen (197Ð for sunmarY).

4.1.?.b Retinoic acid and enbryonic devel-opnenf .

Vitamin A in the alcohol and esler forns have been shown to cause severe

abnormalities in a nurnber of organ systems depending on the dose and tines

of adnÍnistration during prenatal development. lfhile all-Lrans retinoic

acid, a physiologically active metabolite of vitamin A' and 13-cis

retinoic acid ( Ísotretinoin, Accutane), a stereoj.somer of aJ-1- lrans

retinoic acid, have been shown to be more effective in the treatment of

skin disorders and have fewer side effects, they have al-so been reported

to act as potent teratogens and embryotoxins in a numben of species,

including the mouse (Kochhar 1973i Kwasigroch & Kochhar 1980; Webster et

aI 1986; Yasuda el aI 1986; Jarvis et aI 1990), rat (KistIer 1981),

hamster (Shenefe1L 1972t Ïfitey et al t983; lfillhite et aI 1986), chick

(Hart et a1 1990) and nonkey (Fantel et aI 1977:. Yip el 1980). Ïfhile lhe

teratogenic effects of rebinoic acids are very sinitar to bhose of vitamin

A, retlnoic acid appears to be twice as potent as retinoL and retinyl

esters in producing abnormalities since defects could be induced using

only half the former dose (Lorente & MiIler 1978a,b). It has been

suggested (Morrj.ss & Thonson 1974) that the greater teratogenicity of

retinoic acld may occur because vitamin A may not be teratogenic untiL the

capacity to store vitanin A in the ¡oabernal tissue is exceeded, whereas

retinoic aci.d cannot be stored in maternal tissue

4-16

(Dowling & lJald 1960).

A comparatÍve assessment of the toxÍcotogr of vitanin A and retÍnoids

has recentry been compiled by Biesarski (1989). The most prevalenf

anomal j-es attribut,ed to retinoic acid, depending on the timing of

administration of the compound are resorptions (Shenefelt 1972),

craniofacial defects (Willhit,e 1984; SkaIko & Kwasigroch 1985; Webster et

aI 1986; Abbott et aI 1990; Ritchie & !{ebster 1991), heart (TayIor et aI

19gO; Davis & SadIer 1981) Iinb abnormalities (SulÍk & Dehart 1988; Abbott'

et al 1990), and caudaL neural tube defects (Tibbles & lliJ-ey 1988; Alles &

SuI ik 1 gg0). Doses of 7 .5-10 nglkg in the pigtail monkey (Macaca

nemestrina) from day 20 to 44 of gestation resulted in muscufo-skeletaL

and,/or craniofacial defects, in virtuatly alL delivered offspring (Fantel

et aI 1gT7, Newetl-Morris et aI 1980, Yip et al 1980). These abnornalities

included nalformed ears, trypertolerism, exophthalmia, as well as

hypoplasia and apoplasia of facial bones. Doses of 25 nglkg retinoic acid

have led to extensive death in rats following gavaging on day 9 of

gestat,ion (Kist]er 1981), while 120 ng/kg on days 9 or 10 of gestation

have Ied to resorptions of up to 100f as well as craniofacial and

central nen/ous system defects in surviving enbryos treated on day 9 or

day 11 of gestation (Kistler 1981)'

AdminÍstration of retinoic acid on day 7,5 of gestation in hansters Ied

to a diverse range of anonalies covering narìy organ systens and including

spina bifida (Shenefe] f 1gT2). Differences were observed in the forns of

spina bifida between rodents and hamsLers foltowing 80 ng/kg all-trans

retinoic acid (libbres & ÌJiley 1988), whÍch these authors suggested may

reflect species differences in the position of the posterior neuropore in

the neuraxis and Ín the extent to which secondary neurulation contributes

4-17

to the development of the Iumbo-sacral cord segrnents' Treatment of mice

with 50-100 ng/kg have led to anonalies in 40-100f of embryos while

higher doses (Zoomg/ke) of retÍnoic acid on days 8, 9, and 10 of gestation

have 1ed to nearly 100f resorption of conceptuses (Kochhar 1967)' In a

more recent study, (AIIes & Sulik 1990) using lower Ievels (28 mglkg) of

all-trans-retinoic acid, resorptions occurred in 44f of conceptuses' while

7g% oî surviving term fetuses denonstrated spina bifida aperta, which the

authors aùtributed to celt death in subjacent non-neural tissues.

Administration of retinoÍc acid Iater in gestation (days 12-18) have

led t,o anomafies of skeleton, limbs and palate (Kisbler 1981) which have

alsobeenobservedfollowingadministrationofthestereoisomerof

retinoic acid, 13-cis-retinoic acid (1lcisRA, isotretinoin, Accutane) in

nice (sulik & Dehart 1988), while craniofaciat and cardiovascufar defecfs

were observed in chÍck enbryos on day 14 of incubat,ion (Hart et ar 1990)'

4.1 .7. c Behavioural abnormalities'

Many studies have shown that teratogenic agents administ'ered relatÍvely

early in pregnancy during the embryonic period produced Sross structural

mal_formations thaf nay be lethal to the offsprin8. These same agenfs

administered Iater in pregnancy may act as behavioural teratogens' which

may or may not be correlated with histological abnormalities of the brain

Hypervitaminosis A, rnaternal-Iy adninistered during fetal life has been

shown to read to behaviourar abnormarities (Marakhovski 1969; Butcher et ar

1g72i Hutchings & Gaston 1974; Vorhees 1974; Coyle & Singer 1975i Vorhees

etaI1978)andimpairedlearningandnemorisation(Butcheretal1972)

at doses rower than those required to produce congenital abnormarities

Iike exencephaly, cleft palate or linb deformitÍes in animals

(Cotùan 1g53i Giroud & Martinet 1956; Noden 1969).

4- 1 I

Although congenital anomalies need not be associated with behavioural

abnormalit.ies, Langman & Í'leIch ( 1967) have reported norphological def ects

in the cerebral- cortex associated with spasticity, tremors and

hyperactivity in nice, following the naternal administration of 10'000 IU

of vitamin A on days 16 to 18 of gestation There was a decrease in celL

density in the cortex 1O days postnatally, which Índicated either a

reduction in the nunber of cells formed or a degeneration of existÍng

cells. Ataxia has also been associated with histological abnornalities in

hamsters exposed to 2OO'0OO IU of vitamin A on days 15¡ 16 or 17 of

gestation (Robens 1970). In contrast, there was no evídence of

histological defects withín lhe brain Ín rats that showed hypoactivity and

reduced avoidance acquisition (Malakhowski 1969,1971) or swimming maze

deficits one month postnatal-ly after maternal- administraLion of 100,000

IU of vitamin A on days 9 to 11 of gestation (Butcher et al 1972).

Several other studies (Hut,chings & Gaston 1974, Vorhees 1974) have not

shown behavioural abnormalities to be linked with braÍn dysmorpholory

over a range of doses of vitamin A up to 1OO,0OO IU (Vorhees 1974).

More recently Sallenfait and Vannler (1988) denonstrated behavioural

inpairment in rats at the neonatal or adult stage in the absence of any

overt signs of toxicity in offspring following exposure of the fetuses

to 160,000 IU of vitamin A.

4.1.8 Vitanin A and retinoid toxicity in embryonic developrnent in hurnans.

Tl¡e confirnation of teratogenicity of vitanin A in a number of animal

species in nary studies since the original report by Cohlan (1953) has

províded the basis for the determination of abnornalities in hunans- Since

the recognition of the value of the applÍcation of high doses of vitanin A

(K1ignan et aI 1974) and its analogues (ShalÍta et aI 1983) for the

4_19

treatment of dernatological diseases such as acne and psoriasis, as well

as the practice of ingesting rrmega dosesn of vitamin A for possible health

benefits, (Paul-ing et al- 1986), the risk of fet,al abnornaliLies anong

women who use these compounds in the first trinester of pregnancy has

increased.

Only a few of the cases of human birth defects possibly attributed to

high maternal vitamin A ingestion had been published up to 1986, nost

however had been reported to bhe FDA by nedical practitioners and are

sumnarised by Rosa et a1 (1986). Doses aslow as 181000 IU vitamÍn A and

up to 150,000 lU daily before and during early pregnancy, or throughout

pregnancy have been shown to be teratogenic wit,h birth defects

enconpassing a wide range of organ systems Stange et al (1978) reported

that daily doses of 150,000 IU of vitanin A recornnended for acne

treatnent, were ingested by a woman unLil pregnancy was confirmed on day

40. TL¡e newborn infant was microcephalic with multiple malformations of

the CNS including cerebral and aqueductal abnormaLities, as well as small

adrenal glands. Renal abnormalities have also been reported (Bernhardt &

Dorsey 1974) r âs welL as heart defects and cleft palate (Morriss &

Thonson 1974), abnornalities of the external- ear and auditory eanal (Von

Lennep et, at 1985, unpublished reports to FDA), and skelelal anomalies

(unpublished report to the FDA). Accidental overdosing with 500,000 IU of

vitamin A during the second nonth of pregnancy led to a series of defects

including facial palsy, henifacial atrophy and bilateral oculomotor palsy

(Mounoud et aI 197Ð.

DespiLe bhe wÍdespread use of long term negadoses of vitanin A in fertile

wo¡nen, there are no epidemiologÍcal studies that, provide the data

necessary to quantitate the risk for najor malfornatlons following dally

4- 20

fetal exposure to supptements of any dose of vitamin A' Hence

striking findings of teralogenicity onty arose in 1983 following Lhe

narkefing of isotrelinoin' a vit,amin A analogue for oral treatment of

acne, psoriasis and keratotic dermatitÍs (ShatÍta et aI 1983)' The

teratogenic effect of isotrelinoin in hunan pregnancy is nolJ welL

established (Rosa 1983; Braun et aI 1984; Lamner et at 1985; Rosa 1986)

and its use in pregnancy is contraindicated Hence women inadverlently

exposed to it should be counselled for the possibility of abortion (f¡¡'

19g3). Nonetheless, despite the limited scale of information available on

vitanÍn A teratolory, bhe pattern of dysmorphologr reported in infanfs

exposed to isotretinoin during early preSnancy (Rosa 1983; Braun el aI

1984; Lamuer et aI 1985; Rosa et al 1986) is sÍnílar to that following

maternal negadose exposure of vitamÍn A, and includes craniofacial

defects such as cleft palate, nicrotia, central nervous systen

abnormalities includÍng hydrocephalus, microcephaly, cardiac and thymic

abnormalities (Fernhoff & Lammer 1984; Lott eL al 1984; Lamner et aI 1985;

Rosa et aI 19S6). Since isotretinoin and vitamin A (retinol and retinyl

esters) also induce sone quite similar patterns of dysmorphology in

aninals (see 4.1.?), it is probable that similar pathogenetic ¡nechanisms

are involved in inducing the malformations.

There have been reports in the lilerature where isotretinoin ingestion

durÍng the first eighl weeks of pregnancy Led to the delivery of a normal

infant (Kassis et aL 1985). However, white there were no visible

abnorrnalities it is Iikety that postnatally the infant may have suffered

inpaired long tern intellectual or psychotogical development and learning

disabilities, sÍnce behavioural anomalies were common postnatally in

roorphologically nornal rodents following retinoid adninlstration

(Hutchings & Gaston 19?4; Vorhees 19?4). Sínce it has been established

4 -21

that there are similarities in teratogenic outcone between humans and

animals, further observations by physicians nay establish the existence

of Iearning and psychological deficits in infants who were thought to

be exposed to excessive vitamin A or isotretinoin in utero'

The extensive reports of teratology in animals and humans following

exposure to a whole range of retinoids including megadoses of

vitamin A should provide strong motivaLÍon to the manufacturers and

distributors of bhese products to provide striking warning to their

restricted usage during pregnancy'

4.1.9 Mechanis¡ns of vitanin A action'

Teratological experiments using retinol and alI-trans-refinoic acid

clearly demonstrate the effects of the retinoids on a variet'y of enbryonic

populations, which has ted to a nunber of proposals as to the lesion

underlying the fail-ure of the anterior neural bube to close in association

with excess vitamin A. Several Sroups (Langnan & I'lelch 1966, 1967i

ïheodosis & Fraser 19?8; Yasuda et al 1989) have attributed the causes

to a fault derived fron the neuroepithelium. Langman & tIeIch (1966)

observed an accunulation of dividing neuroepitheriar ceÌrs adjacent to

t,he ventricul-ar surface of the anterior neural tube together with

disruption of the internal Limiting rnenbrane and forrnation of bleb-like

cytoplasmic protrusions. These irregularities v¡ere considered to lead to

the brain vesicle contÍnuing to Srow and differentiate so that a larget

everted brain rnass formed, and upon degeneration resul-ted in anencephaly.

These observations were then followed by biochemical studies (Langnan &

welch 1967) which showed that treat¡nent with excess vitanin A on 2 to 3

successive days in nice, inhibited mitosis and DNA synthesis in

neuroepithelial cells of the cerebral cortex, prolonged the cell cycle by

\ -22

\Tft, interfered with differentiatÍon of existing neuroblasts and resulted

in fewer, and abnormal cortical ceIIs. It is unclear how excess vitamin A

causes the accumul-ation of dividÍng neuroepithelial- ceIIs. Langman & lüeIch

(967) suggested that, it might occur through interference with one of the

¡nitotic phases or perhaps through accel-eration of lhe ceLl cycle resulting

in an increased number of cell-s going through mitosis at a given tine'

Iheodosis & Fraser(1978) presented evidence that suggests that vitamin A

affects the enbryonic tj-ssue involved in neurulation by inducÍng

uLtrastructural changes, in the nembranes of the enbryonic cells and their

organelles a few hours after maternal treatment. It is postulated that

these early cytotoxic changes are relevant to the ensuing failure of

neural tube closure and consequently to the development of exencephaly.

These observations and conclusions gain further support from studies

utitÍsing ret.inoic acid. The severe brain malformations observed in

hunans, and the uptake of 14C-a11-trans-retinoic acid by neuroepithelial

celLs (Dencker 1g7Ð, and of Nile blue stain by necroLic neuroepithelial

cells (Sulik et aL 1988) strongly suggested that these cells were

affected by excess vilanin A and its derivatives. In contrast to these

proposals however, several other workers (MarÍn-Padilta 1966; Gee1en 1973i

Morriss 197Ð, suggested that any stimulus which adversely affects

nesenchyrnal proliferation can secondarily affect closure by producing a

Ìack of supporting mesenchyne for the rising fo1ds. Ultrastructural data

(Morriss 1972, Joschko et aI 1989), have demonstrated a paucity of

mesenchymal cells, and the presence of ceII death in the nesenchyme nay

underlie this observation. It has also been suggested t'hat the

biochenical defect underlyÍng this necrosis nay be due to a deficiency of

glycoprotein synthesls essential for biosynthesis and/or naintenance of

menbranes (Geelen 19?3; Theodosis & Fraser 1978; Marir¡'Padilla 1966)'

)-23

llhile several proposals have been made as to the site of action of

excess vitamin A teratogenesis underlying failure of neural fube closuret

a deficiency of neural crest ceIIs is thoughü to underlie isotretinoin-

induced craniofacial defects in mice (tlebster et aI 1986), possibly

through an absence of neural crest cell activity and poor nigration

of branchial arch nesenchyme. In nanmalian embryos, neural crest cells

are initially located in the nargins of the neural folds (Tan & Morriss-

Kay 1985), and as the folds elevate to form the neural tube, the crest

cells migrate primarily under the surface ectoderm to new locatÍons in the

upper face and visceral arches (Nict¡ots 1981 i Johnston & Sulik 1985).

Cephal-ic neural crest cell abnornalities may also be responsible for some

of the nalfornations attributed to alcohol and thatidonide exposure (SuIik

et aI 1988). Hence the above considerations suggest that, the mechanisns

underlying retinoid-induced CNS malformations are currently still poorly

understood, inconclusive and probably differ from the mechanisms affecting

the development of neural crest cel-I-derived structures.

4.1.10 Justífication for vita:lin A study.

Although animal studÍes have demonstrated gross and ceIIuIar

dysnorphofogy, very few have extensively explored the effects of

hypervitaminosis A on the ceII menbrane and subcelLular components of the

neural folds during the critical period of neurogenesis. lIhile Morriss

(1973) has exanined the anterior neural tube during and after completion

of neurulatíon in the rat, to the present authors knowledge this study

reports for the first tÍrne, the ultrastructural effects on the neural

tube, j.mmediately following neurulation on day 1 1.5 of gestation. This

chapter crÍtically examlnes the developing neuroepÍthe1 ium and

underlying mesenctgrne, and attempts to draw conclusÍons as to the possible

sites and nechanisms of action underlylng the teratogenic assault in

relation to previously proposed mechanisms and to others which may

as a result of this investigation'

1I -24

ari se

Although the i¡ vitro culture technique has been shown to be a híghly

suitable system to examine vitamin A induced teratogenicity, in vitro zinc

deficient enbryos do not demonstrate the typicat pattern of dysnorpholory

urùess the enbryos are explanted from nothers who are already zinc

deficient(Recordeta]1985).Hencean.uvivoinvestigationwas

performed so that both manipulations were administered under similar

experimental conditions. This hlas necessary in order that the issue of

whether an interaction occurs between zinc deficiency and

hypervitaminosis A in anímals exposed to both teratogens simuftaneously

might be resolved. The outcone of this investigation is the subject of

chapter 5 of this lhesis.

4.2 MATERIALS AND MEÏ{ODS

4.2.1 Animals and diets.

virgin fenal-e sprague-Dawley rats (180-210g) were praced overnight with

nales of the sane strain, and pregnancy was established by the detectÍon

of sperm in the vaginal smear the following morning (gestation day 0'5)'

hegnant dams s¡ere housed individuatty in stainLess steel and plastic

cages and l.rere assigned to a soybean based diet (llilkins et aI 1972)

containing 100ug of Zn/8 as zinc sulphat'e'

The dams were fed diets in a cycrical pattern in order to ensure high

diet,ary intakes on days I and 9 of gestation, theneby fo]lowÍng the sane

protocol as for the aninats !n the zinc study. The anirnaLs vJere fed a

restricted zinc-replete diet of 129 fron day 0.5 to 5'5 of gestatíon which

l¡as then reduced to 2-3 g/day on days 6.5 and ?.5r and for the following

4-25

three days the dams had free access to both food and water. 0n day 8'5 of

gestation between 8am and 9am, (prior to presentation of ad lib dieLs)

half the animals received a supplement of 100,000 IU of the viÈamin A

ester, vitamin A palmitate in 1ml of sunflower seed oil, or the oil alone'

by stomach intubation. Timing and selection of the dose aduinistered was

based on previous teratological studies performed by Morriss (1973) and

Morris & Steele Qg77). Dams receiving vitamin A palmiEate or the vehicle

srere provided with diet ad libitum following dosing to ensure optimal

intestinal absorption (vtolf 1984).

4.2.2 Tissue preparation and analytical methods.

The dams were sacrificed on day 11.5 of gestation under ether

anaesthesia and blood was obtained by cardiac puncture for serum zínc

analysis by flame absorption spectrophoEometry (Dreosti eE a1 1982). The

embryos hrere removed from the uterus and stored in IIBSS (pI{ 7.3), and

examined for groh'th and morphological develoPment under the dissecting

microscope. The meÈhods by which Ehe embryos hrere examined for growth and

morphological developnent, and by light and electron microscoPy have been

outlined in detail in chapÈer 2. Embryos prepared for scanning electron

microscopy were fixed in 0.1M sodium cacodylaÈe, 27" paxaf.ormaldehyde and

3% glutaraldehyde (ptt Z3) at 4"C overnight and dehydrated through a

series of graded acetones and critically point dried as outlined in

chapter 2. The embryos vùere mounted on stubs, coated with carbon and gold-

palladium and examined with an ETEC scanning electron microscope at 20

KV. The remaining embryos v¡ithin each litter !rere stored at -20oC for

protein estinations (Lowry et al 195I).

4-26

\.2.3 Statisticat anatysis

Continuous growth varÍables and zinc l-evels were analysed by standard

analysis of variance for zinc replete and hypervitaminosis A groups. A

maxj-num l ikel ihood nethod f or discrete norphoJ-ogieal data was used

assuning the data to be binoniall-y distributed and the devÍance to be

approximat,ely dislributed as X2 on the appropriat,e degrees of f reedon.

4.3 RESULTS.

4.3.1 Gror¿th and norphological development.

Day 11.5 embryos exposed to naternaJ. hypervitaminosis A on day g ofgestation díd not show a signfficant reduction 1n any of bhe growth

paraneters examined compared with controLs of the sane age (Table 4.1).

There was al-so no difference i-nzinc levels between the two groups. These

factors were nefrected in the dissecting microscope photograph in Fig.

4.1a where a typical embryo exposed to 100,000 ru of vitamin A on day g of

gestatfon was sinÍlar Ín size to that of the control enbryo on the right.

Although excess vitanin A did not read to a reductÍon in growth

paramelers, norphorogÍcat developnent was impaired (Tabre 4.2). lJhire

dysnorpholory was evident in nearly half ( 46lø) of the hypervitaninosi.s A

group, enbryonic nortality was not affected (TabIe 4.2). Many of the

observed abnormaLlties (37Ð Ínvolved the anterior neural tube with the

nost prevalent consisting of a severe evaginatlon of the neural tube

part'lcularly in the midbraÍn and rostral hlndbrain regions and wÍth sone

forebrain fnvorvenent, in more affected enbryos. This patlern ofabnornality frequently referred to as exencephaly has been reported

prevlously in Wpervitaninosfs A enbryos by Morriss (1gTZ11gT3).

4 -27

Table 4.1. The effect of hypervitaninosis A on gronrth in rat embryos.

Zinc replete : 100'000 IU Vitanin A

Total enbryos-dans

Maternal serum zinc (ug/nl)

Crown-rump length (mm)

Nunber of somites

Embryonic protein (uB)

6t -s

1 .25+0.01

3.68fl.08

22 .5 8+0 .33

2'45 . 40+1 I .6 0

1 06-8

1 .27 70 .01

3.35f .17

21 .44+0.78

269.86+16.92

II Values are means + SEM.

Urilike zinc deficient embryos there was very little variation in the

kind of neural lube defect dísplayed within and between litters of embryos

exposed to excess vitamin A. This is seen in Fig. 4.1b where 3 embryos at

different orientabions from the sane 1Ítter all demonstrate defecbs

characteristic of exencephaly. Several other developmental anomalies were

found to occur more frequently in hyperviLamÍnosis A enbryos conpared

with controls (Table 4.2). These included a signifÍcant increase (8.5f)

in enbryos which failed to rotate into the dorsally convex position

typical of day 11.5 enbryos, while partial or total lack of fusion of

the chorlon and allantoÍs required for placental formation' occcurred in

1gf of treated enbryos and is denonstrated in Fig. 4.1c by the

presence of an unfused allantois. ThÍs enbryo is also representatÍve of

vitanln A enbryos with dysnorphfc eyes, (24.5í), where anophthalnia was a

connon anomaly, as was nicrophthalnia. It Ís posslble however that eye

defects 1.¡ere overestinated in enbryos exposed to excess vitanin A in

this study, as severe foldlng of the anterlor neural tube nay have

led to developed optic placodes being hidden during norphological

exanination. Dysnorphology of ühe branchial arches and oüic placodes

lnduced by hypervitanlnosis A (Table 4.2) were rarely seen in

thts study (1.9í and 2.81 respectively) urùlke prevlous observatÍons

he lefl which was exposed to 100,000

tion was not growth retarded when

ete) embryo of the sane age on theis exencePhal ic, with evaginated

neural folds (NF).

4-28

b. Different orientations of bhree day 11.5 hypervitaminosis A embryos

from the sane ritter of simirar appearance, all showing exencephary

i""*orheads). rnu embryo on the rÍght also has an open caudal neuropore

(p).

c. This day 11.5 byperviLaminosis A embryo shows an unfused al-lantois(A), and a typÍcat ðpen and evaginated cranial neural tube (arrowheads)'

The otic placode (0T) is correctrv *rigned wit'h the second branchial arch

(2) .

Fb, forebraÍni Hbt

f orelinb.

Bars= 10Own.

hindbraini OP, optÍc placode; H, heart; S' somites; F'

NF

Fb

H

F

12.3

\

A1

OT

#"

F

Hv

å

ß

.23

TabI e DE'1

4 -29

rvitaninosis A on norphological development4.2 The effect of hY

in rat enbrYos.

Zinc neplete 100,000 TU VitanÍn A

Total embryo s-dans

Tot,al dead

No. of embryos with any kindof defect

No. of eobryos with cranial-neural tube defects

Eye defects

Ear defects

Branchial arch defects

Heart defects

LÍnb defects

Incomplete flexion

Absence of chorieallantoicfusion

Poor yolk-sac ci.rculation

1 (1.2) :

85-6

6 (7.1)

3 (3.5)

0 (0)

1 (1.2)

1 (1.2)

1 (1.2)

1 (1.2)

1 (1.2)

1 (1.2)

0 (0)

106-8

5 (4.7)

49 (46.2)b

39 (36.8)b

26 (24.5)b

3 (2.8)

2 (1.9)

6 (5.7)

6 (5.7)

9 (8.5)a

20 (18.9)b

12 (11 .3)b

a,Peb

rcentages in parentheses.Significantly less than controls. ap(O.05r bp(O.001

.

(MorrÍss 1973, tlebster et aI 1986) where vitanin A compounds frequently

led to a reductlon j.n branchial arch size and otic placode misalignnent.

The poor representation of these ar¡cnalies in the present study could

possibly be attribuùed to differences in factors such as dosager tinÍng

and retinold compound used. llhile there lJas a trend towards poor heart

and forelinb deveLopment (bolh nearly 6fr) in hypervitaninosis A embryos

(Tabl e 4,2), the results were not signif icant.

Scannlng electron nicroscopy cor¡firned observallons made under the

dissecting nicroscope thaf enbryos exposed to hypervitaninosis A were

generally slnllar Ín size and in morphologlcal developnent to day 11.5

4_30

control enbryos, (see Fie.3.2a), except in the craniat region. Fig.4.2a

shows that the cranial neural- folds of a typical hypervitaninosis A embryo

appeared to develop to fuIl size but were severely evagÍnabed, and

had collapsed onto the cranlum particularly in the mÍdbrain with sone

involvement of the forebrain and rostral hÍndbnain regions. The

appearance of the neural tube suggests that the supporting mesenchyme was

affected, rathen than the neuroepÍthel1um. This observatÍon and suggestion

has been made previously by Morriss (1972). Because of the distortion of

t,he cranium ít was not possible to identify the optic placodes in a

nunber of hypervitaninosls A embryos as seen in Fig. 4.2a, although the

otic vesÍcles $¡ere usually visible. The branchial arches also did not

appear to be affected by hypenvitaminosis A when exanined under the

SEM, although the orientation of the enbryo in Fig. 4.2a does not show

this clearly. In a nunber of enbryos sinilar to FiS. 4.2a, the

presence of the allantois indicated that chorÍeallantoic fusion and hence

placental fornaüion had not occurred.

Neural tube dysnorphologr was often associated wtth changes Ín the

surface and neural ectodern of hypervitaminosis A embryos. Fig.4.2b shows

a region of the surface ectoderm in the nldbrain of a typical control 11.5

day embryo characterised by a relatively dense row of microvilli lining

the ceIl perimeters and a number of microvillf scattered over the surface.

In conparison, the density of microvÍ111 over the midbrain surface of an

experinental embryo (FÍ9. 4.2c) is considerably increased. The ventricular

surface of the neuroepÍthellum which Ís exposed dlrectly to the annlotlc

fluid in exencephalic enbryos also showed obvious abnornalities. Fig. 4.2d

shows that the ventricular surface of the neural tube was covered in very

large, irregular blebs which were nuch nore extensive than those observed

at a conparable slle 1n control embryos (see Fig. 3.3b). A cut wit'h a

Fig 4.2.ti ssue.

4_31

scanning electron micrographs of control and hypervitaminosis A

a. A typical 11.5 day WpervitamÍnosis A embryo showing severeryevaginated neural folds (arrowheads) parbÍcular1y in the midbràin regionwit'h some involvement of the f orebrain and rostral hindbraln The opti"placode does not appear to have developed but the otic pracode Ísunaffected and bhe allantois (A) has not fused. The branchial arches (1)are nomal (al though not cI early visibl e at this orientation) togetherwÍth the heart (H) and forelimb (F). Conpare this embryo wÍth the controlin Fig. 3.2a- Fb, forebrain; Hb, hindbraini S, so¡nites.

b. This nÍcrograph shows a typical region of the surface ectoderm (E) inthe midbrain of a control day 11.5 rat embryo with row s of microvilli(arrowheads) lining the perÍrneters of the ceLls and a few scattered overthe surface.

c. A similar regÍon of the surface ectoderrn (E) 1n the nidbrain of an11.5 day hyperviLaninosis A embryo shows that the density of themicrovilli over the celL surfaces has increased and blebs are alsopresent (arrowheads). Note the large bleb covered Ín nicrovillÍ(asferisk).

d. The ventrÍcular surface of the neuroepitheliun in t,he midbraÍn of an11.5 day hypervitaninosis embryo is covered in irregularly-shapedcytoplasnic projections (or brebs) of varying sizes (asterisks).

erf. The figures show newoepithelÍal (N) and mesenct¡rmal (M) ceIIs froslthe nidbrains of control and hypervitaminosis A unbryo respectively.WhiLe the neuraL tíssue is sÍnÍlar in'both enbryos, the stellate shapedce1I bodies (asterisks) and extensive cytoplasmic network of connectingcell processes or filopodia (arrows) observed in control embryos in e,vJere repraced by more rounded cells (asterisks) of varyÍngotientatlons Ín f, whÍch had l-ost their interconnecting f1lopoctia inthe treated embryo.

Bars=200un, a; 5un, b-f.

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4-32

yazor bl-ade into lhe midbrain region of the craniaL neural Lube of control

and hypervitaminosis A embryos exposed the underlying mesenchyme and

newoepithelium. !thile the newoepithelial cells appeared sinilar in

control (Fie 4.2e) and treated (Fig. 4.21) embryos, the nesenchynal cells\

in the hypervitamÍnosis A embryo appeared smaller and with far fewer

Ínterconnecting cytoplasm ic proj ections.

4.3.2 CelluLar abnornalities

LÍght nicroscopy of a typical affected hypervitaminosis A ernbryo (Fig.

4.3a) showed that ùhe rostral part of the anterÍor neural tube was very

abnormal with neural folds which had failed to rise and fuse in the dorsal

midline of the cranial neural tube. Ttre hindbrain although fused was

irregular in shape being convoluted at both the lumenal and

neuroepithelial-nesenchymal margins. In the sane figure the

neuroepithelial surface exposed to the amniotic fluid showed some

evidence of cytoplasmic Ioss, while in fhe medial region of the

underlying mesenchyme the cells were very sparse, and in the lateral

region cl-ose to the surface ectodern where neural crest cells are

belier¡ed to migrate (Îan & Morriss-Kay 1985), the cells appeared dense

and dark. Higher nagnification (Fie. 4.3b) of the neural tube

close to the level seen in Fig. 4.3a showed that in sone areas of the

neuroepithelÍum which abutted the ventricular lunen the cytoplasm appeared

nwashed outn and lhe cells were shrunken with sone evidence of ceIl debris

being extruded lnto the Iunen In the nesenctyne particularly towards the

midlÍne anea, there vJere few cells and ÍnterconnectÍng cytoplasnic

proJectÍons, however the cells dÍsplayed the typical stellafe shape

chanacterisbic of mesenchyne cells at siniLan sites in control enbryos. In

the lateral reglons of the nesencþne the cells vrere rnore rounded with few

connections, and nargr dense, dark bodies were pnesent both Íntra- and

4-33

Fig.4.3.a. A horizont,al section through the craniun of a typicalaffected hypervitaminosis A embryo, with severely evaginated neural- folds(NF) which had failed to fuse in the dorsal nidtine of the rostral neuraltube, and a convoluted basal surface (arrow heads). The hindbrain hadfused but was irregul-ar ín shape being severely convoluted (arrowheads)along the basal- end of the neuroepitheliun. The underlying mesenchynalcel1 s w ere sparse in som e areas (asterisk) and appeared to havelost t,heir interconnecting filopodia, while Ín the lateral regions thecell-s appeared denseand rounded (¡lack arrows). The area marked by thearrows is seen at hÍgher nagnification in b.

b. Higher magnification of a section narked by arrows in a, but at asIÍghtly deeper 1evel. The neuroepitheliun was mainly unaffected by thetreatnent except in snal-l areas cLose to the ventricular lumen v¡here cellshrinkage (open arrow) was observed. Many of the mesenchymal ceLlsappeared to have lost their fílopodia albhough they maintained theÍrcharacterist,ic steLlate shape (arrows). Large nunbers of cells werelocated close to the surface ectodern (E)in the region where neuralcrest ceI1 mÍgration occurs, and were rounded with condensed fragments(arrowheads) located wibhin the cells or interspersed amongst them.

c. A deeper horizontal section of the craniun of an hypervitaminosis A

embryo at a level where the rostral part of the neural tube has closed.The neuroepitheliurn was smoother but with some distortions of theforebrain and cor¡volutions mainly Ín the nidbrain area (arrowheads), andthe mesenchyme (M) was aLso nore uniformly dense. Note the unusuaLnarrowing of the cranium in the hindbrain region (open anrows). The arrowsnark the area seen at highen magnification in d.

d. Higher nagnification of a section through the forebrairmídbrainregion of an hypervitamÍnosÍs A ernbryo shows that, the neuroepitheliun isrelatively unaffected except at t,he convoluted sites identified 1n c.There appears to be a disruption of cells at these sites (arrowheads).The cells in the nesenctyne showed a Loss of cytoplasmic connections andmany dense, dark bodies located intra- and extra-cellularIy (arrows),partÍcularly in the lateral areas adjacent to the surface ectodern.

N, neuroepithel iun;surface ectoderrn; bv,

M, mesenchyrae; Fb,blood vessef.

forebraini Hb, hindbrain; E,

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4-34

extracell uIar1y.

!then hypervitaminosis A embryos which demonstrated exencephaly were

sectioned at a leveL at which the rostral part of the anterior neural tube

was closed (fig. 4.3c) the neuroepÍthelium displayed a smoothness and

unifornity not seen in sections cut through the exencephalic region Ihe

mesenchyrae was al so nore unif ornly dense, hower¡er there !ras some overall

distortÍon of the cranium in the hindbrain region At higher magnification

(Fig. 4.3d), the neuroepithelial cells appeared relatively unaffected'

how ever the mesenchyme !{as severely abnormal with even rnore densg dark

bodies Ín the laLeral regions than occurred in the evagÍnated areas of the

neural tube seen in Fics. 4.3a and b.

4.3.3 Ultrastructural observations.

Fig. 4.4 depicts a nunber of ul-trastructural features typically observed

in sections of the anterior neural tube of 11.5 day eontrol and

trypervítaminosis A embryos. Although hypervitamÍnosis A appeared bo

affecb the nesenchyne predominantly, some changes were also observed at

certain sites in the neuroepithel iun. At the apical end of the

neuroepithelial cells (Fig. 4.4a) in the mÍdbrain of the embryo prevÍously

seen in Fig. 4.3r cVtoplasmic projections were prominenl and tortuous'

and often had a condensed appearancs These cytoplasnj.c extensions were

much larger and nore irregular than those seen at similar sites in Lhe

neural tube of zinc replete enbryos (fig.3.3b),and are equivalent to the

large, írregular nblebsn visible on the ventricular surface under the SEM

(fig. 4.2d). Langnan and l,lelch (1966) noted sÍ¡¡llar structures and

atlributed them to a decrease in mitosfs wÍth mary neuroepÍthe1ia1 ce1Is

aggregat,lng in the region of the internal lfnitfng nenbrane. In the

present study however, a reduction in the nunber of nltotic cells was nol

4-35

Fig. 4.4 shows a number of representative ultrastructural featuresassocialed wÍth hypervitaminosis A-induced necrosis, within the anteriorneural tube of 11.5 daY embryos'

a. The neuroepithelium (N) aO¡acent to the ventricular lumen in thenidbrain show s extensive ceII disruption (arrows) with many blebsprotruding Ín¡o the ventricular space (arrowheads). CeIl rennants werealso observed in this space (open arrows). MitotÍc cells (asterisk) werevisible and appeared unaffected. L' Iunen

b. At the basat end of the neuroepitheliurn (N) there lras a depletion ofcytoplasmic ribosomes (arrow s) giving some of the celIs a washed outappearance, whiLe the mitochondria appeared condensed. The basal Iamina!üas broken at some sites (arrowheads) and fragnents of neuroepithelialcells are being extruded into the mesenchyne (M).

c,d. Mesenchynal cells (M) fron 11.5 day control and hypervitaminosis A

enbryos respectively. The stellate-shaped control cells inapproximately the sane orientation with interconnectÍng filopodia(arrows) in c, can be conpared with the more rounded, poorly orientatedceIIs which have lost their filopodia in d, and contain hetenolysosones(open arrows) with dead cell remnants. 0ther condensed cell debris(arrowheads) located in the extraceluLlar matrix close fo mesenchymalcell-s appear to be preparing to phagocytose this condensed materiaf.

e. A pyknotic cell (arrowheads) in the mesenchyne of the anlerior neuraltube of an 11.5 day hypervita¡nÍ¡rosis A-treated embryo. The cytoplasm and

nucleus (nu) are condensed, and the mÍtochondrial cristae are severelydamaged (arrows). TLre close proxinity of the necrotic ceÌl to anapparently healthy mesenchyrnal cell (l't) suggests that the dead ce11 Ísabou! to be phagocytosed. ECM, extracellular matrix.

f. A dyÍng mesenchymal cell which had previously phagocytosed a

considerable anount of dead cell debris (aruows) is surrounded by healt'Wcells (M). The cyloplasn, mitochondria (n) and nucleus (ny) are allcondensed and the Iatter 1s irregularly shaped. Abnornally Iargesections of rough endoplasnic retict¡lum are also present (anrowheads).The small, opaque structures nay be prinary lysosones (L). ECM'

extracellrrl ar m atrix.

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4-36

apparent, and mitotic cells (Fie. 4.4a) did not appear to be affected by

excess vitamin A. Despite these changes at the lumenal surface, higher

magnificatÍon (not shown) of newoepiLhelial cetLs near this site did not

reveal any abnormaJ-itÍes of ceIl organelles or nuclei.

In the basaL region of the neuroepÍthelium adjacent to the mesenchyme, in

hypervitaminosÍs A - treated embryos (Fig. 4.4b)r there was a depletion of

cytoplasmic ribosomes giving the cells a washed out appearance' while

the mÍtochondrÍa appeared condensed. At t,hese sites the basal Iamina

was also broken allowing sone of the contents of the neuroepilheliun to

spil1 into the mesenchyme.

Additionat to these lesser neuroepithelial effectsr IEM revealed that

trypervitaninosis A had a sÍgnificant effect on the nesenchyne. Conpared

with the stellate-shaped nesenchymal cells of control enbryos (Fig. 4.4c)

with their extensive connectÍng fÍlopodÍa or cytoplasmic projections,

hypervitaninosis A led to nore rounded cells with fewer fiLopodia naking

connections with neighbouring nesenctrynal cells (Fig. 4.4d)' and in some

secbi.ons t,here w ere aI so f ew er cells. FÍS. 4.4d al so show s that son e

apparently healtty nesenctynal cel-Is contained eLectron dense inclusions

simÍtar to structu¡es observed by Ghadially (1988) in sone pathological

states whlch he referred to as heterolysosones, and which contained lysed

ce1I rennants. This materÍal was probably phagocytosed and digested by

lysosomaJ. enzynes in the ceIl. Extracellular wholer condensed cells and

dead cell remnants were also frequently observed in the mesenchyne of

Ìyper"rritaninosis A enbryos as depicted Ín Fig. 4.4d. Fig. 4.4e shows part

of a typlcal whole dead or dying cell which can be recognised by the

presence of a condensed cytoplasm, condensed, shrunken nucleusr and

swollen nltochondrÍa with broken crfstae. The close proxlnfty of the

4-37

nornal- nesenchymal cells to the condensed cell suggests that Lhe forner

are IÍkely to phagocytose and lyse the dead cell. From the observation of

nunerous micrographs showing cell death in t'his study it appears that

deat,h is also the eventual fate of previously hea]-thy mesenchymal

cells similar to that seen in Fie. 4.4f, which had prevÍousry

engulfed other dead ceII rennants (heterolysosomes) and digested them to

some degree. Already this cell has becone surrounded by other healthy

mesenchymal cel1s and wilI most likely be phagocytosed by these

neighbouring cell s.

Fig. 4.5 shows a number of typÍcaI hypervitaninosis A-induced

ultrastructuraL abnornalities at higher nagnification in the mesenchyme.

Fig. 4.5a is a higher magnification of the two adjacent cells seen in Fig'

4.4e. The upper cell shows the norma.L distribution of cytop]asm with its

polysomal conflguration of ribosomes, whereas the affected ceII below

shows a very condensed state of the cytoplasn although the polysomal

arrangernent of ribosomes Ís still present. The nucleus is also condensed

and shrunken The condensed extracellular rennanls observed in Fig 4'5b

consist of vesiculaled RER which contains ceil naterial within the

cisternae. Condensed mitochondrÍa are also present, and several- prinary

lysosomes of varying sizes appear to be associated with the affected

organelles. tlhile these ce]1 fragments are Iocated in fhe

extracelLular natrix within the mesenchyme, they may condense further

and eventually be phagocytosed by apparently healthy nesenchyrnal cells

such as vras seen in Fig. 4.4d. Higher magnÍfication (Fig'4'5c)of t'he

heterolysoso¡nes Ín the condensed ceII in Fig. 4.4f show partially

dÍgested remnants of other dead cells which were phagocytosed by this

nesenct¡ynal ce]l which still shows sone heatthy cytoplasmic contents'

Fig. 4.5 features differenb aspects of organeller"*nchyme of the anterior neural tube of 11'5 day

ernbryo s.

4-38

disruption in thehypervitaminosis A

a. Higher magnification of a section of Fig. 4.4e, showing the upper cellwith nor¡nal cytoplasmic polyribosomal configuration (arrows) while in thelow er dead or dying cell, t,he cytoplasm and nuclear matrix (nu) arecondensed. The sub-nuclear region of t,he affected cel1 also appears tohave shrunk away from the outer nuclear envelope (asterisks) which isalso disrupted, and the mitochondria (m) have broken cristae.

b. Condensed cell- remnants in the extracelluLar matrix includesvesicuLabed rough endoplasmic reticulun (vr), vacuoles (v)' condensed

cytoplasm (c), nitochðndria (n) and probably nuclear rennants (nu).

Several primary lysosones (L) nay also be present associated with theaffected organelles.

c. Several heterolysosones (arrowheads) within an apparently healthynesenchymal cell containing partially digested remnants of other dead

cel1s. The cell conponents are so heavily condensed and lysed that theyare unidentifiable. m, nitochondria; r, rough endoplasmic reticulum; L'prinary lysosomes.

d. A mesenchymal ce1] at an advanced stage of necrosis showing extrenelycondensed cyboplasm in which the loose ri-bosones have Iost their polysomal

configuration The cytoplasm contains nany structunes (arrowheads) whichmay bã vacuoles, vesicuLated rough endoplasnic reticulum or mitochondrialrenains. Condensed mitochondria (¡n) are also visible.

e. Another nesenchyrnal cel1 at higher magnification shows many affectednitochondria (m). The large mitochondria on the right is about to be

ensheathed by segments of rough endoplasmic reticulum (arrows). A whorlednenbranous body (asterisk) derived fnon invotuting mitochondrÍa is presenb

on the 1eft. r' rough endoplasmic reticulun.

f. IlypervitaninosÍs A also affects cell nenbranes (cm) leading to theirdestruction and Ioss of cytoplasmic content (arrowheads). The mitochondria(m) Ín this cell are severely affected and the nuclear chronatin (arrows)appears nore dispersed.

Bars= 1 um.

I

4-39

The mernbrane components vüere so heavity digested that it was not possible

to distinguish individual organelles. Several other snaller membrane

bound structures present in the cytoplasn appear to be primary

lysosomes, as they are simÍlar to structures depicted as primary

lysosomes by Ghadially (1988). In other cells (Fie. 4.5d'e) exposed

to excess vitamin A, danaged organelles such as mitochondria and Rm

are Ídentifiable. Fig. 4.5d shows remnants of several mitochondria'

however many of the visible renaining sbructures are vesiculated RER'

which have a vacuofated appearance and whÍch in some cases have

sequestered cytoplasrnic remnants in their cislernae and probably lyse lhen

through enzymes released from the polyribosomes on the ensheathing

membranes (GhadialIy 1 988) I Possibty forming aubolysosomes. Loose

oytoptasmic ribosomes seem to have lost their normal polysonal

configuration and appear condensed at sone sites in this cell' In the

mesenchynal cell in Fig. 4.5e mitochondria are severely affected. The

large nitochondria on the rÍght is apparently beconing ensheathed by a

segnent of RER, whiLe a whorLed membranous body, probably derived fron

involutÍng mitochondria, comparable with features observed by Ghadially

(1g88) in cultured nonkey kidney cells can be seen on the left of the

nicrograph. In Fig. 4.5f, the cell rnenbrane has disinlegrated and the

cytoplasnic contents including damaged nftochondria are able to spill out

into the extracellular matrix.

As weLl- aa mesenchynal cells, several other cel-Is associated with the

mesenchyne were also affecled by hypervibaminosis À Erythrocytes in their

imnature nucleated form Iocated within the developing blood vessels v¡ere

often vacuolabed (Fi8. 4.6a), an observation in accord with Glauert et al

(1963). The close proxínÍty of the blood vessels to the disrupted sites in

the neuroepithelÍun suggests that so¡ne of the contents of these vessels

4- 40

Fig. 4.6a show s aff ected erythrocytes from an 1 1.5 day hypervitaminosis A

embryo at various sbages of vacuolation (arrows) witbin the developingblood vessels of the nesenchyme Some of the cell debris wÍthin thesevessels may be derived fron damaged sites in the neuroepÍthel iun(asterisks). Arrowhead marks the site seen ab high magnification in b. bv,blood vessel i Er, nucleated erythrocytes; M' nesenchyme; N'

neuroepithelium; ECM, extracelluJ.ar maLrix.

Fig.4.6b. Higher nagnificatÍon of part of a severely vacuolatederythrocyte marked by arrowhead in a, containing lanel-Iar cupshapedbodÍes (arrowheads). or nilochondria; Er, erythrocyte; vrr vesicuLatedrough endoplasnic reticulun.

FÍg. 4.6c shows several cranial surface ectodermal cells (E) from an11.5 day enbryo exposed to hypervitaminosis A filled with dead ceIIrennants located within heterolysosomes (arrows). Ihe ceI1 debris has beenso heavily lysed that the indÍvidual org¿neIIes are nostlyindÍstinguistrable. Note the presence of microviLli (arrowheads), menbraneand other celL remnants on the surface of, or outside of bhe embryo (openarrows). rmr nucleus; M, nesenchynal cells.

Fie. 4.6d. Another section of the cranial surfacehypervitamínosis A-treated embryo with ectodernal cells (E)

covered in dense mj.crovilli (arrows). Note the presenceremnants (arrow heads) close to the surface, and theheterolysoaomes. nu, nucleus; M, rnesencbynal cells.

Bars=4 tu.

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4- 41

may be of neuroepithelial origin Higher nagnification of one of these

affected erythrocytes (Fig. 4.6b) showed that as well as severe

vacuolation, Iamelfar cup-shaped bodies Ieading to the formation of

autolysosomes were aLso present. GhadÍaIly ( 1988) described these

structures as being derived fron endoplasmie reticuLum which wraps ibself

around organelles to be sequestered. AlternatÍvely, other structures

present in the erythrocytes suggesbs that organeJ-les have dropped into

dilated clsternae of RER again leading to intracisternal sequestration

and autolysosonal fornation, with lysing enzymes being produced by the

polyribosomes on the ensheathing membrane (Ghadially 1 988). The

surface ectoderm of the cranium r{as also affected by hypervifaninosÍs A.

Fig. 4.6c shows several of t,hese cel1s which were filled with

inclusÍons of various densíties. These inclusions consisted of celI

remnants which were probably being digested by lysosomal eraymes, and then

extruded onto the surface of the neural Lube. Sone ceIl debris which had

probably been extruded were located free on the surface of the craniun.

Despite the presence of these large J-ysosomes withln the cells, the

nucl-ear and cytoplasnic contents of the surface ectodermal cells do not

appear to be affected. In another region of the cranium (Fig. 4.6d)

where lysosones w ere not present in the surface ectodermal cells,

there were vast numbers of rnicroviLli projecting above the cranial

surfaces of hypervitaminosis A-treated enbryos. Ihese structures were

seen prevÍous1y under the SEM 1n Fig. 4.2c.

4-42

4.4 DISCUSSION.

ThÍs study is one of the f ew that' examines the ef f ect of t¡y ær-

vitaminosis A on growth, general morpholory and ultrastructure at the

tine when this teratogen specifically affects neural tube closure and

the early stages of developnent of craniofacial structures in the rat.

This is in conbrast to nany reports which examine the anirnals at term

when the full but more general features of hypervitaninosis A

dysmorpholory are evident, but the possible underlying patholog¡l may no

longer be apparent. To the best of the authors knowledge this is the first'

j¡ vivo exanination of the histologlcal and ultrastructural effects of

excess vitamin A imnediately following neural tube closure on day

11.5 of gestation in raüs. In some previous studies (Morriss 1972, 1973',

Morriss & Steele 1974 ,1977) lfmited hÍstological and r.¡Ltrastructural

examination of embryonÍc and extra-enbryonfc tissue in vi.vo and in

enbryo culture prior to neural tube closure has shown so¡ne similarities

in cel1 and organelte damage with those observed following neurulation in

the nuch more extensive present study.

Assessment of excess vitanln A-induced defects followíng neurulatlon

could provide a more accurate indication of the frequency of neural tube

and related abnornalÍties than at term since sone severely affected

animals are llke1y to be resorbed by the end of gestation This

consideratÍon is reflected 1n the low mortality rate observed in t'he

present study, and the conslstently higher lncidence of resorpbions and

fetal loss reported at term (CohIan 1953, 1954). This fs further

supported by the observaü1on that nearly on+f ifth of hypenrltanÍnosis A

enbryos 1n the present study falled to develop adequate fuslon of the

chorion and allantoig Since it ls ttkely that these enbryos would not

develop a vlable placenta, a necessity for adequate nutrltlon 1n older

4-43

these aninals woul-d most likely be lostembryos (New

before they

et aI 1978), nany of

could be exanined.

As in a number of earlier report,s (Kochhar & Johnson 1965, Eckhert &

Hurley 1g7gi Hayes et aI 1981) hypervitaminosis A did not lead to a

reductÍon Ín any of the growth related paraneters exanined in the present

study. This was Ín contrast to several other studies however (Morriss

1972i Morriss & Steele 1974) where excess vÍtanin A in varj.ous forns

Ied to a reduction in embryonic size and somite number, or fetal weight.

The most like1y explanation for the variations in growth outcome 1Íes in

different experlmental condÍtÍons between the studies lncluding animal

straÍns, dose and tlne of vitamin A ad¡ninistration and harvestingr as weIl

as variafions in the mode of vitamin A administration and solubility,

factors which are likely to alter absorption and availabflÍty of fhe

vitanin in utero. ClearIy, in view of the discrepancies between the

findings in different studies, further investigations are required to

establish whether the teratogen has an adverse effect on the growth of the

rat in ubero, and if so, by whal m'echanism(s) this nay occur.

This'study also provides a nore extensj-ve investigation than bhose

available in the pubtished literature fn relation to the celluLar and

subcellular abnornatitÍes of the neural tube inmediately followÍng the

period when neural tube closure is normally completed in rats.

Exencephaly which was obserrred to be the najor hypervltarnlnosls A-

induced abnornallty in this study is characterlsed by nor¡-closure of

the cephalÍc part of the neural tube and fs a confirned neural tube

anonaly of vitanin A teratogenesls, (Cohlan, 1 953, 1954; Morriss 1973).

ThÍs animal nodel provides a sultable corroboration of evenls ln hunans

since exencephaly can be consldered as an early stage in the developnenf

4-44

of human anencephaly (Hanaway & llelch 1970). lJhite few human specÍmens

of exencephaly have been reported, their scarcity mighü inply that the

malfornation is lethal, however Snith et al (1982) suggested that necrosis

of the malfornation occurs at an early gestational age resulfing 1n

anencephaly in berm feLuses.

Growth and development of the neural folds did not appear to be lmpaired

in hypewitaminosis A embryos in this study. Instead, the folds failed to

attaÍn the normal apposfng positions reached prior to fusion (0tShea

1986), spilling over the lateral regions of the cranium, particularly in

the midbrain regÍon wibh sone involvenent of forebrain and rostral

hindbraÍn whlctt prevented fusion of the surface and neural ectoderms and

the co¡oplet,ion of neurulaüion Similar observations have been reported

previously ln rats (Morriss 197Ð and in mÍce (Theodosis & Fraser 19?S).

ThÍs collapsed appearance of the neural folds suggested that the

neuroepitheliun vJas poorly orÍentated rather than extensively da.maged.

However, the lunenal surface of the neuroepitheliun whÍch was constantly

exposed fo high Ievels of vÍtarnin A 1n the amniotlc fluid displayed

extensive abnornal blebbing in the present study, and autophagocytosis has

been reported previously in the neuroeplthelium ln mouse enbryos prior to

neural lube closure (Theodosis & Fraser 1978). It, has been suggested that

the cytoplasrnÍc protruslons whlch distorted the neuroepitheltun occurred

as a consequence of the slowlng of the cel1 cycle leading to an

accumulatlon of nltotic celIs, and hence an increased surface area

(Langnan & ÍIelch 1966).

Ultrastructunal and histological observations 1n the present study did

nol however reveal an increase in nitotÍc cells, hence the author

considers that the abnornallty 1s nore likely to be ühe resul! of a

4-45

direct cytotoxic effect on the exposed neuroepithelial cel-1

menbranes. These nenbrane changes have also been seen in migrating

embryonic cells exposed to excess vitamÍn A for periods as shorf as

two bours (Morríss 19?3), which agaÍn suggests a direct cytoboxic

effect, and which has been associated wifh a loss of mÍgratory ability

(Morriss 1g73, Kwasigroch & Kochhar 1975i MorrÍss 1976). Siroilar

cytoplasnic blebbing has also been reporled in neural crest cells (llebster

et al 1986) following adninistration of isoLretinoin which shows some

simÍ1ar developnental abnorrnalttles lo excess vitamin Ar and also j.n

nesenchymal cells in anencephalic mouse embryos exposed to excess vitamin

A during organogenesis (ÏJaterman 1979).

Other possible direct membrane effects were seen on the surface ectoderm

where the density of mÍcrovilli in vitamÍn A-treated enbryos was in excess

of those in controls. Previously it had been suggested (Harding et al

1988) that increased surface ectodermal processes might be related to the

aninals attenpts to extract addifional zfnc from the environment. The

paucity of mesoderm-derived cells of the developÍng neural Lube

observed previously by Morriss (1973) prior to neunal tube closure was

also observed in this study following t'he period of neurulation,

together with a lack of cytoplasmic connections between renaining

nesenctrynal cells. Furthermore nany of lhe cells in this neural layer in

the midbraln w ere nore sev erely affected than those of the

neuroepithelium at a sÍnilar sile. The necrosis 1n the nesenchyme

consisted mainly of swollen and disorganlsed nitochondria and vacuolated

RER. In addltÍon, severe cell death, and auto- and heterolysosones ï¡ere

observed ln many of these cells in the present study. Although lysosones

cannot be conclusively identified without hlstochemical analysisr fhelr

sinflarity to those observed 1n many pathological tissues (Ghadfatly

1988), enables the author to be sufficiently confÍdent

these structures.

4-46

of the identity of

The reduction in density of nesenchynal celIs at sltes in the cranial

neural tube, where the neuroepithelium was mosl distorted, and the

presence of necrosis in these regions suggests bhat such changes Inay

have contributed to inadequacy of mesenchymal tissue in lhis regÍon Tt¡e

inportance of a sufficient number of mesenchynal cells for cranial neural

tube closure is indtcated by studies on vitanin A-induced exencephaly

(Marir¡Padilla 1966; Morriss 1972i Morriss & Steele 1974). In these

studles, the cranial nesenchymal ceIIs dld not increase their numbers

normaIly, and the neuroepithellum increased in size, bul appeared

irregr:lar and floppy, so that the neural folds did not attain their normal

height and the lateral borders did not nove together and fuser thus

inpeding the conplelion of neurulation. These observations suggesf that

the mesenchyne has a supportive function during lhe first stage of

cranial neurulation as the folds are rlsing, and that the deflciency of

the mesenchyne during this perlod prevents lhe second stage of cranÍal

neurulation fron proceeding nornal1y, hence preventing neural tube

closure.

Since Morriss (1973) reported that the onset of necrosls and cell

loss occurred as rapidly as two hours after excess vitamln A

ad¡nir¡lslration at ühe neural plate stage (day 8) in rat enbryos, this

suggests that excess vita¡nin A exerts a toxic effect directly on the

nesenchymal cells. Furthermore, poor cetl nigratlon (Marlr¡-Pad1l1a 1966i

Morrfss 1972) and a decrease Ín DNA synthesLs (Morriss & Steele 1974)

have also been lnpllcated as causes of poor cephalÍc nesenchyne

nunbers, bub their contributlon at thls level 1s still unresolvedt

4_47

although it does appear that these effects must follow as secondary

to the rapid onset and contÍnued cel1 death seen in this layer of the

neural tube.

The nechanisns associated with the CNS malfornations while poorly

understood, probably differ fron those affecting the neuraL crest ceLl-

derived craniofacial defects (Johnston 19Tù. Howeverr some of the cel1s

observed in the nesenctryme in the midbrair¡hindbrain region in this study'

were probably neuraL crest cells whose nigration to cranio-facial sites

was inpeded by hypervitaminosis A, an observaLion nade prevÍously by

Thorogood et al (1982) in neural crest cel1s i¡ vitro. The frontonasal

processes Íncluding eyes and frontal regions of the braÍn derive from

neural crest celLs in the nÍdbrain (DeMeyer 1975). Neural crest cells

whÍch v{ere identified in bhis study by the sinllarlty Ín the location of

their nigratory pathway to that described previously by Tan and Morriss-

Kay (1985) appeared to migrate between the surface ectoderm and the

primary mesenchyme w here necrosis w as frequently observed. The

presence of necrotic cells in the midbrain mesenchyme may provide a

basis for the large number of optie placode defects observed in the

present study, and may underlie optic dysnorphology observed in

older rat fetuses (Padnanabhan 1981), since brain-eye-face

nalformations are believed to involve deflcient crest cell formation

(Johnston 197Ð. It was not possible to determine whether the fronto-

nasal regions were affected by hypervitaminosis A at this early

stage in gestallon, however the necrosis observed in the mesenchyne nay

interfere with adequate neural crest cell mlgrationr whÍch may provide a

basis for the numerous fronto-nasal abnornalitles reported later in

gestation and in term fetuses (Coh1an 1954; Fantel et aI 1977i Kalter

1960). The absence of neural crest cells at sLnilar sites along a

4-4B

mÍgratory pathway in control embryos may be attrÍbuted to the

observation that Ín normal embryos crest ceJ-l nigration in the nidbraÍrr

hindbrain regÍon which is the last to close occurs early in neurulation

well before neural fold fusion is completed (Morriss & Thorogood 1978,

Nichols 1981), however theÍr lingering presenee in hypervitaminosis A

embryos suggests thaü the teratogen interferes with effÍcient nÍgration

possibly through an effect on the extracellular matrlx, which has been

shown to be involved in efficient crest celL nigration (Thorogood et, al

1982) .

The absence of hypervitaninosis A-induced ear and branchial arch

defects in this study was in contrast to previous observations made ab

approximately the sane time in gestation (Morriss 1972; Morriss & Steele

1977). Ihe dÍscrepancies might be attributed to a delay in the inducbion

of branchial arch and otic anomalies by the teratogen in the present

study' and/or to methodological differences between the studies

including route of adninistration, forn of vitamin A used and solubility

of the vitanin, thus preventing abnormalities from becoming apparent at

this early time Ín gestat,f on. Since the lat,eral structures of the f ace

such as the otemaxillo-nandibulo segnents derfve fron neural crest cells

that originate in the hindbrain reglons of the anterior neural tube and

migrate to the branchiaL arches (DeMeyer 1975), and since necrosis has

been observed in t,he hlndbrain regÍon of the mesenchyne Ín this study, at

a site considered to be in the neural crest celt migratory pathway, it is

likely that a developnental defect nay have been evident if examination

of these trypervit,aninosls A treated embryos had occurred at a tlne later

1n gestat'f on w hen the branchial arch-derived structures Ì{ ere

differentiating lnto their fetal forms.

4_49

In summary the present study supports previous observations wherein

impairment of neural tube closure may be at,tributed to a tack of primary

mesenchyne predominantly in the mÍdbrain, with degrees of involvement of

forebrain and nostral hÍndbrain mesenchyme as we11, leadÍng to a collapse

of the neural folds in this region hlhile the neuroepitheliun was mainly

unaffected, excepl along the apical end, the Írregular, large cytoplasmic

blebs which projected into the ventricular lunen may have interfered with

the second st,age of neurulation, thus preventing fusion of the neural

folds. !{Íth the observed diminution of supportÍng mesenchyne the unfused

neural folds can no longer support themselves and hence collapse, exposing

the ventrÍculan surface of the neuroepithelium thus leading to exencephaly

in rats embryos. Furthermore, the basÍs of craniofacial abnormalities

appear to involve the inpairnent of crest cell nigration and probably also

damage to the crest cells themselves. fn additi.on, light microscopSr has

reveaLed extensive disruption of the cells in the nesenchyne including

blood cells, and both prinary mesenchyrae and probably neural crest eells

which ultrastructurally were seen to be severely danaged with lysosomal

formation, extensive cell and organelle membrane dÍsruptlon, and in nar¡y

cases celI death. The extensive detaÍls at ultrastructural and general

morphological levels derived from the present study considerably advance

our understanding of the finer details associated with hypervitaminosis A.

As vÍtanin A and its analogues are fnequently ingested by young wonen of

child bearing age, these observations strongly potnt to the necessity of

cautlonlng wonen in ühÍs age group agalnst the use of t,hese agents. The

presenü study in rats denonstrates the serfous inpllcations of exposure to

retÍnoid compounds on the developing embryo durlng organogenesÍs.

Since both zfnc deficlency and hypervitanÍnosis A have been shown to

4-50

exert thelr effects on the monphology of the anterior neural tube and

other developÍng structures in the present study, and slnce zinc is

required for adequate mobÍ1lsation of vÍbamin A from the liver (Snith et

al 1973), a further study was perforned in order to establish whether

nanipulalÍon of these two Ínterdependent dÍetary agents causes an

interaction at norphologfcal and ultrastructural 1eveIs.

4-51

4 .5 B IBL IOGRA PTIY

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4 -55

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4-56

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4-57

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4-58

ning electron microscoPY

32, 992-999.- Zinc: A trace element

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;i:îr'r'-""',ï:.:t"i:l;-1""1'"1,'å:,

"T;33i, *:, Grupper, c' ( 1 e? 5) T! e

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97.

Theodo si s, DT' & Fr 'Iy change s in mouse

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Effects of vitamin A on the ¡lnavilur'of nignåio"y neurar crest cells'

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113-125.

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'i"îît":",t"ff::t;flr13;, *#.','

Tcoanier, c1.., & Butcher, RE. ( 1e? 8) rr':relabionship of gestationai- age to vitamin A Índuced postnatal

4-59

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Chen. 238, 541-546'

5-1

CHAPTER 5.

ZINC-VITAMIN A INTERACTIONS AND TERATOGEX{ESTS.

5.1 fntroduction.

0vert similarÍties between the cranial and neural dysnorphology of

embryos exposed to zinc deficiency or hypervitanlnosis A, together with

thelr individual effects on cell viabllity raised the question whether

teratogenesis may be potentiated if the two conditions occurred

concurrently. An inleraction between zlnc Ímpoverishnent and

hypovitaminosis A has been clearly shown for a nunber of parameters

including lnpi.antatÍon sites, nalforned fetuses and plasna vitanin A

levels (Duncan and Hurley 1978). The observatlon that zlnc deficient

rats exhibited Iow plasma vltanín A levels despite adequate dietary

vÍtanin A ingestion and adequate llver stores of vita¡nin A (Smith et al

1973r Duncan and Hurley 197 8) led to the suggestion that an interaction

between zinc and vitanin A was I1ke1y, possibly at the level of the

llver RBP which was significantly reduced, resulting in Ínpaired

nobflisation of vitanin A fron this organ (Snith et aL 19T4). Since

large numbers of fetal abnornalities accompanied these low serun

vitamin A and zÍnc levels lt Ís likely that inadequate levels of bobh

nutrlents nay underlle the inductfon of these fetal anonalles.

Thus, whlle strong evidence pofnts to an Ínteractfon between zinc

deficiency and vltanin A when vftanin A leveIs are 1ow, the possibility

that an lnteractlon ¡nay occur between zinc inpoverishnent and excess

vitanin A has not been widely studled, although llnlted morphological

evldence so far suggests that it does not occur (Eckhert and Hurley

1979). Furtherrnore, although ft, has been proposed thaf vltamln A

5-2

exerts its terabogenic effect through interference with DNA synthesis

leading to altered tissue differentiation and abnormal developmenl

(Kochhar 1 968, Nanda 197 1), and a s1m11ar mechanisn has been

expressed for the teratogenic effects of zinc deflciency (Swenerton et

aI 1969, HurIey et al 1971, Eckhert and Hurley 19TT)' avallable

blochemical dala have failed to support an interaction between zinc

deficiency and excess vitanin A (Eckhert and Hurley 1 979)'

Nevertheless, the invoLvement of these nutrients in a number of

¡netabolic processes comnon to both, prompted the present investiSatÍon

to explore al the cetÌular and subcellular leve1 the posslbility of a

potentÍal interactíon Since adequate levels of zinc (Chvapll 1976) and

vitanin A (l,lo1f 1984) are known to be required f or the proper

naíntenance of menbranes, and several studíes have shown sinilarities

between cellular necrosis and severe menbrane disruption in enbryonic

brain tlssue of zinc defÍcient (Record et aI 1985ar Harding et aI 1988'

Brenert et al 1989) and hypervitaminosls A anÍmals (Morriss 1973,

Iheodosis & Fraser 19?8), these factors point to the likelihood of a

potential shared mechanisn of teratogenic action for the two nutrients.

previous studÍes have directed litt1e attenlion bo the brain, and the

questÍon of an ÍnteractÍon between zinc deflciency and excess vitanln A

affecting neural morphologl has not been examined. Tt¡e purpose of

conblning these two teratogens in the present study was to examine

nicroscoplcally a possÍble interactÍon between hypervitaninosis A and

zÍnc deficiency al the morphologlcal and particularly at the

ultrastructural leve}s 1n the enbryonic rat. The need for such an

investlgaü1on is hightlghted by the recent reports of bfrth defects ln

wonen lngestÍng vitamln A analogues for the treatment of skin disorders

(Stange et al 1978) and by the lncreastng use of vltanin A itself for the

sane purpose by young wonen who faII Ín an age group where

often appears to be marginal (Record eü aI 1985b).

5-3

zínc lntake

5.2 MATERIALS and METHODS.

5.2.1 Anínals and dfets.

Fenale Sprague-Dawley rats (180-2109) were mated overnighl and were

assigned the following morning (gestatfon day 0.5) to two Eroups receiving

either a zinc-defÍcient (less than 0.5 ue/Ð or zÍnc-replete (100 ue/e)

soybean based diet as described in chapter 2. Animals t{ere housed

individually Ín stainless steel and plastic cages and r.¡ere fed ln a

cyclfcal pattern as outlined in Chapter 2 to ensune hlgh diefary intakes

on days I and 9 of gestation, and subsequent neural tube teratogenesls in

the zlnc-deficient aninals (Record et aI 1986). 0n day 8.5 of gestat'ion

between 0800-0900h the zinc-defieient animals were dosed by stonach

intubatlon with 100,000 IU of vitanin A palnitaüe in 1 ml of sunflol{er

seed oi1, a dose prev iously reported to induce neural tube

teratogenesls in rats (Morriss 1972). The renafning anÍnals received the

oil alone.

5.2.2 Tissue preparation and analytical methods.

On day 11.5 of gestatÍon, dans were sacrifÍced, blood was collected for

serum zÍnc analysis by atomic absorption spectroscopy (Dreosti eb aI 1982)

and embryos were renoved for assessment of growth and developnentr and for

detalled exanination of fhe anterlor neural tube by tight and electron

mlcroscopy described in defaÍ1 in Chapter 2.

Continuous growth variables and naternal serun zinc levels l¡ere

analysed by standard analysis of varfance. For dfscrete developnent data'

such as the nunber of dead or nalforned enbryos, a naximun Iikellhood

nethod of estlnatlon was used, which lnvolved treating the nunber of

npositivesn wÍthin each treatnent Sroup as being

(Baker & Nelder 197Ð.

5-4

binomially distributed

5.3 RESULTS.

5.3.1 Grcr¡th and norphological development.

Both zinc deficienü embryos and those in the conbined zinc-

deficient/hypervitaninosis A group showed signÍficant retardalion of all

growth indices compared with controls (Tab1e 5.1). Zinc Ievels in bot'h

these groups w ere significantly different from control s, whil e the

combined treatment group did not differ in its zinc lei¡eIs conpared with

exposure to zinc deficÍency alone. No reduction of any paraneter occurred

in enbryos fron dans treated only with excess vitanin A. The present study

reports for the first tÍme the effect on embryonic gfowth of the

concurrent exposure to excess vitanin A and zinc deficiencyr at Ievels

which were individually teratogenic, but which together were not different

from the zinc defÍcíent levels, suggesting that even at teratogenic levels

acute hypervÍtaninosis A dld not potentiate the gnowth retardatÍon

associated wÍth zlnc deficiencY.

Tab1e 5.1 Effect of Zinc Status and Hypervitaninosis A on Growth in Rats.

Zínc- Zlnc-replete deficlent

Zinc-replete+ Zinc-deficient+IlypervitaninosÍs A If pervitamlnosis A

No. enbryos( dans) 6 1 ( 5) 66 ( 5)Ihternal sertmzinc (ug/nl) 1 .210.0 0.710.1 a

Crcn¡rprtmpleneth(nni 3.79.1 2.810.14

No. somites 22.6!0.4 1T .5¡1 .14tubryonlcproteln (us) 245.\113.6 145.0111a

106 ( 8)

1 .270.1

3.410.2

21 .4$.8

2f 0 .091 I

80 (6)

0 .610.1 a

2.7¡0.24

18.5+1 .1 a

a SignifÍcant effect of ztnc deficiency orùy. p(0.001

129.0+Z6a

1- Values are neans + SEM.

5-5

ÏJithin-litter and between-Litter effects were very much reduced when

anirnals were exposed to both dietary manipulatÍons concurrently' The three

embryos in FÍgs. 5.1a,b and c whÍch represent Lypical embryos in three

different Iitters demonstrate betweer¡Iitter similarities. The naJority

of abnormal enbryos in nost li|ters had this appearance wibh the cranial

reglon being nost affected. These animals were sinilar to the

hypervitaninosis A embryo in the centre of Fig. 5.1d, except t'hat the

zino-defÍcient/hypervitaninosfs A enbryos generally denonstrated nore

severe dysmorpho].ogf and were up Lo 30{l snaller than hypervibaminosis A

embryos. The renaining enbryos in Fig. 5.1d rvere litternates whlch had

been exposed to concurrent dietary nanipulations and which did not

demonstrate the nore typicat pattern of abnornalitÍes usually seen in this

group, but rather showed the variability and characteristic dysmorpholog¡

of zinc-deficient enbrYos.

No inberaction was evi.dent between hypervitaninosis A and zinc deficiency

with nespect to the rate of inplantation, surr¡ival of the conceptus and

certain teraba, although individual treatnents Ied to a raised incidence

of these anonalies in sone cases (TabI e 5'2)' However, with respect to

dysnorphogenesis in genenal, whÍch occurred fn nearly 52f and 46f, of

embryos in the zÍnc-inpoverished and excess vitanin A groups respectivelyt

bhe conblned effect of the nutrlent nanipulations Ied to 93f of enbryos

showing sone klnd of abnormality (Tabl e 5.2). In the case of neural tube

defects which were observed in over 80f of the enbryos, an increase in the

severity of neural dysnorphology often acconpanled this lncrease ln

frequency. Ele defects also were observed bo increase to nearly 39f as a

result of concurrent adminlstratlon, while the frequency of branohlal arch

defects dld nof dlffer fron those observed when zfnc deffcency alone Ì¡as

induced. The fnterference by zinc deficency with chorio-allanboic fuslon

5-6

Fig. 5.1 arb and c shows three 11.5 day embryos fron different littersexposed to zinc-deficiency and hypervitanÍnosis A concurrently, whichdemonstnate betweer¡-Iitter sinilarities. The embryos appear to be

dysmorphÍc in the cranÍa} region only. All three anlmals show exencephaly.The cranial neural folds have not fused (arrows) and have collapsed onfothe cranial surface in the midbrain and hindbrain regi.ons (arrowheads).The optic vesfcles are also not well developed. Fb, forebraÍni 1r2r3,branchial arches; OP, optic placodes; 0T otÍc vesÍcles; Hr heart; Ft

forelinb; S, somites.

Flg. 5.1 d. A day 11.5 hypervitaninosis A embryo (centre) can be conparedwith the three zÍnc-deficient-hypervitanÍnosis A aninals fn Figs.5.1 â,b, and c. Nobe thal dysnorpholory Ís very similan except that theenbryos exposed to both treabmenüs showed nore distortlon of the cranialneural folds. the renalning embryos 1n the figure are litternates exposedto concurrenf dielary manlpulatlons which show the range of dysnorphologrnore characteristic of zfnc-deficlenb titters. Unfused neural folds areindicated by arrows. 1, abnormal nandibular arches; P, pericardium; H,

heart.

Bars=500um.

Tab1e 5.2 Effect of ZÍnc StatDevelopnent in Rats

5-7

and Hypervitaninosis A on Morphologicalus1

zínc2RepI eLe

Zj-ncDef icient

Zfnc replete + Zinc deficient +

hypervitaninosis A hypervitaninosis A

No. enbryosdans.fubryonícloss.Deformedembryos.NeuraI lubedefect s.Eledefect s.Eardefect s.Branchi alarch defects.InconpletefI exion.Impaired chorio-allantoic fusion.

61-5

3(4.e)

3(4.9)

2(3.2)

0( 0)

1(1.6)

0( 0)

2(3.2)

3(4.8)

66-5

6(9.1)

35( 51.5)

26ß9.4)

9(13.6)

?(10.6)

7(10.6)

1 9( 28.8 )

31(46.9)

1 06-8

5( 4.7 )

49( 46.2)

¡g(:6 .8)

26(24.5)

3( 2.8)

2( 1 .9)

9( 8.5 )

20( 18.9)

80-6

4( 5)

7 4(92 .5)

65( 81 .3)

31(38.8)

2(2.5)

5(6.3)

11( 13.8)

21(26.3)

1 N,-b""" in parentheses indicate percentages'2 zíno nepletå group sane as in zlnc deficient study as both

experÍments were performed concurrently'

A

and dorso-Lateral flexÍon appeared to be ameliorated by hypervitaninosis

Table 5.3 displays the deviances or ngoodness of fitn for evaluatÍng the

inportance of the treatnents and thein interactions. tlhen the difference

between deviances (as indicated in Chapter 2) is greater than fhe X2 value

for the approprÍate degrees of freedon, this indicates that the associated

¡nodel does not fit the data we11, and thab further explanatory variables

are requÍred to descríbe the observed variation. Embryonic loss was the

only outcone which was not affected by either treatnent. Branchial arch

defects and inconplete flexÍon are explained by an effect of zinc-

deficiency only, although the data in Tabte 5.2 indicates an amelioration

of effects in the conbined group due bo hypervitaninosis A for the latter

5-8

defect. For other outcones however it nÍght be expected that the

dlfference between deviances would be less t,han X21,0.05 = 3.84 Íf the

zfnc-depletion and hypervitanÍnosis A were actÍng independently of one

another (Model 4 in Table 5.1). However, the other observed devLances

were substantialty higher than this, hence it must be concluded that

the treatments do fnteract wfth one another. These non-independent

(or interactive) effects were observed 1n relatlon to total nunbers of

deforned embryos, and neural tube defects (NTD) where the incidence of

these terata was 39í, 3Tí, and 81f respectiveJ.y. Hence model 4 in Table

5.3 denonstrates that bhe effect on NTD of concurrent treatnent wit'h

zinc defÍciency and excess vltamÍn A is nor¡independent or j.nteractive'

rather than independent or additlve.

Inconplete flexion and inpaired chorio-allantoic fusion also denonstrated

non-Índependent or interactive effects (Table 5.3). The observation thaü

the frequencÍes of impaired fusion when exposed to concurrent zÍnc-

deffciency and hypervitaminosis A (26.31) was much less than those when

only zlnc deflciency was acting (4Tfi), suggested that hypervftanÍnosls

A 1ed to an amelioration of bhese effects. A sinilar aneliorative

trend was indlcated in TabIe 5.2 for inconplete f}exlon, but was

analysed as an effect of zlnc only in Table 5.3b. Alühough previous

workers have denonstrated a reLationshfp between zinc status and vÍtamin A

meüabolisn (Snlth et al 19?3), the flndtngs of the present study appear to

be the first evidence of a possible reclprocal lnteraction

5 .3 .2 l{i.crosco pic observations.

l{hite morphological data ÍndÍcates that co¡nbined zinc deficlency and

excess vitanin A exert an inleractive effect leading to an increase 1n fhe

frequency of varlous developnental anonalles, nicroscoplc observations

5-9

TabIe 5.3 EValuation of the Degree of Independence of the EffecfsZinc Deficiency and l,lypervitaminosis A on MorphologicalDevelopment in Rats.l

of

Deviance2

!Ícdel 1(dev 4)No effectof eithertreatment

(3 df)

Idcdel 2(dev 3)Effect ofzi-ncdef ici encyonLy ( 2 df)

l4cdel 3( dev e) l4cdel 4( dev 1)Effect of Independent

hypervitanÍnosis effects ofA onJ-y both treatnents(2 df) (t df)

hbryonicI ossDef oraedembryos

NTDEledef ect sBranchÍ aIarch defectIncøpleteflexlonInpairedfusion

5.23

129.434

114.254

4o .47 a

9.724

26.614

46.goa

7 0.24b

65.fib

34.27b

1 .23

10.52

22.6gb

87.07c

74 36c

13 .4 Bc

I .95c

25.19c

46 .66c

12.OOe

g.57e

0.27d

1.67

9.76

16.36e

,|fSee chapber 2 for explanation of statistical model used.¿Deviance is assumed lo be distributed as X2

a ff dev 4-dev1>5.99 (critical value for X2 for 2df, p(0.05)r then rejectl,lodel 1.bri a"u l-dev 1>3.84 (critical value for X2 for 1df, p(0.05), indfcatessignificant effect of hypervf taminosis A' then rejecf }dcdel 2.cff dev 2-dev 1>3.84 indÍcates signÍficant effect of zinc defÍciency, then4eject I'l¡del 3.olf dev 1<3.84 accept Model 4 for Índependent effects of both treatments.e If: dev 1>3.84, reject ì4cdel 4, indicates interactive effects of bothtreatrnents, ie. l,fodel 5.

allow a nore precise assessment of the severity of these abnormalities

particularly |n relation to the cranial regÍon which appears to be nost

affected by the concurrent dietary rnanipulatfons. Scanning electron

nicroscopy of the surface features of a typlcal affected 11.5 day enbryo

exposed to zLnc deffciency and excess vltanin A concurrently (Flgs.

5.2a,b) showed that the neural folds $¡ere severely evaginatedr and

dlstorted, and had ' conpletely collapsed onto the remainder of the

craniun exposÍng the neural ectodern. The neural folds had

5- 10

collapsed throughout the midbrain, and in the rostral parb of the

hindbrain down to the level of the mandÍbular arch. The neural tube

caudal to this site was nearly always closed. The forebrain vesicles also

appeared to be underdeveloped, while the site of lhe optic placodes was

often hidden under the collapsed neural folds making il difficult 1n

sone enbryos to determine whether eye developmenb !{as affected by

the treatment. The severity of these abnormalitles appeared greater

than in embryos exposed to zinc-deficÍency or hypervitamlnosis A aIone.

The otic veslcles which in the embryo 1n Fig. 5.2a ÌJere correctly

allgned with the hyoÍd arches, Ì¡ere rarely dysnorphic fn enbryos exposed

concurrently to both treatnents. Flg. 5.2b shows a ventral view of

the same enbryo with the perlcardiun renoved to provide a full view of

the branchial arches. In thls enbryo and most others exposed

to z1-nc deficiency and hypervitamlnosis Ar these arches !üere weII

developed, however in a nunber of affected animals the hyold arches

were fused to the pericardiun. FÍg 5.2b atso denonstrates the distorted

and flattened prosencephalic region seen so frequently Ín exencephallc

enbryos.

Higher nagnification of reglons of the zinc defÍcient-hypervltanÍnosis A

embryo showed that both the exposed neural and surface ectoderms (Figs.

5.2crd, respectfvely) $¡ere very irregular conpared with sinflar surfaces

in controls (Figs. 3.3a,b) and none affected than 1n zinc deficient

(Figs. 3.3c,d,g) and hypervitaninosis A enbryos (Fig. 4.2c,d) at the sane

stage of developnent. The apical surface of the neural ectodern 1n the

nidbraÍn (Flg 5.2c) whlch was constantly exposed to the annfotic fluid

in these enbryos, was covered fn blebs of varlous sizes wfth

associaüed nicrovilll, and long cytoplasnÍc strands which appeared fo

Itnk the blebs logethen The cells of the surface ectodern (Fig. 5.2d)

5-1 1

Fig. 5.2arb. Scanning electron nicrographs of a dorsal and ventrelateralview of a typical day 11.5 zinc-defÍcient-hypervitamlnosis A embryo'showing unfused and severely evaginated cranial neural folds (arrows)completely exposing the ventricular surface (asterisk) to the anniotÍcfluÍd. The neural folds have collapsed onto the cranlal surface in theforebrain, nidbrain and hindbraÍn regions (arrowheads). The presence ofnornal optÍc prinordia cannot be determined because of the collapsedneural folds in that area. The otic vesicles (0T) have not closed, buü theanimal has rotated and has developed a forelimb bud (F). The pericardlunand heart which appeared normal were re¡noved to clearly show the welldeveloped branchial arches (1 rZ13¡. Note that even at thÍs lownagnification the surface ectoderm over the cranlun and branchlal archesappears abnormal. Fb, forebraini Hb, hindbrain; Sr sonftes.

Fig. 5.2c, Ventricular surface in the nidbrain of bhe same embryo showsthat the apical ends of the neuroepithelial cells have large irregularcytoplasmlc projectlons (asterisks) with extenslve branching blebs(asterisks), and occasional long cytoplasnic strands (smal1 arrows)belween blebs, and scattered mÍcrovilli (arrowheads).

FÍg. 5.2d. The cells of the surface ectodern of the midbrain in the sameenbryo while relatfvely snooth were interrupted by huge blebs (asterlsks)with scattered microvilli (arrowheads) and a network of interconnectingcytoplasmLc strands (arrows).

Bars=100un arb; 1oum c,d.

r

t,

'-f

5- 12

also shovred sinilar features, however the individual blebs were larger and

nore widely spaced with a more promÍnent network of cytoplasmÍc sLrands

between them.

When the cranlal neural tubes of embryos exposed to both zinc-deficiency

and trypervitanlnosis A were sectfoned and exanined under the llghl

nÍcroscope, celluLar abnornalities were so severe as lo support a

potentiation rather bhan an additfve effect of bhese two teratogens. Fig.

5.3 shows horizontaf secbÍons through an embryo sínilar to thab seen in

FÍgs. 5.Za and b. At low magnÍfication (Fig. 5.3a) bhe cranial neural

folds were distorted and flattened with nar¡y free neuroepithelial cells in

the region cotnparable to the ventrÍcular 1u¡nen in control enbryos, while

the underJ.ying mesodern appeared depleted of a few nesenchynal cel1st and

contained abnornall-y large, developing blood vessels'

Higher nagnificatÍon of a reglon through the forebrain (Fig. 5'3b) showed

bhat the cells of the neuroepÍthellum $rere no longer closely alÍgned as is

typical of these pseudostratified colunnar cells, but were severely

disordered, with nany loose, rounded cells together wÍth nÍtotic cells

lying free in the region of the ventrlcular lumer¡ The structures which

had been identifÍed as blebs at this site under the SEM (Fig' 5'2c) could

be Ídentified under the llght nicroscope as individual cells rather than

as extenslve cytoplasmlc cell projections. At the basal end of the

neuroeplthellum in the sane sectlon, neuroepÍthelial cells appeared bo be

extruded lnto the mesenchyme, where there lJas a severe paucity of cells

avallable to support the neural folds.

This absence of rnesenchymal cells was also seen at higher nagnlfication

in a sectlon through the midbrain (Fig. 5.3c)r where these cells were thln

and stnetched and had lost their lnterconnecting cyloplasmlc proJections

Fig. 5.3. Horizontal sections through the anteríor neural

day zinc-def icient-hypervitaminosis A exencephal ic embryo'

5- 13

tube of an 1 1.5

a. The neural tube is severely distorted and the neural fotds (NF) have

not fused and are evaginated. The neuroepÍthelium (N) is thÍn and bhe

orientabion of the cells at the apical end Ís highly irregular (arrows),

while bhe basal end is convoluted. There is a paucity of mesenchynal ceLls(M) throughouü the cranial regions and abnornalJ-y large develo-pÍng blood

vessels (bv) oo"upy rnost of the space in the hindbrain (Hb) ab t'hisIevel. Confirnation that these areas are occupied by blood vessels was

made at higher magnÍfication. The sites marked by arrowheads and

asterisks are seen at higher nagnification in b and c respectively'

b. Higher magnification of the area between the arrowheads in theprevious micrograph showing a thin neuroepithelium in which fhe cells have

lost their pseuáostratified colunnar orÍentation with cells and cellfragments (süort arrows) and mÍtotic figures (arrowheads) Iying freeon the surface of the ventricular lumen The convotuted basal end of the

neuroepitheliun appears to contain large empty spaces at some sites(Iong årrows), "nd

probably indicates regions where neuroepithelial cel1

debris has been extruãed info the mesenclryrne. The f ew mesenchynal ceIIs(M) present have a rounded rather Lhan a typical stellate shape and

filopodia are absent. bv, blood vessels'

c. Higher magnification of an area between the asterisks in a, in thenidbrain region of the neural tube showÍng sinilar disorientation withinthe neuroepithelium as was seen in b. In additlon, many dense inclusionsand extracellular debris were also visible (arrows). N' neuroepitheliun;M, nesenchynal celJ.s; Mf, nitotic figure'

d. A horizontal section at a deeper level where the neural folds in the

forebrain region had nearly fused. The neuroepithelium (N) is stillseverely disrupted wÍth free cells and ceI1 fragnents (arrowheads)spilline into the ventrÍcular lumen (L). The basal end of thenèu"o"pÍthelium is still highly convoluted, but the nesenchynalcells il¡l are extremely dense although nany condensed structures(arrows) were visible Ín the lateral regions'

Bars=50um.

-_- NF 1

¡\

ÐA

It

.)t ¡s. ?.

;l ¡'\

a

a

t

.+

d

5- 1 4

or filopodia. In addition bo the severe disruption of the neuroepithelial

cells, many of these cells contained dense inclusions similar to

strucbures identified in previous chapters as phagocytosed dead ceII

rmnants.

A horÍzontal sectÍon through the craniun of the same embryo at a deepen

level whene the neural folds had not collapsed (Fig. 5.3d), and where the

forebrain region had nearly closed, showed the typical disorganlsed

features of surface and neuroepithetÍal celts exposed directly to the

berabogens, however the underlying mesenchyme contained many cel1s at this

level together with nar¡y dark intr+and extracellular bodies, whlch

suggested that the teratogens had caused extensive cell death in this

Iayer of the neural tube.

5.3.3 Ultrastructural Observations.

The patt,ern of necrosis observed in enbryos exposed concurrenfly to

zinc-def iciency and hypervitaminosis A in bhe nesenctryne was sinÍ1ar to'

but nore extensÍve than that observed at comparable sites in zinc-

deficÍent and trypervitaninosis A ani.nals. Ihe transnÍssion electron

nicroscope rer¡ealed that the neuroepithelfal cells which l¡ere seen to be

spil1Íng out into lhe ventricular space under the light nicroscope (Fig.

5.3) had Iost theÍr colunnar orientation and theÍr apical junctional

conplexes (fig. 5.4a), tùhich are characteristic of healfhy

neuroepithelÍat cells (see tr'ig 3.6a). MarV of the nuelei were lrregular

1n shape, however the nucleoli and chronatin appeared norual, as did

the cytoplasn. I,lhile the nore superf Íciat regions of the neural folds

whlch had collapsed and fallen out over the renainfng areas of the

cranium were depleted of mesenchyne cells (see Ffgs. 5.3arbrc), the

section 1n Fig. 5.4b 1s from an area of the nesenchyme at a deeper

5- 15

Figs.5.4a.d.Electronnicrographsofsectionsoftheanberiorneura]-tube of the day 1i.s "i""

deficient-hypervitaminosis A embryo in Fig' 5'3'

a- A section of Lhe disrupted neuroepitheriun (N) where the cells have

lost bheir apicai junctional conplexes causÍng then to Iose their closely

spacedcolumnarorient,ationandtospillintotheventricularlumen(L).The nuclei (nu) were irregular in shape' however the nucleoli and

distribution of chromatin appeared normaf. M' nesenchyme. Mf, mitoticf igur e.

b. A sectÍon of the mesenchyne with many affected mesenchynal cells'The nuclei (nu) 1¡ere abnornally shaped in cells which contained

heterolysosones fiIled with hðavity condensed, lysed cel1 rennants(arrowheads). The extracellular matrix contained Iarge areas of dead cellrennants (asterisks) ranging from light flocculent to electron dense

debris, as weII as whole, dead onoensed ceIIs (d)' Part of the

nesenchymal cefi (¡f1) can be seen at higher magnification in d'

c. Higher nagnification of the extracellular debris seen in the extreme

top right, hand corner of Fig. 5.4b. Some of the identifiable features

include nenbrane bound vacuoles (v)r vacuolated rough endoplasmic

reticufum (vr), mitochondria (n), and cytoplasn (c)' The dense amorphous

structures nay be primary lysosones (Ly). ECM, extracellular matrÍx'

d. Higher nagnification of several heterolysosomes (arrowheads) seen inthe mesenchynai cell (Mr) shows that mosb organelles exposed to zinc-def iciency and lt*"iilaå'inosi.s A during neurulation have been digested'

The only recogãtïaute structures remaining are vacuolated rough

endoplasnic ""iiou:.,rt (asberisks) which probably participate in the lytic

process. nur nucleus.

Figs. 5.4e*f. Electron nicrographs of sections of the anterÍor neural tube

of a sinjl-ar zinc-deficient - hypervibamlnosis A embryo'

e. The nicrograph shows a condensed, vacuolated, dead cel1 (d)

extruded from the ieuroepithelÍum (N) into the extracellular natrixat a slte where the basal famina is disrupted (arrows)' Note the

vacuolation of this cell and the phagocytosed membranes contained

sone of the vacuoles (arrowheads)' BL, basal lanina; M' mesenchymal

f. This micrograph shows a range of ceIl rennants including part of a

condensed whoLe cell (d) in various stages of destruction wíthin fhe

extracellulan space ln the neuroeplthelÍun (N). Several anorphous'

erectron dense siructures which are probably primary rysosones (Ly) are inclose proximity to these cel-I rennants. Vacuolated rough endoplasmic

reticul-um appears to be digesting several nitochondrÍa (arrowheads)' The

broken basal- fånin" (arrõw) provides a site for extrusion of these

rennants lnto the extracellular natrix (ECM) of the nesenchyne' IDt

nitochondria.

g. Lysosones (Ly) wÍth li¡nitin isible (arrows)'

were closely """oti"l"d with cell hyne' vacuoLated

rough endoplasnrã reticutum (vr) ta (n) as well as

cytõprasniô ribosones (c) are also na1 cell'

belng(ECM)

severew lthincel I.

Bars=4un a,b; lurn crd,erfrg.

ECM

ECM

5-16

Ievel- in the mídbrain comparable to Fig. 5.3d' which was observed to

contain numerous cells and many necrotic cell remnants. The pattern of

necrosis observed in enbryos exposed concurrently to zinc defÍciency and

hypervitaninosis A in the mesenchyne was much nore extensive than that

observed at sinflar sites in zinc-defÍcÍent enbryos and even

hypervitaninosis A animal s at neurulation Mar¡y of the cells in the

mesenchyne, sone of which nay be neuraL crest ceIIsr contained

electron-dense material wlthln structures that were probably secondary or

heterolysosomes, as judged by their sinilarity with structures

identified by GhadiaIIy (1988). These lysosomes contained necrotic cell

material which had probably been phagocytosed by the host mesenchymal

cells. The extracelLular matrÍx was also fulI of dead cell rennants

ceLls and other unidentifiable ceIIuIar malerial. Higher magnification

(Fig. 5.4c) of sone of the extracellular remnants seen in Fig. 5.4b

showed that despite bhe severely condensed appearance of much of this

debrÍs, a few nenbranes and organelles had not been entirely digested

and were stÍII recognisable as RER and nltochondria. Digestion of

cell conponenbs 1n the nesenchymal cells by heterolysosomes seen at

higher nagnification Ín Fig. 5.4d, Ieaves nost onganelles

unidentÍfiable after exposure of the neural tube to zinc deficiency

and trypervitaninosÍs A throughoul neurulabion Itre only recognisable

organeJ-1es renaining appear to be vacuolated rough endoplasnlc

reticulun which have thenselves been reported to release lytic enzymes

derived fron ribosones (Ghadially 1988).

The probability thab sone of ühe cell debris Iocated fn the nesenchyne

v¡as derlved fron the neuroepitheliun is conflmed fn Ffg. 5.4e. It¡e

micrograph shows a condensed dead cell belng extruded into the nesenchyne

bhrough breaks Ín the basal lanina which suggests bhat sone of the

5-17

necroticnaterialinthiscelllayerisprobab}ytheremnantsof

neuroepithelial celIs. The micrographs of FÍgs'5'4f and g confirm the

involvement of Iysosones in the destruction of neural tube ceIIs' Fig'

5.4f shows a range of celI rennants in various states of destrucbion in

bhe neuroepitheLium near the junction with the mesenchyme' The loss of

basal Ianina at this site suggests that the dead cel1 remnantswill be

extruded into the nesenchyme. The etectron dense strucbures associated

with the area of extracellular debris are probably primary lysosomes

and are closely associated with sevenely depleted areas of cytoplasm' A

nunber of sections of vacuolated RHì surround some of the mitochondria

and appear to be digesting then. sinilar electron dense structures

are seen to be associated with cell remnants in the mesenchyme at hÍgher

roagnification in Fig. 5.4g. Several appear to be menbrane bound while

another appears to be only partially menbrane bound, whÍch suggests that

prinary lysosomes nay lose lheir menbranes when actively secretÍng theÍr

Iytic contents.

5- 1 I

5.4 DISCUSSION.

This is the first report of the combined effects of vitanin A excess and

zinc inpoverishment imposed at teralogenic levels on embryonÍc body size

as determined by total body protein content' crown-rump length and

somite nunbers. zj:nc deficiency arone at teratogenÍc leveIs has been

consistently reported to lead to reduced embryonic (Record et aI ,|986;

Joschko et al- 1989) and fetal (Eckhert and Hurley 1979; Duncan and Hurley

1g78; Rogers et al 1985) size, while embryonic size and fetal body weighl

were not affected by negadoses of vitamin A (Eckhert and Hurley 1979;

Joschko et aI 1989), nor by vitanin A insufficiency (Duncan and Hurley

1978). Theresultsinthe present study have shown LhaEzinc deficÍency

alone in rat enbryos led to reduced leveIs for all growth parameters

considered, and the conet¡rrent adminlstration of excess vitamin A did not

ameliorate this effect. similar observations were made by Eckhert and

Hurley (19?9) in rat fetuses. Peters et aI (1986) recently reported

sinilar findings in zinc deficient fetuses, but with only marginally hÍgh

(subteratogenÍc) levels of dÍetary vitamin A, while hypovitaminosis A at

narginal and decreased concentrations also had no influence on the reduced

body weight of zj.nc deficient fetuses (Duncan and Hurley 1978). Adequabe

zinc leveLs appear to be of far greater consequence for the efficient

growth of the organisn than vitamin A, which appears to have no apparent

effects on growth in excess or deficiencyt alone or cornblned with zínc'

although some studies referred bo in the previous chapters have

demonstrated a reduction in enbryonie and fetal size associated with

hypervitaminosis A (Morriss 19?2). Hence it appears that whÍIe zinc and

vitanin A nay interact at sone levels they do nob appear to do so at the

level which affects embryonic and fetal body welght'

5-1 9

Although previous workers (Smith et aI 1973) have denonstrated that zinc

deficÍency a.Lters vitanin A netabolism at' the level of the 1Íver by

preventing adequate mobÍlisation of Liver vitanfn A, probably by

Ínterfering with the synthesÍs of the specific retinol transport protein,

ttsP (Smith et aI 1974), this study appears to provide the first evÍdence

of a possible specific interaction between these two nutrients.

The data presented 1n bhis report for concurrent administratÍon of zinc-

deficiency and excess vitamin A appear to faI1 Ínto several categories

indicating differentÍal effects on the developnent of the varfous

morphological features exanÍned. In the first category, when zfnc

deficiency and hypervitaminosis A were induced together, they did not

exert Índependent effects on the embryo Ín relatfon to the anÍnalrs

fallure to rotate and its inability to achÍeve chorÍeallantoic fusion,

but rather concurrent adnÍnÍstration appeared to lead to an aneliorat,lon

of effects which denonstrated a staüistically signÍficant teratogenic

interaction. In the second group, the facial structunes of eye and

bnanchial arches $¡ ere affected deleteriously by the concurrent

adninistration of both teratogens which however acted independently. In

the third category which included the t,otal nunber of malformed enbryos,

and frequency of embryos with neural tube defects the teratogens did not

exert lndependent effecüs buf rather demonsürated an enhanced

effect on the frequency of dysnorphology as well as severe

exacerbatlon of embryonlc anonalles, and when analysed in süatistical

terms there $¡as evidence of an interaction between the two teratogens.

This exacerbation of dysnorphologr was reflected in nany cases by cell

and subcellular dlsruption in bhe Iayers of the anterlor newal tube.

The nost interesting obsen¡atlon nade for both of the paraneters 1n the

5-20

first category is that the interference with enbryonic rotatlon and

chorÍo-allantoic fusion induced by zinc deficiency alone appeaned to be

ameliorated by hypervlbaninosis A leading to fewer embryos beÍng affecled'

This suggests that vitanfn A at high leveIs nay exert a protective

influence on the embryos from the deleberious effects of zlnc deficlency,

although this effect was nob observed for normal or sub-nornal level-s of

vitaminA(Duncan&Hur1ey1978).Partofthedeleteniouseffectofzinc

deficiency on these two parameters nay be associated with reduced plasna

vitanin A levels, since zinc deficency is known to reduce these levels by

50f even when normaf quantities of vitamin A are ingested (Duncan & Hurley

1978), probably because of Ínhibltion of the synthesis of RBP, retinol

being the active forn in which vitanin A fs transported in the plasna to

the tissues (Snith et aI 1974).

Desplte this inabillty of retlr¡oI to pass to bhe üissues, it 1s possible

that vitamin A nay exert a coúpensatory effect on menbranes and ceIl

differentiaüion through the retfnyl ester conponent which is known to be

stored in the llpoprobeÍn fractlon of the naternal clrculation and which

passes to the enbryo when the leve]s of vitamin A ingested are excessive

(Underwood 1 984).

Hence the present author proposes that in the case lJhen severe zinc

defÍciency Iowers plasrna retinol Ievels considerably, the retinyl

esters nay conpensate by ralsing the vÍtanln A to nore nornal levels

and hence nay act Ín bhe embryo on lipid bilayers of the chorlon

and altantois preventfng zinc deffciency fron destabillslng the

menbranes, leadlng to sinilar breakdown and subsequent death of the

cells and tÍssues, as has been observed in thÍs study ln other

strucüures foltowing concurrent exposure to z|nc def|clency and

5-21

trypervitamino sis A. ÌÍhíte this might be an approprÍate explanation f or

Ínteractions whÍch demonstrate a protective nechanisn it does not

appear to explain the independent or the potentiative effects observed

in other structures in this study. Hence the author can onfy suggest

that the diffenences may lie in the teratogens acting on dífferent

nechanisns as well, and that these structures may have different nutrient

requirements at different tines.

The second category of results which íncluded the neural crest derived

structures of eye and branchial arches (Johnston 1975¡ DeMeyer 1975)t

demonstrated that zinc deficiency and hypervitaninosÍs A exerfed

independent effects of both treatnents on the frequency of dysnorpholory

of the optíc placodes, while only zinc deficency had a significant

effect on branchial arches so when concurrent nutrÍent manipulations

were adninlsbered only the zinc conponent of the treatment contributed to

the teratogenic effecL In contrast, Eckhert and HurIey (1979) reported

that only vitanin A treatnent affected the nunber of fetuses with eye

anonalies, while cleft palate which appears to develop fron branchial

arch defects at a later stage in gestation, was affected by the

intake of vita¡oin A but not zÍnc. DespÍte these differences' both

studies determined bhat there was no sÍgnificant interaction between

zinc and vilanin A treafnents Ín relatÍon to the frequency of these

anonal ies.

The dffferences between these studies nay have arÍsen through the

exanination of these paraneüers at differenü tÍmes in gestaü1on. In

addition, the apparently snaller nunber of eye defects seen by Ecld:ert

and Hurley (1979) nay be abtrlbuted to the increased lfkellhood of

resorptlons and death fn teratogenlc lltters when harvested at ternt

5-22

rather than at mid-gestatlon and/or possibly also as a result of the

different treatment regimes. In additÍon, the maternal vitamin A intake

was considerably lower 1n the earlier study (Eckhert & Hurley 1979) and

the lnduction of zinc deficlency was linited to days I t'o 12 of

gestation tlhile plasna zinc Ievels 1¡ere s1¡nflar in both studies a

possÍb]e effecb of zfinc defieiency on enbryonic developnent prior to

the tine of prefnplantatÍon was not consÍdered in the previous study'

which night also contribute to dlfferences in the teratogenic outcones

between the two studies.

Defective branchial arch developnent is believed to result fron

defectlve crest cell migration (Johnston 19TÐ. This might occur through

mechanlsns such as destabíIísation of the extracellular matrlx which Ís

required for adequate crest ceLl migration (NichoIs 1981) or by

destabillsatlon of cytoplasuic projections between the cells which

nay interfere with crest cel} orientatÍon and nay prevent mlgration Ín

this nanner, or through a direct acblon on the crest cells along their

nfgratory pathway whlch was observed in the present study' Tt¡e additive

effect on eye defects whfch occurred as a result of eombining the two

teratogens suggests that zlnc deficlency and excess vltamin A can exert a

teralogenic effect on the sane organ probably by Ínterfering w 1th

adequate crest cell fornation and possibly through several different

nechanims.

In the thlrd group, an lnteracbion between zlnc deficiency and

trypervttamÍnosis A was evident tn relation to the total nunber of enbryos

affected, and also !n the frequency of abnornalltles ln the developing

braln when both nutrlents Ì¡ere adninistered concurrently, which was Ln

dlrect contrast to !he lndependent effecbs denonstrated by the

5-23

admÍnistrabion of the teratogens in the brain of term fetuses by Eckhert

and Hurley (1gTÐ. These workers reported that despite the high incÍdence

of fetal dysnorphologr in the brains of these anlmals no interaction vlas

apparent between the two treatnents. They also noted that apart fron the

brain, a distinction could be nade between the organs and paraneters

affected by the individual treatnent and concluded lhat 1L was unlikely

that the two treatnents acted through a conmon ¡nechanisn. Un1ike the

aforenentioned report, the present study was unable to draw a

distinction between the organ systens which were affecled by one or

other of the tenatogens, except Ín the case of branchlal arches. In

nost cases there appeared to be a contribution to the defect from both

teratogens which in sone structures led to a slrongly interactive

effect either of ameliorallon or potentiatlon. However nost of

these dÍfferences ane probably due to different paraneters being

examined in the two studies, and also that the aninaLs were exanÍned at

different times ln gestatfon. The use of enbryos at ühe tine of

neurulation ls highty relevant to the lnterpretation of the data' as

it reduced the conpltcating effects of conceptlonal loss and/or

conpensatory grolrth which nay have occurred lf fhe conceptuse s lJ ere

examined al the end of gestation This distfnction nay account at least

in part for the apparent Índependenü effect of each nutrient when

conblned, reported by Eckhert and Hurley (197Ð in rat fetuses at term.

Not only was the frequency of teratogenlcfty enhanced 1n the anterior

neural tube, but at the light nicroscope 1wel, enbryos exposed Lo zinc

deflciency and hyper"vltanlnosis A concurrently exhfblted more severe and

extensive necrosis within the cells of the neuroepfthellun of the

developfng neural üube than thaü obsen¡ed 1n enbryos exposed to the

indivfdual teratogens Thls supponts the other observatlons thaü

5-24

concurrent treatnent Ieads to a potentiative rather than an additive

effect on the developing neural tube. tlhereas the effect of zinc

deficÍency alone on the mesenchyme is not especially profound (Record et

at 1985; Handing et aI 1987), that of excess vitamin A in thÍs study and

previously (Morriss 1973) was shown to lead to a paucity of cephalic

nesoderm. When the two breatnents were conbined, a nore extensive and

severe necrosis aIso occurred in lhe nesenclynal ceII s than would be

expected if the two treatnents administered their effecbs independently'

suggesting an exacerbation of necrosis which was synergÍstic rather than

addÍtive. I,thile it appears that each teratogen exhibits a pref erence for

action at a specific site in lhe neural tube, concurrent treatnent

resulted in more extensive and more severe danage to both the nesenchyme

and the neuroepitheliun of the developing neural tuber leadfng to

conplete collapse of the anterior neural tube. This suggests that at the

cellular level- the two teratogens exert their effects synergistically

which accords wíth the observed developmental arpnalfes described in this

study. Also, sínce the ultrastructural data has shown that both nutrients

severely conpromise membranes ln the developÍng neural tube Ít seems

likely thab this night be the site of teratogenic action for these agenls'

eÍther through a conmon or indÍvidual mechanisns dependÍng on lhe

specificÍty of action of the two teratogens on nembrane.

!Jhether the nenbrane nedÍated effect 1s the pnincipal teratogenfc lesion

or fs secondary to sone other mechanisn preventlng neural closure requÍres

further ÍnvestigatÍon Howorer, bhe author feels that in view of lhe

sorerity of lhe ultrastructural damage, and fts appearance close bo the

time of nutrlent manlpulation, lhat at least at thÍs early period Ín

gesüatton, neunulation and organogenesis are sorerely compromised by a

direct menbnane effect, a suggestlon supported by Morriss (1973) who

5-25

obsen¡ed on day 8, two hours following vitamin A adninistrationr that

severe cellu1ar necrosis had occurred in the ectodernal and endodernaf

gern layers. Furthernore, Record et aI (1985a) have proposed from

ultrastructural observatfons in zinc-deficient enbryos that necrosls nay

be responsible for early, preplacental effects on the embryo and suggests

bhat Iater in developnent inhibitÍon of growth is responsible for some

of the more subtle effects. Scobt (1977) has also observed cell death

to be associated with teratogenic responses of a wide range of

compounds. Hence the ultrastructural observations point bo the

underlying nechanism for an interaction between zÍnc deficiency and

hypervilaninosis A to be prinarily related to a direct teratogenic

actlon on the nenbrane, and that nore subtle developnental anomalles

nay be due secondarily to lmpaired DNA synthesls, although Eckhert

and Hurley (19?9) found that reduced DNA synthesls on day 12 o1

gestatÍon appeared to be zlnc-related onlY, as hypervitaminosís A did

not affect the levels of DNA synthesÍsr and the conbined treatment did

not cause the Íncorporatlon of 3H-thymidine lnto DNA to fall beyond

levels assoclated w llh zinc deficiency alone. As their study has been the

only one lo neasr¡re DNA synthesls in enbryos exposed to these concurrent

nutrlent nanipulations, further confirnatory studÍes need to be performed.

In conclusíon, light and electron microscope studles suggest that ceLl

necrosis nay be the underlying defect associated wlth neural lube

dysnorphology 1n rat enbryos exposed to zlnc deficiency and

hypervftaminosts A. When adnlnistered alone, each treatnent appears to act

prinarlly on a separate germ layer of the developing neural tuber but when

admlnlstered concurrenüIy, bolh the neuroeplthellun and nesenchyrne are

affected wlbh an Íncreased severlty suggesting a posltive fnteractlon The

histological evidence 1s supported by gross developnental data, whlch upon

5-26

statisticalanalysisstronglypoÍntsboanon-lndependent,lnteractive

action of zínc deficlency and hypervitaninosls A on neural tube

teratogenesÍs. Ultrastructural studies have highlfghted the effect of bofh

nutrient manipulations on lhe inüegrÍly of membranes, which raises the

possibÍliüy that a significant conponent 1n the lndÍvfdual and comblned

teratogenfcity of zlnc deficÍency and hypervitanlnosis A nay lle in their

separate effects on cell nembrane stabillty'

5-27

5.5 BIBLTæRAI'HY.

Baker, F.I. & Nelder, JA. (1978). The GLIM system. Release 3. GeneralÍsedlinear interactive nodellings. Numerical Algorithms Group, Oxford.

Breuert, JC., DreostÍ, IE. & TulsÍ, RS. (1989). A teratogenic Ínberactionbetween dietary deflciencies of zfnc and follc acÍd in rats: anelectron microscope study. Nutr. Res. 9, 105-112.

Chvapil, M. (1976). Effect of zinc on cells and bÍome.mbranes. Med. CI1n.Nth. An. 60, 799-812.

DeMeyer, Í1. (1975). Medlan faciat nalformatlons and their implicationsfor brain nalfornations. BÍrth Defects: Original Article Serles. 11'155-181.

Dreosti, IE., McMichael, N., Gibson, GT. r Buckley' RA., Hartshorne, J. &

ColIey, DP. (1982). Nutr. Res. 2t 591-602.Duncan, JR. & HurIey, LS. (1978). An interactÍon between zinc and vitamin

A in pregnant and fetal rats. J Nutr. 108, 1431-1438.Eckhert, CD. & Hurley, LS. (1977). Reduced DNA synthesis in zj.ncdeficiency; regional differences Ín eubryonlc rats. J. Nutr. 107, 855-861 .

Ecklrert, CD. & Hurley, LS. (1979). Influence of various levels oftrypervitaninosÍs A and zinc deficiency on teratogenesís and DNA

synthesls in the rat. Teratologr 19, 2T 9-284.Ghadially, FN. (1988) Ultrastrucbural Pathology of the Cell and Mat,rix.Butterworths. Vol. 2' pp589-765.

Harding, 4,J., Dreosti, IE. & Tr:Isi, RS. ( 1987) Teratogenlc effects ofvitanin E and zÍnc deficiency ln the 11-day rat embryo. Nutr. Res. Int.36, 473-481 .

HardÍng, A.J. , Dreosti, IE. & Tr:J.sf , nS. ( 1988) . ZInc def iclency in the 11

day rat embryo: a scanning and transnission electron rnicroscopestudy. Lif e Sci. 42, 889-896.

Hurley, LS. , Gowan, J. & Swenerton, H. ( 1971). Teratogenic effects ofshort-term and transÍtory zinc deficiency in rats. Teratology 4, 199-204.

Johnston, MC. (1975) The neural crest in abnornalíties of the face andbraln. Birth Defects Origfnal Article SerÍes 11, 1-18.

Joschko, l'{4. , Dreosti, IE. & Tr:1si, RS. ( 1989) . Zinc/ vitamin A

interactions and teratogenesis Ín rats: a light and electron microscopestudy. Nutr. Res. 9, 205-216.

Kochhar, DM. (1968). Studies of vltanin A-Índuced teratogenesÍs: eff,ectson enbryonic rnesenctryne and epitheliun, and on ÍncorporatÍon of 3¡lthymidine. Teratolory 1, 299 -310.

Morri.ss, G.M. (1972). lúcrphogenesis of the malformations induced in ratembryos by naternal hypervitanÍnosis A. J. Anat. 113, 241-250.

l"lorriss, GM. ( 1973) The r¡ltrastructural effects of excess naternalvitanfn A in pregnant and fetal rats. J. Nutr. 108'1431-1438.

Nanda, R. (1971). Trttlated thynidÍne labelllng of the palatal processesof rat eobryos with cleft palate lnduced by hypervlüa.ninosis A. Archs.Oral Blol. 16, 435-444.

Nichols, DH. (1981) NeuraI crest fornaülon in the head of the nouseenbryos as observed using a new histological technÍque. J. Enbryol. Exp.Morphol. 64, 105-120.

Peters, AJ., Keen, CL., Lonnerdal, B. & HurIey, LS. (1986). ZÍnc -vitanin A interacfion in pregnant and fetal rats: supplaental vÍtanln A

does not prevent ztnc-deficiency-induced teratogenesis. J. Nuür. f16,1765-1771 .

Record, II. , Dreosti, IE., l,lanuel, St. , Buckley, RA. & Tulsf r RS.

5-28

(1985b). Teratological lnfluence of the feeding cycle in zfnc deficientrats. fn: Trace Elernent Metabotim in l,fan and Anina1s. Mills, et al(eds). Cornmon^¡ealth Agricultural Bureau, Slough. ! ' 210-213.

Record, fR., Dreosti, IE., Tulsi, RS. & Manuel, SJ. ( 1986). Maternalnetabolim and teratogenesis in zlnc-deficlent nabs. Terabology 33, 311-317 .

Record, IX. , Record, SJ. , Dreostir IE. & Rohan, IE. ( 1985c). Dfetary zlncintake of pr+'nenopausal wonen. Hunan Nutr. Appl. Nutr. lpA' 363-369.

Record, n. , Tulsi, RS., Dreosti, IE. & Fraser, FJ. ( 1985a). CellularnecrosÍs in zinc deficient rat embryos. Teratology 32, 397-405.

Rogers, JM., Keen, CL. & Hurtey, LS. (1985) Zínc deficiency in pregnantLong-EVans hooded rats: teratogenicfty and tissue lrace elenents.Teratolory 31, 89-100.

Scott, ILJ. (19TT). Cel1 death and reduced proliferative rate. In:Handbook of Teratology. VoI 2. flÍIson, JG. & Fraser, FD. (eds). P1enunkess, New York. pp 81-98.

Snith, JC. Jr., McDaniel, ¡8., Fan, FF. & HaIstead, JA. (1973). Zinc:a trace element essenti.al in vitanÍn A metabolisn. Science 181, 954-955.

Smith, JE., Brøvn, Ð., & Smith, JC. Jr. ( 1974) The effect of zincdeficiency on the netabollm of retfnol-binding protein in the rat. J.Lab. CIin. Med. 84, 692-697.

Stange, L., Carlstrcm, K. & Eriksson, M. ( 1978). tlypervitanirpsis A inearly hr.¡man pregnancy and nalforoatlons of the central nervous system.Acta Obstet. Gynecol. Scand. 57,289-291.

S¿enerton, H., Shrader, nE. & HurIey, LS. (1969). Zinc defÍcient øbryos:reduced t,hynidine incorporatÍon. Science 166, 1014-1015.

TheodosÍs, DT. & Fraser, FC. ( 1 978). Early changes in the mouseneuroepithelir:n preceding exencephaly induced by Wpervitaminosis A.Teratology 18, 219-232.

llolf, G (1984). Multiple Functions of vltanin A. Physiol. Rev. 64' 873-937 .

6-1

CHAPTER 6

THE EFFECTS OF ETHANOL IN VITRO ON THE DEVH'OPING NERVOUS SYSTEM'

6.1 INTRODUCTION

6.1 .1 Ethanol Metabolisn.

The netaboLisn of ethanol wilI only briefly be outlined as its nany

cornponents and the influences upon it are beyond the scope of this

Introduction For a more detailed account the reader is referred to Loomis

(1986), and to the numerous publications of Lieber, includÍng Lieber

(1g85). Ethanol is a relatively smalI' uncharged nolecule completeJ-y

miscible with waber, while slightly fat soluble. It is mainly absorbed

across the gastrÍc mucosa by diffusion across a concentration gradientt

however it can also be absorbed through the mucous nembrane of the nouth

into lhe blood. The portion of ingested alcohol- that enters the blood via

absorption fron the stonach depends on dilution factors such as the

quantity of food and water ingested as weII as the emptying time of the

stomach when alcohol is present. That part of the alcohol whÍch is not

absorbed fron the stomach is readily absorbed froro the upper srnall

intestine. Frorn the tine that lhe ethanol is transported in the blood to

the liver and kidneys, netabolic and excretory mechanisms begin, resulting

in a continuously shifbing state of equilibriun between the alcohol in the

blood, the tissues and the gastno-intestinal contents' The rate of

ethanol oxidalion has been shown to involve a nunber of factors including

enzyne systems, nutritional status, genetic factors (Vesel1 et aI 1971)'

ingestion of other drugs (Lieber & DeCarli 1970; Sotaniemi et aI 1972),

and liver disease (Da0ruz eb aI 1975i Dow et aL 197ù'

Most of the ingested alcohoL is metabolised through the nechanj'sns

6-z

operating within the liver hepatocyte, which contains 3 pathways for

ethanol- metabolism, each Located in a different subcellular compartrnent

(Lieber 1985), while about 5f is elininated unchanged Ín bodily fluids

such as sweat, breath and urine (Loomis 1986). In alcohol-naive animals

or humans, of the alcohol- metabolised by the Ii.ver, up t,o 98f is

metabolised nainty through the alcohol dehydrogenase (ADH) pathway of the

cytosol, an enzyme that catalyzes the conversion of ethanol to

acetaldehyde (Lieber 1985). This init,ial step is followed by several more

which utilize other enzynes (see Loomis 1986 for details) and which

ultimately leads to the fornation of C0, and Hr0. In the process of

converting each grarn of alcohol to COa and HrO, the body receives 7.1

Kca1,. Lhereby naking alcohol an effective source of energy.

A port,ion of al-cohol is also believed to be oxidised by a second type of

systen that is independent of the ADH catalysed systen (Lieber & DeCarli

1968). The microsonal ethanol-oxidising system (MEOS) located in the

endoplasnic reticulum also converts alcohol to acetaldehyde. Urùike the

ADH system, it is a membrane bound system that utilises a cytochrome

protein to oxidise ethanol, and has been shown Ín animals Lo increase in

activity folJ-owing chronic ingestion of ethanol (Lieber & DeCanli 1970;

Estes & Heinenann 1986). This increase in activiLy appears to be

associated with an increase in various constituents of the snooth

endoplasmic reticulum assocÍated with drug metabolisn including

phospholipÍds, cytochrom" P45O reductase and cytochrom" P45O in ani¡nals

(JoIy et aI 1972), and in alcoholics on after chronic alcohoL Íntake in

hunans (Mezey & Tobon 1971). The third pat,hway of ethanol ¡netabolism is

via the enzyme catalase which occurs primarily in the peroxisones

and nifochondria, but this pathway is not believed by sone to

play a signÍficant role in ethanol metabollsn (Feytmans & Leighton 1973).

6_3

rn the case of chronic alcohor consumpbion, the MEOS and possibry the ADH

andcata}asesystemsareinduced,withanincreasedcapacitytocopewith

a total alcohol load in patients with alcohol dependence (Lieber 1985)' In

the fetus, levels of ADH are low and alcohol elimination is consequently

about50fslowerthaninadults(rdanpaarrHeikkilaetallg72).Therefore'

alcohol is eliminated prinarily by passive dÍffusion back through the

placenta and is oxidised by the mother'

6.1.2 Hr:nan studies'

6.1.2a Introduction.

AlthoughtheassociationofalcoholwÍthdanagetothefetuswaSwell

known fo the Anclent Ïlorrd, with the early Greeks prohibÍting the use of

alcoholbynewlymarriedcouplestopreventtheconceptionofchildren

with defects (llarner & Rosell 1975)' it was not until Lemoine el aI (1968)

Ín France, and Jones and Smith (19?3) in England reported a distinct

pattern of abnornalifÍes associated with infants born of alcoholic

nothers, that it became internationally recognised as the Fetal Alcohol

syndrome (FAS). A patbern of anomalies comprised of prenatal and

postnatal growth rebardation, facial dysmorphology, ongan pathogenesis

and cNS dysfunction currently constitute crj'teria for a diagnosis of the

FAS (CIarren & snith 1978; Streissguth et aÌ 1980b; Clarren 1981)' l'lhile

considerable atlention has now been focused on the effects of rnaternal

intoxication, identification of FAS at birbh is still far from adequate

due to dlfficulties with recognition of some physical anomalies and nental

retardation whlch nay not become apparent until later in childhood'

Furthermore, available human studies designed to deternine the incidence

of FAS have not been consistent however, as many are fraught with

difficulties particularly with regard to assessing the quantity of

alcohol actually consu¡ned by the mobher durlng pregnancy' slnce nost

6-4

underestimate their intake (Lipson 1988)' AIso of concern Ís the lack of

specific criteria for distinguishing the syndrome' While solne

dysnorphologists have described a characteristic facÍal appearance' rnost

concede that they are fþn-specific and can be imitated by a number of

other syndromes (Lipson 1988). In addition, the presence of sorne or one of

the features which normally characterises the FAS in the offspring of

naternal alcohol abusers, or the occurrence of spontaneous abortion

have led to a further group of infants being classified as denonstrating

fetal alcohoL effects (FAE) (Jones & Smith 1g73i Rosett & Weiner 1984)'

A recent estimabe of fhe incidence of FAS is 3-5 per 1000 líve births

among women who consuned arcohor during pregnancy, and up to 90 per 1000

arnong alcoholics, whereas the incidence of FAE is 1-2 per 1000 live

bÍrths and 25 per 1000 for the lwo Sroups respectÍve1y (Abel 1984)'

6.1.2b AIcoho1 and pregnancy wastage'

Harlap & ShÍono (1980) perforned a prospective study involving over

30,000 women and reported that regutar ingestion of 1 or 2 drinks daily

during the first brimester had a significantly increased risk of rnid-

trinester abortion (relative risk 1.98) which increased to a relative risk

of 3.8 in women who exceeded 3 drinks da1Iy. Retrospective studies by

Hanson et aI (1978) and Kline et aI (1980) have also reported an increased

risk for spontaneous abortion prior to the 28th week of gestation i'n wonen

who were moderate or heavy drinkers, and this increased risk could not be

explained by age, parÍty, race' marital status' smoking' or the number of

previous spontaneous or induced aborllons sÍnce control for these

confounding variables dÍd not alter the associatÍon between drinking

during pregnancy and spontaneous abortion Hence KIine et al (1980)

estinated bhat nore tjhan 25l, of pregnant wonen drinkÍng twice a week or

nore are likely to abort, conpared with abouL 141 among women who drink

6-5

less often, and that considering beverages such as wine, beer, and spiribs

they suggested that the minimum harmful dosage l{as one ounce of absoLute

alcohol daity. In another study (Sokol et aI 1980)' an association was

also reported between nalcohol abusen and sponLaneous abortion' but the

Ievel of alcohol which these workers considered as abusive was uncLear'

Late fetar deaths in some instances appears to be pant of a biological

continuum represented in early gestation by spontaneous abortion An

association between maternal alcohol intake and stillbirth rates has been

reported in a prospective study by KaminskÍ et aI (1978). For light

drinkers, the stillbirth rate was 9.9/1000 births which Íncreased to

25.5/1OOO for heavy dninkers while risk factors such as smoking when

combined with heavy alcohol use resulted in even higher stillbirth rates

of 50 .5/ 1000.

6.1.2c Growth retardation and ethanol ingestion'

The average birthweight of children with FAS is 2000g (Abel 1984)

compared with a median birthweight in the United Stabes of over 3000S'

Although AbeI and sokol (19s6) have stated that reduced birthweight 1s the

nost reliable observed effect of prenatal alcohol exposure in humans and

animals, the literature revealed that the outcone of studÍes in hunans has

nob been consÍstent, pnobably due to a number of factors which include the

range of confounding variables which must be considered in epldemiological

studÍes (Mau & Netter 1974), the quantity of alcohol- consumed (Little

1g77i Kaninski et al 1978)' as vrell as the type of beverage (Kuzma & Sokol

1982) and the trimester when the alcohol was consuned (Tennes & Blackard

1978; Little 1977).

l,lhereas Oulebte et aI (1977) did not observe a reductlon in birth weight

in offspring of nothers who drank moderately throughout pregnancyt Mau and

6-6

Nett,er(1974)notedthatmoderatedrinkingledtoagrowthretardationin

newborns, however variabres such as age, parity and smoking $Jere not

conlrolled for in their studY'

In a recent prospective study (Day et al 1989) Iow birthweight and

decreased head circunference were found to be significantly correlated

with exposure to alcohol during the first two months of the first

trimesber. However, Abel and sokol (1986) concruded thaf exposure durin8

the Iatter part of preSnancy appeared to have a Sreater Ímpact on

birthweighl than exposure during early pre8nancyr âs wonen who stopped

drÍnking in rnid-pregnancy had children with lower birthweights and

lengths than those who stopped drinking before 13 weeks (Rosett et

aI 1978; Olegard et a] 1984). In contrast however, Tennes and Blackard

(1980) failed to demonstrate any relationship between drinking patlerns

in each trimester and birthweight'

The quantity of alcohol is also consj.dered to be of significance in

determining whether the birthweight in offspring of drinkÍng mothers 1s

affected. In a large study of over 9000 births, Kaminski et aI (1978)

found no relatÍonship between birthweight and daily alcohol consunption

below45Elnonwasthereasignificantreduclioninbírthweightandhead

circumference in a study by Davis el al (1982) invorvÍng 973 wonen'

although a trend r.¡as observed for these two paraneters in bhe 28 wonen who

drank over 20g of alcohol daily. The data however were not corrected for

confounding variables. In contrast, ÏJright et al (1983) reported an

association between birthweight, infant Iength, head circumference and

prenature delivery in a prospective study which involved 1122 preCnanclest

whereby an excess of 10g of alcohol daily before conception and early in

pregnancyapproxinatelydoubledtherlskofatightinfant.

6_7

Alcohotic beverages such as beer have been reported (Kuzma & sokol 1982)

toleadtosignificantlymoreweightreducedinfantscomparedtothose

borntowomenwhodrankotheralcoholicbeverages,especiallyinfrequent

drinkers.OtherepidemiologicalstudiesÍncludingKaminskietal(1978)

have arso implicated beer as a faclor affecting birthweight. This effect

may however be due to obher factors such as differences in socio-economic

status,orthat,thesenothersdrankmoreheavilythanwomenconsumÍng

othertypesofalcoholicbeverages.AddibionalstudiesarerequiredaS

differences in birthweight have not been related to different alcoholic

beverages in aninals (Abel et al 1981)'

postnatal growth was also reduced in children with FAS in sorne studies'

but not in obhers. smith (1980) has described postnatal catch up growth as

infrequent, although Nitowsky (1980) reported that in a substantial group

of children (25Ð acceleration of growth can be seen during childhood'

However, Ín a study by streissguth et aI (1985) when children identified

with FAS at birth were exanined 10 years later, they continued to exhibit

growthretardationintermsofr.¡eight,heightandheadcircumference.

similar observations were also recently reported in 3 year olds exposed

prenabally to alcohol (¡ay et aI 1991). Postnatal catch-up growth has

been reported to be related to the anount of prenatal alcohol exposure

(4be11984),ratherthantoimpairedfoodutilisation(AbeI1981)'

6.1.2d Ethanol and gross abnornalities'

The typical facial nalfornations have provided the means by which

patients with FAS can be nosb easily categorised and bherefore grouped

intoaratherspecificentity.ThefacialcharacteristicsofFASchildren

have been described by clarren and smith (1978), and include facial bone

hypoplasia with maxillary and nandibular hypoplasia, short palpebral

6-8

fissures,thinupperlipandhypoplasiaoragenesisofthefittrum.The

shortened palpebral fissures are believed to be a consequence of

microphthalmia. short nose with depressed bridge, epicanthar folds'

anteverted nares, cleft tip or palate and posteríor rotation of the ears

are additional but less common facíal defects. As welL as facial

anomalies, dysmorpholory associated with the FAS in humans cover a wíde

range including cNS defects such as microcephaly (Jones et al 1973;

Clarren & Smith 1978; Samson 1986), and skeletal, urinogenital' cutaneous

and cardiac abnormalities of which ventricular septal defects occurred in

up to SOf, of FAS children (Clarren & Smith 1978)'

6.1.2e Ethanol and CNS dysfunction'

Although cNS dysfunction represents one of the nost common features of

FAS, evidence that CNS damage can occur even j'n the absence of other febal

alcohol effects, suggests that the developing brain may be especially

susceptible to damage from alcohol (streissSuth 1986)' The literature

dealing with this topic is extensÍve and the reader is referred to !Jesb

(1986) and !fest and Goodlet (1990) for comprehensive reviews' Numerous

studies have demonstrated thal IQ scores were often well below average in

children born to chronically alcohotic women (Jones et aI 1 974;

Streissguth et aI 1976; Streissguth et aI 197ü. In a sample of 20

patÍents between 9 months and 21 yeans' the average IQ was 65, with 60f of

the patients with an IQ more than 2 standard deviations below the nean

nor¡nal val-ue (streissguth 19?6). Further, clarren & snith (1978) reporled

t,hat when standardised Íntelligence exaninatÍons were administeredr more

than851,ofchildrenborntoalcoholicmotherswereofsubnornal

intelligence. In anobher more recent study (conry 1990) children dÍagnosed

with FAS were found to perform significantly worse on a battery of

Ínteltectual and neurophysiological bests than nornal controls' The degree

6-9

of inpairment in neasured intelligence has been found to be associated

with severitY of the

(Clarren & Smith 1978;

physical anomal-ies associated with FAS and FAE

Abel 1984).

other manifestations of cNS dysfunction include hyperactivity (shaywitz

et aI 1980, Streissguth et aL 19?8), seizures in newborns, irritability'

tremuLousness and poor suck (Havliceck & Childaeva 1977; Clarren & Snilh

1978). l,lhile attention deficits (ShaywiLz eL aI 1980) and poor habi|uation

(Streissguth et aI 1983) were also reported in very young children' mental

retardation and sensory deficits resulting fron prenatal alcohol exposure

were often difficult to detect for several years after birth and fhe

majorÍty of these problems did not become evident untÍI the chitd entered

school (Pearson & Peckham 1g77 Streissguth et aI 1978). At 7 years of

age nany of these children were found to be educallonal-Iy subnornal with

sensory defects which were still present at 11 years, and were greatesL at

16 years (Pearson & Peckham 1977).

The neuropathological alterations ident,ified in children diagnosed with

FAS revealed severe CNS disorganisation which derived fron alterations in

the firsl trimester, the period of greatest neural migrabÍon, and included

cortical disorganisation, agenesÍs of corpus callosum and anterior

commissure, and !¡ere presumably caused by focal areas of cellular

deslruction in the germinal matrices that gÍve rise to these tissues

(ct-arren et a] 1978; Pfeiffer et al 1g7gi clarren 1981; ÏIÍsniewski et aI

1983). In addition, sone of these brains (clarren et at 19?8) had areas of

leptomeningeal neuroglÍal heterotopias-(vÍable ectopic clusters) which

suggested that alcohol nay not only be neurally toxic, but nay also

specifically interrupt the migration of cells fron the germinal rnatrix to

their proper destination (CIarren 1986). Gross neural dysnonphologies

6-10

obser,vedinthesebraj.nsineludednicrocephaty,hydrocephalus,andless

frequentlyanencephalyandmeningomyelocoele(Clarren&Smíth197B),and

ithasbeensuggestedthatalcoholmayinterferewithneuraltubeclosure

(CIarren 1 979).

Clarren (1986) has stated that a simple relationship between the

díagnosisofFAsorFAE,theseverityofbrainrralformatÍonsandthe

consunption pattern of the nother does noL exist. However, it renains

a strong clinical impression that children wÍth aII the phenolypic and

growth deficient features of FAS are likety to be significantJ'y inpaired

in behaviour and intellectual function' whereas children

few clinicaL features of FAS have far nore mild central

dysfunction, if anY at all'

who disPI aY

nervous sYstem

6.1.3 Animal studies'

6.1.3a Acube and chronic effects of ethanol on developnent'

since most alcoholic women are chronic drinkers il has been assumed that

longdurationofexposureboalcoholisaprerequisitetoFAS.Hencemany

experi.nents designed to reproduce FAS in aninals took this into account

and concentrated nostly on chronic alcohoL treatment' The major features

of FAS including growth relardation, craniofacial defects and brain

abnormalities v{ere observed in a number of species (TabIe 6'1)' It is

arso necessary however to consider that drinking patterns anong wonen

vary'andasizeablepercentageofthenareactuallynbingedrinkersn'

a condition which has also been reported to lead to FAS in children of

these wonen (Madden & Jones 1g72i Clarren et aI 19?8; Hermann et al

1g80). These observations have led a nunber of workers bo exanlne the

effects of acute doses of alcohol durÍng the critical early periods of

developrnent in aninals which often resulted in abnornallties of offspring

6-11

Table6.lTeratogenesisofChronicalcoholingeslioninanimalsinvivo.

Species : Major Outcomes : Aulhors

;;;;";-;;;-; ;;;-;;;il ";;;;;-p*;;;;;;;;;-;;;-;- ;;";;-l;il;;;coordination; abnornal flattening of Telford 1957

gyri & shallowness of fissures'

Rats : Low birtrn^reight; decreased litter : Tse & Lee 1975

size; neural tube defects(NTD);abnornal skin.

Mice : CNS, cardiac and eye defects' : Chernoff 1977

il- - - - - - ;;; "; ;;- i ;;;;; ";; ";

- ;;- ;il;; ;;; ";;;l ;il-

-

Rats : Increased resorptions; increased fetal: Hendersen et aInortality; decreased body & organ 1979

weights.

Míce:Fetalwastage;CNS,skeletal,:Randall&Taylorand cardiovascular anomalies' 1979

Rats : Low body and brain weights; reduced : Ïloodson & Ritcheybraín RNA, DNA levels and brain 1979

cell numbers.

Beagles : Reduced litbersize; decreased pup : EIIis & Pick 1980

w eight.

Pigs Potter et al 1980

Pigtailed : Growth retardation; facial anomalies; : clarren et aI 1988

¡nacaques CNS dYsfunction.( Macaca

:::::::ïl--------whichwere also characteristic of the human FAS (Tab1e6.2)' The type of

anomalies induced in the offspring have been observed to depend on the

gestational day of teratogen exposure (sulik et aI 1984). These animal

nodels are especially useful in bhe eLucidation of stages susceptible

to the deleterious effects of alcohol and their pathogenÍc mechanisns.

6.1.3b. Ethanol and cultured embryos.

Sone of the features typical of alcohol-induced teratogenicity observed

in offsprÍng in utero have also been reported in cultured embryos (Brown

: Brain dysmorphology often withoutexternal signs of FAS.

6-12

TabIe 6.2 Teratogenesis of acute alcohol ingestíon in animals j¡ vivo.

SpecÍes : Major teratogenic outcomes : Reference

Chickseggs

Growth retardation; CNS & skeletalabnornalities leading to increasedchick norbaliLy. CeIl necrosis inneural lube and chaotÍc neuroblastformation in pnimitive spinal cord.

Sandor 1 96 8; Sandor &

Etias 1968.

Rats : CNS abnormalilies including : Nakanura & Suzukí 1967

microscoPic degeneration ofbrain tissues.

Rats & : Increase in fetal mortatity; : Sandor & Anels 1971

mice caloboma of iris; ectodactYlY;CNS abnornalibies.

Mice Increased death and malformationsincluding f etal neurologicala bnormal i tie s.

: Chernoff 1977

Rats Felal growth retardation;skeletal retardation

Kronick 1976, KennedY& Persaud 1979.

Mice Exencephaly & other head defects;brain abnotmalÍties ; craniofacialanomalies & Iimb def ects.

: llebster et al 1 980, 1 983;Padnanabhan et aI 1984.

Mice Increased resorptions; craniofacial : Sulik et aI 1981.abnormalities; poor heart development;abnormalities of neuroecloderm.

Mice Open neural tube; degenerating cells: Bannigan & Burke 1982;& necrotic fragments. Bannigan & cottell 1 984.

Mice Exencephaly ; reduced brain weight. SuLÍk et al 1983a' 1984'1 988.

l.'.ice Retardation of mesodermal celL layer: Nakatsuji & Johnson 1984.

Rats Fetal nortality; growth retardation; :

cranioschisis with exencePhalY ;

naxillary hypoplasia ; exophthalnia ;

dÍgital anomalÍes.

Padnanabhan & Muawad

1985.

Rats Reduced febal viability; decreasedbraÍn weight.

: Bonthius & lfest 1989

Rats Thinning and malfor¡nations of thefetal cerebraL cortex

Kotkoskie et at 1 990

et aL 1g7 g, tlynter et al t 983; Beck et al 1984;

vitro culture technique is partÍcularly useful

Priscot,t 198S). Thej¡

in determining whether

6_13

alcohol alone has teratogenic properties, and Íf so whether it exerts its

teratogenic effect through maternally mediated mechanisms, or by an effect

directly on the enbryo itself.

llhen Brown eb aI (1979) cultured rat embryos in ethanol from day 9.5 to

11.5 (the period approximately equivalent to days 20 to 30 of human

embryonic development), growth and developnent were retarded by 5 Lo 7

hours. Developnental reLardation was consistent with the najor features of

FAS including microcephaly and the aut,hors suggested that these structural

deficiencies may be the result of reduced cellular proJ.iferation in bhe

organogenesis phase due to a direct action of ethanol. An effect of

ethanol on embryonic developnent independent of maternal netabolisn was

inplied by the results reported by l{ynber et al (1983) who cultured rat

enbryos in ethanol for specific periods of organogenesis. Marked growth

retardation, panticularly of the head region was observed, and depending

on the dose and in which 6-hour period of organogenesis the embryos were

exposed to ethanol, open neural tubes were also observed in sone anirnals.

Beck et a1 (1984) further denonstrated a direct, debrimenbal effect of a

brief exposure of al-cohol when 9.5 day rat enbryos were cultured for the

finst four hours Ín the posb-drÍnk serun of scotch consuners. The results

demonstrated the growth retarding and teratogenic effects of alcohol

ingestion including non-closure of the anterior neural tube and

mlcrocephaly. Open neural tubes and a range of dysnorphologies which

Íncluded dorsally concave enbryos were observed when I'lÍsbar rats lJere

cultured in ethanol from day 10 to day 12 of gestation (Priscott 1985).

Since embryos at this period of gestation have not yet developed alcohol

dehydrogenase, the aubhor suggested that the abnormalities observed

nay be a direct action of ethanol on the enbryo.

6-14

6.1.3c. Alcohol and CNS dysfunction'

Structural abnormalities of the central nervous system which have been

observed both in vivo and in embryo culture have been associated with

histological changes in sorne studies. Exposure of chick embryos to ethanol

(sandor 1 968) led to cell necrosis in the neuraL tube and chaotic

neuroblast formation in the spinal cord. sulik et aI (1981) have also

reported histological abnormal-ities in the neuroectodern of the developing

brain in mice shortly after exposure to ethanol on day J of gestation when

nice undergo gastrulation or mesodernal fornation which is responsible

for induction and naintenance of the neuroepithelium. Furthermore the

obse¡r¡ed decrease in development of the neural ptate and its derivatives

nay account for the craniofacial malformations observed Ín their study. In

a later study, Sulik eb aI (198S) observed a depositÍon of NiIe blue stain

in regions of neuroepithelium in embryos which were affected by maternal

ebhanol åd'ninistration

BannÍgan & Burke (1982) also neported cel-luLar changes in mice up to 50

hours following ethanol injection on day 9 of gestation, includÍng

degenerating cells and necrotic fragments, whÍch accompanied open defects

of the cranial neural tube. Nakatsuji & Johnson (1984) also reponted

retardation of mesenchynal cell migration in the rnesodermal cell layer

in ¡oouse enbryos following ethanol injections on days 6.5 to 7 of

gestatioru These workers suggested that t,he inhibitfon of morphogenetic

novements during gastrulation may be the primary effect of ethanol in

causing major craniofacial malfornations of FAS.

CNS dysfunction, now routinely descrlbed as a characteristic conponent of

bhe FAS Ín humans remains the most devastating consequence Ín fetuses that

survÍve heavy naternal alcohol consunption (Strelssguth et al 1980a).

6-15

Learning defects and other behavioural impairnents (see Streissguth 1986

for review) are considered to be the nost obvious form of aberrant

brain dysfunction (Abel 1 979;Streissguth et all 980a). Afcohol-induced

effects on learning in aninals have been noted by a number of other

investigators (Vincent 1958; Bond & DiGiusto 1977 i Martin et al 1977:'

Riley et aI 1979) and included inferior learning ability' learning

avoidance behaviour, as well as difficulty with response inhibition and

reversal learning, which Streissguth et aI ( 1 97 8) suggested w ere

behaviours analagous to the inhibited behaviours observed in some FAS

chitdren Hyperacbivity in newborn rats, determined by a wide varieby of

activity neasurements, has also been reporled by several groups as a

consequence of maternal ethanol adninistration (Bond & DiGiusto 1977;

Martin et aI 1978). More recently, long tern behavioural effects

persisting into adulthood following prenatal alcohol exposure have been

demonstratdd in a number of species, and are reviewed extensively by Riley

(1990).

Ethanol- induced CNS dysfunction has also been nanifested as

neurochemÍcal, hormonal and neural anonalies, Ieading to a profound

effect on offspring functÍon even in the absence of external physical

abnornalities (Papara-NÍcholson & Telford 1957i Barnes & llalker 1981;

West et al 1981; Abel et al 1983; West & Hodges - Savola 1983). This is

denonstrated in guinea pigs whose neural development is complete at birbh'

where shallow fissures, abnormalÌy flat ryri and cel-lular lesions in fhe

cortex and basal ganglia were found 1n the offspring of nothers given oral

doses of ethanol, in spfte of the absence of external sbructural ano¡nalies

(Papara - Nichotson & Telford 1957). Delayed myelination of the fetal

brain was also observeO (Rosman & Malone 1976i Jacobsen et al 1979) as

well as structural alterations of specific regions of the brain such as

6-16

neuronal ceII loss (Barnes & Walker 1981) and alteration of the mossy

fibres in the hippocanpus and cerebellun (Bauer-MoffetL & AItman 1977 i

together with alterations in circuitry (llest et

1985) and developmental delays at synapses (VoIkPhilIiPs

aI 1981;

& Cragg 1982),

!lest & Hanre

1984) and in dendritic spÍnes (Davies & Smith 1981; Hammer & Scheibel

1g81) of the pyranidal neurons of the somatic sensori-notor cortex

(Hamner & sheibel 1981). Other studies displayed decreased proteÍn

synthesis (Rawat 1g7Ð and reduced brain serotonin levels (Krsiak eb aI

1977)intheoffspringofa]-coholfedrats.Thesereducedserotonin

levels in brains of the offspring may be particularly relevant' as Brase

and Loh (1975) have Linked reduced levels with hyperactivity 1n hunans'

6.1 .3d Teratogenesis of acetaldehyde'

Although alcohol is certainly the necessary core to lhe productlon of FAS

and the brain anomalies described in humans, it is not' crear if ethanor

itself, or netabolites Iike acetaldehyde and acebate or a conbination are

t,eratogenic Ín hunans. Aninal studies have been performed in an effort to

clarify thÍs uncertainty but with linÍted success' A nunber of j¡ vivo and

j¡ vitro studies (Table 6.3) investigated the effects of acetaldehyde on

developnenbandhavesofararguedbothforandagainstarolefor

acetaldehyde in the genesis of FAS'

6.1.4 Mechanisns of alcohol teratogenesis'

lJhereas the nechanj.sns by which ethanot exerts its widespread teratogenic

effects are not crearry understood, and have not yet been furly

deternined, tt is likely that a nunber of mechanisns are involved' which

are far too extensive to be dealt with fully 1n this report' sone of these

will be briefJ.y considered in thts section, and the reader is referred to

a nunber of cornprehensive reviews (Abel 1984; West et aI 1986; Webster

6_17

TabIe 6.3 Teratogenesis of Acetaldehyde in vivo and in vitro in animals.

Species : Major teratogenic outcomes Reference

CFT. P

mice.T-npaired fetal growth and protein :

content. I-mpaired developnent with NTD

a frequenl anonalY. AcconPanyinghisLological and ultrastructural defects.

0r shea & Kaufman1979, 1981.

Hoodedwistarrats.

Reduced body weight and reduced DNA

synthesis especiallY in fetal CNS;

Increased febal mortalitY.

Dreosti et aI 1 981 .

catn].ce

Significant incnease in NTD andabnormal CNS develoPnent; Reducedsomj.te count; Reduced DNA synthesis.

Thonpson & FoIb 1982.(:Ln vitro)

Sprague-DawIeyrat s.

Decreased growlh and developmentalretardation including total proteincontent and head length. ReducedDNA content above 75 uM.

Canpbell & Fantel1983. (¡a vitno)

Al binowistarnat s

No significant effect on total protein': PriscottDNA, somites, viabÍlity or morphologicalaberrations at 100 & 260 uM. fubryotoxicat 800 uM causing rapid death & necrosis.

1 985.

Sprague-Dawleyrals.

Decreased embryonic head length. No

significant effect on morphologicalscore and crown rump length.

: AIi & Persaud 1988.

1989; RandaLl et aI 1990; Schenker et al 1990) for additional infornation

One of the mechanisms through which ethanol nay exert its teratogenic

effect is inpaired placental transport of nutrients. ïlhile essential

amino acid transport was reduced in feLuses ln some studies (Henderson et

aL 198?; Fisher & Karl 1988) following maternal ethanol exposure, no

reduclion in nutrient transfer was reported by Jones et aI (1981). J¡

vitro sludies (fJynter et al 1983) have also denonstrated reduced growth

and dysmorpholory in embryos assocÍated with inpaired yoJ.k sac circulation

but not exclusively, hence it is unllkely that a nutrÍent defíciency

solely underlÍes aLcohol teratogenesÍs, and thus a direct effecb of

alcohol on enbryonic tissue, possibly through mechanlsms such as ionic

changes within enbraonic menbranes (Michaelis et al 1978) should also be

6-18

considered.

Fetalhypoxiahasalsobeenreportedtobeaconsequenceofethano].

ingeslion because of the excessive demands fon oxygen made by the liver

during the metabotism of ethanol (Ugarte & Val-erøuela 1971) Ieaving other

tissues deprived of necessalxr oxygen Ethanol also leads to a release of

catecholamines fron the adrenals (Pernan 1958) causing vasoconstrictÍon

(Abel 1g82) which exacerbates the oxygen deficiency' A further

contribution to bhe hypoxic effect vJas reported Ín pregnant monkeys given

Iarge doses of ethanol which led to transient collapse of the unbilical

vasculature (Mukheriee & Hodgen 1982). This effect nay also underlie

the abnornally high incidence of spontaneous abortÍon reported during

first and second trimesters in binge drinkers (Yang et al 1986)' Ïfhite it

is likety that hypoxia causes deficits related to the cNS (MÍchaelis

lggo), it is stilI uncertain whether aIl the defects associated with FAS

and FAE can be attrÍbuted to this mechanism'

Maternal ethanol ingesbion also led to increased prostaglandin revels in

the fetus foIIowÍng the release from varÍous tissues (Anton et al 1983;

CoIIieretaIlgs5)'aswellasinterferencewithprincipalcatabolic

enzynes (Pennington et aI 1983). Both actions lead to increased levels of

prostaglandins Ín the fetal circulaLion, which in turn has been shown to

stimulate cAMP productlon which reduced the role of cell division (Pastan

et al 1g,lÐ. Increased levels of brain eAMP couÌd affect the development

of the cNS, as Penníngton (1988) reported that increased prostaglandÍn E

and cAMP Ievels were positively correlated with decreased brain weighf

afber in utero ethanol exposure. Furthermoret the admlnistration of

acetylsalicylic acid, a prostaglandÍn synthesis inhÍbitor' prior to

naternal ethanol administration led to a 5Ol reduction in birfh

6-19

defecbs in newborn mice (Pennington 1988)' Hence' increased prostaglandin

levelsmayalsounderliesomeofthedefectsassocíatedwithFAS.

Ithasalsobeensuggestedthatethanolmayelicitdeleteriouseffects

inuberobyalteringmaterna}andfetalhornonelevelsduringpregnancy.

Halmesnaki ef aI (1987) reporLed that women who Save birth to FAS

children had }ow estradiol and estriol Ievels throughout pregnancy, and

Low levers of progesterone accompanied by high proJ-actin concentrabions

duringweeks16to24,whiledrinkÍngrnotherswhogavebÍrthbonormal

infantsshowednochangesinhormonelevels'However'asyetitisnot

knownwhetherdeviabionsfronnornalnaternalhormoneconcentratlons

duringpregnancycontributetoFAsEthanolhasa]-sobeenshowntoleadto

allerationsinfetalsteroidhormonelevels,whichledtochangesin

hypothalamicnucleiinma].erabsbutnotinfemalesexposedLoethanolin

ulero (Barron et aI 1988). More research is required in order to elucidate

thecontributionofa].terationsofvarÍoushornonesintheaetiologyof

FAS and FAE.

6.1.5. Aims of the study in relation to alcohol teratogenesis'

The ains of the present study were firstry to confirm previous

observations thab ethanol has a direct dose-related effect on embryonic

growth and dysmorphology, and secondly to confirm and extend' for the

first time in the rat å¡ vitro model, the limited studies of the cytotoxic

effectsofethanolinthecranialneuraltubepreviouslyexaminedinthe

mouse embryo ;!¡ vivo. To the best of the authors knowledge, the

cellularabnornalitiesobservedinnicehavenotbeenexaninedatthe

ultrastructurallevelinarryspecies.Hencethethirdandmosbinportant

aim was to describe the alcohol-induced subcellular changes seen in the

cranial neural tube following neurulatlon, which accompany the cytological

6 -20

and morphologÍcal anonalies observed in this study' The likely underlying

mechanismsreSponsibleforthisteratogenicactionwÍlIaIsobe

considered.Theoutcomeoftheseinvestigationsmightenabletheauthorto

specurate as to whether the observed neurar tube patholory is associated

withtheneuraldysmorphology,lowbrainweighLandCNsdysfunctions

reported as features of the FAS in anÍnal-s and humans'

I,lhile the rat in vivo nodel has not proven to be as effective an animal

nodelforalcoholleratogenesisasthenouse,previousinvitrorat

studies have shov¡n considerable sinilarit'ies in ethanol-induced

dysmorpholorytothoseinthemousemodelj¡vivo,aswellasinhunans.

Hence, the use of explanted enbryos in the present and subsequent studies

inthisreportprovÍdesasysteminwhÍchtheembryonÍcenvironmentcan

be controlled, and fetal growth and development examined visuall-y with a

precision not possible in j¡ vivo studies when the time of adninistration

of the agent 1n relation to or8anogenesis Ís criticat in order to induce

maximal dysnorphology during neurulation. In addition, j¡ vÍtro rnodels

enable direct assessment of the teratogenicity of the curtured conpound to

be nade in isolabion, without bhe confounding aspect of maternal

influenees such as metabolite fornation

6.2 MATERIALS AND METTIODS.

Enbryos were removed fron pregnant sprague-Dawley rats for explantation on

day 9.5 of gestation (ttre ¿ay on which spern was delecled in the vaginal

snear was designated day 0.5 of gestation). Aninals were anaesthetised

with dÍethyl ether and maternal blood was collect'ed by cardiac puncture

and imnediately centrÍfuged. The ulerÍ were renoved from the dam' a slÍt

was made along the anti-mesonetrial borders and individual inplantalion

sites $¡ere carefutly excised. Enbryos were explanted under a stereoscopic

dissecting nicroscope by the method

parietal yolk sac and ReicherÈs membrane vJere renoved, leaving intact the

visceral yolk sac, ectoplacental cone and Èhe annion'

embryos at the early headfold stage were added t o cu1 t.ur e

6-2L

of New (1978). Decidua, Erophoblast,

containin g 2.7 m1 of heat inactivaEed rat serum' together

of antibiotics, streptomycin G-sulphate (OO ug/ml) ' and

Groups of 3

bot tIe s each

w irh 100 ul

penicillin-G-sulphate (6ug/m1), made uP to volume with sterile water'

I,lhere appropriate eÈhanol (of the grade used in the liquor industry) Í'sas

diluEed with sterile vrater Eo give a final concenEration of 200 (43'4

mr"r),400 (g6.8 mM),600 (130.2 mM) and 800 (173,6 mM) mg/100nl of serum'

Culture bottles containing media and embryos hlere placed in a water bath

at 37oC and flushed witb a nixture of 5% 02, 57. COZ and 90% N2, after

which they were placed in a roÈaEing incubator set aË 37oC' Following 20

and 40 hours of incubaÈion, botEles were renoved and flushed wíth 207' 02'

57" co. and 7 5% NZ, and 407' 02, 57' co2 a¡ð 557" N, respectively'

After a total culture period of 48 hours, embryos were microscopically

examined for viability, which was judged by the Presence of a contracting

heart, and by blood vessels vrith circulating red blood cells in the yolk

sac. viable ernbryos $rere examined for several neasures of growth

including yolk sac size, crovtn-rumP length and sotoiEe numbers' The

embryos were also assessed for the degree of developroent aÈtained and

abnormalities $¡ere noted quantitatively v¡hile overall assessmenÈ of

embryonic development was performed by use of Ehe scanning electron

microscope. cellular and ultrastructural morphology specifically of tbe

neural tube were examinecl by light and transmission electron microscopy

respectively. Continuous growth variables and discrete development data

were statistically analysed as described in detail in chapter 2'

6 -22

6.3 RESULTS.

6.3.1 . Gro,¡th and morphological development.

Embryos explanted at the headfold stage of development (9.S Aays) (Fig.

6.1a) and culbured Ín rat serun for 48 hours (Figs. 6.1b,c) showed

sinilar development to 11.5 day embryos which had developed in utero

(c.f. Fie.3.1a). Ìlhen conpared with ix vivo control enbryos (see Table

3.1) developnent was not affected by j¡ vitro culture, although there

was a slight reduction in growLh in in vitro controls (TabIe 6.4)

reflected in shorter crov¡n-rump J-engths and reduced somite nunbers.

Embryonlc viability, was rarely affected by ethanol:h vifno even in

embryos that were severely growth retarded and developnentally

compromised.

In the present study a significant effect of dose $ras found for crown-

runp Iength (F 4,76 = 38.0. p(0.001), somite numbers (F4,t6= 38.0 p(0.001)

and yolk sac diameter (f 4,?0 = 23,1. p(0.001). Generally embryos

exposed lo 200 ngl of ethanot did not differ frorn controls in terns

of growth parameters such as crown-runp length and yolk sac dianeter,

although there vùere sígnificantly fewer somites even at this low dose

(TabIe 6.4). At concentrations greater than 200 mBít all 3 determinants

of growth denonstrated a dose-dependent significanl decrease. Conpared

with controls cror"rrFrtunp lengths were reduced by 13fi at 400 mgl ethanol

and by over 40f at 800 mgf. Tt¡ere were also significantly fewer somites

at each concentration of ethanol up to 800 ^g%

where the number of

somites was 12 less than in control enbryos. Yolk sac dÍaneters were not

slgnifÍcantly differenü from controls at the Iowest concenlraLion of 200

ne% elhanol but decreased in a dose-dependent nanner as the

concentratlon of ethanol lncreased from 400 r,9fit with a 25fi reductlon

6-23

1

Tab]e 6.4. Effects of alcohol on Srol.rth on day 11.5 embryos j¡ vitro.

Ethanol Concentration (mef )

20 200 400 600 800

Total no.embryos 16

Crowrrrunp1 ength( nrn) 3.22+0.07No.somites 23.75!0.28YoIk sacdian( rnm) 4.06+0.06

13

3 .23t0 .07

20.31t0.65

4.06+0 . 12

19

2.32t0 .t1,3

16:63!0.96

3.þ?10.{f

20

2.48s0.13

13.1010.87

3.08g0.09

14

1 .80$.17

9.3111 .35

2.92+0 .12

1' Values are neans + SEM.t Crow n-runp length & somite nunbers are only approximate at thisconcentration due to small size of embryo and the failure of the embryosto rotate into a dorsally convex confornation

in diameten at 8OO nef. tÍh1le growth of theyolk sac was significantly

conpromised in ethanol embryos, the vasculature of thÍs structure dÍd not

appear to be affected except ab the highest concentnation

GeneraJ.Iy, morphological data demonstrated that the frequency and/or

severity of abnormalibies w ere dose-related such bhat Íncreasing

concentrations of ethanol led to an increase in dysmorpholory (Table 6.5,

Figs. 6.1d-g). Furthermore, nany affected embryos denonstrated nore than

one dysnorphic feature. The neural tubes of embryos exposed to

concentrations of 400 mg% ethanol and upwards were frequently affecbed

conpared to control day 11.5 enbryos (Table 6.5). These neural anonalies

which included open neural tubes, obserrred in sone enbryos fron 400 to

8OO neí alcohol (FÍgs. 6.1d-e) were sonetimes confÍned to the cranial

reglon 1n sone anÍmals, while in others they extended caudally. Cranial

anonalies also included Iarge fluld-fÍlled vesicles (Fig 6.1d)' as well

as abnornally- shaped cnanla with severely dininished forebraln or

nidbrain reglons, mainly in enbryos cultured in 600 ng! and 800 mgf,

(ftes. 6.1e,f,g)r which !{ere typical of nicrocephaly which has been

6 -2t+

Fig. 6.1. a.

untreated dam.

allantois i An'

Headfotd stage (g.¡ day) rat enbryos removed fronEc, ectoplacental conei Ch, chorion; Vsr yolk sac;

amnion; hf, headfold.

anA,

brc. Lateral and dorsal views respectively of an 11.5 day embryo

cuilured in rat serum for 48 hours. Note the neunal folds have fused along

the entire length of the neural tube. Fb, forebrain! Mb, midbrain; Hb,

hindbrain; OP, optic vesiclesi OT, otic vesicles; 1 1213, branchial arches;

H, heart; F, forelinb buds; Sr sonites.

d. Three 11.5 day enbryos fron the sane culture bottle exposed to 400 mgf

alcohol. The embryo in the centre appears developnental.ly normal-, whereas

the other two show underdeveloped forebrain and nidbrain regions, and

fluid filled vesicles (arrowheads). The neural folds have not fused fromthe hindbraÍn to bhe caudal end of the embryo (arrows indicate rostralpoint of neural tube closure). The enbryos are anophthalmic' themandibular arches (1) are abnornal, while the second and third branchialarches are poorly developed or absent, and the animals have not rotated'

ê,f. Dorso-ventral and dorsal views respectively of three 11'5 day

embryos cul!ured in 6oo n8Í," alcohol. The anlmals show overaLldevelopmental retardation and are microcephalic with abnormal neuraltubes, open either cranially (open arrow), and/or from the hindbrain tothe caudal ends of the embryos (the arrows indicate the rostral-mostpoint of closure). The embryos are also anophthalnicr otic vesíc1es are

not present, and the mandibular arches (1) are fused to the pericardium(arrowhead), while the second and third branchial arches are under-developed. The anÍnals have only partially rotated. Mx, naxillaryprocess; A, allantois; H, heart.

g. Enbryos cultured in 8OO ng9l alcohoL l^Iere even nore severelydãvelopnentally retarded, particularly Ín the cranial region The animalsappeared anencephalic, and sone showed unfused cranial neural folds(árnows) and anóphthaimia, with severely affected nandibuLar arches (1)

fused to the pericardium (arrowheads). The second and t'hird branchialarches were absent and the anÍmals had not rotated. H, heart.

Bars=50oun.

6-25

Table 6.5 Effects of ethanoln on morphological developnent in 11'5 day rat

- :1::I:-"- i * - -- - - - - - -Ethanol- concentration (mgf) Significance of

do se- reL atederfect (p)

0

Total no. enbrYos 16

No. with anY kindof defect 2(12.5)

Arry kind of NTD 1(6 .3)

Open N. T. ar¡rwhere 1 ( 6 .3 )Open cranialN. T. orùy 0( 0)

Ele defects 0( 0)

Ear defects 1(6.3)Branchial- archdefects 1 ( 6.3)

200

13

400

'19

600 goo 2

Heart defectsAbsence offorel imbs

9(69.2)

2(15.4)

2( 15 .4)

0( 0)

0( 0)

0( 0)

0( 0)

0( 0)

0( 0)

6(42.2)

5(38.5)

3(23.1)

1?(89.5)

10ß2.6)

5(26.3)

3( 15.8)

5(26.3)

2( 10.5)

5(26.3)

0( 0)

11(63.2)

1?(89.5)

7(36.8)

6(31.6)

18(90.0)

1 5(75 .0)

8(40.0)

3( 15.0)

11(55.0)

8(40.0)

9(45.0)

2( 10 .0)

16(80.0)

18( 90.0)

10(50.0)

15(75.0)

14

14( 100)

14(100)

7(50.0)

6(42.9)

9(64.3)

7(50.0)

7( 50.0)

7(50.0)

13(92.9)

14(100)

7( 50.0)

11(75.6)

<<0 .00 1

<0.001

<0 .03

<0 .003

<<0 .00 1

<<0 .00 1

<<0.00 1

<<0 .00 1

<0 .00 1

<0.001

<0.002

<<0 .00 1

20

0( 0)

1(6.3)

Incomplete flexion 2(12.5)Inpaired chorio-alLantoíc fusion 0(0)YoIk saccirculation 1 ( 6.3)

1 Percentages gj.ven in parentheses2 F""qr"nJ:.es are only approxÍmate due to dif iculties in idenlifying

anonalies in these very snall embryos'

observed Ín experimental animals at term and in hunan offspring as a

result of in utero alcohol exposure. Many of these enbryos also showed

open caudal neural tubes characteristic of 'spina bifida.

Ear and eye defects were also observed in some enbryos at concentrations

from 400 ngf ethanol¡ aPProachÍng 65Í of embryos in the case of eye

defects and up to 50f for ear defects (TabIe 6.5, Figs. 6.1d'e,f)' The

prinary optic and otic defects observed at this early stage in the

6 -26

deveLopnent of these structures were usually the absence of lhe

respective placode either unilaterally or bilaterally, and narrowing of

the optic stalks. There was also a dose-dependent increase Ín branchial

arch defects with abnormalities occurring in26% of embryos at 400 mgf

and up to 50l, aL 8OO me% of ethanol (Table 6.5). Dysnorpholory occwred

mainly Ín the first branchial- arches which were often underdeveLoped and

smaller than in control enbryos and vJere frequently fused to the

pericardium (Fig. 6.tg). In some cases the second and third arches were

either underdeveloped or absent (Figs. 6.1e,f). Heart defecbs were not

readily apparent at this stage in morphogenesis, with orùy 10f detected

at 600 ngf, which consisted of poor development of bhe primitive heart

tube and possibJ.y up to 50$ affected at 800 tg%. However bhe snall size of

the embryo prevented an accurate determination of heart abnornalities at

this concentration

A number of control enbryos denonstrated inconplete flexion which

suggested that they may have been cultured at a slightly earlier

stage in developnent Lhan other enbryos in this group (Table 6.4). This

was also indicat,ed by fewer sonites in these enbryos after 48 hours Ín

culbure than previously reported. Compared with control s (12.5%) there

was an increase in the number of ethanol-cultured enbryos that failed to

rotate into the dosatly convex conformation, with 43f of enbryos failing

to rotabe at 200 ngf ethanol, and 90f at 400 and 600 mgf' while none had

rotated when exposed to 800 ne%. Enbryos at different stages of

rotation are tllustrated fn FÍgs. 6.1d,9. Impaired chorio-allantoic

fusion was also observed in 39f to 50l of enbryos through the full range

of concentrations of ethanol.

6 -27

6.3.2 Scanning electron microscopy.

Fig. 6.Za is a scanning electron nÍcrograph of a typical j-u vitro 11.5

day control- embryo, w hich al though slightly small er than its ;!-n vivo

counterparts was structurally sinilar to them (c.f. Fig. 3,2a). The embryo

had rotated Ínto bhe dorso-convex posifion, the neural tube had fused for

Íts entire length except in some cases for the caudal-most region of the

tube, and the forebrain vesicles and midbrain were wel-I developed. Three

pairs of branchial arches had developed, and the heart and forelímb buds

$¡ere well- forned. The optic placodes were also present at thÍs stage, and

the otic placodes (not seen in this nicrograph) were aligned with the

second branchial or hyoid arch cLose bo the dorsal midlÍne. Surface

features of control -Ln vitro enbryos de¡nonstrated by a section fron the

midbrain in Fig. 6.2b showed similar characteristics to those observed in

day 11.5 j¡ vÍvo control enbryos (Fig. 3.3a). The indivÍduaI

ectodernal cells were surrounded by a thick border of microvilli along

the perimeters with others scattered over the remaining cel1 surfaces.

Characteristfcs of the cranial neural tube of an in vitro day 10.5 contnol

embryo can be seen ín Fig. 6.2c. At, this stage in development the neural

tube is open at the rostraL and caudal extrenities (latter not seen in the

nicrognaph) with the intervening part of the tube closed. The craniat

point of closure 1n the hindbraÍn can be seen to be V-shaped and the

neural folds are upríghf, snooth and extending t,owards each other. Higher

nagnification of a sectÍon of the midbratn (Fig. 6.2d) of thls 10.5 day

ernbryo shows the Ínterface belween surface ectodern and neuroepithellum.

fn control embryos aü this stage of developnent there Ís a snooth

tnansltion between surface and neuroepÍtheliun which represents the

neural crest regfon where fusÍon of the two sldes of the neural plate

occurs Ín the do¡'sal mldline of the anlnal. At even hígher nagnification

6 -28

Fig. 6.2. a. Scanning electron micrograph of a typical 11.5 day embryo

cultured in rat serum for 48 hours. The embryo is structurally simi-Lar toits;Ln vivo counterpart in Fig. 3.2a. The asterisk marks the site seen athigher magnification in b. Fb, forebrain; Mb, midbrain; 11213, branchialarchesi OP, optic placode; H, heart; F, forelÍmbs; S, somites'

b. A section of midbrain surface ectoderm (E) at the site marked by theasterisk j.n a, showing sinilar featunes to those observed at comparablesites in in vivo enbryos of the same age (see Fig. 3.3b). The cells weresurrounded by a dense perimeter of nicrovilli (arrowheads) with additionalrnicnovilli scattered over the celI surfaces.

c. The micrograph shows a dorsal vj.ew of the cranium of a 10.5 day i4vitno embryo w ith unfused neural fotds (arrowheads) and exposed

ventricular surface (asterisks). The Iarge arrow indicates the most recentpoint of closure of the anterior neuraL tube. The regionnarked by thesmall arrow is seen at higher magnification in d. Mb, midbrain

d. The neural crest region (arrowheads, NC) in the midbrain of the 10.5

day embryo in c, which lies between the surface ectodern (E) and theventricular surface of the neuroepithelium (N) is very snooth whichprobably facíIitates fusion of the neural folds. The asterisk marks theapproximate site seen at higher magnification in e.

e. A seetion of the mÍdbrain surface ectoderm (E) from a

embryo marked by an asterisk in d, showing smaller celIsdist.ribution of microvilli interspersed with blebs (arrowheads).

10.5 daywith a

f. Higher magnification of the neuroepithelial surface of the ventrieularl_umen frorn a 10.5 day enbryo near (N) in d, shows the surface covered invery long roicrovjfli (arrows) and bLebs of varying sizes (asterisks).

g. Dorso-laberal view of an abnorrnal 1 1.5 day enbryo cultured in 600 ngfalcohol for 4B hours denonslrating craniorachischisis. The neural tube had

not fused cranially fron between the forebrain vesicles to the caudal end

of the embryo (arrowheads). Conpare this embryo with the limited neuraltube opening in the controf 10.5 day embryo in c. The asterisk marks theapproxinate site seen aL higher nagnification in h. Fb, forebraini Mxt

maxillary prominence; H, heart.

h. Section of open neural tube fron the mldbrain region of the 11.5 day

enbryo in g, which was exposed to 600 ngÍ alcohol in culture. The neuralcrest area (NC) is very irregular, being covered 1n blebs of varying sizes(asterisks) w hich nay prevent neural tube closurs Large blebs(arrowheads) were aLso located on the ventricular surface of theneuroepÍtheliun (N). Cornpare ùhis figure with a sinilar site in the 10.5

day controf embryo in d. The arrow indÍcates the approximate site of thesection seen at higher nagnificabion in j. E' surface ectodern.

6-29

i. HÍgher magnification of a region of the surface ectoder¡n (E) of the

midbrain in h, fron an 11.5 day enbryo cultured in 600 mgfr, which shows

that the cells are covered in nÍcrovilli which appear to be sínilar tothose at conparable sites in controJ- day 11.5 enbryos in b.

j. Higher magnifícation of the ventricular surface of the neuroepitheliu¡noi an enbryo exposed to 600 mgf ¿coho] in culture, shovrn in h' The

surface in the open neural tube of the midbrain was covered in blebs ofvarious sizes (asterisks), but dÍd not show the long nicrovil-Li observed

at a similar site in the open neural lube of a 10.5 day contro] enbryo inf.

k. A severely affected 11.5 day enbryo cultured Ín 800 mef alcoholshowing overall developmental rebardatÍon and craniaL features which are

indistinguishable. The aninal is microcephalic and the forebrain vesiclesare absent. The neural folds remain unfused (arrows) and the nandibulararches (1) are abnormal and fused to the pericardium (arrowheads),

although the hyoid or second branchÍal arches (2) have developed. The

enbryJ has no¡. rotated. The vertical arrow also narks t,he approxinatepositlon of the section seen in e. Hr heart.

I. A section from approximately bhe nidbrain region of bhe enbryo in kt

cultured Ín 8OO mgf alcohoL showing dense nicrovil-Ii on the surfaceectodermat cells (E), a severely disrupted neural cnest area (NC)' and

I arge blebs ( arrowheads) on lhe ventricular surface of theneuroepithetium (N).

Bars= 1OOum, a,c,g,k;1Oum, drhrli 2tun, brerfriri'

trlt't !¡'

,

tç

_a

6-30

of the midbrain surface ectoderm of this 10.5 day control enbryo (Fig.

6,Ze), the epiLhelial cells were srnaller with dense microvilli around the

perineter and over the ceII surface, which was interspersed with a few

bIebs, while the neuroepithelÍal- surface (Fig. 6.2f) was covened in

large microvílli with marry associated blebs which projected into the

ventricular lumen

Fig. 6.2C shows a typical severely malformed ethanol-treated enbryo

cultured in 600 tg% with sinilar external features to those seen j.n a

number of embryos exposed to 400 mgf. The general ethanol-induced defecùs

have already been described in Fig. 6.1, how ever the scanning

electron micrographs denonstrate more specific aspects of these

anonalies. In many of these enbryos which had not rotated, the neural

tubes remained open from between the forebrain vesicles to the caudaL

end of the enbryos, a defect typical of craniorachischisis observed

previously in ethanol-treated enbryos ;i.-n vivo, and which was unlike the

appearance of less developed 10.5 day embryos (e.f.Fig. 6.2c). At this

stage in developnent ühe optic placodes and the forelimb buds were

frequently not evident, alfhough the branchial- arches had developed in

sone cases. A portion of the neural folds in the nldbrain of the Éne

enbryo (Fie 6.2h) showed the site of fusion of t'he folds to be

heavily blebbed, unlike the relatively smooth appearance of this site Ín

the 10.5 day embryo in Fig. 6.2d.

Higher magnification of a region of the surface ectoderm of t,he

nidbrain (Fig.6.2i) of the sane 11.5 day enbryo cultured in 600 ngl

ethanol showed that the cells were covered Ín large nunbers of

nícrovilli which were i.n excess of those seen at slmflar sltes In control

enbryos of the sane age (FÍ9. 6.2e). The neuroepitheliat cells located

6-31

$rithin the open neural tube (Fig. 6.ej) were covered in Iarge blebs'

with very few microvilli, which once again was very dÍfferent to the

sane surface in control embryos with open neural- tubes at an earlier

stage Ín development (Fig. 6.2f).

FÍgure 6.2k shows a severely affected microcephalíc enbryo typical of

those cuLtured in 800 mgf. The forebrain region is severely abnormal, the

midbrain-hindbrain areas are grossly reduced and the cranial neural tube

had not completely fused. The enbryo had also not developed optic and

otÍc placodes or linb buds and had not rotated. The abnornal- mandibular

anches were fused to the pericardium as were the snaller hyoid arches, and

the third vi.sceral arches were absent. Fie. 6.2I revealed that the

nidbrain surface of the enbryo was also eovered in extensive

nicrovillÍ while the site of fusion of neural folds rì¡as even more

irregular than that observed in 600 mgf ethanol-treated embryos, and the

neuroepithelium was covered in large blebs. The differences j.n structural

and surface appearance of treated enbryos compared with 10.5 day

contnols Índicates strongly that development was not nerely delayed as

a result of ebhanol exposure but thaü the differences could be

attributed to teratogenesis.

6.3.3 CelluIar observaüions.

LÍght microscopy of malformed etharpl-cultured embryos reflected the

dysmorphology observed under the dÍssectlng and Lhe scanning electron

nÍcroscopes. Sections through the closed cranial neural tube of a typical

11.5 day crrltured control enbryo (ftgs. 6.3a,b) were sinflar bo those of

control enbryos of the sane age which developed fn utero (c.f Figs.

3.4a,b). Fig. 6.3a shows a thÍck, smooth neuroepithelfun, wlth an

abundance of surrounding nesenchynal cells wlth snall, developing

6 -32

Fie. 6.3. a,b. HorÍzonlal sections through bhe craniun of an 11'5 day;¡¡vitro embryo showing a thick neuroepithelium (N) of pseudostratÍfÍed

colunnar cefls (long-arrows). At the apical end of the neureoepithelium

which abuts tne vent,ricular Iumen (L), snalL blebs (short arrow s) and

nitotic figures (Mf) can be seen The mesenchyne (M) contaj-ns mar¡y

sterrate-shaped cells with interconnecting firopodia (arrowheads)

supporbed in an extracellular natrix (EcM) which also contains developing

uiããa vessers (bv). E, surface ectodtrm; Fb, forebrain; Hb' híndbrain

c. Horizontal- section through the craniun of an 1 1.5 day embryo cultured

1n 600 ncf alcohol showing a thin, severely affected neuroepithelium (N)'

but a healtkry mesenchyne except for a number of blood vessels (bv)' which

appeared to be abnormllly dilated. The neural folds in the rostral part of

the cranium have fused but are severely distorted, while the neural tube

in the hindbrain region remains open The open neural tube continued tothe caudal end of thã ernbryo, typical of craniorachischisis' Many denset

dark structures were observed w-ittrin the ventricles (shorb arrows), as

well as in the neuroepÍthelium (arrowheads) and adjacent to Ít 1n bhe

nesenchyme (asterisks). Fb, forebraini Hb' hindbrain

d. Higher magnification of a sectíon of neuroepithelium denarcated by

Iong anrolJs in c. The neuroepithellun is severely disrupted with largeextracellul-ar spaces cOntalning ltany condensed ceIl rennants (short

arrows), which are atso locateO witf¡in the cells (Iong arrows)' Many other

condensed particles are located in the nesenclryme (asterÍsks) aL siteswhere the basal ends of the neuroepithelial cells are rþ longer closelyapposed (arrowheads). Some cell debris (open arrows) is also present lnthe ventricular tunen (L). bv, blood vessel'

Bars=50un.

la

a

t.â

\', . t,l.l,J',t"../IsI

ì'..

'l !-

¿

I

,

)

{>

l¡aa" .a

¡)t' ìt, l'

tl

¡a

a!

..,

I

t'¡.)

IaC

tt-

oa.

a. I

/aa

i

I

.¿ -t\t

o

a

kfrÁ'..

b

\\.

¡'i'-l

ttt(

1a'\

f

dÕ.ta

,

B\it

I

\r.-

P

I

l-*.-t

1.. lìt l

ìf

.ty.

- t-

I

.h

6-33

blood vessels interspersed amongst then.

At higher magnification (Fie. 6.3b), the neuroepithel ial cells are

arranged in a negular pattern with very little extracellular space between

them. The surface of the ventricular lumen is relatively smooth and

mitotic cells are Located ab the lunenal- border. At the other end of the

neuroepitheliun, but separated fron it by the basal lamina' the nesenchyme

consísts of stellate-shaped celts with cytoplasmic projections which forn

a dense fibrillar network with which the cells nake contact with each

other and the basal Iamina. SnaII endothelial-lined blood vessels can be

seen lo be interspersed anongst the mesenchymal ceLls.

HorÍzontal secbions through the craniun of ethanol-cuLtured embryos

demonstrated a strong dose-dependent effect of alcohol on cellu1ar

abnormal-ities fro¡n 400 mgf,, which supports a simíIar dose-dependent

rnorphological effect observed under the dissecting and scannÍng electron

nicroscopes llhile enbryos were hardly affected at 200 ng% ethanolt

cellular abnormalities occurred in nore extensive areas of the

neuroepitheliun with increasing ethanol concentrations, while the

mesenchynal celIs did not appear to be affected. Fig. 6.3c shows a

horizontaÌ section through the anterior neural tube of a representative

1 1.5 day enbryo cr:L tured in 600 nC% al cohol. The rostral part of the

anterÍor neural tube has closed but is disborted, whÍle the caudal end

remains open. The nesenchyne is abundant and appears unaffected by the

treatment, except for bhe presence of dark bodles close to the junctlon

with the neuroepithelÍun. Slnllar dark bodies are present throughout the

neuroeplühellum which appeared narrower and cor¡voluted at the junctlon

with the nesenchyne as $¡ell as at some sites in the ventrfcular lunen

Hlgher nagnÍfication of a site in the forebraÍn-nldbrain region (ffg.

6-¡+6.3d) revealed a severely shrunken, thinner neuroepithelial waLl withlarge extracelLt¡lar spaces which often contained ce11 debris, as weII as

dense, dark, cellular inclusions which r{ere consistently seen atconcentratÍons of 400 mgf and above in this study.Neuroepithelial celIrennants appear to be extruded into the ventricular lumen, as well as intothe mesenchyrne. Apart fron this cellutar material, the mesenchynal cel1

nunbers of ethanol-cultured enbryos appeared relatively unaffected

although it is like1y t'hat there were fewer intercellular connections.

ft appears that the consequences of an intact mesenchyne were that

the neural folds although open had not, eollapsed and fallen over the

cranium as was seen in previous chapters in this thesis. At

concentrations of 600 ne% (fig. 6.3d) and g00 mg%, developÍng blood

vessels within the nesenclryme appeared enlarged.

6.3.4 Ultrastructural observations.

J¡ vitro 11.5 day control enbryos denonstrated sfnilar neural tube

features to those observed previously Ín control in vivo animaLs of the

same age (Fis. 3.5). Tt¡e anterior neural tube consists of a thick,pseudostratified columnar epithetÍun (fig. 6.4a) with nost of the

neuroepithelial cells extending fron the ventricular lumen to the basal_

lamina. In the lateral regÍons of the waLls of the anterior neural- tube atthe lumenal- surface where the apical cells and their processes are

nornally closely apposed, mÍtotÍc cells are prevalent (FÍg. 6.4a). Along

lhis surface the apieal cell processes have conspicuous junct,ional

conplexes or ternÍnal bars at their bases, and a tenninal web of ftlaments

extends fro¡o,junctÍon to Junction (not shown). At other sltes along the

neuroepÍthellal cells including the basal end adjacent to the basal Iamina

(Fie. 6.4b) the cytoprasmic nenbranes are closely apposed but are not

held together by junctional conplexes. Mesenclrynal cells nay often 1ie

6_35

Figs. 6.4a-f. El-ecbron micrographs of norrnal neuroepit'helium and

nesenchyme from the anterior neural tube of an 11'5 day cultured embryo'

Nobe the sinilarities in the ceÌl-s and organelles between this in vÍtroembryo and an j¿ vivo animal of the sane age (FiS' 3'6)'

a. The interphase cells of the thick pseudo-stratÍfied columnar

neuroepitheliun (N) are closely apposed with oval shaped nucleÍ (nu)

and dense nucleoli (n). The apícal ends of these cells which abut bhe

ventricul-ar lunen (L) consis! of cytoplasmic processes or blebs(arrowheads) which project into bhÍs space and are held together byjunctional conplexes- (ig). Mibot,ic f igures (Mf) are prevalent in bhisarea.

b. The basal end of the neuroepithelium is supported by a basal Lanina(arrows) but the cells while closely apposed are not connected by

junctÍonal conplexes. Ihe adjacent nesenchynal cells show t'he t'ypicalstellate shape with long cytoplasnic processes (large arrowhead), and

are suspended in an extracelltùar matrix (ECM)'

c. Structures such as nuclei (nu), nitochondrla (m), Gol8l apparatus (g)

and rough endoplasnic reticulun (r) which are surrounded by cytoplasnlcribosomeã (cr) in polysomaL arrangements, all show the characteristics ofnornal in utero interphase organeJ-les observed in the anterfor neuraltubes of untreated enbryos -ü Jivo.

d. occasional dead or dying cetls (d) and phagocytosed cell rennants(arrow) are located in the lateral regions of the neuroepitheliurn (N) ofthe anterlor neural tube of 1 1.5 day jn vÍtro control enbryos'

q Extensive prograrnned cell death or apoptosis (arrows) occurs at fhenosü recent site of closure of the anterior neural tube along the dorsalnidline, fron the sane 1 1.5 day i4 vitre control enbryo featured 1n d.

sone of the dead ceII conponents (arrowheads) appear to have been extrudedinto the ventricuLar lunen (L). lhe asterisk narks the site seen at highermagnification in f. N, neuroepithelir:n; E, sgrface ectoder¡n.

f. Higher nagnÍflcation of apopbotic area narked by asterÍsk in et fron an

11.5 day j.u vitro control enbryo showing dead cell rennants locatedintracelLr.rlarly (arrowheads) and extracellularly (arrows), consisling ofmembranes, cytoplasn, nuclear naterial and organelles in advanced sfagesof degeneratlon Ly, prinary lysosone.

g. E[ectron micrograph of a section of neuroeptthelfun (N) fron fhelateral antenior neural tube, at the interface with the nesenchyne (M)t

fro¡o an 11.5 day enbryo cultured 1n 200 mgf ¿cohol. Tt¡e basal lanina(arrowheads) is intact at this concentrabÍon, however the neuroeptthelialce1ls (N) are shrunken Ieadlng to extracelluLar spaces containing sone

nembrane rennants (arrows). The close proxiniüy of the cytoplasmic vacuole(v) to the extracellular nenbrane remnanls suggests that the ceII ls about

6-36

to phagocYtose this naterial'

h. Electron micrograph of a section of neuroepithelium (N) from the

lateral region or tne anterior neural lube of an 11.5 day enbryo cultwedin 400 lqgf alcohol. CeII shrínkage and death has occurred throughoub thisarea leading to extensive extracellular debris (arrows) as well as so¡ne

ptragocytosed ceII rennants (arrowheads). The asterisk .marks the site seen

at higher magnification in Í'

i. Higher magnification of extracellular debrls indicated by asterisk Ính. The structure on the right appears to be a whole dead on dying cell (d)

with severely condensed cytoplasm and organelles, and vacuoles (v)

containÍng cell remnants. The cLose proxirnity of the discontinuous ceLl

neu¡brane of the dying cell to the ex;racellular rennants (arrows) suggests

that the dying -ce1-1

may be phagocytosing some of these remnants' m'

condensed nitochondria; r, condensed rough endoplasnic reticuluni vrrvesicuLated rough endoplasnic retÍcuLum'

Bars=1Oum, a,d,e,h; 1un, b,crf,gri'

"u!,

üþ'

/(Û

I

6 _37

close to this membrane, and their characteristic pseudopodÍa which nay be

extensive, often contact t,he basal fanina (not, shown), as well as other

rnesenchymal cells which often gives the appearance of an extensive

fibrill-ar network of cells when viewed under the scanning el-ectron

microsco pe.

High nagnification of an interphase celI body of the neuroepithelium (Fig

6.4c) revealed that it characberisticatly contains a Iarge nucleus with

dense chronatin finely dispersedr or sometimes concentrated in patches.

Ihe cytoplasm is f illed with free ribosomes in a polysomal aruangenent

indicative of an active state of proteÍn synthesis, while rough

endoplasmic reüiculum usually consist,s of only short fragments, although

branching cisternae nay be presenb and may even be dilated. The cells also

typically have a prominent Golgi apparatus and coated vesicles can be

seen in fhe cytoplasn nean t,he GoIgÍ, from which Lhey are derived

(Ghadially 1988), but were aÌso found near or fused with Lhe plasma

nembrane (not shown). The mi.tochondria Ín neural tube cel1s generally

displayed cristae with a Iamellar configuratÍon which extend across the

organell e.

Occasionaldead or dying cells and phagocytosed material were observed

1n the Iateral regions of the neuroepitheliun of control enbryos (Fig.

6.4d), and are consídered to be part of normal progra¡uned cell death,

which however, 1s nalnly concentrated along the dorsal aspects of the

anterior neural tube where closure has occurred most recently. This

necrotic naterial nay consist of rnany dead cells or phagocytosed rennants

of celIs contalned 1n ot,her neuroeplthetlal cells (Fig 6.4e). Sone

of these remnants appear to have been extruded lnt,o the ventricular

lunen. The mÍcrograph in Fig. 6.4f shows sone of these cell rennants

6-38

which include membranes, cytoplasm, nuclear material and organelles in

advanced stages of degeneration, and located either Ín vacuol-es within

other cells or free in the extracellulan space. Note that sone of these

bodies range in degrees of condensation The very large osmiophilic

granules associated with an area of degeneration is most likely a primary

Iysosone.

Although sections through the anteri.or neural tube of enbryos cultured in

200ne|/ alcohol showed few cytological abnormalities ab t'he light

microscope level, similar sections observed under the bransmission

eLectron rnicroscope showed some initial signs of cellul-ar disruption

at this concentratlon when compared with control sections. Fig. 6.49

shows a region of the neuroepitheliurn which lies close bo the junclion

with the mesenchyme. I,Ihile the basal famina is unaffected the cells

show evÍdence of shrinkage causing them to puJ.l away fron

neighbouring cel-ls at sites along t,heir length Ieading to the

developnent of large extracellular spaces between the columnar ceI1s.

Vacuolated nembrane fragnents which were vÍsible in these spaces nay be

remnants of ceII organelles extruded by neighbouring necrotÍc ceIIs. There

was also an increase in bhe number of electron dense granules (possible

prirnary Iysosomes) conpared with control enbrlos.

At 400 mg! alcohol (FÍgs.6.4h,Í), necrosÍs slmllar to the cell death

observed malnly in the dorsaL regions and occasÍonally in the lateral

areas of control enbryos, was observed to be nuch ¡nore severe and

extensive in the lateral regions of ethanol-treat,ed enbryos. Urùlke

simllar sltes in controls hower¡er (FÍe. 6.4d), the neuroepithellal cel]s

of treated enbryos (Ffg. 6.4h) were shrunken and hence no longen closely

apposed, with nany of the extracellular spaces filled with necrotic

6_39

material which consisted of recognisabl-e ceI1 organelles (Fie. 6.4i) at

various stages of electron density. This increase in necrosis was similar

to lhe effect seen in zinc-deficient embryos in Chapter I of this thesis.

The anterior neural tube of embryos cultured in 600 ngf alcohol showed a

large variety of cell destructÍon which is lllustnated in Fig. 6.5' and

is representative of the increasÍng severify of necrosis observed in

embryos with increasing concentratíons of ethanol, leading ultimately to

skrinkage and in some cases alnost conplete destruction of the

neuroepithelium particuÌarly in lhe forebrain of the anterior neural tube

(FÍgs. 6.5a,b). Many of the cellular remnants lay free wit'hin the

exbracellular spaces, or were phagocytosed by neighbouring apparently

healthy celIs. Some of these dead celI components $¡ere eventually extruded

into the ventricular lumen (Fig. 6.5a), or Ínto the mesenchyne at sites

where the basal tanina was disnupted (Fig. 6.5c). Highen magnificatfon

(fig. 6.5d) of a site adjacent to the venlricular Iunen in Fig.6.5a

revealed thab extrusion of dead cell naterial inbo the ventricular lumen

occurred at sites where the junctional complexes between the cytoplasnic

processes of the neuroepithelial cells were disrupted. CeIIs in this

region whÍch appeared vacuoÌated at lower magniffcation conlained areas

where the cyloplasn was depleted of organelles and bhe free ribosones had

lost theÍr polysomal configuration

Many neuroeplthel iaI celL s contafned heterolysosones (fig. 6.5e)

described as incluslons under the light microscopêr ÌJlth naüerial that

ranged from Iight membrane fragments to dense bodies confalning

degeneratlng nuclear rennants and cytoplasnlc organelles which

appeared to be phagocytosed by apparently healthy neighbourlng cells.

In thls nicrograph, most of the lndividual onganelles were no longer

Fie. 6.5tube fronhours.

6-40

shows electron micrographs of sections of the anberior neural

an 11.5 day embryo expãsed to 600 ngf ¿coho1 -Ln vitro for 48

a,b. Sections of the neuroepibheliun (N) at two different sites showÍng

extensive cel-I disruption and shrinkage with many extracellular remnants

iop"n arrows) throulhout the neuroepithelium, and phagocytosed ceIIdeùris (long arrows) within obher neuroepithelial ce1ls 1n b. It appears

that bhe basal lanina (short arrows) has been forced away from fhe

neuroepithelium by the vast anount of cell debris which has accumulated

at this site in a. Sorne neuroepithelial rennants Ín a. (asterisk) alsoappear to have been extruded into the ventricular lumen (L)' The arrowhead

in u. narks the region seen at hÍgher magnifÍcation in e.

c. Basal end of the neuroepÍthelium in the same embryo (exposed t'o 600

ßgfi) seen in a. and b, showing extrusion of neuroepithelÍaI rennants intothe extraceLlul-ar matrix (ECM) of the mesenchyme at the site of disruptionof the basal lamÍna (arrows). Note the considerable quantity of celldebris in the nesenctryne (arrowheads), and the large exbracellular spaces

within the neuroepithelium (asterisks)'

d. Higher magnification of the area near the asterisk in a, which shows

a site at the apical end of the neuroepitheliu¡n (N) where a condensed dead

ceII (d) containing heterolysosmes (smaII arrowheads) and other extra-cellular debris (arrows) are being extruded into the venbrÍcularIumen (L) at a site where the junctional complexes of the neuroepithelialcyboplasnic processes (cp) or blebs have been disrupted. Large arrowheads

indicate dead cell remnants in the venbricufar fumen

e. Hlgher magnÍfication of a slte Ín the neuroepÍtheliun narked by the

arrowhead in b. This neuroepithelial ce]] contains numerous

heterolysosones (arrows) filled with dead celL debris of varying densÍty.The orgânelles are so condensed and severely lysed as to be nostlyunrecognisabls Note the presence of danaged mitochondria (m) in the hostce!l, wrrictt suggests thaf it too nay face destruction Dür nucleus.

f. Extrusion of neuroepitheliat cel-I rennants Ínto the extracellularnatrÍx (gcli,t) of the nesenchyne at a site where the basal lanina isOi"""pt"d, (arrowheads). lhe cell debris included vesiculated rough

endoplasmic reticulun (vr)r nitochondria (¡n) and nuclear rennants (nu).

Note the poor integrity of neuroepÍthelÍaI cell nenbranes Ín this region(arrows). Nr neuroepitheliuni BL, basal Ianina'

g. Extraceltular remnants with a flocculent appearance within theneuroepitheliun (N) consisttng of cytoplasmic ribosones (cr) which appear

to be relatively unaffecteã (c.f. cytoplasn in adjacent cells),vesiculated rough endoplasmic reticuLum (arrows), swollen ¡nitochondriawith broken crÍs-tae (ni and probably prlnary lysosones (LV). nu, nucleus.

tr Other indlcatlons of celL necrosls in the neuroepithellun includedswollen and dispersed cyuoplasnlc ribosomes (cr), condensed cytoplasm (c),

nuclear rennanbs (nu), nitochondria wlfh broken cristae (n), fragrnents ofmicrotubules (aruows), and danaged GoIgi Uodles (g)'

Barg=1Oun a,b, c; 2un d, erfrgr h.

f t,;

:l¡ltù

k'ã.

:ri

T;il

/ tr'

\Ì:

,'i

,*o

fn o 3o

r- 1â)

0d

t,

) 1 ì./l a¡

i¡*

JÉ

-::

.1..

I

6-41

identifiable, being severely condensed and rysed. whire the int,egrity of

these phagocytosing neuroepither ial cerr s appeared good at row

magnification, higher magnÍfication revealed that the mitochondrla were

often swollen with broken cristae.

Figure 6.5f shows the junction between the neuroepitheliun and

rnesenchyne where a range of extracellular materÍal is being extruded

through a disrupted basal Lani.na into the extracellular matrix of the

adjacent mesenct¡rme. Ident,ifiable cel1 organelles lncluded condensed

mitochondriar vesiculated RER, and nuclear rennants. CeII remnants

which Iìt ere frequently observed in the extracellular spaces withÍn the

neuroepithelium (Fig. 6.59) usually Íncluded organell-es such as free

ribosomes, vesiculated endoplasmic reticulun often containing celldebris within the cisternae, and swollen nitochondria with broken cristae.

The electron dense structure fn close proxirnity to these necrotic

organelles may be a prlmary lysosome.

Other features of interest relating to cellu1ar necrosis can be seen Ín

Fig. 6.5h' and include loss of the polyribosomal configuration within the

cytoplasn, fragments of mÍcrotubules, and Golgi bodies whÍch were in the

process of being extruded fron a condensed, dead ce1l. Dispersed nuclear

naterÍal and damaged niüochondria r'rere also present 1n thls nf crograph.

6 -\2

6.4 DISCUSSION.

Use of the fu vitro enbryo culture technique demonstrated that ethanol

has a direcb teratogenÍc effect, reflected in impaired growth and

development, in the absence of confoundíng factors such as altered

maternal functi.on, nutrition or metabolisnr. A direct effect by ethanol

on the enbryo would make it particularly vulnerable in utero since at this

stage of development enbryos do not possess ethanol dehydrogenase or

ethanol-oxÍdising acbivitÍes (Pikkarainen & Raiha 1967). Hence the

concentration of ethanol in the enbryo renains high and at any tine may

be higher than in bhe mother, since it lacks the ability to dispose of

ethanol itself, although the MEOS which converts l-ess than 2% of ethanol

to acetaldehyde (Lieber fi DeCarIi 1970) may be induced followíng chronic

exposure to atcohol. However, since previous observations (ïIynter et aI

1983) have shown that short term exposure (6 hours) to ethanol during

neurogenesis leads to slnilar open anterior neuropores in 11.5 day rat

embryos as seen in the present study' it is unlÍkely that these

abnornalities are due to ethanol being netabolised to acetaldehyde. In the

event thaü some acetaldehyde was forned, it wouLd probably be insufficient

to contribuLe significantly to the extensive neural tube defects induced

by direct alcohol exposure.

Use of the i¡ vitro nethod in this study also allowed the

contlnuous observatlon of embryos affected by ethanol at specific

tÍnes such as during and around the time of neurogenesis. J¡ vltro

studies are also parbicularly useful in inducing ethanol

teratogenlcfty slnce the concentrations requlred to lnduce

dysnorpholory 1n rat offspring 1n utero are frequenlly lethaL to both

nother and embryo (unpublished observation) whereas rat enbryos

6_43

tolerate them well in vitro, and they are comparable bo levels observed

in heavy drinkers. Blood ethanol- concentrations vary wideJ-y in humans

depending on the amount of ethanol consumed and serum alcohol levels as

high as 500 to 780 neft have been reported in alcoholics (Hanmond et

a1 1973; Lindblad & Olsen 1976).

ComparÍson of ethanol dysmorpholory in rat embryos ;Ln vitro in present

study, with effects in vivo in a pilot study perforned by author

(unpublished data), as weIl as by others in mice (Sulik et aI 1981;

Bannigan & Burke 19821 Bannigan & Cottell 1984) have demonstrated a

similar pattern of abnormalities which included ce1IuIar and

ultrastructural defects under both experinental conditions. The results of

the present study have shown that ethanol leads directly to growth

rebardation of enbryos in culture, and consequentJ-y may be the agent

responsible for developmental anonalies in alcohol teratogenesis in vivo.

Growth retardation occurred in a dose-dependent manner over a range from

2OO nB% to 800 ^g%

alcohol, which confirmed the work of Wynter et aI

(1983). Hence growth retardation demonstrated in this study by a reduction

in a number of paraneters occurred during the period of organogenesis, and

nay parallet the snall-for-gestational- age phenomenon often reponted in

tern infants witb FAS (Jones et al 1973i Hanson et a1 1976).

The dysmorphologr reported in this study ft vitro which included cranial

abnorrnalities such as exencephaly and nicrocephaly, as weII as oculan and

branchial arch irregularities extends tinited observations reported

previously in aninalsiX vitro (tlynt,er et aI 1983) and similarities in

aninals in vivo (Bannigan & Burke 1982;!Iebster et a1 1983;SuIik 1984a'b;

SulÍk et aI 1988). The particular vulnerability of the developing head

reported in these studies is important because of the frequency of

6-44

craniofacial dysnorphology of the human FAS child' which it has been

suggested, could be a sensitive indicator of Lhe level of alcohol exposure

in utero (Rostand eb aI 1990). Many of the rat embryos wÍth facial

defects observed in the present study also demonstrated open neural

tubes and microcephaly, the latter invol-ving variable reduction of the

rostral parts of the brain

These observed cranial abnormalities correspond with those reported in

humans where clinical correlation of head size with subsequent brain

function has suggested that microcephaly is strongly related to mental

retardatÍon (Crome 197 2). Brown et aI (1979) ha0 previously estinated

that, day 11.5 rat enbryos treated with 300n89l atcohol have a deficiency

of 8.9 x105 cella conpared with controls, and that treated enbryos

were retarded by 5-7 hours gestat,ion This finding together with the

observation Ín the present study of a severe loss of neuroepithelial

cells durÍng neurogenesis suggests that reduced cell nunbers in the

developÍng neural tube may underlie, or at Ieast, contribute to the

often severe nental retardation and behavÍoural abnormalities reported in

children with FAS (AbeI 1984). llhether necrosis and celI Ìoss are also

Ilnked wibh the failure of the neural tube to close is unclear, as not aII

embryos with extensive neuroepithelial cel1 danage denonstrated open

neural tubes and in fact the neural tube was found to be closed in some of

these severely affected embryos even in cases when t'he ethanol

concentraiÍon was 800 ngfr. Hence the nechanism through which neural tube

closure Ís prevented by ethanol exposure during neurogenesis may be

unrelated to the severe necrosis observed in the anterior neural- tube.

To the best of the authorfs knowledge this represents the

whÍch describes the cellulan and ultrastrucbural changes 1n

firsf study

the neural

6-45

tube which occur as a result of ethanol exposure during organogenesis in

the rab in vitro. BannÍgan & Burke (1982) previously described cellular

abnornalities in the anterior neural tube of the nouse embryo in utero

from 6 to 50 hours after ethanol iqjection Both studies demonstrated

sinilar abnornalitÍes however the absence of dead cel-I material in the

neuroepithelium at 50 hours post-ir¡jection suggested that all the cell

debris had been renoved by this tine. A similar absence of dead cells was

observed by the present author Ín an;i-n vivo píIot study when embryos

were examined 96 & 120 hours post-ethanol intubation (unpublished data).

In both cases bhe neuroepithelial wall contained large extracellular

spaces and was severely depleted of cells. In a later study by this

group (Bannigan & Cottell 1984) simÍlar ultrastructural abnorrnaliLies to

those seen Ín the present study were observed up Eo 24 hours following

ebhanol exposure. Dying and fnagnenting cel-1s were observed in the

neuroepithelium of all nouse embryos exanined, and the debris from this

necrosis was also phagocytosed by neighbouring healthy cells.

Extrusion of dead cell naterial from the neuroepitheliun in the present

study probably occurred through fhe large apical cytoplasnic

extensions of the neuroepithelial ceLls abutting the ventricular lumen

of the anberior tube. These cells which contained heterolysosones with

lysed dead neuroepithelial ceII fragnents, appeared to extrude this

material into the lumen probably by a process Like exocytosis. In

addition, the r¡Ltrastructural observations revealed that extracellular

debris in t,he neuroepithelium located closer to the junction with the

mesenchyne appeared to be extruded into it through breaks in the basal

lanina of the neuroepitheliun. The fate of this dead ceII material is

still not cLear, however there was some evidence of necrotic naterial

within the developing nesenchynal blood vessels.

6-46

TheultrastructuraldataobtainedinthisreporthaSenabled

clarification of some of the abnormal cellufar structures idenlified as

cytoplasmic vacuoles reported previously at the Iight microscope leveI

inmice(Banni8an&Burke1982),andwhichhadtedthoseauthorsto

suggesbthatethanolisprimarilytoxictosomeconponentofthecytoplasrn

orthecellmenbrane,withlittteeffectonthenucleus.Sinilar

sLructures to those seen by Bannigan and Burke (1982) were observed in

thepresentstudyatthecellularlevel,howeverfollowing

ullrastructuralexamÍnationthesestructureswerefoundtobe

abnormal organelles such as swollen nitochondria with damaged cristae

and RER with dilafed cisternae. A sinilar observation was made 1n

Iiver tissue followÍng alcohol exposure (Lieber 1982), where vesiculated

RER often contained cell debris and mitochondria were enlarged with

abnormal cristae following alcohol exposure'

Bannigan and Burke (1982) have proposed that the neural tube failed to

close because of growth retardation resulting from the loss of large

nunbers of neuroepithellal cells This nay not be the underlying factor of

impaired neural fold fusion however' as the neuroepithelium in the present

study was severely affected even in embryos where the neural tube had

closed.Analternativeexplanationmaybethattheneuraltubeis

prevented from closing as a result of a very disrupted lunenaL border in

the neural crest region, where fusion of neural folds occurs' since

ethanol nay exert an effect on the extracellular nacrornolecules (surface

coat naterial), which are thought to play a role in neural tube closure

(Sadler 1978, Lee et aI 1976). This explanation is further supported by a

snoother appearance of the lumenal border at the sile of fusion along the

dorsal aspect of lhe craniun in enbryoswith closed neural tubes in bhe

presenb study. These considerations ted bhe present author to propose that

6_47

lhe mechanism by which ethanol interferes with neural tube closure Ís

Índependent of its necrotic cell-depleting action on the neuroepithelium,

which in turn may be linked with the severe nent,al retardation associated

with children of alcoholic mothers (Jones et al 1974; Streissguth et al

1978).

It is difficult to pinpoint a site of acLion and mechanj-sn for ethanoL

teratogenicity from histological and ultrastructural- studies alone. The

effect on mitochondria observed in this study suggests that ethanol nay

interfere wibh oxidative phosphorylation, the neans by which the ceIl

provides enerry for functions such as celI nembrane maintenance and

int,egrity. However, many other mechanisns have been proposed and anong the

celluLar effects considered are increased peroxidative activity

(Dreosti 1984) and free radical generatÍon (Zidenberg-Chenr et al 1988),

interference w ith cytoskeletal cornponents (Hassler & Moran 1 986) '

dininished DNA synthesis and reduced rate of ceLl division (Dreosti et aI

1981; Webster 1989), direcb effects on nenbranes resultfng in excessive

flufdity (Daniel & Evans 1982), and reduced yolk sac developnent (ïüynter

et al 1983). The observation however by (Wynter et aI 1983) that some

enbryos with curvature defects and open anterior neural tubes grew within

yolk sacs w it,h w elL developed circul-ation inpl ies instead that ethanol

exerts its dysmorphic aclion directly on embryonie tissue. This is

supported by the observation that ethanot did not affect the yolk sac of

the embryonic rat aL nid gestation (Jollie 1990). Studies showing

inberference w ith ce11 nigration and subsequent effects on

gastrulation and neural crest populabions also suggest inhlbition of

morphogenetic movement as a slgnÍficant effect of ethanoL on developing

embryos (Nakatsuji 1983; Nakatsuii & Johnson 1p84; Hassler & Moran 1986).

Since there is considerable evidence in support of a nunber of likeJ.y

6-48

mechanisms of action, it is probabJ.e that ethanof teratogenicity is not

mediated by a single factor, but rather t'hab this agent has a

nuttifactorial effect on the developing embryo, which would nake the

organism especially vutnerable to other teratogenic influences relating

to nutritional inbalance or environmental toxins and drugs.

The results of t,his study which relate to the pathogenesis of the FAS

indicate thab alcohol exerts a direct norphoJ.ogical and cyloloxic effect

on the developing mammalian embryo, irrespective of maternal rnetabolism'

and consequently is a teratogenic agent, if not the teratogenic agent

responsible for FAS, and that alcohol exert,s a severe effect on the

developing neural t,ube leading to open neural tubes and nicrocephaly.

Ultrastructurally, neuroepitheliat cell death is a prominent feature of

affected embryos with severely disrupted mitochondria. In addifion' open

neural tubes may be linked with the disruption of basal lanÍna and J.arge

cytoplasnic blebs, while necrosis nay underlie the frequently observed

nicrocephaly and may be the principal factor Ín behavioural abnornalities

and nental retardation, connon features of the FAS

Another agent which is frequenbly associated with alcohol ingestion is

nicoLine. The effects of cigarebte smoking often confound epidemiological

data, and like alcohol this agent has been shown to exhibit growth

rebarding effects and microcephaly in the fetus, as well as behavloural

abnorrnalit,ies and mental rebardation in children. Because smoking and

alcohol consunption often go rrhand in handn in this socÍety, it seened

pertÍnent that the morphological and ultrastructural outcome of nicotine

exposure inrabs be investÍgated and bhis is descrÍbed in the foJ.Iowing

chapter.

6-49

6.5 BBL]OGRAPHY.

AbeI, EL. (1978) Effects of ethanol on pregnant rats and their offspring'

of alcohof on Iearning in rals'

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dífferent periods of development: changes in hippocanpal mossy fibers'Dev. Brain Res. 17, 280-284.tlisniewski, K., Danbska, M., Shar, JH. & QazÍ, a' (1983) A cIinical

neuropathological' study of the fetal alcohol syndrome. Neuropediatrics1 4, 197-201.ÏJoodson, PM. & Ritchey, sJ. (1979) Effect of naternal alcohol consulnption

on fetal brain ceII number and size. Nutr. Rep. Int' 20, 225-228'ÏIright, JT., !Íaterson, J. r Barrison, IE', TopI is, P' et aI' ( 1 983)

Alcohol consumption, pregnancy and lor¿ birth rate. Lanceb 1, 663-665'Wynter, JM., ltalsh, DA., I'lebster, !lS', McEwen, SE., & Lipson' AH' ( 1983)

TeratogenesÍs after acule alcohol exposure in cultured rat embryos'Teratogãn Carcinogen Mutagen 3, 421-428'yang,HY., Shurn, lrc., Ng, HT. & Chen, cF. (1986) Effect of ethanol on

human unbilical artery and vein in vitro. Gynecol' Obstet' Invest' 21t

131-135.Zidenberg-cherr, s., Benak, PA., Hurley' LS. & Keen, cL. (1988) Altered

nineral metabolism: A nechanisn underlying the fetal alcohol syndrone in

6-57

rats. Drug Nutr. Interact' 5, 257'Z14'

7 -1

CHAPTER 7

THE EFFECTS OF NICOTINE TN VITRO ON THE DEVELOPING NERVOUS SYSTEM AND

OTHER STRUCTURES.

7.1 INTRODUCTION.

7 .1 .1 HistorY.

The development of the tobacco habit has been briefly sunnarised from the

account by Ashton and stepney (1982)r which shouÌd be consulted if further

information is required. The use of tobacco snoke prior to the late 15th

century, probably initially for relígious and ritual purposes' but later

also for pleasure played an important role Ín Mayan civiLÍsation and

ultimately spread throughout North and South America. Tobacco smoking

was first observed by Europeans in 1492 in their travels to the New lIJrId

with Columbus. During the 16th century, tobacco cultivation and the

medÍcinal use of the Ieaf became widespread in Europe, although the

recreational use of bobacco was at first largely confined to American

colonists, sailors and inhabitants of the naritine ports' It was thought

by many to be prophylactic against the plague and to cure a variety

of complaÍnbs including headaches, asthma, gout, scabies, ulcers, labour

pains and ironically, even cancer. It was the belief in the medicinal

properties of tobacco whích caused Jean Nicot, the French anbassador to

portugal, after whom the plant was named Nicotiana tabacun, in 1570' to

Íntroduce the leaves to France and to encoura8e their culbivatÍon

In t,he late 16th century, English colonlsts in Virginia began smoking

tobacco and the hablt spread to England. During the 17th century smoking

exlended throughoul Europe, Russia and many other countrles

includlng the Far East, and despite harsh penalties in a nunber of

countries such as Turkey, Persia, Russla and Japan, the habib could nob be

checked. Nicotine was first Ísolafed fron the leaves 1n 1828 by

7-1I

the incidence of nmininal brain dysfunctionrt(Dunn et aL 1977; Mau 1980).

In contrast, in a careful- follow up study of offspring of a large and

homogeneous population of snokÍng mothers, Hardy & Mellits (1972) failed

to detecl any significant difference in eibher physical neasure¡nenbs or

intellectual functioning in 4 and f year olds as compared with non-snoking

rnobhers. Ïlhile nicotine is only one component of cigarette smoke

whÍch may contribute bo the increased incidence of congenÍtaI

abnormalities, a number of biochemical studies described in the following

section implicale it in having a detrimental effect in neural tissue,

which may contribute to the increased incidence of neural tube defects

and behavÍouraL deficits associated with cigarette s¡noking reported in

some human and ani-mal studies.

7 .1.7 Nicotine and enbryonic development in aninals.

7 ,1.7 a Birthweight and perinatal mortality.

The pattern of stillbirths, fetal wastage, neonatal- deaths' reduced

birthweight and slowed developnenb observed in human offspring followÍng

nabernal smoking during pregnancy has been replicated under controlled

conditions in sone animaL models which employed nicotÍne as the agent

(Becker et aI 1968; Becker & Martin 1971; Hamner & Mitchell 1979).

Administration of nicotine between 0.5 and 5nglkg twice daily throughout

pregnancy has been reported bo Iead to increased gestation length and

postnatal mortality, as well as reduced litter size, maternaL food intake

and weight gaÍn (Becker et al 1968; Becker & Martin 1971; Hudson & Tiniras

1g7Z). In contrast, AbeI et al (1979), found no significant effects on

gestatlon length, litbersize or birthweighl for rats injected with 1.5

ng/kg dally when pair-fed controls were incLuded ln bhe experinent'

althougÌr lt was suggested lhat the discrepancy may lie in the lower dosage

used in hÍs study. Abel et al ( 1979) also falled to observe any

1-19

significant differences in nost organ weights of adult rats whose mothers

received nicotine throughout pregnancy, conpared with pair-fed offspring'

which was in accordance with the observations that body weÍghl of

nicotine-exposed offspring are reporled to catch up to thaf of non-exposed

offsprÍng by lhe time of weaning (Becker et al 1968)'

T.l.TbTeratogeniceffectsofcigarettesmokingandnicotine.

In severaL animaL studies a deliberate attenpt has been nade to control

for many of the variables which have previously led to inconsistent

findings with hunans, bub no clear picture has yet energed, aS anÍnal

experiments of cigarette or nicotine exposure and malfornations have

produced confJ-icting resulbs. Nicotine injected into chick enbryos in

doses equivalent to the amount absorbed Ín humans fron smoking one

cigarette has induced vertebral anornalies (Landauer 1960; Upshall 1972),

while skeletal abnormalities have also been reported in mouse neonates

(Nashinura & Nakai 1958; Paulson et aI 1988), with a naxÍmum nalfor¡nation

rate occurring when the aninals were Lreated with nicotlne between days 9

and 11 of gestation in the early study, which suggested that the conpound

interfered with osteogenesis. Ingestion of tobacco plants by pregnant

cattle and pigs also regularly led lo musculo-skeletal anomalies in

the offspring (Menges et aI 1970; Crow & Pike 1973; Keeler et aI 1981)'

In contrast, pregnant rats exposed to smoke equivalent to 30 cigarettes

twice daily did not denonstrate an increased frequency of skeletal

anonalies followÍng fixÍng and staining of fetuses (Reckzeh et aI 197Ð,

nor were there any signlficant teratological effects when pregnant rats

were subjected to varlable doses of nicotine (Lindenschnldt &

Persaud 1980; Persaud 1982).

Atthough studies in chicks (Landauer 1 960; Gatling 1964) reported

7 -20

several abnornalities including cephalic lesions foLlowing injections of

nicotine fnto the embryos, there were no reports of anencephaly or other

nen/ous systen anonalies in these animals, nor in rats exposed to heavy

snoking or nicotine throughout pregnancy (Geller 1959i Becker et aI 1968)'

albhough craniofacial abnornalities have recently been observed in mouse

fetuses (Saad et aI lggù. whereas a number of animal studies reported a

nÍcotine and snoking effect during organogenesis, the effects of which

are fhought t,o be mainly atLributable to nicotÍne inpairment of

placental function, (Becker & King 1966; Hudson & Timiras 1972), several

studies also indicate that fhe embryo is susceptible to the effects

of nicotine prior to and during inplantation and well before the

establishnent of the placenta. Adminislration of nicotine to rats during

the first 5 days of pregnancy has been reported lo suppress embryo growth

(Card & MitchelI lglgi Hanrner & Mitchell 1979; Hanner et aI 197Ð, and ib

has been suggested thab this effect nay in part occur from reduced uterine

blood flow and decreased intrauberÍne oxygen tension (Harnmer et aI 1981;

Mitchell et aI 1983). As well as reduced lubal btood flow' lhis Sroup

(Mitchell & Hanmer 1985) also reponfed that exposure to nicotine in the

rat led to reduced enbryonic cel1 proJ-iferation. Hence iü appears thab

nicotine exerts an effect on the embryo even during the preinplantation

period, and the consequences of delayed developnent persisl beyond the

period of nicotine exposure. Balling and BeÍer (1985), in an ix vifro

study of preimplantabÍon embryos, observed a sÍnilar decrease in

development fron the sÍngle cell stage Ín rabbits, as welf as a decrease

tn DNA synthesis, hence denonstrating t,hat nicotine exerts an effect on

embryonic development which is at least in part not mediated by naternal

nechanisns. These observations occurred at concentratlons of nicotine

hlgher than those that may be expected in the blood of hunans considered

7 -21

Lo be nnor¡naI snokersrf.

Despite lhe absence of overt neural dysnorpholory in rodents, in utero

exposure to nÍcotine or tobacco has been correlated with reduced ceLl

numbers in fetal rat brain and other organs (Haworth & Ford 1972), as wel]

as lower fetal and brain weight (Younoszai et aI 1969; Haworth & Ford

1972). Rat studies that showed reduced DNA and protein leveLs in

developÍng brain suggested developmental delays which persisted lnto the

postnatal perÍod (Hudson et al 1974). Slot,kin et al (1986) showed

equivalent impairment of brain and body weÍght in rat offspring following

maternal nicotine injections during pregnancy ralher Lhan the normal

rrbrain-sparingr situation following non-specific toxic insults (Reunis &

Goldman 1980), which suggests that there is a nore selective action on

braln üissue development in nicotine-exposed group. This was reflected ín

the nicotine group showing greater abnormaliLies Ín ornithine

decarboxylase (a known najor regulator of macronoLecule synthesis during

replication and differentiation ) activity in fetal brain than in other

rat bissues ( slotkin et ar 1 984). A prolonged elevation of

ornithine decarboxylase has been shown previously to be associated with an

overall delay 1n the course of ceLlular naturatlon (Slotkin el al 1984),

and this pattern of ornithine decarboxylase persisted well intopostnafal 1Ífe, days lo weeks after terninatÍon of nicotine exposure

(Slotkin et al 1986). Hence it has been postulated ( Al-Hachim & Mahmoud

1985; Lajtha & Sershen 1986; Seidler et aI 1986) that nicotine exposure

during early fetal developnent regulates growth and developnent of the

central nervous sysüen, eventually translating into various functional and

slructural abnormalities.

These biochenlcal abnormalities nay be tinked to the long-tern effects of

7 -22

snoking and nicotine in pregnant rats whÍch have been reported to lead to

impaired responses in the offspring (Martin & Becker 1972) where a daily

injection of 3mg of nicotine throughout pregnancy led to a learning

deficit in the offspring, a finding similar fo that observed in

epidemiological studies wÍth humans (gutler & Goldstein 1973, Dunn et aI

1976). fn contrast, BertoLini et al (1982) reported a significant Íncrease

in the rate of learning at 60 days postnatally in rat offspring whose

mothers !vere exposed to smoke daily throughout gestation conpared with

controls. No other changes Ín parameters including brain weight were

affected however. The improved avoidance acquisition of the offsprÍng of

these cigarette smoke-exposed dans was considered by these workers as not

due to nícotine, as nicotine exposure during pregnancy had no significant

effect of any kind (Bertolini et aI 1982), and hence Ít was suggested that

the behavioural effect nay be attributed to a nunber of ot,her factors.

7 .1 ,8. Ai-us of the present study.

The conflícting evidence frorn hunans and aninals as outlined above with

respect to the norphological effects of nicotine, particularly in relation

to the development of the central nervous system Ied the author to exanine

whether nicotine is a nervous system teratogen, and whether it nay exert

its effect at the time of neunulation. These questions were investigated

in cult,ured rat embryos as this systen provides a means by which lhe

direct terafogenicity of nicotine per se can be gauged rather than of its

netabolltes which need to be consldered when the naternal environ¡nent is

present.

7 -23

7.2 MATERTALS AND METHoDS.

T .2.1 Aninals.

virgin fenale sprague-Dawtey rats (200-250Ð were placed overnight with

nares of the same strain AnÍnals with positive vaginal smears were housed

individually and the tine of detection of sperm was designated as day 0'5'

of gestation.

7.2,2 Enbryo culture and beratological screening'

A detailed account of the embryo culture technique can be found in

chapter 2 of this thesis. On day 9.5 of gestatÍon, dams were anaesthetised

with diethyl ether, uteri were renoved and enbryos were cleared of

decidual tissue and Reicherts membrane for enbryo curture. Groups of 3

early head-fold embryos were placed in 60mI culture bottles along with

3mI of prewarmed nedium and gassed with oxy8en' nltrogen and carbon

dioxide af, the concentrations previously described. where appropriate

nicotine (freebase, 98- 1OOl,, Signa) was made up to the required

concenbration in sterÍle water in the range of 1ooug/nl to 400u9/mI' and

added to bhe culture bottles. The appropriate range of concentrations

was deternined iniLially in a pilot study where embryos were cultured in

nicotine at levels of O, 25t 1OO' 250 and 500 ugln]'

After forty-eight hours in culture, enbryos were removed and exarnined

under the dissecling nicroscope for signs of impaired growth and gross

dysmorphology. Representative enbryos $tere processed for Iieht and

electron microscopy as described ln chapter 2'

7.2.3 Statisfics.

Continuous growth variables such as crolJIFrulnp length and sonite nunbers

Fon discrete develoPnent

tube defects a naxÍmun

were analysed by standard analysls of variance'

data such as number of enbryos wlth neuraL

T -24

likeLlhood method was used assuning the data to be binonially dfstributed

and the devlance to be approxinately dlst,rÍbuted as X2 on t,he appropriate

degrees of freedon (Baker & Nelder 1 978).

7 .3 RESULTS.

7 .3.1 Grcn¡th and norphological developnent.

Deternlnatlon of appropriate concentrat,ions from an j¡ vitro pilot study

(Fig. T.1a) revealed lhat at a concentration of 25ug/mI, enbryos !ûere

growth reduced conpared with controls, but did not denonstrate ar¡y

dysnorphology. At concentratlons of 100 and 250 uglmL dysnorphology was

dose-dependent whlle at a concentration of 500 uglnl the enbryos $rere

severely growth and developnentally retarded with only a rudlmentary heart

tuber and a heartbeat was nearly always absent. A representatlve enbryo

cultured in 250 ue/nI of nicotine (Fí9. 7.1b) $¡as growth reduced with a

diminished forebrain characteristic of nfcrocephaly. The abnormal

branchial arches were fused to the perlcardium and the poorly

developed caudal region was typical of enbryos which had not rotaüed into

the dorsi-convex position. FolIow ing norphological and ultrastructural

exanination of enbryos Ín the pllot study, concentrations of nicotine fron

100u9/nI to 400 uglnl were considered a suit,able range for use in the

presenb study.

Enbryos explanted on day 9.5 of gesüation and cultured for 48 hours inraf serun containing nÍcotine at varÍous concentratlons showed

sÍgnificant retardatlon of several growth indÍces conpared wilh controls

(Table 7.1) there was a clear and sÍgnlficant dependence on dose for both

crolrrr-rulnp length (r' 4,g1=47.14 p(0.001) and sonite nunber (r 4,g1=67.99

p<0.001). Ttre yolk sac dÍaneter of enbryos cultured ín 100 uglnl was not

different fron conüroIs, hower¡er a sinilar dose-dependent reductj-on 1n

7 -25

slze occurred at doses grealer than 200 ug./nI (F 4, g.1=27.96 p<0.001).

As well as a reductlon in yolk sac sfze, nany enbryos also showed poor

developmenb of the yotk sac vasculature at higher concentrations of

nicotine, when conpared wibh controls (Figs. 7.1crd). The yolk sac !s the

princlpal route of nutrient uptake for pre-placental rat enbryos (Bect

1967), and is equÍvalent to the syncytlotrophoblast in fhe human (I{endell-

Snith & l{illians 1984).

A nunber of the morphological paraneters exanined when the enbryos were

cultured in nÍcotine showed a concentration-related dysmorphologr whlch

Ìras statislÍcally highly significant (Tabte 7.2). 0f particular interest

was the ÍncÍdence of overt anterior neural tube dysnorphologr whfch rose

lron 20l in enbryos treated wÍth nlcotine at 100 uglnl to nearly all

enbryos affected at 300 ug/n].

The nost signifÍcant anonalies of the CNS included narked under-

developnent of the nidbraln and forebrain regÍons with frequent distortion

of the latter. Ele defects were orùy observed at higher concentratlons,

with anonalies including nlcrophthalnÍa and anophthalnÍa, while branchial

arches were frequently distorüed and/or reduced in size 1n up to 86f of

Table 7.1 Effect of nicotÍne on grorth of rat embryos;L¡ v1tro.1

Nicotine Concentration (ug/nI serun)

0 100 200 300 400

Total enbryosCrowtrrunpIength (nn)

No. sonitesYoIk sac dian.( nn)

21

3.3x0.1

22.5¡0.5

4.1to.t

15

I .0g0. 1

20.59.9

4.1t0. t

21

2,6¡0 .1

16 .o$ .9

3 .8t0.1

15

2.2¡0.1

1 1 .4+0.5

14

1 .7$.1

7 .7¡0.6

3.ofl.1

.lI Va1ues are neans + SEM

3.39.1

7 -26

Fig. 7.1. a. Ttre photograph shows the degree of teratogenesis associated

with 11.5 day enbryos cultured for 48 hours at different levels ofnicotine fron O (LHSj, zS, 1oo , 250 to 50O ug/ ml (nHS)'

b. VentraL view of a representative severely dysnorphlc enbryo culturedin 250 ug/mf showing a reduced forebrain (arrow) and abnormal nandibulararches (1) whlch are fused to the pericandlum (arrow heads). H, heart.

crd. yolk sacs (ys) of 11.5 day j¿ vitro embryos cultured 1n normal ratserum and Ín serun containing 100 uglmt nicotlne respectlvely. The

treated enbryos and corresponding yolk sacs were bolh growth retardedand the latter failed to develop the vasculature indicated by the arrowsin the control enbrYo fn c.

Bars=50oun.

7 -27

embryos at 400 ug/nI. Retarded devel-opment of the pnimitive heart tube

was rarely observed except at the highest level- of nicotine, however all

embryos exhÍbited heart beabs. fmpaired dorso-lateral flexion and chorio-

alLantoic placental fonmation were often observed in nÍcotine treated

embryos throughout the range of concentrations.

T .3.? Scanníng electron microscopic observations.

Fig 7.2 demonstrates the typical dysnorphology observed in embryos

exposed to incneasing concentrations of nicotÍne during neurulation.

!lhile the cranial neuropore was nearly a1w ays cl-osed in nicotÍne-

cultured 11.5 day enbryos, they were often microcephalÍc with oddly

shaped rostral portions including under developed nidbrains and forebrains

even at, the lowesü concentration of 100 uglmI (Fig. 7.2a).

Culture of embryos in 200 ug/nl nÍcotine often led to cranial anornali.es

which consisted of hypolasia of the forebrain and in sone cases of the

mÍdbraÍn as weI1. In addition, nandibular arches had developed abnormally

whereas hyoid and third branchial arches appeared to denonstrate

developmental delay. Optic placodes were also absent while otic vesicles

appeared moderately erùarged at this concentration suggesting otic placode

delay. A section of the forebrain surface ectoderur (Fig. 7.2c) revealed an

abundance of short microvilli extending over the entire surface of the

ectodernal cells, with occasional long nicroviLli interspersed anong then.

The densÍty and length of the mlcrovÍIli appeared to increase with

increasing concentration of nicotine (Flg 7.Ze). The surface ectodern of

affected enbryos can be conpared wÍth a sinilar sÍte fn a control enbryo

in FÍg. 6.2b.

As the concentration of nicotine fncreased to 300 ug/nt (Fie.7.2d) tt¡e

craniun was even nore abnornal in appearance, and in sone embryos the

7 -28

Table 7.2 Effect of- nicotine on norphological developnent 1n rat embryos

ån vitro. 1

Significance ofconcentration-related effect

(P)

Total embryos 21 15 21 15 14

Any kind ofdefect5(21,7)9(60.0)16(76'2)15(100'0)14(100'0)<<0'001Neural tubedefecrs o(o) 3(20) 11(52.4) 14(93.3) 14(100'0) <0'001

Ele defecrs 0( o) 0( o) 2(9 .Ð 4(26 .T) 13(92.9) <0.001

Ear def ects o( o) o( o) 2(9.Ð 1(6 '7 ) 4( 28 '6) <0 '024Branchial arch

def ects o(0) 2( 13.3) 6(28.6) 6(40 '0) 12(85 '7) <<0'001

Heart defects o(o) 1rc.7) 3(14.3) 1rc'7) 7(50'0) <0'001

Absence offoreLinbs o(o) 1(6.7 ) 2(9.5) 8(53'3) 13( 92'9) <0'001

Inconpleteflexion 1(4.4) 3(20.0) 8(38.1) 15(100'0)13(92'9) <0'001Impairedfusion 5(21 .7) 9(60.0) 13(61.9) 10(66.7) 12(85'7) <0'003YoIk saccircularÍon 3(14.3) 5(33.3) 13(51.9) 15(100.0)14(100.0) <0.001

;--------I Percentages given in parentheses.

forebraín vesicles had collapsed. AbnormalÍtles of the branchlal arches

were also nore severe at this concentration and included dislortion of the

hypoplastic mandibuLar arch and maxillary component as well as fusion of

the arches to the pericardÍun. Once again the second and third branchial

arches denonstrated developmental de1ay. At bhis concentration oti.c

vesÍcl-e fornatlon also appeared to be delayed, as lndÍcabed by the

perslstence and apparently enlarged opening of thls structure, which

howeven appeared to be correctly aligned with the hyoid arct¡. Anonalles of

the obic vesLcle and branchial arches are demonstrated at hlgher

nagnlflcalion tn Fig.7.2h and can be compared wtth these features in a

control enbryo (FÍg. 7,2e) of the sane age.

Nicotine concentration (ug/nI serum)

o 1OO 200 300 400

Fig. 7.2. a. Ani¡ vÍtro day 11.5 embryo cultured in 100 uglml nicotlnewhich appears rerativerv welL developed except for an underdeveloped

forebrain and midbrain

b. An i¿ vitro day 11.5 enbryo cultured in 200 uglnl nicotine showing

hypoplastic foreUrãtn (asterisks), diminished nidbrain' abnornal

mandÍburar arches and derayed olic vesicle formation (arrow). The opticplacode does not appear to be present. Note that fhe neural foldshave fused, except for the posterior neì'rropore (arrowheads)' The asteriskmarks the approxl¡nate region seen at higher nagnification ln c.

7 -29

c. A section of surface ectoderm fron the forebraÍn regfon narked by an

asterisk in b. The surface was covered in an abundance of nicrovilli inexcess of the numbers observed at a comparable site in control ;iA vitroenbryos (c.f. Fíg. 6.2b).

d. An i¡ vitro day 11.5 embryo exposed to 300 uglml nicot'ine 1n cultureshowing bhe unãerOevetoped forebrain and midbrain characteristic ofnicotine-cultured enbryos. In addÍüion the forebrain in this enbryo has

collapsed aL the síte where the neural folds have fused (arrow). The

nandibular arches and naxillary processes were also frequently severelydysnorphlc and otic veslele fornatlon was delayed (arrowhead). Tt¡e

asterist< narks the appnoxÍnate area seen at hÍgher magnifÍcation in e'

e. A section from the forebrain suface ectoderm narked by an asterÍsk ind. At 3OO ug/ml of nicotine the surface is covered in very long extensÍvenÍcrovillf' and interspersed with blebs (asterisks)'

f.AnÅ¡vltroenbryoculburedin400ug/mlofnicotÍne.Atthisconcentratlon the enbrVo shows severe overall devetopmental retardationThe cranial- regions are indisbÍnguishable fron each other, the site ofneural fold fuãion appears to have collapsed (arrowheads) and t'he

mandibular arches are reduced and very abnormal. The stonodeun (arrow)

has become readily vlsible as a result of trypoplasia of the forebrain and

¡nandlbular arches.

g,h. Higher nagnificatlon of the branchial arch regions of 11'5 day

nõrnal (wf,or" enbryo not shown) and 300 uglml nlcotine-culüured enbryos

respecllvely showlng fusion of the abnornal mandlbular arch to thepenicarOtum (arrowhÀads) of the treated anÍroal, as well as lncompletedevelopnent of the second and third arches. The nicrographs also show thatottc vestcle (arrows) fornaffon appears to be delayed indlcated by thepresence of the erùarged opening of this strucfure in h, compared with theconbrol.

Fb, forebralni Mb, nidbrain; Hb, hindbraini 11213, branchlal archesi Mxt

naxÍllary process; H, hearb; F, Fonelinb bud; S' somLtes'

Bars=10Oun, arb, d, frgr h; 2umr cr ê.

7-30

At the highest, concentration of nicotine (400 ue/mL) the embryos showed

considerable developnental retardation (Fig. 7.2f) and often appeared

microcephalic or anencephalic, with complete absence of the forebrain, and

reduced midbrain and hindbrain regions. ThÍs was often accompanÍed by

collapse of the neural folds at fhe site of fusion along the dorsal aspect

of the neural tube. Hypoplasia of the forebrain and ¡nandibular arches Led

to exposure of the stomodeum or primilive mouth, whÍIe the second and

third branchÍal arches wene frequently absent in bhese embryos.

7 .3.3 CeIluIar observations.

Light microscopy of embryos cultured 1n varying concentrations of

nicotine revealed that the severity of cellulan abnornalitÍes

increased with increasing concentrations of nicotine. Histological

examination of a horizontal section through the cranial- neuraL tube of

a typical 11.5 day conlrol enbryo (Fig. 7.3a) and an embryo of similar

age cultured Ín 300 ueln1 (FÍ8.7.3b) revealed that whÍle fusion of the

anterior neuropore had occurred in both, the neuroepiühelium of the

nicotine cultured enbryo was diminished in thickness with fewer cells

partÍcularly in the lateraL regions of the neural tube and many

large intercellular spaces. These celluLar abnornalities were observed

even at the Iowest concentration of nÍcotine but the effect was l-ess

severe, The nesenchyme was also affected with nany of the celLs in this

Iayer aggregated close to the surface ectoderm, such that nost of the

mesenchynal area appeared enpty at this magnÍfication

Higher magnification (Fig. 7.3c) of a seclion of the neuroepÍthelÍum in

Fig. 7.3b showed a relatively snooth apical surface with snall blebs' and

many large dark intra- and extracelluLar bodies present throughout the

neuroepithelial wall of the forebrain and nidbrain, which suggested that

7 -31

Fig. 7.3. â,b. Horizontal sections through the cranium of an 11'5 day j¡vítro contror enbryo and an embryo curtured in 300 ug/mr nicotinerespectively. The neuroepithelium (N) of the treaLed embryo is dininishedin thickness particularly in the lateral regions of the neural tube' withmany extracellul"" "p""""

in this area (arrows). The mesenchyme (M) was

also affected, as most ceIls appeared to have aggregated close to the

surface ectodern (E), which appeared to consÍst of thicker cells in the

nicotine-treated "rï"yo, while most of the space in this region of the

neural tube wa" oooupi"d by large, abnormal, developing blood vessels(bv). The arrowheads and arrol^rs in b, mark the regions seen at highernagnification in c and d respectively'

c. HÍgher magnification of an area in the neuroepithelium marked by

arrowheads in b, showing severe ceII disruption and shrinkage throughout

the width of ih" n"u"oãpitheliun (N) as well as along the basal end

adjacent to the mesenchyne where the ceLl-s no longer appear to be restingon a basal lamina (openärrows). Despite evidence of severe necrosis which

Íncluded dense extraäellular debris and cell inclusÍons, nitotic figures(Mf) were frequ"ntry visible adjacent to the venlricul-ar surface which was

relatively smooth with only srnall blebs (arrowheads)' bv, blood vessef'

d. Higher magnification of an area ín the mesenchyrne marked by arrows inb. Many of the aggregated mesenchymal cells (M) appeared to have losftheir stellate snifr, orientation, and interconnecling filopodia' Some

intracellular and extraceLlular condensed structures (arrows) can also

be seen. The thickened surface ectoderm (E) contained a nunber ofvacuolated ceIIs and dense inclusions (arrowheads)' N, neuroepit'heliun;bv, blood vessels'

Bar s= 50um.

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T -32

extensive cell death had occurred at these sites.

Higher nagnificatÍon of the underlying mesenchyrne (Fig. 7.3d) showed that

interspersed arnongst the aggregated nesenchymal cells were fragnents of

extracellulan debris, and nany of the ceLls contaÍned dense dark

inclusÍons, sÍmilar to those seen in the neuroepithelÍun. The regÍons in

the nesenchyme which appeared enpty at low magnificatÍon vüere Ín fact

occupied by extrenely lange abnormally developing blood vessels, whose

endothetial cells also showed evj.dence of necrosis, although not, visible

Ín this secbion. These large vesseLs were seen in many of the

nicotine-cultured enbryos. The ce1Is of the surface ectoderm adjacent

to the nesenchyne were also affected by nicoüine and showed evidence of

patholory.

7.3.4 Ultrastructural observations.

Transnission electron microscopy of representative enbryos cultured Ín

nlcotlne for 48 hours during the period of neurogenesls extends bhe

observations of celluIar disruptlon seen under the 1Íght microscope, and

confirms that the anterior neural tube is affecüed by nicotine 1n a dose-

dependent manner at the ultrastructural level, with extensive cel1

deabh in the neuroepithelÍum and with a lesser effect in the nesenchyne.

The mlcrograph of Fig. 7.4a st¡ows a section of neuroeplthelium and

adjacent mesencl¡yne from a lateral regfon of the anterlor neunal tube of

an enbryo cultured 1n 300 ug/nl nicotlne. Many fragnents of dead or dying

cell components were observed 1n the large extracellular spaces

lndlcatlng that cell loss and shrinkage had occurred. Sone of this cell

debris appeared to be extruded inbo the nesenchyne at sltes where the

basal lanlna was broken Dead celL remnants were also presenb Ín other

healtty neuroepithelÍaI cells. At t,his low nagniff catlon the cytoplasn

7 -33

appeared to be vacuolated and the nucleÍ contaÍned very abnornal

nucleoli. Despite the extensive necrosis 1n the lateral neuroepithelium,

mltotic cells were abundant and dÍd not appear to be affected by

nicotine. Higher magnificatlon of neuroepiLhelial celIs fron Lhe sane

region of t,his nÍcotine-cultured enbryo (Fie. 7.4b) showed that the

nvacuolesn seen in the cyloplasm in Fig. 7 .4a Ír ere damaged

mitochondrÍa whose cristae w ere severely disrupted by the

treatnent. Conlrol- chromatin (Fig. 7.4c) denonstrated a clunped appearance

whereas the chronatin in the nicotine-cuLtu¡ed enbryo was dispersed,

a feature indicative of increased protein synthesis. The nucleoli also

had a ribbor¡-lÍke appearance and were sinilar to structures described

as nwanderingrf nucleolÍ observed in germ ceII tumours and Ís suggestive

of overactivity of DNA synthesis (GhadÍal-Iy 1988).

Cellt¡lar disruption was not conf ined to the nesenchymal end of the

newoepltheliun. Tlre apical reþÍon of the neuroeplthelÍal ce1ls whÍch

abuts the ventricular lumen stas also affected in embryos exposed to

300 ug/ul nicotine (Fig. 7.4d), and compared with a sirnilar site in a

control enbryo (Fig. 7.4e) showed extensive disruption of the

mÍtochondria, with sone vaeuolation, whÍle the ceI1 conplexes and the

renaining cytoplasnic organelles did not appear to be affected.

Necrosis was also evÍdent in the nesenchyne of nicotÍne-cultured embryos

(Ffe. 7.4f). tJhile the ¡nesenchymal ceIIs in control embryos (Fig. ?.4e) at,

a similan site, are normalty stellateshaped wlth extensive cytoplasmic

projections, those ln experfnental animals were often condensed and

contained sinilar danaged nitochondrial cristae as seen 1n

neuroepithelial cells, and nany lysosone-Lfke structures assoclated wÍth

cell debris lyfng wlthln the extracellular maürix. At the lower end of the

T -34

Fig. 7.4. a. Electron erior neural tube

of a day 11.5 embryo showing severe

disruption of bhe ne acellular spaces

often contained dead hich was extruded

into the extracellufar natrix (ECM) ) ab sites where

bhe basal lanina was broken (open arrows)' Some apparently healthyceIIs contaÍned dense inclusions (arrowheads). Despite the severenecrosis, nitotic figures (Mf) were still visible. Note the apparent

vacuolation of nany oi th"s" cells. The asterisk marks the site seen athigher magnification in b.

brc. Higher nagnification of several neuroepithelia-[ cells from an

"r'bryo crrltured iñ gOO ug7ml nicotÍne, (region narked by the asterisk in

a), and fron a similar site in an in vitro control animal respectively'Several ultrastructural abnormalitíes which can be attributed to thenicotine treatment include the presence of ribbon-like rrwanderingrl

nucleoli (n), dispersed chronatln and nunerous nitochondria (n) withdanaged cristae. conpare these w ith the normal nucleoli (n),nÍtoc-hondria (m) and clunped chronatin (ch) of control neuroepithelialcells fn c. vtîr vesÍculated rough endoplasmic reticulun.

d, e. Electron micrographs of the apical end of the neuroepÍtheliun froman 11.5 day embryo cullwed in 300 ug/nI nicobine, and conparabte controltlssue respectÍïely. In treated embryos, there was usually Iess ce]lshrÍnkage and extracellular spaces belween the cells at this end of thenewoepitheliun (N), which was also only mitdly blebbed. The mitochondria(n) however were severely affected. The opaque circular structureslocated in areas close to danaged nitochondria may be prinary lysosomes(Ly). Conpare these featunes with conparable structures in e, and note

the absence of nicrofilanents (nf) in d. L' Lurneni Jct junctlonalconplexes; Br blebs.

frg. SectÍons of the nesenchyne (M) from the anterior neural tube of invitro day 11.5 embrryos exposed lo 300 ug/ml nlcotÍne or to nornal- serum

respectivêfy. The nicotine-treated tissue in f, Iies close to thedisrupted basal Lamlna (open arrows) (seen at low nagnificatÍon in a).The extracetlu.Lar matrix (ECM) contained cell debris (arrows) which was

often electron dense, and which was sonetimes assocÍated with lysosonal-tikeslructures(asLerÍsks).Mar¡yofthenitochondria(n)alsocontaineddanaged cristae. Compare with nornal nesenchyne in g. BL, basal lamina;bv, blood vessels.

Bars=4un.

Dr

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aI

ECM

ECM

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7 -35

nicrograph, the origin of some of these cell rer¡nants becomes apparent, as

neuroepithelial cell debris is extruded into the mesenchyme through breaks

in the basal lamina.

Fig. 7.5 shows the range of celLular necrosis typical of enbryos

exposed to 300 ug,/mI nÍcotine observed at higher nagnification Some of

the Íntracellular and exbracellular material (Fig. 7.5arb) whÍch was

seen particularly in the forebrain and mÍdbrain regions of the anterior

neural tube, was often so severely condensed that it was difficult t,o

identÍfy the individual celI conponenfs, although some nuclear and

organelle structures can be recognised Ín Fig. 7.5b. The large size of

the structures contained within the neuroepithelial cells and the healt,ty

appearance of the eells suggests that they are heterolysosomes

phagocytosed by the host cell, rather than autolysosomes, however the

presence of vacuoles ingesting cytoplasn of the host cell suggests that,

this structure may also act âs an autolysosome leading to eventual

destructlon of the host cell as welL.

In some cases entíre whole, dead condensed cells were also observed(Fig.

7.5c). the nuclear and mitochondríal components are readily identifiable,

and the circular opaque nembrane-bound structures are probably

lysosones. Another feature of uIürastruclural- pathology was the

presence of aggregations of cytoplasnic organelles particularly

nitochondrfa and cÍrcular RR (Fle. 7.5d). These organelles appeared to

be rounded up, and surrounded by adjacent apparently healthy

neuroepfthelial cells whfch nay be preparing to phagocytose then. I?¡e

nftochondria nay be the remnants of cells such as $¡ere seen 1n Ffg.

7 .4d.

T -36

Fig.T.S shows a range of nicotine-induced ultrastructural pathologl fronthe anterior neu""I tube of an 11.5 day enbryo cultured i'n 300 uglm}nicotine for 48 hours.

a. Neuroepithel ial cells eontainÍng either cell rennants Iocatedextracellularly (arrowheads), or lntracel-Iular1y within heterolysosones(anrows). Lysls within these structu¡es was often so extensive that theÍndividual cell conponents could not be recognised'

b. Some rnenbrane (arrow) and nuclear conponents (nu) of what nay have

been a condensed whole celL could still be recognised in thisheterolysosome. Severa] peripherally located vacuotes (v) appear to be

ÍngestÍng cytoplasn of the host cel1, suggesting thab this heferolysosonemat also function as an autolysosome leading to the eventual digestion ofthe host celJ.. nu, nucleus.

c,d. WhoIe condensed dead cell and cell organelles locatedextracelluJ.arly withÍn the neuroepitheliun, showing a range of organellesincludÍng nuclei (nu) mitochondria (m), vesiculated rough endoplasnicreticulum (vr) and free cytoplasmic ribosonesr (cr). Note the nenbranebound opaque structures associ.ated wÍth the necrotic naterÍal in c' v¡hichane probably prÍ"nary lysosones (Llr).

e. The extracellular matrÍx (ECM) of the nesenchyne contained a range ofce1l components at various stages of condensation from light f[occulent bo

very dense, and which included cytoplasmfc ribososmes with soneinterspersed organelles (arrows) to whole dead cells (d). These condenseddead cells appear to be preparing to ingest ceII remnants from theextracellular natrÍx eÍther through vacuoles (v) or through disrupted cel1menbranes (arrowhead). bv, blood vessel.

f. HÍgh magnification of a dying mesenchymal cell which appears to have

ingested a nunber of whole, condensed dead cells (arrows) in which then,.,õlet (nu) are cLearly visible, and is phagocytosÍng severely lysed celldebris (arrowheads). Note fhat the mitochondrÍa (m) of the host cell arealso affecfed.

Bars=2um.

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tl .'r

-J

e .r .'

1

î

I ¿

T -37

Ihe mesenclryne also contained a range of dead cell rennants. Fig. T.5e

shows cell debris at varj.ous stages of condensation from lfght flocculent

to electron dense. The close proxÍmity of much of bhis debrÍs which has

already been seen to be broken in many enbryos exanined suggests t,hat, its

origÍn is within the neuroeplthelium. The structure in Fig. 7.5f appears

to be a condensed nesenchynal cell which is acbively phagocytosing

dead ceII remnants. TT¡e cell appears to have previously phagocytosed

other whole condensed cells.

7-38

7 .4 DISq]SSION.

The present study reports for the first time the i¡ vitro effect of

nicotine on embryonic growth and morphological and ultrastructural

developnenb at the tÍme of neurulation Because of the short duration of

the action of this drug (Lichtensteiger et aI 1976, 1982; Svensson &

Engberg 1980; Taylor 1985), the continuous exposure obtained by smoking

can hardly be nimicked by indivídua.L ir¡jections. ü vltro exposure

provides an atternative to nicotine administratfon to whole anÍmals by

way of mi.ni-osmotic pumps, and to exposure in drÍnking water (Peters et

al 1979i Peters 1984) which has been proven to be unpalatable to rats

(Murrin et al 1987; Slotkin et a1 1987). LeveIs of nicotine in

steady smokers (BenowÍtz & Jacob 1984) have been observed to be raised

considerably to peak blood levels of over 50 ng./ mI for a 12 hour period

tn 24 hours and were never completely reduced. Although the leveIs of

nfcotlne required to induce growth retardatÍon and abnornalities in vitro

1n rats were considerably higher in the presenl study than was found Ín

the serun of heavy smokers, the potential of nicotine to act as a nervous

systen terabogen inhumans should not be overlooked. !fhlle the rat does

not appear as sensftive to nicobine as the hunan, at this elevaled scale

of exposure growth retardation was first observed at the lower levels of

the dose range and at slightly higher 1evels, teratogenesfs was lnduced'

and included mÍcrocephaly, branchial arch defects and ceLl death. It 1s

likely that a similan pattern of teratogenesls may follow the growth

retardatlon also seen in humans, bub at a lower dosage level. The

observatfons made in anfnals:Lg vitro 1n fhe present study nay süill be of

direct appllcation to hunans, as 1n cases such as cancer, substances

consldered to be carcinogenic and potentially hazardous nust be less than

1000-fold the leveI shown to be carcinogenlc 1n aninals 1n order to be

7 -39

considered a safe Ievel in humans (Dreosti, personal communication).

Furthermore, while the j¡ vitro systen enables a pattern of nicotine-

induced teratogenesis to be clearly defined' in vÍvo studies have shown

that growth retardation is observed at a sinilar order of magnitude in

animafs (Murrin et aI 198?) to that which has been reported in humans to

induce gnowth retardation. Hence it is possible that differences in t'he fu

vitro systen itself may underlie the lower sensitivity to nÍcotine in

rats, and nay possibly be related to the absence of the maternal

metabolism, ralher than a subsensitivity effect of nicotine in rats. Since

this study has reported a direct teratogenic effect of nicotine per se

on the developing rat neural tube further studies will be perforned which

exanine t,he norphological effects of nicotine Ín vivo.

The in vitro system was utilised in the present study as it aIIows the

investigation of a possible direct teratogenic effect of nicotine. In

addition, since alcohol teratogenesj.s mimics the hunan conditÍon more

readily -h vitro than in vivo in rats, and sj-nce the author also wished to

investígate the concurrent effects of alcohol and nÍcotine described in

the subsequent chapter, this required that the teratogens be examined

under the sane experinental conditions.

The welI established link between intrauterine growth retardafion

(present at tern) and heavy snoking associated with high nicotine

levels Ín humans (Heinonen et aI 1977; Mau & Netter 197\; Haddow et aI

1987i Bosley et aI 1981) and in rodents (Mau & Netler 1974; Dunn et al

1977, Saad et aI 1991) was confirned in this study, and was shown for the

first time to occur as early as the time of neurulation in rats' the

severlty of the effect being dose-related. It has been proposed

previously that nicotine nay exert Íts effect of fet,al growth by neans

7-40

of vasoconstriction, leading to decreased perfusion of fetal tissue

and hypoxia (Mochizuki et al 1984)r or by a growth-inhibiting action

directly on fetal tissue (Suzuki et al 1974). In this context it is

relevant to consider that while the vasculature of the anterior

neural tube is neither adequately developed nor innervated at this

stage of embryogenesis, the yolk sac vasculature however is well

developed. Sínce ib has been established that the yolk sac is the main

nutritive organ of the rodent embryo during much of the period of

organogenesis (Beck et aI 1967) any agent harnful to this membrane can

also be expected to affect fetal growth and developnent.

The present study has denonstrated that yolk sac size was severely

compronised by the presence of nicotÍne in the culture medium. As nicotine

is highly Iipid-soluble, it may exert a direct effect on the

developnent of the yolk sac leading bo inadequate digestion and absorption

of macronolecules vital for adequate nutrient supply of the embryo. A

study usÍng pregnant rhesus monkeys (Suzuki et aI 197 4) supports the

suggeslion that nicotÍne nay exert a growth-inhibiting action directly

on fetal tissue, which is supported by hlgher concentration, longer

half-life and specific fetal organ accumulation In addifion, since

the vascular development of the yolk sac which Ís crÍtica1 for adequate

and efficient oxygen circulation prior to the fornation of a functÍonal

placenta (New 1978) was also compronised, it fs possibJ-e that nicotine

nay interfere with the formation of an adequate vascular transport

systen within bhe yolk sac by an unknown nechanisrn. Hence, lnadequate

nutrition and perfusion which have been observed in rats in culture,

nay be a basis for the very high incidence of snoking-related

spontaneous abortions observed in hunans (Ktine et aI 1977), where bhe

syncyüiotrophoblast (llendell-Snlth & l{illians 1984) ls the human analogue

7 -41

of the endogenously functioning yolk sac Ín rodents.

llhile previous epideniogÍcal studies were unable to determfne with

certainty whether smoking leads to congenltal abnornalities, possibly

because of nethodoLogical and analytical differences betw een studÍes

(Ìferler et aI 1985), the present data have shown considerable nicotin+

related dysnorpholory particularly within the developing nervous systen.

Consequently, it is possible that the incidence of neural tube defects

and cleft palate observed previously in humans and rodents although

1ow, nay have been significant, but have passed undetected because

these anomalÍes r{ere nasked by the hígh incidence of spontaneous

abortion, and early fetal deabh occurrlng in heavy snokers.

llhereas nÍcotine-nedÍated embryonic dysnorphologr appears to affect a

number of organ systems, the teratogenlc effect exerted by this agent, on

the developing nervous system, futher supported by the cellr:l-ar and

ultrastructural observations, may be especially severe, since prevÍous

observations (TJalve et aI 1968), have shown brain tissue in rodents to be

one of fhe principal accumulating regions for thÍs conpound. tlhile

thÍs accunulatlon has previously been acconpanied by reduced leveIs of

brain DNA and proleln during neurogenesls, this suggests that the

growth and the consequences of developnental delays which occur aE that

stage may persist postnatal.Iy (Hudson et, aI 1974). In the present study

the observation of dispersed nuclear chromatin in nicotine-treat,ed enbryos

suggests that, DNA activily 1s erùanced. In additlon, the nucleoIl whlch

are the sites of rfbosonal RNA synthesis, assuned a ribbon-like appearance

1n treated enbryos, whlch had beenobserved previously ín actfve gern cell

tunouns (Ghadially 1988), and which may lndicate an enhanced level of Rl'lA

productlon as weII. It has been postulated that, nicotfne adnlnlstration

7 -42

during early fetal development interferes with early biochemical events

whlch regulate growth and developnent of the central nervous system, and

nay underlfe the various structural and functional abnormalities (41-

Hachim et al 1985; Seidler et al 1986).

Although narry of the surviving off spring of hearry snoking nothers appear

physically normal, psychological and intellectual deficits are apparent in

humans (Rantakiltio 1983; Naeye & Peters 1984) and aninals (Peters eü al

1979i Peters & Tang 1982; LÍchtensteiger & Schlunpf 1985), studies

clearry implicate nicotine as a behavioural teratogen. The

behavioural abnormallties seen in children of mothers who smoked during

pregnancy resembLe the symptons of mÍninal brain dysfunction, which

include short attention span, hyperactivity, poor reading ability,

sensorX¡ deficits and difficulties in social adjustment. Alterations in

central catecholanine neurons may play a role in t,his syndrone (Shaywitz

et al 1976; Raskin et aI 1984), and these systems are typical targets for

nicotÍne fn the adult brain (Lichtensteiger et aI 1988). SpecÍfic

nicotinic binding sites begin to develop as early as gestabfonal day 12 at

sone brain sitesr and are associated wfth catecholanlne nerve celLs

(Lfchtensteiger et al 1982). Hence, the present author suggests that, in

view of the severe nicotine-induced ultrastructural necrosis observed inthe anterior neural lube in thls study close to the t,ine of comnencemenü

of nicotinfc receptor developnent, this destructlon may fnterfere

with the proper developnent of the catecholamine neunons and

assoclated nicotlnlc receptors. ThÍs 1n lurn nay be tinked with the

functlonal abnornalitles characterised by minlmal brain dysfunctÍon

l¡hile 1t is tenpüing to consfder such an assocÍatfon, an

interpretatlon of data along thfs Ilne is stlll difftcult as othen

consl.derations such as the relative role of other neurotransnitter

systems such as central dopamine and

1988) is not yet cIear.

7-43

norepinephrine (Lichtensteiger et al

Although nicotÍne-induced neural tube anomal ies observed in this

study differed morphologically from the open neural tubes associ.ated with

zinc and vÍtanin E deficiencies and vitanin A excesses (Record et aI 1985;

Harding et aI 1988; Joschko et aI 1989), the treatment did nevertheless

induce severe forebraÍn and branchial arch defects. Furthermore, at a

cellular and ultrastructural 1evel, nicotine caused neuroepithelial and

mesenchymal cell death and disnuption sinilar in sone hrays to that

induced by these other nen/ous systen teratogens TL¡e observatlon that the

underlying neural tube cel1 death induced by various agents Ín previous

chapters was sl.milar, while norphologically the neural üubes appeared

very dífferent between the studies, suggests that celL necrosis may

not be the underlying factor responsible for the lack of neural fold

fusÍon observed 1n the other studies. Thus, while nicotine may not

interfere directly wÍth the nechanisn affectÍng neural tube closure, it

may share some conmon mechanisms of action with zJ-nc deficlency,

hypervÍtamj.nosis A, and alcohol,and strengthens the suggestÍon nade above

that these changes nay underlÍe behavioural and leanning disabilÍt1es, as

these features r,Jere aLso reported to be associated wÍth offspring

exposed to these three aforementloned teratogens.

Hence the biochenical defects, reporled prevlously and the

ultrastructwal changes seen in the presenl study whieh reflect nicotine-

induced biochemical changes, not necessarÍly 1n ühe sa¡ne directlon as

reported by Hudson et al (1974)r ¡nây be associated with the abnormal

growth and developnent observed during enbryonic 1ife, and nay be

¡nar¡lfesbed postnatally as lntellectual and behavloural lnpairnent.

7-44

Sone of the nechanlsms underlyÍng bhis nicotine-induced cellular necrosis

shor.ù-d now be considered. Of particular interest is the possibllity that

nicoting-¡nediated neural cell necrosis nay also involve oxidative menbrane

damage which occurs as the resutt of superoxide and hydroxyl radical

formation, (Joschko et aL 1989, Dreosti & Partick 1987) both of which are

known to be hlghly danaging t,o biological tissue including the developÍng

fetus (Dreosti 1986). Producbion of these free radicals could occur in the

rat, embryo at 10.5 days gestation when the electron transport system

becones functional (New 1978), and when the antioxÍdant defence nechanisms

involved in free radical removal are ninimally developed (Dreosti &

Partlck 198?). In addition, free radical nelated injury nay arise

because of the generation of superoxide radicals via the cytochrome P450

enzyne systems, through which nicotine appears to be netabolised in the

ceI1.

Also relevant is the findÍng that post-ischaernic ir\jury followÍng hypoxia

appears to arise from superoxÍde radicals generated upon avaílabi1ity of

molecular oxygen accompanying reperfusion (Granger et al 1981). TttÍs nay

be of fnportance in relatÍon to nicotÍne as although the vascular changes

brought about by nicotine are conplex, anlnal studÍes indicate that the

initial coronarT vasoconstrictÍon leading to hypoxia 1s followed by

inereased coronary blood flow and dilatlon of perÍpheral blood vessels

when the stage of sympathetic ganglionic stinuLation is succeeded by

paralysis (Goodnan & Gitnan 1985). ThÍs sequence of events nay underlle

lhe poor developnenb of yolk sac vasculature observed Ín this study

Ieading to nenbrane liptd danage of cell nenbranes of the neural tube

ceIIs as well as mitochondria which appeared to be nore affected than

oüher organelles, and nay underlle nlcotine-nedÍated neural tube

teratogenesis at the tfne of neurulatlon lÍhile a nunber of suggesbions

have been made as to the rÍkely mechanism underlylng cetl necrosis

on observations made in this study, experimental assessnent

validÍty of these suggestions ls beyond the scope of this study.

T -45

ba sed

of the

llhat the present study has shown is thab reduced intrauüerine growth

assoclated with nicotlne in rats in vivo can also be tnduced in ratenbryos in culture. It has atso provided the first evidence that a

specific component of cigarett,e smoke has a dlrecü teratogenic effect, on

the deveroping newal tube j-n vitro, whÍch ineluded cellular and

ultrastructural abnormalitÍes, and that the severe necrosis observed at

these levels rnight underlie the norphological denonstratlon ofnicrocephaly. The observation that:Ln vivo growth effects can be ninicked

fu vÍtro suggests that the l¡ viüro dysnorpholory night be reproduced l¡JÅre. This suggestion will be investigated shortly by the author. l?¡e

neans by which nicotlne lnterferes wlth normal growth and developnent isas yet unclear, but the influence of nicotlne on many enbryonic

conponents seen 1n the present study suggests a multffactorial orlgÍn

fn sumnary, the findings outlined in thls st,udy denonstrate the rlsk tot'he erobryo assocÍated w ith intraulerine exposure to nicotlne, and

when extrapolated to humans, hfghlight the need for hronen üo exerclse

cautÍon wfth regard to snoking during pregnancy.

7 -46

7.5 BIBLIOGRAPITY

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pregnancy upon the felal heart rate. An. J. Obstet. Gynecol.29r 77-83'-Spira, 4., SpÍra, N., Goujard, J. & Schwetz, D. (1975) Snoklng durÍngpregnancy and placental weight. A nultivariate analysis on 3759 cases.Perinat. Med. 3, 237-241.Surgeon General (19?9) Srnoking and health. Departnent of HeaIth'

Education and ÏleIfare. Publicatlon No. (PHS) 79-50066, US. GovermentPrinling Office, ïJashinglon.Surgeon General (1981) The Health Consequences of Snoking: The changing

clgarette. Departnent of Health and Human Services. Publlcatlon No.

7 -53

(Plts). 81-50156, US. Government Printing office' l{ashlngton.suzuki, K., Horiguchi, T., Comas-Urrutia, c. et a1. (1974) Placental

transfer and distri¡ution of nicotine in the pregnant rhesus monkey. An.

J. Obstet. Gynecol. 119, 253-262-Suzuki, K., Minei, LJ. Johnson, EE. (1980) Effect of nicotine upon

uterine blood flow in the pregnant rhesus monkey. Am. J. Obstet. Gynecol.

136,1009-1013.Svensson, TH. & Engberg, G. (lggO) Effect of nicotine on single cell

activity in the noradrenergic nucLeus locus coerul-eus. Acta. PhysioI.Scand. Suppl. 479' 31-34.Taylor, p(1980) In: The pharmacological Basis of therapeutics. GiIman'

AG, Goodnan, LS. & Gilman, A. (eds). vracnillan, Ner¿ York. pp 211-220.Taylor, p. (1985) Ganglionic stimulation and blocking agents. In: The

Pharmacological Basis of Therapeutics; Gilnan, AG., Goodman, LS., Rall'TI{., Murad, F. (eds). Macmillan PubIÍshing co. New York. pp 215-221,1699.Tjalve, lL, Hansson, E & Schmiterlow, CG. (1968) passage of 14C-nicotine

and its metabolit,es into nice foetuses and placentae. Act,a Pharmacol.ToxicoL. 26, 539-555.Tokuhata, GK. (1968) Snoking Ín relabion to lnfertilÍty and fetal Ioss.

Arch. Environ. HIth. 17t 353-359.UIikorkala, 0., Viinikka, L. & Lehtovirta, P. (1985) Effect of nicotine

on fetal prostacyclin and thromboxane in hunans. Obstet. Gynecol. 66, 102-

105.underwood, P., Kester, K., OtLane, JM. & CaIIagan, D. (1967) Parental

snoking enpirÍcally related lo pregnancy outcorne.Obstet. Gynecol. 29, 1-8Upshail, DG. (gTZ) Correlation of chick embryo leratogenicity with the

nicotinic activity of a serles of tetrahydropyrinidfnes. Teratology 5'287 -294,van den Eeden, sK., Karagas, MR., Daling, JR. & Vaughan, IT.. (1990) A

case-conbrol study of naternal snoking and congenital nalformations.Paediatr. Perinat. Epidemiol. 4, 147-155.llendell-Smith, CP. & Williams, PL. (1984) Basic hunan enbryology.

Pittman, London. pp 58-60.hrerler, MM., Pober, BR. & HoInes, LB. (1985) Snoking and pregnancy.

Teratology 32, 473-481.I'Iingerdl J. & Schoen, HI. (1974) Factors influencing length at birth and

height, at fÍve years. PediatrÍcs 53, 737-741.yerushalmy, J. (1971) tt¡e relationship of parents cigarette smoking to

outcome of pregnancy-implicatÍons as to the problem inferring causationfrom observed aÀsociatÍons. An. J. Epideniol. 93r 443-456.Younszai, MK., Pelosa, J. & Haworth, JC. (1969) Fetat growth retardatlon

in rats exposed to cigarette smoke during pregnancy. Am. J. Obstet'Gynecol. 104, 1207-1214.Zuckerman, BS., Parker, SJ., Hingson, R., Albert, JJ. & Mitchell, J.(1986) Maternal psychoactive substance use during pregnancy. In: Advances

Ín PerÍnatal medicine, Vol. 5. Milunsky, 4., Friednan, EA. & Gluck' L.

(eds). pp 130-135.

8-1

CHAPTER 8

THE EFFECTS OF CONCURRENT ADMIN]STRATTON OF ALCOHOL AND NICOTINE ON

cRqrlni AND DEI/EIOPMEX\IT 0F RAT EMBRYOS.

8.1 INTRODUCTION

Alcohol and cigarette srnoking have been shown to be deLeterious to the

developíng enbryo as reviewed and described in the previous two chapters.

It has been well established in both animal-s and in humans that alcohol

exerts a teratogenic effect during the period of organogenesis which leads

to abnormalÍties collectÍveIy described as the FAS and whÍch as weIl as

growth retardation and cranÍofacÍal defects also includes mental

retardation. In a previous chapter the author has denonstrated for the

firsb tine the ethanol-induced cellular changes in the rat enbryo in

vitro, and bhe accompanying uLtrastructural changes not previously

reponled in ar¡y species. Ethanol exposure during the critical period of

neurogenesis was shown to tead to reduced numbers of neuroepithelial

cells which the author suggests nay lead to inadequate differentiation

later in neuronal developnent and hence nay underlle the nental

retardation and behavioural abnormalities observed in chiLdren born of

alcoholÍc mothers, as welI as in offspring of aninals treated wÍth

ebhanol during gestation

Snoking durÍng pregnancy has also been reported to exert a detnimental

effect on offspring leading to reduced birthweight as well as a higher

Íncldence of fetal and perinatal death. There is also sone evidence that

naternal snoking may lead to an increased Íncfdence of congenltal

malformations (LandesmarrDwyer & Enanuel 197Ð. The previous chapter

demonstraüed nÍcotine-induced growth reduction and norphological

abnormalftles l¡ vitro in rat enbryos, particularly of the craniun and

8-2

neunal tube. Abnornalities included branchial arch defects which lead to

craniofacial dysmorphology including cleft palater âs weLl as

anophthalmia and nicrocephaly. Cellular and ultrastructural

abnormal"ities were observed within the neural tube, particularly in the

neuroepithelium where there was a deficit of ceIIs which nay

differentiate into neurons later in development. This paucity of cells

led bo the suggestion by the author that the intellectual

abnormalities recently reported to be related to maternal smoking may be

due to a paucity of neurons brought about by severe cell death of the

precursor cells during neurogenesis.

Ïlhereas the dangers of alcohol ingeslion or of cigarette snoking during

pregnancy are now wídely recognised, a relationship between them has

rarely been considered despite the report that as many as 24ft of pregnant

women consune alcohol and smoke cigarettes regularly (Coolidee 1984). The

wonen who smoked during pregnancy were generally found to be young, of

middle or lower social classes, less educated and consumers of ¡nore

alcohol than those who did not snoke (Rubin et aI 1988). Atthough several

studies have reporLed a significant relationship between snoking and

drinking behav ior during pregnancy (Kuz na & Kissinger 1 981;

Streissguth et al 1983), Iinited information is avajl-able in humans as to

how a conbination of these agents influences the incidence of

dysnorphologl reported for each agent alone. Martin et al (1977) obser¡¡ed

that noderate rnaternaL alcohol intake coupled wit,h moderate to heavy

cigarette snoking exerted an Ínteractlve and deleterious effect upon

Iearning in newborn infants, which was not predicted when the two drugs

were taken separafely. Malernaf aLcohol and nicotine consumption in

combination resulted in poorer neonatal perfornances on day 2 of life. In

addition, the combined effect of maternal drÍnking and snoking has also

been reported t,o have a greater effect than that

Íncreasing the time newborn infants sleep in

(Landesman-Dwyer et aI 1977).

8-3

of drinking alone in

an atypical position

lJhile a number of animal studies have been performed, dffferences in Lhe

varying doses of ethanol and ni.cotine, different treat¡nent periods, and

duration of exposure (chronic or acute) have led to differÍng results so

far. AbeI et aI (1979) using doses of ethanol and nícoline previously

reported to lead to fetal wastage in animals (Becker et' al 1968; Abel &

DinLcheff 1978), could not confirn an interaction between these agents, an

effect which they attributed to the use of sub-teratogenic levels of

nicotine. Lindenschmidt and Persaud ( 1 9 80) reported no significant

effect on embryonic development and fetal viabilfty following a sÍngle

ir¡jection of ethanol and nicotine on day 9 of gestation in pregnant

Sprague-Dawley rats. In contrast, Peterson et aI (1981) administered

ethanol intraperitoneally to pregnant nice exposed to tobacco s¡noke on

gestational days 6 to 16, which resulted in a signifícant decrease

in fetal weight and length, as well as a highly significant resorption

frequency of 67% in the group exposed to ethanol and tobacco smoke

conpared with frequencj-es of 5l and 3 1% íA the ethanol and tobacco groups

respectively. Hence these workers concluded that the effect,s on fetal

vÍabiJ.ity were potentiated by tobacco smoke, and that ethanol and

tobacco smoke may interact to produce fetotoxiclty. The study however dld

not denonstrate that ethanol was teratogenic which the authors suggesfed

mÍght be at,tributed to a lack of sensitivity of swiss albino mÍce. In

a nore recent study (I,Ioo & Persaud 1988) ethanol also fafled to induce a

teratogenic effect Ín rat embryos exposed to this agent on days9-12t

whlle those in ühe nicotÍne and nÍcotine/alcohol groups demonstrated a

range of abnormalities which were significantly dlfferent from controls,

although there was no difference between the groups.

concluded that the concentration of alcohol used in

inadequate to influence embryogenesis.

8-4

These authors

this study was

The following study was performed to examÍne whether a potentiating

effect of ethanol and nicotine occurs in relation to embryonic growth

and developnent during neurogenesis. The author was pronpted to

investigate this issue in view of the often similar teratogenic effects

attributed to ethanol and nicotine both norphol ogÍcalIy and

ultrastructuraJ-Iy described in the previous two chapters, and the

inconcl-usive evidence available in relation to the combined effects of

these agents in previous animal studies, as well as reports of an

association between snokj.ng and drinking behaviour in hunans.

8.2 MATERIALS AND I"ÍETIIODS

The procedures followed in Lhis study for embryo cultivation and recovery

as well as tissue processing were previously outlined in section 7.2 of

this thesis and described in detait in Chapter 2. The culture procedure

also did not deviate from the previous study except bhat 400 nef of

alcohol and 200 uglml of nicotine were added to the cuLture nedlum.

Alcohol was added at the last noment imnediately following the lnitÍal

gassing to avoid loss of this agent through evaporation

As seen in the previous two chapt,ers, the large variation in frequencies

of different abnornalities wfthin the different organ systens nade it

difficult to select levels of t,he two agents whfch would enable a

potential interaction to be lnvestigated for all organ systems. Hence

levels were chosen which were partÍcularly suited to investigate the

effects on the neural tube. The frequency of neural anonaliesr as well as

the severity of cellular dlsorganisaLf on I.¡ ere considered when selectlng

8-5

appropriate concentrations of alcohoL and nicotine for this study.

Continuous growth variables such as crown-rump lengthr somite numbers

and yolk sac diameber were analysed by standard analysis of variance,

and Tukey tests for nultiple comparisons between means. For discrete

developnent data, a maximun likelihood nethod of estimation vùas used

which involved treating the number of affected embryos wÍthin each

treatment group as being binomÍally distributed.

8.3 RE.SULTS

8.3.1 Growth and morphologicaJ- observations.

Table 8.1 shows that there was a significant effect of nicoline and

alcohol on cro$¡n-rump Iength (t3,90=40.37 p(0.001), so¡nite number

(F3,69=64.09 p(0.001) and yolk sac dianeter (F3,90=14.36 p(0.001). The

means of all treatnent groups f or crowtFrump length and somite nunbers

were significantly different fron the control Sroup and from each other.

Eor crown-runp length there was a 289l and 14f reduction for alcohol

and nicotine groups respectively which decreased to 42í for the

conbined group, while for somite numbers, there was a 28ll and 15f,

decrease for these two Sroups, which decreased by 47fi in the conbined

group. These val-ues suggest that alcohol and nicotine exerb an

additive effecl on growth retardation when administered concurrently.

In contrast, comparisons between neans indicates that for yolk sac

diameter only the alcohol group had a signiflcant effect on this

parameter as only alcohol was signtficantly different to controls

(15í reductlon), but was nob different lo the combined alcohol-

nicotine group (191), Hence 200 ug/ml nÍcotine did not have a significant

effect on the yolk sac at t,his concentrabion either alone or in

comblnation w ith alcohol.

8-6

Table 8.1 Effect of a.Lcohol and nicotine on embryonic growth in rats.1'2

Control s +Al c +Ni c +Alc/+Nic

No. of embryos

Crown-runp Iength

No. of somibes

Yol-k sac dian.

12

3.22+0.114

22.67 +0 .[Oa

3.p0+0.134

20 17

2 .I210 . o 8b 2.77 lo .o6c

16.47+0.84b 19.41fl.65o

3.33$.0?b' o 3.64+o.07ar c

45

1 .87+0.06d

1 1 .49J0 .43d

3.16+0.07b

2Values are neans t SE:M.

Means are significantly different from aLl others not sharingthe same superscripts. (Tukey tesbs for multiple conparisons p(0.05).

TabIe 8.2 demonstrates the frequency of dysmorphology of a nunber of

structures exposed individually and concurrently to 400 ng$ aLcohol and

200 ug/nJ- nicotine. The nunber of affected enbryos over aII organ systens

wasvery hlgh in aLI 3 treatrnent groups. Since theleveIs of alcohol and

nicotine v¡ere selected in order to examine the combined effects of these

agents firstly on the neural tube, these doses were not always the nost

suitable for the exanination of other structures. The small incidence of

ear and heart anonalies observed in all groups (Table 8.2) led to no

significant effect of either alcohol or nicotine alone or in combination

(Table 8.3). AIt,hough the frequency of heart defects was very low when

exposed to the individual teratogens, combining the treatments appeared Lo

lead to an exacerbation of effects, which however could not be adequately

Ínterpreted when analysed statistÍcally (Table 8.3).

Neura] tube defects occurred ín65l and 4TÍ of embryos in the alcohol

and nfcotine groups respecüively, and 1n T1l of enbryos in the combined

group. These values indicate that there was an effect of both drugs on

neural tube defects, which was confirned by significant differences

between deviances in Table 8.3¡ which also showed t,hat, alcohol and

nicotfne have an additive effect when adnÍntstered concurrently.

TabI e 8 .2

8-7

dev elo pmentEffect of alcohol and nicotine on morphologicalin rat embryos. l

Control- s +Al- c +Nic +Al c/ NÍc

No. of embryosNo. of affectedenbryosNo. with NTD.No. with openneural tubesNo. with opencranial neuraltube onlyEye defects

Ear defectsBranchial archdefect s

Heart defectsAbsent/ reducedforel Ínbs

fncomplete flexionAbsence of chorio-allantoic fusÍonPoor yolk-sacci rcul ation

2

3(25.0)0( 0)

1( 8.3)

0(0)

0( 0)

0( 0)

0(0)

0( 0)

1( 8.3)

2(16.7)

2(16.7)

1(8.3)

20

19(95.0)13(65.0)

9( 45.0)

6(30.0)

6(30.0)

4( 20 .0)

5(25.0)

1(5.0)

1 9( 95 .0 )

18( 90.0)

8( 40.0)

16(80.0)

1T

12(70.6)B(47.1)

1(5.9)

0( 0)

2( 11.8)

1(5.9)

2(11.8)

1(5.9)

3( 17.6)

1 0( 58 .8)

5(29.4)

6( 35.3)

45

44( 97.8)32(71 .1)

3(6.7)

3( 6.7 )

23$1.1)

6( 13 .3)

14(31.1)

11(24.4)

44( 97.8)

41(91 .1)

34(75.6)

42( 93.3)1

^ Nunbers in parentheses indicate percentages.¿ NTD refer to open neural tubes, microcephaly, bnormal and/or collapsed

forebrain vesi.cles and flabtened midbrains.

Open neural tubes were observed in 45l of alcohol enbryos and only 6l of

nicot,ine enbryos. The dissecting nicroscope photograph in Fig. 8.1a shows

a dorso-lateral view of an alcohol enbryo on the left hand sÍde with an

open neural- lube from behind lhe midbrain to the caudal end of the

embryo typical of craniorachÍschisis. This embryo also exhibited

forebrain vesÍcles which were splayed thus compressing the optic pits. Ttre

two embryos on the rlght hand side cuLtured in 200 uglnt nicotine, have

neural folds which had fused, although the enbryo on the rÍght l¡as

nicrocephalic. The nicotÍne-treated ernbryo in the centre was retatively

unaffected, although the allantois had failed to fuse to lhe chorion.

llhen the treatnents were combined, there appeared to be an amelÍoratlon

8-8

of alcohol-induced open neural tubes, although this effect could not

be shown statistically (Table 8.3), probably because of convergence

probrens wÍthin the program rerating to the lange alcohol and very

smal-l nicot'ine effects. Similar difficulties arose with the analysis of

data relating to embryos with open cranial neural tubes only.

Figs.8.1b and c, show ventral- and dorsal views respectively of

microcephalic embryos cultured in alcohol and nicotine concurrently with

hypoplastic forebraÍns, abnormal nandibular arches and enlarged

pericardia. The dorsal regions of the neural tube appear normal however,

and the neural folds have fused along Lhe entire length of the enbryo.

The enbryos have only partially rotated into the dorsi-convex positÍon

Alcohol and nicotine also exerted teratogenic effects on chorieallantolc

fusion, dorsi-convex flexion and yolk sac circulat,ion and, when combined,

showed an incneased Íncidence above that seen for the individual groups.

Thís was reflected in the statisticar analysis (Tabre g.3), which

denonstrated that the individual treatnents exerted a signÍficant effect

of both agents on dysnorpholory which was additive when aLcohol- and

nicotÍne were adninistered concurrently.

EVe' branchial arch and forelinb defects could be nainly att,rfbuted toarcohol with respective frequencies of 30Í, 25% and 95{n, and 12fi, 1zl and

18l of aninals respectively denonstratÍng these terata in the nicotine

group. The co¡nbined effect of alcohol and nicotÍne led to an increase lnincidence of eye defects (51%), branchlal arch defects (31f) and forelinbdefects (98f). These observaüions were confÍrned statistÍcalIy Ín

TabIe 8.3. DÍfferences between devlances denonstrated a strong alcohol

effect fon all three terata, with orùy a trend towards an effect on eye

defects by nlcotfne at this concentratfon

8-9

Fig. 8.1. a. Day 11.5 embryos cultured in 400 ngf alcohol (tert) and 200

"ä7;i nicotine (äentre and right). The neural tube of the alcohol- breated

embryo is open caudal to the midbrain (arrowheads). The forebrain is also

abnormal leading to conpression of the optíc vesicles (arrow) and theanimal has not rotated. The nicotine-treated embryo on the right has a

closed neura]. tube throughout its entire length, however it isnicrocephalic and has not rotated, while the embryo Ín the centre isrelatively unaffected despite the lack of placental formatÍon indicated by

the presence of the unfused al-Iantois (A)'

b, c. Thnee day 11.5 æbryos at different orientations exposed

concurrently t,o 20ó ue/m1 nicotine and 400 ngf ¿cohol in vitro' A dorsal

view in b, shows that the neural folds had fused throughout the length ofthe neural tube (asterisks). A ventral víew in c, shows thaL the embrlos

were microcephalÍc wíth severely reduced fronto-nasal regions (arrow)

and abnorrnaL nandibutar arches which were fused to the pericardium(arrowheads). OT, otic vesicles; 1, mandibular arch; H' heart'

Bars=1rnm.

t

t

TabIe I .3 Evaluation of the dealcohol and nicotine.

8- 10

çree of independence of the effects of

Deviance2

I4cdel 1

(DevÍance 4)No effect ofAIc or Nic.

( ¡or)

I.4odeI 2(Deviance 3)Effect of Alconly.

( 2df)

l',lcde1 3(Deviance 2)effect of Nicon1y.

( 2df)

I'{c del 4(Deviance 1 )Inde pendenteffects ofAIc and Nic.

( 1df)

24.064

15.06

11 .37

1g.07a

4.14

g.354

9 .16

7 2.484

30.764

21 .7 6a

45.154

9.93b

12.68

5 .81

4.16

1 .85

4.78

0.87

5.51b

8. 14b

5.45b

ß.29c

5.35

6 .93

14.52e

7 .12c

3.7 u

7o .6 gc

26 .46c

13.14c

39.45c

1 .06

*

*

0 .97 d

0.05d

1.72

o.1gd

0.83d

2 .3od

o.g2d

dNo. with NTD.No. with openneural- tubesNo. with opencranial N. T.

Ele defects

Ear defectsBranchial archdef ect s

Heart defectsAbsent/ reducedfor eI imbsIncmpletefl exionAbsence ofchorio-aIIanb.fusÍonPoor yolk-sacci rcul ation,|

]See chapber 2 for explanatÍon of statistical nodel used.

loevianoå is ássuned to be distributed as X2.-Convergence problems assocÍated with zero effect of nicotine or large

effect of aIcohol.aIf Dev 4-Dev 1 >5.99 (critical vafue for X2 for zdf, p<0.05), then. rejecf l.'l¡de1 1.brr ¡"i l-Dev 1 >3.84 (critical value for X2 for 1df, p(0.05), índicates

sÍgnjJicant effect of nfcotine, then reject lbdel 2.c1f Dev 2-Dev 1 >3.84,indicates signifieanl effect of aIcohol, reject. I,lodel 3.dtf Dev 1 <3.84, accept Model 4 for independent effects of

trea tnent s.

Sone of the morphological features are reflected 1n the scanning electron

micrographs in FÍg. 8.2. A typical enbryo exposed to 200 uglmI of nicoflne

(Fig. 8.2a) demonstrated fused neural folds and was nlcrocephallcr with

reduced forebrain vesicles and flattened midbrains. The enbryo falled to

8- 1 1

rotate into bhe normal- dorsi-convex positíon and showed a dysmorphic

appearance overaLl. The hyoid and third nandibular arches had not

developed, and there was no evidence of the optic placode. lfhile a few

of the enbryos exposed to 400 nCÍ alcohol had open cranial neural tubes,

more conmonly at t,h1s concentration the neural tubes demonstrated

craniorachischisis (FÍ9. 8.2b), wÍth the neural folds failing to fuse

from behind the midbrain. In addition, the forebrain vesicles of sone

embryos were curiously splayed. At, higher nagnification (Fig B.2c) the

forebrain vesicles of eubryos with this defect appear to have collapsed

and separatedr !tith the surface ectoderm falling into the ventricular

space between the vesicles.

The enbryo Ín FiS. 8.2d is fypical of those exposed to alcohol and

nicot,ine concurrently. The craniun was severely dysnorphíc, but the

neural folds had fused bhroughout the length of the neural tube,

branchial arches w ere abnorrnal and the embryo had not rot,ated. Higher

magnification (Fig. 8.2e) showed thaf the cranium was nicnocephalic with

an absence of forebrain vesicles and reduced mÍdbrain The mandibular

arches were fused t,o each other and to the pericardium, whire bhe

second and third pairs had not differentiated. The positÍon of the

forebrain vesicles in relation to the mandibular arches suggests that

palatal defects may have occurred if the embryo had been examined later

in gestation. Other cranÍal defects lncluded the absence of optÍc

placodes and Ín some embrXlos, inconplete closure of the otic vesieles.

The surface ectodern 1n the forebrain region were conpared for each

treatment group and were also found to be affected by alcohol and nicotine

aIone, and when conbÍned. Dense short nicrovÍIIÍ and sone blebs are

frequently obse¡¡¡ed in day 11.5 control enbryos on a snooth surface in

8- 12

Fíg.8.2. a, A dorsal view of a typical invitro 11.5 day embryo culbured

in 200 ug/m] of nicotine. The animal is generally dysmorphic' and although

bhe neural tube is closed, fusion did not appear to have occurred smoothly

"i "or" sites (arrowheads). The animal is also nicrocephalic with reduced

foretlrain (arror¿s) and flattened rnidbrain, and has not rotated inbo the

normal dorso-convex position. l'lhereas the mandibular arch ( 1) appears

normal, the second ànd third branchiat arches have not developed' H'

heart.

b. Dorso-Iateral view of anin vitro 11.5 day embryo cultured Ín 400 ngf

a1cohol. The neural folds have not fused from the hindbrain to the caudal

end of the embryo (arrows). The cranial neural tube is also abnormal withdistorted forebrain vesicles which appear to be severely splayed (curved

arrows), and a flattened midbrain (Mb). The enbryo has not rotated'

c. Dorsal vier.¡ of bhe craniu¡n of the salne enbryo. The nsplayedn

appearance seen in b, can be. attributed to collapse of the neural folds inthe forebraÍn region (arrows). The open caudal neural tube is indicated by

the arrowhead. Mb' midbraini H, heart'

d,e.Int,hisembryowhichwasexposedtonÍcotineandalcoholconcurrently at Ievels of 200 ug/¡rt and 400 ngf, respectively, the animal

was growth retarded with a severely dygrorphic cranium. The enbryo was

nicrocephatic with severe hypoplasia of the forebrain (asterisks) ' Despite

these neural tube abnormalities, the neural- folds had fused throughout the

enbryo. The mandibular arch ( 1) !'ras also affected while the second and

third arches are not prorninent, and the embryo had not rotated'Higher magniffcation of the cranial region in e' shows that thenandibular arch is fused to the pericardium (arrowheads)' compare the

branchial arches with those of a normal embryo in Fig. 6.2e- Note thatthe obÍc vesicle (OT) is normal and aligned with the second branchialarcn (Z) which like bhe third branchial arch (3) had onJ-y comnenced todifferentiate in mar¡y of these embryos. Fb, forebraini Mb, midbrain; Mx,

maxillary processi OT, otic placode; ys' yolk sac'

f,g,h,i. surface features of bhe forebrain of 11.5 day æbryos culturedin normal serum (f), 200 ug/rnl nicotine (g), 400 nSÍ alcohol (h)' and the

two treatments 'conbined

aÍ). The surface of control enbryos (f) was

covered j.n microviifi (arrows) with a few blebs Ínterspersed (asterisks)'whereas the forebrain of nicotine treated embryos showed sinilar bub nore

abundanb nicrovilli (arrows) located on irregular ectoderrnaL ceLls'In atcohol-treated embryos the ectodernal cells $¡ere nore regularr but

were covered 1n long thin nicrovilli (arrows). llhen the treatnentswere conbined, the fòrebraÍn surface v¡as covered in vast numbers of veryIong, thick nicrovÍIIi (arrows).

Bars= 10Ourn. a, br cr d, e; 3um. f r gr hr i.

d

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this area of the

exposed to 200

nyriads of short

relatively smooth

8- 13

craniun (FÍg 8.2f). The forebrain surface of enbryos

ugln1 of nicotine (FiB. 8.29) were often covered with

microvilli on irregular raised cells, while

cells with large blebs seen in embryos cultured in

replaced by very long and dense microvilli on

the

400

theng% aIcohol, were

surface ectoderm of enbryos exposed to both treatments.

Fig. 8.3 shows light microscope secbions of the craniun of embryos

exposed lo 200 ug/ml nicotine (a'b)' 400 mg$ al-cohol (c'0) and 200 ug/ml

nicotine-400 ngft alcohol (e,f). The levels at which the horizontal

sections were cut was slightly different in each treatment group and also

in relation to conbrol sections (Ffe. 6.3) due t,o the difficulties in

determining conparabJ-e depths of cutting because of the dysmorphic shapes

of these enbryo s.

Figs. 8.3a and c demonstrate the typical appearances of horizontal

sections through the craniuns of embryos exposed to 200 ug/mI nicotine and

4OO neí atcohol respectively. In the nicotine embryos al this

concentration (Fig. 8.3a), there was a dininution of the forebrain and an

absence of welI defined optic vesicles. Even in deeper sections (not

shown) the optic vesicles were not apparent. The mesenchyne

contained Iarge areas which appeared devoid of cells as seen

prevÍously in Fie 7.3b. In contrast, the alcohol-treated enbryo (Fig

8.3c) appeared microcephallc with conpressed optic vesicles and unfused

neural folds in the hÍndbrain region In both sections the shape of the

neural tubes reflected the reduced forebrafn areas observed 1n scanning

electron ni.crographs. Hlgher nagnificatÍon of sectfons of the cranlal

neural tube in Flgs 8.3b and d, revealed thaü nicotine (Fie8.3b) and

alcohol (Fig 8.3d) exposure led to extracellular spaces and dense, dark

8- 1 4

cell debris wit,hin the neuroepitheliuur, with sone remnants in the

ventricular Iumen, especially in embryos exposed to nicotine. While the

nesenchymal cells appeared unaffected in embryos exposed to either

treatment, abnormally large blood vessels Iô¡ ere present in bhe

extracellular matrix of the nicotine-cuLtured embryos (Fies. $.Ja,b)r

whÍch appeared to force the compression of mesenchynal celLs into the

space adjacent to the surface ectoderm.

The overallshape of the cranium in embryos exposed to alcohol- and

nicotine simul-taneously (Figs. 8.3 e,f) showed that the forebrain vesicles

were narrow conpared with controls, and the neural tube in the mÍdbrain-

hindbrain region !tas abnornal in shape, while the basal end of the

neuroepithelium was convoluted and the thickness of the neuroepitheliun

was i.rregular. Ttre degree of cell shrinkage and intra -and exbracellular

debrls was j.ncreased in enbryos exposed to both treatments and occurred

to a lesser degree Ín the nesenchyme as well (FÍ9. 8.3f).

8.3.2. Ultrastructural observations.

Figs. 8.4a and 8.4b depict the general features of lhe neuroepitheliun

of enbryos exposed to 200 ug/ml of nicotine in culture at the

lumen end and at the mesenchymal end respectively. The principal.

feature is a narrowlng of the neuroepithelial wall with considerable

celt shrinkage and l-oss Ieading to Iarge extracelluLar spaces and

Ín sorne cases intracellular inclusions which are probably condensed dead

cell rennants. lhe nucleoli denonstrate the ribbor¡-Ilke wandering nucleoli

sinilar to structures seen in the nucleus of gern cell tunours by

Ghadlalty (1988), and which v¡ere descrÍbed in Chapter 7. Desplte the

cell disruption, nltotic cells were stilt observed close to the lumen or

apical end of the neuroepfthetlun (not shown).

8- 1 5

FÍg. 8.3 shows horizontaL sectÍons through the cranium of nicotine (a,b),

atcõfrot (c,d), and nicotine-al"cohoI (e,f) treated 11'5 day ¿4 vitroenbryos. sections shown at higher rnagnification in b,d,f are narked by

arro$¡s in ar cr e.

a,b. Horizontal sections through the cranium of an 11,5 day embryo

exposed t,o 200 ug/nI nicotine. Light microscopy reveals that there is an

abÃence of optic vesicles in the forebrain region. The thickness of theneuroepitheliun is reasonably comparable to controls (see Fig' 6'3) aL

this concentration of nicotine, however b, shows that there is ceIIshrinkage and loss Ieading to extraceLlular spaces (asterisks) which oftencontained dense ceII remnants (arrows) which were also observed ín otherhealthy cells. The mesenctrymal cells vJere aggregated lowards the surfaceectoderm (E), and much of Lhis neural tube layer appeared empty, althoughhigher magnificalion Í.n b, revealed that these spaces were occupied by

targe, developing blood vessels. There appeared bo be sone loss ofintercellular cytoplasmic connections betl¡een the mesenchynal celIs.

c, d. fubryos exposed to 400 mgf alcohol appeared microcephalic withcompressed optic vesicle area (o) and nininal ventriculan space. The

neurat folds (¡W) frave not fused in bhe hindbrain region except at therostral end whÍch 1s marked by the arrowhead. The neuroepithelium was

convoluted at the basal surface (thin arrows) with large extracellularspaces (asterisks) and sone cell debris located mainly with other ceLls(ùnict arrows). The mesenchymal ce1ls appeared to have lost some of LheÍrinterconnecting filopodia, buL did not appear to be dying.

erf. The cranial shape of enbryos cultured in 200 ug/n1 nÍcotine and 400

ngi alcohol concurrently, was abnotual as the forebrairmidbrain regionwas reduced in nagnitude (doubLe-ended arrow). The cranial neural foldshave fused in thÍÀ ernbryo although there is extensive disruption of theneuroepitheliun at its basal end (arrowheads) and large extracellulanspaces (ast,erisks) and nany dense extracellulan and intracellular ceIIremnants (arrows). Despite the severe breakdown of the neuroepitheliun'nitotic figures are stll1 visible. The surface ectodern has been tornaway fron the mesenchyne during processing (arrowheads).

bv, blood vessel-si L, ventricular luneni B, blebsi Fb, forebraini Hb'

hindbrain; N, neuroepitheliun; M, mesenchyne; Mf, nitotÍc figures.

Bar s=50um.

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8-16

Comparable observatíons were made in enbryos exposed to 400 n8%

alcohot. Fig. 8.4c shows a region of the neuroepithelial wall at

the apical end from an ethanol-treated embryo, where cells abut the

ventricular lumen of the anterior neural tube. Sone cell debrÍs was

present in the extracellular spaces, which once again were considerable.

At the basal end of the neuroepithel ium (Fig. 8.4d) where the

neuroepithelial ceIIs abub the nesenchyne, cell debris was found within

this layer and within the large extracellul-ar spaces seen more often at

this end of the neuroepithelial wal-l. Condensed ¡nitochondria and circular

endoplasnic reticulum were slill discernable amongst' the cell debris.

Higher nagnification of the extracellulan matrix in the region adjacent to

the neuroepithelium (Fig. 8.4e) shows that the basal Iamina has been

disrupted and the neuroepithelial cell remnants have been extruded into

the nesenchyme. The neuroepithelium of enbryos cuLlured in alcohol-

and nicotine concurrently (figs. 8.4f,9) showed cel1 shrinkage and

loss leading to the fornation of many large extracellufar spaces as

r.J ell as extracellular and intracell-uIar dead celI remnants, w hich

appeared to be phagocytosed by heatthy cells. The apical surface of the

neuroepithelial cells projected into the venbricular l-umen' which also

contained extruded dead ceIl debris (Fie 8.4f). Ribbon-lÍke nucleoli were

also observed in the nuclei of sone of these cells. At the basaL end of

the neuroepithelium (Fie 8.4e) the cel1 borders were highly irregular

and dead ceII materÍal was present in nesenchynal ceLls close to bhis

j uncfÍon.

Higher nagnÍfication (Fig 8.5a) of an area of neuroepÍtheliun seen Ín

Fig. 8.4f, shows extracellular debrfs with remnants of cells which

consLst of danaged nitochondria which have lost their cristae'

polyribosones, REn and probably condensed nuclear components. The

Fig. 8.4 show sneural tube of(a,b), 4oo mgf(f,e).

8_ 17

sone of the ultrastructural features of the anteriorll.5dayinvitroembryosculturedin200ug/mlnicotÍneafcãnoi 1c,O,e), and 200 uglml nicotine + 400 mgf alcohol

arb. A section of Lhe neuroepithelir.un abutting the ventricular lwen (L)

in a, and adjacenb to the extracellular natrix (ECM) of the mesenchyme'

Large extracellular spaces were observed bhroughoul the neuroepithelial

""rT t""terisks) which were particularly extensive al the basal end of the

nueroepitheliun in b, as well as some intracelluLan Ínclusions (arrows) 'The basal lamina (SL) appears to be disrupted at some sites (arrowheads),

and ribbon-like, wandering nucleoli (n) are also visible.

c,d. ALcohoL - induced cell necrosis in the neuroepitheliun showÍng the

apicat and basal ends respectively. Both regions showed ceII shrinkage and

disruption leading to the formation of extracelluJ.ar spaces (asterisks)which often contaiñed condensed cell remnants (arrows). Phagocytosed celldebrls (arrowheads) was also present within other neuroepithelial cells.

e. This electron micrograph shol'¡s a range of ceII debris tocated in bhe

extracellu.Lar mabrix (ECM) adjacent to a site where bhe basal lamína has

been disruptecl (arrowhead) and some cytoplasmic ribosomes (cr) are beingextruded from the neuroepithelium. he condensed vacuolated ce1I (arrows)

nay be preparing to phagocytose surrounding organelles'

îrg. Features of the neuroepitheliun of nicotine-alcohol cultured embryos

shõwing the apical or lumenal, and basal ends respectively. The neuralcell_s shov¡ed extensive cell destruction and hence extracell-uIar space

beyond that observed for the indÍvidual treatnents and which oftencontained condensed, dead cells (Iong arrovs). Some apparently healthycells contained nunerous heterolysosonest which were filled withcondensed, Iysed naterial (arrowheads). Note the distortion of the nucleiin these celLs (short arrows) r âs well as the presence of wandering

nucleoli (n) in others. CeIl remnants were also present in the ventricularIunen in f (open arrow) adiacent to the apical blebs (B). TLre basal end ofthe neuroepitheliunwas highly convoluted (maII arrq¿head), and the basal

Iamina was also disrupted at bhese slles. ftre asterÍsk in f, and gr mark

the sites seen at higher nagnificalion in FÍg. 8.5. L,Itmen; ECM'

extracellular matrix; M, nesenchyrnal cell.

Bars=1Oun a, b, c, f,g; 2um dr e.

ECM

...,1.1,'d

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llECM

:,t:.1,. -

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L

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8-18

heavily condensed structure in this nrÍcrograph appears to consÍsb of

an aggregate of ceII organelles, which are probably mitochondria, while

the large, circular opaque structure is probably a Iysosome. 0f

partÍcular interest is the close proxinity of this extracelluLar debris

to the ventricular Iumen. It appears to be separated from it only by a

junctional complex. In a sinilar micrograph in FÍ9. 8.5b cut at a

sIÍght1y different depth, the junctional- conplex appears to have parted

and has now becone associated with the membrane surrounding the

exbracell-ufar material- i.nstead, allowing the extracellular debris to

be extruded from the neuroepithelÍun into the lumen. Note the

affected mitochondría in the adjacent neuroepithelial cells. Higher

nagnification (Fig. 8.¡c) clearly shows the remaÍns of the nitochondrÍa

which have lost their cnistae, and outer membranes. Interspersed

anongst these is cytoplasm with vÍsible polyribosomes. The dense

structure consists of very condensed cel1 organeÌ1es and the amorphous

region of this nay be a lysosome.

The nicrograph in Fig. 8.5d depicts part of a healthy neuroepithelial

ceII which has phagocytosed the celluIar remains of other cells As with

much of the phagocytosed naterial it was not possible to distinguish

individual celI components whict¡ suggesls that nuch of this cell debris

nay have been digesled by the host cell, or the lysosonal systen of the

engulfed cell. At the basal end of the neuroepitheliun in Fig.8.5er the

basal lanÍna is disrupted and neuroepithelial cel-1 rennants located in the

large extracelLular spaces are aboub to be extruded into the nesenchyme.

The nicrograph in Fig. 8.5f reveals the nore typical differences between

cytoplasn and nuclei in affected and non-affected cells exposed to

nicotlne and alcohol concurrently. The cell on the left appears nornal

B- 19

Fig. 8.5 shovls a range of ceLlular necrosis at higher magnificationwithin the neuroepittreliunm of 11.5 day in vitro embryos cultured in 200

uglnt nicotine and 400 mgÍ alcohol- concurrently'

â,b. Higher magnification of a site in Fig. 8.4f marked by Lhe asterisk'showing ã condensed, dead on dying ceII (arrows) containing a range ofdamageã cell organelles adjacent to the ventricular Iumen (L) at differentstagãs of tysis. The organelles are prevented from being extruded

into the lumen by the junctional conplex (arrowhead) between two apposing

neuroepithelial ceIIs in a. In b. however, which is from the same sitein the neuroepithelium, but at a different teveI, the neuroepithelialcells appear to have formed junctions (arrowheads) with membranes of the

dying celIs enabling its contents to be extruded into the ventricularfumen Note the severely affected nitochondria (n) in the neuroepithelialcells (N) in b.

c. organelles within the condensed, dying cell in b, can be clearlyidentified as nitochondria (m) and condensed cytoplasmic ribosomes (cr) inthis nicrograph. The cell also contaj-ns heterolysosomes (arrows) filledwith phagoèytosed, severely Iysed cel1 remnants. The structure on

the 1ef t may be a primary lysosome (LV) containing lytic erìzynes only'

d. A heterolysosome (arrows) contained within an apparently healthyneuroepitheliaL cetl (N), where cell organelles are mainly unrecognisable'The presence of cytoplasmic ribosomes within vacuoles (arrowheads) around

the perimeter of the lysosome suggests thaL it may also be digesting partof the host celI.

e. Higher magnification of a site in Fig. 8.4g marked by an asterisk.Neuroepithelial cell debris was also removed from the neuroepitirelium (N)

at the basal end by extrusion into the extracellular matrix (ECM) of themesenchyme at sites where the basal lamina was disrupted (arrows)' Note

the large extraceLluJ-ar space (asterisk) in lhe neuroepithelium'

f. High magnification of the typical appearanee of cytoplasm and nucleus(nu) of affected (N.r) and unaffected N2r Na neuroepithetial cells fnom a

nicotin+aIcoho1 tieated aninal. The-typi-cal polysomal configuration ofcytoplasmic ribosomes (arrø¡s) in N, has been Iost in N, and the cytoplasmhas becone condensed. The clunped aþ earance of the heterochromatin in thenucleus of the normal ceII (N:) (arrowheads) has also been replaced by

dispersed chronatin in N, whicJr is Índicative of a metabolÍcally activesta te.

Bars= 1un.

f.4', i :,L

t '¡

T, ECM I

T t \æ.

8- 20

with cytoprasm showÍng the typicar poryrÍbosonar confÍguration, whereas inthe affected cell on the right the ribosomes are condensed and have lostt'heir porysomaì. configuration There are al-so differences between thenuclei' fn the apparently normal neuroepithelial ce1l, the chronatin iscJ-umped which is indicative of netabolicaì-ly Ínactive cerls, whereas inthe affected cell the chnomatin is dispersed which is characterist,ic ofa net'abolically active state. At the top of this cerl is a section offragmented nucreorus, which is arso characteristic of a verymetabolically active stat,e.

8-21

8.4 DISCUSSION.

The results of the present study indicate thaü af the concentrations of

nicotine and alcohol used in this experiment, rat enbryos exposed to these

agents individually from day 9.5 to 11.5 of gestation showed significant

reductions in crovün-rump length and somite nunbers. When exposed to

ethanol and nlcotine concurrently there Ìùas a further reduction in these

parameters, which vJas additive. fn cont,rast, yolk sac dianeters were

significantly reduced by alcohol onIy, as the alcohol and alcohol-

nicotine groups Ìrere not different from each other. This suggesbed

that the concentration of nicotine used in this study was below the

threshold required to Índuce an effect on this growth parameter. ft was

difficult to make a direct comparÍson between the results observed in

the present study and the few which had previously examined the effects

on gnowth when these two agents wene combined, rnainly because of the

variatÍon between t,he studies in the species used, route of drug

adninÍstration, time of aninal examination and most important,Iy dosage

1evels.

I'Ihen Abel

(6e/Ke/ day)

et al (1979) exposed pregnant rats to high levels of ethanol

and low levels of nicotine (1.5 ng/Ke/day) aL the sa¡ne

t,he reduction in blrth weight in this group suggested that

alcohol contributed to this effect and thab the dose of nicotine used

lneffective in producÍng a growth effect. A similar observation

reported by the aubhor in the present study but only ln relation to

sac di.ameter, which suggesbs thaf nicotine does nol interfere with

sac growth al the sane level as it does wiLh enbryonlc growttr.

tin e,

only

}JAS

was

yol k

yolk

Murrin et aI (1987) also found a sinilar lack of effect on growth and

developnent at this level of nicotfne (1.5 ne/ke/ day) in rats, atthough Ít

8-22

was in the range received by humans smoking one pack of cigarettes per

day, and produced plasma leveIs of nÍcotine in rats similar to the

Ievels found in hunans (Benowitz & Jacob 1984), and which clinical

studies have shown leads to growth retardation in utero (Kullander &

Ka1len19T 1; Andrews & McGarry 1972; ). Hence it appears that the rat is

far Iess aensitive to the teratogenic effects of nicotine than are

humans. This concLusion was support'ed by the higher nicotine

ler¡eIs requÍred to induce growth retardation and dysnorpholory in the

presenb study and in one reported by lloo and Persaud (1988). In their

studyr êrbryos ÍJere exposed to intermediate IeveIs of nicotine (16.7

nC/Ke) and a low level of ethanol (I.t{ e/KÐ from days 9 to 12 of

gestation, the level of reduction in growth parameters this time suggested

that the concentrabion of alcohol was inadequate to influence

embryogenesis. Yet another dubious outcome of the concomitant exposure to

alcohol and nicotlne was reported by Persaud (1982), whose entÍre study

was composed of only an alcohol group and three alcohol-nicotine groups.

Although higher doses of nicotÍne of up to 28.4 ne/Kg daily for 7 days via

mini-osnobic punps and moderate ÌeveLs of alcohol in a liquid diet were

used, they did not observe growth retardation, or devel-opmentaJ-

dÍsburbances in term fetuses.

Persaud (1982) did however observe 21 f resorptions in the alcohol

group and up Eo 11.5% 1n the alcohol-nicotine group, which although not

signifÍcant Índicated that there was a trend t,owards amelÍoration due to

nicotlne, These observations did not support clinical studies which had

reported t,hat smokÍng potentÍat,es the effect of alcohol (Littf e et aI

1976; KaninskÍ et, al 1978), Ieading to an increase 1n sti[births and

a greater reduction in birthweight associated with maternal smoklng and

drinking conpared to naternal drinking alone. l{hile Peterson et al (1981)

8_23

observed a high resorption frequency and growth retandation in the

combined alcohol-nicotine group indicating a potentiation of effects

as was observed in the present study, and which supported clinical

data, the absence of a significant growth effect in al-cohol and nicotine

groups and the presence of only one malfornation in both of these groups'

suggests thaù the doses of these two agents were sub-teratogenic. As

discussed by these authors it is likely that extraneous physical factors

may have led to the reduced grovrth in the combined group. Peterson et al

(1981) suggested that the lolr growth effect in nice in alcohol (trend

onfy) and al-cohol-snoke groups was due to undernourishmentr âs al-cohol

causes animals to be conatose for 4 hours. This is support,ed by the

observabion ( Schoenaker et aI 1 980) that ethanol-treated animals

if adequalely nourished do not produce low birthweight fetuses.

Furthenmore, Peterson et aI (1980) suggested that the potentiation of the

Low growth effect in the alcohol-smoke group nay be atùributed to

asphyxiation from smoke inhalation, whereas animals in the individual

groups were exposed to only one of these factors. The associated

high leveI of resorplion in the alcohol group (311) and even higher

level in the alcohoL-snoke group (67%), but low leveL in the snoke group

nay also occur as the result of a stress reaction rather than fron an

effect of the agents thenselves.

In the present study where alcohol and nicotine are added to the culture

nedium, confounding issues such as a nalnourishnent and asphyxlation

are ellninated and hence the potentiatÍon of a grovlth retardation

in the alcohol-nicotine group can be nore confídently attrlbuted

to a direct effect of these two agents on the developÍng embryo.

Some suggestions relating lo the 1ikely mechar¡lsns underlying growth

retardatlon derive from the observations made by Brown et aI (197Ð that

8-24

ethanol retards gnowth in utero by inhibiting cell proliferation and that

tobacco smoke (nicotine), lowers the amount of oxygen available for fetal

glucose metabolisn, thus decreasing the anount of available enerry needed

for enbryonic growth and differentiation (Robkin & Cockroff 1978).

As the present study was primarily concerned with the dysnorphic and

ultrasfructural abnormalitÍes of bhe neuraL tube, the doses of nicotine

and alcohol were chosen in accordance wÍth this consideration Nicotine

level-s of 200 ug/mI were considered appropriate as although neunal

dysnorpholory assoclated wÍth nicotine rarely consisted of open neural-

tubes, nícrocephalic embryos with hypoplastic forebrains and sonetimes

reduced midbrains and hindbrains were frequently observed, together with

neuraL tube ceII death. ConcentratÍons of 400 ngÍ atcohol were chosen as a

suitable teratogenic level for aLcohoL as Ít appeared to be the threshold

dose for open neural fubes and neural necrosis. In utillslng

concentrations of these two agents appropriate to demonstrate neural tube

defects when administered al-one or in combination, these IeveIs were not

necessarily appropriate for exanining abnormalities of structures such as

forelimbs, and processes such as enbryonic rotation. The incidences of

anonalies of these systems r4rere so high at the chosen level- of aIcohol,

that iL complet,ely sr¡ramped the moderate effect of nicotÍne makÍng it,

dffficult to adequately assess the dysnorphic action of nicotine on these

para¡neters in the combined group. Since the affected embryos generally

demonstrated an absence of forelinbs rather than abnornalÍties of this

structure, and enbryos which failed to rotate into the dorsi-convex

positfon often had only achieved the rotational positlon sínilar to 10.5

day enbryos, the present aufhor suggests that these anonalies are more

lndicative of an alcohol-nicotine-induced developmental delay rather than

an enhanced teratogenlc susceptfbillty.

8-25

The high incidence of abnornalÍties and the leveI of dysmorphology of

the neurat tube and other developing structures $thich have been attributed

to conconitant exposure to alcohol and nicotine in the present study have

not been reported previously. The absence of very l-ow Ievels of

dysmorpholory in sone previous studies nust be atbributed to a number of

causes íncluding the use of sub-teratogenic I evel s of al cohol and

nicotine, and possibly also to the high nortality rates observed in some

treatment groups which nay mask a higher incidence of fetal abnormalitles.

This study also provided the first adequately illust,rated evidence

of dysmorpholory in nicotine-alcohol treated enbryos using t,eratogenic

doses of both agents. At the gross level and under the scanning electron

microscope, enbryos exposed to nicotine and alcohol concurrently appeared

to exhibit a dysmorphic effecb for a number of structures including

branchÍal arches and optic prinordÍa, which were often more severe than

that, observed when the treatnents were administered alone. These

observations are difficult to compare with obher studies once again

because of the bias towards an alcohol (Abel eb aI 1979) or nlcotine

(lloo & Persaud 1988) effect in the comblned groups, due lo t,he use of

subteraLogenic levels of one of these agenls in each study. In addition,

several other studies using either a single combÍned treatment of

ethanol and nicotine in noderate doses early in gestation (Lindenschnidl

& Persaud 1980), or a large dose of nicobine and noderate levels of

alcohol (Persaud 1982), did not lead to a significant deleterfous

effect on fetal development. Hence, the signÍficance of these findings

cannoü be deternined, because nelther the alcohol nor nÍcotine groups

dlffered signÍficantly fron the salÍne controls.

the data relating to neural tube defecbs Índicated a strong effect on

8-26

neural dysnorpholory for both agents, whÍch were shown to exert their

teratogenic effects independently when conbined. l'{hen an attempt was nade

to analyse neural tube defecbs according bo whether the embryos

demonstrated open cranial neuraÌ tubes only, or whether they exhibited

open neural- tubes arrywhere along the enbryo, Ít was not possible to do so

due to convergence problems associated wÍth zero or low levels of

nicotine, although the frequency of open neural- tubes in the nicotine

alcohol gnoup suggested that there v¡as a trend towards an anelioration of

an alcohol effect. In a study which examined specifically the effects of

these agents on the neural tube, Ìüoo and Persaud (1988) observed that

forebrain, midbrain and hindbrain defects in enbryos exposed to nicotine

were r¡ct significantly different to those exposed to alcohol-nicotine, but

differed from controJ.s, and since the ethanol group did not differ from

saline controls the results of exposure to the combination of alcohol and

nicotine were almost due solely to the nicotine. The differences between

the outcome in their experiment and the present one can probably be

attributed to exposure of the embryos to subteratogenic leveIs of alcohol

Ín the forner study.

The detrinental effects of alcohol and nicotine combined on neural tube

norphologr were acconpanled by changes to the cranial surface where there

was an incnease in the numbers of rnicrovÍ111 conpared wÍth controls and

with the individual treatnents. heviously this increase fn nicrovllli has

been observed in deficient states of nlcronutrients (Harding et aI 1988;

Brenert et al 1989) and was atüributed to a conpensatory nechanism to

extract nore of these nutrienls from the environment. However the

occurrence of the phenonenon in the presence of noxious agents such as

nicotlne and alcohol could also indicate thaü the cells are

extendfng thenselves i.n search of nutrients that are prevent,ed fron

8-27

reaching the embryo as a result of interference wÍth yolk sac

developnent which was observed in these anírnal- s. Alternatively, the

presence of additional microvilli in dysmorphic 11.5 day embryos, wbich

were of a similar density to leveLs observed in younger prenaLal rats

(TuIsi et al 1991), suggests thaL the mechanisns t,hat lead to normal

dÍminutlon of ecLodernal mícrovilli with progressing embryonic/fetal age

(HoIbrook & Odland 1975i TuIsi et al 1991) may be interfered with as a

result of exposure to embryotoxins such as nicobine and aLcohol.

This study al so denonstrated f or the f irst, time the cel-I ul-ar and

ultrastructural effects of combining nicotine and alcohoI. The independent

effects of the two agents reflected in dysmorphology of the neural tube

and ot,her systens was also reflected in neural tube cell death. The degree

of disrupt,ion observed in the neuroepithelÍun of enbryos exposed to

nicotÍne and alcohol simultaneously appeared to be addibive. This suggests

that nicotine and alcohol may induce neural cell death through different

mechanisms, possibly through hypoxia in the case of nicotÍne, and through

interference wj.th prostaglandin synthesis in the case of aIcohol.

AIt,ernatively, these agents may each exert their teratogenic effect,s

directly on the neuroepithellal cellg The nuclear effects of dispersed

chronatin characleristic of an active metabolÍc slate (Ghadialty 1988) and

meandening nucleolÍ seen in nicotine.-treated aninals was also observed

in alcohol-nicotÍne embryos, along with severe nltochondrial

disruption, also seen in alcohol-treated enbryos. SÍnce lhe neural

tubes were not exanined until 48 hours after the connencement of

culture, sone of these observallons may sÍmply be part of the general

pattern of necrosis of developing cel1s, rather than nechanlsns

specific to the lndividual teratogens, although the nuclear effects

8-28

observed may be nicotine-specific as they were only reported in embryos

exposed to t,his agent. The mechanisns by which the necrotic remains of

neuroepÍtheIial ceII s w ere removed from the neuroepithelium w ere

similar to t,hose observed in previous studies in this thesis. Dead cel-l

remnants Ì{ere extruded into the ventricular Iunen, probably following

disruption of the junctional complexes or inbo the nesenchyme through

breaks in the basal Lanina.

Neural tube celL death has been suggested as a likely mechanism

underlying open neural tubes by a number of workers (Scott 1977i Record et

aI 1985). However, several observabions nade by the present author appear

to refute this association fn parficular, day 11.5 enbryos exposed bo

nicotine always demonstrate closed neural tubes but often show severe

neuroepithelial necrosi.s. In addition, aLcohol-treated enbryos which may

have closed neural tubes in the forebrain region st,ill demonstrate severe

cel-I death at this si.te, as do others with completely cLosed neural

tubes, or tubes that appear to have collapsed following initial fusion

A possible explanation for the above alternatives nay be related to

the rapidity with which the individual teratogen causes celI death. If

lhe onset is rapid and occurs early fn neurogenesis, the neural folds fail

fo fuse and exencephaly is evident by day 11.5 of gestation. However,

if cell death occurs at a slower rate the lube may close initially and

when sufficient necrosÍs has occurred the closed tube collapses, and

finally if cell death does not occur until late fn neurogenesls neural

tube closure 1s unaffected as 1n lhe case of nicotine-treated enbryos.

AIternatlvely, the author suggests that neural tube cell death nay in

fact be an additlonal buü potent effect of the teratogen, unrelated to,

buf Ín some cases concomitant with open neural tubes, and thab necrosis

8-29

may be more related to the mental- retardabion and behavioural

abnormalities observed in offspring exposed prenatally to these agents,

either aLone or in combination Clinical studies have shown that infants

born to nothers who both drink and smoke have been reported to perform

significantly worse on a simple operant learning task (Martin et aI

1977) and to have a weaker suck (Martin et aI 1978) compared to those

born to mothers who either drink or snoke heavily but do not do both.

Functional abnormalities were also reported in rats (Martin et aI 1982)

where an i-nteraction was obse¡r¡ed in which the conbined effect of nicotine

and alcohol was greater than would have been predÍcted fron the effect of

either agent alone.

While there has been a lack of consÍstency between available studies

which examÍne the like1y polentiation of the effects of concomitant

exposure to alcohol and snoking on developnent in utero, the present study

strongly indicates that concurrent exposure of these agents leads to an

additional effect on the nervous systen which although not interactive

Íncreased the frequency of cranial dysrnorphology and neural cell death.

These observations lead to the concl-usion that the nechanis¡ns underlying

alcohol and nicotine teratogenesis act Índependently. Furthernore, fron

the present findings and previous Iimited clinical studies r¿hich indicated

a polentiaLÍon of poor perfornance on learning and behavÍour, Íf alcohol

and smoking are part of ühe J.ifestyle of the pregnant woman, thenearly

indicatÍons are that nornal prenatal development nay be further at rÍsk if

both practices are pursued during pregnancy. fn view of the profound

social and econonic inplÍcations of the conbined effects of snoking and

drfnking, further, carefully focused studies deserve conslderation

8-30

8.5 BIBLIOGRAPHY

AbeI, EL. & Dinbcheff, BA. (1978). Effects of prenatal alcohol exposureon growth and developnent in rats. J. Pharnacol. Exp Ther. 207 t 916-921.Abe1, 9.., Dintcheff, BA. & Day, N. (1979). Effects of in uteno exposure

to aLcohol, nicotine and alcohol plus nicotine on growth and developnentin rabs. Neurobehav. Toxicol. 1 ' 153-159.Andrews, J.& McGarrity, JM. (1972) A conmunity study of smoking in

pregnancy. J. Obstet. Gynecol. Br. Conmonw. 79, 1057-1073.Becker, RF., Little, CRD. & King, JE. (1968) Experinental studÍes on

nicotine absorption in rats during pregnancy. Am. J. Obstet. Gynecol. 100,

957 -968.Benowitz, NL. & Jacob, P. (1984). NÍcotine and carbon monoxide intake

from high-and 1ow-yieId cigarettes. CIin Pharmacol. Ther. 36' 265-210.Brenert, JC., Dreosti, IE. & Tutsi, RS. (1989). A t,eratogenic interactÍon

between dietary deficiencies of zÍnc and folic acid in rats: an electronmicroscope study. Nutr. Res. 9, 105-112.Brown, NA., GouIding, EH. & Fabro, S. (1979). Ethanol embryotoxicity:

effects on mammalian enbryos in vitro. Science 206, 573'575.Coolidge, JS. (1984). Prenatal risk factors. hegnant Ì¡onenrs awareness

of these and lheir behavior. Ugeskr. Laeg. 146, 218'222.GhadiaIly, FN. (1988) Ultrasbructural pathologl of the cell and rnalrix.

Vo1. 1. Butterworths, London. pp. 1-55.Harding, AJ., Dreosti, IE. & Tulsi, RS. (1988).Zinc deficiency in the 11

day rat embryo: a scannfng and transmission electron microscope study.Life Sci. 42, 889-896.HoIbrook, AK. & OdIand, GF. (1975) The fine structure of the developing

human epidernis: light, scanning and transnission electron nicroscopy ofthe perldern. Invest. Dermatol. 656, 16-38.Kaminski, M., Fumeau, C. & Schwartz, D. (1978). AIcohoI consumption Ín

pregnant wonen and the oulcome of pregnancy. Alcoho1 2, 155-164.KuIlander, S. & Kallen, B. (1971) A prospectlve study of smoking and

pregnancy. Acta Obstet. Gynecol. Scand. 50, 83-94.Kuzna, JhI. & Kissinger, DG. (1981). Patterns of alcohol and cigarette use

in pregnany. Neurobehav. Toxicol. Teratol. lr 211-221.Landesnan-Dwyer,S. & Enrnanuel, I. (1979). Snoking during pregnancy.

Teratologr 19¡ 119-126.Landesnan-Dwyer, S,KelIer, LS. & Streissguth, AP. (1977). Naturalísbic

observation of high and low risk newborng AIcoho1 2, 177-178.Lindenschnidt,RR. & Persaud, TVN. (1980) Effect, of ethanol and nicobine

in bhe pregnant rat. Res. Connun Chen Pathol. Pharnacol. Zlr 195-198.L1tü1e, RE., SchuItz, FP. & MandeIl, W. (1976). DrinkÍng during

pregnancy. J. Stud. AIc. 37, 375-379,Martin, JC., Martfn, DC., Chao, S. & Shores, P. ( 1 982) Interaclive

effects of chronlc matennal ethano] and nlcotine exposure upon offspringdevelopment and function Neurobehav. Toxicol. Teratol. 4, 293-298.Mart,ln, JG., MartÍn'DC., Lund, C. & Streissguth, S. (1977) Maternal

alcohol ÍngestÍon and cigarette smoking and their effects upon newbornconditfonlng. AIcohol. Clin Exp. Res. 1, 243-247.Martin, JC., Martin, DC., SÍgnan, G. & Radow, B. (1978) Maternal ethanol

consunption and hyperactÍvity 1n cross-fostered offsprfng. Physiol.Psychol. 6, 362-365.Morrlss, GM. (1973). The ultrastruclural effects of excess maternal

vitanln A on the prinitive streak rat embryos. J. EnbryoI. Exp. MorphoI.30, 219-242.

8-3t

Murrin, LC., Ferren, JR., Zeug, hr. & Hatey, NJ. (1987). Nicotineadministration to rats:Methodological considerations. Life Sci. 40, 1699-1708.Peterson, KL., Heninger, RW. & SeegmiIler, RE. ( 1981). FetotoxÍcity

following chronic prenatal treabnent of mfce with tobacco smoke andethanol. Bull. Environm. Contan. Toxicol. 26, 813-819.Persaud, TVN. ( 1982). Further studies on the interactÍon of ethanol and

nicotine Ín the pregnanf rat. Res. Conmun. Chen. Pathol. Pharnacol. 3T ¡

313-316.Record, IR., Tulsi, RS., DreostÍ, IE. & Fraser, FJ. (1985) CeIlular

necrosis in zinc-deficient rat embryos. Teratology 32r 397-405.Robkin, MA. & Cockcroft, DL. (1978) The effect of carbon monoxide on

glucose metabolism and growth of rat embryos. Teratology 18, 337-342.Rubin, DH., KrasilnÍkoff, PA., Leventhal, JM. et al. (1988) Cigarette

smoking and alcohol consumption during pregnancy by Danish wonen and theÍrspouses. A potential source of fetal morbÍdity. An. J. Drug A1c. Abuse.14, 405-417.Schoemaker, WJ., Koda, LY., !Jiener, SG. & B1oom, FE. (1980). Animal

nodels of fetal alcohol syndrone (FAS): Toxicity of teratogenÍcity.Teratolory 21, 694.Streissguth, AP., Darby, BL., Barr, HM. et a]. (1983) ComparÍson of

drinking and smoking patterns during pregnancy over a sfx year interval.An. J. Obstet. Gynecol. 145,716-724,Tulsi, RS., Harding, AJ., Joschko, MA. & Dreosti, IE. ( 1 991 ) A

quantitative and morphologfcal study of ectodermal microvilli in ten areasÍn the control and experimental prenatal rat. Teratolory ( in press).tloo, ND. & Persaud, IVN. (1988). Rat embryogenesis following exposure to

alcohol and nicotine. Acta Anat. 131, 122-126.

9-1

CHAPTER 9

THE IN VTTRO ACTION OF SALTCYLIC ACID ON RAT EMBRYOS.

9.1 INTRODUCTION

9.1.1 HistorY

Since the history of the salicylates Ís extensive and dates back to the

hippocratic era, only a brief account w ilI be presented in this

Introduction. Because many of the relevant references are original

publications of the late 19th, and early 20th centuries and are therefore

difficult to obtaln and many are also in foreign languages, they have not

been viewed by the authon. A comprehensive review of the history of

salicylates to the time of developnent of conmercial aspirin, together

with the relevant references can be found in Rainsford (1984).

It was known nearly 2500 years ago that various plant extracts including

poplar, willow and myrtle juices had analgesic and anfi-inflamnatory

properties, cbaracteristics of modern day sallcylate compounds, and were

a particularly useful renedy for the treatnent of eye diseases and pain

in childblrth. In the first century, the Ro¡nans and Greeks used

salicylate-containing preparations derlved from extracts of willow leaves,

or poplar bark infusions for the treatment of pain, whlle the ash of

wilLow bark was effective in the treatnent of corns, skin diseases, gout

and earache. In the second century AD the antiseplic properties of the

salicylat,es Ín willow Ieaves were recognised for the treatnent of various

skin conditions including wounds and ul-cers. Hence by tbis tlne there

were nar¡y therapeutic applications of salicylate-contalning plants, of

which a number are slill appropriate today. These plants were also used

from the early days of cÍvilisation in Europe, the Middle East, AsÍa and

America. For exanple, in China preparations of the bark of the poplar

9-2tree have been used for centuries for the treatnent of rheumatic fever,

coldsr haenorrhages, goitre and as a general antÍseptÍc for wounds and

abscesses, whiLe the North AnerÍcan rndians considered wintergreen as an

exceptional herb for therapeutic use.

The antipyret,ic properties of salicylate-containing wil1ow bark was

reported by Stone in 1763 following the first clinical trial of a

salicylate-containing preparation for the treatnenl of fever from maLaria

which was still endemic in Britain During the 19th century intense

interest üras shown in the therapeutic developnent of the saJ-icylates

including the preparation of salicylic acid finst fron natural sources of

salicylaLe, and raber by chenical synthesis, pioneered by Kolbe and

Lautemann in 1860. It was used extensively as an antiseptic to replace the

more noxious phenor, and as an anti-pyretic agent from 1g75, when Buss

used it on patients wiLh typhoid fever, and for the treatment of acut,e

joint rheunatÍsn, chronic rheumatism and gout. Buss also recognised the

synptoms of overdose fotlowing oral ingestion of up to 49 of sal_icylÍcacid, and t,he gastric irritancy accompanying rarge doses in rabbits.

Acetylation of salicylic acÍd to produce aspirin in an unpure form was

performed by Von Gerhardt in 1853, and purified by Von GiIm at, fnnsbruck

in 1860. commercial production of acetyl salicylic acid (ASA) was

initiated by the Bayer compar\y in Erberf erd, Germany, Ín 1g99 af ter itvJas recognised that the high doses of sodiun salicylate and salicylic acid

enployed at the time had noxious effects including nausea, vomiting and

serious gastro-int,estinal symptons. SÍnce the chemicaÌ name was

difficult to pronounce, and sounded sÍmilar to the salicylÍc acid itwas designed to replace, Dreser recalled that natural salícylic acid had

been prepared from plants of the spirea family, hence the term

9-3lrspirinn, to w hich he added the nArt f or acetyl. Aspirin was eventually

marketed in Ausbralia in 1915 by a pharmacist George Nicholas, whose

firm Nicholas Pty Ltd marketed aspÍrin under the welL known trade name

of nAspron. Today, aspirin rates next to alcohol as the nost consumed drug

in the world, with over 40,000 tons of aspirÍn consumed in the worLd each

year (Laurence & Bennet,t, 1 982).

9.1 .2 Metabolisn of salicylate compounds.

The term ttsali-cylatesn is used to describe a group of drugs which have incommon a 2-hydroxy benzoaLe radical, and sevenal reviews deal adnirably

with the subject of absorptÍon, netabolism and distribubion (Smith &

Snith 1966; Barnett et aI 1982; Rainsford 1 984). Aspirin and saticylicacid are both noderately weak acids and are fairly highly lipid soluble

with poor aqueous solubility (RaÍnsford 1984). Oral_ty admÍnislered

ASA is absorbed by passive diffusion partry in the stonach, buL to a

large extent in the smalr intestine (rtowen et ar 19g5). The gastro-

intestinal absorption of the salicylates Ís governed by a nunber ofproperties including the pH of the intestinal lunen and the surface area

of the gastric and intestinal tract available fon absorption (Rainsford

1984). Ihe weakly acidic properties of all the salicylates neans that they

are largely unionised in the acid environnent of the stonach, which

facilitat'es absorption because the uncharged molecules of weakly acidicdrugs are able to penetrate lipid roenbranes with relative ease (Rang &

DaIe 1987). lÍhen these drugs pass into the intestine in appreciabt_e

quantities, the anount absorbed witr be a large proportion of the

total fron the gastro-intest,inal tract because the surface area

available for absorption in the intestÍnal tract is nuch greater than

that in the stomach (Levy et al 1961), and at arnost neutrar pH lhegreater solubllity ln water aids in dispersal of these drugs on the

9-4

absorbing surface, thereby enhancing absorption (Rang & Dale 1987).

Most of the absorbed aspirÍn is hydrolysed to salicylic acid and acetic

acid by eslerases in the gastno-intestinal tract, blood' liver' kidney and

other organs (Spenney & Nowell 1979i Rainsford et aI 1980; Rylance &

Lrallace 1981). Aspirln esterases have been identified in the gastric

nucosa of humans (Trnavsky & Zaehar 197il, and have been isolated and

characterised from this tissue in rabbits (Spenney & Nowell 1979), rats

and pigs (Rainsford el aI 1979¡ unpublished studies). lüith low therapeutic

doses, nost of lhe salicylates in the plasma are protein bound, but with

higher concentrations nore is available for action in the tissues

(Rang & DaIe 1987). The plasma half-Iife of ASA Ís about 15 mÍnutes,

while that of salicylic acid Ís longer and dose-dependentr being 2-3 hours

after 600 ng, and 6-12 hours after Iarger doses, while at toxic doses'

the half-life of salicytic acid may increase to 15 lo 2O hours (Flower &

Moncada 1985).

The prÍncipal plasma constituent bindlng salicylate and aspirin is

albumin (Levy 1978) and there are two inportant consequences of the

drug bÍnding to this plasma protein Finslly, the binding capacity of

albumin varies with age and disease states, and is markedly reduced in

the eIderly, j.n neonates and in those suffering from rheumatoid arthritis'

al-cohol liver dÍsease, or acute or severe infection, as a consequence of

altered binding properties, and/or reduced plaslla concentrations of

albumin in the circulabion (Brodie & Boobis 1978; Levy 1978; ltindorfer

et al 19?8). Hence these Índividuals may have a much greater proportion

of rfreen (ie-unbound) drug relative to that which is bound, since the

ilfreen drug concentrations determine the occurrence of some therapeutic

effects as !reLl- as side-effects. The tendency for this to occur will

9-5

relative to bodY weightdepend upon the anount of drug which is ingested'

or surface area, since bhe capacity of individuats bo bind the drug to

plasma proteins is linited (Windorfer et aI 1978)' Secondly, when

saticylate binds to albumin, there is an associated nelease of tryptophan'

urate, snall peptides and cerbain non-esterified fatty acids (Dawkins et

aI 19?0; McArthur eL al 197 Ð, and it has been postulated that'

salicylate-induced displacenent of these peptides and tryptophan from

albumin couLd account in Partr for the anti-inflamnatory actions of the

drug (McArthur et at 1g7ü. Hence, these authors suggested that it is the

amount of drug bound to plasna albunin which determines the therapeutic

actions of salicylate (McArthur et aI 1971). Thus it appears that both

drug binding to albumin and the amount of free drug determine the

therapeutic effects and sid+effects of the salicylates.

A najor proportion of aspÍrin is hydrolysed to salicylic acid and acetic

acids in the gastro-intestinal tract, blood, 1iver, kidney and other

organs by the proposed aspirin esterases (Rylance & I'laIlace 1981;

Rainsford 1g80). However, aspirin also acetylates a variety of proteins in

various tissues and conçonents of the blood (Caterson et al 1978) and from

this acetylabion, salicylate is released. Sallcylabe is then netabolised

and hence renoved from the body by a number of parallel and competing

pathways (Thiessen 1982) involving a serles of liver microsomal and

mibochondrial enzynes (Rainsford 1984) to form salicyluric aeid, salicylic

phenolic glucuronides, acylglucuronide and gentisic acid (See RaÍnsford

1984 and Barnett et al 1982 for revlews). The general pattern of aspirin

and salicylate netabolism Ís slnilar Ín hunans and laboratory animals

albhough the relative proportions of the netabolites formed nay vary

somewhat in different species (fwanoto et aI 1982). I{hile sone biliary

excretion of sallcylate nebaboli.tes occurs in nost specles, the bulk of

9-6

the netabotites along with a fraction of the unchanged salicylic acid

excreted in bhe urine (Iwamoto el al 1982)'

are

placental transfer of salicyl-ate Is rapid, because of Ùheir high tipid

solubility coefficient (Boutos et aI 1972). However fetal blood

concentrations do not reach naternal blood levels for 6O to 90 mÍnutes

after intravenous admlnÍstration in humans at term (Noschel et aI 1972) or

earlj_er in gestation (Elis et aI 19?8). This slow equilibration may be

attributed to the observation thab almost 751 of the drug Ís bound to

plasma proteins and is thus not free for rapid diffusion fron naternal to

fetal plasna (BouJ.os et al 1g72). Furthermore' it was observed (PaLnesano

& Cassidy 1969; Levy & Garrettson 1974) that salicylate elininatÍon is

slow because of the immaturity of the glucuronídation and renal excretory

pathways. This results in a higher concentration in the fetus and

increases the length of exposure to the drug (PalmÍsano & Cassady 1969).

9.1.3 pharmacological and blochemical actions of the salicylates.

The prÍncipal pharmacological actions of the salicylate drugs are as

anti-inflannatory, analgesic and anti-pyretic agents. The literature

describtng these actions and others is extensÍve (see reviews Barnett et

aI 1982; Rainsford 1984) and wíll onty be summarised brÍefly in t'his

thesis. AspirÍn (and its hydrolysis product salicylic acid), is thought to

exert its naJor pharmacologÍcal effects by inhibition of prostaglandin

biosynthesis (Vane 1971; Flower & Vane 197Ð although by different

nechanisros (Laurence & Bennetb 1987). The najor effect of aspfrin is on

bhe cyclo-oxygenase enzyne system responsible for the primary step in the

netabolic conversion of bhe precursor arachidonic acid, to bhe unstable

cyclic endoperoxidases. Hence administratÍon of these agents can lead to

inhibition of the entire prostaglandin cascade and therefore

9-7

inhibition of the production of the unstable intermediary substance as

well as thromboxane and al-1 prostaglandins (Heymann 1986). Prostaglandins

and the synthetic pathways responsible for their production have been

identified in virtually all mannalian tissues, including fetaL tissues,

and their physiological effect depends on continued synthesis, since they

are rapidly catabolised and are not stored in tissues.

Aspirin has an analgesic effect particularly usefuL for 1ow intensity

pain, such as headache, myalgia, arthralgia and other pains arising fron

integumental structures. Chronic use of salicylate drugs does noL lead to

tolerance or addiction (FIower et aI 1985). The analgesic dose range is

between 0.3 and 19, 3-4 times daiIy. Pant of the analgesia arises from

actions on subcorüical siües of the CNS, probably the hypothalanus. In

addÍtion, salicylate may inhibit plasmin, and thereby bradykinin

formation Penipheral chemoreceptors which are stimulated by kinins to

produce paÍn are blocked by salicylates. hostaglandin synthesis is

blocked because of cyclo-oxygenase inhibltion, therefore the sensitivity

of the receptors to pain producÍng substances Ís greatly reduced (Moncada

et al 1985).

SaJ-Ícylate also has antÍpyretic properties, and lowers the body

tenperature in cases of fever by a direcf action on the hypothalanic

thermoreceptive region and the temperatune regulating centre concerned

with heat production and heab 1oss. The Íncreased heat loss occurs due to

perÍpheral vasodilation, and to Íncreased sweating. The nechanis¡n of

antlpyretic action while uncertain, appears to block pyrogen-induced

prostaglandin synthesis (lfoodbury et al 197Ð. In contrast, however large

doses of salicylates can actually induce a pynetic effect as oxidatÍve

phosphorylat,ion is lnhibited by large doses of salicylates, and the energy

9-8

nornally used for ATP production is dissipated as heat (l{oodbury eü al

1975).

salicylates when adninistered Ín doses of 5-89 daily are used for

the treatment of inflammabory diseases including rheunatoid arthrifis.

The increased capillary permeability which is characteristic of

the inflammatory state is reduced by salicylates, hence preventing

edema, cellular exudation and pain These effects appear to be due to a

number of nechanisms including inhibition of prostaglandin synthesis'

and of plasnin, a proteolytic erøyme which nay actÍvate kinin formation'

as w eI1 as inhibilion of leukocyte migration and phagocytosis

during which conpounds such as histanine, and 5-tVdroxy-tryptophan may

often be released, and stabilisabion of lysosomal merobranes' thus

preventing the escape of lysosomal enzymes into the cytoplasn and

damage to cell structwes (Moncada et al- 1985).

Aspirin exhibits a number of other physiological effectsl some of which

are not always advantageous, but will onty be briefly al-luded to in this

section. The reader is referred to Laurence & Bennett (1987), and GiInan

et aI (1985) for nore detailed accounts of these actions. fn medium or

large therapeutÍc doses, aspirin dÍrectly stimuLates the respiratory

centre leading to respiratory alkalosis which is nornally conpensated by

Íncreased urinary bicarbonate el imination. In toxic doses how orer,

salicylates cause respiratory depressÍon and a combination of

uncompensated respÍratory and metabolic acldosls. Salicylate also has an

effect on carbohydrate metabolism. At noderate doses salicylate can lower

the blood sugar, by increasing peripheral ulilÍsation of glucose and has

been used in the treatnent of diabetes mellitus. I'lÍth heavy doses howevert

hyperglycaenia can occur, perhaps due to depression of aerobic glycolysist

9-9

increased hepatic glycogenolysis and increased adrenal cortical activity'

At Iow doses (1ess Lhan 2g/day) salicylate causes the retention of uric

acid, whereas at high doses (S-8glOay) tne renal tubular reabsorption of

uric acid Ís reduced and salicylate is useful Ín depleting uric acid

leve1s in patients suffering from gout, although few patients with this

disease can tolerate the hÍgh doses required. SalÍcylate is also useful

in reducing blood plabelet adhesiveness which results in prolongation of

bleeding time, an action of aspirin vaLuabLe in occlusive arterial

disease, while at doses in excess of 58r aspirin or sodiurn salicylate can

induce hypo prot,hronbinaemia.

Aspirin also has an effect on endocrlne functlon In addition to

stinulating adrenaLin release, ASA increases the plasna adrenocortico-

steroid Ievels by sbimulabing the hypothalamus to increase the release of

adrenocorticotrophic hornone and also interferes with thyroid hornone

binding by competition for binding sÍtes. In addition, aspirin can l-ead

to epigastric distress, nausea and voniting, as well as to exacerbation

of peptic ulcer synptons, gastro-intestinal haenorrhage and blood Ioss in

some sensitive patÍents on prolonged salicylate therapy' Since

prostaglandins are essential to naintain cellular tntegrity in tbe

gastrointestinal tract, inhibition of prostaglandin synthesis nay

lead to danage of gastrointestinal epithelium.

There is no evidence that occasional use of snall doses of ASA durÍng

pregnancy is harmf ul-. How everr if taken negularly during t'he last

trinesler of pregnancy, aspirin may conLribute to prolonged gestation and

Labour as well as increased naternal blood loss during dellvery. ASA nay

also cause a variety of diffÍculties if ingested in Iarge enough doses

during criülcal periods of early geslation. Sone data show a strong

9- 1o

correlation between consunption of large doses of acetyl salicylic acid

during the fÍrst 16 weeks of pregnancy, and the incidence of fetal

nalfornations. This Íssue wÍ11 be explored nore fulIy in Lhe following

sections.

9.1.4 The effects of aspirin on enbryonic development in aninals.

9 .1 .4a. Gror¿th

Administration of ASA and related salicylate conpounds to pregnant rats

have led to a reductÍon in growth parameters (KoshhakjÍ & Schubert

1973i DePass & l,leaver 1982; Greenaway et al 1982; Cicurel & Schrnid 1988),

with a similar observation reported in cats (Khera 1976)r but not in

nice (Guy & Sueheston 1986). Maternal- administration of a dose of

450-500me/ke of aspirin or Na salicylate in rats led to a significant

reduction in fetal weÍght (Goldnan & Yakovac 1963; McGarrity et af 1981i

DePass & Ïleaver 1982) and length (DePass & tleaver 1982). A simil-ar

decrease in growth paraneters in vitro including length, somite number,

and protein content was shown to be dose-dependent at concentrat.ions of

400, 600 and 800 uelml of Na salicylate when embryos l¡ere cultured for 24

hours on day 10.5 of gestation (Greenaway et aI 1982). In another study,

while protein content and crown-runp Iength of cultured enbryos were

significantly decreased in aspirin-treated groups, exposure to sinilar

concentratÍons of its major met,abol ite, sal icylic acid, did not

significantly affect growth (Yokoyama et aI 1984).

9.1.4b Enbryonic and fetal wastage

SalÍcylate compounds have also been shown to lead to enbryonic and fetal

death, following daily dietary adninistration of doses of aspÍrin Ín rats

(Brown & l{est 1964) ranging fron 50 ng/Kg/day throughout pregnancy, whlle

all fetuses were killed by treatnent wfth 600ne/Ke/daily fron day 6 of

9-11

gestation (Obbink & DoLderup 1964). I,lÍlson et al (1977) administered

aspirin orally to rats and monkeys during the post-Ínplantation period and

reported an incidence of 34f dead or resorbed rat fet,uses on day 20 of

gestation at 150 mg/kg, but with a considerably lower level in nonkeys at

a comparable dose and time of gestation These differences Ín enbryonic

and fetal wastage between species appeared to be nelated to the hÍgher

levels of unbound salicylic acid in rab embryos, which matched unbound

leveIs in maternal plasna. Hence the rat embryos were exposed lo a

considerably greater level and duration of dosage of salicylic acid than

were nonkey enbryos ab equivalent maternal- doses of aspirin A nunber of

other studies have reporled an Íncreased incidence of resorptions

follov¡Íng asplrin adninistratÍon, including DePass & l,leaver (1982) who

observed a significant increase in the nunber of resorbed fetuses in

FÍscher and llistar rats at 625 nglkg and 500 ng/kg respectively,

conpared with controls, while a lower dose of 400 mglKg daily in early

gestat,ion in dogs induced similar effects (Robertson et al 197Ð. Goldman

& Yakovac (1963) observed that an injection of 500 ng/Kg of Na

salicylate, a nore soluble form of aspirin, on days p, 10, or 11 in

pregnant rats also 1ed to an lncrease in felal death, but they did not

report any sÍgnifÍcant dÍfferences in naternal morüality or early

resorptions. A higher incidence of fetaL death has also been reported when

aspirin administration was restricted to later in gestation (Eriksson

197 1) .

9.1.4c. Aspirln and dysnorpholory

The teratogenicity of the salicylates was first descrlbed by tlarkany and

lakacs (1959) who found that adninistration of nethyl or sodiun salicylate

to pregnant rats on days !, 10 or 11 of gestation resulted 1n a spectrum

of congenital abnornalitles extending over a nunber of organ systens.

9'1 2

These included malfornatÍons of lhe skeleton, viscera and palate, as weII

as the CNS, where anonalies included cranj.orachischisÍs, exencephaly,

hydrocephaly, hindbrain defects, ocular defects and spina bifida. Simil-ar

abnormalities have been reported in a nunber of other studies following

the naternal- adninistration of salicylate compounds in rats (GoIdman &

Yakovac 1963i Bertone & Monie 1965, McColL et, al 1965i McGarrity et al-

1981) as well as in guinea pigs (Kronka & Hoar 1973) and cats (Khera

1976). Larsson & Eriksson (1966) were abl-e to induce anonalies of the ribs

and vertebrae in fhe offspring of Ajax mice treated wit,h Na salicylate on

day 9 of gestation, as welÌ as vesseL abnormalities when treatnent was

delayed until day 15, while Trasler (1965) induced cteft tips in the

offspring of these aninals by administering 500 ng/Kg of asplrin over a

24 hour period between the 8th and 10th days of gestation TeratogenicÍty

was also reported in 50f of the offspring of dogs folJ-owing a large dose

of aspirin (400 nelk8lday) between 23 and 30 days of pregnancy,

which included cleft palate, micrognathÍa, and cardÍovascular and tail

anonalj.es. ÏIhile salicylateinduced teralogenesis has been observed

in nost species, congenital abnormalities were not observed in rabbits

in early studies (EarIey & Hayden 1964; McColI et aI 1967), which was

attrÍbuted to the different netabolism and rate of excrebion (Bray et al

1948) as weLl as the degree of plasma binding of salicylate in this

species (Kucera et aI 1968). More recent studies however (Rainsford 1984)

have reported teratogenic anonalies in bobh rabbits and ferrets.

A teratogenlc dose-response relationship has been reported by Kinnel et

aI (1971) following doses of aspirin in the range of 250 to 1000 nglkg inpregnant rafs at each treatnent tine between days 9 and 11 of gestatlon

An extenslve range of abnormallties was reported inctuding defecfs of the

cNs and face, including eye and palate, as well as anonalÍes which

9-13

occurred before lhe peak tirne of organogenesis for that organ such as

skelebal defects which included polydactyly of the hÍndfeet, and kidney

abnornalities which led the authors to suggest that the cel-lu1ar

precursors of bhese systems were affecbed at the biochemical l-eve1

resulting Ín abnormal early differentiation Fol1owÍng administration of

625 ng/kg of aspirin to pregnant rats on day 1 1 of gestatÍon, Klein et al

(1981) has reported a unique pattern of cytotoxicity in embryonic

hindlimb bud r¡ith ce1l death Iocal-ised in the pre-axial mesodern of the

developing rat hindlimb, and has subsequently speculated that thÍs

necrotic event Ís responsible for the observed polydactyly in this

structure. Cybotoxicity of aspirÍn has been previously reported in cells

of adult gastric mucosa of mice (Hingson & Ito 1971), rats (Rainsford &

Brune 1978) and ferret,s (Pfeiffer & lleibel 1973).

Several j¡ vitro studles in rats have reported a nunber of congenital

abnormalities similar to those jX vivo, particularly in relation to the

CNS where defects were described both at a gross and cellr¡Lar level. JI}

vitro salicylate-induced neural tube defects were fÍrst reporled by

McGarrity et aL (1981). In a more detailed study Greenaway et aI

(1982) observed nalformations of the developing nervous systen

following the culture of rat enbryos in 400, 600 or 800 uglnl of Na

salÍcylate between days 10.5 and 11.5 of gestation, as well as a dos+

dependent reducbion in gnowth paraneters. Ano¡nalies included open

neural tubes, necrosis of the mesenehyne and dilation or thinnÍng of fhe

neural ectoderm, which were suggestive of exencephaly in rat fetuses

exposed to salicylate conpounds in vivo. Yokoyama eü al (1984) also

reported salicylate-induced abnormalities foltow íng 72 hours of culture

with exposure Iinit,ed to less than 4 hourg The predoninant defects of the

fetuses on day 14.5 of gestation were edenatous anonaLies of the face and

9- 14

tait following exposure to aspirin and l-ocalised malfornations such as

clef t Iip and curly or short t,ail following exposure to t'he netabolite

salicylic acíd.

Several studies have denonstrated the Iong term effects of maternal

aspirin administration on offspring. Butcher et al- (1972) investigated

the learning abÍlity of rats exposed to subteratogenic amounts of aspirin

and concl-uded that thÍs compound which is known to be teratogenic to the

central nervous system of animals, Ied to psychological inpaÍrment in

offspring, evenwhen administered in anounts that do not lead to gross

malformations. Kinmel et al (1974) also reported íncreased activity in rat

offspring following exposure to doses of aspirÍn too low to produee

differential birthweight effects. In a more recenb study Vorhees et aI

(1982) have shown a psychoteratogenic effect postnatal-Iy fol1owÍng a

single hÍgh dose of aspirin on day 1 1 of gestation.

A nunber of studies have been performed in an atùempb to determine

the causative agent of aspirin teratogenicity. The observation that

berzoÍc acid, a widely used food preservative (Furia 1 968) significantly

Íncreased t,he frequency of maLformations when 250ng/kg of aspirin were

used (Kinnel et aI 1971), implicated salicylic acid in this role, since

benzoic acid nodified the metabolÍsn of aspirin and thus increased the

concentration and persistence of salicylic acid Ín plasnar preventing

further netabolisn to sallcylurlc acid (Levy et aI 1969). This was

further supporled by the observation that salicyJ.ic acid was the najor

netabollte that reached the enbryo after oral adnlnistration of aspirin

(wÍt,h or without benzoÍc acÍd) (KÍnnel et aI 1971). These observations

together wlth the knowledge that aspirin can readlJ.y undergo hydrolysls

to forn salicyllc acid (Snith & Snifh 1966), and that aspirin and

9-15

sal-icylic acid induced sÍmilar abnormalities Ín day 20 rat febuses

(Kost¡ajkÍ & Schulert 1973) also led these workers to conclude that' the

teratogenÍc effect of aspirin is due to its najor metabollte'

The teratogenic action of salicylate compounds was thought to occur

through maternally nediafed metabolic facbors, and was possibly rel-ated to

maternal stress (Goldman & Yakovac 1963)r oF to food restriclion during

pregnancy (Beatl & Klein 1977) as aspirin administration greatly increased

t,he formation of congenttal malformations in rats (96%) on restricbed

dÍets, when compared wibh nornally f ed control s Q\ft). Qther

experinents (Kímnel et aI 197 1) Ínvol-ving fluorÍnetrÍc technlques

where enbryoníc and plasna leveIs of salicylales were assessed showed

thaf salicylat,es crossed the rat placenta and were thus avaílable for

direct action on the enbryo. In addibion, more recent j¡ vitro culture

studies (McGarrity et aI 1981; Greenaway et at 1982; Yokoyana eb al

1984) have shown conclusively bhat this class of drug has a direct effect

on the rat embryo in the absence of any maternal influence.

Tk¡e rnajor netabolic consequences of salicylate poÍsoning capable of

acbing direcbly on the enbryo is the cytotoxic uncoupling of oxidative

phosphorylabion (Smith 1959i Bostrom et at 1964), which has been

reported to be linked wíth depressed synthesis of acid muco-

polysaccharides (Larsson et aI 1 963). Hence the teratogenic effect of

sal lcylates in nice has been suggested to be related to the

depression of acid rnucopolysaccharide synthesÍs (Larsson et af 1963i

Larsson & Bostron 196Ð, whleh is particularly relevant since the presence

of acid mucopolysaccharides in embryonic tissue nighf have an Ínportant

function in growth and differentiation (Balazs & Holmgren 1949)' and

abnornaLities have been reported in vascular and skeletal tissuest

which nornally conlain high concentratÍons of bhese

1956).

9- 16

compounds (Meyer el al-

Another possible mechanism underlying salicylate-induced dysnorpholory may

be rel-ated to the drugs effect on reducing RNA polynerase activify in 13

and 16 day fetuses leading to an inhibition of RNA synthesis (Janakedevi &

Srnith 1969) and wilh interference with biosynfhesÍs of nucleic acids and

protein synthesis (Janakadevi & Smith 1970). It has also been suggested

that the observed dysnorpholory nay be nediated through disturbed

prostaglandÍn synthesis (Vane 197 1) since aspirin, a prostaglandÍn

synthesls inhibitor interferes with fetal vascular physfology (Rudolph

1981 ) .

9.1 .5 Sal icylabe con pounds and embryonic deveJ.opment in humans.

!lhile the metabolic pathways for sallcylate netabolism ane the same Ín

rats and humans (Levy & Leonards 1966), data pertaÍning to human

studies do not indicate as cl-ear a correlation between aspirÍn

ingestion and unfavourable pregnancy outcome, although several

retrospective studies do suggest such a trend. Collins & Iurner (975)

and Turner & Coltfns (197il found significantly decreased birthweights

as weII as increased fetal wastage ln wonen who chronicaJ.Iy ingested

saIÍcylates throughout pregnancy. PerÍnatal mortality was observed to

be associated with these low birthweight infants. These findings Ied

these workers to conclude that the regular ingestion of aspirin by

these wonen was detrinental to the welfare of Lheir babies. In contrast

however, Shapiro et al (1976) using the collaborative perinatal project

previously described by Slone et at (t976)' could find no evldence

that, prenatal- aspirin is a cause of stillbirths, neonatal deaths, or

reduced birthweight.

9-17

In a retrospective study by Nelson and Forfar (971), aspirin ingestion

during the first 28 days of pregnancy occurred in a significantly higher

proportion of nothers of infants with congenital malformations than in

conLrols. Abnornalities included hydrocephalus and congenital heart

disease as well as talipes, achondroplasia, mongolism, congenital

dislocatÍon of the hip, and hydroceLe. Richards (1972) also perforned a

rebrospective study of 883 cases and found that significanbly nore

mothers with malformed infants had ingested salicylates during the first

16 weeks of pregnancy. I'Iomen w ere matched f or such variables as aget

parity, social class and area of residence. Ïlhi1e an overall lncrease of

congenital abnornalities was observed in the study, talipes was the only

anomaly whose incidence was significantly lncreased by aspirÍn ingestion.

Richards(1972) however, indicated that the study was not able to

separate the effects of aspirin fron Lhose of the underlying condltion

for which the drug was taken In a four-year retrospective study, Saxen

(1975) reported that the eonsumption of aspirin during the first

trinester occurred nearly three times as frequently in rnothers ofinfants

wfth oral clefts and nearÌy four tines (19.8í) as frequently in mothers of

infants with both cleft lip and palate' as in normal infants.

Several other reports have been published which point to a possible

association between congenital abnormalities and salicylate ÍngesbÍon

during pregnancy. McNieI (1973) report,ed on 8 individual cases which

inplicated aspirin ingestion durÍng pregnancy as a likely cause of

congenital nalformatÍons Ín hunans, with anornalies that encompassed a wlde

range of organ systeros and Íncluded skeletal defects especially

polydactyly, cardÍovascular ar¡ornalÍes and in one case anencephaly.

McNleI (1973) suggested bhat ingestion of aspirin Ín dosages of 650n9'

4 tines daily for 5 days, by wonen during a critical perlod of gestation

9-18

nay result in congenital defects. Since most of the organ sysbems

are differentiated and formed by 60 days of gestation (Millen 1963), it

is like1y that the critical period would occur wibhin this tÍne.

Although Turner & Cotlins (1975) did not report a significanb increase

in salÍcylat+induced abnornalities, major congenital nalformations were

observed in 6 of 144 infants of mothers who regularly took sallcylates

during pregnancy.

In contrasb, Crombie (1970) sall no difference between the nunber of

congenital abnormalities and nornal infants in women fssued with aspirin

prescriptions during early pregnancy. The authors concluded that any

relationship between drug consunption and a congenifal abnomality is

indirect and possÍbly more related to the norbld conditions for whÍch fhe

drugs were given A further prospective study (Slone et al 1976) involvÍng

50,000 mother-child paírs also found no evidence that aspirin ingestion in

conventional doses during pregnancy was associated with congenital

nalformations, however these workers dÍd not discounb the posslbillty that

grossly excessive exposure to aspirin nay be teratogenic. Many factors

have undoubtedly contrÍbuted to the discrepant results in hunan studiest

sone of which according to Streissguth et aI (1987) include differences in

the brimester of drug use, the dose studied, the type of statistical

analyses employed, and the degree to whlch other confounding factors

were controlLed. Recent evidence has also suggested that maternal aspirfn

ingestion during the first half of pregnancy was significantly relaled

to IQ and attention decrenents in 421 exposed children at 4 years of age

even at doses when structural anonalies or lntrauterine growth was not,

affected (Streissguth et al 1987), and continued to be signifÍcant even

when adJustnenüs were made for a variety of potentially confounding

factors. 0f particular interest was lhe observation that this asplrin

9- 19

effect was sÍgnificanbly greater for girls than boys. This study led the

authors to suggest that naternal aspirin use during pregnancy can have

long tern effects on exposed ehildren and bhat these effects reflectfunctional decrenents in the absence of physical growth decrenen¡s inexposed offspring. A much larger study however (Klebanoff & Berendes

1 988) which involved nearly 20,OO0 pregnancies refuted these

salicylate-nediated effects on the IQ of 4 year olds and in fact reported

that Lhe mean IQ of children exposed to aspirin was sÍgnificantlyhigher than that, of unexposed children, even after adjustnents were made

for rnultiple social, denographic and other confounders. The effect ofprenatal aspirÍn exposure also did not vary by Ínfant sex, and hence itwas concluded that an adverse effect of aspirin exposure on rQ was

urù ike1y.

The presenl i¡ vitro study was perforned to confÍrm and extend prevlous

observations thal salicylic acÍd has a direct effect on the developing

embryonic nervous systen, and to study for the first time the effect of

this aspirin metabolite on the ultrastructure of the nervous system at the

tine of neurogenesis. It, is hoped bhat fhe ultrastructural data nay

contrÍbute to a better understanding of t,he factors underJ-ying neural

dysnorpholory in the developing enbryo.

9 -20

9.2 MATMIALS AND MEITIODS.

9 .2 .1 Ani¡nal- s

Virgin fenale Sprague-Dawley rats (180-230e) were housed overnight with

males of the same strain Tt¡e tine of detection of spern 1n vaginal snears

was designated as day 0.5 of gestation and animals with positive smears

were placed in cages v¡ith free access to food and water.

9.2.2 fubryo cuLtwe and teratological screeni.ng

0n day 9.5 of gestati.on, inplantation sites were removed from the uterÍ

of dams under diethyl ether anaesthesía and enbryos were dissected free of

decidua, parietal yolk sacs and Reicherts nembranes. Groups of three early

headfold enbryos were placed in 60mI culture bottles along with 3m1 of

prewarned medium and gassed with oxygen, nitrogen and carbon dioxide as

described in chapter 2 of this thesis. Where appropriate, salicylic acid

was added to the medium at concentrations ranging from 100 ug/nI to 300

uglnI, which Iies wÍthin the range known to occur in adult and fetal blood

after ingestion of aspirin as an analgesic by pnegnant women TL¡e bottles

were rotat,ed for 48 hours where upon enbryos $Jere removed and exanined

under the dÍssectÍng microscope for evÍdence of inpaired growth and

dysnorpholory. The methods by which embryos were processed for Iight and

el-ectron rnicroscopy are described in chapter 2.

9 .2 .3 Sta li stÍ cs

Continuous gnowth variables such as crown-rump length and somite

nunbers ÍJ ere analysed by standard analysis of varlance. For discret,e

development data, such as the nunber of enbryos wilh neural tube defects,

a naximum IikelÍhood nethod was used, assuning the data to be binonial.ly

distrÍbuted and the deviance to be approxinately dfstributed as

X2 on the appropriate degrees of freedom (Baker & Nelder 1978).

9 -21

9.3 RESULTS.

9.3.1 Growth

Enbryos cultured for 48 hours fron day 9.5 of gestation in rat serun

containing salicylic acid at concentrations ranging from 100 ug/ml to 300

ug/nl showed a significant and dos+dependent growth retardation as

determined by several growth indices conpared with controls (TabIe 9.1),

which confÍrms previous findings i¡ vitro in animals (Yokoyama et aI

1984). There was a clear and signÍficant dependence on dose for

crown-rump Iength [Fç3,60) =1]1.0 p(0.001Jr somite numbers [F(3,60) =132.5

p(0.001J, as well- as yoJ.k sac dÍaneter [F(3,60) =32.7 p(0.001J.

Table 9.1. Effectcul ture. ?t io vitro salicylic acid on growth of rat embryos 1n

Salicylic acid concentration (ue/n])

0 100 200 300

Total embryosCroln-runplength ( rnn)

No. of somitesYoIk sacdianeter (nrn)

15

3 .2 8+0 .07

23 .37 70 .41

4.17+0.07

17

3. 1 010 .07

21 .66+0.51

3.8810.10

15

2.60+0.08

17 .23!1 .01

3.56$.13

7

1 .4510.08

7 .29+0.42

3.02$.071 Values are means + SEM.

9.3.2 Morphological development.

A nunber of the norphologÍcal parameters exanined when the enbryos vJere

cultured in sallcylic acld showed a concentration-related dysnorpholog¡

(Table 9.2. Figs. 9.1 a-c). The nunber of dead enbryos was not recorded in

t,his tablg as enbryonÍc viabÍIfty deternined by the presence of a

heartbeat, was found 1n aII experinental anÍmals regardless of whether the

enbryos $Iere grollth retarded and/or abnornal.

Tabl-e 9.2 Effect of in vitrgdevelopmenb in rats.

9-22

salicylic acid on norphological1

SalicyJ-ic acid concentration (ug/nL)

0 100 200 300

Total mbryos

Any kind of defect

NTD

Ele def ect s

Ear defectsBranchial archdefect s

Heart defects

Absent f orel inb

fncornplete fLexion

Inpaired fusionYoIk saccÍ rcul atÍon

5

2(13.3)

0( 0)

0( 0)

1(6.7)

0( 0)

0( 0)

0( 0)

1(6.7)

0( 0)

2( 13.3)

17

6( 15.3)

1(5.9)

0( 0)

6(0)

0( 0)

0( 0)

1(5.9)

4( 23.5)

4( 23.5)

2(11.6)

15

1 5( 100)

15(100)

8(53.3)

1(6 .7)

3( 20)

3( 20)

10(66.7)

15(100)

6( 40)

8(53.3)

17

17( 100)

17( 100)

15( 88.2)

17(100)

7(41.2)

1 5( 88 .2)

17( 100)

17( 100)

17( 100)

17( 100)

1 Percentages given Ín parentheses

Most enbryos exposed to 100 uglml salicylic acid did not appear to be

affected norphologically except for j.ncomplete rot,ation which was observed

in over 20f of the enbryos and which was often acconpanÍed by a lack of

chorio-allanboic fusÍon As very few other abnormalities were observed at

this concentration, these parameters are likely to be representative of an

induced developnental delay ratber than teratogenesÍs.

There $¡as a sharp Íncrease in the incidence of open anterior neunal tubes

aE 200 ug/ml or nore with nost embryos affect,ed. The nost frequently

observed neural tube anomalies conslst,ed of interruption of neural

closure, which either involved the cranial region aIone, reported

prevíously following exposure to salicylate salts (Greenaway et aL 1982),

orwhich additionally invol-ved the neural tube caudal to the craniun, a

9-23

precondition of cranioraschÍschisis observed in aspirin-treated animals at

term (!'larkany & Takacs 1959; Kimmel et aI 1971; McGarrity et aI 1981).

Craniofacial structures including eyes and branchial arches were also

affected by salicylic acÍd exposure. Eye defects which consisted of

compression and narrowing of the optic vesicles, and in some cases absence

of the optic placodes, occurred only at higher concenbrations and affected

up to 88f of breated ernbryos. Branchial arch defects which included the

absence of mandibular arches, or fusion of these slructures to the

pericardÍun, occurred in 41f of enbryos at 300 uglml. These anomalies are

represented later in development by cleft palate and cleft, lip in enbryos

foll-owing exposure to aspÍrin or salicyllc acid durÍng organogenesis

(DePass & hreaver 1982; Yokoyama et al 1984).

Cardiac abnormal-ities rose to 881 at 300 ug/nl salÍcyIÍc acid, with nany

embryos denonstrating prinitive ntube-liken hearts, although the

pericardÍa were not affected, nor was contractility. Yolk sac circulation

was also severely iupaired in over 50ß of enbryos exposed to 200 ug/nl(fig.9.1d), and in all aninals at the higher concentratlon. The absence

of forelinb buds which occurred 1n all enbryos exanined nay also be

indicative of developnental deIay.

Scannlng electron mÍcrographs of typical salicylic acid-ct¡ltured enbryos

(Fig. 9.2) showed that as the concentration of lhis agent, increased,

the severity of gross general embryonic dysnorpholory also lncreased

(Figs. 9.2 a,d). llhlle the neural tube was closed in embryos cultured

in 100 ullnl sal1cyI1c acidr there was sone underdevelopnent of the

forebraln and nidbrain compared with controL anfnals of the sane age

(Fig. 6.Za), and 1n some enbryos there was partfal collapse of the dorsal

neural tube' which suggest'ed thinnlng of the neuroepfthelium in this

9 -24

Figs.9.1.a,b,c.Invitroday11'5embryosexposedtoincreasingconcentrations of salicylic acid. The enbryo in a. exposed to 100 uglml

appears similar to control jn vitro enbryos of the same age (c'f' Fie'O.ia) except that there vras a smalL degree of cranial underdevelopment ofthe forebrain - nidbrain region and dorsi-convex rotation was incomplete.The embryos in b, exposed t,o 200 uglnl demonstrated exencephaly' The

cranial neural folds (NF) had not fused except for the region between the

forebrain vesÍcIes (asterisk). The enbryo was also growth retardedt and

had nob rotated into the dorsi-convex positlon and forelinb buds had nob

developed. The embryos in c, exposed to 3oo ug/ml showed overall growth

and developmental retardation, with neural tubes that were open from above

the forebrain vesicles (arrows) often to bhe caudal end of the embryo' and

lrere often distorted caudal to the hindbrain (arrowheads). Many of the

embryos which faÍled to rotate into the dorsi-convex position also failedto develop a placenta as indicated by the presence of an unfused

al Iantoi s.

d. Yotk sac (ys) conlaining affected embryos exposed tosaticylic acid. Circulatlon was severely retarded with theabsence of visceral blood vessels (c.f. control ln FÍg. 7.1c).

200 uglmIcomplete

Fb, forebrain i Mb, ¡oidbrain i Hbt

vesicles i 1 12 13 , branchial arches;ectoplacental cone.

Bars= 1 nn.

hindbrain;H, heart;

optic pits; 0T'forelimb buds;

OP,Errt

oti cEct

9 -25

region. Examination of the cranium of an embryo cultured in 200 uI/ml

salicylic acid (Figs.9.2d,e) revealed that the anterior neural tube was

severely evaginated, typical of exencephaly, with closure confined to a

small region at the nidbrain-hindbrain junction. Apart fron this site of

closure, many embryos had neural tubes which were open from rostral- to

caudal- extremes (Figs. 9.1b, c).

As well as structural abnornalities salicylic acid al-so had an effect on

the surface features of the craniun as shown in Figs. 9.2crf. At a

site on the forebrain of an in vitro 11.5 day embryo (Fie. 9.2b) the

surface ectoderm was covered in microvilli. In contrast, at 100 uglnl

saticylic acid (Fig. 9.2c), the forebrain consisted of relatively snooth

cells rimmed with microvÍ11i with clumps of blebs within the perimeters.

As the concenbration of salicylic acid increased t,o 200 uglmI, the

ectodernal surface in the forebrain region was covered in very large,

irregular blebs (fig. 9.2f), which were also observed on the ventnicular

surface of the fonebrain (Fig. 9.2Ð. Whereas several of these micrographs

demonstrate the general features of surface cells of the cranium in day

1 1 .5 ernbryos, there w ere how ever some variations between embryos.

9.3.3 Cellular observations

Light nicroscopy of typical embryos cultured in sal-icylic acid (Fig.

9.3) revealed thal the gross structural abnormalities denonstrated in

FÍg. 9.2 were often acconpanied by extensÍve cellulan changes in some

enbryos but not in others. Horizontal- sections through the cranj.un of an

embnyo cultured in 200 uglnI (Figs. 9.3 arb,c) revealed t,hat, the neural

folds had not fused throughout the cranlum. T?¡e anferior neunal tube was

dlstorted leading to conpression of the optic pfts. DespÍte the dysnorphic

appearance of the craniun, the neuroepit,heliun appeared relatlvely

9 -26

Fig. g.2. a. scanning electron micrograph of an 11.5 day i¡ vitro enbryo

cullured Ín 100 uclnI salicylic acid for 48 hours. overall developnent of

the anj-maL was unaffected except for incomplete developnent of the

forebrain vesicles (arrows), and bhe neural tube appeared to have

coltapsed sIight,ly (arrowheads) at the site of fusion of the neuralfolds in the hinàbrain region. white the enbryo had nearly completed

rotation, the posterior neuropore (P) reruained opert The asterisk narks

the approximate site seen at' higher magnification in c'

b. Surface ectoder¡n from the forebrain region of an 11'5 day control j¡vitro enbryo covered in nicrovilli (arrows) and small btebs (asterisks) '

c. A similar site fron the forebrain region of the affected embryo in a'

shows that exposure to 100 uglnI salicylic acid leads Lo relatively snooth

cells with large clunps of blebs (asterisks) at some sites.

d. Day 11.5 unrotated exencephalic embryo cultured Ín 200 ug/nl salicyllcacid. The cranial neural folds whieh had not fused (arrows) had collapsedonbo the cranial surface in the forebrain region (arrowhead) exposinS the

ventnicular surface (asterisk) to the a¡rniotic fluid' The neural tube ofthis embryo had also not fused in the caudal regions, holever only the

open posterior neuropore (P) is visible at this orientation. Note thep""""no" of the first I fl and second (2) branchial arches' The black and

white dots mark the approxi.mate sites seen at higher nagnification in f'and g. respectively. H' heart.

e. A dorsal view of the ventricular surface seen in d.

developed, but exencephalic cranium. NC, neural crest; ot

Fb, forebraini Mb, midbrain; Hb, hindbraín; H' heant'

show ing a w eI 1optic vesicles;

f, g. Higher magnification of the surface ectoderm and the ventricular

"u"i""" respecfively of the forebrain of the 11.5 day salicylic acid-treated embryo aL sites marked by black and while dots in d' BoÙh

surfaces appear similar with Iarge Írregular clumps of blebs (arrows)'

Compare the surface ectoderm with that from a sinilar site Ín the controlembryo in b.

Bars=100urn ar d, er 2un b, er fr 8.

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9 -27unaffected except for some irregularifies in the midbrain region whichcottl-d probabry be attributed to the dÍstortion and partial colLapse of the

neuroe pith erium in exence phal ic embryos. Large, abnornally devel0 pingblood vessels occupied most of the nesenchyme, confining the nesenchymal

cerls to areas adjacent to the surface ectoderm. Higher nagnification ofsites around bhe optic pits (Fi8. g.3b) and the hindbraÍn (Fig. g.3c)

reveal-ed that the colunnar neuroepithelial ceLls were tightry packed,

smooth and reratÍvery unaffected, with l-inited osmiophilic granules, which

were probably rysed ceI1 rennants interspersed amongst them.

FiS. 9.3d revealed that following exposure to 300 ugln1 salicylic acidt'he anterior neural tube of this 11.5 day enbryo was compretely open

and distorLed particularly in the regÍon of the forebraÍn The neurar tube

was smaller than in controls (c.f. FÍg.6.3a) and there was extensivecelLuLan pathologr. The neuroepitherium appeared t,hin, with narry

extracellular spaces and osmÍophilic granules, while the nesenchyne was

less dense with ceLls confined to the regions near surface ectodern,and developing blood vessels whÍch were abnornally large, occupfed much

of the space in lhis neural tube tayer.

Higher magnification of the forebrain area (Fig. 9.3e) at the level ofthe optic ptt Ín thls enbryo exposed to 3OO ug,/ml salicylic acid reveaLed

only mild blebblng of the ventricular surface. The neuroepitheriar wallwas severery shrunken and thin, with extensive extracellular spaces and

dead cells tocated both intra- and extracellularly. The osniophilicgranules observed at 10w magnification appeared to be dead ceLlrennants' The nesenchyme was also affected in a sinilar nanner, but to a

lesser degree,wit,h f el¡er cerrs and fewer firopodia and sone lntra- andexfnacellular osnÍophilic granules. The ect,odernal cells also

9 -28

Fig.9.3. Horizon|al- sections through the cranium of 11.5 day in vitroemUryos cultured in 200 ug/mI (arb,c) and 100 uglmI (d,e,f) of salicylicacid during neurulation

a. The anterior neural tube is distorted causÍng compression of bhe opticpits, and the neural folds have not fused throughout the craniun. The

neuroepitheliun appears to be unaffected except for sone bhickening in themidbrain regíon (arror¿s). Large, abnormally developing blood vesselsoccupy nost of the nesenchyne causing aggregation of the nesenchymal ceIIstov¡aràs the surface ectodern. The arrowheads and asterisks nark the sÍtesseen at higher nagnification in b. and c. respectively.

b. Higher nagnification of a region around the optic pit narked by

arrc[^¡heads in a, shows thaü the neuroepitheliun is snooth and relativelyunaffected, with very Iíttle cell necrosis (arronrs) and mitotic fÍguresare visible. The nesenchyroe shows limited evidence of cell patholoryin this area (arrowheads).

c. Hígher nagnÍfication of a section fron the hindbrain marked by

asterisks Ín a. The cell s of the neuroepithelir.rm are tightly packed

except at the apical end which abubs the ventri-cular ltmenr wherecondensed cell fragnents can be observed (anrolus). Some omiophilicgrangles (arrowheads), probably lysed cell- remnants, were observed withinthe neuroepitheliu¡n and nesenchyne.

d. The cranial neural tube is open throughout as the neural folds havefailed bo fuse. The forebnain vesicles (long arrows) appear reduced and

the optic pits ane conpressed. The neuroepithelir.¡n is severely shrunkenand thin, and convoluted at the basal end (arrowheads) which abuts themesenchyme. The nesenchymal ceLls were confined to regions near thesurface ectoderm as most of the space lJas occupied by large blood vessels.The asterisks and short arrows mark Lhe sltes seen at higher magnÍficationÍn e. and f. respectivelY.

e. Higher magnÍfication of a site in the forebrain in dr marked by

asterisks, sho+ing extensive celI shrinkage and extracellulan spaces asweLL as dense, omiophil ic ceLl debris (arrowheads) nairùy in theneuroepithelÍum, but with some ceLl remnanbs in the nesenchyme aswell. The surface ectodermal cells also appeared to be disrupfedwith large extracellular spaces (arrol¡s).

f. Higher magnification of a site in the hindbrain in d' narked by shortarrotrs, showing increased extracellular spaces (asterisks) and condensedceII debris (arrowheads) within a thin neuroepitheliun. The site abubtingthe ventricular lumen appears to be particularly affected (arrows),however mÍtotic fÍgures are abundant.

Fb, forebrain; Hb, hindbrain; N, neuroepltheliun; M' nesenchyne;NF, neural folds; o, optic vesicle; bv, blood vessel; Mfr miboticf tgure; E, surface ectodern; L' ventricular lumer¡-

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9 -29

demonstrated some disruption at this concentration.

At the hindbrain end of the neural tube (Fig. 9.3f), cells exposed

to 300 uglnI salicylic acid demonstrated simiLar disruption to

elsewhere in the rostraf neural tube, but in addition' the blebs

Iining the ventricular surface which apppeared healthy in the forebrain

region, formed a dense dark labtice-like lining typical of shrunken,

dead tissue. These cells abutting Lhe lumen also appeared to contain many

vacuoles, and the basaf surface bounding the nesenchyne was highly

convoluted. I^Ihile present at concentrations of 200 and 300 ug/nl

sal icyl-ic acid, the severity of these effects al so increased w ith

concentraLion Despite the severity of the ceLlular abnormalities induced

by sal icylic acid, mitotic celIs w ere very apparent in the

neuroepithelium bordering the ventricular lumen throughout the anterior

neural tube and did not appear to be affected.

9.3.4 Ultrastructural observatÍons.

The cetlular abnormalities revealed by llght' nicroscopy lrere further

investigated by transmissÍon electron nicroscopy in order to establish the

pafhology underlying these changes. The appearance of the

ultrastructunal patholory was sinilar at all 3 levels of salicylic acÍd

Ínvestigated, however there was a dose-dependent effect rel-ated to

the severity of the pathology. Ultrastructural- exaninalion of

representative anterlor neural tubes from a number of embryos confirmed

observations nade at the light nicroscope 1evel, t'hat salicylic acid

exerts its teratogenic effect nainly on the neuroepitheliun, with a lesser

effect on the underlyÍng nesenctyme. tlhile there was a small a¡nount of

cellular disruption at 100 uglrnl, the neural pathology was consÍderably

enhanced at concentrafions of 200 and 300 uglnI salicyllc acfd,

9-30

although as seen at the light microscope level (Fie.9.3), not aI1

dysmorphic enbryos demonstrated neural necrosis. Affeeted neuroepitheliun

showed a variety of abnormalities including dead and dying ceIls,

intraceLlular and extracellular materÍa1, as w eI1 as w ashed out

neuroepithelial cells fro¡o which the cytopJ.asmic contents appeared to have

1 each ed.

Some of these features which are illustrated in Fig.9.4, have been

extracted from a number of different embryos similan to, and including

the enbryo seen at the l ight microscope level in Figs. 9.3 d-f. Fig. 9.4a

shows a section of the neuroepithelÍum in the forebrain of an enbryo

exposed t,o 300 uglmI salicylic acid. The neuroepithelial wal1 is

thÍn, with extracel-lular spaces and a variety of dead cell debris

Iocated within other cells which had apparently phagocytosed these

remnant s.

At higher magnification (Fie. 9.4b) , the dead celI intra- and

extraceLlular material was seen to consist of nuclear and cytoplasnic

rennants, including rough endoplasic reticulrm, nÍtochondria which v¡ere

often condensed, and free rÍbosones. The very electron dense structure in

the nicrograph nay be a prinary lysosome. The lumenal end of the

neuroepithellun appeared also to be affected by salicylic acid exposìlre.

The micrograph in Fig. 9.4c sho,ws the surface to be extrmely blebbed and

condensed, wÍth large extracellular spaces, suggesting t,hat cell

shrinkage and loss of some cells has occurred. The severely fragnented

appearance of the ceLls in this region as well as the large

extracellular spaces suggest,s thaf the blebs are all that renains of these

cell s.

The large vacuolar structures observed under the light micnoscope often

9-31

Fig. 9.4 denonstrates some of the ultrastruclural features associated

wlth salicylic acid-induced neural necrosis in 11'5 day j¡ vitro enbryos

exposed to 300 uglmI during neurufation'

a. section of the forebrain neuroepitheliun adjacent to the ventricularIu.nen showing extracel-Iular spaces containing ceII debris (arrows) ' and

phagocytosed, Ìysed cel-I rennants (arrowheads). Note the large vacuole (v)

associated with the extracellular naterial'

b. Higher magnification of the vacuole site (v) in a, showing a range of

danaged organelles including cytoplasmic riboso¡nes (cr)' mit'ochondria(m) which were either condensed, or swollen with broken cristae, and

vesiculated rough endoplasrnic reticulum (vr). The adjacent neuroepithelialceII appears tã Ue surrounding the celI debris (arrows) which may be

eventually absorbed into the vacuole and lysed by the cell.

c. section of ühe hindbrain neuroepithelir'm showing large, irregular,condensed(probablydying)cytoplasmicprojections(orblebs)(aslerisks)protuding inbo the ventricufar Iunen, large extracellula¡ spaces' and

nu¡nerous heterolysosomes (arrowheads). Note the abnornal shape of one of

the nuclsl (nu) in the micrograPh'

d. The basal end of the neuroepithelir-m showing abnormal whole cellswhich appear to be severely depleted of cytoplasm (arrows) ' and

å*t"ã""riri""' debris of similar appearance (large arrowheads). Note the

distorted basat lamina in this area (s¡nall arrowheads). The asterisk rnarks

t,he site seen at higher rnagnif icatlon in e'

e. Higher magnification of the site marked by the asterisk in d' shot¿s

that, the celL membranes of affected cells had disintegrated (arror¿s),

Ieading to the dispersal and loss of cytoplasnic ribosomes, and

o"g"n"Il"". Note the condensed appearance of mitochondria (m) in adiacent

cell s.

f. NeuroepithelÍaI cell debris is extruded fron cells with dispersed

cytoptasmic ribosones and condensed nitochondria ( m) into the

extracellular matrÍx of the rnesenchyne at sites where the basal Ianina has

been disrupted (arro^rs). Arrowheads indicate regions where the cellnembranes have broken.

N, neuroepitheliun; ECM, extracellular natrix; L, Venticular Iunen; BL,

basal Ianinai bv, blood vessels.

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9 -32lying close to the nesenel¡rne hrere seen to be neuroepÍtheliat ceLls whÍchr,v ere sev erely de pl eLed of cy to pl asm and orga neL l es (Fig. 9.4 d). High ernagnification (Fi8'9.4e) revealed that cerr me¡nbranes were broken,hence allowing the cytoprasm and poryribosomes to rreachn out fromthese cells leaving large extracellur.ar spaces. rn sone cases (Fig. g.4f)neuroepithelial cel-1 debrÍs was extruded into extraceLLurar matrÍx of themesenchyme through breaks in the basar. ranina, leaving nainly organelles,particularry rough endoprasmic reticulun, and condensed mitochondnia. Thenesenchyne also contained sone dead ceLl rennants, but were considerablyless affected by salicylic acid than was the neuroepitheliun.

9-33

9.4 DISCUSSTON

This study which consists of materÍal recently subnitted for publication

(Joschko et aI 1991), demonstrates for the first time the ultrastructuraL

patholory induced by salicylic acid, a metabolite of aspirin in the tissue

of the developing rab neural tube, and confirms and extends salicylic

acid-induced growth and norphological anomalies reported previously. The

malfornations observed in embryos exposed to salicylic acid in vitro in

this study demonstrated a dose-related dysmorphofogy, and were similar to

anonalies reported in rats j¿ vivo (l,larkany & Takacs 1959; Kimmel et aI.,

19711, McGarrity et al., 1981) folLowing aspÍrin administration. This

suggests that sal.icylic acid exerts a teratogenÍc effect directly on the

developing enbryo, an effect which is probabJ.y masked j¡ vivo by maternal

¡nedia tion.

These observations also support previous suggestions (Kimne1 et al., 1971;

Koshakji and Schulert, 1972) bhab the metabolite is a causative agent Ín

aspirin teratogenesis, which is further strerrgthened by the observat,ions

reported by Yokoyama et aI (1984) of differential effects of aspirin and

salicylic acid. These workers showed that a short exposure to aspirin led

fo edenatous anomalies of the face and t,ail which they suggested was

related to a decline of systemic physiological functions since a

functionaL reduction of yolk sac denonstrated by degenerative changes in

thal t,issue, as well as decreased growth parameters were observed. fn

contrast shont exposure to the netabolite, salicylÍc acid 1ed to

localised nalformations such as cleft lip and curly or short tail, in the

absence of these nore generalfsed effects. l'lhile growth and yolk sac

developnent were observed to be lnpaired at levels of 200 ug/ml inthe

present study it is likely that salicylic acÍd itself induces these

9-34

effects when the level is sufficientì.y high and the perÍod of exposure

sufficiently lengthy.

Fron the present study it would appear that rat embryos are more

sensitive to salicylic acid, the netabolite of aspirin, rather than to it,s

sodiun salts, as the Ievel-s required to induce a teratogenic effect

following in vitro exposure to salicylÍc acid was in the range of 100 to

300 uglnI, whereas Na salicylate-induced teratogenicity occurred in the

range of 200 ug/ml to 800 ug/mÌ (lleGarrily et aI 1981, Greenaway et aI

1982), and showed comparable dose-dependent neural tube anomalies.

Ï.lhile initial doses used in rats in the present study v¡ere approximately

five times higher than human therapeutic leveLs of aspirin, Levy et aI

(1969) reported that these doses produced plasma salicylic acid levels of

39-42 ngft in humans which are similar to the free serun salicylate levels

observed in rats. Hence the leveIs of salicylic acid used in the present

study are simiÌar to the human serum range, hence extrapolation of rat

data to the hunan conditÍon would seem appropriate and especially relevant

since it is known that fhe netabolic pathways of aspirin are the sane in

both species (Levy & Leonards 1 966).

The present observations also confirm previous reports both i¡ vivo

(Ïlarkany & Takacs, 1959; Kimrnel et aI., 1971; DePass & ÏIeaver, 1982) and

Àn vÍtro (Mcgarrity et aI 1981; Greenaway et aI 1982; Yokoyana et al 1984)

that aspirin or its derivatives led to growth retardation in a dose-

dependent nanner. llhile nost studÍe.s have denonstrateci this effect in rat

fetuses at ferm, j¡ vitro studies perforned during the critical period of

organogenesis have the advantage of demonstrating the tenporal

relationshÍp between aspirin administ,ration and growth reductÍon. These

studles in rats nay be of parfÍcular rel-evance and inportance as some

9 -35

human studies have implicated aspÍrin as a causative agent for reduced

birthweight (Turner & Collins, 197Ð,

Vühile these workers (Collins & Turner 1975, Turner & Coltins (197Ð also

found significantly increased fetal wastage which could also be relaLed t,o

chronic ingestion of salicylates throughout pregnancy, not aIl human

studÍes have denonstrated this det,rimental- association including Shapiro

et al (1976) who found no evidence that prenatal aspirin ingestion Ì¡as a

cause of stiIlbÍrt,hs, neonatal deaths or reduced birthweight. Since the

embryos were removed on day 11.5 of gestation in the present study, it was

not' possible to deternine whether the embryos t,hat demonstrated severe

neural tube dysmorphology wouLd have survived to term, however it islikely that a good number would not in view of the severiLy of the

defects reported here, and observations from prevÍous studies. Brown and

llest ( 1964) reported f etal- death at tern when up to 500 ni/ke w ere

administered, doses which have been reported previously (Kinme1 et aI

1971) to generate equivalent serun levels to those in which the embryos

were curtured in the present study. rn a prevÍous i¡ vitro study,

Greenaway et al (1982) also expressed the opinion that they did not

expect their study to be generaJ-1y compatible wilh viability at

tern. Ihese observations led the present author to suggest that severe

salicylic acid-induced dysmorphology such as neural tube defects nay also

be inconpatible to life in hunan fetuses and hence may be the underlyÍng

cause of sone of the stillbirths and fetal deaths reported in hunans.

WhlÌe aspÍrÍn-induced teratogenesis has been fÍrnly estabLished inaninals (Ìlarkany & Takacs 1959; Goldnan & Yakovac 1961; McGarrity et al

1981; Guy & Sucheston 1986)r the results fron hunan studles are not

consistent. The varj-ations in outcome in epidemiological reports nay occur

9-36

because of diffenences in methodology and anal-yses between studies, as

well as confounding personal and environmental factors Íncluding naternaL

exposure to alcohol and nicotine. Despite these inconsistencies, aspirin

should not be underestimated as a possibLe nervous systen teratogen Ín

humans. Even if teratogenesis Ís not evident when aspirin is administered

alone, its effects nay be exacerbat,ed if it is ingested with conpounds

which modif! its metabolism. Agents such as the comnonly occurring food

additive, benzoíc acid which increased the concentration and persistence

of salicylic acid in the plasma of maternal rats, also increased the level

of teratogenesis of the nervous systen and ot,her organs in tern fetuses

conpared with aspirin alone, when adninistered during the cnitical period

of neurogenesis (days 9-11) (Kinnel et al 1971). Since the netabolic

pafhway for salicylate metabolisn is the sane in both humans and rats

(Levy & Leonards 1966), berøoic acid nay exert a sinilar effect, in hunans.

Hence the knowledge that not only might aspirin and/or Íts derivalives

be teratogenic but that the effect may be exacerbated by other cornpounds

to which the mother may be exposed to during pregnancy, should

encourage healfh workers to alert their pregnant patients to the possible

dangers of these conpounds t,o their unborn children

Some of the salicylic acid-induced anomal-ies of the branchial arches

reported in this study -ln vit,ro on day 11.5 of gestation which involved

abnormalÍties or absence of the mandibular arch, (which differentiates

into the naxillary arch by day 12 of gestatÍon in rats), was

represented in previous Ín vivo studies at term as cleft 1ip and palate

(DePass & tleaver 1982) fotlowing acetylsalicylic acid exposure throughout,

pregnancy, as well as creft lip in fetuses j.¡ vitro on day 14.5 of

gestation (Yokoyana et aI 1984) following short periods of exposure to

sallcylic acid or aspirÍn. Ïrrhile the doses of salicytic acid were

9 -37

comparable with Ievels used in the present study, the duration of

exposure in the study by Yokoyarna et aI (1984) was only 4 hours' which

suggests that branchial arches may be more sensitive to the effects of

salicylic acid or aspirin laLer Ín organogenesis rather than earlier.

Depass and Weaver (1982) also reported cardiac abnormalities at term

which Ì,¡ere apparent as early as day 11.5 in the present study' with

nany embryos demonstrating only simple ntube-liker hearts, although

the pericardia did not appear to be affected.

Tkre most frequently observed neural tube anomalies were related to the

interruption of neural tube closure, which either involved the cranial

region alone which has been reported previously following culture of

embryos in sodiun salicylate (McGarrity et aI 1981; Greenaway et al 1982)

orwhich additionally involved the neural tube caudal to the cranium' a

precondition of craniorachischisis, observed in aspirin-treated animals at

tern (Ïfarkany and Takacs 1959; Kimnel et al 1971; McGarrity et al 1981).

It is likely that the ¡nore severe neuraL tube nalfornations observed in

the present study nay not be eompatible with vÍabllity at tern. The

absence of chorío-allantoic fusion in many of the embryos at higher

concentrations also makes survival unlikely as the embryo is dependent on

an active placenta for its nutritional requirenents later ín development

(New 1978).

The ceLlular necrosis described in the neural tube of these salicylic

acid-cultured enbryos did not appear to be associated with programned cell

death or apoptosis, which is a normal consequence of differentiatlon

(Schluter 1973, Geelen & Langman 1977), as necrotÍc cells induced by

salicylic acid were apparent throughout the entire neuroepithelium Ínstead

of being confined to the sites of closure along the neural tube. It, is

9-3I

likely that the severe cell shrinkage, and large condensed blebs adjacent

bo the ventricular lumen, together with the extensive cell loss

throughout the neuroepithelÍum, nûay interfere wilh fusion of the neural

folds. The severe neural tube dysmorphologr demonstrated in the present

study was also similar to abnormalities induced by zinc and vitamin E

deficiencies (Record et al 1985a; Harding et al 1988) and vitamin A

excesses (Joschko et aI 1989). Necrotic cells were also apparenL in the

cranial neural tubes of enbryos exposed to these teraüogens.

Histologically, the dead and dying cells in sal-icylic acid-induced embryos

showed a similar pattern of disruption, and could be related to the

histological abnormaLities observed by Greenaway et al (t982) acconpanying

Na salicylate-induced gross dysmorpholory which included a neuroepithelium

reduced Ín thickness, and necrotic cells sloughing into the lumen

Although cell death is not necessarily a prerequisite for a teratogenic

response, such events have been reported (ScoLL 1977) to occur as a nesult

of a wide range of treatnents. Aspirin-induced dysmorphology has been

linked previously to a cellular abnornalÍty in the adult gastric mucosa of

several species (HÍngson & Ito 1973i Pfeiffer & V{eibel 1973i Rainsford &

Brune 19?8), and also in embryonic hindlimb, as Klein et aL (1981)

reported an unique pattern of cytotoxicity in the hindlÍnb bud where cell

death was localised to the preaxial nesodern of this structure in ral

fetuses. These workers speculabed bhat, this necroLic episode v{as

responsible for the subsequent polydactly. Hence the severe oel-I death

observed Ín the open anterior neural tube in this study nay underlÍe the

Ínability of the neural Lube to close, however the absence of a

sÍgnificanbly severe ceLl necrosis acconpanying exencephaly in some

salicylic acid-treated enbryos in the present study, together with the

observabÍon in nicotÍne-treaLed enbryos (see chapter 7) that cell death

9-39

occurs even when the neural folds have fused, strongly suggests that

necrosis may be an additional teratogenic effect unrelated to exencephaly

bub which may underl-ie more functional disabilities, possibly with long

term consequences.

Long term effects of prenatal aspirin administration have been reported

in rat offspring and included impaired learning abÍlity (BuLcher et aI

1g72) as well as postnatal developmental delays (Vorhees et al 1982).

Furthermore, a recent epidemiological report (Streissguth et al 1987) also

showed that maternal aspirin ingestion during the first half of pregnancy

was significantly related to IQ scores and attention deficibs in four year

o1d children These observations led the author to propose that while the

presence of severe celluLar disruption and blebbing along the dorsal

margins of the neural tube at the site of neural tube fusion may be

involved in faÍ1ed neurulation, the often severe necrosis and ceIl Ioss

throughout the renaining neuroepithelium may be Iinked with previously

observed intellectual and behavioural abnonnalities.

Several possibilities exÍst to account, for the nechanisns whÍch underlie

neural tube dysnorphotogy. As the yolk sac and vascular development were

Ínpaired in many embryos exposed to salicylic acid in culture, in a

sÍmilar nanner to that observed previously in nicotine-cultured enbryos

(Joschko et al 1991, chapter 7 of this thesis), and since lheyolk sac is

crÍbical for adequate nutrition and oxygen uptake at this tine in

enbryonic developnent (New, 1978), sone of the neural tube pathology nay

derive from an inability of the yolk sac to neet the denands for essential

nutrients. However, while this nay underlie the poor growth and overall

developnental retardatÍon associated with salicylate teratogenesis, 1f is

unlikely that this is the prÍmary mode of salicylic acid-induced

9-40

teratogenesis i.n view of the different patterns of dysmorphology and

neural tube patholoSl between nicotine and salÍcylíc acid-induced embryos'

Al-ternatively it is likely that, salicylic acid exerls a direct cytotoxic

effect on celI membranes, which were observed to be seriously dísturbed at

sone sites in the neuroepithelium, particularly at sites adjacent to the

nesenchyrne, where the basal Iamina was also frequently seen to be

disrupted. It is at these sites that necroti-c neuroepithelial debrÍs'

which is not digested by heterolysosomes loeated within apparently healthy

ceIls, can be removed from bhe neuroepithelium and extruded into the

mesenchyne. This nethod of renoval of neuroepithelial debris has been

previously reported in nicotine-cultured enbryos (Joschko et aI 1991a)'

and in other chapters of this bhesis'

Prostaglandins have also been implicated in adequate fetal developmenf

since they are known to have a large nurnber of desirable effects some of

which occur at the celLular 1evel (HorrobÍn 1980)' Aspirin, l-ike zinc

deficiency leads to an inhibition of prostaglandin synthesis (Horrobin &

cunnane 1980; MeLz 1981), and since both of these experimentally induced

conditions produces neural tube dysmorpholory such as exencephaly at Lern

(Metz, 1981; llarkany & Takacs, 1g5Ð, and open neural tubes accompanied by

severe cell death in the developing nervous system (Record et al 1985;

Joschko et a] 1989), these observations could suggest a role for

prostaglandins in the adequate development and closure of the neural tube'

The present study has shown for the first time the salicylic acid-induced

ultrastrucbural- defects which underl-ie ühe gross and ce1lular disruption

observed both i! vÍvo and in vitro in lhe anterior neural tube of the

developÍng rat embryo. The principat disruption consisted of conplete and

severe necrosis of neuroepithelial celIs at the lumen in the region

9-41

where neural tube closure normally occurs, as well as broken celL

menbranes and basal laminae adjacent to the nesenchyme. The author

suggests that the disrupted necrotic lumenal surface may underlie the

failure of the neural folds to fuse and that necrosis outside of these

sites may be related to more functional dysmorphology including

behavioural and learning deficits. The nechanisms fon sal-icylic acid-

induced teratology are unresolved but nay involve an action of the

teratogen on cells involved in the closure of the anterior neuropore

possibly through one or several nechanisms including a direct cytotoxic

effect, yolk sac-mediabed nutritional deficiencies, and interference with

prostaglandin synthesis. The findings in the present study demonstrate

that the developíng rat embryo is at risk fron salicylic acid exposure

and that when related to hurnans, severe disruption to the developing

neural tube may lead to exencephaly and inpaired development and I.Q.

postnatally.

In the following chapter the author will exanine the potential

teratogenic Ínteraction which night arise when salicylÍc acid is conbined

with alcohol in culture. In additÍon to the experimental evidence ouLlined

in this chapter, as well as in chapter 6, there are also several inportant

clinical justifications for j.nvestigating such a combination which will be

discussed in chapter 10.

9-42

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9-44

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9-45

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Saxen, L. (19?5) Associations between oral clefts and drugs Laken duringpregnancy. Int. J. Epidemiol-. 4' 37-44.SchIuter, G. (1973) Ultrastructural observations on cell necrosis during

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acid - New uses for an old drug. Barnett, HJM., HÍrsh' J. & Mustard' JF.(eds) Raven hess, New York. pp 49-62.Tras1er, DG. (1965) Aspirirrinduced cleft lip and other malformations in

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distribution and enbryotoxicity of acetylsalicylic acid in pregnant ratsand rhesus nonkeys. Toxicot. Appl. Pharmacol. 41, 67-68.Wilson, A, Karitzky, D., Gasteiger, U. & Stehr, K. (1978) Investigations

on saLicylate prolein binding in newborns and infants. Eur. J. Pediatr.127 ¡ 163-172.Woodburyr DM. (197 5) Analgesics-antipyretics, Anli-inflannatory agents,

and drugs enployed in the therapy of gout. In: The PharnacoLogical Basisof Therapeutics. Goodnan, LS. and Gilnan, A. (eds). MacMillan, New York.pp 325-358.lÍy1J-ie, AH. (1981) CeI] death: a neÌ¡ classification separating apoptosis

from necrosis. In: CeIl death in bfology and pathology. Bowen, ID. &

Lockshin, RA. (eds). Chapman & HaIl' London. pp 9-34.Yokoyana,4., Takakubo, F., Veno, K.r Igarishl, T. et al., (1984)

Teratogenicity of aspirÍn and Íts ¡netabolite, sallcyllc acid, in cultured

9-47

rab embryos. Res. Comnun Chem. Pathol. Pharmacol'46' 77-91'

10- 1

CHAPTER 1O

THE TERATOGENIC EFFECTS ON CULT{JRED RAT EMBRYOS OF CONCURRENT EXPOSURE TO

ALCOHOL AND SALICYLTC ACID.

1 0.1 INTRODUCTION

lfhereas the teratogenic properties of alcohol in both animals and humans

have been well established (Jones et aI 1973, Sulik et aL 1981, Webster et

aI 1980, 1983), and the potential for salicylates to induce teratogenesis

during developnent is weLl known (Ïlarkany & Takacs 1959, McGarrity et aI

1981), very few studÍes have examined the norphological effect that these

two agents exert when adninistered in combination during embryogenesis

(Randall & Anton 1984; Guy & Sucheston 1986).

To the best of the authors knowledge however, there are no studies which

have examined the effect that concurrent ad¡ninistrafÍon of these agents

exerts on the ceÌIuIar and ultrastructural profiles of the developÍng

neural- tube. The fact that Australians rank fourth in the world in total

per capita eonsunption of alcohol and first in the consunption of beer

(Senate Standing Co¡nmittee 1977) and that aspirin is readily available as

an over-the-counter medicatÍon, means that there is a significant,

potential for an interaction between alcohol and aspirÍn

Aspirin, which is a popular analgesic is frequently self-administered and

hence is a likely drug to be ingested by pregnant women evenwith aIcohol,

as it is reputed to be an innocuous agent when taken Ín modenation. Its

effect in excessive doses combined with heavy drinking on enbryonÍc

developnent is largely unknown. However, since ethanoL interacts with

drugs at a nunber of sites lncluding the gastroÍntestinal tract, liver and

braÍn (Desnond & Crotty 1987) and since both alcohol and salÍcylic acid

freely cross the placenta (BouIos et aI 1972), and are present at higher

concentrations in ühe developing individual early in uterine Iife when

1 0-2

the enbryo lacks the enzyme systens required to metabolise these agents,

investigation of this issue j.s worthy of considerable at,tention

The Iimited data available to date is however conflÍcting. Randall and

Anton (1984) demonstrated that subteratogenic doses (150 ne/kÐ of aspirin

on gestational day 10 in nice actually sÍgnificantly redueed the number of

malformed pups caused by maternal ethanol administration by 50Í, and

reduced perinabal nortal-ity to control- leveIs. These observations led

the authors to conclude that aspirin pretreatment was able to ant,agonise

the del-elerious effects of acute alcohol administration on perinatal

mortality and norphoJ-ogical development, ie. the effect was amel-iorative.

In contrast, Guy and Sucheston (1986) reported that offspring from

et'hanol- mice pretreated with aspirin exhibited more external and visceral-

nalfonmatÍons and reduced fet,al weight without affecting resorptions and

perÍnatal. mortality. These authors concluded that the results indicated an

additive effect of aspirin and ethanol on the developing mouse fetus. fn

an even nore recent study, Bonthius and West (1989) denonstrated that

aspirin augnents alcohol-induced microencephaly and concluded that the

vJorsening of effecls was due to an interaction between aspirÍn and

alcohol. Despite dÍfferences between the two nore recent studies, both Guy

and Sucheston (1986) and Bonthius and Vlest (1989) suggest that aspinin

nay worsen some of the teralogenic effects of al-cohoL. Since nany of the

pharmacological actions of aspirin are nedÍated through prostaglandin

synthesis inhibition (Vane 1978), Randalt and Anton (1984) suggested that,

prost'aglandins nay play a role in the etiologr of aspirin-related bÍrth

defe cl s.

This study was perforned in order to examÍne the effect t,hat concurrent

adninistration of ethanol and salicylic acid, a netabolite of aspirin has

10-3

on morphological and ultrastructural development in rats inmediately

following the period of neurogenesÍs. In additÍon to the convincing

teratogenic effects attributed to both alcohol and salicylic acfd Ín

animals which have been reported in chapters 6 and 9 of this thesis, the

knowledge thaL 50í of pregnant women ingest aspÍrin at sonetime during

their pregnancy (HiIl e! aI 1977, Streissguth et at 1987), and Lhat

alcohol which is consumed by up to 75í of aII pregnant Ì¡omen (Rosetl et aI

1976; streissguth et al 1987) is berieved to be Lhe mosb com¡non

ernrironnentaL cause of mental retardation in !lestern society, provides

clinical justification for investigating a potential interaction between

aleohol and aspirÍn Ín inducing nervous system anonalies. Fwthermore, the

ingestion of aspirin is an event whÍch is direct,ly Iinked wÍth alcohol

consunption, as the forner is a commonly used method to prevent and treat

al cohol- induced hangovers.

The in vitro model was considered suitable for this study as Ít al-Iows

any direct cytotoxic effects of al-cohol and salicylic acid to be confÍrned

and also ensures thab the comparison of embryos for morphologÍcal

assessment were all at the same devel-opmental stage when exposed to the

teratogens. Furthermore, whÍre the j¡ vivo rat model_ of arcohol

teratogenesis is not as effectÍve as the mouse model, it seens thab

ethanol-cuL lured rat embryos show simirar morphologicar and

ultrastructural abnornalÍties to those observed in the nouse nodel of FAS.

10.2 MATERIALS AND METHODS.

Details of enbnyo culture, teraüological screening and tissue processlng

and exa¡ninaüion have previously been outllned in sectÍo n 9.2 of t,hls

thesfsr and described fully in Chapter 2. Investigation of teratogenfc

effects over a range of concentratlons of sallcylic acid in chapüen !, and

10-4

alcohol in chapter 6 led the author to seÌect a concentration of 150 ug/nl

and 400 ngfr to be bhe most suitable levels of salicylic acid and alcohol

respectively at which to investigate neunar tube dysnorphology and

necrosis. Hence 9.5 day embryos srere eÍt,her cuLtured for 48 hours innornal serum' or serun containing either 150 ug/n1 salicylic acid or 400

ngfr alcohol, or both.

Once again alcohol was added to the contents of the culture bottles just

prior to them being placed in the incubator in order to avoid reducing the

concentratÍon of this agent through evaporatÍon Statistical analysis was

perforned as outlined in section 8.2 and described fully in chapter 2 of

t,his thesis.

10.3 RESULTS.

10.3 . t . Gror.¡th and norphological observations.

Treatroent effects ona number of growth parameters are shown in Table

10.1. Analysis of varÍance Índicated that, there was a sÍgnifÍcant effect

of alcohol and salicylic acid on cror.rn-rump length (î3r77 = ZO.3Z,

p(0.001), somite number (t3,2, = 22.35t p(0.001) and yolk sac diameter

(F3,77 = 10,23, p(0.001). Iukey test s ldentif ied that onty the control v s

sallcyIic acid gnoups and the alcohol vs alcohol-sallcy1ic acid groups

were nob significantly dlfferent fron each other for crown-rump Iength and

sonlte number. This indicates that only alcohol had an effect on these

growth paraneters. rn the case of yolk-sac dianeters, there was no

difference between the neans of any groups except between controls and the

alcohol-salicylic acid group. The results indicate that alcohol and

salicylic acid do not have an effect on yolk sac dÍaneter indivídualty but

when conbined they exert an j.nteractive effect.

10-5

alcohol and salicylÍc acid on embryonic growth inTable 10.1 Effect ofrats.1'2

Control s +A]-3-c. + SaI +AI c. /+SaI .

No. of ernbryos 1 1

Crow n- rumplengbh 1.06+0.064

No. of somites 23.27!0.334YoI k- sa cdianeter 1.82+0.084

20

2.1210.08b

16 .4710 . 84b

3 .3310 .07 a

21

2.6910.104

20.05+0.694

3.4910.124

29

2.02+0 .0 9b

14 .6?fl .? 2b

3.06+0.08a'b

1 V"l,r"" are neans + SEM.2 M""n" are signifîcantly different fron all others not sharingthe sane superscripts.(Tukey tests for nuLtiple cornparisons p(0.05).3 E".u-Jo.s *..c- 3iqwrè qs l\ese- ; T*bl'.- 9'l

The results in Table 10.2 indicate the incidence of abnormalities in

control enbryos as well as in embryos exposed to 150ug/mI salicylic acid

and 4OO ngft atcohol alone, and in conbination. Levels of alcohol and

salicylic acid were selected from sLudies described in chapters 6 and 9

respectively which were appropriate in bhe first instance to assess the

teratogenic effect of these agents on the developing nervous system. For

nost of the strucbures exanined, the incidence of abnormalities observed

for the individual agents increased when the two teratogens were co¡nbÍned.

Most enbryos in the atcohoL and alcohol-saJ-icyJ-ic acÍd groups demonstrated

sone kind of abnor¡aality (95íl and 931 respectively) while 76l of embryos

exposed to 150 uglnI salicylic aeid were affected, conpared with controls

where 2 enbryos had not fully rotat,ed while another showed nelarded

forelimb bud development.

In relation to neural tube defects which occurred ín 65l and 52f of

enbryos |n the alcohol and salicylic acid groups respectively' concurrent

treatments Ied to 791 of embryos affected. 0f Lhese, open neural tubes

þJere observed approxinately equally in the Sroups exposed to the

indÍvidual agents, (45í for alcohol and 43f for salicylic acid), and when

10-6

the treatments were combined lhe incidence of open neural tubes was

reduced to 38Í (Table 1 0.2).

Some of the embryos in the sal-icylic acid group (Fig. 10'1a) and the

alcohol group (Fig. 10.1b) exhibited open neural tubes which were either

confined to the cranial region, characteristic of exencephaly, or to

sites caudal to the nidbrain, typical of craniorachischisis ' and

occasionally were open for the entire length of the enbryo. tlhen the

treatnents were conbined fewer embryos exhibited open neural tubes, while

nore v{ere nicrocephal-ic with severely reduced forebrains (Fig. 10.1c).

FÍg. 10.1c also shows that branchial arches were abnormal, especialLy the

Table 10.2 Effect of alcohol and sadeveloPment in rat embrYos. {icVIic acid on morPhological

Control s +ALC +SAL +ALC/+SAL

No. of enbryosNo. of affectedembryos

No. with NTD2No. with openneural tubes

Ele defects

Ear defectsBranchialarch defects

Heart defectsAbsent/ reducedforelinb budsIncornpleteflexionAbsence of chorio-allantoic fusÍonPoor yolksaccircul ation

11

3(zl .2)

0( 0)

0( 0)

0( 0)

0(0)

0(0)

0( 0)

1(9.1)

2(18.2)

0( 0)

0( 0)

20

19(95.0)

13(65.0)

9(45.0)

6(30.0)

4(20.0)

S(es.o)

1(5.0)

19(95.0)

18(90.0)

8(40.0)

16(80.0)

21

16(76.2)

11$2.4)

9(42.9)

4(19.1)

4(19.1)

3( 14.3)

2(9.5)

12(57 ,1)

14( 66 .? )

8(38.1)

12(57 .1)

29

27 (93.1)

23(]9.3)

11(37.9)

12(41.4)

8(27 .6)

14(48.3)

6e0.7)

26(89.7)

25(86.2)

12( 41 .4)

26(80.7)

1 Nr-b""" in parentheses indicate pencentages.2 Wt¡ incLudås open neural tubes, hypoplastic forebrains, abnormally

fused neural tubes and nicrocephaly.

1 0-7

Fig. 10.1. a. Variations between embryos cultured in 150 ug/ml ofsatlcylic acid duning neurulabion include unfused neural folds(arrowireads) either in the cranial region or caudal to this site, rotatedor unrotated enbryos, and tack of placental formation indicated by thepresence of an unfused allantois (A).

b. Embryos cultured in 400 ngí alcohol during neurulation denonstratedhypopla"iio forebrain vesicles (arrows), unfused neural folds (arrowheads)

and had rarely rotaLed into the dorsi-convex position All three embryos

ane from the same culture bottte. Note that the aninal in the cenfreappears unaffected.

c,d. Ventral and dorsal views respectively of two 11.5 day embryoscultured in 150 ug/mI salicylic acÍd and 400 mgf, alcohol concunrentlydurÍng neuruLation. The enbryos showed overall growth and developnentalretarãation, and wene microcephalic with hypoplastic forebrain vesicfes(asterisks), and the rnandibular arches were often fused to the pericardium(large arrowheads). The neural folds are dístorted in lhe cranial regioni""io"heads), (c.f. Fig. 6.Zc), and bhe optic placodes (0P) arecompressed. In the enbryo on the right the neural folds have notoorfl"t"Iy fused throughout, the Iength of the aninal whÍch frequently had

not rotated into the donsi-convex position. H, hearti Mx, maxillaryprocess.

e. Dorsal view of a 10.5 day fu vitro enbryo cullured Ín normal serun for24 hours at an earlier stage in development. A conparisonwith a. and d.

clearly shows thal the effects of salicylic acid and alcohol areteratogenically induced and not jusf due to developmental de1ay. ArrowsÍndicate unfused neural fo1ds. 0P, optÍc placode.

f. Day 11.5 yolk sacs and enbnyos cultured in 150 ug/nl salicylic acldand 400 ngfr alcohol concurrently showing an absence of developed yolk saccirculation (c.f. Fig.7.1c). ys, yolk sac; Am, amnion; Ec, ectoplacentalcone.

Bars= 1 nm.

10- I

mandibular arch which was often fused in the midl-ine and sornetines to the

pericardium. A dorsal view of the same ernbryos (Fig. 10.1d) reveal- ed

unusual fusion of the neural tube (compare with Fig. 6.1c) which led to a

nanroÍJing or conpression of the optic vesicles (eye defects were observed

i.n over 401l of embryos exposed to alcohol and salicylic acid concurrently,

Table 10.2) and non-closure of the cranial neural tube.

As seen in Table 10.2 and in Fig. 10.1 nany embryos failed to rotate into

the dorsi-convex position. In the alcohol and salicylic acid groups, 90{"

and nearly 67í respectively exhibited this abnormality, whereas 86f of the

conbined treatnent group were affected. Enbryos from aII these treatment

groups also displayed an absence of chorio-al-IantoÍc fusion with all three

groups showing approximately equal numbers of animaLs affected (40tr, J8Í,

41Ð. A comparison of these treated embryos with a labe 10.5 day i¡ vitro

control embryo (Fig. 10.1e) in which the cranial neural folds have not

fused shows tha! the abnornalitles induced by alcohol and salicylic acid

are indicative of teratogenesis rather than of a developmental delay

alone.

Poor yolk sac circulation was also observed in a large nunber of embryos

in all three groups (80l , 571 e 90Í respectÍvely). The absence of a vlable

cfrculation over the surface of the yolk sac in alcohol-sallcyIic acid

treated embryos seen in FÍg. 10.1f, should be compared with the healthy

yolk sac clrculation of normal day 11.5 embryos (Fie.?.1c).

Table 10.3 displays the deviances or ngoodness of fiün for evaluatingthe

inportance of ühe treatments and the outcone of conbÍning the treatnents.

l{hile there}Jere no significant effecbs of either treatnenb on ear and

heart defects in the conblned group, there $¡aa a trend for an alcohol

effect relating to ear defects and a sallcylic acid effect relating to

10-9

heart abnormalibies. Differences between deviances indicated that there

were strong effects of alcohol and salicylic acid when the treatment,s r{ere

combined on neural tube defects, incornplete flexion, absence of chorio-

alLantoic fusion and poor yolk sac circulation, however there was no

interaction between these agents for any of these paraneters and alcohol

TabIe 10.3 Evaluation of the degree of independencealcohol and salicylic acid on rat embryos.

of1

the effects of

Deviance2

Model 1

(Dev Íance 4)No effect ofeither trnent.

( sor)

Model 2 lttodel 3 }4cdel 4(Dev iance 3) (0ev Íance 2) (Dev iance 1 )Effect of Effect, of Independentalcohol orùy. sal/acid only. effects of both.

( 2df) ( 2df) ( 1df)

No. wit,h NTD

No. with openneural tubes

Ele defect,s

Ear defectsBranchial archdef ect s

Heart defectsAbse nbl reducedf orel f¡rbsInconpletefI exLonAbsence ofchorio-alLantoi cfusfonPoor yolk-saccÍ rcul alion

24.464

9.944

9.584

5.02

14.194

5.07

34.044

21 314

g.2Ta

1 2 .34b

8.63b

3 .58

3.29

2.86

3 .83

8.32b

7 .36b

T .fib

19.250

8.99c

I . o?c

3 .56

13.040

1 .62

32.82c

19.58c

0.1?d

5.45e

o.o2d

0.35

o.ogd

0.38

2.zgd

2.gld

2.5gd7 .61c

b 28,gto33 .854 13 .50 0.06da

'See chapter 2 for explanation of statÍsticql model used.zDeviancè is assuned to be distributed as X2.a ff Dev 4-Dev 1>5.99 (critical value for NZ for 2df, p(0.05), thenbreject l{cdel 1 .

rf Dev J-Dev 1>3.84 (criticar value for x2 for 1df, p(0.05), indicatessignÍficant effect of sal1cyIÍc acid, therefore reject l,lcdel 2.ff Dev 2-Dev 1 >3.84 indicates significant effect of alcohol, therefore

reject l,fodel 3.ff Devl <3.84 accept Model 4 for independent effects of both

freatnents.e ff Devl>3.84 reJect Model 4-indicates interactive effects of bothtrea tnent s.

c

d

and salicylic acid

i nde pe nde ntly.

l0- 10

were shown to exert their teratogenic effects

fn relation to open neural tubes specifically, there was an interaclive

effect' which suggested that combÍning the treatments led to an

anel ioratÍon of the frequency of open neural tubes. Eye defects and

branchial arch defects both showed a sÍgnifÍcant alcohol effect, in the

conbined treatment group wÍth a trend lowards salicylic acid exertlng an

effect on the forner abnornality. The nor¡signifÍcant values for salicylic

acid are probably due to 150 ug/ nI being too low a dose to induce these

anonalie s.

The scanning electron micrographs in FÍg.10.2 demonstrate sone of the

structural abnornalities associat,ed wÍth alcohol and sallcylic acÍd

adninistration, as $Iell as the features of Lhe neural and surface

ectoderms. Since the structural and surface abnormalities of enbryos

exposed t,o 400 ng% al-coho1 have already been described in detail in

chapter6, the reader is referred to Figs.6.2 and 6.3 for exaninaüion of

these features. The enbryo Ín FÍg. 10.2a is typical of 11.5 day enbryos

exposed to 150 ug,/nl of salicylic acid. The anÍmal has not, rotated, Ít

lacks forelimb buds and the second and thÍrd brancbial arches ane

underveloped. The forebrain vesicles are reduced in size and the neural

folds have not fused fron a slte behind the forebrain vesicles üo the

caudal end of the enbtyo. Ihe pericardiun has been renoved fron the enbryo

to reveal a pninitive heart tube.

Abnornal ÍtÍes w ere observed in both the surface ectodern and

neuroecfoderro of the ventrícular lunen In Fig. 10.2b, the s¡oooth surface

of the ectodernal cells of the forebrafrnidbrain area which are covered

fn nicrovitli typical.ly found 1n control enbryos also shows neruptionsn as

10- 1 1

well as a number of blebs interspersed amongst the microvilli. These

eruptions appear sÍmilar to structures described as intracelluLar yolk on

the endodernal ceIIs of chick enbryos (EngIand & Vlakely 1986). The

neuroectodern in the open nidbra:.n (Fig. 10.2c) was severely disrupted

wibh extensive protrusions, blebbing and budding into Lhe ventricular

lumen The uneven surface suggests that the junctional complexes between

lhe cells may be broken, and that some of these probrusions may be cell

rennants which have been extruded into bhe ventricular space.

The embryo in Figs.10.2 dre and f has been exposed to the combined

treatnent of 400 ne% al-cohol and 150 ug/mI salicylic acid, and is shown at

differenl orientations. A dorso-lateral vi.ew (Fig.l0.2d) denonstrates the

peculiar shape of the cranium frequently observed in rat enbryos exposed

to both these agents. The anj.nal is mÍcrocephalic with an hypoplastic

forebrain regÍon and an open neural tube caudal to the hindbrain.

gpicalty it has failed to rotate into the dorsi-convex position and the

presence of the allantois denonstrates that fusion to the chorion with

subsequent placental fornation has not occurued. Ttre otic placodes were

also not present. The ventro-lateral aspect of the same embryo in Fig.

10.2e reveals a sptaying of the forebrain vesicles rostral to the level of

the opt,lc sulcus. ThÍs arrangement leads to a conpression of the optic

pÍts as seen in Fig 10.1d. The branchÍal arches were very affected,

partÍcularly the nandibular arch which was distorted and fused to the

perÍcardiun. The second and third branchial arch es $¡ere often

underdev el oped.

At higher nagniflcatlon of the forebrain and mfdbrain regions of the

cranfum (FÍe.10.2f), it appears lhat the neural folds had fused and then

collapsed possibly causlng the sr¡rface ectodern to spllt in the nidbrain

10-12

embrYo cultured in 150 ug/mI ofe not fused frorn a site behind the

d marks the site seen at higher2,3, branchÍal arches; H' heart'

region of the embrYo in a' atrist<, showÍng manY microvillis' intersPersed with blebs

s) were also visible'

c.Theapicalendsoftheneuroepithelialcellsintheopennidbraln(markedbyarrowheadina),}¡asseverelydisruptedwÍthextensiveblebbingand budding (arrows) inbo the ventrj-cular space' Long cytoplasmic strands

(arrowheads) appear to rojecb froro the surface eciodermal cells (E) to

the ventricular surface'

d,e,f.Differentorientationsofanll'5day-invitroembryoculturedin15oug/mlsalicylicacid"ld4o0neíalcoholconcurrentlyduringneurulation. The embryo is microceptraiic and the neuraL folds have not

f used f ron ¡er,ino rhe- hindbrain ."?'"?:Jå 11î;#St,:.io"i|J"I";"'i;i:

dorsi-convex Posi es(

the yolk sac (Ys ds

higher nagnificat sP(

sife seen at high in i'

irregular (arrows) and are covered in btebs (arrowheads)' These

similaribies confirn that the cells between bhe forebrain vesicles are

surface ectodernal ceIIs'

region (approximate site narkedsmooth cells covered with IongPersed with short nicrovillÍ

s (asterisk).

Barg=100un a, d, e, f. Bars= 5um b' c' g' h' Í'

,+ N,Fb ñ--*'

+

a

\\

*,f

t./

.J

Êr'

a

t

t

a

a

, Lù¿

¿aI ï

I

t

a)'t{ +

/¿

j

., rl,

\ ,lI

'a

I

'aa

t

,

ta

a

ò

. raaia

l' I

10_13

region. InitiaL examination under the SEM of the highly irregular cells

betw een the f orebra j.n vesÍcles suggested that this surf ace vras

neuroectoderm. However, sinilarities between the surface ectoderm from lhe

Iateral region of the forebrainvesicles (Fig 10.2g) with a section at a

corresponding location between the vesicl-es (fig. 10.2h) confirmed t,hat

the latter surface was in fact surface epithel iun rather than

neuroectoderm. The cells were extrenely affected by the alcohol-salicylic

acid conbinatÍon, and lacked the more typicaÌ snooth forebrain

appearance. Instead the cells were raised and irregular with nany

cytoplasmic blebs of varying sÍzes superimposed on their surfaces. In

contrast, the neuroepitheliuro in the regÍon of open hindbrain (Fig. 10.2i)

showed an unchanacteristically smooth bleb-free surface but with long

processes interspersed with microvilli and cell debris.

FÍ9. 10.3a shows a horizontaL section through t,he cranÍum of a typical

affected enbryo cultured in 150 uglml salicylic acid for 48 hours. The

embryo is exencephalic with severe distortÍon of the forebrain including

bhe optic vesÍcles, and poorly aligned neural folds Ín the hindbrain

region. Ihe neuroepÍtheliun is reduced in thicloress and the basaL nargin

of the neuroectodern which is anchored by the basal lanina was convoluted

and hence lacked the typical snooth appearance observed in contnol

sections (c.f. Figs.6.3a,b). The nesenchyne appeared rerativery

unaffected at this magnification, with an abundance of stellate - shaped

cells. The only abnor¡nal1ty appeared to be in the regÍon rostral lo the

opfic vesicr e where the nesenchyne had noved anay fron the

newoepitheliun. Fig. 10.3b shows a section of the neural tube around the

opfic vesicle at higher nagnification. There fs a Iarge anounü of cell

disruptfonr shninkage and intracellular and extracellular condensed

naterial within the neuroepithelium whÍch was typÍcat of the disruption

10- 14

throughout the regions of the cranium at this level. The ¡nesenchyme also

dernonstrated condensed Ínclusions, however as seen in this photograph celIdeath was usual-ly located close to the neuroepitheliun and naÍn1y in the

forebrain region. The irregular basal end of the neuroepitheliun was

clearly visible, while snall blebs extended Ínto the ventricular lunen

which contaÍned fragments of ceII debris.

A similar section through the cranium of an embryo cultured Ín 400 ngf

al cohol- (ri.g. 10.3c) al so demonstrated exencephaly with severe conpression

of the optic vesicles. The density of the neuroepÍthelÍum was Írregularwith a convoluted basar area abutting the nesenchyme, and enpty spaces

between the cell-s particularly Ín the forebrain regÍon The nesenchymal

cells generalJ-y appeared nornaL and abundant but were conpressed towards

the surface ectoderm due to the presence of a large, abnornally developing

blood vessel (confir¡ned at higher magnification, not shown). Similar blood

vesseLs lJere observed previously in enbryos cultured in nicotine (see Fig.

7 .3). Sepanation of t,he nesenchyme f rom the surf ace ectodern in thehÍndbrain region in Fig 10.3c is artefactual. HÍgher magnification (rig.1 0.3d) reveal ed considerabl e neuroepithel iaI ceI1 disruption incl uding

shrÍnkage leading to abnornally Iarge int,racellular spaces vrhich were

often filled with condensed ceII rennants, as well as cell inclusions. The

convolutlons along the basal surface obsen¡ed at Iow nagnification could

be seen clearly to be due to a lack of continuity of the surface and isprobably associated wf th disnuption of the basal lanina The nesenctrymal

cells 1n this reglon appeared reLatively unaffected.

When the two treatnents were combfned (Fig. 10.3e), sections at a sinilarlevel through the craniun revealed that the neural folds in the forebrain

regionhad closed but the hÍndbrain anea remained open. This patt,ern of

10- 1 5

10.3. a. Horizontal section through the craniun of an 11.5 day embryo

cultured in 150 ug/nl salicylic acid during neurulation. At this level the

neural folds (NFl have not fused and are severely distorted in theforebrain region, and are incorrectly aligned in the hindbrain (shortarrow). The neuroepitheliun in the midbrain - hindbrain region is thickand irregular (long arrows) along lhe basaL margin The mesenchyme appeans

dense and relatively unaffected at this magnificabion, except for severalnempty spacesn (asterisks) which nay be the síte of abnormal developingUfoo¿ vessels. Arrowheads mark the site seen ab higher nagnification in b.

Hb, hindbraÍn; N, neuroepitheliun; M, mesenchymei or optic vesicles'

b. Higher magnÍfication of a section in the forebrain marked by

arrowheads in a, showing extracellular spaces often containing cell debrisand dense inclusions (arrowheads) in the neuroepithelium (N)' as wel-l- as

cell remnants Ín the ventricular fumen (smaIl arrow). There appeared tobe fewer fiLopodia connecting mesenchymal celIs (M)' and dense ceÌIremnants were also visible (large arrows). Mf, mitotic figures'

c. Horizontal sectÍon through the craniun of an 1 1 '5 day ernbryo culturedin 400 ng1, alcohol during neurulation. The neural folds (NF) in theforebrai-n have not fused and there is severe distortion and reduction ofthe forebrain vesicles and compressed optic pits (o). The nÍdbrain -hfndbraÍn neuroepithelium (N) is of varying thickness and convoluted alongthe basal margins (long arrows). The hindbrain is distorted near the poinfof closure (sñort arrow) with cell debris in the lumen at' t'his site. The

nesenchyne (M) is filted with stellate-shaped cells, and also confains a

Iarge, àbnormat, developing blood vessel (bv). The nesenchynal spacenrarked by an asterisk is a processing artefact. Anrowheads nark the siteseen at higher nagnificatÍon in d.

d. Higher magnification of a sÍte around the optic pÍts marked by

arrowheads. The neuroepitheliun (N) contained nunerous dead cell remnantseÍther in extracellular spaces or within other ceIls (arrowheads).Cytoplasmic UIebs (B) proiect into the ventricular lumen (L), and thebasal end of the neuroeplthelium is also disrupted (short arrows). The

mesenchynal cells (M) were only occasionally affected (large arrow). bv,

blood vessel i Mf, milotic figure.

e. Horizontal sectÍon through the craniun of an 11.5 day embryo cultunedÍn 150 rC/ßI salicylic acid and 4OO n1lr alcohol concurrently' The

forebrain vesicles (thin arrows) were distorted with conpressed optic pits(o). The neuroepitheltum (N) was reduced in thickness, and dÍsruptedfhroughoub especfally at its basal end (thick arrows), and the neuralfolds (NF) had failed lo fuse fn the hindbraÍn area. The nesenchyme (M)

appeared unaffected except at those sites where the neuroepitheliun was

dÍsrupted. Arrowheads nark the site seen at higher nagnificatlon in f.

f. Hlgher magnifÍcaLion of a sectÍon of the forebrain at the level of theoptic veslcles narked by arrowheads fn e, shows severe cell disruptfonleadlng to large extraòe1luIar spaces wlthin the neuroepttheliun (N)

containÍng cett rennants (arrowheads). Large blebs (B) proiect lnto the

10- 16

Iumen which also contains cell debris (short arrows)' Many mesenchymalcells (M) close to the neuroepithellum also contain dense Ínclusíons (long

arrows) whlch probably had their orlgin in the neuroepLtheliun. Note thatcell remnants are even present wlthin the blood vessels (bv)'

Bars=100un a, c, e, 50urn br d, f.

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10- 17

opening is typical of craniorachischisi.s, as the neural folds in many of

fhese aninals failed to fuse throughout the caudal aspects of the embryos.

Although the neural folds in the region of the forebrain had fused, the

histological appearance of this area shows that the forebrain had sunk

between Lhe vesicles causing severe compression of the optic pits.

Histological analysis clearly explains the pattern of abnormality observed

under the scanning electron (Figs. 10.2c,d,e) and dissecting (rigs.

10.1crd) ni.croscopes. The density of the neuroepithelium was severely

reduced and the basal surface was highly convoluted with regions where

neuroepithelial cells appeared to nspilln into ühe nesenchyne which

contained abundant ceI1s. (This nspillagen although not clearly seen inthis mlcrograph was confirned at high nagnÍfÍcation at the IightnÍcroscope 1evel, and is demonstrat,ed rater in t,his chapber). Higher

magnification of an area in ühe forebrain (Fig 10.3f) shows that t,he

degree of ce]1 debris, shrÍnkage and general disruption within the

neuroepÍthelium was severely exacerbated by conconitanl exposure to both

agents. Much of this cell debris was observed to be present in the

ventricular lunen and also appeared to extend into the nesenchyne.

Affected mesenchymal cells containing inclusions v¡ere nainly observed to

occur in the regions close to the neuroepithetir.¡n in the forebrain region

This close proxfnity and the dfsruption of the basal end of the

neuroepÍthellun suggests that some of the condensed cell debrfs withinnesenct¡ynal cells nlght be extruded remnants fron the neuroepithelium.

A dose of 150 ug,/nJ- sallcyllc acid was chosen as it provided a suitable

level of gross neural tube teratogenesis, as well as evidence of celldeath. The fmportant characterÍstics of satlcyllc acid-lnduced patholory

have already been descrlbed Ín detail in chapter p. Signlficant features

of cellular disruption assocfated wlth exposure to 150 ue/ nI salicylic

10- 1 I

acid frorn day 9.5 of gestabion for 48 hours are shown in Figs. 10.4a and

b. The major abnormalities occurred within the neuroepithelium wÍth cell

debris in the mesenchyne generally confined Lo the proximity of the

neuroepitheliurn. Disruption and shrinkage of the neuroepithelial cells led

to the appearance of large extracelluLar spaces, with cytoplasmic

projections (blebs) protruding into the ventricular lumen, while many

cells contained electron-dense inclusions (Fig. 10.4a). Fragments of, or

whoLe dead cells were aLso observed within the extracellular spaces. The

basaL end of the neuroepithelium rÁras of ten affected, (Fig. 10.4b) becoming

disrupted and irregular, reflecting the convoLuted appearance observed at

the light nicroscope 1evel. Fragnents of cells appeared to project into

the mesenchymal matrix which was filled with cell debris close to the

neunoepithelium. The basal ends of some of the neuroepithelial ceLls

displayed evidence of cytoplasmic leaching whÍIe the mitochondria renained

intact, as observed previously in similar sections in salicytic acid-

treated enbryos al higher nagnifications.

Highen nagnification (Fie. 10.4c) of an area of bhe micrograph in Fig.

10.4b showing the neuroepitheliat-mesenchymal bonder, revealed that the

basal lamina was completely disrupted and cell conponents including

mitochondria were in the process of being extruded inüo the extracelluLar

rnesenchynal natrix or vJere cLose to doing so. The irregularity of ceIls

along this border has been linked with the disruption of the basal lanina

suggesting that the basal lanina nay be involved in the maj.ntenance of the

snooth shape of the neural tube (Turner 1990). Fig. 10.4d demonstrates the

presence of celL debris free in the rnesenctrymal natrix as well as in blood

vessels. 0n1y a small number of mesenchynal cel-Is appeared to be affected

and these were usually located close to the neuroepitheliun. The naJor

abnornaLities appeared to be disruption of the nitochondria and the

10- 1 9

Fwtheanteriorneuraltubesfromseveralll.5dayem 150 ug/n1 salicYlic ac tion' The

ne as disrupted with extra ontainingde ragments (arrow s) in a, b w ere aI so

Iocated in the neuroepithelium (arro ¡head) in dispersed

cytoplasm were located ab the basal end of the neuroepitheliun (asterisks

in b). The mesenchyne also showed evidence of cell remnants (short arrows)

in b, l-ocated wiùnin lhe extracellular matrix (ECM)' Note the nitotÍciieu"" (Mf)in a, adjacent to the ventricular lumen (L)' and lhe presence

of blebs (B) Protruding into it'

c. Higher nagnification of lhe basal end of the neuroepithelium (N)

adjacãnt to the mesenchyne (M), near the whibe asLerisk in b' The

nicrograph shows thaù the basal lanina has been disrupted at various sites(arrows) allowing neuroepithetial cell debris t'o pass into bhe

extracellular matiix (EcM). Conpare parb of the apparently healthyneuroepithelial cel] (N) with adjacent affected cells containing damaged

nibochðndria (m) and Ieached cytoplasm (asterisk). Both lhe affected cellsappear to be extruding their contenbs into the extracellular natrix'

d. Mesenchymal- contents of celIs in the anterior neural- tube of an 1 1'5

day enbryo cultured in 1 50 ug/nl salicylic acid. Most affected mesenchynal

cei.Is tùl vüere usually close to the neuroepithelium (N), and oftencontained large heterolysosones (Ly) with Iysed cell debris' damaged

nitochondria (m) and abnormally shaped nuclei (nu)' The extracellularmatrix (EcM) and blood vessels (bv) often conbained dead celf rennants(arrows), The opaque structures associated with the endothelial- cells (En)

of blood vessel" ù"y be primary lysosomes (arrowheads)' BL, basal lamina'

e,f. The nicrographs show the lunenal and basal ends respectively of the

neuroepibhelium (¡¡) from the anterior neural tube of day 11'5 enbryoscultured fn 150 uglnI salicylic acid and 400 uglnl aJ-cohol concurrently'Cell disruption f"d to exlensive ceII debris located in largeextracellul.ar spaces (asterisks) as welL as heterolysosomes fil-Ied wÍthIysed cell remnants (arros¡s). one cell i.n e, appears to be Ín the process

of phagocytosing nenbranes and other cell contents (Iarge arrowheads). The

lumenal border in e, was also very disrupled wÍLh condensed cytoplasmicprocesses or blebs (short arror,¡s) projecting Ínto the venbricular lunen(l) ,hich also contained dead cell conponents (snall arrowheads). Despitethe extensive necrosis, mltotic figures (Mf) can stiIl be seen' Si¡nilarstrucbures were obsen¡ed at the basal end of the neuroepithelium in fr and

I ncl uded exlensiv e extracellul ar debri s (asteri sk) and heterolysosones(arrows). Ttre ceII remnants appear lo be Ín the process of being extrudedinto the extraceLlufar natrix (ECM) of the mesenchyme at sÍtes where lhebasal Ianina is disrupted (arrowheads). The circular structures of varyingdensities (marked by X) 1n e. and f. may be prinary lysosones'

Bars=5un arb,erf; 2un crd.

tr-' r'

qv'

ECM

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-

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1 0-20

fornation of heterolysosomes containing cytoplasmic and other condensed

cell- rennants. The reader is referred to Figs.8.4 and 8.5 for lhe major

ultrastruetunal- abnornaÌitj.es associated with exposure to 400 ngf aIcohol.

l{hen embryos r^Jere exposed to both agents in culture over 48 hours durÍng

neurogenesis, the anterior neural tube was severely affecLed throughout at

the level examined. Sect,ions of neuroepithelium fron the forebrain region

show an exacerbation of the ce]l necrosis observed with the individual

agenls. Many celÌs were severely disrupted and shrunken with large

extracellular spaces often fil1ed wit,h cel1 debris of varying eLectron

densiby, whí1e other cel-1s contained lysed cell debris probably

phagocytosed cell rennants contained within heterolysosones (Fig. 10.4e).

Condensed cell- remnants were located along the ventricular lumen and some

cell components had been extruded into the lumen, $rhile others at the

basal end (Fig 10.4f) with its highly irregular border, appeared contained

by the basal lamina.

Higher magnification (Fie, 10.5a) of cel-1 components revealed lhaf the

combined treatment led to a breakdown of nitochondria and a reduction in

the density of the polyribosomes while the nuclei generally renained

intact. Figs. 10.5b,c,d show the t,ype and degree of celluIar dlsrupLion

assocÍated with conconitant exposure to alcohol and salicylic acid.

Disruption of the celI conponents was very severe and most of the

extracelLular debris was no longer distinguishabte as specifÍc organelLes

(fig 10.5b), although there was some evidence of disintegrat,ing

nitochondria, as welL as free polyribosomes, while the heavÍIy condensed

material probably consisted of nuclear remains. It is likely that some of

the extracellular rennants in Fig. 10.5c were being phagocytosed by other

healthy cel1s. The opaque structures associated with disintegratlng

1 0-21

Fig.lo.5showsanumberofultrastructuralfeaturegobservedintheneuroepitheliumofaffecteddayll.5]nvitroenbryosculturedinl50uglnl sattcytic acid and 400 nef alcohol concurrently'

a. The rn icr ogr aPhneuroepitheliat cells (

losb their cristae. Pos

well as heterolYsosome(cr). Note bhab struclures such as nucl-ei i

(nb) are not affected at the early stages of cell disintegration' Note

also ceI1 remnanbs Iocated extracellularly'

b. This mÍcrograph shows a number of cells at varíous late stages of

necrosis. nenainini sections of cell membranes (short arrows) facilitateindividuat ce]ls, which ondria (m)

ear (nu) naterial (N1)r ribosomes

"o"or""(N3), while oth omPretelY

Ies (N4).

c. A range of extracelluLar rennants which are nostly unidentifiable' The

heafthy ãeLI (N) appears to be phagocytosing some of this debris at two

sites (arrows). The opaque struciures âssocÍated with danaged nitochondria(n) and vesicufated rough endoplasmic reticulum (vr) nay be lysosomes (LY)

as sone can be seen to be membrane bcund'

d. The large extent of organelle dlsruption in this cell which stÍIIcontains healthy cytoplasn toward ¡ one end (asterisk) suggests that fhe

cell is digesting its own orSaneLles (autophagocytosis) rather than

conponents of othãr ceIIs. Cytoplasmic componenls (arrows) can be seen tobe noving into the autolysosone whlch is fiLled wlbh nembrane componenbs

whÍch are unidentifiable (arrowhead;). Note the swolLen mitochondria (m)

in the adjacent cell.

erf. The neans by whlch necrotÍc neuroepithelial cell components nay be

less affected. A section of Ftg' 10'4shows extrusÍon of cell rennants inlnto the extracellular natrfx (ECM)

the neuroepÍtheliun at sites where the basal fanina 1s severely disrupted(arrows). BL, basal lanÍna; nr condensed mÍtochondrlai cr¡ cytoplasnicribosomes; nu, nuclear comPonents'

Bar s= 1 un.

L.;f

-

i", .i,.

ECMI

+

*

,iÂso

10-22

organeLles and cytoplasn in this ¡nicrograph are probably lysosomes. In

contrast, the nenbrane fragnents in Fig. 10.5d are Iocated within the

cells in t,his micrograph and are probably exanples of autophagocytosis

where the celI 1s digesting itself rather than rennants of oüher cel-ls.

As iL has been observed in previous chapters, the micrographs Ín Figs.

10.5e and f, indicate that dead ceLl rennants are re¡ûoved fron the

neuroepÍthelium by several mechanisns. Fig. 10.5e shows a large amount of

dead cel1 materiaL whlch also contains lysosonÈ1Íke structures located in

close proximity to the Iumen end of the neuroepilheJ-ium, at sÍtes

associated with junctional conplexes. The cor:fusion in this area suggests

that' the junctÍonar complexes may be preparing to break apart. At the

lunen end in Fig. 10.5f, the basal lamina has parted at some sites

al-lowing the neuroepithelíal contenbs to spílI into the mesenchynal

matrix.

10-23

10.4 DISCUSSION.

The results of this study demonstrate that orùy alcohol had an effect on

crown-rump length and somite numbers. This was reinforced when the two

treatmenLs were combined as there was no further reduction in these growth

paraneters, which supports similar effects on fetal weighl in mice

reported previously by RandalI and Anton (1984). In contrast to these

findings Guy and sucheston (1986) Oi¿ not observe a significant

dÍfference in febal weight in mice exposed to alcohol only, however

pretreatnent with 250 ne/kg aspirÍn Ied to significantly lower fetal

weighl in the aspirin-alcohol group than in all other Eroups' with a

corresponding lower brain weight. The authons attnibuted these differences

between studies to variations in strain, dose level' tiningof doser or

Lreatnent protocol. Bonthius and llest (1989) also neasured body weights

during the rapid brain growth period postnatatly in rats and saw no

difference in body weights compared with controls regardless of whether

treatnent was alcohol or salicylic aci.d, alone, or in conbination, as all

groups gained weight uniformly. The differences between observaLions Ín

their study and the present one could be attributed to very different

metabolisn in developing animals at nid-gestatíon compared with

postnatally.

The absence of an effect of salicylic acid on growth at 150 uglml in the

present study, while affectÍng teratogenesls, suggests lhat a higher

threshold level of salicylic acld is required for affect,ing growth than

for inducing dysmorphogenesis. This suggestion is supported by Cicurel and

Schnid (1988) who induced nalfornations in rat embryos at a lower level of

aspirin (1S0 ug/nI) than was required to reduce crown-rump lengths (4OO

uglnl). Previous reports (Butcher et al 1972l' Kinmel et aI 1974) h4ve also

shown that behavÍoural teratogenesis nay be induced in aninal nodels

10-24

exposed to aspirin at levefs below the threshold for producing deficits in

either bodyweight or anatonical rnalfonmations'

l¡hite the IeveI of salicylic acid appeared to be subthreshold for

affecting growth parameters such as crown-rump Iength and somite number,

yolk sac dianeter was also not affected by salicytic acid or alcohol alone

at the levels used in the present study, however, when the treatments were

combined there was a significant reduction in yolk sac size. This suggests

that there may be an interactive effect of the agents on yolk sac diameter

when alcohol and aspirin are adrninistered sinultaneously.

The results of bhe present study denonstrate that rat embryos are

advensely affected oven a number of organ systems by ethanol exposure when

the embryos are also cultured in salicyJ-Íc acid. Contrary to observations

by Randall and Anbon (1984) where aspirin reduced the frequency of

mal- f ornations due to al cohol- in mouse f etuses, t,he present study show s

that the individual teratogenic effects of alcohol and aspirin during

embryogenesis wene greaten when the treatments were combined for all

structures except for open neural tubes. Statistical analysis (see Table

10.3) indÍcated that the Ievel of augnentation in the combined breatnenb

group was additive rather than ì.nberactive, indicating that bhe two agents

exerted their effects independently. 0f specÍal interest in relation to

neural lube anomalies is the observation that w hi-l-e the two agents appear

to exert their teratogenic effects independently when all neural tube

defects were grouped collectively (see Tables 10.2, 10.3) the al-cohoI-

salicylic acid group demonstrated an interaction which was ameliorative

when only open neural tubes were considered. Hence this suggests that

concurrent exposure to alcohoL and salicylic acid nay provide protection

to the embryo, enablÍng the neural folds to fuse, and thus reducing the

10-25

frequency of this life threatening abnormality'

Several other Sroups (Guy & Sucheston 1986, Bonthius & t'lest 1989) have

described a worsenÍng of dysmorphology due to aspirin pretreatnent of

al-cohol embryos, alLhough the conditions of the studies varied, which may

have contribuled Lo differences in some of the conclusions reached between

them. Bonthius and Ïlest (1989) observed lhat aspirin rather than having a

protective effect, worsened brain growth restriction induced by alcohol

in prenatal raLs, and since salicylic acid alone had no significant effect

on total, brain weight, these workers atlributed the worsening of the

alcohol- induced microencephaly by saI icytic acid to be due to an

interaction, rather than to an additive effect of salicylic acid and

alcohol. since these authors exposed neonates to alcohol and salicylic

acid during the brain growth spurt period rather than during enbryogenesis

as in the present study, and measured neural tube defects in terms of

diminished brain weights, these very different experinental regimes nay

account for the differenb conclusions reached. In contrast, Guy and

sucheston (1986) arrived at a similar conclusion to that in the present

study where an increase in the frequency of a range of abnormalities was

obser¡¡ed to be greater in the combined treatment Sroups than Ín the groups

exposed to aspirin or alcohol aLone. The incidence and severity of fetal

defects in their study, which included visceral, skeletal and external

abnormalities in term fetuses were greater in offspring of pregnant mice

treated with aspirin and alcohol together, than in those treaLed with each

agent alone. However, unlike the present study no neural tube anonalies

were reported. One of the particularly significant and likely explanations

for this difference between studies may lie in the fact that Guy and

Suchesbon (1986) did not expose mice to aspirin untiL day 8 of gestation

which is equivalenb to prenatal day 10 in rats, and Ís a time when fusion

10-26

of the anlerior neuraf folds is completed. The observation in the present

study that the severity of the additional teratogenic effect on the neural

tube brought about by combinÍng the treatments was additive rather than

interactive probabLy indicates that both agents exert their effects

through independent, rather than through the same mechanism.

rn another study however which used subteratogenic levels (150 nc/ke) of

salicylic acid in mice (Randall & Anbon 1984), alcohol fetuses which were

exposed to pretreatment w ith saI icylic acid during embryogenesis

denonstrated an anelioration of the prenatal mortality and

dysnorphogenesis attributed to ethanol exposure' without affecting these

measures itself. Since large doses of salicylic acid have been shown to

exhibit dysnorphology (lfarkany & Takacs 1959) and behavioural

teratogenesis (Randall et aI 1983) in rodents, it would appear that at the

Iower dose of 150 ng/kg in mice, salicylic acid does not alter fetal

development. The lowest single dose reported to cause teratogenic effects

in the nouse is 500 ng/kg (Larsson & Erikson 1966). Hence it Ís Iike1y

that if the dose of saticylic acid wene increased this salicylÍc acid-

induced amelioration of alcohol teratogenesis night disappear and come

into line with the additive effecls of both agents observed in lhe present

study.

Apart from neunal tube defects, other systems exposed to salicylic acid

and alcohol and examined for teratogenesis demonstrated similar additive

effects. These abnormal ities inctuded forelfmb defectsr inco¡nplete

flexion, absence of chorÍo-allantoic fusion and poor yolk sac círculation

Statistical analysis indicated thab forelÍnb defects and lncomplete

flexion showed strong alcohol effects and weaker but significant salicylic

acid effects, while yolk sac circulation was affected nore by salicylic

10-27

acid than alcohol, and chorio-aflantoic fusion was affected approximately

equally by both teratogens.

fhe frequency of eye and branchial arch defects both showed strong

effects of alcohol only. It would appear that in order to induce these

defects in a significant number of embryos, higher doses of salicylic acid

are required. These observations suggest that these structures are nore

resistant to bhe effecls of salÍcy1ic acid at the l-evels in the culture

medium, than are others such as the neural tube. Alternatively, it is also

Iike1y that these l-evels of salieylic acid may induce significant numbers

of eye and arch defects if the embryos were exposed at slightly different

times. Since maximal eye defects are induced prion to naximal neural tube

defects and branchial arch defects al a later tine in organogenesis

(Ìlilson 197Ð, the culture period nay have excluded the optimum times for

inducing these defecbs. However, due to the extensive exposure of the

enbryos from days 9.5 to 11.5 of gestation, this alternative is less

Iike1y to explain these low leveLs of defects. It nay however explain the

non-significant frequencies of a range of abnornalities examined by Guy

and Sucheston (1986)r who administered acute doses of aLcohol and

salicytic acid on day I of gestation Ín mice, although optimun development

of the structures which they examined does not occur untiL later in

gestation (llilson 1973).

These differences in frequency of organs affected by each treatnent

indicate thab different organ systens appear to vary in sensitivity to the

different agents which may also be related to t,he difference in tining of

the teratogenic assault on each systen (I'lilson 197 3). These variations

make Ínüerpretation of norphological dat,a between studies particularly

difflcult. The difference in tine of exanination of the terata between

1 0-28

studies has also contributed t,o diffÍcuIties in comparing previous data

with that in the present sfudy as no other studies have examined the

outcorne of salicylic acid and aLcohol at the criticaÌ time following

neurogenesis Furthermore, it, is difficult for the author to conpare the

teratogenic outcomes of concurrent ethanol-salicylic acid administration

between studíes when different organs and,/or structures are examined.

To the best of the authors knowledge, this study demonstrates for the

first time histological- and ultrastruclural defects associated wilh

concurrent treatment of alcohol- and salicylic acid. In addition to the

unique dysmorphology associated with the cranium in this study'

hisbologicalexanination further supported the notion of an adverse effect

of both salicylic acid and alcohol conbined on the developing neural bube.

!{hile the overall shape of the neural bube did not differ significantly

fron treatment to treatment, the ernbryos lJere observed to be more

microcephalic in the combined treatment group with open neural tubes in

sone cases, which occurred either in the forebrain' midbrain or bindbrain

regions, or more caudally, and the neuroepithelial waII appeared thínner

and the forebraÍn was reduced in size and distortedr especially in the

region of the optic pits.

CeIlular disruption, while evident in the neuroepitheliun of alcohol

embryos and also salicylic acid-treated embryos appeared to increase in

embryos exposed to both agents together. CelI death Ieading to dense

extraceflular and inbracellular debris was nore pronounced in the conbined

group and the neuroepithelium appeared thinner as Iarge extracellular

spaces becane evident. Both the apical and basal ends of the

neuroepíthelium were affected, wÍth blebs protruding into the ventricular

Iunen and severe disruption of the basal lanina. Blebbing has previously

10-29

been proposed to occur due to teralogenicalJ.y induced breaks in the

junctional complexes following sodium valproate administration (Turner et

aI 1990). this proposal may underlie similar observations in the present

study and others in this thesis, where disruption of junctional- complexes

provided a means by which neuroepithelial dead cel-l remnants were extruded

into the ventricular lumen.

Alterations in the basal Iarnina have frequently been assocj.ated with

retinoic acid-induced interruptÍon of neural tube closure (Yasuda et aI

1987), and nay contribute to this defect within the present study. The

abnormal shape of the newal- tube especÍally in the forebraÍn region nay

be associated with Lhis disruption, as the basal lamÍna may serve as a

support for the morphogenic bending and shaping of the neural tube. In

this study however, the basal lanina of bhe neuroepithelium was often

highly irregular and dÍscontinuous in treated enbryos both with and

without open neural tubes, and the neuroepithelium was often markedly

disorganised, with loss of the norrnal palÍsade architecture and celluLar

polarity. These observations suggest that the intact basal Lanina may be

necessary for general architecture wibhin the cranÍaI Iayers and thal

dÍsruption of it nay occur even in the absence of an open neural tube.

fn view of the observation of leaking of cytoplasmic contents into the

extracell-ular space of the neuroepitheliun it is Iikely that one of the

nechanÍsns by which ethanol and salicyì.ic acid act in combination is to

exerl a direct cytotoxic effect on cel1 membranesr causÍng their breakdown

and subsequent dispersal of cel-l contents into the surrounding spaces.

Previous studies have shown both sallcylic acid (Leonards & Levy 1973i

Klein et at 1981) and ethanol (Guth et al 1983) t,o be cytotoxic, and their

concurrent presence may exacerbate this effect as ethanol decreases the

10-30

anount of aspirin esterase causing aspirin to ¡netabolise more slowly in

the presence of ethanol (Gupta et aI 1979) such that the Iikelihood of

menbrane disruptÍon and ceII death Ís greatly enhanced.

Another Iike1y ¡aechanisn of action with this drug conbination is an

imbalance in prostaglandin (PG) fevels. Since both ethanol (Rotrosen et

aI 1980; Horrobin 1980) and saticylic acid (Smith & I'üillis 1971; Vane

1971) change the rate of PG synthesis it is possible that an inbalance in

PG leveIs nay be at leasb partially responsible for the fetal darnage.

Horrobin (1980) has suggested that a PGEI deficiency could be responsible

for FAS. The role of prostaglandins in fetal developnent is currently

under investigation.

Another physiological paraneter altered by both salicylÍc acid and

alcohol is acid-base balance. Ethanol has been deternined to cause a

netabolic acidosis (Lieber 1984), and together with salicylic acid, the

conbined direcb effect could be detrinentaL or could contribute to hypoxia

as a result of the Bohr effect (Mann et aI 1975). Hypoxla is an FAS

nechanism suggested by several investigators to be due to either a change

in placental function resulting from ethanol danage (Amankwah & Kaufmann

1984) or to contraction of unbilÍcal vessels caused directly by ethanol.

Umbilical vessel constriction is also caused by salicylic acid (Klein et,

aI 1 9 81).

Reduction in sÍze of yolk sac and abnornal developnent of vascuLature has

been reported in this study and previously in nlcofine embryos elsewhere

(Joschko et al 1991) and in Chapter 7 of this thesi.s, and following Na

valproate adninistnation (Coakley & Brown 1986). Since the rodent enbryo

ls dependent on this transient yolk sac placenta for both nutrltlon and

protection during early stages of developmenf it, is possible that danage

t'o this structure couLd subsequentry read to a cascade of

embryo including alterations in developing neurar systens.

1o-3 t

effects in lhe

VlhiLe cerr deat'h has been proposed as a mechanism underlying open neuraL

tubes by previous investigators (Record et ar 19g5), the author has

observed in the present and previous chapters that these two teratogenÍc

effects nay nco-existn wÍthin the same enbryo, but each nay aLso occur insome erabryos in isolation as a result of ethanoL and salicylic acidexposure. Hence the author proposes that these abnormalities may be

assocj-ated with Índividual underlying rnechanisns of action. Since both

drugs are assocÍated with ¡nicrocephaly, which appeared to be more sevene

when t,he drugs were conbined in the present study, the aut,hor proposes

thaü the severe cell- deat,h associated with t,he anterior neural tube may

lead t'o a depletion of stem cells which differentiate into neurons lat,erin development, and consequently may underlie the severe nentalretardation associated with ethanol and aspirin Whether factors such as

the disruption of junctÍonal complexes and bleb fornation contribut,e tothe faiLure of neurulation requÍres further extensive invesLigation The

results of thÍs study suggest very strongly that, there ismore thanoneunderlying factor in the teratogenesis of the developing neural tube.

rn sumnary it would appear that alcohol- and derÍvatives of aspirin when

conbined lead to an additive effect on teratogenesis of a nunber ofdeveloping structures, suggesting that these agents nay act throughindependent nechanisms. rn the case of open neural t,ubes however, aLcohol

and saricylic acid appear to denonstrate an interactÍve effect which was

protective, leading to an arnerioration of this defect,, produclng fewer

and possibly Less l-ife threatening abnornalities. These observationssuggest that' the two teratogens may exert their neurotoxic effects through

1 o-32

similar nechanisns. Severe neural ceÌI death was not consistently Iinked

with exencephaly and open neural- tubes alone, and hence iü is 1ikely that,

thÍs cytotoxic effect nay be an additÍonaL anonaly caused by exposure to

aLcohol and salicylic acid, and possibly one of the underlying causes ofpostnatal intellectual and behavioural deficits reported in animals and

hunans.

10-33

10.5 BIsL IOGRA PHY.

Anankwah, KS. & Kaufnan, RF. (1984) Ultrastructure of human placenta:Effects of maternal drinking. Gynecol. 0bsteL. fnvest. 18, 311-316.Bonthius, DI. & !fest, JR (1989). Aspirin augments alcohol in restricting

brain growth in the neonatal rat. NeurotoxÍcof. Teratol-. 11, 135-143.Boulos, BM., AInond, CH., Davis, LE. & Hammer, M. (1972) Placental

tnansfer of salicylates under constant naternal blood levels. Arch.Intern. Pharmacodyn. Ther. 196, 357-362.Butcher, RE., Vorhees, CV. & Kimnel, CA. (1972) Learning inpairment from

naternal salicylate treatment in rats. Nature: Nevr BíoI. 236, 211-212.Cicurel, L. & Schnid, BP. (1988) Postimplantation embryo culture for the

assessnent of the teratogenic potential and potency of conpounds.Experientia 44, 833-840.Coakley, ME. & Brown, NA. ( t986). Valproic acid teratogeniciby in whole

embryo culture is not prevenbed by zinc supplementation. Biochem.Pharmacol. 35, 1052-1055.Desmond, PV. & Crotty, ME, (1987). Alcohol and drug interactions. J.

Gastroenterol. Hepatol. 2, 271-218.England, MA. & I'Iakely, J. ( t ggO) Scanning electron microscopaJ- and

histochenical study of the endoderm in the earJ.y chick embryo. Scan.Electr. Microsc. 3, 1 1 83-1193.Gupta, JD., Gruca, M. & AbIett, Ï1. (1979) Effecl of other drugs and

chemicals on the degradation of aspirin j¡ vitro: possible extrapolationto in vivo metabolism of aspirin. Eur. J. Drug Metab. Pharmacokinet.2,103-108.Guth, PH., PauI son, G. & Hirabayashi, K. ( 1 983). Prostagl andi n

cytoprotection Prostaglandin does not protect against aspirin-or alcohol-induced red blood cetl henolysis. Dig. Dis. Sci. 28, 903-907.Guy, JF. & Sucheston, ME. (1986). Teratogenic effects on the CD-I mouse

ernbryo exposed to concurrent doses of ethanol and aspirÍn Teratology 34,249-261.HiIl, RM., Craig, JP., Chaney, MD., Tennyson, LM. & Mc0u1ley, LB. (1977)

Ut,ilization of over-Lhe-counter drugs during pregnancy. Clin. 0bstet.Gynecol . 20, 381-394.Horrobin, DFA. ( 1980). A biochenical basis for alcoholism and alcohol-

Índuced damage including the fetal alcohol syndrone and cirrhosis:Interference with essential fatty acid and prostaglandin netabolisn. Med.Hypotheses 6, 929-942.Jones, KL., Smith, Dll., UIteland, CN. & Streissguth, AP. (1973). Pattern

of malformations in offspring of chronic alcoholic nothers. Lancet 1,1267 -127 1 .

Joschko, MA., Dreosti, IE. & Tu1si, RS. (1991) The teratogenic effects ofnicotine in vÍtno in rats: A tight and electron nicroscope study.Neurotoxieol. Teratol. 13' 307-316.Kinnel, CA., Vlilson, JG. & Schumacher, tU. ( 1971) Studies on metabol-isn

and identification of the causative agenL in aspirin teratogenesj.s inrats. Teratology 4, 15-24.KinmeI, CA., Butcher, RE., Vorhees, CV. & Schumacher, HJ. (1974) Metal-

saIt potentia tion of sal icylate- induced terafogene sis and behaviouraLchanges Ín rats. Teratology 10, 293-300.KIein, KL., Scott, ÏIJ. & Wilson, JG. (1981). Aspirin-induced

teratogenesis: A unique pattern of celL death and subsequent polydactylyin the rat. J. Exp. ZooI. 216, 107-112.Larsson, KS. & ErÍkson, M. (1966). Salicylate-induced fet,al death and

nalfornations in two nouse strains. Acta Paediatr. Scand. 55, 569-576.

10-34

Leonards, JR & Levy, G. (19?3). Gastrointestinal- blood loss from aspirinand sodiuro salicytaie tablets in nan Ctin Pharmacol. Ther. 14, 62-66.Lieber, CS. (19-84). Metabolism and roeLabolic effects of aIcohoI. Med.

C] in. Nth. An. 6 8, 3-3 1 .

Mann, LI., Bhakthavathsulan, a, Líu, M. & Makow sJ. i, P. (197 Ð. Placentaltransport of aLcohol and its effect on ¡naternal and fetal acid-basebalance. Am. J. Obstet. Gynecol. 122,837 -844'Randall, CL. & Anton, RF. (1984). Aspirin reduces alcohol-induced

prenatal mortality and nalfornations in mice. Alcohol: Clin Exp' Res' B,

513-515.Randall, CL., Anton, RF., Hoffmeyer, GE. & !laItis, cJ. (t983). Prenatal

aspirin: effect on ambulation and one-ltay avoidance in mice' Soc'

Neurosci. Symp. Part 1,9, 666.Record, IR., Tulsi, RS., Dreosti, IE. & Fraser, FJ. ( 1985) Cellular

necrosis in zinc-deficÍent rat enbryos. TeratologV 32, 397-405.Rosett, HL., Oueltebte, EM., t'liener, L. ( 1976) A pilot prospective study

of the fetal alcohol syndrome ab lhe Boston City Hospital. Part 1'

l4aternal drinking. Ann. NY. Acad. Sci.273,118-122'Rotrosen, J., Mandio, D., Segarnick, D., Traficante, LJ. & Gershon, S.

(1980). Ethanol and prostaglandin E1: Biochemical and behaviouralinberactions. LÍfe Sci. 26, 1867-1876.Senabe Standing Committee on Sociat Welfare (1977) Drug problems in

Australia : an inboxicated society. Aust. Govt. Publ. Sewice' Canberra.Smith,JB. & ÏliI1is, AL. (1971). Aspirin severely inhibits prostaglandin

productÍon in hunan platelets. Nature: New Biol. 231, 235-237.Streissguth, 4., Treder, RP., Barr, HM., Shepard, TH. et aI., ( 1987)

Aspirin and acetaminophen use by pregnant wornen and subsequent child IQ

and attention decrernents. Teratolory 35, 211-219.suItk, KK., Johnson, Mc. & InIebb, MA. (1981). Fetal alcohol syndrome:

Enbryogenesis in a mouse model. Science 214, 936-938.Turner, S., Sucheston, ME., DePhilip, RM. & Paulson, RB. (1990).

Teratogenic effects on the neuroepithelium of the CD-1 mouse embryoexposed in utero to sodiun valproate. Teratology 41, 421-442.Väne, JR (1971). Inhibition of prostaglandin synthesis as a meohanisn of

actÍon for aspirin-like drugs. Nature: New BioI. 231' 232-235.trIarkany, J. & Takacs, E. ( 1959). ExperÍnental productÍon--ef,-congenital

nalfornations Ín rats by salicylate poisoning. An. J. Patho1.35' 315-331.l{ebster, }lS, 1,lalsh, DA., McEwen, SE" & lipson, AH. ( 1980). TeratogenesÍs

after acute alcohol exposure in inbred and outbred mice. Neurobehav.Toxicol. 2, 227-234.Irlebster, WS., I,laIsh, DA., McEwen, SE. & Lipson, AH. ( 1983)' Some

teratogenic properties of ethanol and acetaldehyde in C57BL/6J mice:Inplicat,ions for the study of the fetal alcohol syndrone. Teratology 27,231-243.Ílilson, JG. ( 1gTÐ. Environment and Birth Def ects. Academic Press, New

York.Yasuda, Y., KonishÍ, H., Kihara, T. & Taninura, T. (1987). Developmental

anonalies induced by afl-trans-retinoic acid Ín fetal mice: II. Inductionof abnornal neuroepitheliun. Teratolory 35, 355-366.

11-1

CHAPTER 1 1

Gn\IERAL DISCUSSTON

11.1 fntroduction.

llhile the vulnerability of mother and developing fetus to nutritÍonal and

environmental factors has been recognised and the overaLl dysmorphology

associated with many teraLogens has been described, limited studies have

been performed t,o date that attenpt to examine and relate this

dysmorpholory with cel-luIar and ultrastructural abnormal ities. I,ùhiIe thÍs

thesis has described the effects on growth and the overaLl dysmorphologr

in rats associated with exposure to various enbryotoxins, either alone or

in combination, it has been mainly concerned with the assessnent of the

risk to brain development, and consequently the embryos were exanined

immediately f ollowing the ncritical- periodn f or neurogenesÍs (l'lilson 1973)

when the developing neural tube is nost vulnerable to teratogenÍc insult.

An Ín depth examínation of a large sample of embryos provÍded the author

wÍth extensive information regarding neural tube dysmorphology, and

related ceLlul-ar and uLtrastructural changes induced by fhe various toxic

agents applied. Many previous teratogenic studies examined gross

dysmorphology in term fetuses fron the tine of neurogenesis, which

provides a good assessnent of overall abnornalities but generally

patholory is no longer apparent. In nost cases the general dysmorphology

of the agents utilised in this thesÍs have been studied to sone extent by

previous workers, however there is a pauciLy of infornation relat,lng

specifically fo neural tube defects and acconpanying pathology of Lhe

nervous system, particuJ.arly at the ultrastructural Ievel. Hence the

neural- tube was studied in detail at the norphological and subcellular

Ievels bo confinm, extend or report for

five selected agents exerted on the

neurul-ation.

11-2

the first tine the effect that fhe

developing neural tube during

In addition, siniLarities between some of the abnormalities seen in rats

exposed to Índividual agents in t,his thesis, together with simil-ar

previous observations in the Iiterature, pronpted the author to examine

the effects on neurulation of combining several of the agents. This

stnategr which to date has been largely unexplored, was adopted in order

to detenmine whether concurrent exposure to teratogens increased the

frequency of mal-formations and the severity of necrosis in an additive or

interactive nanner.

SÍnce the author has already discussed extensively the najor and

Ímportant findings relating to each of the teratogens alone' or in

combination in the individual chapters of this thesis, the purpose of the

presenl chapter is to integrate the various aspects of dysnorphology

induced by the five teratogens investigated and to relate the findings to

those of previous workers where applicable. The discussion wil-l deal

fÍrstly wÍth the overall dysmorphology, but particularly with defects

pertaining to the anterior neural tube, induced by the indivÍduaI and also

the conbined teratogens. In addition it wiII consider various aspects

relating to the pathological changes observed in the neural tube as a

resul-t of exposure to these agents, including the relationship of induced

pathology to nornal physiological cel1 death, and the possible

inplications of experimentally derÍved necrosis in relation to neural tube

dysnorphology. Fina1ly, where possible, the relevance and clinical

implications of the observatÍon reported iJ vivo and i¡ vitro in rats in

this thesis wiLl be consldered in relation bo parallel conditions in

1 1-3

humans.

It was the authors intention to perforn all experinents in this thesis in

vÍtro using the embryo culture method. This technique was considered to be

an extremely useful model for the examination of teratogenesis since the

authors interests lay specifically in the investigation of neural tube

defects, and the culture nethod allows for comparable growth and

devefopmenL Lo lhat of embryos of the sane age developed in utero from

the headfold stage to the25-somite stage (New et aL 1976)' The 48 hour

period which these developnental stages encompass constitute the rrcritical

periodr for neurogenesis in rats (I¿Iilson 1973). Hence the author

considered thal the progress and completion of neurulation and the effects

of teratogens on this stage of development could be followed extremely

well using this technique. Furthernore, the observation that previ'ous in

vitro teratogenic studies in the 1ÍLerature using embryotoxÍns such as

alcohol and Na salicylabe demonstrated sinilar abnornalities at' the

norphological and celÌular IeveI s (Greenaway et aI 1982, Ìlynt'er et al-

1983) to those reported;L¡ vivo further strengthened Lhe rationale for

using bhe embrYo culture sYstem'

This rnethod was particularly useful as il enabled the author to deternine

whether the agent exerted ils toxio effect directly on the embryo rather

than through naternal mediation and it also allowed for the identification

of the teratogenic potential of particular coutpounds or metabolites of

conpounds to be assessed, such as ethanoL and salicylic acid which in

utero would be nasked by the processes of maternal netabolisn' The

technique also ensures t,hat aIl enbryos are at the same developmental

stage at the connencement of culture (and also aIlows for any abnornal

enbryos at this tine to be discarded) since tlming ls crltical for the

11-4

successfuL induction of a partÍcular defect. Fina)-Iy it allows Lhe

progress of enbryos to be monitored so that factors such as the time of

embryonic death or the tirne course of developmental anonalies can be

a scer tai ne d.

Not a1I studies in this thesis could be performed i¡ vitro however. Zinc

deficiency was induced in vivo as previous investigations in our

laboratory (Record et al 1985) faited to induce malformations in enbryos

cultured in zinc deficient medium, probably because of the retention of

some zinc tightly bound lo the large alpha-2 macroglobulin molecules

(Parisi & Vallee 1970, Parry 1977) which accounts for approximately 20f of

circulating zinc. This zinc component is probably filtered out fron the

embryonic environnent in utero by the presence of an intact Reicherts

menbrane which it has been suggested (Record, PhD thesis) acts as a

barrier to high nolecular weíght fracLions such as alpha-2 macroglobulin

in the serum thus preventing access to the bound zinc source, a vj-ew

shared by Webster (1986, personal connunication).

In order Lo minimise the serum zinc conponent associated with lhis

macronolecule the author attempted unsuccessfully using ultrafiltration

to remove the renaining zinc conponent from the serum (Joschko, 1985'

unpublished results). Atthough successful in reducing the serun zinc

levels in sone cases to as low as 0.18 ug ZnlnI (control levels approx.

1.2ugZn/nI), nost of the embryos did not grow and develop normally or

abnormally, but rather died. The author concluded that this night be

attributed to the additional- loss of important lower nolecular weight

nulrients such as afbunin and even insulin, essential for adequate growth

and developnent. The pink colouration on the filtratÍon mernbrane suggested

that the resj-due contained haemoglobin (mw 64000) which was of a sinilar

11-5

molecular weight to albumin (mw 65000). Even lower molecular weight

proteins such as insulin might also be retained if the pore size was

suffieiently reduced by lhe buÍId up of residue. Although difficulties

arise onJ.y with ü vitro zinc deficiency studies, inorder to make data

from hypervitarninosis A investigations comparable with these studies so

that a potential interaction between the two teratogens might also be

ínvestigated, it was necessary to perform these studies j¡ vivo.

1 1 .2 General norphologica)- observations.

11.2a Zinc deficiency and hypervitaminosis A'

Since HurIey and Swenerton (1966) first reported that a maternal dietary

zinc insufficiency was highly teratogenic in nats, many subsequent studies

have invesligated both the occurrence and the aetiology of the defects

observed, however the najorÍty of studies examined dysmorphologies at

tern, far from the period of organogenesis when patholory is Iikely lo be

absent. The observations in chapter 3 of this thesis provided a nore

detailed account of the effects of zinc insufficiency during neurulat'ion

than has been reported before,and has focused on the adverse effects of

zinc deficiency on the developing anLerior neural tube. Att,ention shoul-d

be drawn to the fact that the study also confirmed previous observations

in our laborabory (Record et al 1985a, Harding et al 1988) Lhat reduced

embryonic size and increased dysnorphogenesis are directly correlated with

dininished naternal serum zj,nc 1evels. Furthermore in the present study

the observation that embryonic growth is severely retarded by in utero

zinc impoverishment denonstrates that growth retandabion occurs as early

as the time of neurogenesis.

As in previous reports (Hurley & Schrader 1972, Record et aI 1986) many

embryos exposed to a zine deficient environment in the present study

11-6

demonstrated severe dysnorphology of the central nervous system and

associated cranlofaciaL structures, however unlÍke in sone other studies

(Duncan & Hurley 1978) there was an absence of a high incidence of

intrauterine death, which the present author at,tributed to the renoval of

the embryos at mid-gesbation when the rat embryo is not yet dependenL on a

functioning placenta for adequate growth and nutrition (New et ar 1976).

Since chorieallanloic fusion, a requirener¡t f or placental fornabion was

frequently absent or in¡nired in dysmorphic enbryos in Lhis study, erobryos

developing in utero after this tÍme wourd probably fail to survive.

MorphologÍcal and nicroscopic evidence revealed that whÍIe nost embryonic

structures examined were affected to some extent by exposure to

insufficienL zinc Ievels, the developing nervous system appeared to be the

rnost frequently and severely affected. In view of the observations of a

reduced level of nucleic acid synthesis in the brainof zinc deficient

fetuses conpared with other body regions (Eckhert & HurIey 1977), fhe

present author proposed that biochemical factors might account for this

increased nervous system vulnerability. The vast range of abnormalities

which was characteristically associated with the developing neural tube

and which incruded open neurar tubes, nicrocephary, hydrocephary and

cranial cysls is lhought to occur as a consequence of large changes inserum zinc l-evels during organogenesis due to the cycJ.ing of food Íntake

by zinc deficient daros (Record et al 1985a). Record et aI (1986) reported

that a specific nalfornation is Iinked intimately to the tining of the

feeding and fasting st,ages of the cycle. By fasting the dams on days 6 and

f of gestation, the present author v'¡as able to induce low serum zinc

levels during neurogenesÍs, which was reflected in the high incidence of

brain and eye anomalies, since these organs are deveLoplng naxinaltybetween days 8 and 11 of gestation (Hurley et al ,197D.

11_7

Ultrastructural observations suggested that zinc deficiency teratogenesis

may be tissue specific, exerting its effect predominantly on the

neuroepithelium. Tissue specificity of a teratogen has been reported

previously by Morriss (19'12, 1973), as weIl as in chapter 4 of this

thesis, when hypervitaninosis A appeared to preferentially affect the

mesodermal germ Iayer with a minimal effect on the neuroepithelium. The

severe cell death associated wiLh zj-nc deficiency in the present study is

simiLar to that observed for a nunber of teratogens including alcohol

(Bannigan & Burke 1982, SuIik et aI 1988), folic acid deficiency (Brenent

et al- 1989), cadmium (Webster & Messerle 1980) and arsenic (Morrisey &

Mottet 1983), âs well as many others described by Scott (1977), The

present author observed extensive necrosj-s evidenced by heterolysosones

and extracell-uIar membrane remnants, located throughout the

neuroepithelium and particularly in the mitotically active latenal regions

of the anterior neural tube, unlíke physiotogicaL cell dealh which was

rnainly confined to the sites of neural tube closure located dorsally along

the enbryonic rostro-caudal axis. SimjLar observations of necrosis in the

Iateral regions of the anterior neural tube in zinc deficient embryos in a

pnevious study by Record eb aI (1985b),led the present author to conclude

t,hat the extensive celL death observed in experimental animals previously

(Joschko et aI 1989), and in this study was not part of the programmed

cel-I death seen routinely in control animals but rather that it reflected

the teratogenic outcome of zinc inpoverishment in rapidly prolÍferating

regions of bhe preplacental enbryo. The present author was unable to

consistently distinguish between physiologicaÌ and teratogenicalJ.y-induced

celI death based on specific patterns of destrucLion as described by

Ïly1l1e (1981), and suggesbed instead that if a distinction could be nade'

it shoul-d be based on a different locus for each kind of ceII death.

11-B

The mechanj.sm through which zinc deficiency exerts iLs teratogenic effecton preplacentaL embryos has not yet been elucidated, however a nunber ofstudÍes have suggested that it night be biochemically derived. l\IhiLe there

is strong evidence which suggests that zinc-deficiency-reratedteratogenesis may involve an action on the cell cycle,a considerationwhich is extensively discussed in chapter 3, the appearance of thedysmorphology of the neural folds and of t,he urtrastructuraL me¡nbrane

defects suggests that zinc impoverishment may int,erfere with neuraL tube

closwe by an actÍon on ceLl and organelle nenbranes, illustrated by the

distorted and disrupted lumenal- border of the neuroepitheliun. Thismembrane mediated acLion is particularly likery since an absence of zÍnchas been shown previ.ously to compronise nenbrane integrity (chvapil 1976),

Hence the author proposes that, the biocheuicaL arterations associated withzinc deficient teratogenesis may be a secondary consequence to a directaction on cell and organerle membranes leading to their disruption and

destabilisation, and subsequent rapid and severe onset of celr death.

Ïr¡hire the present resurts appear to support membrane destruction and

subsequent cel1 death as being closely correlated wiLh neural tubedysnorphology Índuced by zinc deficiency, this study was the first of a

nunber perforned by the author, and further studies described in l_aterchapters cast some doubt on the validity of this association

Hypervitaminosis A was shown in chapter 4 of this thesis to act as a

potent' neurar teratogen at the norphological, ceLlular and subcellurarleveLs' The present study confirned the hypervitaminosis A-inducedexencephaly and other morphological defects particularly craniofacial,which have usuarly been reported in tern fetuses (Geeren et al lgTg) when

the full but more generar features of hypervitaninosi.s A dysnorphoJ-ogy are

11-9

evÍdent, but underlying pathology may be absent. Exencephaly which is a

confirmed neural- tube defect of vitamin A teratogenesis (Cohlan 1953,

1954, Morriss 1973), and equivalent to human anencephaly (Hanaway & I'leIch

19TO) was consistently observed in hypervitaninosis A embryos as reported

in chapter 4. Fron the appearance of the cranÍum growth and development of

the neural folds did not seem to be impaired, as they had become elevated

in the nornal manner, but had failed to fuse, and had then col-lapsed over

the lateral regions of the craniun, as seen prev j.ously in rats (Morrj.ss

197Ð and mice (TheodosÍs & Fraser 1978). The collapsed appearance of the

neural folds suggested that the neuroepithelium was poorly orientated

rather than extensively damaged, although some abnormal blebbing was

observed al-ong the lumenal surface of the neuroepithelium. Mieroscopic

studies showed that there was an associated paucity of mesenchymal cells'

cel-1 death and abnormally-shaped cel-Is in the underlying mesodermal Serm

1ayer, which Morriss (1973) concluded played a supportive tissue rol-e to

the overlying neuroepithelium. The possibility that abnormalities of the

nesenchyme could lead to the collapsed appearance of the neural folds is

supporLed by the observation !hat in zinc-deficÍent enbryosr where the

nesenchyne r^¡as obserrred to be mininal.ty affected, the neural f olds often

failed to fuse, but rarely collapsed onto the surface ectoderm.

This is the first study to examine the cellular and ultrastructuraL

appearance of the anterior neural lube in hypervitamÍnosis A rat enbryos

írnmediately following neurulation on day 11.5 of gestation The relatively

unaffected neuroepitheliun, and the paucity of mesenchymal cells in the

underlying nesenchyne and associated pathology was sinÍIar to obserr¡atlons

nade following exposure to excess vitanin A palmitate 1n rat enbryonlc and

extra-enbryonlc tissue prior to neural tube closure (Morriss 1972, 1973).

11-10

Other cells which may be affected by hypervitaminosis A are neuraL crest

cells and developing erythrocytes. Identification of the approximate sÍte

of neural crest cells which migrat,e laterally through the ¡nesenchyme

close to the surface ectodern to structures such as the branchial" arches,

led to the observation of nany dead cells and phagocylosed cell remnants

along the nigratory pathway which may underlie abnormal facial structures

including clefb palate reported elsewhere Ín term fetuses. Another feature

comnonÌy observed in the present study, which confirms observatÍons made

by Glauert et aI (1963) was the presence of vacuolated erythrocytes within

developing blood vessels in the mesenchyme. fn the present study' these

vessels also contained cel-I debrÍs which were probably remnants of

neuroepithelial or nesenchymal cel1s. This observation might represent a

means of disposal of neural bube cell rennanls as the endothelial lining

of the developing blood vessels was sometines observed to be disrupted.

Despite zinc deficiency and hypervitaminosis A predoninantly affecting

the neuroepitheliun and nesenchyme respectively, the type of cell death

observed in the different germ cell layers of affected embryonic neuraL

tubeswas similar, with primary lysosornes assocj.ated with damaged

organelles such as nitochondria r âs well as the presence of

heterolysosomes indicative rather of Type 1 or Type 2 cell death accordÍng

to Schr"¡eichel & Merker (1973) (see section 11.3a). These workers reported

thaf hypervitaminosis A l-ed to type 2 necrosis. They also stated that by

the tÍne lysosomes are present a distinction between types 1 and 2 cell

death is difficult which probably explains why the present author

considered both types to be represented.

It is likely thab excess vitamin A exerts ils toxic effect direcfly on

the nesenchynal cells, since Morriss (1973) reported that the onset of

11-11

necrosis and cel-I Ioss occurred as rapidly as two hours af¿er excess

vitanin A adninistration at, the neural plate stage in rat enbryos. rn

addit,ion, a decrease in DNA synthesÍs (Morriss & steele 1924) and poor

cell migration (Marin-Padilla 1966) have aLso been implicated as causes of

poor cephalic nesenchyme numbers however considering the rapidity of the

effect it appears that these factors nust foLlow secondarj.Iy to ¡he rapid

onset of cell- death in this layer of the neural tube. Hence the extensive

details at the ultrastructural and general morphological levels reported

in chapter 4 contribute significantly to our understanding of the finer

details associated with hypervitaninosis A. The observations nade in that

study also strongly indicate that young women of chtld bearing age who

frequently ingest vitamin A need to be warned of the serious potential

risks that reLinoid exposure nay lead to ín their young unborn infants.

fn chapter 5 sone overt similarities between zinc-deficient and

hypervitaminosis A-induced dysmorpholory of the central nen¡ous system and

other developing structures together wÍth the previous observation that,

zinc and vitamin A interact at thelevel of the liver (Smith et aI 1gT3)

led the present author to investigate whether nanÍputatlon of these two

dÍetary agents causes an interaction in utero at norphological and

ultrastructural IeveIs. Linited nacroscopic evidence (Eckhert & Hurley

1979) had suggested that this nay not be sor however lit,tle attention was

paid directly to the brain nor had the question of an interaction been

studied in detail.

This is the first report of the combined effects of hypervitaminosis A

and zlnc deficiency at teratogenic levels on enbryonic body size. The

study shor¡ed that, the growth retardation produced by zinc deficiency alone

was not, aneliorated by hypervÍtaminosis A. Hence adequat,e zínc IeveLs

11-12

appear to be of far greater Ímportance for the efficient growth of the

embryos than vitanin A, at least durÍng organogenesis, when rapid

prol if eration is at an optimum (f oner 1970).

Chapler 5 also reponls for Lhe first tine on the norphological and

u.l-trastnuctural effeets of combining zinc deficiency and hypervitarninosis

A at the tÍme immediately following the period of neurogenesÍs. The nost

inportant finding made in this study was that the combined nutrient

nanipulations led t,o a positive interaction wíth regard to the frequency

of ovenall dysnorphology and specifically of neural t,ube abnornalities,

wibh nany enbryos de¡nonstrating severe exencephaly wÍth extensive cranial

coIJ.apse.

Although exencephaly was reported previously in term fetuses following

exposure to zinc deficiency and hypervitaninosis A (Eckhert & Hurley

1979), unlike the present study these workers reported that, these agents

exerted an additive effect on neural- tube abnorrnalities. The present

author feels that the absence of an interaction on neural tube defects

despÍte the presence of exencephaly at term may be attributed to reduced

fetal viability at thÍs time as severe defects of this kind are likely t,o

lead to fewer survivÍng embryos. 0n the other hand, the frequency of

defects of structures such as eyes and branchial arches was addÍtive in

embryos in t,he present study and in fetuses at term (Eckhert & Hurley

1979). Different responses of different structures led the present author

lo suggest that bhe neural t,ube nay be more sensitive than cranio-faciaL

slructures to the comblned effects of zÍnc deficieney and excess vitanln A

and that, fetuses are more like1y to survive with facial defects than with

dlsrupted neural tissue.

AnoLher inberesting observatlon in this study was that, embryonic rotatfon

11-13

and chorj_o-allantoic fusion induced by zinc deficÍency alone appeared to

be aneliorated by hypervitaninosis A Ieading to fewer embryos being

affected. This suggests thal vÍtamin A at high leveIs nay exert a

protective influence on the embryos fron these deleterious effects of zinc

deficiency although this effect has not been observed for nornal or sub-

normal- levels of vitamin A (Duncan & Hurley 1978)'

TLre morphologicaÌ interaction on neural tube defects in embryos exposed

to bobh treatnents was supported by an apparent interaction at the

cel-IuIar and ultrastructural levels as the frequency and severity of

organelLe disruption and ceII death in the neuroepitheliun and mesenchyrne

and a paucity of cells in the latter germ cell layer led to a potentÍative

rather than an additive effect. HistologÍcaJ-Iy, Lhe effects vùere so

extensive as to show a severe disordering of surface ectodermal and

neuroepithelial eeLls at the ventricular lumen which suggested that even

the junctionaL complexes at the lunenal ends of Lhe neuroepithelium were

di srupted.

In the light of these histologÍcal and ultrastructural observations the

present study indicates that while each treatnent appeared to act

primarily on a separate germ layer of bhe developÍng neural- tube both the

neuroepithelium and mesenchyme are affected with an increased severity

when both treatments were applied together, suggesting a positive

interaction ThÍs is in accord with the observed developmental anonalÍes

described in thÍs study. The ultrastructural data have shown that both

nutrÍents sevenely co¡npronise nembranes in the developing neural tube

(Chvapit 1976, Morriss 1972) and it would appear that this nlght be the

site of teratogenic action for these agents, either through a common or

individual nechanism(s) depending on the specificity of action of the two

11-14

teratogens on membranes. Whether the membrane-mediated effect is the

principal t.eratogenic lesj-on or is secondary to some other mechanism

preventing neural tube closure requires further investigation. However,

the author feels that in view of the severity of the cellular and

ultrastructural disruption, and their appearance cfose to the tine of

nutrient manipulalion, that at least at thls early period in gestatÍon,

neurul-ation and organogenesis are severely conpronised by a direct

nenbrane effect.

The importance of the present findÍngs in rat embryos nay be especially

relevant in view of the recent reports of birth defects in women consuming

vitamin A and its anaì-ogues for the treatnent of skin disorders by young

wonen who falI in an age group where zinc intake often appears to be

marginal.

11.2b AIcohol and nicobine.

In contrast to the nutritionally-derived teratogens described in previous

chapters the remaining agents examined in this thesis are reguJ-ar1y used

for social on therapeutic purposes. AIcohol, nicotine and aspirin are all

conpounds whÍch are known to enùail risks in hunans whether to pregnancy

or to other aspects of healt,h. In chapter 6 the author confÍrmed the

alcohol-Iinked growth retardation seen in nany previous studies in mÍce in

vivo, and in limited studies in ratsj¡ vitro reviewed recently by !{ebster

(1989). The results of Lhe present study showed a dose-related range of

malformations that included abnormalities observed in nice followÍng acute

exposure to alcohol during neurogenesis (llebster et aI 1983). Sone embryos

exposed to the higher doses of alcohol denonstrated an absence of most or

all of the forebrain, which would resuLt in severe abnormalities of median

facial structures at tern. Nervous system abnormalities observed 1n the

11-15

present study incLuded microcephaly and neura.l- tubes that were open caudal

to the midbrain' typical of craniorachischÍsis, or in the rostral pant of

the neural tube whích corresponds to the serious human malformation of

anencephaly. These neuraL defect,s were not merely a reflection of

embryonic developmental delay as v{as seen by comparison with normal 10.5

day enbryos where neural tube closure follows a particuLar crani.o-caudal

sequence of neurulation (Morriss & New 1979), which was dÍsrupted in

embryos exposed to alcohol.

Ihe present study described nore extensively and in more detait than

previously :Ln vivo, alcohol--induced cranial cell death w hich was observed

fon the first time to the authorfs knowledge, in microcephalic enbryos and

those with open neural tubes folJ.owing neurulation in rats in vitro. The

major features of the anterior neural tube included a thin neuroepÍthelium

with large extracellular spaces containÍng swol-Len nitochondria with

disrupted cristae, and other cerl rennants, as w er1 as numerous

heterolysosmes. Previous reports (Schembri 1981 cited in !Iebster 1989,

Bannigan & Burke 1982, Bannigan & Coltelf 1984, Sulik et al- 1988) have

demonstrated sinilar abnornalities in nouse embryos in vivo. The

sinilarities in dysurorphology and cellular defects during organogenesis

between rat embryos in vitro in thÍs study, and the :Ln vivo nouse model

for hunan FAS indicates that the rat enbryo culture may be a suitable

alternative model, and nay be especially useful in the assessnent of

nechanisns of aLcohol teratogenesis.

Alcohol' like other teratogens (Scott 1977) was observed in this st,udy to

be cytotoxic, an observation rnade previously in mouse enbryos (Bannigan &

Burke 1982, Bannigan & cottelr 1984, surlk et ar 1988), and may be

involved in teratogenesis. Possibly it underlies the nicrocephaly seen in

11-16

many aÌcohol embryos in this study and previously in mice (Sutik &

Johnston 1983) and, since mj.crocephaly is often Iinked with behavioural

abnormaLities and nental retardation, conmon features of the FAS (Clarren

& Smith 1978) it may be the principal factor underlying the long-Lerm

legacy of maternal alcohol ingestion.

The study also demonstrated that alcohol appears to exert a direct

norphological and cytotoxic effect on ernbryonic tissue, and is a

teratogenic agent in rat embryos ;iq vitro, and hence may be the teratogen

responsible for the extensive symptoms which constitute the FAS in hunans.

The presenL author has clearty shown thatalcohol per se caninduce FAS

synptoms of consÍderabl-e severity, as the 11.5 day enbryo racks the

enzymes required to netabol-ise alcohol to acetaldehyde. Since sone studies

however have shown that acetal-dehyde is also teratogenic (llebster et aI

1983, 0tshea & Kaufman 1979, 1981), it is likety that both agents

contribute to the induction of FAS

The observation lhat ceII death beyond physiological. levels occurred inenbryos that did not demonstrate open neural- tubes, but rather were

mÍcnocephalic, led the present author to suggest that, open neural- tubes

may not be attributed to necrosis, but rather to other mechanisms whieh

may underl-ie this abnormality, such as disruption of basal lamina and

large cytoplasmic blebs at the lumenal end of the neuroepitheliun

including the neural crest region. The author further proposes that the

often severe alcohol effect on rnembranes such as nÍtochondrial cristae nay

have the potential- to be a principal factor Ín Ínpairnenb of postnatal

factors such as behaviour and nental capacity, connon features of FAS.

An agent which is frequently associated with alcohol ingestion fsnicotine. Like aIcohol, this agenb has been shown t,o exhibít sinilar

11-17

grollth and morphological effects in the fetus, and to have serious post-

natat af f ects in children. As these lw o agents f requenlly go frhand Ín

handn socially, Ít seemed pertinent to investigate the rnorphologÍcaI and

ultrastructural outcone of nicotine exposure alone, and conbined with

aI cohol.

The study in chapter 7 reported for the first tine the i¿ vitro effect of

nicotine on embryonic growth and morphological development at, the time of

neurogenesis and has provided the first celluIar and ultrastructural-

evidence that nicotine acts as a nervous system teratogen. It also

confirms the previously well established intrauterine growth retardation

present at tern assocj.ated with heavy smokÍng and nicotine intake in

humans (Haddow et aL 1989, BosIey et al- 1981) and Ín rodents (Hudson &

Timiras 1972, Hammer & Mitchell 1979), and shows that t,his retardati.on was

dose-related and occurs as early as the time of neurulation inrabs. At

this tine in embryonic development when the enbryo becomes dependent on an

adequate vascul-ature system and placental formation, gror.rth retardation

may be attributed to bhe poor developnent of the yolk sac vascuLature

which is critical for adequate and efficient oxygen circulation prior to

the formation of a functional placenta (New 1978), and the absence of

chorio-alLantoic fusion, anonali.es which were frequently observed in the

present study. Since the yolk sac is the nain nutritive organ of the

rodent enbryo during much of the perÍod of organogenesis (Beck et al 1967)

any agent harmful- to this membrane can also be expected to affect fetal

growth and development.

YoIk sac sÍze was also severely reduced by Lhe presence of nicoline 1n

t,he cuLture mediun. As nicotlne is highly 1Ípid-soluble it nay exert a

dlrect effect on yolk sac development leading to Ínadequate digestion and

11-18

absorption of nutrÍents vital for ernbryogenesi-s. Hence the author proposes

that the very high incidence of smoking-related spontaneous abortions

observed in hunans (Ktine et aL 1977) nay arise fron the inadequate

nutrition and perfusion as was observed in rats in culture in the presenl

study, and w hÍch in the hunan embryo may originabe in the

syncytÍotrophoblast (l{endelt-Smith & fIilIÍams 1984), tne human analogue of

the endogenously functioning yolk sac in rodents'

The present data in chapter 7 have also shown considerable nicotine-

related dysnorphoJ-ory within the developing rat nervous system. Hence it

Ís likely that detection of only a low incidence of NTD and cleft palate

previously in hunans and rodents nay be atbributed to a masking of effects

by the high incidence of spontaneous abortÍon and early fetal death

reported in ner¡borns of heavy snokers (l'lerler et al 1985). Although

nicoline-induced neural tube anomalies were norphologically less apparent

lhan the open neural tubes associated with other beratogens investigated

Ín this study, nevertheless lhe treatnent did lead to nicrocephalic

enbryos, with hypoplastic forebrains, reduced olfactory placodes and

branchial arch defects. However, at a cellu1ar and ultrastructural level'

nÍcotine caused neuroepithelial and nesenchynal cel-l damage not urùike

that induced by zinc deficiency, alcohol and salicyJ-ic acid. Thus while

nicobine may not interfere directty with the mechanisms affecting neural

tube closure, it probably shares sone conmon mechanisms of actions with

these other teratogens.

The leratogenic effect of nicotine on the developÍng nervous system nay

be especially severe because brain tissue in rodents has been shown to be

one of the principal accunulating regions for this compound (Tia1ve et aI

1968). Ïlhile this accunulation has previously been acconpanied by reduced

11_19

Ievels of brain DNA and protein during neurogenesis (Hudson et al 1984),

Ín the present study the appearance of dispersed nuclear chromat,in in

nicotine-treated embryos suggests that DNA replicative activity is

enhanced, alLhough this is not consistent with reduced embryonic size

observed in affected animals. In addition Lhe sites of ribosomal RNA

synthesis took on an unusual- ribbon-like appearance sinilar to those

previously observed in active germ cell tumours (Ghadially 1988), which

may point to an enhanced Ievel of RNA production as wel-l. These nuclear

ul-trastructural changes require f urther investigation biochemically in

view of the apparent discrepancy between these observations and those

reported previously (Hudson et aÌ 1984).

Although many of the swvÍving of f spring of heavy smoking mothers appear

physically norual, psychological and Íntellectua1 defici.ts are apparent in

bot'h hunans (Fogelman 1980, Naeye & Peters 1984) and in animals (Peters et,

al 1979). These functional abnormalities may be related to structural

anornalies in the central nervous system which may arise from the cellul-ar

disruption and ce11 death within the dweloping neural tube as obser¡¡ed

for the first tÍme foll-owing neuruLation Ín the present study. The

nicrocephalic appearance of narV nicotine-treated enbryos appears to be

linked with thinning of the rostral neuroepitheliun and dininished cell

numbers, a consequence of severe and extensive celI death. Several

possible underlying mechanisns have been extensively discussed in chapter

7, however the means by which development is Ínpeded is unclear, although

evidence suggests a nultifactorial orÍgin 0f particular intenest is the

possibility that the nicotine-¡nediated neural necrosis may involve

oxÍdative menbrane danage, which is thought to be involved in zlnc

def icient and ethanol- t,reated embryos (Dreost,i & Partick 1987). These

enbryos deroonstrat,e similar menbrane disruption to nicotine-treated

11-20

enbryos, incJ-uding those of mitochondrial cristae and which occurs as the

result of superoxide and hydroxyl radical formati.on, both of which are

known to be highly damaging to biological tÍssue including the developing

felus (Dreosti 1986).

In conclusion, the findings of this study demonstrate Lhe risk to t,he

embryo assocÍated with intrauterine exposure to nicotine, and when

extrapolat,ed to humans, highlights the need for wonen to exercise caution

with regand Lo sraoking during pregnancy.

In chapter 8 the author investigated whether concurrent exposure to

alcohol and nÍcotine in rat embryos woul-d lead Lo an exacerbat,Íon of

teratogenesis. Levels of nicotine and alcohoL were chosen to maxinise

information pertaining to a possible interaction of neural tube

dysmorpholory and necrosis, rather than of other structures such as eye,

branchial arch and forelimb which demonstrated mainly alcohol effects at

lhe leve1s chosen The study showed for the firsb time a growth reduction

al 11.5 days Ín rat enbryos exposed to nicotine and alcohol, and the

reduction was at a leveI which indicated an additive rather than a

synergistic effect of these agenfs. The observation that the level of

nicotine used was insufficient bo induce an effect, onyolk sac dÍaneter

although crown-runp length and somite number were reduced, confirrns

observations reported by Abel (1984a) t,hat nicotine does not interfere

wilh yolk sac growth ab the same level as it does with enbrryonic growth.

To the best of the authors knowledge, this is the first tine that a

norphological effect of comblning alcohol and nicotine on enbryonic

development has been successfuLly shown. The linited studies report,ed

previously generally utilÍsed leveLs of either alcohol or nÍcotine, that,

failed to induce teratogenesis and consequently the observed dysmorpholory

11-21

was attributed to either an alcohol or nicotine effect, but not both. It

can be concluded fron observations made in the present study Lhat

different embryonic structwes are sensitive to different levels of each

of these teratogens, hence doses of aÌcohoÌ and nicotine were selected

that were observed in the previous chapters to specifically demonstrate

neural teratogenesis. Morphological features of the neural tubes of many

affected enbryos showed that nany embryonic forebrains were unusuaJ-1y

Itsplayedtt and m icrocephal ic, w ith compressed optíc stalks and vesicl es.

The present study showed that the effect of combining the treatments on

neural tube defects was additive rather than synergistic, although the

frequency of alcohol-affected embryos with open neural tubes caudaL to

the midbrain, showed a trend towards anel-ioration when nicotine was also

included in the nediun Persaud (1982) had also reported a trend towards

amelioration of the frequency of alcohol-induced resorptions when also

exposed to nicoLine. In contrast, Peterson et al (1981) have shown an

interactÍve effect of nicotine and alcohol on the frequency of cleft

palate in term mice. 1,lhiIe it is tÍkeIy that the discrepant results

rela¡ing to whether two agents in conbinatíon act additively or

synergisticaJ.ly probably related to some degree to differences between

studies and parameters examined, the present author considers that

assessnent, and interpretation of conbined treaLnent data is highly conplex

and requires extensive further investigation

This sludy also shows for the first tine cellular and ultrastructural

changes observed in embryos exposed conconitantly to ethanol and nicotine.

Ihe subcellutar features of the cranial neural tube reported in chapters 6

and ? persisted in the conbined treatnent groupsr and reflected the

pattern of dysnorphology by demonstrating a IeveI of celluIan necrosis

11-22

which also appeared additive. These observations suggest that alcohol and

nicotine-mediated teratogenesis are independent of each other and hence

probably utilise different nechanisms to induce abnormalitÍes. This

proposal was supported by Lhe observation that the nicotine-mediated

chromatin and nucleol-i abnormalities reported Ín chapter 7 were similar

to those j.n neural tube tissue of alcohol--nicotÍne embryos, suggesting

lhaf DNA and RNA are affected by nicotine but not by a1cohol.

In conclusion the present study strongly indicates that, concurrent

exposure of lhese agents Ieads bo an additive effect on the nen/ous system

which although not interactive increased the frequency of cranial

dysnorphology and neural cell death above the levels observed for each

teratogen alone.

11.2c Alcohol and sal-icylic acid.

Teralogenicity of Lhe salicylates was first described by ÏIarkany and

Takacs (1959) in rat fetuses in which a spectrum of congenital

abnormalities $¡as observed extending over a nunber of organ systems. A

number of subsequent reports have verÍfied these early observations. Most

studÍes have investigated aspirÍn or its Na salts, but few have examÍned

the teratogenic effects of its metabolltes. The results reported in

chapt'er 9 demonstrate for the fÍrst time the uLtrastructural pathology

induced by salicylic acid, a metabolÍte of aspirin in the tissue of the

developing rat neural tube, and recognise simÍlarities between asplrin,

its Na salts and salicylic acid, on growth and norphological developnent.

When compared to growth in control enbryos, a signÍficant dose-dependent

decrease in a nunber of growth paraneters was reported, a finding which

confirned previous in vlvo (warkany & Takacs 1959, KinneL et at 1971,

DePass & tleaver 1982) and in vitro reports (Greenaway et, ar 1!82, yokoyana

11-23

et aI 1984) lhat aspirin or its derivatives led to growth retardation in a

dose-dependent nanner. The malfornations observed in embryos exposed to

salicylic acid in vitro in the present st,udy demonstrated a dos+related

dysnorpholory, which were similar to anomalies reported in rats i! vivo

(Kinmel et aI 1971, McGarrity et al 1 978). Induction of similar

malfornations in cultured enbryos to bhose j¡ vivo suggets thal salicylic

acid exerts a teratogenic effect directÌy on the developing embryo rather

than through maternal mediation. Furthermore the i¡ vitro system

demonstrated that this metabolite is a causative agent Ín aspirin

teratogenesis, which confirns previous suggestions nade following i¡ vivo

investigations (Kimne1 et al 1971, Koshakii & Schulerb 1972). The present

studies in rats may be of particular relevance and importance as sone

human studies have inpticated aspirin as a causative agent for reduced

birthweight (Turner & CoIIins 1975).

!,lhile there was a signif icant increase j-n overall dysmorpholory including

êVêr branchial arch and heart abnornalities, bhe neural tube was

especially vuLnerable and many neural tube anomalies were reÌated fo the

interruption of neural tube closure which either involved the cranial

region aLone, resulting in exencephaly and which has been reported

previously in only a few enbrryos folÌowing cultwe of embryos in sodium

salicylate (Greenaway et al 1982), or which additionally involved the

neural tube caudal to the craniun, a precondition of craniorachischisis in

term animals, observed in the offspring of aspirin-treated rat's (llarkany &

Takacs 1959, Kimmel et al 1971). The nore severe neural tube malformations

denonstrated ín this study at nid-gestation may not be conpatible with

life, hence neural tube defects nay be an underlying cause of some of the

Iarge numbers of pregnancies that do not reach term. The absence of

chorio-allantoic fusÍon in many of the severely dysnorphic enbtryos further

11-24

suggests that survivaL to term is unlikely as the fetus is dependent on an

active placenta for its nutrÍtional- requirenents Iater in development (New

1978).

The ceLlufar necrosÍs observed in the neural tube of these sal-Ícylic

acid-cultured embryos did not appear to be associated with progranmed ceÌl

death or apoptosÍs, a nornal consequence of differentiatÍon (Geelen &

Langman 1977) which occurs predominantly along the dorsal aspects of the

neural tube, but rather necrotic cells were apparent fhroughout the entire

neuroepitheliun of the anterior neural- tube.

Aspirin-induced dysmorphology has been linked previously to a celluLan

abnormal-ity in the adult gastric mucosa of several specÍes (Hingson & Ito

1973, Pfeiffer & !'leibel 1973, Rainsford & Brune 1978), and also in

enbryonic hindlimb where Klein et aI (1981) reported an unique pattern of

cytotoxicity where ce1I death was observed. Despite these associations in

the l-iterature, the author feeLs that whÍte cell death in the embryonÍc

neural tube night underlie some dysmorphology such as thinning of the

neuroepitheliun and consequent nicrocephaly, exencephaly nay not be

attributed to this necrosis. This conclusion was made following the

observation that in sone embryos in which the neural folds failed to fuse,

the neuroepithelium did not display disruption and necrosis. These

observations led the author to propose that other adverse effects of the

teratogens such as the extensive blebbing at the apical end of the

neuroepithelÍal ceIIs including neural crest sites where fusion occurs nay

have interfered with neural tube closure.

It has been reported in hunans (StreÍssguth et' al- 1987) that young

children exposed to naternal aspirin ingesbion durÍng the first half of

pregnancy $¡ere sÍgnificantly related to 1ow IQ scoresr and attention

11-25

deficits, while aninal studies (Butcher et aÌ 1972) have shown irnpaired

learning in ral offspring. Observations such as these led the present

author to propose that the necrosis observed at dÍfferent sÍtes in t,he

anterior neural tube may be functionally different. Possibly the presence

of severe cellular disruption and blebbing along the dorsal- margins of the

neural tube at the site of neural tube fusion may interfere wÍbh

neurulation, while necrosis and cell Ioss throughout, the renaining

neuroepitheliwn may be linked with inadequate structural plasticity and

possible associated structural and behavioural deficits. The possible

mechanisns which might underlie neural tube dysmorphology have been

considered in sone detaÍI in chapter p, and will only be briefly alluded

to. Mechanj.sms such as prostaglandin synthesis inhibition is of particuLar

interest as aspirin, Iike zinc deficiency led to an inhibÍtion of

prostaglandin synlhesis (Horrobin & Cunnane 1 980, l{eLz 1 981).

Prostaglandins have been inplicated in adequate fetal- development since

they are known to have a large nunber of desirable effects, some of which

occur at the celluLar l-evel (Horrobin 1980). In addition, since both zinc

deficiency and aspirÍn adninistration produce neural tube dysnorpholory

such as exencephaly at tern, and open neural tubes acconpanied by severe

celÌ death in the developing nervous system, these observations could

suggest a role for prostaglandins in the adequate development and closure

of bhe anterior neural- tube.

The fÍndings in the present sbudy denonstrate that t,he developing rat

enbryo is at risk fron salicylic acÍd exposure, and that when related to

humans naternal aspirin ingestion may contrÍbute to the Iack of viability

of many fetuses and may Iead to brain abnornalities, impalred developnenl

and IQ postnatally.

11-26

The knowledge that 509l of pregnant !ronen Íngest aspirin during pregnancy

and up Lo 75l¡ consume alcohof (Streissguth ef aI 1987) which is believed

Lo be the most commonenvironmental cause of mental retardation in the

1,lestern worÌd, provided the auLhor with a clinical juslification for

investigating the teratogenic outcome of cornbining these agents on the

developing nervous system. The resulLs of the study descrÍbed in chapter

10 showed that only alcohol reduced crowrFrump length and somite nunbers

at the doses used in this study as no further reduction occurred when the

two agents were combined. Hence the absence of a salicylic acid-induced

effect on growth while affecting teratogenesis indicated that different

threshold levels of this agent are required to induce growth retardation

and dysnorphology. This this is in accord with previ.ous reports in animals

which have shown thal behavioural teratogenesfs occurs at aspirin levels

below the threshoLd for producing deficits in body weight (Kinuet et al

1974) or structural abnormalities (Butcher et al 1972). In the case of

yolk sac diameter, leve1s of alcohol and salicylic acid were subthreshold'

however Ín tbe conbined group there was a significant reduction in this

parameter, suggesting that al-cohol and salicylic acid inLeract at the

Ievel of the yolk sac. Guy and Sucheston (1986) also observed a

significantly Iower fetal weight which corresponded with a lower brain

weÍght in the aspiripalcohol Sroup conpared with al-] other groups.

In the present study rat embryos denonstrated abnornalities including

neural tube defects at frequencles w hich w ere additive' w hich suggesfs

that alcohol and salicylic acÍd exert their effects independently. These

obserr¡atÍons !ùere contrary to a previous study however, (Randall & Anton

1984) where aspirin Ín faet reduced (ameliorated) the frequency of

malfornations due to alcohot in nouse fetuses. Several other studies

however have aLso described a worsening of neural tube defects due to

aspirin Pretreatment of alcohol- embryos (Guy & Sucheston 1986'

interest to note that while overall-

11-27

Bonthius &

neural tubeWest 1989). rt is of

dysnorphology which Íncluded mÍcrocephaly and open neural fubes was

additive Ín the conbined alcohol-salicylic acid group, the frequency of

open neural- tubes aIone, was ameliorated in the combined groupt

indicating that probably an interaction occurred between the teratogens

leading to few er enbryos with unfused cranÍaI neural tubes. The

a¡nelioration of an afcohol effect on the brain by salicylic acid can also

be demonstrated by the ability of aspirin to ninimise the symptoms of

aI coh ol- lnduced hangover s.

In a nunber of embryos that were microcephalic, the neural lubes appeared

bo have collapsed after fusion particularly in the forebrain region with

optic stalks and vesicles that were usuafly compressed. This abnormal

norphological appearance was often accompanied by severe cell disruption

and death, which t,o the authors knowledge has been reported here for the

firsl tirne, along with the unique neural tube dysmorphology.

CelI death l-eadÍng to extensive extracellular and intracellular debris

was nore pronounced in bhe combined group where the neuroepithelÍun

appeared thinner as large extracellular spaces became evident' and

junctional complexes and basaL lanina were disrupted. Extensive blebbÍng

was observed aLong the ventricular lunen and probably provides a neans by

which neuroepithelial cell rennants are removed from this cell Iayer.

Mesenchynal cel-1s near the opbic vesicles and blood vessels were also

affected and others contained heterolysosones and ceLl debrls

respectively. The aubhor has proposed that several nechanisns of

teratogenic action may be operative. These include a direct cytotoxic

effecl on cell nenbranes, or an inbalance in prostaglandin Ieve1s or even

inadequate nutrition due to an affected yolk sac' These

further intensive investigation'

11-28

proposals require

11.3 Cell death and neural teratogens'

11.3a Physiological cel-I death vs necrosis'

One of the important contributions that' this thesis makes to teratology

is the observation of pattern and variety of extensive cell deat'h in the

embryonic neural tube and associated cranial- structures such as the optic

prinordia, when enbryos are exposed to various teratogens during

neurogenesis. Extensive necrosis was observed in the neural tube at

optimal doses for alL teratogens examined both aLone and in combination

To the best of the authors knowledge this is the first tine that necrosi's

at the ultrastructural level assoeiabed with exposure to nicotine,

salÍcylic acid and combinations of zinc deficiency-hypervitaminosis A'

alcohol-nicotine and alcohol-salicylic acid have been reported. In

addition the study has also extended prevj-ous observations relating fo

cell death foLlowing exposure to zinc defÍciency, hypervitaninosis A and

alcohol aLone. cell death is widely recognised to be a najor phenonenon in

the normal developnent of both vertebrates and invertebrates' Ílhile nany

reports and reviews are avajLable on the role of cell death in normal and

pathological tÍssue a complete analysis of this extensive topic cannot be

undentaken here. Hence the author proposes to discuss the observations

nade in lhe present thesis in reLation to two opposfng theories relaüing

to developnentaL cell death. Scweichel and Merker (1973) proposed bhat

bhere are bhree types of celI death in developing tissues which were

characteristic of physiological cell death, and thal exposure to certain

enbryotoxic agents could lead to unscheduled cell death or necrosis which

denonstrated features of one of these three types of ceII death. These

could be dÍstinguished fron one another accordÍng to the roles played by

11-29

Iysosones. In contrast WyIIie (1981) nade a distinction between the

sequence of events leading t,o physiological cel-I death (programmed ceII

death or apopt,osis ) and teratogenically-induced cel1 death (necrosis).

The author concluded from observations that were reported earlier by TuIsi

et al (1989) and in this thesis, that for the teratogens examined, no such

dist,inction could be nade between apoptosis and necrosis. A similar

conclusion was reached by Clarke (1990) in his extensive review of the

many publications dealing with developmental cell- death. In fact CIarke

(1990) concluded that Schweichel and Merkerrs (1973) cell types should be

accepted, but should include an additional subset of type l.

UnLÍke Schweichel and Merker (1973) who reported that one of the 3 types

of cel-L death coul-d be associated with different embryotoxins, the present

author noted fron examination of aIl 5 agents in this thesis that usually

type 1 and type 2 cell- death were present in the neural tube of embryos

treated with each of the teratogens. In type 1 cell death, condensation of

nucfeus and cytoplasm occurs, followed by cell fragnentation and

phagocytosis of these rennants Ieading to degradation in heterolysosomes.

This pattern was observed in the developing neural tube in association

wÍth exposure to all five agents exanined in this thesÍs, either alone or

in combination, and uas reported prevÍously in nervous system tissue

(Schweichel 1972). IVpe 2 cell- death was characberised by the fornatÍon of

autophagic vacuoles which probably play a najor role in cell destruction

Schweichel and Merker (1973) considered that prinary lysosomes probably

discharged their contents of hydrolytic enzynes into the autophagic

vacuoles. It is likeIy that soroe of the vacuoles containing cell remnants

iJ.lustrated in thÍs thesis were in fact aulophagic J.ysosones rather than

heterolysosnes, as higher nagnification of apparently healthy ceIIs showed

disintegration of cytoplasnic contents such as nitochondria, rough

11-30

endoplasmic reticulum and polyribosones. Sites of destruction were

f requentJ-y associated with what the present author believes were pri.nary

lysosomes, based on sinjLarities with structures identified as Iysosomes

by Ghadially (1988). It was generally diffÍcult for the author to

distinguish between types 1 and 2 cell death in pathological neuraÌ tissue

probably because the neural tube was not examined until 48 hours after

initial exposure to the teratogen, at which time much of the ceII debris

appeared as inclusions in other celIs. This difficulty is not surprising

since Schweichel and Merker (1973) stated that a definite norphological

distinction between types 1 and 2 was no longer possible fron the time

Íncl-usions were present. Features such as loss of junctional conplexes and

blebbing which indicate changes Ín the plasma membrane were also

frequently obsenred in the present study, associated with the presence of

inclusions. These abnormalities have been reported previously (Fox 1977b,

Peluso et al 1980) and were considered as a conponent of type 2 celt

death.

TVpe 3 ceIl death which consisted of non-lysosonal disÍntegration whereby

the cellular structures disintegrate into snal-Ler and snaller fragments

was rarely identified in pathological neural tube tissue in this thesis

except in salicylÍc acid-induced ceIl death. In addition to type 1 and/or

type 2, type 3 necrosis was observed in regions of the neuroepithelium

adjacent to the basal larnina, where cells appeared devoid of organelles,

ceLl nembranes were disrupted and only snall fragments of cytoplasn

appeared to renain. The presence of more than one of the 3 celI types

described by Schweichel and Merker (1973) fs not considered as unusual as

in his review CLarke (1990) observed thal on nar¡y occasions more than one

of these ceLL death types occurred in enbryonic tissue.

11-31

11.3b Mechanisms of disposaL of neuroepithelial cell debris.

Although a number of studies have reported on enbryonic cell deathr very

few have reported on the means by which enbryonic tissue disposes of the

remnants of physiological and teratogenically-induced cel1 death. A nunber

of disposal routes for pathological tissue have been observed in the

neural tube of enbryos i.n this thesis. CelI debris Iocated within the

neuroepitheliun was observed to be extruded into the ventricular lumen

through disrupted cell rnembranes of debris-filled blebs which protrude

into the lumen, as seen in zÍnc-deficient embryos. In addition, the

appearance of celI debris encl-osed in nvacuole-1Íken structures within the

Lunenal space suggesls that these might be whole blebs which had been

pinched off fron the apical neuroepithelial surface. Ïfhite evidence of

this mechanisn ü¡as illustrated in alcohol-salicylic acid-treated embryos

in chapter 10, the author observed vacuolated cell remnants Ín the

ventrÍcu]ar lumen of the neural tube of enbryos exposed to all teratogens

exanined Ín this thesis.

Loss of junctional cornplexes aLso appeared to provide an additional

mechanism by which neuroepithelial celI debrÍs may be extruded into the

ventricular lumen, and evidence of Lhis mechanisn was also observed in

chapter 10. Turner et aI (1990) have lÍnked the fornation of irregular

blebs to the dÍsruption of junct,ional complexes following exposure to Na

val proate.

At the basal end of the neuroepithelium exposure to aII fhe teratogens

exanined aIone, and in conbination, Ied to broken basal laminae in

affected embryos, through which neuroepÍthelial- ceLl debris was observed

to pass into the underlyíng mesenchyne. This was lllustrat'ed in this

thesis in embryos exposed to nÍcotine, salicylic acid and alcohol-

11-32

salicylic acid.

previous workers (Duband & Thiery 1982; OrShea & Liu 1987) have proposed

that the basal lamina plays a role in general architecture of the neural

tube, and that its disruption may underlie the failure of the neural tube

to close. The observations Ín chapter 7 however, where nicotine exposure

frequently led to the disruption of the basal lamina in embryos in which

lhe neural tubes had fused, tend to undermine this proposal. Instead, the

present author suggests that the basal lanina is disrupted as a result of

membrane sensitivity to teratogenic action and once brokent

neuroepithetiat cell debris passes into the mesenchyne where together with

mesenchynal cel] rennants, it may be disposed of via the developing blood

vesseLs. ceII debris was observed within these vessels in embryos exposed

to nicotine and to hypervitaminosis A. This is consistent with the

observation in hypervitaminosis A enbryos that the endothelial IÍnings of

the developing blood vessels were disrupted, allowing access to the

necrotic cell remnants.

A finat mechanÍsrn to be considered derives fron the observation that sone

cell debris was phagocylosed by surface ectodermal celIs(illustrated in

hyperviLaninosis A embryos in chapter J¡), and extruded onto the cranial

surface (observed in zinc-deficient enbryos in chapter 3). A previous

investigation by Manusek et aI (1969) denonstrafed that remnants of

epidernal celLs were extruded onto the body surface of chick embryos'

ExtrusÍon of celI debris via the surface ectoderm would explaÍn the

presence of blebs and apparently free malerial on the developing craniun

followÍng exposure to the teratogens Ín this thesis'

It would appear that a dlsllnction coufd also be nade as to the mechanisrn

of disposal between the two kinds of ceIl death. PhysioJ-ogicaL cell death

11-33

may be disposed of simply by extrusion into the ventricular lunen, as the

basal lamina was not observed to be broken in control enbryosr whereas

teratogenicalty-induced ceII debris Ís removed fron the neuroepithelium at

both apical and basal- ends. Hence the present author concludes that

following extensive teratogenically-induced cell death in the anterior

neural tube, ce11 debris located within t,he extraceLlular spaces of the

neuroepithelium migrate to either Lhe apical or basal- ends depending on

the proximity of these sites to cell- debris. At the ventricular end cell

debris is renoved either via blebs through exocytosis or by means of the

blebs pinching off into the ventricular lumen, or vj.a loss of junctional

conplexes, and passes frorn the anterior neural tube into the amníotic

fluid. At the basal lanina, breaks in this nenbrane possibly by rneans of a

direct effect of the teratogen, allow cell debris to be extruded into the

mesenchyme, where some may be phagocytosed by mesenchynal cel-ls and/or

taken up by developing blood vessels, or even phagocytosed by sunface

ectodermal cells and extruded from the embryo through these cells.

WhÍIe it was difficulL to distinguish between normal programned ceLl

death and pathological cel1 death in terms of pattenns of celluLar

disintegrati.on, the author felt nore confidenb in assuming one or other

type of cell death from the location of the affected cells in the waII of

the neural tube. The teratogenic agents were seen to produce an extension

of pCD areas vJhich are predoninantly associ.ated with areas along the

dorsal aspecb (the sites of fusion) of the neural tube. The sites observed

under the electron microscope conpty lJith bhose seen in control lDouse

enbryos following administrabion of Nite bLue sulphate dye (Sulik et al-

1988). In the present study, ultrastnucbural examination revealed that 1n

teratogenically exposed enbryos an extension of ceI1 death was heavily

concentrated in the forebrain and optic vesieles, which corresponded to

11-34

sites of concentrated Nile blue sLain in alcohol-treated nouse enbryos of

the equivalent age (Sulik et al 1988). According to Pexieder (1975) the

dye accunulates in cytolysosomes and phagosonesr structures which were

proninent in bhe present st,udy folLowing exposure to aIl of 5 agents in

the present study, and to a lesser extent in control neural tubes.

11.3c. Microvilli and neural teratogenesis.

ÏlhÍIe the assocÍation of blebs with ectoderm has already been considered,

another significant feature associated with these sites was observed by

the author at the ectodermal surface and neuroectoderm of 11.5 day

affected embryos regardless of whether the teratogens $,ere nutrient

deficiencÍes, excesses, or cytotoxic agents. The surface ecLoderm of 11.5

day embryos exposed to the agents exanined in this thesis denonstrated

consíderably more nÍcrovilIi than controls of the same age' and ofben

showed similar densities of microvilti to 10.5 day controls.

Microvilli are reported to increase the tofal absorptive area of the cell

which greatly increases its transcutaneous transport (Alberts et al 1989)'

an important function of the prenatal mannalian ectodern (Lind et aI 1972,

Rulter 1988). A recent report in our Iaboratory (Tulsi et al 1991) has

shown that, Ín rats, surface ectodernal nicroviLli diminish in numbers

durÍng prenatal Iife, an observation in accord with Holbrook and 0dland

(97Ð who reported a diminution of microvilli size and density in humans

as parturition approaches. Previous studies in this laboratory (Harding et

al 1988, Joschko et al 1989) have also reported extensive ectoderrnal

nlcrovilli in enbryos exposed to zinc deficiency alone, and conbined with

hypervitanonsis A. These workers also observed that when the rat enbryo is

exposed to the dleLary nanipulatÍons, usually a change in the denslty and

morpholory of the ectodernal nicrovilli of the affected enbryos reflects

11-3¡

the severity of dysnorphology of the neural tube and possibly other

organs. This association !J as confÍrned in this thesis. 0ther

investigators too have obser¡¡ed an increase in epithelial microvilli int'he prenatal rat under experimental conditions. Schupbach and Schroeder

(1986) found that, in experinentally induced cl-eft patate in the rat there

was a characteristic increase in the population of microvill-i in the

palatal epithelial cell-s of the niddle el-evation area. An abundance of

heterogenous microvilli have also been reported on malignant tumour cells

but their role remains unclear (!larhol ef aI 1982, Engstrand & England

1987).

HardÍng et aI (1988) speculated that the increase in microvillÍ reflected

an attempt by zÍnc-deficient embryonic cells to extract as much zinc as

possible fron the annj.otic fIuid. fn this thesis however, nutrit,ionaldeficiencies and excesses as weLl as cytotoxic agents alike led to a

relative nincreasen in surface ectodermal microvill-i in 11.5 day embryos

compared with sane age controls, but exhibited conparable densities to

10.5 day controls. These observations led the present author to propose

that' rat'her t,han reflecting an attempt by the enbryonic zinc deficientectodermal cells to extract as nuch zinc as possible fron the amniolic

fluid by apparently increasing the nunber of nicrovÍ11i, the nexcessivert

nunbers of nicrovilli conpared with sane age controls nay in fact be due

to interference by the zinc deficÍent state with the processes which

nornally lead to shedding of nicrovilli in older embryos as they become

nore reliant on placental than transcutaneous transfer of adequate

nutrition and oxygenation. Hence, failure of these ernbryos to lose

nicrovillÍ nay be a response of a cell populat,ion under stress which could

occur if yolk sac and placental developnent were interfered wÍth, as þ¡as

clearly observed Ín embryos exposed to aIcohol, nicotine and salicylic

1 1_36

acÍd, and may al-so occur in zinc deficient and hypervitaminosis A enbryost

aSnanyfailedtodemonslratechorio-allantoicfusion.

!Íhile their rnechanisns of formation and exact functional role rernain

unclear the densify of the ectodernal microviLri population may serve as a

faírIy reliable marker of the severity of underLying dysnorphologies.

1 1 .3d Neural tube dysmorphologr and cell death'

Since nany teratological investigations have demonstraled embryotoxic

insult during organogenesis often produced within a few hours or days' as

welL as obvious signs of cell necrosis in tissue destined to be

dysnorplrÍc, it wou.l-d be reasonable to attribute the maLfornations to this

cytotoxicity. scott (1g77) has noted that the severi'ty of cel-I death is

lÍnked with the enbryos ultinate fate in as much as it probably determines

whether an embryo develops normally or is growth retarded, nalformed or

even dies.Our Iaboratory (Record et aI 1985b, Harding et aI 1988t Joschko

et al 1989) has observed cellular necrosis at sites in zinc deficient

embryos associated w it,h abnornal- branchiaL arches and exencephaly' This

associatlon Ied Record et aI (1985b) to conclude bhat ceIIular necrosis

was largely responsible for the terata, as the foci of necrosis correlated

weIl with the anornalies observed'

The present aulhor also observed neural tube necrosis in enbryos where

the anterior neural tube had fatled to fuse, however the severity of cell

death in this region was not always linked with the degree of

dysnorpholory observed but could vary from noderate to severe in enbryos

that often showed severe exencephaly. Furtherroore, several inconsistent

observations in various chapters of this thesis led the author to consider

that necrosis may not be the orùy underlying factor in the failure of the

anterj.or neural tube to fuse. For exanple there $¡as a lack of

11_37

neuroepithelial necrosis in severely exencephalic hypervitamÍnosis A

enbryos in chapter 4 which was in contrast to severe neuroepithelial

necrosis in other exencephalic embryos such as those exposed to zinc

deficiency (chapter l) and salicylic acid (chapter 9). Hence whÍIe a

sinilar morphological defect of the neural tube occurred j.n enbryos

exposed fo these agents, reduced celL nunbers and necrosis occurred

predoninantly in the nesencþmaI cel1 layer in hypervitamÍnosis A-treated

embryos. In addition, it was observed in chapter 9 that open neural tubes

can occur at lower concentrations than celL necrosis. SalicyJ.ic acid led

to severely exencephalic embryos at 150 and 200 ug/nI but with litt1e

accompanying cell death, while at higher concentrations exencephaJ.y was

acconpanied by severe necrosis.

Other inconsistencies observed in this thesis which weaken the Iink

between necrosis and dysnorphology, at least of the developing anterion

neural tube, include the observation that nicotine-induced enbryos in

chapter f denonstrated severe neuroepithelial pathology. This however was

not accompanied by exencephaly, but rather the neural Lube had fused,

although matry of the enbryos were microcephalic, wÍth a visibly thinner

neuroepitheliun. Hence in this case necrosis appeared to lead to a reduced

nu¡nber of viable neuroepithelial cells, a probable cause of microcephaly.

Furthermore sone enbryos exposed to zinc defieiency and alcohol also

denonstrated fused cranial neural folds and were severely microcephalic

with associated neerosis in the neuroepithelium. These inconsistencies Ied

the author to have sone reservatÍon in at,tributing anterior neural tube

dysnorphology, particularly neural fold fusion defects, to neuroepithelial

cell necrosis as the underlying cause of this defect. These reservations

were reflected previously by Scott (.|977) who pointed out that, not all

ernbryonic necroses precede dysnorphologies, nor do all t,eratogens induce

11-38

necrosis. Hence the present author suggests that anomalies such as

nicrocephaly rather than exencephaly nay be associated w ifh

neuroepiLhelial ceII death and consequently fewer neural tube cells. It

is of interest thaù hypervitamÍnosis A, the only teratogen in thÍs study

that did not demonstrate embryos with extensive neuroepithelial cell-

death, also did not demonstrate any mÍcrocephaly but the affected embryos

were characterised by exencephaly, and Ioss of nesenchynal ceIIs.

It is not surprisÍng that an agent which destroys cells or reduces their

rate of proliferation couLd produce a teratogenic effect. The rate of cell

prol iferation has been effectively neasured by the i.ncorporation of 3H-

t,hymidine into DNA, and many studies have denonstrated a reduction in the

incorporation of labelted precursor in tissues observed to be nal-formed.

Kochhar (1968) noted fron exaninaLion of histological sections that t'he

mass of cells forming the palatal process vtas nuch snalfer than in

controls, and that 3H-thynidÍne autoradiographs showed a reduced nunber of

labelled cells in agreement with the snaller size. Similar reponts of a

reduction in the incorporation of labelled precursor into DNA in enbryos

observed to be malformed has been reported by Dreosti (1983). In contrast,

other groups have suggested that a direct correlation between the

Íneidence of enbryotoxicity and the anti-proliferative or cylotoxic effect

of a teratogen does not always exist (Fern 1964, Gibson & Becker 1968).

Furthermore, tissue-excess deformities such as polydactyly have been

induced by agents such as cytosine arabinoside, whÍch have been shown to

reduce ceII numbers (Scott et aI 197Ð.

since brain slze has been reported to be reduced by 8'9X109 ceLls

foLlowing exposure to ethanol (Brown et aL 1979) in enbryos denonstratlng

nfcrocepbaly, necrosis seens to be a Ilkely nechanism associated wfth this

11-39

anonal-y. It is also of interest that aII teratogens examined excepL

hypervitaminosis A demonstrated Íntellectual and behavioural deficits in

surviving offspring both in animals and humans (see individual

Introductions of chapters for appropriate references). Hence it is very

Iikely that neural tube necrosis might underlie nicrocephaly and

subsequent postnaLal deficiencies rather than exencephaly. These

observations point to cell death not being the mechanism by which neural

tubes fail to fuse and in some cases even to collapse. Instead the author

proposes that structwes such as the excessively large teratogenicaJ-1y-

induced blebs observed on the apical surface of the neuroepithelial cellst

includÍng tbe neural crest region (see alcohol and zinc chapters) where

fusion of the neural folds occurs, nay be involved in the productionof

this dysmorphic feabure. Simil-ar structures were observed in enbryos with

neural tube defects fol-lowing exposure to the anti-convulsant agent' Na

valproate (Turner et aI 1990). The author proposes that as the neural fold

cells proliferate and rise becoming closely apposed for fusion' the

ngeographyn of the site of fusion is so severely affected by the blebs

thal fusion cannot proceed. Furthermore, depending on how severely the

mesenchyne is affected by the teratogen this will determÍne how severely

the exencephalic neural folds will have collapsed onbo the surface

ectoderrn since the mesenchyne is considered the supportive layer of the

neural tube by some workers. In addition, disruption of the basal lamina

nay also contribute bo faiLure of the neunal tube to close as this

structure is considered inportant in rnorphogenetic shaping of the neural

tube (OtShea & Liu 1987). Although Turner el al (1990) reporled dÍsrupted

basal lanlna Ín embryos with neural tube defects following exposure to Na

valproate, it Ís urùikely that these observatÍons are linked' as results

obtained tn chapter J tend to refute this as nfcotine exposure frequently

11-40

Ied to the disruption of the basal lamina even though the neural tube had

fuse d.

1 1 .4 TeratogenesÍs and the human condition.

The nunber of agents confj.rmed as teratogens in humans is surprisingly

sr¡alL in contrast to a very extensive list in aninals (Shepard 1989). The

apparenl discrepancy between observations in experimental and clinical

studies can probably be attributed to a range of factors involved in the

inductÍon of congenital ano¡nalies which includes the Level at which

enbryos and fetuses are exposed to a particul-ar teratogen, and most

irnportantly, the precise period in norphogenesis when the agents are

adninisbered, as welL as the appropriate genetic susceptibility lo react.

It is the inabilily to accurately monÍtor features such as exposure times

and leveIs in humans that has cast, considerable doubt on the fenatogenic

potency of many agents whereas these fact,ors are readÍIy controlled under

experimental conditÍons. llhile extrapoJ-ation of animal studies bo humans

requires caution, it seens appropriate to rel-abe the observations

descrÍbed in this thesis to the human condition, particularly since the

enbryos gIere exanined followÍng neurogenesis on day 11.5¡ which is

equivalent to days 30-40 in humans. The present author hopes that

observations made in rats at t,his significant tine in gesbation may

provide further insight into the basis for some of the observations in

human offspring at term and postnalally.

It was stated in chapter 1 that around 509l of hunan pregnancies result Ín

embryonic and fetal Loss. ExanÍnatÍon of 11.5 day enbryos in this thesis

exposed to each teratogen alone or Ín conblnation frequently demonstrated

an absence of chorlo-alLantoic fusÍon, a necessary requirenent for

adequate placental formation and subsequent nutrition 1n fhe growing

11-41

fetus. Few aninal studies have denonstrated this anomaly, and hence

failure to form a placenta may weII underlie some of the spontaneous

abortions and fetaL losses reported to be associated with mabernaL zinc

deficiency, retinoids, alcohol and aspirin exposure in humans. It is aLso

likely that severe structuraL anomalies may also play an extensive role in

Low viability IeveIs in humans, as around 751 of strucburally abnormal

enbryos and fetuses never reach fhe viable stage. By exanining embryos at

mid-gestation it was shown that a large proportion of aninals exposed to

the teratogens in this thesis denonstrated abnor¡nalities such as cranial

neural tube dysnorphology which were often very severe. StudÍes in

animals whÍch examine fetuses at Lern generally show far few er

maLformations when exposed to the sane teratogens but often with

considerable evidence of resorptions. These observations tend to indicate

that abnormaliLies such as neural- tube defects mÍght reduce viability in

aninal studies and consequently also in humans. The exencephal-ic enbryos

often associated with zinc deficiency in this thesis nay be associated

with the obsen¡ation of anencephalic infants in regions of the world where

zinc deficiency is prevalenL, and in offspring of mothers who suffer fron

acrodernabÍtis enteropathica Ihe addit,ionaL observation that children in

areas of low dietary zinc intake were often slow learners (Pfeiffer &

Barnes 1981)r IIìâV be linked with the offen severe neuroepithelial ceII

loss observed Ín the cranial neural tubes of zinc deficient embryos.

Perhaps a similar association occurs in human offspring, however to the

authorts knowledge there are no studÍes to date in which the brains of

intellectually inpaired neonates or young children exposed to a zÍnc

deficient environment in utero have been examlned for structuraL or

biochemical- abnornali tie s.

Although the author also demonstrated that rat enbryos exposed to

11-42

hypervitaminosis A were severeJ-y exencephalic' so far despite the

widespread use of long term doses of vitamin A in fertile wonen at Ievels

around those used in the study descrÍbed in chapter 4, no epiderniological

studies have provided t,he data to quantitat'e the risk for major

malfornations followÍng in utero exposure. l^lhíte some cases have been

documented Lhat describe newborn infants with craniofacial abnormalities

and microcephaly (Mounoud et aI 1975, Stange et al 1978)' Íf the hunan

nervous system defect is as severe as that in rat enbryos the absence of

recorded cases nay be attributed to a lack of viability in humans. 0n the

other hand, ingestion of vitanÍn A at high leveIs, early in pregnancy

supposedly for the pronotion of health nay be overlooked as a possible

explanation of an abnormaf infant by epidemiologists and health carè

personnel and by the woman henself. To the authors knowledge, long terrn

intellectual and behavioural abnormalities have not yet been reported in

aninals or human offspring following exposure to excess vitamin A. An

absence of this abnormality would further support the authorrs contention

that this kind of postnatal impairnent may be linked wibh early

neuroepÍtheIial celI death, as hypervitaminosis A exposure ütas observed to

predoninantly affect the underlying nesenchyne, with onJ-y a mininaL effect

on the neuroepitheliun.

The author is also unaware of any cases of abnormalities induced by a

nutritional deficiency of zlnc and an excess of vltanln A having been

recorded in hunans. Nevertheless lhe severity of the interaction between

these agents on the developing neruous system both at the norphological

and nicroscopic Ievels at nid-gestation should alert wonen to bhe

potential hazards of Íngesting vitanin A at nfaddÍshn Ievels particularly

since nargÍnal zLnc deficiency may not be detected, and should both

sltuations arise durÍng pregnancy, the comblned effects of these agents in

animals were so devastating as to conclude

unl ikely in humans.

11-43

that fetal vÍability would be

The patterns of abnornalitÍes that constitute the human FAS or FAE have

generally been reported in a nouse rnodel, and the present observations in

rats in viLro have denonstrated many of these features including reduced

growth, microcephaly and craniofacial deficifg which the author feels

provides a suitable alternative nodel for the hunan FAS. The observation

of spontaneous abortion in wonen who ingest alcohol regularly during the

first trimester and late fetal deaths may be associated with the inabilÍty

of ernbryos to develop a viable placenta, a conclusion based on the

observaLion that in many affected embryos chorio-allantoic fusion had not

occurred. fn additÍon, this study also demonstrated growth retardaLÍon in

rat embryos at the line of neurogenesis that I¡ras comparable to Ìevels

observed in hunans. The average birthweight of chÍldren wÍth FAS is 20009,

a 33í reduction on median birthweight (AbeI 1984b).The embryos exposed to

600 mef aLcohol in this study, the equivalent of serum alcohol levels

reported in alcoholics, demonstrated a growth-retardation of 33-351

measured by crown-rump length and yolk sac dianeter at rnid-gestation.

Despite the association of the range of fetal abnornalities which

conslitute the FAS with heavy drinkers, binge drinking in hunans has been

cLassified as a npossiblert teratogen (Shepard 1986). The present author

considers that the observations in chapter 6 of this thesis strengthen the

argument for binge drinking to be classified as a definite humart neural

teratogen. Exposure to alcohol in this study $¡as effective onJ.y for 20

hours during neurogenesis, as the enbryos t{ere ne-oxygenated at fhis

time, thus brÍngÍng about the evaporation of ar¡r renaining alcohol in the

rnediu¡n. ThÍs time period is simllar to the tine that alcohol takes to be

11-44

metâbolised following a bout of binge drinking in humans, and was shown to

lead to ¡nany similar features including microcephaly and cranio-facial

defects usually associated with the FAS following long term exposure to

alcohol in mice and hunans examined at term.

Microcephaly has been confirned as a typical feature of FAS in humans and

animals, and was observed in many enbryos in chapter 6, along with severe

neuraL tube necrosj.s. These observations led the author to propose that

bhis abnormality nay result fron neuroepithelial cel1 death which Ieads to

cell loss and subsequent thinning of the neuroepithelium especially in the

forebrain region, and may underlie the long term intellectual and

behavioral disabilities so frequenfly reported in chjLdren of alcoholics.

Nicotine is considered to be one of the principal conponents of cigarette

snoke responsible for the well known deleterious effects of reduced

birthweight and embryonic and fetal Ioss during developnent. The

observations in chapter I clearly clemonstrate thaf nicotine is a nervous

systen teratogen which acts directly on enbryonic tissue to Índuce

craniofaciaL and neuraÌ tube dysmorpholory which was accompanied by severe

cranial- neural tube necrosls. Prevlous epideniological studies however

have been unable to deternine wlth certainty longlasting effects of in

utero exposure to snoking and nicotine other than growth retardation,

possibty because of the nunerous confounding variables tha! cannot be

controLled for Ín humans.

The well known dose-dependenb relationship of naternal suoking with

reduced birLhweight was reported by Simpson (1957) and subsequent,ly

confirmed by many others who have taken into account nany of tt¡e potential

confounding variables. This growth reduction was observed in rat enbryos

as early as day 11.5 of gestation and described in chapter f of this

11-45

thesis. Nicotine-induced fetal hypoxia has been att'ributed to the

impairment of utero-placental circulation caused by vasoconstricbion of

uterine vessels which has been proposed as a mechanisrn through whÍch

cigarette smoking nay cause fetal grow th retardation In the present study

both yolk sac vasculature and chorj.o-allantoic fusion were impaired or

absent in some embryos thus providing likely mechanisns for an hypoxÍc-

based growth retardabion and also for the increased spontaneous abortions

reporLed to be linked with maternal- snoking (Himnelberger et aI 1978,

Hemroinki et aI 1983). fncreased spontaneous abortions in pregnant heavy

smokers may explain why nicotine-exposed infants have nob shown

considerable overt dysmorphology at, birth. The present author contends

thaL severe central nervous abnormalities such as microcephaly and

anencephaly reported in sone studies (Naeye 1978, KeIsey et aI 1978, Evans

et al- 1979) may not be observed in nany offspring of mothers who smoked

heavily during pregnancy as these fetuses could not be sustained during

further development either direcbly because of the severity of neural tube

abnormalities or indirectly due to severe hypoxia. Hence the nicotine-

mediated enbryonic dysmorpholory exerted by this agent on the developing

nervous systen, together with cel-Iular and ultrastructural observations,

nay in fact be especially severe. These ce1Iul-ar and uftrastructuraL

abnormalities especially in the forebrain rnay underlie psychological and

intell-ectual- deficits which are apparent in humans (RantakalIio 1978r

Naeye & Peters 1984), with syroptons characteristic of minÍmal brain

dysfunction. Aninal studies have also cJ.early indicated nicotÍne as a

behavioural teralogen Hence the findings outlÍned demonstrate the risk to

the enbryo associated with intrauterine exposure to nicotine, and when

extrapolated to hunans, highlighb the need for women to exercise caution

with regard to snoking during pregnancy.

11-46

I'lhile aLcohol and nicotine have been shown to lead to growth retardation,

reduced viability and gross behavioural dysnorpholory indÍvidually the

observation that nearly 251 of pnegnant wonen both consume alcohol and

smoke cigarettes regularly (Coolidge 1984) led the author to investigate

just how severe the outco¡ne of these conbined activities might be on

developnent. The results of this study when extrapolated to lhe hurnan

condition suggesbs that the combined effects of nicoline and alcohol on

neuraf dysnorpholory and associated cellular necrosis are additive rather

than interactive, suggestÍng that, these agents exert their teratogenic

effects independently. In view of the extensive demonstration of

rnicrocephaly and neural tube cell death appearing rrhand in handn in many

affect,ed enbryos, the authors previous contention of a link between these

abnornalibÍes is strengthened. Furtherroore, it allows for the proposal

that cell death nay underlie at least some of the inteLlectual and

behavioural abnornalities reported in several epidemiological studies in

hunan offspring exposed to heavy drinking and snoking in utero. Martin et

aI (1977) have reported an interactive and deleterj.ous effect upon

learning in rats following exposure t.o nicotine and alcohol together,

which was not predicted when these two agents were administered

separately. Too few studies have been perforned to date to confirn this

findÍng of an interactÍon at the behavÍouraI Ievel- and its possibLe

inplications in relation to different, findings in aninals.

The reporl that, two-lhirds of pregnant wonen ingest aspirin during

pregnancy (Hill 1973, Forfar & Nelson 1973) is of considerable concern as

a nunber of reports have associated high analgesic inbake wit,h the

inductlon of congenital abnormalities such as CNS defects and other

anonalies in the offspring of sone pregnant l¡onen, but as is the case for

nany other teratogens there are sone reports which refute an assocj-atlon

between the use of these drugs in pregnancy and

11-)7

adverse effects in

offsprine (Shapiro et aI 1976, stone et aI 1976). Since the pathways for

salicylate netabofism are the same for humans and rabs (Levy & Leonards

1966), the author wouLd expect that an exLrapolation of observations nade

in rats to the hunan condibÍon is particularly iusLifÍable.

In the present study the cLear evidence shows that in rats, salicylic

acid, â Eetabolite of aspirin leads to cranial and neural tube

dysnorphology such as eye defects and exencephaly. DaLa pertaining to a

correlation between aspirin ingestion and unfavourable pregnancy outcome

has not been as clear in humans and very few reports of anencephalyr the

equivalent of exencephaly in rats, has been documented. This nay be

attributed to early embryonic loss of affected embryos as the condition is

not compatible with life, although hydrocephalus, a Lesser neural tube

defect which Ís generally not 1Ífe threatening has been reported (NeIson &

Forf ar 197 i. Increased f etal wastage vJas reported in women who

chronically ingested salicylates during pregnancy (CoIIins & Turner 1975¡

Turner & Collins 1975) and perinatal mortality was observed to be

associated with these low birthweight infants. Allhough these resul-ts are

contraindicated in sone studies which nay be confounded by a large range

of variables, the present studies woul-d indicate thab infants of wonen who

ingest salicylates are vuLnerable to congenital abnormalities including

central nervous systen defects partÍcularly in view of the observations

that rats and humans netabofise salicylate in a sinilar manner.

The presence of severe necrosj.s particularly in bhe forebraln may also

underlie the observatÍon that naternal aspirin Íngestion durlng the first

half of pregnancy was related to IQ and attention decrenents Ín 4 year old

children, even when structural abnornalities or lnbrauterine growth were

11-48

absent (Streissguth et aI 1987). Further investigations of fhis nature

need to be carried out as Klebanoff & Berendes (1988) showed that maternal

aspirin exposure actually increased IQ above the Ievels reponted in

unexposed children

In chapter 10 the author described the effects of conbining alcohol with

salicylic acid in rat enbryos j¡ vitro during neunogenesis. Vlhile t'he

results denonstrated that these agents had an additive effect on the

frequencies of all parameters examined, except for open neural tubes,

which appeared to be protected by the eombined treatment, to ühe authorts

knowledge epidemiological studies have not been performed t'hat

specifically examine the co¡nbined effect of alcohol and aspÍrin on hunan

offsprÍng. However several hunan studies have reported synergistic effects

of aspirin and alcohol on gastric mucosal irritation (Hayes 1981),

pancreatic duct permeabilit,y (Reber et aI 1979) and bleeding tine (Deykin

et aI 1982). These observations in humans, together with those reported in

this thesis suggest that concurrent alcohol and aspirin ingesbion may also

Iead lo avoidable deleterious effects in human febal tissue as well.

1 1 .5 Conclusions.

J¡ vivo studies Ín bhis thesis have considerably extended previous

observations of teratogen-induced effects of zÍ-nc deficiency and

hypervitanÍnosis A, particularly in relation to neural bube necrosis. -IIt

vitro studies which exposed enbryos to aLcohol, nicotine and salicylÍc

acid at the tine of neurogenesis have shown that all three agents act as

nervous sysLen teratogens and exert direct effects on the developfng

neural lube resulting in extensÍve neural bube dysnorphology and cel1

death.

Several other studies reported have shown for the first ti¡ne in rat

11-49

enbryos that naternal dietary zinc deprivation conbined with excess

vitamin A induces severe synergistic effects on neural tube dysnorphology

and ceII necrosis. In addition, aLcohol conbined wit,h nÍcotine has an

additive effect on dysnorpholory and neural tube necrosj-s suggesting that

these two leratogens may exert their effects through different mechanisms.

A similar pattern for general dysnorphology was observed in alcohol-

salicylic acid treated embryos, however the frequency of open neural tubes

appeared bo be ameLiorated by concurrent adnÍnistration of lhese agents,

whiLe the effects of alcohol and saficylic acid on neural tube ceII death

appeared to be synengistic. These interactive effects suggests that

al-cohol and salicylic acid may exert Lheir newotoxÍc effects through the

sane mechanism/s.

The exanination of neural tube cytotoxicity induced by each teratogen was

extended to include the patterns and location of celL death, organelles

affected, and mechanisns of removal of celL debris fron the

neuroepitheLiun. The lack of association between the severity of neural

dysnorphology and cell death in sone embryos, J-ed the aut,hor to propose

that necrotic cell death may be linked with nicocephaly and long tern

Íntellectual and behavioural deficits, rather than neural tube defects

such as exencephaly, as proposed by several previous workers. Furthermore,

it Ís suggested that exencephaly and open neural tubes observed in the

present studies and previously, may arise as a result of severe blebbing

at the sites of neural fold fusion and/or by other mechanisms yet to be

elucidated, while the disruption of the basal lamfna is not llkely to be

involved.

1 1- 50

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11-54

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alterations in the caudal neural tube of the delayed Splotch embryo. Dev.

Brain Res. 37 , 11-20 -

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foLLowing chronic prenataL treatnent of mice lrÍth tobacco smoke andethanol. BulI. Envir. Contam. Toxicol. 26, 813-819.Peterson, WF., Morese, KN. & Kattreiden, DF. (1 965) Snoking and

prematurity. A prelimÍnary report based on lhe study of 7,7 40 Caucasians.Obstet. Gynecol. 26, 775-779.Pexieder, T. (197Ð CeIl death in the roorphogenesis and teratogenesis of

the heart. Adv. Anat. tubryo]. CeLl BioI. 6-99.Pfeiffer, CC. & Barnes, B. (1981) Rote of zinc, nanganese, chronium, and

vitanin deficiencies in birth defects. Int. J. Environ. Stud. 17,43-56.Pfeiffen, CJ. & ÍIeÍbe1, J. (1973) The gastric mucosal- responses to

acetylsalicylic acid in Lhe ferret. An ul-trastructuraL study. An. J. Dig.Dis. 1 8, 834-846 .

Rainsford, I(D. & Brune, K (1978) Setective cytotoxlc actÍons of aspirinon parietal cells: A principal factor in the early slages of aspirininduced gastric danage. Arch. Toxicol. 40' 143-150.Randall, CL. & Ant,on, RF. (1984) Aspirin reduces alcohol-induced prenatal

rnortality and malformations in mice. A1cohol: Ctin. Exp. Res. 8' 513-515.Rantakillio, P. (1978) ReLationship of maternaL smoking to norbidity and

mortality of the chÍld up to bhe age of five. Acta. Paediatr. Scand. 67,621-631.Reber, HA, Mosley, JG. & Fox, JN. (1979) Effecùs of aspirin plus ethanol

on the pancreatic duct mucosal barrier. Surg. Forun J0, 414-416.Record, IR. ( 1986) PhD thesis. Dept. Anatony and Hislolory, University of

Adel aide.Record, IR., Dreosti, IE. & TuIsi, RS. (1985a) J¡ vitro developnent of

zinc deficient and replebe rat enbryos. Aust. J. Exp. BÍoI. Med. Sci.63r65-7 1 .Record, IR., Dreosti, IE., Tul si, RS. & Manuel, SJ. ( 1 986) Mat,ernal

netabolisn and teratogenesis in zinc-deficient ratg TeratologY 33' 311-317 .Record, IR., TuIsi, RS., Dreosti, IE. & Fraser, FJ. (1985b) Cellular

necrosis in zinc-deficient rat embryos. TeratologY 32t 397-405.Rutter, N. (1988) The i¡nmature skin. Brit. Med. BulI. 44, 957-970.ScLùuter, G. (97Ð Ul trastructural observations on ceII necrosls during

fornation of the neural tube in nouse embryos. Z. Anat. EntwÍckl-Gesch.

11-55

141 , 251-264.Schupbach, PM. & Schroeder, HE. (lgg0) Fat,e of unfused medial edge

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epithelialen ScheiteLleiste wahrendder Extrenitatenentwicklung beiRattenfeten. Z. Anat. Entwickl- Gesch 136, 192-203,SchweicheJ-, JU. & Merker, HJ. (t973) The morphology ofvarious types of

celL death in prenatal tissues. Teratolory 7 , 253-266.Scott, HJ. (1977) Ce]l death and reduced pro].Íferative rate. In: Handbook

of reratology. 11Iilson, JG. & Fraser, FD. (eds). plenun press, New york.pp 81-98.scott, !rrJ., Ritter, EJ. & lùilson, JG. (t975) studies on the induction of

polydactyly in rats with cytosine arabinoside. Dev. Bior. 15, 103-111.Shapiro, S., Monson, RR., Kaufman, DW., Siskind, V. et â1. , (1gT6)

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Abstra ct.

TuI si, RS., Harding, AJ., Joschko, MA. & Dreosti, IE. ( 1 991 ) A

11-56

quantitative and norphologica-L study of ectodermal microvilli in ten areasin the control and experimental prenataJ- rat. Teratolory ( in press).Turner, S., Sucheston, ME., DePhilip, RM. & PauIson, RB. (1990)

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teratogenic properties of ethanol and acetaldehyde in C57BL/6J nice:ïnplications f or the study of the fet,al alcohol syndrome. Terat,ology 27,231-243.Vf endelL-Smith, CP. & !üiI1iams, If,. ( 1984) BasÍc hunan enbryolory. pittman,London. pp 58-60.I,lerler, MM., Pober, BR. & Holmes, LB. ( 1985) snoking and pregnancy.

Teratology 32, 473-481.ÏlÍIson, JG. ( 1973) Environnent and Birth Defects. Academic Press, New

York.!lylIie, AH. (lggl) Ce]1 death: a new classifÍcation separating apoptosis

from necrosis. In: CelI death in biology and pathology. Bowen, fD. &Lockshin, RA. (eds) Chapnan & HaII, London pp 9-34.VIynter, J M., Ìlalsh, DA., Ïlebster, ÏJ S., McEwen, SE. & Lipson, AH. ( 1983)

Teratogenesis after acute alcohol exposure Ín cultured rat enbryos.Teratogen. Mutagen. Carcinogen. 3, 421-428.Yokoyama, A., Takakubo, F., Ueno, K., Igarishi, T. et âf., ( 1 984)

Teratogenicity of aspÍrin and its netabolÍte, salicy1lc acid, in cultwedrat embryos. Res. Conmun. Chem. Pathol. Pharmacol. 46. 77-91.

APPENDIX 1

PUBLTCATIONS AND ABSTRACTS

M.A. Joschko, I.E. Dreosti and R.S. Tulsi (1989) Zinc/vitamin a interactions and

teratogenesis in rats: A light and electron microscope study.

Nutrition Research, v. 9 (2), pp. 205–216, February 1989

NOTE: This publication is included in the print copy of the thesis

held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1016/S0271-5317(89)80007-7

M.A. Joschko, I.E. Dreosti and R.S. Tulsi (1991) The teratogenic effects of nicotine in

vitro in rats: A light and electron microscope study.

Neurotoxicology and Teratology, v. 13 (3), pp. 307–316, May–June 1991

NOTE: This publication is included in the print copy of the thesis

held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1016/0892-0362(91)90076-9

1

Teratolosy (Subnittecl)

THE TERATOGENIC EFFECTS OF SALICYLIC ACID ON TEE DEVELOPING

NENVOUS SYSTEM IN RATS IN VITRO.

Marion A. Joschkol rZ Ívor E. Dreostil and Ra¡n S. TuIsi2

lCSlnO (Ausbralia), Division Of Hr:nan Nutrition, Adelaide'

South AustralÍa' 5000

2Department of Anatorny and Hisüolory, University of Adelaide,

souLh Austraria' 5ooo

2 Tables, I Figures.

ABBREl/IATED TITLE: Salicylic acid and teratogenesÍs 1n rat embryos'

SBID PROOFS I0: Dr. IE. DreosliCSIRO (Australia)DÍvision of Human NutrÍtionAdel aideSouth Australia.5000.(08) 2241837.

2

ABSTRACT

JoScHKo, M.A.' I.E. DREoSTI and n.S. TIJLSI. The teratogenic effects of

salicytic acid on the deveJ'oping neryous system in raLs'

TERATOLOCY-------- 1 991 '

Aspiriningestioninhunansandanimal.shasbeenreçortedtoleadtoa

rangeofundesÍrableoutcomesincludingfetaldeath,growthreLardation

and congenital abnormalities. Rat ernbryos were cultured for 48 hours in

100.400 uglml of salicylic acid, a metabolite of aspirin, from days 9.5-

11,5 of gestation. When compared to growth in controL enbryos' a

signifÍcant dose-dependent decrease in cro}fn-rump lengths, sonite nurnbers

and yolk sac dianebers was observed. There was also a signifÍcant incnease

inovera]-ldysmorphologyincludinEeYelbranchialarchandheart

anomaLies and an absence of forelimb buds. The neural tubò was especÍalIy

vulnerableandhadfrequentlyfailedLoclose.Cellularand

ultrastructural exanination revealed extensive ceLl death in the

neuroepitheliun, with a Iesser effecb on the nesenchymal cells' Large'

condensed blebs projected into the ventricular lumen, and cell mernbranes

as well as the basal lanina were severely disrupted, with atl cyboplasmic

organelles affected in dying celLs. It is likely thab the extensive ceII

necrosis and blebbing in the developing neuroepithelium at the sÍte of

neural tube fusion may be involved in faÍIed neurulation' while necrosis

at other sites in the cranÍaI neuroepÍthelium nay be Iinked with

previouslyreportedintellectualandbehaviouralabnormalities.

3

INTRODUCTION

Administration of salícyJ-ate colnpounds has been shown in enbryonic rats

(Koshakji and SchuIert, '72., DePass and Weaver, , 82., Greenaway et al,

'82) and cats (Khera, rl6)t but not mÍce (Guy et aL, '86) to tead to a

reducbion in growth paraneters, while Turner and Collins (t75), unlike

Shapiro et al ('76), have reported reduced birthweighb and stillbirth

following aspÍrin ingestÍon during pregnancy in hurnans. SalÍcyJ.ate

compounds were also beratogenic Ín a number of animal studies leading to

nalformations of the central nervous systern (CNS), skeleton, viscera and

pal ate (I,larkany and Takacs, t59; McColl et al ,65; TrasJ- er, ,65i Greenaw ay

et aI; t82). Dysnorpholory of the CNS Ín rats included craniorachischisis,

exencephary and hindbrain def ects in v j.vo (vlarkany and Takacs, ,59i

GoLdrnan and Yakovac, t63; McGarrity et a1., tT8), as well as defective

cIosure of, t,he anterior neural tube, reduced ecLoderm, and rnesodermal

necrosis in vitro (l'lcGarrÍty et aI., t81i Greenaway et aI., t82).

Salicylate co¡npounds are readily hydrolysed to salicylic acid (Levy,

t78), which is considered to be the causative agent in aspirin

terafogenicity (Kirnnel et aI., '71; Koshakji and SchuIert, ,72). Ear1y

studies (Goldman and Yakovac, t63) concluded that the teratogenic action

of salicylate compounds occurred through naternally mediated netabolic

factors, however nore recent fluorimetric Lechniques (KimroeI et aI. , ,71)

and in vitro culture roethods (McGarrity et ar., tB1; Greenaway et ar.,t82) have denonstrated that t,his class of drug has a direct affect on the

rat enbryo in the absence of any maternal Ínfluence.

ÏlhÍLe the metabolic pathways for salÍcylate rnetabolisn are the same 1n

rats and humans (Levy and Leonards, t66) ¿ata pentalning to human studies

4

does not indicate as clear a correlation between aspirin ingestion and

congenitalmalformationsasoccurswithrats,alfhoughseveral

retrospective studies do suggest such a brend. Excess aspirin use has been

reported anong mothers of children wiLh cNS defects (Richards, '69, Nelson

and Forf ar, t7 1) as welI as with oral clef ts (Saxen, t75)' In contrast,

several prospeclive studies did not support increased congenilal

abnormalities following excess aspirin exposure during pregnancy (CoIIins

and Turner, t75, slone et â1., t76). White recent evidence has also

suggested that maternaL aspirin ingestion was significantly related to IQ

and attention decrements in exposed children, even at doses where

structural anomalies or j.ntrauterine growth were not affected (Pytkowicz-

streissguth, t87), Klebanoff and Berendes, (t88) however refuLed this

r eI ati onsh i P.

severaL proposafs have been made to explain lhe mechanisns of action of

aspirin on the cNS in utero. It has been suggested that the observed

dysnorphology may be mediated through disturbed prostaglandin synthesis

(Vane 171), or interference with oxidative phosphorylatÍon (Bost'ram et al

t64) or biosynthesis of nucleic acids and proteins (Janakadevi and Smith

'70).

The present in vitro study was performed to confirn previous observations

that salicylic acid has a direct affect on the developing embryonÍc

nervous system, ancl to study for the first time the effect of salicylic

acid on the uI trastructure of t,he nenvous system ab lhe tirne of

neurogenesis. It is hoped that the ultrastrucbural data may contribute to

a better understanding of the factors underlying neural dysnorpholgy in

the developing embrYo.

5

METHOD

A nirnal s

Virgin female Sprague-Dawley rats (180-230e) were housed overnight with

nales of the sarne strain The time of detection of spern in vaginal smears

was designated as day 0.5 of gestat,ion and animal s w ith posit,ive smears

were placed in cages with free access to food and waler.

fubryo culture and leratological screening.

On day 9,5 of gestation implantation sites were renoved from the uteri of

dams under diethyl ether anaesthesia and embryos were dissected free of

decidua, parietal yolk sacs and Reicherts membranes. Groups of three early

headfold enbryos were placed in 60 mL culture boLtles along with 3nls of

prewarned nedium and gassed with oxygen, nÍtrogen and carbon dioxide as

previously described by Record et aI, (t85a). Where appropriate, sal.icylic

acid was added to the rnedium at various concentrations ranging fron:100

ug,/mL to 400 uglmI, wtrich corresponds to the IeveIs known fo occur in

adult and fetal blood after ingestion of aspirin as an analgesic by

pregnant !üomen The bottles were rotated for 4B hours whereupon embryos

were renoved and examined under the dissecting microscope for evidence of

inpaired growth and dysmorphology. The embryos were then processed for

1Íght and electron microscopy as described previously (Joschko et al.,

'gg) .

Stafi stics.

Conlinuous growth variables such as crowrFrurnp Iength and sonite nu¡nbers

were analysed by standard analysis of variance. For discrete development

dat,a such as nu¡nber of enbryos with neural tube defects, a naximun

likelihood nethod was used, assuning the data to be binonially dÍstrÍbufed

6

and lhe deviance to be approximatety distributed as X2 on the appropriate

degrees of freedom (Baker and Nel-der' t78)'

RESULTS

The range of concentrations of salicylic acid used in the present study

for which abnormalÍties were observed was 100-400 ug/ml, however it was

diff j.cult to quantitatively assess embryonic grow th and dysmorpholory at

bhehighestlevelasdevelopmentwassoseverelyimpairedthatinmany

embryos individual structures were not identifiable' Enbryonic viability

determined by the presence of a heartbeat was not affecLed in any of the

experimental animals regard]ess of whet,her the enbryoS were growlh

retarded and/or abnormal at this time in deveLoprnent'

Day 11.5 erobryos cultured for 48 hours in salicylic acid showed

significanf grow th retardation conpared with controLs (Table 1)'

as determined by several growth Índices, which confirned previous findings

in vivo and invitro (yokoyama et a1., t84; Guy and swenerlon, t86)' There

lras a clear and significant dependence on dose for crown-runp l-ength

IF(3,60) = 131.0, P(0.001]' somite nunbers IF(3,60) =1J2'5t p(0'001Jr as

w eII as yolk sac dianeter IF13 ,6 o) = 32'7 ' p(0'0011'

(TabIe 1 near here)

A number of morphological parameters examÍned when the ernbryos were

cultured in salicytÍc acid showed a concentratÍon-related dysrnorpholry

(TabLe 2). Mosb embryos exposed to 100 ug/mI salicylic acÍd did not appear

to be affected nonphologicalLy except for inconptete rotation which was

observed in over 20f of the embrTos and which was often accompanied by a

Iack of chorio-allantoic fusion. As very few other abnormalities were

nI

observed at this concentration, these parameters are Iikely to be

representaLive of an induced developmental delay rather than

teratogenesis. There was a sharp increase in the incidence of open

anterior neural tubes at 200 ug/ mI or nore with rnosL enbryos affected

(Fig.1). The most frequently observed neural lube anomalies consisted of

interruption of neural tube closune, which either involved the cranial

region alone, or additionally involved the neural tube caudal lo the

cra ni um.

(lable 2 near here, Fig. 1 near here)

Craniofacial structures including eyes and branchial- arches were also

affected by salicyJ.ic acid exposure. Eye defects whích consÍsted of

compression and narrowing of the optic vesicles, and in some cases absence

of the optic pÌacodes, occurred only at higher concentrations and affected

up to 100t of treaLed embryos. Branchial arch defects which included the

absence of nandibular arches, or fusion of these structures to the

pericardiun, occurred in 411 of embryos at 100 uglnl. These anonalies are

represented later in developnent by cleft palate and cleft lip in embryos

foLlowÍng exposure to aspirín or saJ.icylic acid during organogenesis

(DePass and lleaver, t82; Yokoyana el aI., t84).

Cardiac abnornalities rose to 88f at 300 uglml salicylic acid, with many

enbryos demonsLrating primitive ntube-liken hearts, although the

perÍcardia were not affected, nor was contractility. YoLk sac circulafion

was also severely impaired inover 501 of embryos exposed to 200 ug/mI,

and in all animals al the higher concentration. The absence of forelinb

buds which occurred in all embryos exanined may also be indÍcative of

developnental delay.

I

SomeoftheteratologicatfeaturesobservedinwholeembryosinFigurel

were refÌlected in the histologicat sections (Fig' 2) of the cranial neural

tubes of salicylic acÍd-treated enbryos. TLre different appearance of the

sectÍons of cornparable Ievels in the craniun of control and salicylic

acid-treated animals in Figure 2, can be attrÍbuted to the compression of

the optic area and poor cephalic flexure in affected enbryos in t'he

forebrainandhindbrainareaSrespecbively.Inrepresentativeembryos

exposed to 300 ug/nl the neuraL folds had not fused and the neural tube

was severely distorted. The neuroepithelial walL was thin and there was a

l-argedegreeofcetlulardisorj.entation,withdark,irregulartissue

projecting into fhe ventricular Iumen of bhe neural tube' cells in the

neuroepitheliumandmesenchymer+erelessdenselypackedthanincontrol

enbryos, leading to targe extracelLular spaces' while large, dark

intracellular bodies !Iere also present within the neuroepithelíum'

Despite the severity of teratogenesis, nitotis did not appear to be

affected and niLotic figureswere visible at the lumenal nargin of the

neuroepithelium. The underlying mesenchyme contained ceIls which l¡ere

of ten closely apposed to the surface ectode¡'n whil-e most of the space in

this germ }ayer was occupied by large, abnormal, developing blood vessels.

(Fie.2 near here)

ultrastructural exanination of the anterior neural tube of affected

enbryos reveaÌed that the main affect of sal.icylic acid was on the

neuroepitheliun, and that the severity of necrosis was dose-dependent' At

100 ug/ml there was little evidence of dead or dying cells except in fhe

rostral part of the telencephalon which was consj'stent with normal

erobryonic development. Aü higher concentrations of salicylic acid (Fie' 3)

the thin neuroepithelial waI1 with its shrunken cells contained Iarge

9

extracellular spaces, and at the apical end condensed cyboplasmic blebs

pnojected into theventricufar Iumen. A comparison of this region in an

affecled embryo can be nade with a similar site in the control in Figune

3a.

The dark bodies observed previousJ-y at the cell-u-l-ar level appeared to be

membrane bound bodies or heterolysosomes containing a variety of dead cell

debris, within ot,her apparently healthy ceLls which appeared Lo have

phagocytosed these remnants. While the dead ceLl material contained

within these structures was often too condensed bo identify as individual

organelles, extracellular debris could be identified as degenerating

cytoplasmic organel-les including condensed or swollen mj.tochondria,

vesiculated rough endoplasmÍc retÍculum, and free ribososroes. Electron-

dense amcrphous structures which were frequently associated with sites of

necrosis may be primary lysosomes.

In regions of the neuroepithelium al the basaL end adjacent to bhe

mesenchyme, ceIl s w ere often sw oIIen, cell m embranes w ere severely

disrupted enabling t,he cytoplasrnic contents to leak from these cells into

extracellular spaces, and Ín sone cases into the nesenchyme through

disrupted regions of the neuroepithelial basal lamina.

DISCUSSION

This study demonstrates for the first Lime bhe uLtrastructural patholory

Índuced by salicylic acid, a metabolÍte of aspirin in the tissue of the

developÍng rat neural tube, and confirros and extends salicylic acid-

induced growth and norphological anonalies neported previously. The

malformations observed in embryos exposed to salicylic acfd 1n vitro

10

in this study demonstrated a dose-related dysmorphology, and were sinilar

Lo anonaLies reported in rats in vÍvo (Vlarkany and Takacs, r59; Kimmel et

aI., t71; McGarrity et al., t78) f olLowing aspirin administration. This

suggests thal salicylic acid exerts a teratogenic effect directly on the

devetoping embryo rather than through malernal mediation, and confirns

previous suggestions (Kinme1 eb aI. , '71i Koshakj i and Schu1ert, '72) that

this netabotite is a causative agent in aspirin teratogenesis. This

proposal is further strengthened by observations made by Yokoyana et al.,

lrgt¡) of differential effects of aspirin and its netabolite, whereby short

term exposure to salicyJ-ic acid 1ed to localised malforrnations such as

cleft Iip and curly or short tail, whereas aspirin appeared to induce

edenatous anonalies of the face, which was attributed by these workers fo

a decLine of systemic physiological- functions.

l¡hile initial doses used in rats in the present study were approximately

f ive times higher than human therapeutic level-s of aspi.rin' plasma or

serun salicyLic acid leveÌs at 6 and 12 hours post administration are

comparable in both species (Woodbury, '73), The doses that produced

malfornatÍons in vitro in Lhis report were lower than in some other

sLudies, which may be attributed to differences in experimental design

including the time, and route of drug adminislration, or differences in

the teratogenic activity of aspirin, its netabolites and othen salicylate

compounds used in the various studies.

The present observations also confirm previous report,s both in vivo

(l.Jarkany and Takacs, t59; Kimnel et al', r? 1; DePass and 1¡¡eaver, t62) and

in vÍtro (Greenaway et a).., t82; Yokoyama et âf., t84) that aspirin or its

derivatives led to gnowth retardatíon in a dose-dependent nanner. While

most sbudies have demonstrated this effect in rat fetuses at term, in

11

vitro studies performed during the cribical- period of organogenesis have

the advantage of demonstrafing bhe temporal relationship between aspirin

administration and growth reduction. These studies in rabs nay be of

particular reLevance and importance as sone hunan studies have implicated

aspirin as a causative agent for reduced birthweight (Turner and Collins,

'75).

I'lhiI e aspirin- induced teratogenesi s has been f irmly establ ished in

animals (!larkany and Takacs, t59i Goldnan and Yakovac, 163; Greenaway eb

aI., t82; Guy et a1., t86)' the resuLts from hurnan studies are not

consisLent. The variations in outcone in epideniological reponts may occur

because of differences in methodology and analyses between studies, as

well as confounding personal and envÍronnental factors. Despite these

inconsistencies, aspirin should not be underestimated as a possible

nervous system teratogen in hunans. Even if teratogenesÍs is not evident

when aspirin is administered alone, its effects nay be exacerbated if it

is ingested with conpounds which nodif! Íts metabolisn Agents such as the

comrnonly occurning food addibÍve, benzoic acid which increased the

concentration and persÍstence of salicylic acid in Lhe plasma of naternaL

rats, also increased the level of teratogenesis of the nervous system and

other ongans Ín term fetuses conpared with aspirin alone' when

administered during the critical period of neurogenesis (days 9-11)

(Kiromel et al., '71). The higher Ievels of dysroorphology reported Ín the

presenl study compared with those where aspirin or sodÍum salicylate were

used also indicate that salicylic acid is a nore pofent teratogen than

aspirin or ils sodluro salt.

The nost frequently observed neural tube ano¡nalles were related to the

fallure of the neural tube to fuse, either in the cranlal region aIone,

12

which has been reported previously foltowing cul-ture of enbryos in sodium

salicylabe (McGarrity et â1., t81; Greenaway et a1.r t82), or which

additionally involved the neuraL tube caudal to t'he cranium' a

precondition of craniorachischisis, observed in aspirin-treated aninals at

term (Warkany and Takacs, t5gi Kimmel et al' , \1)' It is likely that the

more severe neural- tube malfornatj.ons observed in the present sbudy may

not be compatible with viabÍlity at term. The absence of chorio-al-lantoÍc

fusion in many of the enbryos at higher concentratÍons also nakes survival

unlikely as the animal- in utero is dependenb on an active placenta for its

nutritional requirenents Iater in development (New, t78).

The cellul-ar necrosis described in the neural tube of these saLicylic

acid-cultured embryos did not appear to be assocj-ated wÍth progranmed cell

death or apoptosis, which is a normal consequence of differentiation

(schluter, t73i Geelen and Langman, 177), as necroLic cells induced by

saticylic acid were apparent throughout the entire newoepithelium instead

of being confined to the site of closure along the roid-dorsal aspect of

the anterior neural tube. It is likely that the severe ce]l shrinkage' and

Iarge condensed bl-ebs adjacent to the ventricular lurnen, together with the

extensive celL loss throughout the neuroepÍthelium, nay interfere with

fusion of the neural foIds. The severe neural tube dysnorphology

demonstrated in the present study was also sinilar to abnormalities

índuced by zinc and vitamin E deficiencies (Record et al', t85a; Harding

et aI., r8B) and vitamin A excesses (Joschko el aI., r89). Necrotic cel-ls

wer,e also apparent Ín the cranial neural tubes of embryos exposed to these

teratogens, and the neuroepitheliurn in salicylic acid-induced ernbryos in

the present study showed a sÍmilar patLern of cytotoxicÍty which could

aLso be rel-aled to the histologicat abnormalities observed by Greenaway et

13

aI. , ( '82) accomparving Na salicyi-ate-induced gross dysrnorphology.

Although cell death is not necessarily a prerequisite for a teratogenic

response, such events have been reported (Scobt, t77) to occur as a resull

of a wide range of Lreatnents. AspÍrin induced dysnorphology has been

linked previously to a cellul-ar abnornality in the adult gastric mucosa of

-several species (Hingson and lto, t73i Pf eiffer and Weibe] , t73i Rainsford

and Brune, tJB)r and also in embryonic hindlimb, as KIein eù al., (r81)

reported an unique pattern of cytotoxÍcity in tbe hindlimb bud where celI

death was localised to the preaxial nesoderm of bhis structure in rat

fetuses. The absence of severe cel1 death t,hroughout the open anterior

neural tube in some salicylic acid-treated exencephaJ-ic enbryos in the

present study suggests that severe neural fube necrosis, except in regÍons

adjacent to sites of neural Lube closure, may be an additional teratogenic

effect,, poSsÍbly unrelated to neural lube dysrnorpholory.

It has been reported in a recent epidemiological sbudy (PytkowÍcz-

Streissguth et al., t87) that maternal aspirin ingestion during the first

half of pregnancy was significantly related to low IQ scores, and

attention deficits in four year old children, while Butcher et al., (,72)

reported Ínpaired learning ability in rat offspring exposed to maternal

aspirin These observations led the authors to propose that the necrosis

observed at different sites in the anterion neural tube nay be

functionally different. The presence of severe cellular disruption and

blebbing along the dorsal nargins of the neural Lube al the site of neural

tube fusion may interfere with neuruLation, whÍIe necrosls and cell loss

throughout the renainÍng neuroepithelium nay be linked with intellectual

and behavioural abnormal ities.

14

Several possibilities exist to account for the nechanisns which underlie

neural tube dysmorpholory. As the yolk sac and vascular development w ere

inpaired Ín narry enbryos exposed to salicylic acid in cuJ.ture, similar to

the nicotine-induced effects seen previously (Joschko et, aI., t91), and

since the yolk sac is crÍtical for adequate nutrition and oxygen uptake at

this time in embryonic development (New, tf8)r some of the neural lube

pathology nay derive from an inability of the yolk sac to neet the demands

for'essential nutrients. However, it is unlikely that this is the primary

mode of saLicylic acid-induced teratogenesis in view of the dÍfferent

patterns of dysmorphology between nicotine and salicylic acid-induced

embryos. Alt,ernatively it is likely that salicylic acid exerts a direct

cytotoxic effect on cel-l- rnenbranes, which were observed to be seriously

disturbed at sone sites in the neuroepilheliun, particularly at sites

adjacent to the nesenchyne, where Lhe basal Ianina was also frequently

seen to be disrupled. It is at these sites that necrotic neuroepithelial

debris, which is not digested by heterolysosones located wiLhin apparently

healthy ceIIs, can be removed from the newoepithelium and extruded into

the nesenchyme. This method of removal of neuroepithelial debris has been

previously observed in nicotine-cultured embryos (Joschko et al., t91).

Prostaglandins have also been implicated Ín adequate fetal development

since they are known to have a large number of desirable effects some of

which occur at the celluIar level (Horrobin, t80).AspirÍn, like zinc

deficiency J.eads to an inhibÍtion of prostaglandin synthesis (Horrobin and

Cunnane, t80; Metz, t81), and since bot,h of these experimentally induced

conditions produces neural tube dysmorpholory such as exencephaly at tern

(Metz, t81 i Warkany and Takacs, t59), and open neural tubes accompanied by

severe cell death in the developing nervous system (Record et aI., t85;

15

Joschko et al., tB9), these observations could suggest a role for

prostaglandins in the adequate development and cl-osure of the neural tube.

The fÍndÍngs denonstrate that the developing rat embryo is at risk from

salicylic acid exposure, and that, when related to humans, maternal aspirin

ingestion nay lead to brain abnormalitÍes, impaired developnent and IQ

po sl na t,al Iy.

ACKNOITTLEDcEMENTS

The authors wish bo thank Dr. P. Baghursl of CSIRO., Division of Hunan

Nutribion for his st,atisbical advi.ce, Mr R. Murphy of AdelaÍde University

for photographic assistance, and Mr A. Partridge for typing this

manuscr ipt.

16

LITENATURE CTTED

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Generali sed LÍnear Interactive ModelL Íng. Num eri cal Al gorithm s Group'

Oxford.

Bostron, H., K. Berntsen and M.I\1.

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Whitehouse ( 1 96 4) Bioch e¡o ical

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Charnock, J.S., L.J. 0pit, and B.S. Hetzel (1962) An evaluation of the

effect of salicylate on oxidative phosphorylatÍon in rat-Iiver

mitochondria. Biochem. J., 83: 602-606.

Co1).ins, E, and G. Turner (197ù Maternal effects of regular salicylate

ingestion in pregnancy. the Lancet., 2: 335-337

DePass, L.R, and E.V. l.leaver (1982) Conparison of teratogenic effecls of

aspirin and hydroxyurea in the FÍscher 344 and Wistar strains. J. Toxicol.

Envj.ron. Hea1th., 10: 297-305.

Goldman, 4.S., and W.C. Yakovac (1963) The

teratogenicity by rnaternal immobilization in

Ther. , 142: 351 -357 .

Greenaway, J.C., T.H. Shepard., A.G. FanteI, and

Sodiu¡n salicylate teratogenÍcity in vitro. Teratology.,

II. Some effects on sul phate-35s

enhancenent of salicylate

the rat. J. Pharnacol. exP.

M. R. Juchau ( 1 982)

167 -17 1 .26

Guy, J.F., and M.E. Sucheston (1986) TeratogenÍc effects on the cD-1

mouse embryo exposed t,o concurrenl doses of ethanol and aspirin.

Teratolory., l4: 249-261.

17

Horrobin, D.F. ( 1 9BO) A biochenical basis for aI coholism and aI cohol-

induced damage including the fetal alcohol- syndrome and cirrhosis:

rnterference with essentÍar fatty acid and prostag)'andin metabolism. Med'

Hypotheses., 6 : 929-942-

Horrobin, D.F., and s.c. cunnane (1980) Inüeractions between zinc'

essential fatty acids and prostaglandins. Med' tlypotheses', 6: 277-296'

Janakidevi, K., and M.J.H. Smith (1970) Effects of salicylate on RNA

polynerase activity and on the Íncorporabion of orotic acid and thymidine

into the nucLeic acids of rat foebuses invitro' J' Pharm' Pharnac" 22z

249-252.

Joschko, M.4., f. E. Dreosti, and R' S'

interactions and teratogenesis in rals:a

study. Nutr. Res. , 9z 205-216.

TuI si ( 1 989 ) zinc/ vitan in A

lieht and eleclron mÍcroscoPe

Joschko, M.4., r. E. Dreosti, and R. S. Tul si ( 1 991 ) The teratogenic

effects of nicotine in vitro in rats: a light and electron microscope

study. Neurotoxicol. Teratol., 13: 307-316.

Khera, K.S. (19?6) Teratogerricity studies with methotrexate, aroinopterint

and acetylsal.icylic acid in doroestic cats. Teratologl. I 14: 21-28'

Kimroel, C.4., J.G.!IiIson, and H.J. Schunacher (197 1) St'udies on

roetabolisn and identif ication of the causative agent Ín aspirin

teratogenesis in rats. Teratology., 4: 15-24.

Klebanoff, M.4., and H.Ï1. Berendes (1988) Aspirin exposure during the

firsb 20 weeks of gestationand IQ atfouryears of age. TeratoIoBY.r 37:

249-255.

18

Ko shakj i, R. P. ' and

salicylabe beratolory

A.R. Schulerb (1972) Biochemical" mechanj-sms of

in the rat. Biochen. Pharmacol., 222 407-416.

Larsson,K.S., and M. Eriksson (1966) Salicylate induced fefal death and

malforrnations in two mouse strains. Acta Paediatrica Scandinavica., 55:

569-576.

Levy, C. (1978) Clinical pharmacokinetics of aspirin Pediatnics (Suppl).,

62: 86T -872.

Levy, G., and J.R. Leonards (1966) Absorption, metabol-isn and excretion

of salicylates. In: The SaIicyIates. M.J.H. Smith and P.K. Smith' eds.

InterscÍence, New York, Pp. 5-48.

MeLz, SA- (1981) Anti-inflammatory agenLs as inhibitors of

syntbesis in man. Vred. CLin. Nth. Arnerica. , 65: 713-7Tl .

prostagl andi n

GI obus, and S. Robi nso n ( 1 96 5) Effect of som e

rat fetus. Toxicol. Appl. Pharmacol.,on the developing

McG arrity, C. '

(Abstract).

Sarnani, NJ, and Beck, F. (1978) J. AnaL.' 127: 646.

McGarrity, C., Samani, NJ., Beck, F. and Gulamhusein, A. (1981) The

effect of sodium salicylate on the rat embrao in culture: an invitro nodel

for t,he morphological assessmenb of teratogeniciby. J. Anat., 1332 257-

269.

M., and J.0. Forfar (197 1) Association between

McCo11, J.D., M.

therapeutic agents

7 z 409-417.

Nel so n, M.

a dmi nÍ stered

Br. Med. J.,

dr ugs

fetus.durÍng pregnancy

1: 523-527.

and congenital abnormalities of the

19

New, D.4.1. (1 978) whore-embryo cuLture and bhe study of namnalian

embryos during organogenesis. BioI. Rev., 532 81-122.

Pytkowicz-Streissguth, 4., R.P. Treder., H.M. Barr., T.H. Shepard., V,I.A.

BIeyer., P.D. Sampson, and D.C. MarLin ('1987) Aspirin and acetaminophen

use by pregnant women and subsequent child IQ and attention decrements.

Teratolory., 35: 211-219.

Record, I.R., R.S. Tulsi., f.E. Dreosti, and F.J. Fraser (1985a) In vitro

deveropnent of zinc-deficient and replete rat embryos. Aust. J. exp. BÍot.

Med. Sci., 6 3: 6 5-7 1,

Record, I. R., R. S. Tul si., f. E. Dreosti,

necrosis in zÍnc-deficient rat enbryos.

and F.J. Fraser (1985b) Cettul_ar

Terabology., 32: 397-40l-.

Richards, ï.D.G. ( 1 96 9) Congenital maI forn ations

soc. Med., 23:

and env ironmental

218-225.influences in pregnancy. Brit. J. prev.

(197il Associations between oral crefts and drugs t,aken during

Int. J. EpidemÍo1., 4: 3T-44.

Saxen, L.

pregnancy.

Shapiro, S., R.R. Monson., D.Vl. Kaufman.,

D. Slone (1976). Perinatal nortatity and

aspirin taken during pregnancy. Lancet., 1:

V. Siskind., 0.P. Heinonen and

birth-weight Ín relation to

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sJ-one, D.v., 0. P. Heinonen., R.R. Monson., D.hr. Kaufman, and s. Shapiro

(1976) Aspirin and congenitar maLformalions. Lancel., 1: 1373-1375.

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( 1966).

20

TrasIer, D.G. (1965) AspirÍn-induced cleft Iip and other nalformations in

mice. Lancet. , 1: 606-607.

Turner, G., and E. Collins (19?5) Fefal effects of negular salicylate

ingestion in pregnancy. Lancet, 2z 338-339.

vane, J.R (1g71) Inhibition of prostaglandin synthesis as a nechanisrn of

action for aspirin like drugs. Nature New BioI. ' 2312 232-235,

llarkany, J., and Takacs, E (1959) Experimental production of congenital

rnarf ormations in rats by saticyrate poisoning' Am' J' Pat'hol-' ' 352 315-

331 .

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Pharmacological Basis of Therapeutics'

MacMillan, New York, PP 312'34U,

and antipyretics. In: The

L. S.Goodrnan and A. GiInan' eds.

21

TABLE 1

EFFECT OF IN VITRO S&TCYLTC ACID OI'I GRChITH OF RAT EMBRYOS ]N CULTIJRE.

TREATI"IENTlSalicyJ-ic Acid Concentration (uS/mI)

0 100 200 300

Total erobraos 15

Crown- rum p

leneth (nn)

No. of somÍtes

Yolk sacdiameter (rnn)

3.28t0.07

23.37t0.41

17

3.10r0.07

21 .66l¡0.51

15

2.60r0.08

17 .23¡1 .01

17

1 .45t0.08

7 .29t0.42

4.17ú.07 3.88¿0.10 3.56:10.13 3.02¿0.07

Values are means + SEM.

22

TABLE 2

EFFECT OF IN VITRO SALICYLIC ACID ON I'ORPHOLOGICAL DEVELOB'IENT IN RATS.

TREATMENT

SaIicy1ic acid concentration (ue/nI)o 100 200 300

SIGNIFICANCE OF

C0NCEXIITRATION

RH,ATED EFFECT (P)

Total enbryos 15 17 15 17

Ileuraldef ecf s

tube1 15( 100)

B( 53.3)

3( 20)

3Q0)

<0 .00 1

<0 .00 1

<<0 .00 1

<0 .00 1

Ele defecLs

Branchial archdef ect s

Heart defects

0( 0)

0( 0)

1(5.9)

0( 0)

0( 0)

0( 0)

0( 0)

0( 0)

17( 100)

1r(88.2)

7 (41 .2)

1 5( 88.2)

Absence off orel irnb buds

Incompletef1 exion

ï-mpairedfusion

Yolk saccircul ation

0(0) 1(5.9) r0(66.7) 17(100) <0.001

1(6.7) 4(23.Ð 15(100) 17(100),. <0.001

0(0) 4(23.5) 6(40) 17(1oo) <o.oo1

2(13.Ð 2(11.6) 8( 53.3) 17( 100) <0.001

lPercentages given in parentheses

23

FIG. 'I

FIG. 2

LEGENDS TO FIGURES

a. 11.5 day control enbryo. the neural foLds have fused along the

entire tength of the neural tube. b. 11.5 day exencephalic enbryo

exposed to 200 ug/mI salÍcylic acid. The cranial neural folds had

not fused except in the region between the forebrain vesicles

(arrow). The enbryo had not completed rotation, and only the

mandibu.l-ar arches ( 1) had developed. c. 11 .5 day embryos exposed to

300 uglml salicylic acid, with open neuraL tubes which extend fron

behind t,he forebrain vesicles (arrows) to the caudal end of the

enbryos. The neural tube was distorLed in the hindbrain region (ar

rowheads). Note thaü the allantois had not fused. d. YoIk sac of an

11.5 day control embryo with well developed blood vessels (arrows).

e. Abnormal 11.5 day yolk sac and embryo exposed to 200 uglml

salicylic acid with poorly developed blood vessels. Fb, forebrain;

Mb, midbrain; Hb, HÍndbrain; 0P, opbic placode; 0T, otic vesicle;

11213, branchial arches; H, heart; F, forelimb bud; YS, yolk sac.

Bars= 500um âr€i 1mm b,c,d.

a. Horizontal section through cranial neural tube of an 11.5 day

control embryo. b. Higher nagnification al a site near the arrolr Ín

a, showing mitotic figures (arrowheads) and closely packed cells in

the neuroepithelium. c. Horizontal seclion through the cranium of

an 11.5 day enbryo cultured in 300 ug/nJ. salicylic acid. The neural

folds have failed to fuse. The opbic vesicles are compressed and

the neuroepithelium is severely shrunken and thin Tt¡e cells in the

mesenchyme are confined Lo regions near the surface ectodern and

nost of the space is occupled by large, abnormal, developing blood

FIG.3

2\

vessers. d. Higher magnification of the neuroepitheLiun at a site

near the arnow Ín c, showing lange extracellurar spaces and

condensed, dead ceLl debris within the neuroepitherial walr and

projecting into the ventricular lumen (arrows). Numerous nitotic

figures are visible (arrowheads). Fb, forebraini Mb, midbrain; Hb,

hindbrain; N, neuroepitheliun; M, mesenchyne; E, surface ectoderm;

L, ventricular Iumen; NF, neural folds; 0, optic vesicle; bv, blood

vessel. Bars=50um.

a. Electron nicrograph of a section of the cranial portion of an

11.5 day controL embryo at the apicar end of the neuroepithelÍum,

with cytoplasmic projections (or blebs) (arrows) adjacent to the

ventricurar lunen. The ceLrs are in cLose proxinity and are

connected by junctional complexes at the apicar ends. b. A simirar

section of an 11.5 day ernbryo exposed to 300 ug/rn1 saricylic acid,

showing large irregular, condensed brebs (arrows) protruding into

the ventricular lunen, extensj.ve extracellular spaces and numerous

heteroJ.ysosomes (arrowheads). Note the nucreus has lost its normal

columnar orientation. c. A range of celt debris consisting of

cytoplasmic organelLes from the neuroepithelium of an 11.5 day

embtao cultured in 3oo uglmI salicylic acid. (The arrows indicate

sites where cell menbranes are disrupted). d. Neuroepitherial cell

debris consisting of dispersed cytoplasmic rÍbosornes and condensed

nitochondria, was extruded from cell-s at the basal end of the

neuroepithrium into the extracellurar natrix of the mesenchyne, at

sites where the basal Lamina has been disrupted (arrowhead). Arrows

indicate regions of severe celr menbrane disruption. N,

neuroepithelÍun; L, ventricular lumen; M, nesenchyne; BL, basal_

25

laminai ic, junctional complex; n' nucleusi m' miLochondria; vPr

vesiculated rough endoplasmic relÍculum; cr' cyboplasmic ribosones;

r, rough endopJ-asmic reticulum; Ly, lysosome' Bars= 4um' â' €i 1um'

b, c, d'

F i:i

I

l:ìq.z

f

t

I

a

I

Ì.¡

+

NF---

Fb

NF

t-_l

f'\. .a

;. 1

-¡

þv -.i:tr:a''

.ì

Li¿ I

ìji,tt\,.1I ¡rt.ì

c

lI

.- Itì

d

l'

Ii¡

I ix,z\L

\

Teratology 36, p151 , 1987 .

THE COMBINED EFFECTS OF ZINC DEFICIENCY AND HYPERVITAMINOSIS A ON THE

DEVELOPING CENTRAL NERVOUS SYSTEM IN RATS

M.A. Joschko *+ I.E. DreosEi,* and R.S. Tulsi+

'tCSIRO Division of Human NuEriEion, Adelaide, S'A' 5000*Department of Anatony and Histology, university of Adelaide' Adelaide 5000

Zinc an¿ vitamin A are known to play a role in cell membrane stabil-isation. Zinc is also required for the mobilísagion of vitamin A from Ehe

liver. Dietary manipulation of both nut.rients leads to a ÈeraEogenic effecEin -animals and subsequent impairrnenE of neural tube development' The

present Study invesEigated wheEher a teratogenic interaction occurs between

low maternal zinc "t"1n" and hypervttaminosis A during Pregnancy in raEs'

Electron microscopy was used t,o examine the individual and combined effectsof these t.".tt"r,is on norphology of the neural tube and its cellularmembrane conponenf.s.

Scanning electron nicroscopy of Il-day-old zinc-deficienE embryos ofEen

revealed extensively open neural tubes, as well aS anencephaly or micro-cephaly. Blebbing of the neuroepithelíurn r¡ras common. TreatmenE with excess

.rìlami" A led to õpen neural Eubes mainly confined to craníal regions. llanyembryos r¡Iere exencãphalic, wiEh neural folds severely evaginated and heavilybtebbed. When the treatments \¡Iere superimposed, teratology was enhanced'The neural tube was often extensively open and evaginated cranially.Exencephaly or anencephaly \./aS observed and special sensory organs \¡Iere

frequently affecEed.

Transrnission elect.ron micrographs of zinc deficient, 1l-day-old embryos

revealed severe neuroepichelial cell death, and disrupted cell- and míto-chondrial membranes in some living cells. The supporÈing mesenchynal cissuewas less affected. Ilypervitaninosis A exerted its teraEogenic assault nain-ly on Ehe nesenchyne, with a lesser insulE on neuroepithelium abutEing the

lumen. CeII death was common and severe swelling and breakdown of roiEochon-drial membranes vlas observed in many living cells. ConcurrenE EreaEmenf

with boEh Eeratogens showed extensive cell death in the neuroepiEhelium and

mesenchyme, and an enhanced degree of nembrane disrupEion.

This sEudy demonsEraEes E,he involvemenE of membrane damage and celldeath in the tera¡ogenesis associaEed with in uEero zínc deficiency and

hyperviEarninosis A. Superimposition of Ehe EreaEmenE.s leads Eo an additiverather than an inEeractive effecL on Èhe developing cent,ral nervous sysEem

making a common mechanism of action unlikely.

M.A. Joschko, I.E. Dreosti and R.S. Tulsi (1988) An in vitro study of the effects of

nicotine on the developing central nervous system in rats.

Journal of Anatomy, v. 161, pp. 259, 1988

NOTE: This publication is included in the print copy of the thesis

held in the University of Adelaide Library.

M.A. Joschko, I.E. Dreosti and R.S. Tulsi (1990) The teratogenic effects of nicotine in

the developing central nervous system in rats: an in vitro study, In: The Australian

Teratology Society, Inc. 7th Annual Scientific Meeting, December 6–8, 1989.

Teratology, v. 42 (3), pp. 329, September 1990

NOTE: This publication is included in the print copy of the thesis

held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1002/tera.1420420317