studies on The Genotoxic Effects of Indian Varieties of Silicate Dusts

( SUMMARY )

THESIS SUBMITTED FOR THE DEGREE

OF

Doctor of Philosophq IN

Zoologtj ( FACULTY OF LIFE SCIENCES )

OF

ALIGARH MUSLIM UNIVERSITY ALIGARH - 202002 (INDIA)

BY

MOHD. ASLAM M. Phil (Alig.)

INDUSTRIAL TOXICOLOGY RESEARCH CENTRE

LUCKNOW (INDIA) MARCH, 1990

Asbestos i s extensively used in i n d u s t r i a l and

commercial app l ica t ions because of i t s h e a t and f i r e

r e s i s t a n t p r o p e r t i e s . The consequences of developing

lung diseases in occupat ional ly exposed populat ion by

asbestos are well documented. Therefore, i ndus t r i e s are

exploring other mineral f ib res as s u b s t i t u t e s for

asbestos t o . meet out increas ing demand for cheap and

r e l i a b l e safer m a t e r i a l s . Among the promising

s u b s t i t u t e s , wo l l a s ton i t e received wide a t t e n t i o n and

i s under t r i a l in Ind ia . I t i s a nonmetalic na tu ra l ly

occurring monocalcium s i l i c a t e . In view of i t s

app l ica t ion p o t e n t i a l , i t i s e s s e n t i a l to evolve i t s

safe ty by experimental s t u d i e s .

In the p resen t s tudy, tox ico log ica l evaluat ion of

three v a r i e t i e s of wol l a s ton i t e of Indian o r i g i n ,

namely kemoli t A-60, keraolit-N and kemoli t ASB-3 were

ca r r i ed out . The r e s u l t s obtained from these s tud ies

were compared with c h r y s o t i l e , one of the most toxic

form of asbestos , in order to p r e d i c t t he i r r e l a t i v e

toxic p o t e n t i a l s . The dus t -e ry throcyte i n t e r ac t i on

suggests t ha t dust can damage membranes ind ica t ing i t s

po t en t i a l of in v i t r o t o x i c i t y . Apart from indica t ing

membrane tox ic i ty of dust, the p re sen t study on

erythrocyte toxicant in t e rac t ion a l so ind ica t e s t h a t

h'uman erythrocytes are an ideal t e s t system for

tox icologica l evaluat ion of p a r t i c u l a t e a i r p o l l u t a n t s .

The r e l a t i v e hemolytic a c t i v i t y of wol l a s ton i t e dusts

show t h a t kemolit A-60 has most hemolytic p o t e n t i a l

than the other two var ie t i e s . Although i t was l e s s

toxic than chrysoti le which i s in agreement with the

previous reports. The ly s i s of erythrocytes by these

dusts were found to be dose and time dependent.

Wollastonite dust induced l y s i s , unlike chrysot i le , i t

remained unaffected by EDTA treatment but was

s igni f icnal ty reduced by BSA and PVP. The poss ib le role

of calcium in the mechanism of wollastonite induced

hemolysis was not confirmed by EDTA treatment, vfliich

might be due to the inactivation of calcium released

from the surface of the dust. The decrease in the

ac t iv i ty of wol lastontie after BSA and PVP treatment

showed that the chemical interaction of the dust

surface with c e l l membrane may be involved. Acid,

a lkal i and water treatments a l so change the henolyt ic

potential which may be due to change in the structure

of the f ibres .

The induction of l i p i d peroxidation (LPO) in

erythrocyte and low speed supernatant of hemolysate by

Xemolit A-60 was higher in magnitude than the kemolit-N

and kemolit ASB-3. However, i t was less than t h a t of

c h r y s o t i l e . The less induction of LPO by wo l l a s ton i t e

may be due to the d i f ferences in chemical composition

of wo l l a s ton i t e than t h a t of c h r y s o t i l e . The induct ion

of peroxidat ive damage of polyunsaturated f a t t y acids

of erythrocytes suggests tha t involvement of f ree

r ad i ca l s in the t o x i c i t y caused by these dus t s . I t was

a l so observed tha t g lu ta th ione depleted during the

course of l i p i d peroxidat ion which ind ica te the

r e l a t i onsh ip between g lu ta th ione and LPO.

The scanning e lec t ron microscopic s tud ies showed

d i s t o r a t i o n and deformation of e ry th rocy tes . The

deformity appeared as invagination or c renat ion of

e ry th rocy tes . These s tud ie s have a l so suggested l e s s

t o x i c i t y of wo l l a s ton i t e as ccsipared t o c h r y s o t i l e . The

ciiange in the morphology of the ce l l may be due to the

abnormal inf lux of calcium or in t e rac t ion of calcium

from the surface of dus t with the s i a l i c acid of

erythrocyte membrane.

Although, i t i s well documented tha t e ry throcytes

are good model for tox ico log ica l evaluation of d u s t s .

Besides i t s s u i t a b i l i t y , in the absence of nucleus and

i n t r a c e l l u l a r o rgane l l e s , f indings on erythrocytes may

be d i f f i c u l t to ex t rapo la te to more organised c e l l

types l i k e macrophages and bepatocytes e t c . In view of

the well organised c e l l type, hepatocytes were taken as

a model for cy to tox ic i ty evlauat ion.

The re lease of l a c t a t e dehydrogenase (LDH) has

been considered as a convenient parameter for the

cy to toxic i ty assessment in hepatocytes and other wel l

organised c e l l s . Therefore, the effects of Indian

v a r i e t i e s of wo l l a s ton t i e on the LEH released from the

hepatocytes were measured. Results show tha t kemoli t A-

60 was more prone to re lease LEH than kemolit-N and

kemolit ASB-3 from hepa tocy tes . However, the r e l ea se of

th i s enzyme was less than in ch ryso t i l e t reatment .

As previously mentioned t h a t wol las ton i te dusts

induce peroxidation in human ery throcytes , hepatocytes

were a l so used for l i p i d peroxidat ion study to support

and know the s u i t a b i l i t y of the previous c e l l system.

The induction of l i p i d peroxidat ion by wo l l a s ton i t e

dusts shows i t s concentrat ion and time dependence. I t

has a l so been found t h a t mineral f i b r e s induced

membrane damage, enhanced l i p i d peroxidt ion, r e s u l t s in

c e l l u l a r depletion of g lu ta th ione (GSH) and subsequent

l y s i s of c e l l s . I t i s bel ieved tha t GSH p r o t e c t s

aga isn t LPO in metabol ica l ly ac t ive c e l l s . "this

indica tes towards the casual r e l a t i onsh ip between LPO

and c e l l u l a r l y s i s . Here a l s o , involvement of free

rad ica l s support the previous f indings with

e ry throcytes .

I t i s well documented tha t mammalian c e l l s have

evolved numerous mechanisms to prevent themselves in

tox ic i ty e l i c i t e d by xenobiotics and t h e i r by products

of c e l l u l a r metabolism. Glutathione, an endogenous

p ro tec t ive system, i s p resen t in high concentrat ion in

most mammalian ce l l s as reduced g lu ta th ione . I t acts as

a nucleophi l ic "scavenger" of numerous compounds and

the i r metabolties a l so ac t as cofactors in GSH

peroxidase-mediated des t ruc t ion of hydrogen peroxide.

In addi t ion to the t h i o l redox system, severa l

endogenous ant ioxidants a l so serve as p r o t e c t i v e agent

agains t oxidat ive damage. Among them, the l i p o p h i l i c

an t iox idant , OC - tocopherol i s the primary p ro t ec t i ve

factor aga ins t such a t tack on c e l l u l a r meitibranes. The

present study a l so demonstrated t h a t i>^-tocopherol

i n h i b i t LPO induced by mineral f i b r e s , v*iich ind ica tes

the termination of propagat ive process .

Since a balanced i n t r a c e l l u l a r t h i o l redox system

i s important for c e l l v i a b i l i t y , i t s r e l a t i o n s h i p with

LPO in the presence of C>d-tocopherol was a l s o

inves t iga ted . The prevention of LPO and subsequent

efflux of GSH a f t e r supplementing 0{-tocopherol was

observed. This shows t h a t addi t ion of 0^-tocopherol

i n h i b i t the LPO process via the donation of hydrogen

atom to the l i p i d r a d i c a l s forming the hydroperoxide

and a lcohol . Methionine, which i s known to s t imula te

GSH synthesis in l i v e r , when added to the incubation

medium resul ted in increase in the GSH leve l during the

t h i rd and subsequent hours of incubation in comparison

to f i r s t and second hours , while l i p i d peroxidat ion was

i n h i b i t e d . This a l so shows tha t GHS plays an important

r o l e in maintaining the i n t e g r i t y of the c e l l . I t has

been suggested tha t LPO does not proceed u n t i l OO -

tocopherol has been s u b s t a n t i a l l y depleted. In s p i t e of

the lack of 0^-tocopherol prevention of the efflux or

oxidat ion of GSH, most evidence based on inh ib i t i on of

LPO would suggest t ha t the e f f ec t of Od-tocopherol on

GSH biosynthesis i s secondary to i t s prevention of

increased oxidative potential within the c e l l . The

present results strongly support the hypothesis that

GSH def ic ient hepatocytes can undrgo LPO rapidly enough

to destroy the c e l l s .

For genotoxic potential human peripheral blood

lymphocytes were used. They offer an unique opportunity

to assess the inpact of environmental pollutants

d irect ly at the leve l of the workers as well as in

laboratory conditions. Lymphocytes provide a readily

avai lable source of c e l l s that can be stimulated to

undergo mitotic divis ion in culture. The mitot ical ly

dividing ce l l s can be arrested in metaphase with the

help of mitogen added in culture medium. These

metaphase c e l l s help in. studies on chromosomal

aberrations and s i s t e r chromatid exchange which are the

markers of genotoxicity.

The type and pe r s i s t ance of les ions induced in

DNA by various agents w i l l determine the degree of

cytogenetic damage observed a f t e r mitogen s t imula t ion .

Wollastonite t rea ted lymphocytes have shown increased

frequencies of chromosomal aber ra t ions and s i s t e r

chromatid exchanges. Among the wol las ton i t e samples

tes ted , kemolit A-60 was more prone to i,nduce

chromosomal aberra t ions and SCEs and kemoli t ASB-3 was

less t ox i c . In a conparat ive study with c h r y s o t i l e ,

kemolit A-60 was found to be l e s s tox ic than

ch ryso t i l e . The changes in t h e i r frequencies were

concentrat ion dependent. The main abnormali t ies were

chromatid and chromosome gaps. In the case of chemical

l es ions , the c e l l s have to be s t imulated with mitogen

to pass through S-phase. Only misrepaired les ions

during S-phase can be expressed as aber ra t ions and

s i s t e r chromatid exchanges. Most s i n g l e s t rand breaks

are repa i red immediately v ^ i l e the remaining f rac t ion

slowly r e s u l t s in the formation of gap followed by

f i l l i n g and l i g a t i o n . Double s t rand breaks are a l s o

repaired slowly. In such a case a double s t rand break

in GX chromosome should give r i s e t o the chromosome

type of aber ra t ions and in G , should give r i s e to the

chromatid type of a b e r r a t i o n s . So, i t i s c l e a r from

chromosomal aberf-ation s tud ies that^ the chromosomes

are damaged by mineral /^El|^^i^'"T56foj4s>:\and a f t e r

r e p l i c a t i o n .

8

S i s t e r chromatid exchange (SCE) process

presumably involves DNA breakage and reunion of the DNA

rep l ica t ion products a t the apparently homologus l o c i .

The increased frequencies of SCE a lso ind ica te the

involvement of DNA r e p l i c a t i o n product with the mineral

f ibres suspended in the cul ture medium, supports the

findings of chromosomal aberra t ions as discussed

e a r l i e r . The method 5-bromo-2-deoxyuridine (BrdU)-

label l ing for SCE analys is can a lso be used

simultaneously for inves t iga t ion of c e l l k i n e t i c s . The

d i s t r i b u t i o n of f i r s t (Mj_), second (M; ) , t h i r d and

subsequent (Mo.) mi tos i s in cul ture supplemented with

BrdU shows t h a t a l l the mineral f ib res t es ted did a l t e r

c e l l k ine t i c s which indica tes c e l l cycle delay.

Therefore, the c e l l cycle delay observed in

experimental cu l tu res may be mainly due to the

treatment of the mineral f i b r e s . The c e l l cycle delay,

in addit ion to the production of chromosomal

aberra t ions and SCEs, may represent another leve l of

b io logica l damage incurred due to the t reatment of

dus t . However, the induced delay may be cor re la ted to

the dose of mineral f ib res and was re la ted with the

degree of SCE induct ion . The enhanced SCE r a t e in

t rea ted c e l l suggests t ha t DNA lesions for SCE

formation are increased per genome with the increase

in treatment dose b u t no t in a s t r a i g h t way. I t may be

due to the random d i s t r i b u t i o n of mineral f i b re s in

9

culture vials or in suspension. So, SCE and chromosomal

aberrations may be coincidental with regard to their

induction and chromosomal localization. Both probably

reflect responses to lesions in TMA or to defeciencies

or errors in the repair of such lesions.

A significant increase in lipid peroxidation

(LPO) induced by wollas tonites in hximan erythrocytes

and isolated rat hepatocytes has been already

discussed. Few reports have provided evidences for a

relationship between lipid peroxidation and

genotoxicity. Further, it supports the possibility that

DNA reacts with either free radicals needed for lipid

peroxidation or intermediate and final products of the

LPO process. It is already reported in the literature

that superoxide radicals are involved in the genesis of

ciiromosomal aberrations and sister chromatid exchanges.

This was prevented by the addition of superoxide

dismutase to the culture medium. Recently, it has been

reported that the generation of singlet oxygen in the

medium creates problem to the cells. It increases the

duration of cell cycle which could be situated at the

level of G phase of the first mitotic division. These

investigations have provided evidences that free

radicals generated by wollastonite dusts may be one of

the factors directly or indirectly involved in causing

cJiromosomal aberrations and sister chromatid exchanges

and cell cycle delay.

10

The present s tud ies have, thus, es tabl ished the

fact tha t wol las toni te dus t smaples are both cy to toxic

and genotoxic though the i r t o x i c i t y was less in

conparison to ch ryso t i l e dust . Among the wo l l a s ton i t e

v a r i e t i e s , kemolit ASB-3 l i e s a t the bottom of the

ladder followed by kenol i t -N and kemolit A-60 in order

of increasing t o x i c i t y . The r e s u l t s reported in t h i s

d i s s e r t a t i o n c l ea r l y indicate t h a t free r a d i c a l s

generated by wol las ton i t e scimples may be one of the

factors responsible for dust induced t o x i c i t y . This

information may prove useful in advocating and

implementing p r o t e c t i v e measures to safeguard tJie

hea l th of p o t e n t i a l l y vulnerable persons a f t e r long

exposure.

studies on The Genotoxic Effects of Indian Varieties of Silicate Dusts

THESIS SUBMITTED FOR THE DEGREE OF

Doctor of Philosophy IN

Zoologq ( FACULTY OF LIFE SCIENCES )

OF

ALIGARH MUSLIM UNIVERSITY ALIGARH - 202002 (INDIA)

BY

MOHD. ASLAM M. Phil (AHg.)

INDUSTRIAL TOXICOLOGY RESEARCH CENTRE

LUCKNOW (INDIA) MARCH, 1990

T4010

To

MY SISTER -NUZHAT'

CERTIFICATE

This 1? to certify that the work embodied in this thesis entitled Studies on the Genotoxic Effects of Indian Varieties of Silicate Dusts has been carried out by Mr. Mohd. Asian under our supervision.

He has fulfilled the requirements for the degree of Doctor of Philosophy in Zoology of Aligarh Muslim University. Aligarh. regarding the nature and prescribed period of investigational work. The work included in this thesis has not been submitted for any other degree and unless otherwise stated. IS all original.

Prof.(Dr.) Ather H. Siddiqi Ph.D.(Alig.).Ph.D.(Purdue)

Dean Faculty of Life Sciences Aligarh Muslim University ALIGARH-202 002

Dr.(Mrs.) Qamar Rahman Ph.D.

Scientist-in-Charge Fibre Toxicity Division

Industrial Toxicology Research Centre. LUCKNOW-226 001

ACKNOWLEDGEMENTS

I t i s more t h a n k f u l t h a n m e l o d r a m a t i c t o l i k e n

Dr. Qamar Rahman t o " k i n d l y l i g h t " which g u i d e d me i n

t h e c o n d u c t i o n of t h i s work . Sine p r o v i d e d me w i t h

n e c e s s a r y m o o i o e u v r a b i l i t y and f reedom to work on t h e

p r o g r e s s . She t r i e d v e r y h a r d t o mould me from an

immature s t u d e n t i n t o a r e s e a r c h e r . My e x p r e s s i o n of

t h a n k s i s o n l y a f r a i l of my i n d e b t e d n e s s t o h e r .

I t i s my p r i v i l e g e t o e x p r e s s my p r o f o u n d s e n s e

of g r a t i t u d e t o P ro f . A t h e r H. S i d d i q i , Dean, F a c u l t y

of L i f e S c i e n c e s , A l i g a r h Muslim U n i v e r s i t y , A l i g a r h

f o r h i s g u i d a n c e , e v e r l a s t i n g e n c o u r a g e m e n t and

u n s t i n t e d s u p p o r t t h r o u g h o u t t h e c o u r s e of p r e s e n t

i n v e s t i g a t i o n .

I am d e e p l y i n d e b t e d and t h a n k f u l t o Dr. M.U.

Beg, A s s i s t a n t D i r e c t o r , I n d u s t r i a l T o x i c o l o g y R e s e a r c h

C e n t r e , Lucknow, f o r h i s u n t i r i n g h e l p , c o n s t a n t

e n c o u r a g e m e n t and a e s t h e t i c s u g g e s t i o n s .

My r e g a r d s and h o n o u r a r e due t o P r o f . P.K. Ray,

D i r e c t o r , I n d u s t r i a l T o x i c o l o g y R e s e a r c h C e n t r e ,

Lucknow and P ro f . Mumtaz A. Khan, Cha i rman , Depa r tmen t

of Zoology , A.M.U., A l i g a r h f o r p r o v i d i n g l a b o r a t o r y

f a c i l i t i e s and c o - o p e r a t i o n d u r i n g t h e p e ^ r s u i t of t h i s

work .

My s i n c e r e t h a n k s a r e due t o P r o f . Usman M.

Adhami, Dr . T a r i q M. Haqq i , Dr . Sher A l i , Dr . Waseem

F a r i d i and Dr. Anjum Ara who a s my t e a c h e r s i n t r o d u c e d

me i n t h e f a s c i n a t i n g f i e l d of g e n e t i c s .

I t h a s been a p r i v i l e g e t o c a r r y o u t t h i s work i n

an e n v i r o n m e n t f u l l of s m a r t , i n t e l l i g e n t and c o ­

o p e r a t i v e c o l l e a g u e s and f r i e n d s . Words would be

i n a d e q u a t e i n e x p r e s s i n g my t h a n k f u l n e s s f o r t h e i r k i n d

a s s i s t a n c e and f r i e n d l y d i s c u s s i o n s .

My s i n c e r e t h a n k s a r e a l s o due t o Mr. Mchd.

Ashqu in f o r h i s u n f l i n c h i n g c o - o p e r a t i o n i n t h e smooth

r u n n i n g of t h i s work .

I am g r a t e f u l t o Dr. K . J . Ahmad, A s s i s t a n t

D i r e c t o r and Dr. M. L a i , N a t i o n a l B o t a n i c a l R e s e a r c h

I n s t i t u t e , Lucknow f o r g i v i n g me t h e r e q u i r e d h e l p t o

c a r r y o u t s c a n n i n g e l e c t r o n m i c r o s c o p i c s t u d i e s . ojm.

I/also thankful to Mr. Amrit Lai and Mr. Umesh

Prasad for immaculate typing and Mr. Ram Lai for

photography. Financial support in the form of research

fellowship from Department of Environment, Government

of U.P. , Lucknow, is gratefully acknowledged.

Lastly, words fail to come by which I can express

my sincere regards and appreciation to my parents,

family members and relatives for their blessings, good

wishes, affection and constant encouragement which have

been the source of inspiration throughout the course of

present study. HdJ^MoM

(MOHD. ASLAM)

C O N T E N T S P a g e

1-30

1-23

FOREWORD

ABBREVIATIONS

REVIEW OF LITERATURE

A. Genetic Toxicology

- Introduction - Role of genetic toxicology in health

effects testing - History of genetic toxicology - Target of genotoxic agents in cells - Genotoxicity of silicate dusts

B. Wollastonite 23-30

- Introduction - Use - Hazardous properties of wollas tonite - Epidemiology - Experimental studies

- In vivo - In vitro

CHAPTER - I 3 1 - 5 1

STUDIES ON HEMOLYSIS AND L I P I D PEROXIDATION INDUCED BY INDIAN WOLLASTONITE DUSTS USING HUMAN ERYTHROCYTES: IN VITRO

CHAPTER - I I 5 2 - 7 5

STUDIES ON CYTOTOXIC EFFECTS OF INDIAN WOLL­ASTONITE DUSTS USING ISOLATED RAT HEPATOCY-T E S : IN VITRO

CHAPTER - I I I 7 6 - 9 1

STUDIES ON CYTOGENETIC EFFECTS OF INDIAN WOLLASTONITE DUSTS USING HUMAN LYMPHOCYTES: IN VITRO

SUMMARY AND CONCLUSION 92-101

BIBLIOGRAPHY 102-124 * * * * *

FOREWORD

Genes i s of t h e Work

D u s t s h a v e a lways been c o n s i d e r e d t o be an

u n w a r r a n t e d c o n s t i t u e n t of a i r , even though p r e s e n t i n

t r a c e a m o u n t s . However , w i t h i n c r e a s i n g i n d u s t r i a l and

a g r i c u l t u r a l p r o d u c t i o n , f u e l b u r n i n g and o t h e r t y p e s

of human a c t i v i t y , l a r g e amounts of v a r i o u s

p a r t i c u l a t e s a r e b e i n g i n t r o d u c e d i n t o t h e a i r i n h i g h

c o n c e n t r a t i o n s s o t h a t d u s t s h a v e come t o assume an

a l a r m i n g s t a t u s as one of t h e major a i r p o l l u t a n t s .

I n d e v e l o p i n g c o u n t r i e s l i k e I n d i a , whe re l a r g e

s c a l e u t i l i z a t i o n of m i n e r a l s o u r c e s a r e t h e s u r e

t h r u s t f o r t h e economic p r o g r e s s , p r o b l e m c o n c e r n i n g

t h e o c c u p a t i o n a l e x p o s u r e t o d u s t i s t h e m o s t a c u t e

h e a l t h h a z a r d . The d u s t a s s o c i a t e d d i s e a s e s h a v e b e e n

known s i n c e l o n g t i m e . Asthma, p u l m o n a r y f i b r o s i s a n d

b r o n c h o g e n i c c a r c i n o m a s a r e t h e main h e a l t h h a z a r d s

among p o p u l a t i o n s e x p o s e d t o a s b e s t o s a n d / o r o t h e r

m i n e r a l f i b r e s . Because of t h e w e l l documented

d e l e t e r i o u s e f f e c t s o f a s b e s t o s on t h e l u n g , i n d u s t r i e s

h a v e been d e v e l o p i n g o t h e r m i n e r a l f i b r e s a s a

s u b s t i t u t e f o r a s b e s t o s t o mee t an i n c r e a s i n g n e e d f o r

cheap and r e l i a b l e m a t e r i a l s . A l t h o u g h , t h e r e i s no

s u b s t i t u t e v ^ i c h c o u l d h a v e a l l t h e p r o p e r t i e s

a t t r i b u t e d t o a s b e s t o s , b u t s i n c e a l l p r o p e r t i e s a r e

n e v e r r e q u i r e d t o g e t h e r , s u b s t i t u t e s e x i s t f o r many

a p p l i c a t i o n s and v a r i o u s f r a c t i o n s of a s b e s t o s f i b r e s

l i

can be fabr icated by combination as s u b s t i t u t e

mater ia ls . Among the s u b s t i t u t e s , wol las toni te i s

extensively used, p r imar i ly , in the ceramic and the

p l a s t i c indus t r ies and how nowadays i t has a lso been

advocated for replacement of asbestos in ce i l i ng , f loor

t i l e s , e t c . Recently i t has a lso been used in bone

surgery ( O n o e j t a l . , 1988; Kitsuzi e t a l . , 1989).

Considering, i t s wide appl ica t ion , de ta i l ed research

and developmental s tudies on wol las toni te dust t o x i c i t y

are urgently needed to take su i t ab le an t i c ipa to ry

action to safeguard publ ic hea l t h . To achieve t h i s

objective indepth s tudies on the b io logica l r e a c t i v i t y

of wol las toni te dust in s h o r t term, _in v i t r o model

system were undertaken. In th i s d i s s e r t a t i o n , the

author used human erythrocytes and i so la ted r a t

hepatocytes for hemolytic and cytotoxic evaluat ions

while per iphera l human blood lymphocytes were used for

cytogene t i c s tud i e s .

Aims and Scope of the Investigation

As no r epo r t ex i s t s on the t ox i c i t y of Indian

va r i e t i e s of wo l l a s ton i t e , p resen t inves t iga t ions were

car r ied out to obtain informations regarding the

b io logica l r e a c t i v i t y of Indian wol las toni te mineral

f i b r e s . For achieving a t the preliminary idea, in

v i t r o studies on hemolysis of erythrocytes by following

i i i

the release of hemoglobin under i so tonic condit ions

could be an ideal parameter for comparative t ox i c i t y of

various dusts in terms of t h e i r r e l a t i v e capacity to

damage biomembranes. Therefore, t h i s parameter was

u t i l i z ed to screen the hemolytic potency of various

s i l i c a t e dus t s . Our s tudies ind ica te t h a t wo l l a s ton i t e

ddsts are less hemolytic to tha t of ch ryso t i l e

asbestos . To subs t an t i a t e the findings on the

hemolysis of erythrocytes , fur ther s tudies were

conducted with hepatocytes , which are considered to be

metabolically most ac t ive c e l l s , for cytotoxic

evaluation. The re lease of cytoplasmic enzyme ( i . e .

l ac ta te dehydrogenase) from the c e l l s , a convenient

parameter, was measured to screen the a l t e r a t i o n in

membrane permeabi l i ty .

Peroxidative damage of polyunsaturated f a t t y

acids of membrane l i p i d s has been considered to be a

major cause of membrane damage due to t o x i c i t y of

xenobiotics. Therefore, in the p resen t study l i p i d

peroxidation in human erythrocytes and i so la ted r a t

hepatocytes was a lso s tudied. Glutathione (reduced)

content was estimated in view of i t s p ro t ec t i ve ro l e in

oxidative damage leading to c e l l u l a r l y s i s . Further

s tudies on human per iphera l blood lymphocytes were

conducted for cytogenetic evaluations using chromosomal

aberra t ions and s i s t e r chromatid exchanges as genet ic

markers.

IV

Presentation of Thesis •

This dissertation starts with the review of

literature pertaining to the genotoxic studies carried

out so far with silicate dusts using different cell

systems, including proXaryotic and eukaryotic cells.

It also comprises of thorough literature search on

wollastonite, including epidemiological studies, animal

experimentation and in vitro studies. Review of

literature is followed by individual chapters dealing

with the specific aspects of the problem and detailed

description of the methodology involved. All the

chapters are subdivided into introduction, materials

and methods, results and discussion. Dissertation

concludes with summary, where the findings are

discussed in totality and their significance in the

understanding of the mechanism/causative factor

responsible for membrane damage is highlighted.

Conclusion

Industries are striving to explore other mineral

fibres as possible replacement of asbestos due to its

recognised health hazards. Among the promising

subtitutes, wollastonite has drawn great attention.

For the toxicological screening studies of mineral

fibres, short term i^ vitro model system are preferred

over _in vivo exper-imental animal model systems, which

s normally require a longer time per iod. In t h i

d i s s e r t a t i o n , wol las toni te samples are evaluated for

the i r toxicological po t en t i a l using d i f fe ren t _in v i t r o

ce l l systems.

The present f indings, thus, evidence the f ac t

t ha t the wol las toni te dusts are hemolytic, cytotoxic

and genotoxic. However, t he i r t o x i c i t y was less than

ch ryso t i l e , the most toxic form of asbes tos . Among the

wol las toni te samples, kemolit ASB-3 was the l e a s t

tox ic , followed by kemolit-N and kemolit A-6 0. The

information presented in th i s thes i s may be useful to

take preventive measures to safeguard the hea l th of

exposed workers.

ABBREVIATIONS

AAF

BP

BSA

CHO

IXih

EDTA

EGTA

F i g

GSH

Hb

HI

h

HCl

HGPRT

LPO

/ i g

Lt t i

ml

rrM

mg

man

MI

MDA

M

N

A c e t y l a m i n o f l u o r e n e

B e n z o p y r e n e

B o v i n e s e r u m a l b i a m i n

C h i n e s e h a m s t e r o v a r y

D e o x y r i b o n u c l e i c a c i d

E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d

E t h y l e n e g l y c o l b i s t e t r a a c e t i c a c i d

F i g u r e

G l u t a t h i o n e r e d u c e d

H e m o g l o b i n

H e m o l y t i c I n d e x

H o u r

H y d r o c h l o r i c a c i d

H y p o x a n t h i n e g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e

L i p i d p e r o x i d a t i o n

M i c r o g r a m

Lactate dehydrogenase

Milliliter

Millimole

Milligram

Minute

Mitotic Index

Malonaldehyde

Molar

Normal

NADH

NaOH

nrn

nmole

OD

PVP

PRI

RBC

RNA

rpm

RPMC

SCE

SEM

SHE

TEA

TCA

TDR H

UDS

UICC

w/v

Nicotinamide adenine dinucleotide redu­ced

Sodium hydroxide

Nanometer

Nanomole

Optical Density

Polyvinylpyrrolidone

Proliferative rate index

Red blood cell

Ribonucleic acid

Revolution per minute

Rat pleural mesothelial cell

Sister chromatid exchange

Scanning electron microscopy

Syrian hamster embryo

Thiobarbituric acid

Trichloroacetic acid

Tritiated thymidine

Unscheduled DNA synthesis

Union Internationale Contrele Cancer

Weigh t/volume

REVIEW OF LITERATURE

INTRODUCTION

The f i e l d of t o x i c o l o g y d e a l s w i t h t h e e f f e c t s of

a g e n t s on l i v i n g s y s t e m w i t h t h e p u r p o s e of d e f i n i n g

human h e a l t h e f f e c t s . Over t h e p a s t 20 y e a r s ,

t o x i c o l o g y h a s p r o v i d e d t h e p r i m a r y s o u r c e of d a t a on

h e a l t h e f f e c t s f o r c h e m i c a l s a f e t y e v a l u a t i o n s on

e x i s t i n g and new p r o d u c t s . W i t h o u t t h i s i n f o r m a t i o n ,

many p o t e n t i a l l y h a z a r d o u s c h e m i c a l s would h a v e been

i d e n t i f i e d o n l y t h r o u g h human e x p e r i e n c e .

Animal models a r e g e n e r a l l y u s e d a s human

s u r r o g a t e s i n t o x i c o l o g i c e v a l u a t i o n s . Animal models

a r e b i o a s s a y s t h a t i n v o l v e t h e u s e of a s p e c i e s

b e l i e v e d t o e j c h i b i t a t o x i c r e s p o n s e s i m i l a r t o t h a t o f

humans unde r e x p o s u r e c o n d i t i o n s r e l e v a n t t o human

e x p o s u r e s . Mammals a r e t h e mos t common t e s t o r g a n i s m s

e n p l o y e d . The c h o i c e of a mammalian s p e c i e s depends on

two f a c t o r s : i t s s u i t a b i l i t y as a model f o r t h e human

e x p e r i e n c e , t h a t i s , i t s c l o s e a n a t o m i c and p h y s i o l o g i c

r e s e m b l a n c e t o humans, and s e c o n d l y , economic

considerations, such as availability and cost of the

animals.

Toxicity assessment is clearly an inexact

science,and extrapolation of effects observed in animal

models exposed to chemicals under laboratory conditions

to human is tenous. Reliable estimates of human risk

also depend upon an understanding of the cellular and

molecular mechnaisms that underlie toxic phenomena

expressed at the whole organism level, and on adequate

methods of quantifying both the true observed dose of a

chemical (as well as the amount reaching the target

site) and the magnitude of the toxic response.

The Role of Genetic Toxicology in Health Effects Tes ting

It is well known that many chemicals of natural

or synthetic origin have adverse effects on living

organisms including man. Such toxic effects result in

more or less immediate reactions to the exposure.

However, classical toxicology has usually ignored those

effects which do not result in more or less immediate

reactions but in highly delayed responses. Genetic

toxicology identifies and analyzes the action of agents

with toxicity directed towards the hereditary

ccmponents of living system. Induction of genetic

damage may result in a very rapid response within the

exposed cells but expression can come late, and in the

genetic context late means generations after the

or ig ina l ' inc ident . Some toxicants damage the genet ic

complex a t concentrations a lso producing acute

nonspecific cy to toxic i ty and death. The primary

object ive of genetic toxicology, however, i s to de tec t

and analyse the hazard po ten t i a l of those agents t h a t

are highly spec i f i c for in te rac t ions with nucle ic acids

and produce a l t e r a t i o n s in genetic elements a t subtoxic

concentra t ions . Compound exposures for genet ic

toxicology s tudies range frc«n acute to chronic , thus

f a l l ing in to three major temporal subdivis ions of

toxicology t e s t i n g .

Agents tha t produce a l t e r a t i o n s in the nucleic

acids and associated ccxnponents a t subtoxic exposure

l eve l s , r e su l t i ng in modified h e r e d i t a r y c h a r a c t e r i s ­

t i c s or EWA inac t iva t ion , are c l a s s i f i e d as genotoxic

substances, v^ich usual ly have common chemical or

physical p roper t i es tha t f a c i l i t a t e i n t e r a c t i o n with

nucleic a c id s . Infact , the un ive r sa l i t y of the t a rge t

molecule i s the key to the d i s c i p l i n e of genet ic

toxicology.

History of Genetic Toxicolgy

Genetic toxicology evolved from the i n i t i a l

s tudies of gene mutabi l i ty demonstrated f i r s t by Muller

(1927) using r ad ia t ion , followed almost 20 years l a t e r

by Auerbach ejt a l . (1947) using chemicals. Both of

these inves t iga tors conducted the i r s tud ies using

submammalian spec ies , but within the next 20 years ,

genetic changes in animals induced by rad ia t ion and

chemicals were demonstrated by Cattanach (1966) and

Russell (19 51) a t Oak Ridge. This work created the

awareness t h a t some of the "hereditary" diseases

observed in human populations might be environmental in

or ig in (Bridges et. a l . , 1979 and Russel l , 1977). Proof

in the early 1940s t h a t deoxyribonucleic acid (r»JA) i s

the he red i t a ry ma te r i a l , and the subsequent e luc ida t ion

of i t s primary, secondary, and t e r t i a r y s t r uc tu r e by

Watson and Crick in the ear ly 1950s, opened up new

avenues of research in to the mechanisms of mutagenesis.

Genetic toxicology was recognized as a d i s c i p l i n e

in 1969 when the Environmental Mutagen Society was

founded under the leadership of Dr. Alexander

Hollaender and severa l other gene t i c i s t s who were

concerned about the p o t e n t i a l genetic inpact associa ted

with the p r o l i f e r a t i o n of man-made chemicals in the

environment* The concept of genetic toxicology was

c lear ly cons i s t en t with the intense concern for

environmental p ro t ec t i on t h a t prevai led a t t h a t time.

Occurring simultaneously with the growing concern

by gene t i c i s t s over environmental mutagens were the

reports from severa l independent groups of

inves t iga tors showing a c o r r e l a t i v e r e l a t i o n s h i p

between mammalian carcinogens and mutagens (Ames, 19 79;

Bridges, 19 76; Brusick, 1978; McCann e t a l . , 19 7 5;

Sugimura, e_t al. , 19 76) . Earlier attempts to support

such a correlation experimentally failed because of the

limitations inherent in the genetic assays available

(Brusick, 1977). The concept of mutagenic carcinogens

was however, revitalized, following the introduction of

procarcinogen activation using host-media ted (Gabridge

and Legator, 1969) and in vitro microsome activation

systems {Mailing, 19 71) developed from 19 69 through

1971. The fulfilment of this relationship was the

identification of mammalian cancer genes (oncogenes)

and they demonstrated the presence of mutations in the

activated forms of these genes (Reddy et al. , 1982).

Thus, genetic toxicology has played a dual role

in safety evaluation programmes. One of its functions

is the implementation of testing and risk assessment

methods to define the impact of genotoxic agents found

in the environment. The presence of these agents may

alter the integrity of the human gene pool. The second

function is the application of genetic methodologies in

the detection and mechanistic understanding of

carcinogenic chemicals. In this latter regard, genetic

toxicology has been applied as a front-line screen for

potential carcinogens.

Targets of Genotoxic Agents in Cells

Living organisms are divided into two large

categories These are prokaryotes like bacteria where

the genetic mater ia l , i . e . DNA,is free within the c e l l .

In eukaryotes the genetic information i s cased in to

compartments. Most of the genetic mater ia l is located

in the nucleus and i t i s arranged in complex molecular

s t r u c t u r e s , the chromosomes, cons i s t ing of DNA and

spec i f i c p ro t e in s . Additional genet ic information can

be found in other organelles l i k e mitochondria, the

s i t e of the ce l lu l a r r e sp i ra to ry a c t i v i t y . The genetic

information in prokaryotes and eukaryotes i s laid down

in the base sequence of DNA and consequently, DNA i s

the uniform and ubiquitous genetic mater ia l of a l l

c e l l u l a r organisms. However, when i t comes to genetic

damage, DNA i s not the only chemical t a r g e t . Specially

the highly complex s p a t i a l s t u r c t u r e of the genetic

elements in eukaryotes and a l s o the regular

d i s t r i b u t i o n of genetic elements during c e l l divis ion

requires addi t ional molecules and provides addi t ional

t a r g e t s : chromatin (ENA+protein) , spindle f ib re

apparatus, for the orderly d i s t r i b u t i o n of chromosome

during c e l l divis ion (pro te ins , membranes) and a l so

organel ls whose s t r u c t u r a l and funct ional i n t eg r i t y

require i n t a c t membranes {I»TA, p r o t e i n s , membranes) .

Each one of the d i f fe ren t cons t i tuen t s i s a primary

t a r g e t for disruption of genet ic i n t e g r i t y . In addit ion

to d i r e c t e f fec t s , agents can i n t e r f e r e with various

metabolic react ions l ike those involved in r ep l i ca t ion

of DNA, expression of genetic information in the

process of t ranscr ibing ENA base sequences, into DNA

sequences and f ina l ly in the formation of functional

and speci f ic p r o t e i n s . All t h i s i s ca l led

macromolecular synthesis which follows the genetic blue

p r i n t accurately. Faulty enzymes may commit mistakes in

macromolecular synthesis which wi l l r e s u l t in increased

genetic damage and mutation.

The type of chemical reac t ions found in genotoxic

or mutagenic agents are numerous but the most prcaninent

ones are a lky la t ions , deaminations and a lso rad ica l

react ions with DNA i t s e l f . Also important but not very

frequent are chemicals which are analogs of na tura l

compounds of DtsiA. They may be incorporated in to DNA

and may cause higher r a t e s of errors during r ep l i ca t i on

and t ranscr ip t ion than t h e i r na tura l coun te r -par t s .

Considerable knowledge has been elaborated on such

d i r e c t effects on ENA. Much less defined are chemical

reac t ions with other components of chromosomes and

chromatin s t r uc tu r e in general . The l e a s t understood

ef fec ts are those exerted ind i r ec t ly via in ter ference

with the enzymatic machinery of KsfA metabolism. And

f ina l ly , hardly anything i s ye t known about chemicals

which i n t e r a c t with membrane l i p i d and cause genet ic

effects via l i p id membranes as primary t a r g e t .

Genotoxlclty of S i l i c a t e Dusts

In recent years considerable progress has been

made in understanding the process of chemical

8

carcinogenesis . I t i s now c lear tha t chemicals can

cause or f a c i l i t a t e oncogenesis by more than one

mechanism (Berenblum, 1979). For example, many organic

chemicals are carcinogeneic only subsequent to t he i r

conversion, by ce l l u l a r enzymes, to e l e c t r o p h i l i c

species tha t can then reac t with c e l l u l a r nucleophiles

including various s i t e s on the ENA molecules (Lutz,

1979) . The modified DNA produced by these agents

var ies with the chemical proper t ies of the carcinogen

involved. I t i s widely believed t h a t such chemical-MA

in te rac t ions may be the i n i t i a l factor in chemical

carcinogenesis . The modified DMA i s thus considered

"dcimaged" and the chemicals which effect such reac t ions

are generally label led "genotoxins".

Many inves t iga t ions have been car r ied out on the

genotoxic ef fec ts of s i l i c a t e dusts with prokaryot ic

and eukaryotic c e l l systems. Few negative and in

con t r a s t many pos i t i ve responses with d i f f e r en t c e l l

systems are ava i lab le in l i t e r a t u r e . Here, the presen t

author has attempted to review the ava i lab le l i t e r a t u r e

on genotoxic aspect of s i l i c a t e dus t s .

Chamberlain and Tarmy (19 77) have used

prokaryotic c e l l s to study the genotoxic p o t e n t i a l of a

number of mineral f i b r e s , including UICC reference

s tandard samples, Canadian chryso t i l e B, amosite,

an thophyl l i t e , c roc ido l i t e , SFA chryso t i l e and two

samples of g lass f ib res , to produce back mutations in

Salmon el Ija typh imuriuin and Wh damage in E. coli WP 2

bacteria. They observed that above listed mineral

fibres are inactive in both systems even when the

metabolic activation system (rat liver homogenate

supernatant, S 9 fraction) was employed at the maximum

dust concentration of 500 /ug/plate. Similar results

with asbestos fibre were also noticed by Light and Wei

(198 0) on Salmonella typhimurium who reported that

neither UICC Canadian chrysotile B nor UICC crocidolite

have reverted to histidine dependence at the same

concentration as used by Chamberlain and Tarmy (1977).

In view of the above findings, the lack of positive

response could be, at least in part, ascribed to the

fact that asbestos fibres are not taken up by bacterial

cells, results in negative genotoxic response to

prokaryotic cells.

In contrast to bacterial cells, studies conducted

with cultured mammalian cells have showed that asbestos

and other mineral fibres are readily phagocytized by

lung macrophages (Miller, 1980) and Chinese hamster

ovary (CHO) cells (Huang, et al, 1978). Although it is

evidenced that mineral fibres are engulfed by mammalian

cells, they did not cause genotoxicity in those cells.

An initial study conducted by Kaplan ejt al. (1980)

with cultured rat pleural mesothelial cells showed that

UICC Rhodesian chrysotile A did not elevate the level

of sister chromatid exchange (SCE) to that of control

10

up to the concentration of 5 Aag/ml for a period of 32

hours.

A comparative study conducted with a number of

mineral fibres,including NIEHS reference samples, by

Hart jet al. (1980) showed that none of the mineral

fibres produced DNA damage. The damage was assayed by

a series of experiments including unscheduled DNA

synthesis in human fibroblasts, endonuclease sensitive

sites, single and double strand breaks by means of

alXaline and neutral sucrose gradient sedimentation,

respectively. Mossman e_t ajL. (1983) revealed no

evidence of E*IA strand breaks in cultured hamster

tracheal cells assayed by alkaline elution technique

treated with UICC chrysotile and crocidolite. Similar

negative results in human bronchial organ cultures

treated with UICC chrysotile, crocidolite and amosite

were obtained by Lechnaer e_t al. (198 3) in respect to

E»JA strand breaks. Although, these fibres were highly

cytotoxic to both cell types i.e. cultured hamster

tracheal cells and human bronchial organ cultures.

As discussed above asbestos has evoked negative

response, while many reports are available which

support the genotoxic nature of silicate mineral

fibres. Sincock and Seabright (1975) first

demonstrated that both chromatid and chromosomal

changes occurred in CHO cells exposed to SFA Canadian

chrysotile and UICC crocidolite. However, no

11

aberration was observed in cells exposed, under

identical condition (10/ug/ml), to glass fibres and

glass powder for 5 days before the cytogenetic studies.

Lavappa et a/l. (19 75) reported that a sample of

Rhodesian chrysotile A produced breaks in a dose-

dependent manner in Syrian hamster embryo cells When

applied at the concentration of 0.1 to 100 Aig/ml for 6-

9 hours. It is noteworthy that the same fibre

preparation when administered in vivo did not induce

either chromosomal aberrations (oral administration in

monkey) or micro nuclei (intraperitoneal administration

in mouse) in bone marrow cells, (Lavappa e_t al. , 1975).

The above findings are supported by Rita and Reddy

(1986) who observed that chronic oral administration

of chrysotile (Indian) failed to induce chromosomal

aberrations and abnormal sperms in mice.

In 19 78 Huang et al. have administered

crocidolite to Chinese hamster lung cells and observed

induction of chromosomal aberrations. They also

reported, a statistically significant, increase in the

numbr of 6-thioguanine resistant cells at the dose of 8

jag/ml, indicating forward mutation at the hypoxanthine

guanine phosphoribosyl transferase (HGPRT) locus. AT

the lower (5/ug/ml) or higher (100/ug/ml) concentra­

tions no mutation was noticed. In later study Huang

(19 79) obtained additional evidence that NIEHS

chrysotile, crocidolite and amosite were mutagenic to

HGPRT locus of Chinese hamster lung cells. The level

12

of induced mutations were low and the conclusions

s t rongly depend on the method of s t a t i s t i c a l analysis

employed. However, evidence for chromosomal changes in

cultured rodent c e l l s following administrat ion of

asbestos f ibres has been reported in other s tudies as

well (Sincock, 1977*, Price-Jones £ t a]^. , 1980).

Price-Jones e_t a_l. (1980) showed tha t ne i ther

UICC c roc ido l i t e nor Min-U-Sil s i l i c a induced SCE when

applied up to 15^g/ml of concentrat ion to CHO V79-4

ce l l s for 30 hours . Under the same protocol , potassium

chromate has eschibited pos i t i ve response as an inducer

of SCE a t the concentrat ion of 50(^g/ml. However,

higher doses of both the f ib res caused s t a t i s t i c a l l y

s ign i f i can t e levat ion of chromosomal aber ra t ions . The

above findings indica te t h a t the UICC asbestos f ibres

are e i the r negative or weak to produce SCE in mammalian

c e l l s .

Livingston ejt ^ . (1980) employing CHO c e l l s ,

reported tha t UICC c r o c i d o l i t e and amosite produce

marginal bu t s i g n i f i c a n t increase of SCE ra t e when the

c e l l s were exposed for 64 hours a t the f ib re

concentration of 10 /ug/ml. They a l so noticed t h a t

c roc ido l i t e was more e f fec t ive than the amosite and SCE

ra t e was predominantly to the la rger chromosomes.

Babu e^ al^. (1981) have used Indian va r i e ty of

chryso t i l e for i t s in v i t r o cytogenetic effects on

Chinese hamster ovary c e l l s . They exposed the ce l l s

13

to different concentrations of asbestos for 24 hours

and observed elevated rate of SCE even at the lowest

concentration (0.1 /ug/ml) . However, the linear

relationship between SCE and asbestos concentration was

not seen.

It has been reported by Sincock et al. (198 2)

that UICC crocidolite and SFA chrysotile alter the

morphology of cultured mouse 3T3 cells in a manner

similar to neoplastic transformation. DiPaolo et. al.

(198 2) have observed a very low level of morphological

transformation with UICC crocidolite, anthophyllite,

amosite and Candadian chrysotile B in Syrian hamster

embryo cells. In a later study Oshimura et al.

(1984) have reported that neither chrysotile nor

crocidolite asbestos induced any detectable gene

mutation at doses which caused cytotoxicity and

transformation in Syrian hamster embryo (SHE) cells.

Hesterberg et al. (1985) have found no significant

elevation of SCE frequency to that of control at any

concentration (0.5, 1.0 and 2.0^g/ml) of chrysotile

asbestos in SHE cells.

To gain a better understanding as to how asbestos

was causing transformation, anaphases were analyzed

after asbestos treatment. A very significant increase

in the incidence of anaphase abnormalities, such as

lagging chromosomes, sticky chromosomes and chromoscme

bridges were observed during the first division in SHE

cells after asbestos treatment (Hesterberg and Barrett,

14

1985). All of these abnormalities could r e s u l t in

missegregation of chromosomes which takes place by

d i r ec t in te rac t ion with the chromosomes, or

microtubules, microfilaments, and/or other s t r u c t u r a l

pro te ins of the spindle apparatus, since i t i s known

that asbestos binds to serum albumin and other

p r o t e i n s . In a l a t e r study worked out by Kelsey ejt a l .

(1986b) showed a l t e r a t i o n in CHO ploidy by fibrous

e r ion i t e and c roc i do l i t e asbestos while a s l i g h t

increase in SCE in synchronous CHO ce l l s were a l s o

reported by e r i o n i t e , unlike c roc ido l i t e or Min-U-Sil

quar tz . The cytogenet ic ef fec ts of e r i o n i t e , UICC

croc ido l i t e and UICC chryso t i l e on V-79 ce l l s showed a

s ign i f i can t reduction in d iploid ce l l s with an

accompanying increase in aneuploid and polyploid c e l l s

(Palekar £ t £ l . , 1987). An increase in aneuploidy with

a l l doses except the low dose, 10 yug/ml. Chromatid

aberrat ions were a l so observed in the c roc ido l i t e and

chrysot i le t r ea ted cul tures and were espec ia l ly

pronounced a t a dose of 100/ug/ml of ch ryso t i l e . This

suggests tha t e r i o n i t e was more reac t ive than the other

two minerals in producing aneuploidy. Thus,

aneuploidy, which r e s u l t s due to the missegregation

during the anaphase, appeared to be mechanist ical ly

important for the induction of transformation. The

clas togenic p o t e n t i a l of e r i o n i t e and c roc ido l i t e

asbestos f ibres was reduced by pre-exposing with

15

complete cul ture medium (with serum) in synchronous

Chinese hamster ovary f ib rob las t s (Kelsey _et a l . ,

1986b). Trosic et a l . (1986) have reported dose

dependent effect of asbestos when incubated with lung

f ib rob las t s of Chinese hamster V-79 c e l l s . I t seems

tha t the chemical composition of asbestos i s

responsible for the changes described ra the r than the

s i z e , shape and diameter of asbestos p a r t i c l e s . Palekar

e t a l . (1987) have observed t h a t the c las togenic effect

of e r i o n i t e was weaker but s t a t i s t i c a l l y s ign i f i can t a t

the dose of 100^g/ml in V-79 c e l l s . Scanning e lec t ron

microscopy showed t h a t chromosomes were frequently

entangled with, adhered to,and severed or pierced by

long and thin curv i l inear ch ryso t i l e with occasional

chrcxnatin f ib re threading over the p a r t l y severed

asbes tos , but much less frequent were found in

ch ryso t i l e t reated ce l l s (Wang e^ a^«» 1987). The

above findings were confirmed by transmission electron

microscopy (TEM) with ch ryso t i l e and c r o c i d o l i t e .

Different physicochemical p rope r t i e s of these f ib res

a lso play a role in the d i r e c t , i n t r i c a t e but d i f fe ren t

in te rac t ions with chromosomes as well as cytoplasm of

p l eu ra l mesothelial c e l l s (Wang e^ a^«» 1987).

With respect t o cultured human c e l l s , however,

the evidence for chromosomal ef fec ts i s cont radic tory .

Valerio et al^. (1980) have shown tha t freshly i so la ted

lymphocytes from normal adul t males and females undergo

chromosomal changes when exposd for 48 hours and 72

16

"hours with UICC Rhodesian chryso t i l e A a t 100 Aig/ml*

Increase in numerical a l t e r a t i ons a f t e r 48 hours and

chromatid and chromosome breaks a f te r 48 hours and 72

hours , were s t a t i s t i c a l l y s ign i f i can t . At 72 hours,

the chromatid breaks were most s i gn i f i c an t and the

number of numerical a l t e r a t i o n s were lower than 48

hours of exposure. Sincock e_t a_l. (198 2) showed t h a t

ne i ther cul tured human lymphoblasts nor human

f ib rob las t s have exhibi ted chromosomal aber ra t ions when

exposed to e i t he r UICC c roc ido l i t e , SFA ch ryso t i l e , or

glass f ibres ( lO^g/ml to 100 /ug/ml) a t 48 hours and 7 2

hours . The above findings are in con t ra s t to CHO ce l l s

exposed under the same regime showed numerous

chromnoscxnal changes. Both CHO and human c e l l l ines

are inh ib i ted to a s imi la r extent with respec t to r a t e

of growth by f i b r e preparat ion (Sincock et aJ.., 1982).

In 198 2, Lemaire ejt a^. have observed resumption of

c e l l p r o l i f e r a t i o n in human embryonic f ib rob las t s

exposed to c h r y s o t i l e B asbestos as evidenced by

elevated thymidine (TDR- H) incorporat ion. All the

tes ted UICC asbestos samples have a l t e r e d thymidine

incorporation in a dose dependent manner and to

d i f f e ren t maximum l e v e l s . Chemically modified

chryso t i l e samples, which re ta ined the f ibres

s t r u c t u r e , proved t h a t the chemical or physical surface

c h a r a c t e r i s t i c s of ch ryso t i l e are important in i t s

in te rac t ion with ceJLl membrane. Iji v i t r o cytogenetic

17

studies of amosite, chrysotile and crocidolite asbestos

have shown the induction of chromosonal abnormalities

and an elevated rate of SCE in mammalian cells (Rom ejt

al., 1983). In the later investigation, Lemaire e_t al.

(1986) have studied the effect of UICC chrysotile B

asbestos on human embryonic lung fibroblasts and

reported 3 to 5 fold increase of TDRrH incorporation in

comparison to control cultures at 48 hours of

incubation.

Moreover, continuous exposure of fibroblast

cultures to chrysotile (10 Ajg/ml) for longer periods

of time led to sustai"ned increase of T D R 3 H

incorporation and resiamption of cell proliferation. It

is suggested that increased TDR H incorporation is

directly related to the effectiveness of asbestos in

inhibiting the growth of lung fibroblasts. This

measurement of TDR^ H incorporation may r^resent a

sensitive means for rapid assessment of biological

activity of asbestos. The increase was dose dependent

with optimal doses as low as 10 g/ml at the cell

density of SXld^ fibroblasts per culture.

Kelsey et al . (1986a) have reported that neither

erionite nor crocidolite was mutagenic in human

lymphobias toid cell lines. However, erionite fibres

induced in vitro cytogenetic changes similar to thoe

caused by asbestiform mineral dusts. In vitro

cytogenetic studies conducted by Rahman e_t aJL. (1988 b)

using peripheral blood lymphocytes, showed increased

18

chromosomal aberra t ions and SCE frequencies aga ins t

con t ro l . The increase was dose dependent bu t not in a

l inear fashion. The p a t t e r n of induction of

cytogenetic changes by UICC reference sample and Indian

chryso t i l e was almost the same.

Rom £ t a l . (198 3) have reported for the f i r s t

time a marginal increase r a t e of SCE in c i r cu l a to ry

lymphocytes of exposed workers aga ins t the i r respec t ive

cont ro ls . The frequencies of SCE was increased with

the years of exposure. They have a lso observed a

s ign i f i can t associa t ion between SCE r a t e and smoking

af te r cont ro l l ing for years of asbestos exposure and

age. In vivo s tudies on pe r iphera l blood lymphocytes

of exposed workers, were a l s o conducted by Rahman et^

a l . (1988a). They r ^ o r t e d an elevated r a t e of SCE

and chromosomal aber ra t ions in asbestos exposed workers

in con5)arison to control populat ion and the frequency

was higher in smokers than non-smokers.

Co-carcinogenicity of Mineral Fibres

Attenpts were made to study the in t e rac t ion of

p a r t i c u l a t e s with genotoxic agents which may play an

important ro le in the induction of carcinogenesis .

Studies have shown t h a t NIEHS c h r y s o t i l e , if applied

p r i o r to ben2o(a)pyrene (BP), increased the leve l of

I»JA binding and enhancement of the cytotoxic e f fec ts of

19

the hydrocarbon to cul tured human f ib rob las t s (Daniel

e t al^. , 1980). Similar observations of enhancement of

BP-DNA binding by preincubation of the c e l l cu l tures

with NIEHS "intermediate" ch ryso t i l e in SHE c e l l s was

a l so observed by Eneanya ejt a_l. , 19 79. Later Di

Paolo ejt a l . (1982) reported the syne rg i s t i c effects

beteen asbestos f ib res and BP in respect to SHE c e l l

transformation. The simultaneous addi t ion of NIEHS

chryso t i l e and BP to normal human f ib rob las t s did not

r e s u l t in e i ther increased BP. metabolism (Daniel e_t

a l . , 1980; Hart e t a l . , 1980; Stephens ejt a l . , 1983),

a l t e r e d BP-raetabolite p r o f i l e s , cy to tox ic i ty , (Daniel

ejt a l . , 1980; Hart ejt aJ^. , 1980), BP-DNA binding leve ls

( H a r t £ t aj.. , 1980; Daniel ejt a^. , 1980; Stephens e_t

a l . , 1983), or in s u b s t a n t i a l l y a l t e r e d BP-

deoxyribonucleoside adducts p r o f i l e (Stephens et a l . ,

1983) . Chromosomal changes were observed a f t e r

treatment of r a t p l eu ra l mesothel ial c e l l s (RPMC) with

ch ryso t i l e and benzo-3,4-pyrene, the y i e l d of

metaphases was dose dependent and reached 59% a t a BP

dose of 2 Aig/ml and 21% a t 7 g /ml ch ryso t i l e f i b r e s .

The frequency decreased with fur ther increase in f i b re

concentrat ion (Jaurand et aJL., 1986). Achard et a l ,

(1987) observed t h a t a t t a p u l g i t e did not induce

s i gn i f i c an t increase in the number of SCEs per

metaphase a t the concentrat ion of 10 or 20 /ug/ml in r a t

p l eu ra l mesothelial c e l l s while c r o c i d o l i t e f ib res and

BP in combination enhanced SCEs s i n g i f i c a n t l y . They

20

a lso reported tha t BP so lub i l ized in acetone has higher

SCEs than in dime thylsulph oxide (DMSO). A c lear

assoc ia t ion between c iga re t t e smoking and asbestos

exposure in the s e n s i t i v i t y of lymphocytes to BP was

observed by Kelsey et a_l. (1986a), Who showed tha t the

lymphocytes i so la ted from asbestos exposed workers,

smoking c i g a r e t t e s , were s i g n i f i c a n t l y more suscept ib le

td the induction of SCE by in v i t r o exposure to BP than

of non-smokers. Reiss e_t ajL. (1983), have used l ive r

e p i t h e l i a l ce l l s to study the co-mutagenicity of

chryso t i l e asbestos and benzo( a) pyrene. They observed

t h a t exposure to chryso t i l e alone did not increase the

mutant incidence whereas benzo( a)pyrene (BP) was

mutagenic, simultaneous exposure of the c e l l s t o

ch ryso t i l e and BP resu l ted in an enhanced mutant

recovery ccaipared to exposure to e i ther of those

substances alone. However, ch ryso t i l e did not enhance

the mutagenicity of the carcinogen, N-methyl-N'-nitro-

N-nitrosoguanidine.

Recently, Kimizuka et aj.. (198 7) demonstrated a

co-carcinogenic effect of ch ryso t i l e asbestos and BP.

The in te rac t ion between a nitrosamine, cadmium and

c roc ido l i t e was observed in r a t s . Animals receiving

in t r a t r achea l i n s t i l l a t i o n of a l l components showed an

apparent synergism, though on the other hand the

combination of cadmium and c r o c i d o l i t e alone caused no

increase in bronchial carcinomas (Harrison and Heath,

21

1986). Fischer (1989) has used Chinese hamster

pe r i tonea l ce l l s and reported t h a t the asbestos f ibres

namely f ibres ch ryso t i l e , c roc ido l i t e and amosite as

well as glass f ibres a l so produced weak but s i gn i f i can t

increase in SCE r a t e s , while cadmium chloride applied

singly caused c lear dose-dependent e leva t ions . The

combination of c roc ido l i t e and chryso t i l e with the

three other mutagens did not r e s u l t in grea ter than

addi t ive SCE l e v e l s . He concluded tha t the genetic

events producing SCE are probably less important than

other genotoxic effects for the induction of h e r i t a b l e

changes leading to the appearance of cancer c e l l s .

Few studies on genotoxic effects of mineral

f ibres were carr ied out by means of a well es tabl ished

model based upon the measurement of unscheduled DNA

synthesis (UDS) in primary cu l tu re of i so la ted r a t

hepatocytes . Denizeau et. al^. (198 5a) have used 2-

acetylaminofluorene (AAF), a known genotoxic compound,

to study the co-carcinogenic a c t i v i t y of ch ryso t i l e .

They reported tha t ch ryso t i l e asbestos f ib res were

unable to modulate the 2-acetylaminofluorene induced

unscheduled DNA synthesis in primary cul tures of r a t

hepatocytes. The i n t r i n s i c capaci ty of na tura l

( a t t apu lg i t e and s e p i o l i t e ) and syn the t ic (xonot l i te )

mineral f ibres to induce unscheduled EftJA synthesis

(UDS) was f i r s t examined. None of the f ib re types

showed detectable UDS-eliciting a c t i v i t y . Also, the

poss ible modulation of the c e l l u l a r response to

22

genotoxic agents was examined by exposing the ce l l s to

mixtures of 2-acetylaminofluorene (0.05 and 0.25yug/ml)

and f ib res (1.0 and 10 «g/ml) . In these experiments,

the UES response was s ign i f i can t ly diminished in the

presece of xonot l i t e . This phenomenon may r e f l e c t

changes in the uptake and/or metabolism of AAF or may

r e s u l t from an inh ib i t ion of DNA r e p a i r processes, the

l a t t e r suggesting a possible co-carcinogenic p o t e n t i a l

for th i s synthet ic s i l i c a t e (Denizeau ejt aj.. , 1985b,c).

In addi t ion , Denizeau ejt aj^. Cl98 5d) have studied

genotoxici ty of ch ryso t i l e asbestos UICC B and

xonot l i t e in the presence of N, N-dimethylnitrosamine

(EMN), a genotoxic component of tobacco smoke, using

primary cu l tu re of r a t hepatocytes . No s ign i f i can t

change in thymidine incorporation in presence of DMN

was observed. I t was a lso not iced tha t ne i ther

ch ryso t i l e nor xonot l i te f ib res displayed s ign i f i can t

binding capacity ( a t the end of the incubation period

up to 5 hours ) , the r ad ioac t i v i t y was s t i l l associated

with the soluble phase. This behaviour contras ts with

t h a t of BP which, under the same condit ions, i s

s i gn i f i can t ly adsorbed onto c h r y s o t i l e . These findings

support the hypothesis tha t an increased s u s c e p t i b i l i t y

of asbestos exposed individuals to polyaromatic

hydrocarbon-induced cancer r e s u l t s from an enhanced

s e n s i t i v i t y to the induction of gene t ic damage ra ther

than to an asbestos induced d i f f e r e n t i a l c e l l u l a r

23

m e t a b o l i c c a p a c i t y . These s t u d i e s and o t h e r s may

i n d i c a t e t h a t m i n e r a l f i b r e s a c t a t t h e l e v e l of a c o -

c a r c i n o g e n o r p r o m o t e r w i t h r e s p e c t t o t h e i r o n c o g e n i c

e f f e c t s .

WOLLASTONITE

INTRODUCTION

W o l l a s t o n i t e i s a n o n m e t a l l i c n a t u r a l l y o c c u r r i n g

monocalcium s i l i c a t e . I t o c c u r s as g r a n u l a r

( a c i c u l a r ) o r f i b r o u s c r y s t a l s w i t h c h e m i c a l fo rmula

CaSiOo. The b a s i c s t r u c t u r e of t h i s m i n e r a l i s s i l i c o n

oxygen ( S i ^ P g ) , which i s s i m i l a r b u t n o t i d e n t i c a l t o

t h e p y r o x e n e c h a i n . The n a t u r a l c l e a v a g e of t h i s

m i n e r a l c a u s e s i t t o form f i b r o u s f r a g m e n t s . The

c l e a v a g e of mined m i n e r a l masses r e s u l t s i n a c i c u l a r o r

f i b r o u s v a r i e t i e s . The t h i c k n e s s o r t h i n n e s s of t h e

f i b r e i s d i r e c t l y r e l a t e d t o t h e i r c h e m i c a l cCTnposit ion

which v a r i e s f rom sample t o s a m p l e . The s t r u c t u r e of

i t i m p a r t s h i g h s t r e n g t h t o t h e m i n e r a l which i s most

i m p o r t a n t f o r many of i t s u s e s . P u r e w o l l a s t o n i t e

c o n t a i n s 48.3% CaO and 57.7% S i O ^ . The r e s e r v e s of

n a t u r a l l y o c c u r r i n g w o l l a s t o n i t e a r e c o n t i n u o u s l y b e i n g

e x p l o i t e d m a i n l y i n t h e U n i t e d S t a t e s and F i n l a n d ,

a l t h o u g h t h e m i n e r a l i s mined l e s s e x t e n s i v e l y i n

s e v e r a l o t h e r c o u n t r i e s l i k e , Mexico , Kenya, I n d i a ,

24

USSR etc. Synthetic varieties of wollastonite are also

available commercially. They are produced by

hydrothermal reaction between quartz and calcite.

Use

Because of its heat and fire resistant

properties, it is used primarily in the ceramic and

plastic industries. It has also been advocated as a

replacement for asbestos in ceiling,floor tiles and in

break lining, etc. Ono e_t a_l« (1988) have used it as

wollastonite-apatite fibrin mixture in bone defect

fillers. Recently, it has also been used in bone

surgery under the load bearing conditions (Kitsugi et

al.{1989).

Hazardous Properties of Wollastx>nite

Epidemiological studies

The attention paid to the hazardous nature of

naturally occurring mineral fibers other than asbestos

has been known to be justified. Various studies have

been undertaken on the health hazards of some types of

calcium silicate. Preliminary results from a cchort of

104 workers exposed to wollastonite in some instances,

among them four chest radiographs showed abnormalities

consistent with early pneumoconiosis. ( Boehlecke ejt

al., 1980). Subsequently, Hahon £t al.. (1980) found

25

induction of in ter feron by influenza vi rus in mammalian

ce l l cu l ture by wol l a s ton i t e , but the mineral per se

did not induce in te r fe ron . Shasby ejt a l . (19 79) found

an increased prevalence of chronic bronchi t i s among

wollas ton i te worlcers. Huuskonen et. al. (1980) "have

reported t h a t the prevalence of lung f ib ros i s in case

of limes tone-wollas ton i t e workers, exposed a t l e a s t for

10 years , was about one th i rd whereas the disease in

case of anthophyl l i te asbestos quarry workers (exposed

a t l e a s t for 5 years) was 50%. The r e su l t s from a

ccSiort of 19 2 male and 46 female workers exposed to

wol las toni te in a Finish limestone quarry showed no

indica t ion of increased malignancies (Huuskonen,

198 3a) . One malignant mesenchymal tumor of the

retroperitoneum, a neoplasm d i f f i c u l t to d is t inguish

from mesothelioma, was observed in a non smoking woman.

No r e l i a b l e information on smoking hab i t s of the cohort

or concentrat ions of wol l a s ton i t e encountered by these

workers was ava i l ab le . A c l i n i c a l study was a lso

car r ied out by Huuskonen ejt a l . (1983b) in workers

exposed to wol las ton i te for a t l e a s t 10 years

comprised a t o t a l of 46 men. Their sputum specimens

were normal v^ i l e spirometry and ni t rogen s ingle breath

t e s t indicated the p o s s i b i l i t y of small airways

d isease , three of the f i f t een non-smokers showed

chronic b ronch i t i s , fourteen s l i g h t lung f ib ros i s and

th i r t een s l i g h t b i l a t e r a l p l eu ra l thickening.

26

Experimental Studies

In vivo

Schepers ejt aJ.. (19 55) exposed guinea pigs, rats

and hamsters to two varieties of commercial hydrous

calcium silicate dusts (Ca;j SiO^ and Ca3 SiO^ ) by

irihalation route.Some animals were exposed for a

maximum of 36 months and some dust induced

peribronchiolar fibrosis and epithelization of

atelectatic alveoli was seen. In the period 1965 to

19 70, P.F. Holt at Reading University, U.K. (personal

communication) found no pathological effects of dust

when they undertook an inhalation study using guinea

pigs which were exposed to finely divided calcium

silicate powder prepared from insulation material.

Intrapleural implantation of four Canadian wollasto-

nites (8 um fibre length) of various grades into rats

resulted in smaller numbers of sarcomas (5 tumors in

20 rats, 2 tumors in 25 rats# 3 tumors in 21 rats and

no tumors in 24 rats) in conparison to crocidolite (10

tumors in 29 animals) (Stanton ^ jL. , 1977). Rodents

receiving no implants or pleural implants of non

fibrous dusts also developed neoplasms. Intraperitoneal

administration of wollastonite (20 mg, 5%) did not

cause tumors in rats although very low amounts (0.05-

0.5 mg) of various types of asbestos led to tumor

incidence of 20 to 8 0% (Pott £t al. , 1987). In vivo

biological reactivity of three respirable samples of

27

calcium silicate was compared with chrysotile and

titanium dioxide in rat lung using numerical,

cytological and biochemical parameters (Richards e_t

a_l. , 1981). Differential dose dependent effects were

produced by composites, which were all more reactive

than titanium dioxide initially although after

clearance many of the induced alterations by two

composites were practically reversible (Richards ejt

al. , 1981). Bolton e_t a^. (1986) have also examined the

effects of respirable dust from three commercially

produced calcium silicates in experimental rats by long

term inhalation and injection techniques. No

discernible effects were found on the length of

survival of the animals as conpared to controls after

one year of exposure to a dust cloud of 10 mg/m . No

pulmonary lesions were found that appeared associated

with the inhalation of calcium silicate per se.

In vitro

Wollastonite f ibres of various dimensions and

sources have been evaluated in a number of iri v i t r o

s tudies to assess the i r cytotoxic p o t e n t i a l as conpared

to asbestos and non f ibrous , " ine r t " p a r t i c l e s

(reviewed in lARC Monographs, 1987). Potts e_t a l .

(1978) have shown the hemolytic a c t i v i t y of

wol las ton i te on r a t erythrocytes i ^ v i t r o which may be

28

corre la ted i t s f ibrogenic l ty _ln vivo. Wright e_t a l .

(1980) have found t h a t three separa te samples of

calcium s i l i c a t e produced marked hemolysis with sheep

erythrocytes and they were a l so cytotoxic towards P388

D c e l l s , a permanent c e l l l i n e of transformed mouse

ce l l s with many ch a rac t e r i s t i c s of macrophages. Hunt e t

a l . (1981) have tested the b io log ica l r e a c t i v i t y of

three smaples of calcium s i l i c a t e s (A-C) J^ v i t r o

using r abb i t erythrocytes and a lveolar macrophages and

have compared with t ha t of ch ryso t i l e and titanium

dioxide. All ccroposites were l ess reac t ive than

ch ryso t i l e and more than t i tanium dioxide . The order of

r e a c t i v i t y of each individual composite was d i f fe ren t

in the t e s t system. Like ch ryso t i l e , composite-A was

highly hemolytic and i t s p o t e n t i a l increased upon

sonicat ion. Heat treatment a l so produced a l t e r a t i o n s in

the i r hemolytic p o t e n t i a l . Skaug and Gylseth (198 3)

have reported tha t synthet ic calcium s i l i c a t e s induced

higher hemolysis in comparison to na tura l ones, the

differences are accentuated by weak u l t r a son ica t ion of

the minerals . Calcium chela tors l i k e EDTA and EGTA did

not change hemolytic a c t i v i t y , while an increased

hemolysis was observed vAien calcium (30 mM) was added

in the incubation medium. In addi t ion , wol las toni te was

found to be far less hemolytic in red blood ce l l s than

asbestos (Hefner and Gehring, 1975; Vallyathan e t a l . ,

1984).

29

Calcium silicates (A-C) tested by Hunt e_t al.

(1981) were equally active in reducing alveolar

macrophage viability and like chrysotile, calcium

silicate B interfered with collagen deposition in lung

fibroblast cultures. Skaug e_t aj.. (1984) have used

peritoneal macrophages for J^ vitro cytotoxicity of

five calcium silicate (three synthetic and two natural)

and reported their toxicity by the selective release of

^-glucuronidase from the cells against one synthetic and

two naturally occurring calcium silciate mineral

fibres. Pailes ejt a_l. (1984) have exposed alveolar

macropahges in culture to chrysotile asbestos,

wollastonite or latex for 72 hours. No effect on

oxygen consumption or cellular volume was observed when

treated with as little as 25 /ug asbestos/ml for 24

hours. Iri vitro biological activity of wollastonite

shows that these natural mineral fibres induce effects

on pulmonary macrophages that may stimulate events

occurring in the lung following dust exposure, such as

inpaired phagocytic capacity of the exposed

macrophages, and serum complement activation, as

measured by dose-related increase in pulmonary

macrophage chemotaxis (Warheit e_t al^., 1984).

Skaug and Haugen (1989) have taken four types of

cells for the cytotoxic studies of asbestos and calcium

silicates and concluded that calcium silicates are less

toxic than asbestos fibres in the lung epithelial cell

30

types. No difference in ce l l spec i f ic cy to tox ic i ty was

observed. As no repor t ex is t s on the t o x i c i t y of Indian

wol las toni te present invest igat ions were undertaken for

the cytotoxic and genotoxic s tud ies .

STUDIES ON HEMOLYSIS AND LIPID PEROXIDATION INDUCED BY INDIAN WOLLASTONITE DUSTS USING HUMAN ERYTHRO­CYTES: IN VITRO

CHAPTER-I

INTRODUCTION

P a r t i c u l a t e a i r p o l l u t a n t s i n o c c u p a t i o n a l

a t m o s p h e r e p o s e a g r a v e h e a l t h h a z a r d t o exposed

p o p u l a t i o n ( H u n t e r , 1 9 6 9 ) . I n o r d e r t o s a f e g u a r d t h e i r

h e a l t h s i m p l e J ^ v i t r o model s y s t e m s , which h a v e t h e

d i s t i n c t j a d v a n t a g e s o v e r xn v i v o e x p e r i m e n t s , a r e

r e q u r i e d t o a s s e s s t h e e x t e n t of t o x i c i t y and

c a r c i n o g e n i c i t y of i n d u s t r i a l t o x i c a n t s . Towards t h i s

a p p r o a c h , ijn v i t r o s h o r t - t e r m model s y s t e m s a r e s i m p l e

and u s e f u l p r e d i c t o r s of l o n g - t e r m t o x i c i t y and

p a t h o g e n i c i t y of n a t u r a l and s y n t h e t i c m i n e r a l d u s t s ,

when u s e d t o e v a l u a t e s p e c i f i c b i o l o g i c a l end p o i n t .

A d d i t i o n a l l y , t h e y a r e i n v a l u a b l e f o r d e t e r m i n i n g

c e l l u l a r and m o l e c u l a r mechanisms of m i n e r a l i n d u c e d

i n j u r y t h a t may be c r i t i c a l f o r d i s e a s e . T h e s e s h o r t -

term t e s t s h a v e even p r o v e d h e l p f u l i n p r e d i c t i n g

g e n o t o x i c , c a r c i n o g e n i c and f i b r o g e n i c n a t u r e of

m i n e r a l d u s t s (Rahman £ t a^ . , 19 7 7 ) .

E r y t h r o c y t e s p r o v i d e an e x c e l l e n t o p p o r t u n i t y t o

a s s e s s t h e membrane t o x i c a n t i n t e r a c t i o n . One of t h e

32

simplest test is the measurement of red cell lysis in

isotonic conditions. The biologically active

particulates may induce membrane damage (Harington et

al., 1971; Rahman e_t aJ . , 1974; Skaug and Gylseth,

1983; Singh et al. , 1983a,b; Perderiset ejt aa. , 1989;

Iguchi and Kojo, 1989) resulting in the release of

hemoglobin, which can be measured spectrophotometri-

cally. The experience gained in different laboratories

clearly indicate a positive correlation between

hemolytic activity and cytotoxicity of various dusts

(Gabor and Anca, 1974; Chvapil ejt aA. , 1976; Hunt ejt

al., 1981; Skaug £t al., 1984).

Lipid peroxidation of biomembranes is one of the

precipitating factors in tissue injury (Dianzani and

Ugazio, 1978; Sharma and KrishnaMurti, 1980). Erythrpr

cytes are also good systems to study lipid peroxidation

even in the absence of exogenous catalyst in the assay

mixture. In the case of erythrocytes, this process has

been implicated in several physiological alterations

and pathological conditions (Nitowsky et ajL., 1962;

Gross and Melhorn, 1972) . It has been also reported

that hemolysis is proportional to the rate of lipid

peroxidation (Bunyan et al., 1960; Mengel and Kann,

1966; Singh and Rahman, 1987; Iguchi and Kojo, 1989).

Since no report exists on the toxic potential of Indian

wollastonite dusts, a series of experiments were

carried out to assess their biologial reactivity. In

33

the present study, Kuman erythrocytes were used to

evaluate the hemolytic potential, lipid peroxidation

and morphological changes induced by wollastonite dusts

and results were compared with known toxic variety of

asbestos, chrysotile.

MATERIALS AND METHODS

Source of Silicate Dust

Wollastonite samples were supplied by Director,

Wolkem Private Ltd., Udaipur, Rajasthan, India, and

Indian chrysotile was obtained from Andhra Pradesh

Mining Corporation Ltd., Hyderabad, which was mined

from Cuddapah, Andhra Pradesh, Idnia.

Dust Preparation

The particle size of wollastonite smaples and

chrysotile, less than 30 mji used in present studies,

was prepared by the procedure of Zaidi (1969). In

brief, wollastonites were ground to a fine powder, the

individual particles obtained by this method can be

assessed in terms of their ability to pass through a

sieve mesh of one size, and finally particle size was

determined by microsocope.

Preparation of Washed Erythrocytes

Blood was collected in heparinized vials, from

healthy donors, at local blood bank, Lucknow, India.

34

Plasma was separated by centrifuging the blood a t 1500

rpm for 15 minutes. Erythrocytes (red blood eels) were

washed three times with 0.156 M sodium chloride and

suspended in r e q u i s i t e concentrat ion in incubation

buffer .

Hemolytic Studies

The manner of assessing the hemolytic potency

was the same as employed by Rahman e t a l . (1974). The

desired eimount of dusts were suspended in 0.01 M Tr i s -

HCl buffer (pH 7.35) in 0.156 M sodium ch lo r ide . This

suspension was then added to erythrocyte suspension to

a f i n a l concentration of 0.2%. This mixture (dust +

erythrocytes) was then incubated a t 37 "C for 120

minutes in a metabolic shalcer with moderate shaking,

a l iquots were taken and centrifuged a t 1500 rpm for 10

minutes. The content of hemoglobin released in

supernatant was determined by SP-800 Spectrophotometer

a t 545 nm using a f r a g i l i t y con t ro l . Hundred percent

hemolysis was obtained by using d i s t i l l e d water instead

of buffer .

Lipid Peroxidation Assay

Washed red blood c e l l s (erythrocytes) were

suspended in 10 mM potassium phosphate buffer (pH 7 .4) ,

containing 0.15 M potassium chlor ide for i n t a c t c e l l s

35

in r e q u i s i t e concentft ion. Red blood ce l l s were

heitiolyzed by freezing and thawing the sample four times

in two volume of 10 mM potassium phosphate buffer (pH

7.4) containing 0.05 M potassium chlor ide . A calcula ted

amount of sol id potassium chloride to 0.15 M in

so lu t ion was added. The hemolysate was centrifuged a t

1500 rpm for 15 minutes to obtain a low speed

supernatant of the hemolysate.

Lipid peroxidat ion of polyunsaturated f a t t y acids

in erythrocyte membranes was determined by the method

of Ernster and Nordenbrand (1967). The incubation

mixture contained e i t h e r the i n t a c t erythrocyte c e l l

suspension or the low speed supernatant of hemolysate

in a so lu t ion of 10 mM phosphate buffer, pH 7.4,

containing 0.15 M potassium chloride and known

concentration of wol las ton i te sanples (1 ,2 ,3 ,4 and 5

mg/ml). The f lasks were incubated for 120 minutes a t

37"C in a continuous shaking water bath . At the end of

incubation, 1 ml of a l i quo t was added to 1 ml, of 10%

(w/v) t r i ch lo roace t i c acid (TCA) and 1 ml of 0.67%

(w/v) th ioba rb i tu r i c acid (TEA), kept in a boi l ing

water bath for 15 minutes, cooled in ice and

centrifuged a t 1500 rpm for 15 minutes. The supernatant

was read a t 535 nm using a reagent blank. Malonaldehyde

formation (production) was calcula ted assuming an

ex t inc t ion coef f i c i en t of 1.56 x 10^ M'-^/cm (Wil ls ,

1969).

36

Glutathione (reduced) Estimation

Glutathione content in the erythrocytes was

estimated according to Sedlack and Ludsay (1968).

Erythrocyte (0.2 ml) was lyzed with 1.8 ml of 1 g / l i t r e

EDTA (ethylene diamine t e t r a a c e t i c acid) so lu t ion and 3

ml of p r ec ip i t a t i ng reagent (1.67 g metaphosphoric

aqid, 0.2 g EDTA, 30 g sodium chlor ide , water to 100

ml) was added. After mixing, the so lu t ion was allowed

to stand for f ive minutes before being f i l t e r e d . Two ml

of f i l t e r a t e was added to 4 ml of disodium hydorgen

phosphate (0.1 M, pH 8 . 0 ) , and 1 ml of DTNB reagent (40

mg, 5 ,5-di th iobis - ( 2-nitrobenzoic acid) was dissolved

in 100 ml of 10 g / l i t r e sodium c i t r a t e ) . A blank was

prepared from 1.2 ml of p r e c i p i t a t i n g reagent, 0.8- ml

of EDTA solut ion, 4 ml of disodium hydrogen phosphate

and 1 ml of DTNB reagent . The color was read a t 412 nm.

Protein Estimation

Protein was determined by the method of Lowry ejt

a l . (1951) using bovine serum albumin as standard.

Treatment of Wollastonite Samples

(a) 50 mg of dry wol las ton i te samples were separa te ly

incubated with 5 ml of bovine serum albumin (10 mg/ml),

polyvinylpyrrolidone (10 mg/ml) and ethylene

diaminetetra ace t i c acid (EDTA 1 M/lOOO) for 3 hours a t

37

37°C. The dusts were removed by centr i fugat ion a t 2000

rpm for 15 minutes, dried and tes ted for hemolytic

a c i t i v i t y . (b) 100 mg of wol las toni tes were incubated

a t ST'C for 24 hours with 50 ml of 0.1 N NaOH, 0.1 N

HCl and d i s t i l l e d water, followed by exhaustively

washing with water, dried and tes ted for hemolytic

a c t i v i t y .

Scanning Electron Microscopic (SEM) Studies

Treatment of erythrocytes

To t r e a t erythrocyte (1% c e l l suspension) with

wollas t on i t e , the f i b re suspension was added to the

ce l l suspension a t the f ina l concentrat ion of 2 mg/ml

in 0.01 M Tris-HCl s a l i ne (pH-7.35), and incubated a t

37°C in a metabolic shaker with moderate shaking. After

120 minutes of incubation, erythrocyte-wol las t o n i t e

suspension (mixture) was added to f i xa t i ve .

Fixation eind dehydration of erythrocytes

Fixat ion was carr ied out by the addi t ion of 1.5

ml of 2.5% solut ion of glutaraldehyde in 0.1 M

cacodylate buffer, pH-7.4, to 0.5 ml of a l iquot of

ery throcyte-wol las toni te suspension a t room temperature

for 60 minutes. After f ixa t ion , the ce l l s were washed

with 0.1 M cacodylate buffer , pH-7.4, and pos t f ixa t ion

was done in 1% osmium tetraoxide in 0.1 M cacodylate

buffer, pH-7.4 for 60 minutes. Following

38

c e n t r i f u g a t i o n , t h e - p e l l e t was washed w i t h t h r e e

changes of d o u b l e d i s t i l l e d w a t e r . The c e l l s w e r e

d e h y d r a t e d by wash ing s u c c e s s i v e l y w i t h 3 0 , 50 , 70, 8 0 ,

9 0 , 95 and 100% g r a d e d a l c c h o l f o r 20 m i n u t e s i n each

c o n c e n t r a t i o n .

Mounting and Scanning o f E r y t h r o c y t e s

A f t e r c o m p l e t e d e h y d r a t i o n e r y t h r o c y t e s w e r e

suspended i n a b s o l u t e a l c o h o l . A d r o p of i t was

p i p e t t e d i n t o a s q u a r e p i e c e of c o v e r s l i p and d r i e d .

The g l a s s p i e c e s w e r e mounted on a c o p p e r s t u b . The

s t u b s were c o a t e d on JEOL JFC 1100 i o n s p u t t e r c o a t e r

f o r 12 m i n t u e s and were examined u n d e r JEOL JSM-3 5

Scann ing E l e c t r o n M i c r o s c o p e a t 10 KV. P h o t o g r a p h s

were t a k e n w i t h a 120 p r o b e and 125 ASA p h o t o g r a p h i c

f i l m . S i m u l t a n e o u s l y , c o n t r o l s c o n t a i n i n g o n l y c e l l s

were a l s o r u n w i t h each e x p e r i m e n t .

S t a t i s t i c a l A n a l y s i s

Values r e p r e s e n t mean +_ SE of s i x d e t e r m i n a n t s .

E x p e r i m e n t s were r e p e a t e d f o u r t i m e s .

RESULTS

CoiH)arative Hemoly t i c A c t i v i t y of C h r y s o t i l e and W o l l a s t o n i t e Samples

F i g u r e 1 shows l y t i c p o t e n t i a l of c h r y s o t i l e and

w o l l a s t o n i t e s m a p l e s , namely , k e m o l i t A-60 k e m o l i t - N

80

70

(U

0 'Ji

u

€

1

CO - H

CO > 1

0 e

60

50

40

30

K-vv:::i i

1 1

i \ \ \ \ \ N

k' t H

t "s

1 •

o 1

< -P -H H

i

T \ \ ' ^\\

1 ^ ^ >.

' i ^ 4

. '•J

1

i

iz;

0

T

C O

< ^J

i- l

0 E 0)

,T

Treatments

F ig . 1: Comparative hemolytic a c t i v i t y of ch ryso t i l e and wol las ton i t e samples.

39

and kemolit ASB-3. Among the evaluated wol las ton t ie

samples kemolit A-60 shows maximum degree of hemolysis

aga ins t other two v a r i e t i e s but i t was less hemolytic

than ch ryso t i l e , while kemolit ASB-3 was the l e a s t

hemolytic. The order of the i r hemolytic p o t e n t i a l was

as follows: ch ryso t i l e > kemolit A-60 > kemolit-N >

keftiolit ASB-3. Hemolysis was decreased by 40, 49 and

54% for kemolit A-60, kemolit-N and kemolit ASB-3,

repseci tvely in comparison to c h r y s o t i l e .

Effect of Dust Concentrations on Hemolytic Act iv i ty

Figure 2 records the re lease of hemoglobin caused

by d i f f e r en t concentrat ions of three v a i r e t i e s of

wol las toni te a f t e r 120 minutes of incubat ion. At the

dust concentration of 10 mg/ml kemolit-N showed maximum

lys i s of 85% while kemolit A-60 and kemolit ASB-3

ejdiibited maximum lys i s of 82% and 73%, respec t ive ly a t

the above concentrat ion. Complete l y s i s of erythrocytes

was not achieved, bu t lys i s advanced with the

concentrat ion with a l l the v a r i e t i e s of wol las ton i t e

upto 10 mg/ml in dose dependent manner. Further

increase in wol las ton i te concentration did not lead to

any increase in hemolysis, presiomably due to adsorpt ion

of hemoglobin on the dust (wol las tont ie) sur face .

Relative Hemolytic Index (HT) of Different Dusts

The r e l a t i v e hemolytic index of wol las ton t ie and

chryso t i l e dusts is r ^ r e s e n t e d in Figure 3. Among the

90

0) 6 0 -H 01 > i

o E 0)

3 0

I J-

/ X

j/y

/ / / //f

i'^ y^-^

/ / /

/ - T -

5

-4 Kemolit A-60

Keraolit-N

Kemolit ASB-3

10

Dust Concentration

Fig . 2: Effect of dus t concentrat ions on hemolytic a c t i v i t y

3.3

I ^ 2 . 8^ M

0)

c •H o

-H

> i

i 0)

2 . 3

1.8-;

1 . 3 i

.8

a; <-< •H -P O

' 'n -*:—c—T—r—c

O vD I

-H H

0 E

. i

\ -. \ \ N \ ^

\ \ \ ^;- o

•H

e

.• J

-• \ \ \ \ N

f > x \ \ -\ \

m CQ CO

< •p

i

:-\

\~\\-.\ :-

, - : ; , : ^X,\ \ s \ sj

."• xX \ ' X \ X \XX^ ^ \ • ^ x : ^ ^ \ ^ ^

-X,V\\X^\

sXX\x \ \

xxX .,v^x3 KX'-X-.X-vj

Fig. 3: Relative hemolytic index of different dusts. HI = Amount of dust (mg/ml) needed for 50% hemolysis of 0.2% RBC. The time of incubation was 120 min except chrysotile where it was 10 min.

40

evaluated dus ts , ch ryso t i l e (Indian) showed h ighes t

hemolytic a c t i v i t y and kemolit ASB-3 (wol las toni te)

exhibited lowest. The mos t hemolytic wol las ton i te was

kemolit A-60 followed by kemolit-N and kemolit ASB-3.

However, the hemolytic a c t i v i t y of wol las ton i te dusts

was found less when compared to ch ryso t i l e .

Kinetics of Hemolysis of Erythrocytes by Wollastonite

The increased r a t e s of hemolysis as a function of

contact time of erythrocytes with the wol las ton i te

namely kemolit A-60, kemolit-N and kemolit ASB-3 are

depicted in Figures 4, 5 and 6, r espec t ive ly . The

resu l t s of these experiments showed t h a t a t the h i g h e s t

dust concentration of 15 mg/ml maximum hemolysis was

observed a t 60 minutes of incubation. No increase was

observed a f t e r 60 minutes a t the above concentrat ion,

v*\ile a t the concentrat ion of 2, 5 and 10 mg/ml

hemolysis was found to be time dependent and increased

as recorded up to 120 minutes, except kemolit ASB-3 a t

10 mg/ml dust concentra t ion.

Effect of Treatments of f«ollastonite Dusts on Hemolysis

The effect of various treatments of dusts on

the i r hemolytic a c t i v i t y i s recorded in Figure 7. The

ethylene d iaminete t raace t ic acid (EDTA) t reatment shows

marginal change in hemolysis while bovine serum albumin

(BSA) and polyvinylpyrrolidone (PVP) d r a s t i c a l l y

CO - H

CO >1

o e 0)

9 0 -

60

I .l----

I 30

I. J 10 mg

""'^15 mg

I 5 mg

J 2 mg

I / /

/ /

o<f-10 30

— I —

60 120

Incubation time (mins)

Fig . 4: Kinet ics of human erythrocyte l y s i s a t d i f f e r en t concentrat ions of Kemolit A-60

90t-

[0 -H 0) > i

iH 0 E 0)

60

30

I-I

(

/I • /

/ r'

/ / .1

j—-- J- 1 1 0 mg

15 mg

i 5 mg

/

/ / / / / /

/

I---

_j--

L -

j/-

J - -

r

0*-10

— I —

30 60

Incuba t ion time (mins)

J 2 mg

120

F i g . 5: K i n e t i c s of human e r y t h r o c y t e l y s i s a t d i f f e r e n t c o n c e n t r a t i o n s of Kemolit-N

90-

> 1 i-H 0 e 0)

60-

30

l---

T'

'^^ J 0 -''• X: ——J 10 mg

"4 15 mg

T ^____—e 5 mg

/ ,/" l-—-

> -

_J_—- L-- .J 2 mg

/ /

10 30 60

Incuba t ion time (mins)

120

F i g . 6: K i n e t i c s of human e r y t h r o c y t e l y s i s a t d i f f e r e n t c o n c e n t r a t i o n s of Kemolit-ASB-3

0) > i

rH O

e

dp

[ jKenol i t ASB-3

5>jKemolit.-N

r iKemol i t A-60

Treatments

F ig . 7: Effect of treatments of wol las ton i te samples on hemolysis.

41

reduced the hemolysis, although PVP was less effective

than BSA. Acid, alkali and water treatment

significantly reduce hemolysis. The alkali was more

effective in reducing lysis than acid and water. The

order of hemolysis after treatment with acid, alkali

and water was as follows: acid > water > alkali.

Effect of Increasing Erythrocyte Concentration on Lipid Peroxidation

Values of malonaldehyde formed in two hours upon

increasing red cell concentration in incubation mediiom

is given in Figure 8. Maximum malonaldehyde was

produced in 2% cell suspension. At 2.5% cell

suspension, there was a decrease in the malonaldehyde

production to a significant degree which could be

attributed to inhibition accorded by the high

concentration of susbstrate or physiological

antioxidants. An effect of hemoglobin levels also can

not be ruled out at this stage.

Effect of Calcium (metal) on Erythrocyte Lipid Peroxidation

Since iron (metal) stimulates lipid peroxidation

of erythrocytes, it became interesting to study v^iether

metal ion can stimulate, potentiate or antagonised the

catalytic effect or not. Under this heading Ca is

taken for study as it is a constituent of wollastonite.

c

0)

0 l-I

a. E < a 2 u 0)

1.5'

1.0

0.5 1

I

T

T'

L-^-\

H

0 t-

0.1 0.2 0.5 1.0

— 1 1 —

1.5 2.0 2.5

Erythrocytes concentration

Fig. 8: Effect of increasing erythrocytes concentra­tion on lipid peroxidation.

42

Varying concentrations of Ca were added to the

incubation mixture containing intact cells. After two

hours of incubation malonaldehyde production was

measured. Data given in Figure 9 shows that at the

concentration tested were not effective in inducing

lipid peroxidation.

Effect of Hemoglobin (Hb) on Erythrocyte Lipid Peroxidation

When varying concentrations of hemoglobin were

added in the reaction mixture for lipid peroxidation of

erythrocytes, a biphasic response was observed. Figure

10 indicates that initially with the increase in the

concentration of hemoglobin up to 100 ug, there was

increase in malonaldehyde value in intact cells. Above

the supplementation of this concentration in incubation

mixtures, malonaldehyde values were decreased. At 1 mg

of hemoglobin concentration, lipid peroxidation process

was even lower than control values. The malonaldehyde

value was decreased by 14% in intact cells against

control values. This effect of hemoglobin may be one of

the factors involved in the decreased malonaldehyde

formation beyond 2.5% cell suspension.

Effect of Wollastonite and Qirysotile on Erythrocyte Lipid Peroxidation

Figures 11 and 12 shows that like chrysotile,

wollastonites were also effective in stimulating lipid

0.2405 I J

CM

-H 0 . (1) +> 0

2400

E

O

s 0)

H

0 .2395

0 .2 390

0.2385^

I I \

10

t

50 100 500 1000

Concentration ( JUM)

Fig. 9: Effect of calcium on erythrocyte lipid peroxidation.

. 3 2

0.3CM CM

c

O u o> 0 . 2 8 -

m 0) o e ."^O

0.24

/ r

25

-J I

\

— I —

50

~T

100 250

Concen t r a t i on { xig)

F i g . 10 : E f f e c t of exogenous hemoglobin (Hb) e r y t h r o c y t e l i p i d p e r o x i d a t i o n .

500

on

0 . 6

0.5

rA . X--

1

T

0.2

Amount of dust (mg/ml)

T C h r y s o t i l e

5 T Q> • TKemolit A-60

^ '. J - T jKemolit-N i* 0.4. ^ I - ^"' T O T J Kemolit ASB-3 2

Fig. 11: Effect of dust on lipid peroxidation in intact erythrocyte.

l . i " T Chrysot i le

1.0-

. 9 0)

4-»

c u

6

m <u

i-i O E

. 6 - T •

.

1 ! : . . ^ - : j -J Kemolit A-60

-"1 i

' --" Kemolit-N

^ Kemolit ASB-3

i . 4

0 1 2 3 4 5 Amount of dust (mg/ml)

F ig . 12: Induction of l i p i d peroxidat ion in low speed supernatant of hemolysate by d i f f e r en t dus t .

43

peroxidation in i n t a c t ce l l s and low speed supernatant

of heinolysate. Among the wol las toni tes tes ted , kemolit

A-60 showed maximum induction of l i p id peroxidation of

i n t a c t ce l l s and low speed supernatant of hemolysate,

while kemolit ASB-3 was l e a s t e f fec t ive . However, the

magnitude of induction of l i p i d peroxidat ion by

kemolit A-60 was lower as compared to ch ryso t i l e . A

concentration dependent increase in malonaldehyde

formation was a l so observed in i n t a c t ce l l s and low

speed supernatant of hemolysate with tes ted samples. At

5 mg/ml dust concentrat ion, malonaldehyde formation in

i n t a c t ce l l s was increased by 86%, 68% and 5 5% and in

low speed supernatant of hemolysate by 75%, 69% and 54%

for kemolit A-60, kemolit-N and kemolit ASB-3,

respect ively agains t con t ro l . When the values were

compared with ch ryso t i l e , l i p id peroxidat ion was

decreased by 16%, 24% and 31% in i n t a c t c e l l s and by

20%, 23% and 31% in low speed supernatant of hemolysate.

Kinetics of Hemolysis and Lipid Peroxidation in Erythrocytes

Table 1 represents time course of l i p i d

peroxidation in r e l a t i o n to hemolysis. Malonaldehyde

formation and hemolysis was increased with incubation

time in dust t rea ted e ry throcytes . The malonaldehyde

formation increased by 355%, 295% and 28 0% for kemoli t

A-60, kemolit-N and kemolit ASB-3 and 150%, 146% and

w CQ

<: En

CO 0) 4-» >1 u o u

>1 k 0)

c -H

c o

- H - p

• H X o u Q) Cu

-H ft

- H

C fO

U3 - H

U l > 1

r - l 0 E <u

M

4-1 0

en u

•r-(

-P 0)

c

CO

CO

a>

2!

-H

0)

o I

•P - H

i (1)

CQ

< -P -H rH

i

IZl

•H

0 e

o VD

I < -P -H rH

i (D

C 0

•P 0) C

c

0)

. o r-Q H r-

o rH

n

o

!Zi

H

n (n rH

rH

r o <N

o 04

a\ <n

t^ i n (N rH

00 CM v£> CO

CN <-{ i n ^

• Q •

Z

<T> O

• O ' t

CM

00 •

"* ' t

( N

as •

"<* VO

i n

o 00

• Q •

Z

o CM

• o i n

m (Ti •

<-i VO

<T\ rH

• i n r

o o o CM

• rH

E "oi E in

1

c 0

• H •p (0

^ c d) 0 c 0 0

•p

•p d

C -H

E 0)

+ 73

0) 0)

5 e >H

MH

C 0) >

- H

• 0) r-\

C P ^

QJ D

rH fO > d (0

rtJ -P o (U •p Q)

TJ

-P 0 z i Q Z Z

44

162%, repsecti-vely at. 120 mlntues incubation in

comparison to 30 minutes. Results show tha t both l i p i d

peroxidation and hemolysis are time dependent in the

case of wol las ton i te mineral f i b r e s . These data a l s o

support the higher toxic nature of kemolit A-60 than

kemolit-N and kemolit ABS-3 in bringing the hemolysis

and peroxidat ive damage of erythrocytes membrane.

Effect of Wollastonite and Oirysoti le Dust on Glutathione Content of Erythrocytes

The gluta thione (reduced) content of erythrocytes

incubated with dust i s depicted in Figure 13. These

values also show t h a t the ch ryso t i l e i s the most tox ic ,

\«^ile kemolit ASB-3 i s the l e a s t toxic of the dusts

t es ted . The l a t t e r was 43% l e s s toxic than

chryso t i l e . The values for kemolit ASB-3, kemolit-N,

kemolit and A-60 and ch ryso t i l e were 63.75, 46.27,

50.73 and 36.16%, respec t ive ly in comparison to control

vAiich was considered as 100%. The deple t ion of

glutathione (reduced) c l ea r ly indica tes a l t e r a t i o n in

meiribrane a r ch i t ec tu re , r e s u l t i n g in the efflux or

oxidation of g lu ta th ione (reduced). I t s deplet ion may

be one of the fac to r responsible for l i p id

peroxida t ion.

Kinetics of Glutathione Content of Erythrocytes During the Course of Lipid Peroxidation

I t i s already reportd t h a t g luta thione (reduced)

acts as a strong i n h i b i t o r of l i p id peroxidat ion . In

> u

o o

X CO

o CD

e

59

54.-

49

44

39

34

29

c

i

J

^ ^•^ \ \

• * «

m I

CO CO

< -p

r-i c e 0)

o I

<

0)

X

w

. \ N

0)

•s -P O

I u

6 .\--.

Treatment 'F ig . 13: Effect of wol las ton i te anci ch ryso t i l e dust on

g lu ta th ione (reduced) content of erythrocytes

45

view of the fact t ha t i t p ro tec t s the ce l l s aga ins t

l ip id peroxidation and since g lu ta th ione content is

d i r ec t l y r e l a t ed to functional i n t e g r i t y of

erythrocytes , glutathione content of the system

undergoing l ip id peroxidation a t d i f fe ren t time

in te rva l s were s tudied. Data recorded in Table 2

indica te t h a t as l i p id peroxidat ion progresses

g luta th ione content of i n t a c t ce l l s decreases

s ign i f i can t ly . The decrease was very prominent in the

f i r s t 30 mintues and then more gradual. At 120 minutes,

when the malonaldehyde value was 200, 19 0, 169 and 154%

for ch ryso t i l e , kemolit A-60, kemolit-N and kemolit

ASB-3 respect ive ly while gluta thione content in the

system was decreased by about 45, 32, 25 and 19%

agains t the i r respect ive con t ro l s , which was

considered as 100%

Scanning Electron Microscopic (SEM) St-udies

Scanning electron microscopy c l ea r ly showed t h a t

addi t ion of wol las toni te to erythrocytes led to

d i s t o r t i o n and deformation of ce l l s (Figures 15-17).

Main abnormality appeared as invaginat ion or crenat ion

of erythrocyte membrane. Wollastonite f ib res were

i n i t i a l l y associated with erythrocyte membranes and

port ions of i t were wrapped around the f i b r e s . The

nature of membrane damage was s imi la r with a l l the

tes ted wol las ton i te samples. However, i n t a c t c e l l s were

(N

w CQ

< EH

c o

-H

(0

-o •H X 0 u 0)

T) •H fX

•H

o (U CO

^

B u 0)

01

c ^ 3

T) CO

•p > 1 o o U

u (U

M-l 0

•p c (1) -p c o u

u

c c

• H

-p

3

cn

0

CO 0

-ri

0)

c

c -H 0) -p 0 ^ a

w <: -p • H •H

0 e Q)

tit;

-H

0)

o I

- H

CO

-p -H

i o

•H

0 E Q)

O vO

I

•P - H iH 0 e

c 0 •p 0) d fC 6 -H

c

CO H CO >1

r-l

i 0)

i n

CO

in

Q

Q

O

o CN

CN

O CO

O

o

O O

o

i n

o CN

00 r •

n i n

o r • i n

"*

o o •

00 00

o o •

r n

in in

<-{ CO

<3 CO CO rH

CO in 00 CM

(Ti CM ON CO

o i n rg i-{

i n o VO CO

CN r~ i n •<t

o CN

• rH

e cn E in

1

C 0

-H +J (0 ^ -p c 0) u

•p c C

e

•P

•d

0) V

5 E 0 u

U-l

c Q) >

-H

• (U

i H

cn^ CO

-H

<u D

rtJ •p

u 0) •p

<u C H T! 0 0

P CO

fl > C

•p c z

0) Q

Fig . 14: Scanning e lec t ron micrograph of control human erythrocytes ( X 2000)

F ig . 15: Scanning e lec t ron micrograph of Icemolit ASB-3 t rea ted human erythrocytes ( X 2000)

Fig . 16: Scanning e lec t ron micrograph of kemolit-N t rea ted human erythrocytes ( X 2000)

F ig . 17: Scanning e lec t ron micrograph of kemolit-60 t rea ted human erythrocytes ( X 2000)

46

s t i l l found in wol las toni te t rea ted samples, while

untreated ce l l s exhibited a normal biconcave morphology

(Figure l 4 ) .

DISCUSSION

In the present study Indian va r i e t i e s of

wol las toni te samples were evaluated for t he i r toxic

po ten t i a l using human erythrocytes . These samples

showed less hemolytic po ten i t a l than chryso t i l e which

is in agreement with the e a r l i e r findings of Potts et

a l . (1978) and Hunt ejt a l . (1981). The order of

hemolysis was as follows: chryso t i l e > kemolit A-60 >

kemolit-N > kemolit ASB-3. Tn v i t r o rupture of red

ce l l membrane has been extrapolated to ind ica te the in

vivo cy to toxic i ty of dusts (Rahman £ t aj.. , 1977).

Therefore, i t i s l i ke ly t ha t wol las ton i te dusts are

l ess toxic ^ v i t r o and may account for the incidence

of less f ib ros i s among the workers occupational ly

engaged in the wol las toni te industry a t Udaipur and

other p laces . Wollastonite induced hemolsyis seems to

d i f fe r from chryso t i l e asbestos in ce r t a in aspects .

Here, EDTA treatment was inef fec t ive in reducing l y s i s ,

unlike chryso t i le (Harington ejt a_l., 1975), indicat ing

t h a t the metal hydroxyl layer i s not a causat ive factor

in membrane damage as reported by Skaug and Gylseth

(1983). The chelator treatment i s l i ke ly to inac t iva te

47

calcium released from the mineral to the solu t ion , but

i t i s not c lear whether th i s treatment inac t iva tes high

calcium concentrat ions on the surface of the p a r t i c l e s

responsible for loca l react ion with erythrocytes or

not. The BSA and PVP have inhibi ted hemolysis due to

the adsorption of macromolecules on the surface of

mimeral f ibres (Perder i se t ejt aJ . , 1988, Jaurand e_t

a l . , 1979; Singh ejt ajL. , 1983a) which indica te t h a t

hemolysis i s s t rongly dependent on the surface s t a t e of

the mineral f ibres (Perder i se t ejt a_l. , 1989). There are

p o s s i b i l i t i e s tha t hemolysis might be due to the

abnormal influx of calcium as suggested by Skaug and

Gylseth (1983) . The other p o s s i b i l i t y could be the

leaching of s i l i c i c acid from these f ibres in the

incubation buffer and damaging erythrocyte membrane.

But our s o l u b i l i t y data (unpublished) do not support

tJiis hypothesis . In the case of these f i b r e s , there was

no d i r e c t cor re la t ion between the s o l u b i l i t y of s i l i c i c

acid and hemolysis. I t appears t ha t the t a rge t

molecules involved in hemolysis are d i f f e ren t . The

effect of acid, a l k a l i , and water treatments of dust on

hemolysis was s imi la r , as noticed previously, to quartz

and s l a t e dusts (Clark and Holt, 1960; Singh et_ a l . ,

1983b) .Acid and water treatments are known to enhance

metal d i sso lu t ion to a s ign i f i can t degree (Clark and

Holt, 1960). Thus a l t e r i n g the surface o r i en ta t ion of

dust which may be responsible for hemolytic a c t i v i t y .

But with a l k a l i treatment, reduced ly s i s might be due

48

to the non-ava i l ab i l i ty of s i l i c o n which probably forms

silicon-OH groups with a l k a l i a t the dust surface

(Clark and Holt, 1960).

The erythrocytes are a very good system to study

l ip id peroxidation process even in the absence of

exogenous ca ta lys t s in the assay mixture. The induction

of l i p i d peroxidation with chryso t i l e was found higher

in magnitude than wol las ton i te . This difference may be

a t t r i b u t a b l e to the difference in the i r chemical

corrposition. The less peroxidat ive damage of i n t a c t

erythrocytes was observed as conpared to low speed

supernatant of hemolysate presumably due to the f a c t

tha t free rad ica ls can not pene t ra te the s t rong

hydrophobic zones of biomolecules (Demopoulos, 19 73) .

The i n t a c t c e l l membrane acts as a b u i l t - i n - r e s i s t a n c e

of peroxidat ive degradation, thereby protect ing the

vulnerable polyunsaturated f a t t y acids from free

r ad ica l a t tack , which shows t h a t with the progress of

the peroxidation react ion erythrocytes become f r a g i l e .

Lipid peroxidation in i n t a c t ce l l s increased with the

time of incubation indica t ing t h a t a poss ib le

re la t ionsh ip may e x i s t between the two processes , i . e . ,

l ip id peroxidation and hemolysis as described by Singh

and Rahman (1987). Kennedy jet a_l. (.1989) have

demonstrated tha t hemolysis was decreased by the

hydrogen peroxide scavenger, ca t a l a se , by p re t r ea t ing

s i l i c a t e s with the iron chelators or by adding the

49

lipid soluble hydroxyl radicals (OH) scavenger to the

medium which also indicate towards the correlation

between hemolysis and lipid peroxidation.

Generation of free radicals in the biological

system is facilitated by the reduction of molecular

oxygen through monovalent pathway and depend largely on

the participation of semiquinones such as flavin,

ubiquinone and vitamin K including certain iron

chelators and heme iron. According to some workers the

superoxide anion radical per se is not the direct

initiating factor of lipid peroxidation (Frong £t al.,

1983; Fridovich, 1981; McCay and Poyer, 1976). It is

believed that superoxide radical upon reacting with Hj O;

leads to the formation of hydroxyl radical (OH) by a

typical Haber and Weiss reaction mechanism (Haber and

Weiss, 1934). Hydroxyl radicals have been found to be

extremely potent initiators of peroxidation reactions

and have a longer half life than superoxide radicals.

O^ + ^z^-S. - OH* + OH* + O^

The occurrence of Haber-Weiss reaction in

biological system, in the presence of catalysts, has

already been established (Koppenol and Butler, 19 77;

Rigo £t aj.. , 19 77) .

Regardless of the cellular and subcellular

systems, the foremost requirement in lipid peroxidation

is the molecular oxygen (Bus and Gibson, 19 79) from

50

which superoxide radicals and hydroxyl radicals are

generated by the catalytic action of chelated and non

chelated iron complexes found in the biological system.

With erythrocytes, a special situation exists

especially because of the high concentration of

hemoglobin contained in it. Evidence has been obtained

that oxyhemoglobin oxidises xenobiotics to free

radical, converting itself to methemoglobin through a

peroxidative activity (Mason and Chignell, 1982).

Action of reduced glutathione in inhibiting

lipid peroxidation of erythrocytes is of special

significance. It seems probable that during the course

of lipid peroxidation reaction reduced glutathione acts

as a protective factor by elicitating the reactions of

the enzymes of glutathione peroxidase system (Bus and

Gibson, 1979) that scavenge free radicals. Depletion of

glutathione content of erythrocytes with the progress

in lipid peroxidation supports this finding. It is also

likely that reduced glutathione, during the process of

lipid peroxidation conjugates with the intermediates of

lipid peroxidation or that the glutathione regenerating

enzymatic machinary of erythrocytes get inactivated.

The scanning electorn microscopic studies of

wollastonite treated erythtoryctes show deformity of

cell indicating alteration in cell membrane. It is well

known that erythrocyte membrane undergoes changes

resulting in the alterations in permeability,

characteristic of mammalian cells, ultimately leading

51

to lysis, besides involving alteration in the

biochemical profile of the various enzymes of

erythrocytes on exposure to toxic dusts (Harington et

al», 1975; Rahman £t. aj.. , 1977). The cell deformation

and concomitant redistribution of membrane components

could be integral to the hemolytic process. It is also

possible that normal transport of Na and K ions is

adversely affected by redistribution of membrane

glycoproteins (Deuticke, 1977; Gladen and Sullivan,

1979). The altered membrane properties may be related

to calcium sialic acid binding property (Jaques ejt ajL. ,

1977) or as discussed earlier, abnormal influx of

calcium into the cell as noticed in sickle cell anemia

(Burris, 1980). Present study, also gives a clear

indication of the involvement of lipid peroxidation in

dust induced cytotoxicity and that this parameter could

be used as a valuable tool in evaluating the membrane

toxicity of xenobiotics. Results of further studies on

wollastonite induced toxicity are given in forthcoming

chapters.

STUDIES ON CYTOTOXIC EFFECTS OF INDIAN WOLLASTONITE DUSTS USING ISOLATED RAT HEPATOCYTES: IN VITRO STUDY

CHAPTER-II

INTRODUCTION

S u s p e n s i o n s of i s o l a t e d h e p a t o c y t e s a r e now

w i d e l y b e i n g u s e d i n an i n c r e a s i n g number o f

b i o c h e m i c a l and p h a r m a c o t o x i c o l o g i c a l i n v e s t i g a t i o n s

( F r y and B r i d g e s , 1 9 7 9 ; K l a a s s e n and S t a c e y , 1 9 8 2 ) .

C u l t u r e s of h ^ a t o c y t e s h a v e s e r v e d a s sound b i o l o g i c a l

t o o l s f o r t h e s t u d y of c a r c i n o g e n e s i s iji v i t r o (Borek

and W i l l i a m s , 1 9 8 0 ) . S i n c e t h e y seem t o r e t a i n many o f

t h e e s s e n t i a l p r o p e r t i e s of t h e i n t a c t t i s s u e ,

i n c l u d i n g s i m i l a r p e r m e a b i l i t y c h a r a c t e r i s t i c s . The

s u p e r i o r i t y of t h e s e c e l l s r e s t s m a i n l y upon t h e i r

e p i t h e l i a l c h a r a c t e r s and w e l l d e v e l o p e d m e t a b o l i c

a c t i v a t i o n mechan i sms . I s o l a t e d h e p a t o c y t e s h a v e a l s o

been u s e d i n t h e s t u d y of x e n o b i o t i c m e t a b o l i s m and

c y t o t o x i c i t y . T h e e f f e c t s of drugs and c h e m i c a l s on t h e

c e l l u l a r m e t a b o l i s m h a v e p r o v e d t o b e o f v a l u e f o r

f u r t h e r e l u c i d a t i o n of many a s p e c t s of t h i s p r o c e s s .

H e p a t o c y t e s from a d u l t r a t s , i n p a r t i c u l a r , h a v e

p r o v e n t o be a u s e f u l model f o r s t u d i e s of x e n o b i o t i c

53

transformation (Guzelian et aj^. , 1977; Croci and

Williams, 1985) .

The i so la ted and cul tured hepatocytes are a l so

being increasingly used for s tudies involving

carcinogenesis and mutagenesis (William e_t a_l., 1982;

Mitchell et. al., 1983) . They have a l so been used for

the evaluation of cytotoxic (Fleury e_t £ l . , 1983;

Denizeau, et. a_l., 1985), carcinogenic (Rahman and

Casciano, 198 5) and genotoxic p o t e n t i a l (Denizeau et

a l . , 1985a,b,c,d) of mineral f i b r e s . Therefore, in the

present study, hepatocytes were se lec ted as model

system for evaluating the cy to toxic i ty of wollas t o n i t e ,

an asbestos s u b s t i t u t e .

Cytotoxici ty i s usual ly evaluated by the

measurement of cy toso l ic enzyme leakage (Mitchell and

Acosta, 1981; McQueen and Williams, 1982; Tyson e t a l . ,

, 1983; Qiao et a j . . , 1988; Nakamura et a_l. , 198 5) and/or

determination of c e l l v i a b i l i t y by trypan blue

exclusion t e s t . Lactate dehydrogenase (LEH) has been

considered the most r epresen ta t ive of the cytosol ic

enzymes l ibera ted in to the medium during mentsrane

damage. I t has been considered for severa l years t h a t

l ip id peroxidation (LPO) may be a cause of c e l l injury

induced by xenobiot ics . The p o t e n t i a l l y damaging

effects of LPO, per se , are known to be of a ser ious

nature, pa r t i cu l a r ly to membrane s t ruc tu res (Tappel,

1973). Invest igat ions have a l so been car r ied out in to

the re la t ionsh ip between LPO and concentrat ion of GSH.

54

MATERIALS AND METHODL

M a t e r i a l s

Collagenase type IV, sodium pyruvate. Vitamin E <pC

- tocopherol ) , ethylene glycol bis-N, N, N, N ' - t e t r a -

acet ic acid and trypan blue were procured from Sigma

Ch*emical Company, S t . Louis, U.S.A. 2-Thiobarbi tur ic

acid from B.D.H. Chemical Company,England. Other

chemicals were procured from e i ther Glaxo Laboratories ,

Bombay or Sisco Research Laboratory, Bombay and were

used without fur ther p u r f i c i a t i o n . P e r i s t a l t i c pump,

thermostatic water . bath with moderate shaker,

centrifuge and carbogen gas cylinder were the basic

requirements for perfusion and i so l a t i on of v iab le

c e l l s .

Source of Dust and Pr^aration of Part ic le Size

Source and method for p a r t i c l e s i z e preparat ion

was same as described in the previous Chapter.

Method of Cell Isolation •

The technique described i s based on l i ve r

perfusion with collagenase af te r removal of Ca** by

preperfusion with a chelator (Moldeus e t a l . , 1978)

having cer ta in modificat ions.

55

S o l u t i o n s P r ^ a r a t i o n

T h r e e d i f f e r e n t b u f f e r s w e r e u s e d c o n s e c u t i v e l y

d u r i n g i s o l a t i o n .

B u f f e r A

I t was mod i f i ed Hanks b u f f e r , pH 7 . 4 , ( N a C l - 8 . 0

g, * K C l - 0 . 4 g, MgS04.. 7 HjiO-0.2 g, Na^^HPO^. 2H;^O-0.06 g,

KH^PC^O.Oe gm, NaHC03-2.19 g i n a l i t e r ) c o n t a i n i n g 0 . 5

mM EGTA and 2% BSA.

B u f f e r B

I t was t h e same m o d i f i e d Hanks b u f f e r c o n t a i n i n g ±±

0.12% c o l l a g e n a s e and 4 mM Ca .

Buffer C

K r e b s - H e n s e l e i t b u f f e r , p H - 7 . 4 , ( N a C l - 6 . 9 ; KCl-

0 .36 g; KH;^PO^-0.13 g , MgSO^. 7H2,O-0.29 5 g; CaCl;^.H;iO -

0 . 3 7 5 g; NaHC03 - 2 . 0 g i n a volume of one l i t r e )

c o n t a i n i n g 2% a l b u m i n .

A l l t h e s o l u t i o n s w e r e b u b b l e d w i t h c a r b o g e n g a s

and warmed up t o 37 "C p r i o r t o u s e .

S u r g i c a l Procedure

A l b i n o r a t s w e i g h i n g 2 0 0 - 2 5 0 grams w e r e u s e d

t h r o u g h o u t t h e s t u d y . The r a t s w e r e a n e s t h e t i z e d w i t h

56

ether and the per i tonea l cavi ty was opened by a mid

ventral inc i s ion .

A loose l i g a t u r e was applied around the p o r t a l

vein, an oblique incis ion was then made in the

mesenteric p a r t of the vein (vena mesenteric supe r io r ) ,

and the cannula was immediately inse r t ed . The cannula

wafe f i r s t fixed with a clamp and then secured with

l i g a t u r e . The perfusate flow ra t e was then adjusted to

l e t the l ive r resume i t s normal shape.

Perfusion and Washing Procedure

The perfusion was started with buffer A. To avoid

perfusing bubbles into the liver, the buffer was

permitted to drip out of the cannula before cannulation

of the vein. The vena cava is immediatly cut below the

right renal branch and cannula ted. The suture around

the vena cava is then tightened and flow rate was

increased to 20 ml per minute for 4 minutes. The liver

should swell at this point. In some cases gentle

massaging of the liver may be necessary for complete

clearing.

The flow is then switdied to the 37 "C buffer B

(collagenase solution) for 6 min. During this period

the liver is covered with sterile cotton wetted with

sterile saline. At the end of perfusion, the liver

appeared swollen and pale, but no blebs were seen on

the surface.

57

I so la t ion of Hepatocytes

The l iver was excised by f i r s t removing the

vent r ic le and i n t e s t i n e followed by l ibe ra t ion of the

l i ve r from the diaphragm and, f i n a l l y , cut t ing any

remaining attachments to veins or t i ssues in the

abdomen. The l ive r was then immersed in buffer C in a

beaker, the capsula was cut open and the ce l l s were

dispersed with a pa i r of sc i ssors and gent le s t i r r i n g

movements. Within 2 minutes, a t ambient temperature,

the dispersed ce l l s were f i l t e r e d through cotton gauze

to remove remaining connective t i s s u e and clumps of

c e l l s . The f i l t r a t e was col lected in a beaker. Within

2-3 minutes, a t ambient temperature, the ce l l s s e t t l e d

to form a loose p e l l e t and the supernatant was removed

by asp i ra t ion . The ce l l s were brought to 50 ml with

buffer and resuspended by p ipe t t ing with a wide bore

p i e t t e and were then transfered in to centrifuge tubes.

The ce l l s were centrifuged a t 50 x g for 9 0 seconds and

the supernatant was discarded. The p e l l e t was gently

resuspend in suspending medium with a wide bore

p i p e t t e . The preparat ion should be pr imar i ly a s ingle

ce l l suspension.

Viabi l i ty of Isolated Hqpatocytes

Viab i l i ty of c e l l suspension was rout ine ly

estimated by trypan blue s ta in ing p r io r to each

58

experiment. The v i a b i l i t y of the ce l l s used in the

present study was 80-90%.

Trypan Blue Exclusion Test

The number of v iable ce l l s in each batch was

estimated by counting a suspension tha t had been

dxluted 100 fold in Krebs-Henselei t ' s buffer containing

0.5% trypan blue. Within 5 minutes, dead ce l l s take up

dye iden t i f i ed by s ta ined nuc le i .

Incubation of Isolated Hepatocytes with Dusts

Cells were suspended a t the f i na l concentration

of 1x10 ce l l s /ml . Cell suspensions were incubated with

dust saitples of known concentration a t 37 °C in a

metabolic shaker with moderate shaking. Aliquots were

taken a t r equ i s i t e time in te rva l s and est imations were

car r ied out.

Lactate Dehydrogenase Assay

The a c t i v i t y of l a c t a t e dehydrogenase was

monitored in an a l iquo t of c e l l free medium by the

method of Wootton (1964). The c e l l free medium was

obtained by centr i fugat ion a t 1200 rpm for 5 minutes.

In br ief , 2.7 ml of phosphate buffer (0.1 M, pH 7.4)

was taken in a cuvet, 0.1 ml of c e l l free medium and

59

0.1 ml NADH (0.002 M) was mixed. Add 0 .1 ml of sodium

pyruva te (O.OlM) and read the o p t i c a l dens i ty a t 30

second i n t e r v a l s for 3 minutes a t 340 nm, us ing

Spec t ron ic 2000 Spectrophotometer ( Bausch and Lomb) .

Zero the ins t rument wi th a blank of 0.1 ml c e l l f r e e

medium, 2.7 ml phosphate bu f f e r and 0.1 ml NADH

s o l u t i o n , mixed and pe rmi t t ed to s t and a t room tempera­

t u r e .

A u n i t of LDH a c t i v i t y i s def ined as an o p t i c a l

d e n s i t y change of 0.001 p e r min, m u l t i p l i e d by a

tempera ture dependent of c o e f f i c i e n t of 1.25, s i n c e t h e

r e a c t i o n was performed a t 25° i n s t e a d of the s t a n d a r d

37° .

Lipid Peroxidation

Malonaldehyde (MDA) format ion was measured in

c o n t r o l and t r e a t e d h e p a t o c y t e s suspens ion by t h e

procedure of Wright et al_. (1981) . 1 ml of a l i q u o t was

taken and r e a c t i o n was t e rmina ted by t h e a d d i t i o n of

0.3 ml 5N HCl and 1 ml 10% TCA ( t r i c h l o r o a c e t i c a c i d ) .

Mixture was shaken and 1 ml 2% t h i o b a r b i t u r i c ac id

(TEA) was added. I t was incubated a t 90 "C for 20

minutes , cooled in i c e , c e n t r i f u g e d a t 2000 rpm fo r 10

minu tes . Pink colour was read us ing Spec t ron ic 2000

Spectrophotometer a t 535 nm a g a i n s t a r e a g e n t b l ank .

60

Glutathione Reduced (GSH) Estimation

I n t r a c e l l u l a r g luta thione (reduced) was estimated

in the c e l l p e l l e t s of control and t rea ted hepatocytes

by the method of Sedlack and Ludsay (1968). Cells were

lysed with 1.8 ml of EDTA solut ion (1 g / l i t r e ) and 3 ml

of p r e c i p i t a t i n g reagent (1.67 g metaphosphoric acid,

02. g EDTA, 30 g NaCl dissolved in d i s t i l l e d water,

made up the volume with d i s t i l l e d water to 100 ml) was

added. After mixing, the solut ion was allowed to stand

for f ive minutes before being f i l t e r e d . Two ml of

f i l t r a t e was added to 4 ml of disodiura hydrogen

phosphate (0.1 M, pH-8.0) and 1 ml of DTNB reagent (40

mg of 5, 5 ' - d i t h i o b i s ( 2-ni trobenzoic acid) i s dissolved

in 100 ml of l O g / l i t r e of sodium c i t r a t e ) . A blank was

prepared from 1.2 ml of p r e c i p i t a t i n g reagent 0.8 ml

of EDTA solu t ion , 4 ml of disodium hydrogen phosphate

and 1 ml of DTNB reagent . The colour was immediately

read a t 412 nm with the help of Spectronic 2000.

S t a t i s t i c a l Analysis

Values represent mean +_ S.E. of s ix determinants .

Experiments were repeated four t i n ^ s .

RESULTS

E f f e c t o f Dust C o n c e n t r a t i o n on L a c t a t e Dehydrogenase R e l e a s e d frcm t h e I s o l a t e d Rat H e p a t o c y t e s

Lactate dehydrogenase re leased from hepatocytes

t reated with wol las ton i te and ch ryso t i l e with d i f f e r en t

61

dust concentrat ions are given in Figure 18. Results

show tha t LEH released from the hepatocytes increased

with dust concentrat ion up to 100 yug/ml with a l l the

v a r i e t i e s . I t shows i t s concentrat ion dependent re lease

of the enzyme but a t 500 ng/ml a decrease in LDH

a c t i v i t y was observed. The exact cause of the low

acH:ivity of enzyme i s not c l e a r . I t i s assumed t h a t

the p ro te in (enzyme or any other pro te in) adsorb on the

surface of the mineral f ib res r e s u l t s in the low

a c t i v i t y of the enzyme. The a c t i v i t y was 93, 87, 86 ,

88% a t 50Ckig/ml when cortpared to the value a t 100

>ug/ml, which was considered, as 100% for ch ryso t i l e ,

kemolit A-60, kemolit-N-and kemolit ASB-3, respec t ­

ive ly .

Kinetics of Lactate Dehydrogenase Released from the Isolated Rat Hepatocytes

The re lease of l a c t a t e dehydrogenase from

hepatocytes t rea ted with ch ryso t i l e and wol las ton i te

dusts a t d i f f e ren t time in t e rva l s i s depicted in Figure

19. The r e su l t s show s i g n i f i c a n t re lease of l a c t a t e

dehydrogenase (Lie) from hepatocytes a t 3 hours of

incubation with these dus t s . The re lease of enzyme was

time-dependent and increased with incubation time. The

ch ryso t i l e , k e n o l i t A-60, kemolit-N and kemolit ASB-3

relesed about 418, 303, 234 and 177% more LCe,

respec t ive ly , over t he i r controls a t 2 hours of

. to

i- l (S)

u

o

I

0) CO (0 0)

i H 0) S-i

g

40€^

3Cr(i&

20aDO

lOQ©-

'-^+ C h r y s o t i l e

,' / A ^^ y

^' ^^ y • - ' 1 - ^

/ ' /

/ y

/ •

r ^

x V / , • " • ' "

y ^ > •

.'

^ ^ ^ /x r ^

^^ ^

^ '

---"

^ .

J-

_^

'--B K e m o l i t A-60

• ^ Kemol i t -N

^"^ K e m o l i t ASB-3

10 — I —

50 100 500

Dus t c o n c e n t r a t i o n ( u g / m l )

Cell concentration 1 X Incubation time - 3

1 0 ^ c e l l s ml

F i g . 1 8 : E f f e c t of d u s t c o n c e n t r a t i o n on LDH r e l e a s e d from i s o l a t e d r a t h e p a t o c y t e s .

3000'-j

1

^ 2500 (Q

^.

c

H

W (0 0) rH

2000

1500

1000

500

J"'

-' > / /

J - - -

J- '

_j Kemolit A-60 Chrysotile

T Kemolit-N

J Kemolit ASB-3

Control

Wi 1 2

Incubation time (h)

Cell concentration ^ 1 X 10 cells/ml

Du s t concentration - 100 /ug/ml

Fig. 19: Kinetics of LDH released from i soa l t ed r a t hepatocytes

62

Incubation. While a t 3 hours of incubation, maximum

LDH was estimated with kemolit A-60. I t was marginally

above the chryso t i l e , which might be due to the

adsorption of prote in (enzyme) on mineral f i b r e s .

Effect of Dust Concentration on Lipid Peroxidation in Isolated Rat Hepatocytes

Isola ted hepatocytes incubated with d i f f e ren t

dust concentrat ions, for 3 hours , to measure the

malonaldehyde formation by th ioba rb i tu r i c acid reac tan t

method are depicted in Figure 20. Results show

s ign i f i can t increase of malonaldehyde formation by a l l

the tes ted samples upto the concentrat ion of 100 ^g/ml

aga ins t control . This increase was dose-dependent,

while fur ther increase in dust concentrat ion caused no

effect on l ip id peroxidat ion. At the dust concentra­

t ion of 100 Aig/ml, maximum values of malonaldehyde were

observed and they were increased by 490, 514, 586 and

927% for kemolit ASB-3, kemolit-N, kemolit A-60 and

dhryso t i le , respect ively aga ins t cont ro l . At the above

compared cocnentration, malonaldehyde formation was

353, 341, 284, and 269% higher for kemolit ASB-3,

kemolit-N, kemolit A-60 and ch ryso t i l e sample,

respec t ive ly in comparison to the values of 10 /ug/ml

concentrat ion. At 500yug/ml of dust concentrat ion, a

s l i g h t decrease in malonaldehyde formation in

comparison to 100 ^g/ml was observed, vi^ich has l imited

the use of 100/(g/ml of dus t only for fur ther s t u d i e s .

E C

in CO in

(0

Q O

C -H

0) CT> C rO

F i g . 2 0 :

.4^

3

.2

1.

0+

I-

J

.L

—J- C h r y s o t i l e

T k e m o l i t A-60

- ^ k e m o l i t - N

k e m o l i t ASB-3

10 50 100

Dus t c o n c e n t r a t i o n

500

(/ug/ml cell suspension)

Cell concentration- 1 X 10 cells/ml Incubation time - 3 h

Effect of dust concentration on LPO in isolated rat hepatocytes.

63

Kinetics of Lipid Peroxidation in I so la ted Rat Hepatocytes

The l ip id peroxidation in isola ted hepatocytes

induced by wollas toni te and chryso t i l e dust a t

d i f fe ren t time in tervals are depicted in Figure 21 .

Results c lea r ly show tha t l i p id peroxidation increased

with the incubation time, indicat ing time dependent

peroxidation of polyunsaturated f a t t y acids (PUFA).

Here a l so the induction of LPO in hepatocytes by these

dusts a t d i f fe ren t time in te rva l s followed the same

order as oberved, in previous chapter, with human

ery throcytes . The peroxidation of PUFA was increased

by 413, 448, 504 and 720% for kemolit ASB-3, kemolit-N,

kemolit A-60 and chryso t i l e , respect ive ly , aga ins t

cont ro l . The malonaldehyde formation was decreased by

43, 38 and 30% for kemolit ASB-3, kemolit-N and kemolit

A-60 respec t ive ly , when ccattpared to ch ryso t i l e . These

findings indicate less peroxidat ive damage of

hepatocytes by wol las toni te than ch ryso t i l e .

Conparative Lipid Peroxidation in Iso la ted Rat Hepatocytes Induced by Different Ehist Samples

The effect of ch ryso t i l e and wol las ton i te samples

on l i p i d peroxidation of r a t hepatocytes a f t e r three

hours of incubation i s depicted in Figure 22. A

s ign i f i can t increase in l i p id peroxidation was observed

by a l l these dus ts . The order of l i p i d peroxidation

induced by these dusts i s as follows: ch ryso t i l e

. 5

e c in

If)

+ 0

O

c •H

C

u

44

3,

.2,i

I-

J •

J -

S=''i I-O . A ^

60 120

J C h r y s o t i l e

4-Kemol i t A-60 K e m o l i t - N

K e m o l i t ASB-3

-J

18 0

I n c u b a t i o n t i m e ( i n m i n u t e s )

Dus t c o n c e n t r a t i o n - 1 0 0 / u g / m l

C e l l c o n c e n t r a t i o n - I X 1 0 ^ c e l l s / m l

F i g . 2 1 : K i n e t i c s o f LPO i n i s o l a t e d r a t h e p a t o c y t e s

(U l-l -r-'

c CO

u

B c in

in

•P ro

C

•i-t

0)

c

2r

CO I

m en <

0 e 0)

X

-r-'

o e

O VO I

<

•rJ i- l

i

J

0 1-1 4-'

c 8

• • " , '

. . Xi

4

. . \ .N 1

\-^:i % ^

S - 1

.1

.

1

1 ^ \

^ ^ 1

^ ' • ^ ^ ^ 1 - 1

1

^ T

- , > •.

1 1

Treatments Incubation time - 3 h

Dust concentration -Fig. 22: Comparative LPO in i so l a t ed r a t

induced by d i f fe ren t dusts

100 ug/ml

hepatocytes

64

(Indian) > kemolitA-6D > kemollt-N > kemollt ASB-3.

The r e su l t s showed the less induction of l i p id

peroxidation by wol las ton i te than c h r y s o t i l e . More­

over, kemolit A-60, kemolit-N and kemolit ASB-3 induced

about 38, 40 and 49% less l i p id peroxidat ion respect ­

ively incorrparison to ch ryso t i l e .

Kinetics of Glutathione (Reduced) Content of I so la ted Rat Hepatocytes

Effect of time on gluta thione (reduced) (GSH)

content of hepatocytes incubated with and without

mineral f ibres i s recorded in Figure 23. Results

c l ea r ly show t h a t deplet ion of i n t r a c e l l u l a r

glutathione i s time dependent. The -values decrease with

the time of incubation. A s ign i f i can t deplet ion of

i n t r a c e l l u l a r GSH in dusts t rea ted ce l l s was seen

v^tiile a marginal and ins ign i f i can t decrease was a lso

noticed in untreated c e l l s . At 3 hours of incubation,

GSH was depleted by 53, 42, 51 and 3 5% for c h r y s o t i l e ,

kemolit A-60, kemolit-N and kemolit ASB-3, respec t ive ly

in comparison to con t ro l . • These values, a t 3 hours of

incubation were less in comparison to t h e i r respec t ive

values a t i hour of incubation and were decreased by 6,

28, 38, 23, 32% in con t ro l , kemolit ASB-3, kemolit-N,

kemolit A-60 and ch ryso t i l e , respec t ive ly . This

c lea r ly indica tes a time dependent deplet ion of

i n t r a c e l l u l a r g lu ta th ione (reduced) content of

49-

E (0

V

o

a:

a) i-i i

39

341

29-i

2 4 i

--I

•I • I

• • • I .

t

---L

-4.

- - - - -J Untreated,

•I

191 — — 0 1 2 3

Incubation time (h)

Dust concentrat ion 100 Aig/ml Fig . 23: Kinet ics of g luta th ione (reduced) content of

i so l a t ed r a t hepatocytes

^i Kemolit ASB-3

""••4 Kemolit A-60

"-T Kenolit-N Chrysoti le

65

hepatocytes. The depletion of g lu ta th ione Is also

re la ted to the t ox i c i t y of the mineral f i b r e s .

Effect of Chrysotile and Wollastonite Dusts on Glutathione (Reduced) Content of Isolated Rat Hepato­cytes

The values of i n t r a c e l l u l a r g lu ta th ione content

in* i so la ted r a t hepatocytes incubated with ch ryso t i l e

and wol las toni te dus ts , a t the f ina l concentration of

100 yug/ml, for 3 hours are depicted in Figure 24. I t is

clear from the r e s u l t s tha t g lu ta th ione (reduced)

depleted s ign i f i can t ly frcffii the t rea ted hepatocytes in

comparison to t he i r respect ive control (un t rea ted) .

The order of the i r GSH depletion was as follows:

chryso t i le > kemolit-N > kemolit A-60 > kemolit ASB-3

and i t was decreased by 57, 54, 48 and 42% respect ively

in comparison to con t ro l . I t i s again evident from the

resu l t s tha t the chryso t i l e i s the most toxic and

kemolit ASB-3 the l e a s t toxic mineral f i b re amongst

those used in the p resen t study. The GSH content

agains t chryso t i le i s 135, 107, and 121% more for

kemolit ASB-3, kemolit-N and kemolit A-60, respect­

ively.

Effect of Vitamin E (©(.-tocopherol) Concentration on Lipid Peroxidation in Isolated Rat Hepatocytes

0^-tocopheral i s the most b io log ica l ly ac t ive and

abundant of the four ioC, ^ , ^ and / ) tocopherol

m

49-

(D U

-P (0 0)

+-' c D

03 iH

0)

o vo

o

X

0)

o

F i g . 24;

444

3 9 -

34-

29 i

24 -!

19

r ^

-1

ro I

CQ CO

<

o E

h

o E

o VD

J <

t - l

i

X

Treatments

Incubation time

Dust concentration

0)

-w 4-' C 0)

3 h

100 Aig/ml

Effect of c h r y s t i l e and wol las ton i te dusts on gluta thione (reduced) content of i so la ted r a t hepatocytes

66

conpounds collectively referred to as vitamin E. This

membrane - associated chain breaking phenolic

antioxidant is generally recognised as one of the major

antioxidants present within biological systems.

Vitamin E functions as trap for lipid peroxy (LOO) and

other radicals, effectively inhibiting the peroxidation

of cellular membranes.

In view of its antioxidant properties, different

concenrations of CKL"tocopherol was taken to find the

most effective concentration for the follow up studies.

Figure 25 shows that 0.1 mM concentration of

tocopherol is suitable for the studies in our systems.

Below this levelo(rtocopherol may protect poorly and

will be rapidly consumed. At higher concentration of

oG-tocopherol the oxidation chains can be terminated and

the rate of removal of o<!-tocopherol will be low. This

will in turn minimize the need to regnerate reduced oC-

tocopherol and to remove any lipid peroxides by

auxiliary protective mechanisms.

Effect of (?C-tocopherol on Lipid Peroxidation in Hepatocytes Induced by Mineral Fibres

It is well documented that lipid peroxidation is

a free radical generated process. oC-tocopherol is the

principal biological defence against lipid

peroxidation. It is located in biomenibranes and has

the capacity to scavenge free radicals. The lipid

. 4

E C

IT)

IT)

Q

C •K

<U

c m

. 3

. 2

. 1

OD

Chrysoti le t rea ted - ^ i Control-s- B

1

\ \ \ /

.01 .05 .1 .25 .5 1.0 2.5 5.0

Concentration of oC-tocopherol

Incubation time - 3 h

Dust concentration - 100/ug/ml

Fig . 25: Effect of OC-tocopherol (vitamin E) concentrat ion on LPO in i so la t ed r a t hepatocytes

67

peroxidation in hepalocytes induced by mineral f Lbreb

(dusts) a t 3 hours of incubation with and without oL~

tocopherol i s given in Figure 26. The r e s u l t s c l ea r ly

show tha t 06- tocophero l , which i s a free rad ica l

scavenger, i s very much effect ive in p ro t ec t ing the

peroxidat ive damage of membrane l i p i d s . I t was noticed

tl\at ^ - tocophero l p r o t e c t s l i p id peroxidation to a very

great extent . The values of malonaldehyde, a TEA

reac tan t species formation, was observed c lose to the

control values . This indica tes tha t (Krtocopherol i s

ccsnpletely p ro tec t ing the l i p i d peroxidat ion by

scavengering the free r a d i c a l s .

Effect of OC-tocopherol on Glutathione (Reduced) Content of HepatocyteS

The r e s u l t s of g lu ta th ione content of hepatocytes

incubated with dusts in the presence and absence of e><^--

tocopherol are depicted in Figure 27. The values

c lea r ly show t h a t cC-tocopherol i s inef fec t ive in

preventing the efflux or oxidation of i n t r a c e l l u l a r

gluta thione (reduced) (GSH).'

Effect of Methionine on Lipid Peroxidation in Isolated Rat Hpatocytes

Methionine i s a sulphur containing amino ac id . L-

methionine condenses with ATP, forming S-adenosyl-

methionine or "act ive methionine". The ac t iva ted S-

51

without (X.- tocopherol A

with CC- tocopherol B

e d

in CO

in

D

O c

<u c (0

6 1,

Treatments

X . J

I . . .

-^^

^ ^ >1L

I ='> ^«.%3_

1X4 "0 (U -> to 0)

b c D

CO 1

ffi CO < +i •H t-i 0 e 0) !

Fig. 26:

Incubation time Dust concentrat ion

Effect of oC-tocopherol on induced by dus ts .

2; 1

+J -H tH

0 g 0) u:

o l

o \o 1 <: •»->

-H .H 0

e

-

-

o n LPO

0) i H -r! 4-» 0 X > i

u 6

3 h 100 Aig/ml

i n h e p a t o c y t e s

0)

<J) o

0) tH 0 E

Without fX-tocopherol ,

With (X.-tocopherol I

Treatments

Fig. 27:

Incubation time Dust concentrat ion -

Effect of OC - tocopherol on GSH hepatocytes t r ea t ed with dus t s .

3 h

100 ^g /ml

content of

68

methyl group may transfer to various acceptor compounds

and therefore also involved in biosynthesis of

cysteine. Cysteine is linked with N-terminal of

glutamate in glutathione, an atypical tripeptide. In

view of its correlation with glutathione,methioneine

was used, as an exogenous source, for the study.

Figure 28 shows the conparative lipid

peroxidation in the presence and absence of methionine

caused by dusts. Results clearly indicate that

methionine has inhibited lipid peroxidation in

wollastonite and chrysotile treated cells. The

decrease in malonaldehyde formation was very

significant. It was inhibited by 72, 62, 64 and 74%

for kemolit ASB-3, kemolit-N, kemolit A-60 and

chrysotile, respectively, in methionine containing dust

treated cells when compared to dust treated cells only.

A slight decrease in lipid peroxidation of control

cells, in the presence of methionine, was also noticed,

although the value was not up to the significant level.

It shows the relationship between the malonaldehyde

formation and methionine present in incubation medium.

Effect of Methionine on Glutathione Content of Hepatocytes Treated with Dusts

Figure 29 shews effect of methionine on GSH

content of hepatocytes incubated with dust for 3 hours.

Results clearly indicate that addition of methionine

E C

in n in

c -H

to

Fig . 28

. 5

> 1

,1 .

With methionine A Without methionine B

Treatment ^

* , '>.)

i . \

i 3

T .

cX, N

v ' - ^ N : ^ ^

JL Nf S^

-o 0) •p (0 0) ij c D

n 1 cb w < •p -M i-l

0 e

a 1

•p

•H •-H 0 E <u »<::

1 J

-si

Ni

A

\

Incubation time Dust concentration

Effect of methionine on LPO in i s o l a t e d hepatocytes induced by dusts

H E

o vo 1

< 4-> -H .-1

0 E Ui

-

0) iH •rJ

4J 0 CO

6

3 h 100 ^ g / m l

r a t

V

^

0) i-i

0 E

Without methionine 1_,'

Wxth methioninej -. -

Treatments

-0 0) +> (0 0)

+> c D

n 1

CQ to < ^J -H i H

0)

13 1

•P -H ^ g 0) K

o VO 1

< ^J -H >H

i^

Incubation time

Dust concentration

•P

o

3 h

F i g . 2 9 :

l O O ^ g / m l

Effect of methionine on g lu ta th ione (reduced) content of hepatocyte t rea ted with dusts

69

i nc reases the GSH c o n t e n t in coitparison t o the c e l l s

incubated w i thou t me th ion ine . The i n c r e a s e in GSH

con ten t in h e p a t o c y t e s incubated wi th meth ionine was

139, 126, 126, 115% for k e m o l i t ASB-3, kemol i t -N,

kemol i t A-60 and c h r y s o t i l e , r e s p e c t i v e l y when the

c e l l s w i thou t meth ion ine were cons ide red as 100%.

These values a l s o show the maximum l e v e l of GSH for

kemol i t ASB-3 and minimum for c h r y s o t i l e .

DISCUSSION

I s o l a t e d h e p a t o c y t e s have been used over r e c e n t

years in va r ious f i e l d s of i n v e s t i g a t i o n s i n c l u d i n g

c e l l u l a r t o x i c i t y . The h e p a t o c y t e s p rov ide a system

which i s b iochemica l ly q u i t e s i m i l a r t o i ^ v ivo i s

gene ra l l y accep t ed . Since the i s o l a t i o n of the

hepa tocy t e s i nc ludes r a t h e r d r a s t i c p rocedures such as

p r o t e o l y t i c d i g e s t i o n , d i s r u p t i o n by t e a r i n g and

sed imenta t ion of the c e l l s under subopt imal oxygenat ion

yftiich may r e s u l t in damaging the c e l l s . The most

popular t e s t f o r d e t e r m i n a t i o n of c e l l v i a b i l i t y i s

trypan b lue exc lus ion t e s t . I t i s very easy and r a p i d

to perform under l i g h t microscope a t t h e beg inning of

the exper iment . This i s only an i n d i c a t i o n of a l t e r e d

p e r m e a b i l i t y of the p e r i c e l l u l a r membrane b u t no t a

v a l i d index of me tabo l i c competence (Krebs e_t a l » ,

19 79 ) .

70

The oxidation of NADH added to the ce l l

suspension or the presence of cytosol ic enzymes ( e . g . ,

l a c t a t e dehydrogenase) in the ex t r ace l l u l a r medium are

a lso indica tors of plasma membrane damage. Giao e_t a l .

(1988), therefore, have suggested t h a t the estimation

of i n t r a c e l l u l a r l ac t a t e dehydrogenase (Lffl) a c t i v i t y

i s a d i r e c t and simple measure of cy to tox ic i ty .

Furthermore, a l inear co r re la t ion (r=0.92) between the

i n t r a c e l l u l a r LEH and the number of trypan blue

excluded c e l l s in 25 l ive r preparat ions has previously

been reported by Jauregui et aj.. (1981). The number of

viable c e l l s thus determined should be regarded as

viable average equivalents , since p a r t l y v iable / in jured

ce l l s may r e t a in p a r t of t he i r LEH content .

The continuously increasing but almost iden t ica l

percentage of ce l l s re leas ing LEH and taking up trypan

blue indica tes tha t the b a r r i e r function of the plasma

membrane collapses in a ce r t a in number of c e l l s which

accept trypan blue. Based on these r e s u l t s i s the

concept t h a t the majority of c e l l s or a l l of them s e t

free a small amount of t h e i r LDH, which appears to be

very irtprobable. Only those c e l l s which are ser ious ly

damaged seem to lose t he i r t o t a l LEftJ content . The

escape of LDH should be an indica tor for the re lease of

many other proteins from the cytoplasm of hepatocytes

and might be viewed as an expression of a beginning of

ce l l nec ros i s .

71

The occurrence of l ip id peroxidation (LPO) in

isola ted hepatocytes was f i r s t demonstrated by Hogberg

e_t aJL. (19 753). LPO in i so la ted r a t hepatocytes induced

by asbestos mineral f ib res has already been reported

{Rahman and Casciano, 1985) . They have corre la ted LPO

with the carc inogenic i ty of mineral f ibres and

concluded tha t free r a d i c a l s , superoxide, are involved

in the tox ic i ty of the f i b r e s . This a lso supports

present f indings. This process , LPO, was associated

with a decrease in hepatocyte gluta thione (GSH)

content. Previous s tud ies have provided s t i l l more

evidences for the existence of these changes in

ce l lu l a r biochemistry (Hogberg ejt al^., 1975b) and have

associated them with c e l l u l a r injury (Hogberg £ t a l . ,

1975c, Hogberg and kr i s to fe rson , 1977). I t has been

suggested tha t the drug-induced LPO in i so la t ed

hepatocytes, r a ther than a loss of GSH, i s d i r e c t l y

responsible for subsequent c e l l u l a r damage (Anundi ejt

a l . , 1978, 19 79).

I t has been found tha t mineral f ibres induced

damage, enhanced l i p i d peroxidation, and r e s u l t s in

ce l l u l a r depletion of GSH and subsequent c e l l u l a r

l y s i s . Of fundamental importance for the proposed

toxicological mechanism i s the bel ief t h a t GSH pro tec t s

agains t LPO in the metabolically ac t ive c e l l s . A

causal r e la t ionsh ip between LPO and c e l l u l a r l y s i s was

indicated by the t i g h t coupling between these two

events .

72

Cell injury may be i n i t i a t e d by one or several

possible mechanisms, each p a r t i c u l a r to the i n i t i a l

i n su l t and following d i s t i n c t pathway. After an

extensive i n s u l t , the stages along the pathway pass a

point of i r r e v e r s i b l e damage from which recovery i s

impossible. This i r r e v e r s i b l e reac t ion is character­

ised in general by a loss of membrane i n t e g r i t y and

collapse of ce l l morphology (Trump £ t a_l. , 1981).

LPO is a se l f propagating process of membrane

l ip id degeneration to numerous by-products. In br ief ,

the mechanism of LPO involves the abs t rac t ion of

hydrogen atom from membrane l i p id by an i n i t i a t i n g

factor (pr imar i ly oxygen free rad ica l s ) crea t ing a

l ip id a lkyl rad ica l L*(Kappus, 1985). Addition of

oxygen to th i s rad ica l produces the l i p i d peroxy and

SLlivyl r ad ica l s (LOO* and LO ) vAiich can themselves

abs t r ac t hydrogen from other l i p i d s , thereby

propagating the process .

Mammalian ce l l s have evolved numerous mechanisms

to e i the r prevent or ever t injurious events t h a t might

r e su l t from toxic chemicals and normal qxidat ive by­

products of c e l l u l a r metabolism. Foremost, among these

endogenous p ro tec t ive systems i s the g luta th ione redox

system. Glutathione i s present in high concentrat ions

(general ly in millimolar range) in most mammalian c e l l s

as reduced glutathione (GSH) , with minor f rac t ions made

up of oxidised gluta th ione (GSSG), mixed d isu l f ides of

73

GSH and other c e l l u l a r t h i o l s , and minor amounts of

other th ioethers (Kosower and Kosower, 1983). GSH acts

both as a nucleophil ic "scavenger" of numerous

compounds and the i r metaboli tes, via enzymatic and

chemical mechanisms, forming th ioe ther bonds with

e l ec t roph i l i c cent res , and as a cofactor in the GSH

peroxidase mediated des t ruc t ion of H ^ ^ . The depletion

of g luta th ione levels can readi ly impair the ce l l s

defense aga ins t the toxic actions of such compounds and

against the oxidat ive damage from H^Oo, and may lead to

rapid demise of the c e l l and ce l l death. (Reed and

Far iss , 1984, Moldeus and Quanguan, 1987).

In addi t ion to the t h io l redox system, several

endogenous ant ioxidants serve to p ro tecc t the c e l l

against oxidat ive damage (Sies and Cadenas, 1983).

Both the metabolism of cer ta in foreign compounds,

p r inc ipa l ly redox ac t ive compounds, and the enzymatic

ca ta lys i s of Ho Op may r e s u l t u l t imate ly in the

generation of the oxygen r ad ica l s , superoxide anion

(O^ ) and hydroxyl rad ica l (OH). The l i p o p h i l i c

ant ioxidant , 0^-tocopherol (Vitamin E) , i s the primary

p ro tec t ive fac tor aga ins t such at tack on c e l l u l a r

membranes. C'C-tocopherol i nh ib i t s LPO process via the

donation of hydrogen atom to the l i p id r a d i c a l s ,

forming the hydroperoxide (LOOH) and the alccihol (LOH)

and thereby terminating the propagative process (McCay,

1985). I t i s therefore termed as a chain breaking

ant ioxidant .

74

Since a balanced . Intracellular thiol redox sys tern

is important for cell viability, its relationship to

o(r tocopherol during protection against mineral fibres

induced oxidative stress was also investigated. The

prevention of lipid peroxidation and subsequent efflux

of GSH precursors by supplementation of PC-tocopherol

levels from endogenous sources has already been

reported (Pascoe ejt a]^. , 1987). Although this action

surprisingly does not affect the efflux or oxidation of

intracellular GSH, it does maintain intracellular and

mitochondrial GSH at initial levels and subsequently

prevents the loss of ProSH (Reed et a_l. , 198 7). In

these studies intracellular GSH and ProSH levels appear

to be dependent on the content of cellular cK- -

tocopherol. Methionine is known to stimulate GSH

synthesis in liver (Hogberg and Kristoferson, 1977).

Anundi et al_. (1979) have shwon that addition of

methionine in incubation medium results in increased

GSH during the third and fourth hours of incubation

while MDA accumulation and cell lysis were inhibited.

In the present study similar effects as reported

by Rahman and Casciano (1985) were obtained with OC-

tocopherol. By addition of this naturally occurring

antioxidant, it was possible to preserve the cells in a

seemingly intact state- for up to four hours, despite

the fact that GSH was depleted. A similar conclusion,

as Anundi et_ al . (1979), can be drawn from the

experiments where methionine was added.

75

The (?<--tocopherol content of i so la ted hepatocytes

underwent a gradual, time dependent depletion during

incubation in a vitamin E free Krebs-Henseleit buffer

(Sandy e_t ^ . , 1988). A s imilar decrease in c e l l u l a r

oir tocopherol had previously been described for

hepatocytes incubated in vitamin E free modified

F i sne r ' s medium (Far iss ejt a_l. , 1985). This

consumption of cX-tocopherol may be a r e s u l t of the

incubation condit ions, which may r e s u l t in a moderate

back ground r a t e of LPO within control hepatocytes . I t

has been suggested t h a t LPO does not proceed u n t i l DC ~

tocopherol has been s u b s t a n t i a l l y depleted (Cadenas e t

a l . , 1984; Hi l l and Burk, 1984). The mechanism of o(-

tocopherol mediated maintenance of i n t r a c e l l u l a r GSH

levels in -Oiis model i s not c l e a r . A d i r e c t act ion of

p^:-tocopherol on the GSH biosynthe t ic process appears

equally unl ikely , s ince none of the enzymes a re

membrane associated. In s p i t e of the lack of

tocopherol prevention of the efflux or oxidation of GSH

i t s e l f , most evidence based on i t s mechanism of LPO

inh ib i t ion and re la ted membrane damage, would suggest

tha t the effect of c?C-tocopherol on GSH biosynthesis i s

secondary to i t s prevention of increased oxida t ive

po ten t i a l within the c e l l . I t can thus be summarised

tha t our r e su l t s s t rongly support the thesis t h a t GSH

def ic ien t i so la ted hepatocytes can undergo LPO rapidly

enough to destroy the c e l l s .

CYTOGENETIC EFFECTS OF WOLLASTONITE ON HUMAN LYMPHO­CYTES: IN VITRO STUDY

CHAPTER-III

INDTRODOCTION

There is a growing concern about the possible

mutagenic and carcinogenic effects of genotoxic agents

to which human populations are exposed either

occupationally, accidently or by life style. Both

occupational and environmental monitoring are the areas

v^ere genetic toxicology can be applied at a massive

scale. Unfortunately, there are relatively few direct

methods to measure mutations or other forms of induced

damage in humans exposed to potential mutagens and

carcinogens which may be correlated to their _in vitro

toxicity. Infact, genetic toxicology assay offers an

unique opportunity to assess the impact of environmental

pollutants directly at the level of the workers as well

as in laboratory, since the methodology is common in

several situations.

Human peripheral blood lymphocytes provide a

readily available source of cells. They are initially

in a uniform Go stage that can be made to undergo

77

mitot ic d iv is ion in cu l tu re , and a l so provide raetaphase

stage for chromosomal aberra t ions and s i s t e r chromatid

exchanges. These are the bases of most extensively

employed t e s t s to assess the genetic effects in exposed

persons or i_n v i t r o t rea ted c u l t u r e s . Few repor ts on

genotoxici ty of s i l i c a t e dus ts , asbestos, using

stV.mulated per iphera l blood lymphocytes of exposed

persons (Rom ^ al.. , 1983, Rahman e t a l . , 1988a) and in

in v i t r o t rea ted lymphocyte cu l tures (Valerio et ajL. ,

1980, Casey 1983, Rahman e t a l . , 1988b) are ava i l ab l e .

But no repor t ex i s t s on genotoxici ty of wollas t on i t e ,

i . e . , calcium s i l i c a t e . Although, previous s tud ies with

wol las ton i te have exhibi ted i t s cy to tox ic i ty towards

human erythrocytes and i so la ted r a t hepatocytes . In

view of the e a r l i e r s t ud i e s , wol las ton i te was evaluated

for i t s genotoxic p o t e n t i a l and compared to Indian

va r i e ty of ch ryso t i l e , using human per iphera l blood

lymphocytes, Jji v i t r o . Chromosomal aberra t ions and

s i s t e r chromatid exchange was considered as a monitor

for genotoxicity evaluat ions .

MATERIALS AND METHODS

P r e p a r a t i o n o f Dust Samples

Wollastonite was obtained from Director , Wblkem

Pvt. Ltd. Udaipur, Rajasthan, India . Indian ch ryso t i l e

78

was obtained from AnShra Pradesh Mining Corporation

Ltd . , Hydrabad Which was mined from Cuddapah, Andhra

Pradesh, India . The p a r t i c l e s i ze below 30 um used in

the presen t study was prepared by the method of Zaidi

(19 69) . Dust was kept a t 80 °C in an oven for 24 hours .

The suspensions of d i f f e r en t concentrations were

p r ^ a r e d by s e r i a l d i l u t i ons method in autoclaved normal

s a l i n e .

Chemicals

5' bromo-2-deoxyuridine, bisbenzeimide (Hoechst

33258), colchicine and medium RPMI-1640 were purchased

from Sigma Chemicals Conpany,St. Louis, U.S.A., f e t a l

calf serum from Sera Lab U.K., L-glutamine, p e n i c i l l i n ,

streptomycin and phytohemagglutinin-M (PHA-M) were

purchased from Gibco Laborator ies , Grand Island, New

York, U.S.A. Other chemicals used were of ana ly t i ca l

grade with high p u r i t y .

Cytogenetic Study

Culture ccBiditions for blood

Peripheral venous blood was col lected from 30

years old volunteer donar (blood group, B +ve) in a

heparinized tube under s t e r i l i z e d condi t ions . THe whole

blood was cultured following the method of Moorhead e_t

a l . (1960). In shor t , whole blood lymphocyte cul ture was

79

done in RPMI-1640 medium supplemented with f e t a l calf

serum (20%), L-glutamine (0.03%), p e n i c i l l i n (100

lU/ml) , streptomycin (lOO^g/ml), phytohemagglutinin-

M(3%), 5'-brcmo-2'-deoxyuridine (5/ug/ml) and blood (0.3

ml). Dust suspension of di f ferent concentrat ions ( 0 . 1 ,

1, 10 and 100 y«g/ml) were prepared by s e r i a l d i l u t i on

method, equivalent amount of normal s a l i n e was added in

control cu l tu re s . All cul ture via ls were wrapped with

aluminium f o i l and incubated a t 37 °C in 5% COjj and 9 5%

O^ atmosphere.

Harvesting of blood cultures and s l ide preparation

Cultures were harvested a t 48 hours , to study

chromosomal aberra t ions and 72 hours for s i s t e r

chromatid exchange (SCE) ana lys i s . Colchicine (0.1

^g/ml) treatment was given 3 hours p r i o r to harvest ing

to a r r e s t the ce l l s in metaphase. The cul tures were

gently mixed and reincubated for fur ther 3 hours a t 37

"C in 5% COg and 9 5% O; , atmosphere.

After a t o t a l of 48 hours or 72 hours incubation

a t 37 °C, the contents of the cu l tu re v i a l s were

decanted in to 15 ml centr i fuge tube. The v ia l s r insed

with 2 ml of medium which was t ransferred to the

centr i fuge tubes. The tubes were centrifuged a t 1200

rpm for 10 minutes. The supernatant was aspi ra ted and

the c e l l p e l l e t resuspended in 5 ml of hypotonic

80

solut ion (0.075 M KCl) a t 37 °C for 20 minutes. Again

centr ifugat ion of the mater ia l was car r ied out for 10

minutes a t 1200 rpm, supernatant was discarded and ce l l s

were fixed in freshly prepared methanol: g l a c i a l a ce t i c

acid (3:1) and kept a t 4 °C for 30 minutes. The ce l l

suspension was washed three times in the f i x a t i v e .

Slides were prepared by flame drying technique.

Staining

Sl ides were s ta ined for chromosomal aberra t ions

with 4% Giemsa in phosphate buffer (pH-6.8) for 10-15

minutes. Staining for s i s t e r chromatid exchange (SCE)

analysis was performed according to the technique of

Perry and Wolff (19 74) with s l i g h t modif icat ion. Sl ides

were s ta ined with Hoechst 33258 (50 /ug/ml) for 15

minutes, and then exposed to b r i g h t sun l igh t in the

presence of 2 SSC for 2 hours , followed by s t a i n i n g

with 4% Giemsa in phosphate buffer (pH- 6.8) . The

s l ides were coded to avoid scoring bios and analyzed for

the experimental end po in t . Only metaphases with 46

chromosomes were scored and the metaphases were not

se lec ted for technical qua l i ty provided t h a t there was

good s i s t e r chrcanatid d i f f e r e n t i a t i o n . Hundred well

spread metaphases were scored for chromosomal

aberrat ions and 50 well spread metaphases with good

d i f f e ren t i a t ion were scored for SCE a n a l y s i s .

81

Mitotic index

For counting the mitotic Index, cultures were

maintained, slides were coded and randomised for

screening. A minimum of 4500 cells (nuclei) per

concentration were counted for calculating the mitotic

index.

Number of metaphases x 100 % Mitotic index =

Number of total cells count

Analysis of cell cycle kinetics

Slides were also scored for cell cycle kinetics.

To evaluate cellular replication, the frequencies of the

first, second, third and subsequent metaphase cells from

each of the cultures were counted. Those cells whose

DNA had replicated exclusively before the addition of

BrdU (5'-bromo-2-deoxyuridine) could not be distingu­

ished from cells at first metaphase, and those that had

gone through three or nore cell cycles were ranked as

third mitosis.

In the first division cells (Mj^ ) all the

chromoscxnes were stained dark, whereas in the third or

subsequent division cells (M3^they were lightly stained.

In the second division cells (M; ) all the chromosomes

showed differential staining, i.e., one darkly and one

lightly stained chrcxnosomes.

82

Proliferation rate index

Pro l i f e r a t i on r a t e index (PRI) was calculated from

ce l l cycle Xinetics by the method of Krishna ejt a l .

(1986) applying the formula of

100

Where Mi , M^ and M represent proport ions of

f i r s t , second and th i rd plus-subsequent d iv is ion

metaphases respec t ive ly .

S t a t i s t i c a l Analysis

The differences between the control and

ejcperimental ce l l s were s t a t i s t i c a l l y evaluated with t-

tes t .

RESULTS

E f f e c t o f Dust Concentra t ion on Chromosonial A b e r r a t i o n s

Results depicted in Table 3 c l ea r l y show t h a t dus t

concentrat ion i s an iit^jortant fac tor for chromoscxnal

aber ra t ions . The percent aberrant metaphases increased

with the concentration but not in a l i n e a r fashion.

Maximum number of aberrant metaphases were scanned a t

100 ug/ml of dust concentration. Chrysot i le , which i s

n

CO 0) CO nj ^ a. (0 •p (U 6 +J

c (0 ^1 )-( 0) ^ fd

m 0 •P c 0) u ^ 0)

a< c 0 c 0

-H v fl v V c Q) O C 0 o + CO D

X>

1 M-l 1 0

1 -p 1 U 1 (U 1 M-l 1 (4-1 1 M

CO (U CO (d ^ CU fl? V 0)

s -p c (0 u u 0

JQ < •P c 0) 0 h 0) &

-1 C

c

0 -H CO "d

6^

o vO 1 < -P •H n ^

0 e 0) i<

1 +> •H i-i 0 e 0) fc^

CO 1

OQ CO <: -p -H rH 0 e 0) t«5

C 0 • P ^ (0 rH

0 V < . 3 C 01 a 0) 3

c 0 u

(S

CN

r-i

fN

o •

o

VO 0^ CN

i n 00

VO

n n vo

o

o o o

83

most toxic form of asbestos, was most effective in

producing chromosomal aberrations and kemolit ASB-3 was

the least. The order of their toxicity was as follows:

dhrysotile > kemolit A-60 > kemolit-N > kemolit ASB-3,

supporting the previous studies carried out with human

erythrocytes and isolated rat hepatocytes.

Types of Chromosomal Aberrations Induced by Different Dusts

In the present observation, types of chromosomal

aberrations were studied and the results are given in

Table 4. The most common abnormalities were chromatid

gaps, chromatid breaks and chromosome gaps (Figures 30,

33, 31). It was found that chromosomal aberrations were

high when conpared to Indian variety of chrysotile than

wollastonite at all dose treatments. In chrysotile

treated cultures, some serious alterations like

dicentrics, exchange type and chromosome breaks (Figures

35, 34, 32) were observed v iile these abnormalities were

absent in wollastonite treated cultures. The main

abnormalities were chromatid gaps and chromatid breaks.

Studies on Mitotic Index (MI) in Relation to Dust Concentratin

The mitotic index with different dust

concentration is given in Table 5. The mitotic index

decreased with the increasing dust concentration in dose

F i g . 30: Metaphase chromosomes chromatid gap.

Arrow i n d i c a t e s

F i g . 3 1 : Metaphase chromosomes ch r omos ome gap.

Arrow i n d i c a t e s

F i g . 32: Metaphase chromosomes: chromosome b reak .

Arrow i n d i c a t e s

F i g . 3 3 : Metaphase chromosomes chrcmat id b r eak .

Arrow i n d i c a t e s

F i g . 34 : Metaphase chromosomes: exchange type a b e r r a t i o n .

Arrow i n d i c a t e s

F i g . 35: Metaphase chromosomes d i c e n t r i c chrcmosome.

Arrow i n d i c a t e s

30 "- ^ " V k^^^ ^ I ^if^^

31 / \

>,^l^'^

32

7 •• ^ , -K *V ' -r * »

Vi

/'. l^V 1' ^ J^ J^ .A

35 V •" ^^

CO

CO 3

T(

-P C

<u

^ M-l 0

CO (U

s EH

M-4 I W -H -O

> i

- a <u o 3

C • H

CO

c 0

- H •P

u u 0)

e o

E 0 u

l4-t

o •Ji

EH

olp

CO C 0

•H

(0

0)

c a> ^§ u -P X

d

o -H Q

0) S O CO > :

0 rd e <u 0 < u XI

e 0

§ 2. 0 01 u

-0 -H

• ^ • ^ rO (0

0 u u ^

•p

0 01 )-l

V c 0)

s fO (U

>-( EH

*— rH

H ^ 3.

I I I I I I I I I I I I I r-(

I I I I • I I I I I I I I -H I

I I l i l t I I I I CS

CN r>j (N CNJ f S CM

I I I I I rH CN (N n ^ »* t t

CO CN CN rO n (N rf - t <N n n •<* •^ CN CN C J CS ^

n I

OQ

W

< •P -,-( <-\ 0 E (U

.-H O O O • • • •

O rH O O

13

r-l O O O

O

I

p •H

c -H

c

0)

i-i O O O

O >-< O "H 0 E 0)

be:

o o r-l O

O f-* O •-•

(U

o o

•r-l •p c >1 u

iH O O O

o o

in

Cd

CQ <

X 0)

C • H

U -H

-H

e c o

e - H 4-»

-O C

c 0

•H ^ - » rO U •P c (1) o c 0 u • i J

J)

-a

c

u 0)

m w

to u 3 0

u

0

CO

-p

ttp

«t-i to 0 0)

to u (d <u ^

15

«4-l to O (U

to

(0

0)

s <*A 0

u Q)

• ^

D 12

•O 0) V< 0 u CO

rH i H

0) u

c c

-H +- ' , - . m r-l

c en 0) a c 0 u

r-» in »« "H r

TJ" rj* ^ ^ d

iH C>4 M •<* VO •H O O 00 VO CJ CM <N rH rH

0 0 0 0 0 0 0 0 0 0 in in in in in ^ ' ^1^ ^3* ^ ^ 'sj*

0> ON r^ r o 0^ • • • • • in in in in ^

(yi <yi in in in in in oQ vo ';!' CN fS <N CN fS

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 in in in in in

n I m CO

0

e

r» • n O r 5j" ^ ^ CO

vD CO o o> in • • • • • ^ ^ <^ ro CO

iH 00 CO O VO •-H ON <T» 00 ^ CN rH r-t iH iH

r^ CO o in r-» o ON 00 t^ "^ CM r-l iH iH i-(

0 0 0 0 0 0 0 0 0 0 in in in in in ^^* ^^* ^ ^ ' ^ * ^4*

0 0 0 0 0 0 0 0 0 0 in in in in in ^' 5* T ^* ^*

00 r^ ^ --I vD • • « • • in in in in •<*

ON 00 vD o m • • • • •

in in in in Tj-

o in o in o ON 0 0 f^ i ^ f* CM OJ CN CM <N

ON o o o in in ON 00 i^ ^ CS r 4 CM CM CM

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 in in in in in

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 in in in in in

O rH O O O

o o -H o o M O

h O rH O O O

O O i-H o O nH O

O

0 e 0) hi

O ,H O o O

O O r-l O O -H O

84

dependent manner but not in the l inear order . I t a l so

decreased a t 72 hours when compared to 48 hours of

treated cu l t u r e s . This shows i t s time dependence, which

might be due to the t ox i c i t y of mineral f i b r e s .

Anfelysis of Cel l Cycle Kinet ics in Relation to Dust Concentration

To evaluate c e l l u l a r p r o l i f e r a t i o n , the frequen­

cies of f i r s t ( M ^ ) , second (M ,) / t h i rd and subsequent

(M^f) metaphase c e l l s (Figures 37-39) from each cu l tu re

were scanned and the r e s u l t s are given in Table 6. An

increase in Mj and M; metaphases and decrease in M3

metaphase was observed when coiipared to control with a l l

va r ie t i e s of dus t a t t e s ted concentra t ions . The f i r s t

and second metaphases increased with the dust

concentration while th i rd metaphase ce l l s decreased

indicat ing involvement of dust concentrat ion during

ce l lu la r r e p l i c a t i o n .

Determination of P ro l i f e ra t ion Rate Index (PRI)

Results of p r o l i f e r a t i o n r a t e index (PRI) are

represented in Table 7, which c l ea r ly indica te t h a t as

the dust concentrat ion increases the PRI values

decrease. The PRI was less by 0 .51, 0.52, and 0.56 for

kemolit ASB-3. kemolit-N and kemolit A-60, respec t ive ly

a t 100 yug/ml dus t concentration when compared to t h e i r

control cu l t u r e s . From th is type of r e l a t i o n s h i p , i t

Fig. 36: Metaphase showing s i s t e r chromatid exchanges.

Fig. 37: Metaphase showing f i r s t c e l l cycle .

Fig. 38: Metaphase shwoing second c e l l cycle .

Fig. 39: Metaphase showing th i rd ce l l cycle .

^ < : ^

i " )l

•

36

• C V "

37

?

38 39

c o -H

rd

s C 0 -H -P

m

c

-H

CO

u •H +J 0)

c -H

a> r-{ U >1 o

0)

o

0

en •H CO

>l

c

dP

u -p

c <u o c 0 o -p CO 3 -o

CO 0) CO

m ^ a 5 0) s IH 0

u 0)

M-l CO 0 0)

CO "O ^ (0 <U <D s: u ja Q, o e fd u 3 +J CO

c 0 -H • p ^

+ M 6 CO - P - ^ 3 C en Q 0) 3

c 0 u

r-» in t^ in t^ in in "* CN

vo n CO (30 r^ in in 't CO <N

vo o CO f m in in n n CN

00 I OQ CO <

-H

O e

in r>- CO t~" "* CN CM (N CN) ro

(Ti 0\ vO O "1 <N CN ro CO 00

[ fS O 00 00 CN 00 ^ 00 00

00 00 i^ 00 <y> iH r^ CN CS 00

m CO iH ON CO iH rH OJ 00 00

r CO O f rH iH Ol 00 ^

1 O O O O O 1 O O O O O 1 CN CM CN CM <N

z 1, 4-»

i Q)

o o o o o o o o o o CM <N CM OS OJ

O

1 << 4-"

rH

o o o o o o o o o o fvj CM CM CM CM

O rH O O O

O O M O O .H O

O rH O O O

O O rH O O

O rH O O O • • • • •

O O iH O O rH O

r 1

w ^ < in

0

^^ M 05

a< ^-' X 0) -o c

•H

Q) +> m u c 0

-H -P (0 ^ 0)

M-4 0) •H 0) rH V 0 >1 V O 0 ^ 0 - . -C Q) 0^

51, i H

c 0) -a 0) 0)

3^ (U 0) ^ ^

+ yj 0) tJ

u c C (ti 0 ^ •-{

0) 0

•H C T3 0

0 C

^6

o I

•P

<U

• H

0

e be;

c o

-H

- H a C en (D a o v > C 0 u

Q

O CO

in

o <Ti in

in

o CM CN in

o

o o o

o

85

could be deduced t h a t d u s t concen t r a t i on t r i g g e r s on

enhancement of the d u r a t i o n of the c e l l u l a r c y c l e , most

l i k e l y a t the l e v e l of the G phase .

Frequency of S i s t e r Chromatid Exchange (SCE) in Dust Trea ted Lymphocytes

^ Table 8 shows the d i s t r i b u t i o n of the s i s t e r

chromatid exchange pe r c e l l of the c o n t r o l and a f t e r

t r ea tment with dus t s (F igure 3 6 ) . I t t u rns ou t t h a t t he

frequency of SCE o c c u r r i n g per c e l l i s s h i f t e d towards

the increased va lue when the c e l l s a r e t r e a t e d wi th

d u s t s . The r a t e of SCE was inc reased wi th d u s t

concen t r a t i on b u t n o t dose dependent in l i n e a r o r d e r .

I t was found t h a t SCE induced by w o l l a s t o n i t e s was low

when con5)ared t o I n d i a n v a r i e t y of c h r y s o t i l e d u s t a t

a l l dose t r e a t m e n t s , i t a l s o ejchibits low t o x i c i t y of

w o l l a s t o n i t e .

DISCUSSION

In the p r e s e n t s tudy w o l l a s t o n i t e samples have

shown t h e i r g e n o t o x i c i t y towards c u l t u r e d lymphocytes .

To e x p l a i n i t s mode of a c t i o n , i t has been sugges ted

t h a t e i t h e r i t w i l l a c t d i r e c t l y on the chromat in

m a t e r i a l (Lavappa et. a_l. , 19 75) o r due t o an i n d i r e c t

e f f e c t on lysosomal membranes r e s u l t i n g in t h e r e l e a s e

TABLE - 8

F r e q u e n c y of s i s t e r c h r o m a t i d e x c h a n g e (SCE) i n d u s t t r e a t e d lymphocytes

T r e a t m e n t (yug/ml)

Number of Metaphses S c o r e d

S C E s / C e l l (Mean + S.D.)

0.0. Kemolit ASB-3

0.1 1.0 10.0

100.0

50

50 50 50 50

3.30+0.81

4.66+1.15 4.98+1.50 5.48+1.19 5.84+0.93

0.0 Kemolit-N

0.1 1.0

10.0 100.0

50

50 50 50 50

3.22+0.74

4.82+1.22 5.30+1.36 5.60+1.36 5.60+1.54

0.0 Kemolit A-60

0.1 1.0

10.0 100.0

50

50 50 50 50

3.42+1.69

4.98+1.38 5.40+1.68 6.22+1.01 6.98+1.22

0.0 Chrysotile (Indian)

0.1 1.0

10.0 100.0

50

50 50 50 50

3.00+1.17

5.07+1.66 8.00+1.61 9.20+1.99 10.99+2.26

86

of lysosomal DNase and thereby affect ing chromatin

(All ison, 1973).

The type and pers i s tence of les ions induced in EWA

by various agents w i l l determine the degree of

cytogenetic damage observed following mitogen

s t imula t ion . In the case of chemical induced l e s ions ,

the c e l l s must pass through an S-phase in order to be

transformed in to aber ra t ions . Generally, such an S-

phase does not take place u n t i l the ce l l s are st imulated

with mitogen in v i t r o . Only few les ions t ha t remain can

be expressed as aberra t ions or s i s t e r chromatid

exchanges, if they are misrepaired, when they p^ss

through S-phase iri v i t r o . Most s ing le s t rand breaks are

repai red immediately by r e s t i t u t i o n . The remaining

f rac t ion of s ingle s t rand breaks i s repaired slowly, by

an excis ion repa i r pathway by formation of gap, followed

by f i l l i n g and l i g a t i o n . Double s t rand breaks are

repaired slowly. The prec ise mechanism by which t h i s i s

done i s not c l e a r . The chromosomal r ep l i ca t ion and

segregation have shown t h a t E»JA in eukaryotic

chromosomes i s in the form of s ing le double h e l i x

molecule running a l l through the chromosomes. In such a

case a double s t rand break in a G chrcxnosome (before

rep l ica t ion) should give r i s e to the chromosome type of

aber ra t ions , and a double s t rand break in G; ( i . e . a f t e r

r ep l i ca t ion) should give r i s e to the chromatid type of

aber ra t ion .

87

S i s t e r chromatid exchanges (SCEs) represent the

interchange of DNA rep l ica t ion products a t apparently

homologous l o c i . The exchange process presumably

involves DNA breakage and reunion. The halogenated

nucleot ide , 5 '-bromo-2-deoxyuridine ( BrdU) has largely

supplemented thymidine for the purpose of d i f f e r e n t i a l l y

l abe l l ing s i s t e r chromatids. When incorporated into ENA

or chromatin, BrdU can quench the fluorescence of DNA

binding dyes such as 33258 Hoechst (La t t , 19 7 3 , ) . In

p r i n c i p l e , SCE can be observed in any c e l l t ha t has

coitpleted two or the f i r s t two r ep l i ca t i ons in the

presence of BrdU.

The method of BrdU-label l ing for SCE analysis can

a l so be used simultaneously for inves t iga t ion of

c e l l u l a r k i n e t i c s with great s e n s i t i v i t y (Beck and Obe,

1979, Craig-Holmes and Shaw, 1976, Tice et. a l . , 1976,

Chen et aJL., 1981, Abel, 1987). Study and d i s t r i b u t i o n

of f i r s t (Mj^), second (M ) , th i rd and fur ther (M^

mitosis in cul tures t reated with BrdU alone and BrdU

plus mineral f ibres (dus t ) , a t various concentrat ions,

c l ea r ly show tha t a l l the mineral f ib res did a l t e r c e l l

k i n e t i c s , i . e . , causing c e l l cycle delay of various

degrees. Therefore, the c e l l cycle delay observed in

the experimental cul tures may be mainly due to the

treatment of dus t . Cell cycle delay, in addi t ion to the

production of SCE, may represent another level of

b io log ica l damage incurred due to the treatment of dus t .

However, the data obtained do not allow to assess which

88

speci f ic stages of the ce l l cycle were a l t e r e d by dus t .

Although, a very s ign i f i can t increase in the incidence

of anaphase abnormali t ies , such as lagging chromosomes,

s t icky chromosomes and chromosome br idges , have been

reported during the f i r s t c e l l d iv is ion a f t e r asbestos

treatment (Hesterberg and Barre t t , 1985). All of these

abrJormalities could r e s u l t in missegregation of

chromosomes which takes place by d i r e c t i n t e rac t ion with

the chromosomes or microtubules, microfilaments, or

other s t r u c t u r a l prote ins of the spindle appratus may

r e s u l t in c e l l cycle delay. The extent of c e l l cycle

delay may be corre la ted with the dose of mineral f i b r e s .

The higher the dose, the more delay in c e l l cyc le .

However, the induced delay varied considerably among the

mineral f ibers studied and was re la ted with the degree

of induction of SCE. I t was a lso d i r e c t l y r e l a t ed to

the p r o l i f e r a t i o n r a t e index (PRI).

SCEs have been well documented as a s e n s i t i v e

indicator to de tec t mutagenesis and carcinogenesis

(Perry and Evans, 1975; Wolff e t a l . , 1977; Abel, 1987).

The enhanced SCE r a t e in t reated c e l l s suggests t ha t DNA

lesions for SCE formation are increased per genome with

the increase in dose of treatment bu t not in a s t r a i g h t

way. I t might be due to the random d i s t r i b u t i o n of

wol las toni te f ibres in cul ture v ia l s or in suspension

(Rahman e_t aJL., 1988b) . I t has been suggested t h a t SCEs

are e f f i c i en t ly induced by those substances tha t form

89

covalent adducts to the DNA or otherwise interfere with

ENA metabolism or repair (Perry and Evans, 19 75; Wolff

^ sa. , 1977; Perry, 1980; Latt, 1981; Natara jan et al. ,

1981; Carrano and Thompsom, 1982).

A number of studies, in which SCEs and

chromosomal aberrations induced by various physical and

chemical agents are available. in many cases SCEs could

be induced at doses insufficient to give rise to

chromsomal aberrations (Wolff, 1982; Deen et aJ . , 1989).

This suggests that SCE induction is a much more

sensitive cellular response than the production of

chromoscmal aberrations. Despite early indications that

many chromosome breaks occur at the site of SCE, thus

suggesting a causal relationship between the phenomena

(Ueda e_t al., 19 76). Later evidence suggests that the

common location of SCE and chromosome breaks is probably

coincidental (Galloway and Wolff, 1979). Shiraishi

(198 2) suggests that the two phenomena are the

reflection of different cellular processes. So, while

SCEs and chromosomal aberrations may be coincidental

with regard to their induction and chromosomal

localization, both probably reflect responses to lesions

in DNA, or to deficiencies or errors in the repair of

such lesions. Different mechanisms are likely involved

in their formation (Wolff, 1982).

In previous chapters significant increase in lipid

peroxidation (LPO) in human erythrocytes and rat

hepatocytes have been reported. LPO is a chain reaction

90

process tha t involves the p a r t i c i p a t i o n of free rad ica l

species , and has been shown to induce a l t e r a t i o n s in

several c e l l u l a r functions (S la t e r , 1984; Pryor e_t aJ . ,

1976; COTiporti, 1985; Halliwell and Gutteridge, 1984;

1985; 1986). Furthermore, recent repor ts have provided

evidence for a r e l a t ionsh ip between LPO process and

genotoxici ty as well as carcinogenici ty (Ames et ajL. ,

1982; Ames, 1983; Kensler and Trush, 1984; Vaca e t a l . ,

1988).

There are many theore t i ca l p o s s i b i l i t i e s for the

in te rac t ion between JXih and LPO. A major problem in

elucidat ing the mechanisms behind the proposed

genotoxicity of the LPO process i s , however, the

discr iminat ion between EWA damage caused by the d i r e c t

act ion of free r ad ica l s needed for LPO and damage

caused by the reac t ion of DNA with intermediate and

f ina l products of the LPO process (Vaca ejt al^. , 1988).

The i n d i r e c t act ion of LPO induced ENA damage on

carcinogenesis may have several mechanisms. Thus, LPO

induced membrane damage may lead to increased leve ls of

c a t a l y t i c a l l y ac t ive iron (Vladimirov, 1986) and to

increased concentrat ions of reac t ive oxygen spec ies ,

which in turn may i n t e r a c t with DNA and thereby ac t as

i n i t i a t o r s and/or cancer prcaxoters (Greenstock, 1986).

Emerit et a_l. (198 2) provided evidence t h a t

generation of Oo (superoxide) rad ica ls in the cu l tu re

system r e su l t s in an increase of chromosome breaks and

91

SCEs in human lymphocytes. This could be almost en t i r e ly

prevented by addition of superoxide dismutase (SOD) to

the cu l tu re medium. I t indica tes involvement of Oj

r ad ica l in chromosanal aberrat ions and SCEs. I t has

a lso been reported tha t the generation of s i n g l e t oxygen

in the medium creates problems to the c e l l s . I t

increases the duration of c e l l cycle which could be

s i t ua t ed a t the leve l of the Gj, phase of the f i r s t

c e l l u l a r divis ion (Decuyper-Debergh ejt a_l. , 1989).

Indeed, i t i s a t t h i s s tage tha t les ions induced by

s i n g l e t oxygen in the lymphcoytes could be repaired by

the excision r ^ a i r pathway before DNA rep l i ca t i on

(Friedberg, 1985; Decuyper-Debergh et aj^. , 1987).

However, the increase in the durat ion of the c e l l cycle

may well be s i tua ted during the DNA rep l i ca t i on phase as

has been shown when eukaryotic c e l l s are i r r ad i a t ed with

UV l i g h t (Hall and Mount, 1981). I t may be poss ib le t h a t

not only s i n g l e t oxygen or superoxide r ad ica l i t s e l f bu t

other formed r ad i ca l s , l ike hydroxyl (OH*), for which no

e f f i c i e n t scavenging e x i s t s , are the cause of DNA

damage. This damage may r e s u l t in chromosomal aberra­

t ions and s i s t e r chromatid exchanges.

Further s tudies for the exact mechanisms or as to

which spec i f i c free rad ica l s are responsible , for

enhanced degree of chromosomal aber ra t ions and SCEs, a re

needed to extrapolate the nature of woUastoni te (India)

induced t ox i c i t y .

SUMMARY AND CONCLUSION

Asbestos i s extensively used in i n d u s t r i a l and

coximiercial appl ica t ions because of i t s hea t and f i r e

r e s i s t a n t p r o p e r t i e s . The consequences of developing

lung diseases in occupationally exposed population by

asbestos are well documented. Therefore, indus t r ies are

exploring other mineral f ib res as subs t i t u t e s for

asbestos to meet out increasing demand for cheap and

r e l i a b l e safer ma te r i a l s . Among the promising

s u b s t i t u t e s , wol las ton i te received wide a t t en t ion and

i s under t r i a l in India . I t i s a nonmetalic na tu ra l ly

occurring monocalcium s i l i c a t e . In view of i t s

app l ica t ion p o t e n t i a l , i t i s e s s e n t i a l to evolve i t s

sa fe ty by experimental s t u d i e s .

In the present study, tox ico logica l evaluation of

three v a r i e t i e s of wol las ton i te of Indian o r ig in ,

namely kemolit A-60, kemolit-N and kemolit ASB-3 were

car r ied out . The r e s u l t s obtained from these s tudies

were compared with ch ryso t i l e , one of the most toxic

93

form of asbestos , in order to p r e d i c t the i r r e l a t i ve

toxic p o t e n t i a l s . The dus t -erythrocyte in te rac t ion

suggests tha t dust can damage membranes indicat ing i t s

po t en t i a l of in v i t r o t ox i c i t y . Apart from indicat ing

membrane tox ic i ty of dust, the p resen t study on

erythrocyte toxicant in te rac t ion a l so indica tes tha t

human erythrocytes are an ideal t e s t system for

toxicologica l evaluation of p a r t i c u l a t e a i r p o l l u t a n t s .

The r e l a t i v e hemolytic a c t i v i t y of wol las toni te dusts

show t h a t kemolit A-60 has most hemolytic po t en t i a l

than the other two v a r i e t i e s . Although i t was less

toxic than chryso t i l e which i s in agreement with the

previous r epo r t s . The lys i s of erythrocytes by these

dusts were found to be dose and time dependent.

Wollastonite dus t induced l y s i s , unl ike ch ryso t i l e , i t

remained unaffected by EDTA treatment but was

s i gn i f i cna l t y reduced by BSA and PVP. The poss ib le ro l e

of calcium in the mechanism of wol las ton i te induced

hemolysis was not confirmed by EDTA treatment, which

might be due to the inac t iva t ion of calcium released

from the surface of the dust . The decrease in the

a c t i v i t y of wol las ton t ie af ter BSA and PVP treatment

showed t h a t the chemical in te rac t ion of the dust

surface with c e l l membrane may be involved. Acid,

a l k a l i and water treatments a lso change the hemolytic

p o t e n t i a l which may be due to change in the s t r uc tu r e

of the f i b r e s .

94

The induction of lipid peroxidation (LPO) in

erythrocyte and low speed supernatant of hemolysate by

kemolit A-60 was higher in magnitude than the kemolit-N

and kemolit ASB-3. However, it was less than that of

chrysotile. The less induction of LPO by wollastonite

may be due to the differences in chemical composition

of wollastonite than that of chrysotile. The induction

of peroxidative damage of polyunsaturated fatty acids

of erythrocytes suggests that involvement of free

radicals in the toxicity caused by these dusts. It was

also observed that glutathione depleted during the

course of lipid peroxidation which indicate the

relationship between glutathione and LPO.

The scanning electron microscopic studies showed

distoration and deformation of erythrocytes. The

deformity appeared as invagination or crenation of

erythrocytes. These studies have also suggested less

toxicity of wollastonite as compared to chrysotile. The

ciiange in the morphology of the cell may be due to the

abnormal influx of calcium or interaction of calcium

from the surface of dust with the sialic acid of

erythrocyte membrane.

Although, it is well documented that erythrocytes

are good model for toxicological evaluation of dusts.

Besides its suitability, in the absence of nucleus and

intracellular organelles, findings on erythrocytes may

be difficult to extrapolate to more organised cell

types like macrophages and hepatocytes etc. In view of

95

the well organised cell type, hepatocytes were taken as

a model for cytotoxicity evlauation.

The release of lactate dehydrogenase (LEH) has

been considered as a convenient parameter for the

cytotoxicity assessment in hepatocytes and other well

organised cells. Therefore, the effects of Indian

varieties of wollastontie on the LEH released from the

hepatocytes were measured. Results show that kemolit A-

60 was more prone to release LEH than kemolit-N and

kemolit ASB-3 from hepatocytes. However, the release of

this enzyme was less than in chrysotile treatment.

As previously mentioned that wollastonite dusts

induce peroxidation in human erythrocytes, hepatocytes

were also used for lipid peroxidation study to support

and know the suitability of the previous cell system.

The induction of lipid peroxidation by wollastonite

dusts shows its concentration and time dependence. It

has also been found that mineral fibres induced

membrane damage, enhanced lipid peroxidtion, results in

cellular depletion of glutathione (GSH) and subsequent

lysis of cells. It is believed that GSH protects

agaisnt LPO in metabolically active cells. Hiis

indicates towards the casual relationship between LPO

and cellular lysis. Here also, involvement of free

radicals support the previous findings with

erythrocytes.

96

I t i s well documented tha t mammalian ce l l s have

evolved numerous mechanisms to prevent themselves in

tox ic i ty e l i c i t e d by xenobiotics and t h e i r by products

of c e l l u l a r metabolism. Glutathione, an endogenous

p ro tec t ive system, i s present in high concentrat ion in

most mconmalian ce l l s as reduced g lu ta th ione . I t acts as

a nucleophi l ic "scavenger" of numerous conpounds and

the i r metabolties a l so ac t as cofactors in GSH

peroxidase-mediated des t ruct ion of hydrogen peroxide.

In addit ion to the th io l redox system, several

endogenous ant ioxidants a lso serve as p ro t ec t ive agent

agains t oxidative damage. Among them, the l i p o p h i l i c

ant ioxidant , ^ - t o c o p h e r o l i s the primary p ro tec t ive

factor against such at tack on c e l l u l a r membranes. The

present study a lso demonstrated t h a t p^-tocopherol

i r ihibi t LPO induced by mineral f i b r e s , which indica tes

the termination of propagative process .

Since a balanced i n t r a c e l l u l a r t h i o l redox system

i s important for c e l l v i a b i l i t y , i t s r e l a t i onsh ip with

LPO in the presence of (X^-tocopherol was a l so

inves t iga ted . The prevention of LPO and subsequent

efflux of GSH af te r supplementing 0^-tocopherol was

observed. This shows tha t addi t ion of 0(^-tocopherol

i r ihibi t the LPO process via the donation of hydrogen

atom to the l i p id rad ica ls forming the hydroperoxide

and a lcohol . Methionine, which i s known to s t imula te

GSH synthesis in l i v e r , when added to the incubation

97

medium resul ted in increase in the GSH level during the

th i rd and subsequent hours of incubation in comparison

to f i r s t and second hours , while l i p id peroxidation was

inh ib i ted . This a lso shows tha t GHS plays an important

role in maintaining the i n t eg r i t y of the c e l l . I t has

been suggested tha t LPO does not proceed u n t i l 0(j -

tocopherol has been s u b s t a n t i a l l y depleted. In s p i t e of

the lack of 0</-tocopherol prevention of the efflux or

oxidation of GSH, most evidence based on inh ib i t i on of

LPO would suggest t h a t the ef fect of <X^-tocopherol on

GSH biosynthesis i s secondary to i t s prevention of

increased oxidat ive p o t e n t i a l within the c e l l . The-

presen t r e su l t s s t rongly support the hypothesis t h a t

GSH def i c i en t hepatocytes can undrgo LPO rapidly enough

to destroy the c e l l s .

For genotoxic p o t e n t i a l human per iphera l blood

lymphocytes were used. They offer an unique opportunity

to assess the in5)act of environmental po l lu t an t s

d i r e c t l y a t the leve l of the workers as well as in

laboratory condi t ions . Lymphocytes provide a read i ly

ava i lab le source of c e l l s t h a t can be s t imulated t o

undergo mi to t ic d iv i s ion in cu l tu re . The m i t o t i c a l l y

dividing ce l l s can be ar res ted in metaphase with the

help of mitogen added in cul ture medium. These

metaphase c e l l s he lp in. s tudies on chromostxnal

aberra t ions and s i s t e r chromatid exchange which are the

markers of genotoxici ty .

98

The type and persistance of lesions induced in

DNA by various agents will determine the degree of

cytogenetic damage observed after mitogen stimulation.

Wollastonite treated lymphocytes have shown increased

frequencies of chromosomal aberrations and sister

chromatid exchanges. Among the wollastonite samples

tested, kemolit A-60 was more prone to induce

chromosomal aberrations and SCEs and kemolit ASB-3 was

less toxic. In a comparative study with chrysotile,

kemolit A-60 was found to be less toxic than

chrysotile. The changes in their frequencies were

concentration dependent. The main abnormalities were

chromatid and chromosome gaps. In the case of chemical

lesions, the cells have to be stimulated with mitogen

to pass through S-phase. Only misrepaired lesions

during S-phase can be expressed as aberrations and

sister chromatid exchanges. Most single strand breaks

are repaired immediately v iile the remaining fraction

slowly results in the formation of gap followed by

filling and ligation. Double strand breaks are also

repaired slowly. In such a case a double strand break

in Gj|_ chromosome should give rise to the chromosome

type of aberrations and in G;^should give rise to the

chromatid type of aberrations. So, it is clear from

chromosomal aberration studies that the chromosomes

are damaged by mineral fibres before and after

replication.

99

Si s t e r chromatid exchange (SCE) process

presumably involves DNA breakage and reunion of the DNA

rep l ica t ion products a t the apparently homologus l o c i .

The increased frequencies of SCE a l so indica te the

involvement of DNA rep l ica t ion product with the mineral

f ibres suspended in the cul ture medium, supports the

findings of chromosomal aberrat ions as discussed

e a r l i e r . The method 5-bromo-2-deoxyuridine ( Brdu)-

label l ing for SCE analysis can a l so be used

simultaneously for inves t iga t ion of c e l l k i n e t i c s . The

d i s t r i bu t i on of f i r s t (Mj), second (M^) / th i rd and

subsequent (M^^) mitosis in cul ture supplemented with

BrdU shows t h a t a l l the mineral f ibres tes ted did a l t e r

c e l l k ine t i c s which indicates c e l l cycle delay.

Therefore, the c e l l cycle delay observed in

experimental cul tures may be mainly due to the

treatment of the mineral f i b r e s . The c e l l cycle delay,

in addi t ion to the production of chromosomal

aberra t ions and SCEs, may represent another level of

b io logica l damage incurred due to the treatment of

dus t . However, the induced delay may be cor re la ted to

the dose of mineral f ibres and was re la ted with the

degree of SCE induction. The enhanced SCE r a t e in

t rea ted c e l l suggests tha t DNA lesions for SCE

formation are increased per genome with the increase

in t r e a ^ e n t dose but not in a s t r a i g h t way. I t may be

due to the random d i s t r i bu t i on of mineral f ib res in

100

cul ture v ia l s or in suspension. So, SCE and chromosomal

aberra t ions may be coincidental with regard to t he i r

induction and chromosomal l oca l i za t i on . Both probably

r e f l e c t responses to les ions in E*JA or to defeciencies

or e r rors in the repa i r of such l e s ions .

A s ign i f i can t increase in l i p i d peroxidat ion

(LPO) induced by wollas ton i t e s in human erythrocytes

and i so la ted r a t hepatocytes has been already

discussed. Few repor t s have provided evidences for a

r e l a t ionsh ip between l i p i d peroxidat ion and

genotoxicxty. Further, i t supports the p o s s i b i l i t y t h a t

DNA reacts with e i t he r free rad ica ls needed for l i p i d

peroxidation or intermediate and f ina l products of the

LPO process . I t i s already reported in the l i t e r a t u r e

t ha t superoxide r ad ica l s are involved in the genesis of

chromosomal aberra t ions and s i s t e r chromatid exchanges.

This was prevented by the addi t ion of superoxide

dismutase to the cu l tu re medium. Recently, i t has been

reported tha t the generation of s i n g l e t oxygen in the

medium creates problem to the c e l l s . I t increases the

duration of c e l l cycle which could be s i t ua t ed a t the

level of G- phase of the f i r s t mi to t ic d iv i s ion . These

inves t iga t ions have provided evidences t h a t free

rad ica ls generated by wol las ton i te dusts may be one of

the factors d i r e c t l y or i nd i r ec t l y involved in causing

cJiromosomal aberra t ions and s i s t e r chromatid exchanges

and c e l l cycle delay.

101

The present s tud ies have, thus, es tabl ished iJne

fact t h a t wol las ton i te dust smaples are both cytotoxic

and genotoxic though the i r t ox i c i t y was less in

contparison to ch ryso t i l e dust . Among the wol las ton i te

v a r i e t i e s , kemolit ASB-3 l i e s a t the bottom of the

ladder followed by kemolit-N and kemolit A-60 in order

of increasing t o x i c i t y . The r e s u l t s reported in t h i s

d i s s e r t a t i o n c l ea r l y indica te t h a t free r ad ica l s

generated by wol las ton i te samples may be one of the

factors responsible for dust induced t o x i c i t y . This

information may prove useful in advocating and

implementing p r o t e c t i v e measures to safeguard the

heal th of p o t e n t i a l l y vulnerable persons a f t e r long

exposure.

BIBLIOGRAPHY

102

Abel, G. (1987) . Chromoscrae damage induced in human lymphocytes by 5-methoxypsoralen and 8-niethoxypsoralen p l u s UV-A. Mutat . R e s . , 19 0, 68 .

Achard, S. , P e r d e r i s e t , M. and Jaurand , M.C. (1987) . S i s t e r chromatid exchanges i n r a t p l e u r a l m e s o t h e l i a l c e l l s t r e a t e d wi th c r o c i d o l i t e , a t t a p u l g i t e or benzo 3 ,4 -pyrene . Br. J . I n d . Med., 44 , 281 .

Allison, A.C. (1973). In: Biological effects of asbestos, lARC Sci. Publ. No. 8, p. 89.

Ames, B.N. (1979). Identifying environmental chemicals causing mutations and cancer. Science, 204, 58 7.

Ames, B.N. (1983) . D ie t a ry ca rc inogens and a n t i c a r c i n o g e n s . Oxygen r a d i c a l s and d e g e n e r a t i v e d i s e a s e s . Sc inece , 221 , 1256.

Ames, B.N., H o l l s t e i n , M.C. and C a t h e a r t , R. (1982) . I n : L ip id p e r o x i d e s in Biology and Medicine, K Yogi, ( E d . ) , p . 339, Academic P r e s s , New York.

Anundi, I . , Hogberg, J . and Stead, A.H. (1979) . G lu t a th ione d e p l e t i o n in i s o l a t e d h e p a t o c y t e s . I t s r e l a t i o n to l i p i d p e r o x i d a t i o n and c e l l damage. Acta . Pharmacol. Tox ico l . , 45 , 4 5 .

Anundi, I . , K r i s t o f e r s o n , A. and Hogberg, J . (1978) . I n : I n d u s t r i a l and Environmental X e n o b i o t i c s . In v i t r o ve r sus ^ v ivo Bio t ransformat ion and t o x i c i t y J . R . Fouts and I . Gut ( E d s . ) , p . 277, Excerpta Medic a, Ames t e r dam.

AuerbacJh, C , Robson, J .M. and Car r , J . G. (1947) . The chemical p r o d u c t i o n of m u t a t i o n s . Sc i ence , 105, 243 .

1 0 3

Babu, K.A. , Nigam, S . K . , Lakkad , B . C . , B h a t t , D.K. , K a r n i l , A . B . , T h a k o r e , K.N. , Kashyap, S .K. and c h a t t e r j e e , S.K. ( 1 9 8 1 ) . E f f e c t of c h r y s o t i l e a s b e s t o s (AP-1) on s i s t e r ch roamt id exchanges i n C h i n e s e h a m s t e r o v a r y c e l l s . E n v i r o n . R e s . , 2 4 , 3 2 5 .

Beck, B. and Obe, G. ( 1 9 7 9 ) . S i s t e r c h r o m a t i d e x c h a n g e i n human l e u k o c y t e chromosomes: S p o n t a n e o u s and i n d u c e d f r e q u e n c i e s i n e a r l y and l a t e r e p l i c a t i n g c e l l s _iii v i t r o . Hum • G e n e t . , 4 9 , 5 1 .

Berenblum, I . ( 1 9 7 9 ) . I n : C a r c i n o g e n e s i s : I d e n t i f i c a t i o n and Mechanisms of A c t i o n , A.C. G r i f f i n and C.R. Shaw, ( E d s . ) , p . 2 5 , Raven P r e s s , new York .

B o e h l e c k e , B . , Shasby , D.M. , P e t e r s e n , M.R., Hodous , T.K. and Merchan t , J . A . ( 1 9 8 0 ) . I n : " P r o c e e d i n g s , N a t i o n a l Workshop, S u b s t i t u t e s f o r A s b e s t o s , EPA 5 6 0 / 3 - 8 0 - 0 0 1 1980 , A r l i n g t o n , V a . , J u l y 1 4 - 1 6 , 1 9 8 0 , " , p . 4 7 5 , U . S . E n v i r o n m e n t a l P r o t e c t i o n Agency, R e s e a r c h T r i a n g l e P a r k , N . C .

B o l t o n , R . E . , Add i son , J . , D a v i s , J . M . G . , Dona ldson , K . , J o n e s , A . D . , M i l l e r , B.G. and W r i g h t , A. ( 1 9 8 6 ) . E f f e c t of i n h a l a t i o n of d u s t s from c a l c i u m s i l i c a t e i n s u l a t i o n m a t e r i a l s i n l a b o r a t o r y r a t s . E n v i r o n . R e s . , 39 , 2 6 .

Borek , E. and W i l l i a m s , G. M. ( 1 9 8 0 ) . D i f f e r e n t i a t i o n and c a r c i n o g e n e s i s i n l i v e r c e l l c u l t u r e s . Ann. N.Y. Acad. S c i . , 349 , 4 2 9 .

B r i d g e s , B.A. ( 1 9 7 6 ) . S h o r t - t e r m s c r e e n i n g t e s t s f o r c a r c i n o g e n s . N a t u r e , 2 6 1 , 1 9 5 .

B r i d g e s , G. A. , Bochkov, N . P . and J a n s e n , J . D . ( 1 9 7 9 ) . G e n e t i c m o n i t o r i n g of human p o p u l a t i o n s a c c i d e n t a l l y e x p o s e d t o a s u s p e c t e d m u t a g e n i c c h e m i c a l . I n t e r n a t i o n a l Connmission f o r P r o t e c t i o n a g a i n s t E n v i r o n m e n t a l Mutagens and C a r c i n o g e n s , P u b l i c a t i o n No. 1 , M u t a t . R e s . , 6 4 , 5 7 .

.04

B r u s i c k , D . J . (19 7 7 ) . I n : T o x i c o l o g y A n n u a l , C .L . Winek, ( E d . ) , V o l . 2 , p . 7 9 , Marce l Dekker , New York.

B r u s i c k , D . J . ( 1 9 7 8 ) . The r o l e of s h o r t t e rm t e s t i n g i n c a r c i n o g e n d t e c t i o n . CheTiosphere, 5 , 40 3 .

Bunyan, J . , Green, J . , Edwin, E . E . a n d D i p l o c k , A .T . ( 1 9 6 0 ) .

* S t u d i e s on v i t a m i n E. 5 . L i p i d p e r o x i d a t i o n i n d i a l u r i c a c i d i n d c u e d h e m o l y s i s of v i t a m i n E. d e f i c i e n t e r y t h r o c y t e s . Biochem. J . , 77, 4 7 .

B u r r i s , S .M. , W h i t e , J . G. and E a t o n , J .W. 91980)-. I n : Red Blood C e l l and Lens M e t a b o l i s m . S .K. S r i v a s t a v a , ( E d . ) , p . 4 9 , E l a v i e r / N o r t h -H o l l a n d , Amsterdom. A

Bus, J . S . and Gibson , J . E . ( 1 9 7 9 ) . I n : Review i n B i o c h e m i c a l T o x i c o l o g y . E. Hodgson, J . r . Bend and R.M. P h i l p o t , ( E d s . ) V o l . 1 , p . 1 2 5 , E l s e v i e r , New York.

C a d e n a s , E . , G i n s b e r g , M., Robe, U. , and S i e s , H. ( 1 9 8 4 ) . E v a l u a t i o n of o C " t o c o p h e r o l a n t i o x i d a n t a c t i v i t y i n mic rosomal l i p i d p e r o x i d a t i o n as d e t e c t e d by low l e v e l c h e m i l u m i n e s c e n c e . Biochem. J . , 2 2 3 , 7 5 5 .

C a r r a n o , A.V. and Thompson, L.H. ( 1 9 8 2 ) . S i s t e r c h r o m a t i d exchange and gene m u t a t i o n s . C y t o g e n e t . C e l l G e n e t . , 3 3 , 5 7 .

Casey , G. ( 1 9 8 3 ) . S i s t e r c h r o m a t i d exchange and c e l l k i n e t i c s i n CHO-K 1 c e l l s , human f i b r o b l a s t s nad l y m p h o b l a s t o i d c e l l s exposed xn v i t r o t o a s b e s t o s and g l a s s f i b r e s . M u t a t . R e s . , 116 , 3 6 9 .

C a t t a n a c h , B.M. ( 1 9 6 6 ) . C h e m i c a l l y i n d u c e d m u t a t i o n s i n m i c e . M u t a t . R e s . 3 , 3 4 6 .

C h a n t o e r l a i n , M. and Tarmy, E.M. ( 1 9 7 7 ) . A s b e s t o s and g l a s s f i b r e s i n b a c t e r i a l m u t a t i o n t e s t s . M u t a t . R e s . , 4 3 , 1 5 9 .

105

Chao, E . S . , Dunbar, D. and Karainsky, L.S. (1988) . I n t r a c e l l u l a r l a c t a t e dehydrogenase c o n c e n t r a t i o n as an index of c y t o t o x i c i t y in r a t h e p a t o c y t e pr imary c u l t u r e . Ce l l Bio l . Tox ico l . , 4_, 1 .

Chen, H.H., Hsuch, J . L . , S i r i a n n i , S.R. and Huang, C.C. (1981) . Induc t ion of s i s t e r chromatid exchanges and c e l l cyc le delay in c u l t u r e d mammalian c e l l s t r e a t e d with e i g h t organophosphorus p e s t i c i d e s . Mutat . R e s . , 8 8 , 307.

»

Chvapi l , M., Stankova, L. and Malshet, V. (1976) . Lipid p e r o x i d a t i o n as one of the mechanisms of s i l i c a f i b r o g e n i c i t y : S tudies wi th e r y t h r o c y t e s . Environ. R e s . , 1 1 , 78 .

C l a rk , S.G. and H o l t , P.F . , (1960) . D i s s o l u t i o n of c h r y s o t i l e a sbes to s in wa te r , a c i d and a l k a l i . Nature , 18 5, 237.

Comporti , M. (1985) . Biology of d i s e a s e , l i p i d p e r o x i d a t i o n and c e l l u l a r damage in t o x i c l i v e r i n j u r y . Lab. Inves t . , 53 , 599.

Craig-Holmes,A.P. and shaw, M.W., (1976) . Cel l c y c l e a n a l y s i s i n asynchronous c u l t u r e s u s ing t h e BrdU-Hoechst t e c h n i q u e . Exp. C e l l . Res. , 99, 79 .

C r o c i , T. and Wil l iams, G.M. (1985) . A c t i v i t i e s of s e v e r a l phase I and phase I I x e n o b i o t i c b i o t r a n s f o r m a t i b n enzymes in c u l t u r e d h e p a t o c y t e s frc«n male and female r a t s . Biochem. Pharmacol . , 34 , 3029.

D a n i e l , F . B . , Beach, C.A. and Har t , R.W. (1980) . I n : The in v i t r o Ef fec t s of Mineral Dusts , R.C. Brown, I . P . Gormley, M. Chamberlain and R. Da v i e s , ( E d s . ) , p . 255. Academic P re s s , New York.

Decuyper-Debergh, D. , P i e t t e , J . , Lauren t , C. and Van de V o r s t , A. (1989) . Cyto tox ic and genotoxic e f f e c t s of e x t r a c e l l u l a r genera ted s i n g l e t oxygen in human lymphocytes i n v i t r o . Mutat . R e s . , 225, 1 1 .

106

Decuyper -Debergh , D. , P i e t t e , J . and Van de V o r s t , A. ( 1 9 8 7 ) . S i n g l e t oxygen i n d u c e d m u t a t i o n s i n M13 Lac Z p h a s e mh. EMBO, J . , 6 , 3 1 5 5 .

Deen, D . F . , Morgan, W . F . , T o f i l o n , P . J . and B a r c e l l o s -Hoff, M . F . ( 1 9 8 9 ) . Measurement of s i s t e r c h r o a m t i d e x c h a n g e s and t h e i r r e l a t i o n s h i p t o DNA damage, r e p a i r and c e l l k i l l i n g . P h a r m a c o l . T h e r . , 4 2 , 349 .

Demopoulos, H . B . , ( 1 9 7 3 ) . The b a s i s of f r e e r a d i c a l p a t h o l o g y . Fed . P r o c . 32, 18 5 9 .

Den izeau , F . , Mar ion , M., C h e v a l i e r , G. and C o t e , M.G. (1985) . U l t r a s t r u c t u r a l s t u d y of m i n e r a l f i b r e u p t a k e by h e p a t o c y t e s i n v i t r o . T o x i c o l . L e t t . , 2 6 , 1 9 .

Den izeau , F . , Mar ion , M. , C h e v a l i e r , G. and C o t e , M.G. (1985a) . I n a b i l i t y of c h r y s o t i l e a s b e s t o s f i b r e s t o m o d u l a t e t h e 2 - a c e t y l a m i n o f l u o r e n e - i n d u c e d UDS i n p r i m a r y c u l t u r e s of r a t h e p a t o c y t e s . M u t a t . R e s . , 1 5 5 , 8 3 .

Den i z ea u , F . , Mar ion , M. , Q i e v a l i e r , G. and C o t e , M.G. ( 1 9 8 5 b ) . Absence of g e n o t o x i c e f f e c t s of n o n a s b e s t o s m i n e r a l f i b r e s . C e l l B i o l . T o x i c o l . , 1 , 2 3 .

D e n i z e a u , F . , Mar ion , M. , C h e v a l i e r , G. and C o t e , M.G. ( 1 9 8 5 c ) . G e n o t o x i c i t y of d i m e t h y l n i t r o s amine i n t h e p r e s e n c e of c h r y s o t i l e a s b e s t o s UICC B a n d x o n o t l i t e . C a r c i n o g e n e s i s , 6 , 1 8 1 5 .

Den i zeau , F . , Mar ion , M., F l e u r y , J . , C h e v a l i e r , G. and C o t e , M.G. (198 5 d ) . I n : I n v i t r o E f f e c t s of M i n e r a l D u s t s , E .G.Beck nd J . Bignon, ( E d s . ) , p . 2 6 1 , Sp r i n g e r - V e r l a g , B e r l i n . H e i d e l b e r g . ^ ^

D e u t i c k e , B. ( 1 9 7 7 ) . P r o p e r t i e s and s t r u c t u r a l b a s i s of s i m p l e d i f f u s i o n pa thways i n t h e e r y t h r o c y t e membrane. Rev. P h y s i o l . Biochem. P h a r m a c o l . , 7 8 , 1 .

107

D i a n z a n i , M.U. and Ugaz io , G. ( 1 9 7 8 ) . I n : B i o c h e m i c a l Mechanisms of L i v e r I n j u r y , T . F . S l a t e r , ( E d . ) , p . 6 6 9 , Academic P r e s s , London.

D i P a o l o , J . A . , DeMar in i s , A . J . and D o n i g e r , J . ( 1 9 8 2 ) . A s b e s t o s and benzo{ a) p y r e n e s y n e r g i s m i n t h e t r a n s f o r m a t i o n of S y r i a n h a m s t e r ertibryo c e l l s . J_^ E n v i r o n . P a t h o l . T o x i c o l . , 5 , 5 3 5 .

E m e r i t , I . , Keck, M., Levy , A . , F e i n g o l d , J . and M i c h e l s o n , A.M. ( 1 9 8 2 ) . A c t i v a t e d o x y t e n s p e c i e s a t t h e o r i g i n of chronosome b r e a k a g e and s i s t e r c h r o m a t i d e x c h a n g e s . M u t a t . R e s . , 1 0 3 , 1 6 5 .

E n e a n y a , D . T . , D a n i e l , F . B . and H a r t , R.W. ( 1 9 7 9 ) . E f f e c t of a s b e s t o s ( c h r y s o t i l e i n t e r m e d i a t e ) on t h e m e t a b o l i s m of benzo( a) p y r e n e i n S y r i a n h a m s t e r eiribryo c e l l s . P r e s e n t e d a t Am.Sc i . P h a r m a c o l . E x p t t . T h e r o p . M e e t i n g . Hous ton , TX.

E r n s t e r , L . and N o r d e n b r a n d , K. ( 1 9 6 7 ) . I n : Methods i n enzymology . R .W.Es tabrook and M.E. P u l l m a n , ( E d s . ) , V o l . 1 0 , p . 4 7 4 , Academic P r e s s , New York .

F a r i s s , M.W., P a s c o e , G. A. and Reed, D . J . ( 1 9 8 5 ) . V i t a m i n E r e v e r s a l o f t h e e f f e c t of e x t r a c e l l u l a r c a l c i u m on c h e m i c a l l y i n d u c e d t o x i c i t y i n h e p a t o c y t e s . S c i e n c e , 277 , 5 7 1 .

F i s h e r , A.B. ( 1 9 8 9 ) . I n : E f f e c t s of M i n e r a l Dus t s on C e l l s , B.T. Mosman and R.O. Begin , ( E s . ) 1 4 9 . , S | p r i n g e r - V e r l a g . B e r l i n ^ H e i d e l b e r g .

F l e u r y , J . , C o t e , M.G. , Mar ion , M., C h e v a l i e r , G. and Den izeau , F . ( 1 9 8 3 ) . I n t e r a c t i o n s of a s b e s t o s f i b r e s w i t h h e p a t o c y t e s i n v i t r o : An u l t r a s t r u c t u r a l s t u d y . T o x i c o l . L e t t . , 1 9 , 1 5 .

108

Fong, K.L., McCay, P. B., Poyer, J.L. Keele, B.B. and Mishra H.(1973). Evidence that peroxidation of lysosomal membranes is inhibited by hydroxyl free radicals produced during flavin enzyme activity. J_j_ Biol. Chem., 248, 7792.

Fr idov ich , I . (1981) . In : Membrane Moleculer , Toxins and C e l l s , K. Bloch, L. Boi ls and D. Tosteson, ( E d s . ) , p . 105.

Friedberg, E.G. (1985). In: DNA Repair, E.C. Friedberg, (Ed), p. 459, Freeman, San Francisco, CA.

Fry, J . R . , and Br idges , J.W. (1979) . Use of pr imary h e p a t o c y t e c u l t u r e s in b iochemical tox ico logy . Rev. Biochem. Tox ico l . 1, 201.

Gabor, S. and Anea, Z. (1974) . E f f ec t of s i l i c a on l i p i d p e r o x i d a t i o n in the r ed c e l l s . I n t e r n . Arch. Occup. H l t h . , 32^, 327.

Gabridge, M.G. and Lega to r , M.S. (1969) . A h o s t media ted a s say for the d e t e c t i o n of mutagenic compounds. P roc . Soc. Exp. B i o l . Med., 130, 8 3 1 .

Galloway, S.M. and Wolff, S. (1979) . The r e l a t i o n between chemica l ly induced s i s t e r chromatid exchanges and chromatid breakage , Mutat . Res. 6 1 , 297.

Gladen, B.E. , S u l l i v a n , D.W. (1979) . I n : Progress in CJiemical and B io log i ca l Research , S.E. Lux, V . T . , Marches i , And C.F. Fox, ( E d s . ) , Vol. 30, p . 503, Alan R. L i s s , I n c , New York.

Greenstock, C.L. (1986). In: Free Radicals, Aging and Degenerative Diseases, J.E. Johnson Jr., R. Walford, D. Harman and J. Miquel, (Eds.), 197, Liss, New York.

Gross, S. and Melhorn, D.K. (1972). Vitamin E., red cell lipids and red cell stability in prematurity. Ann. N. Y. Acad. Sci., 203, 141.

109

Guzelian, P . S . , B i s s e l l , D.M. and Meyer, U.A. (1977) . Drug metabolism in a d u l t r a t h e p a t o c y t e s in p r imary monolayer c u l t u r e . Gas t r o e n t e r o l o g y , 72, 1232.

H a l l , J . and Mount, D. (1981) , Mechanism of DNA r e p l i c a t i o n and mutagenesis in u l t r a v i o l e t i r r a d i a t e d b a c t e r i a and mammalian c e l l s . Prog. Nucle ic Acid. Res. Mol. B i o l . , 25, 53 .

Haber, F . and Weiss, J . (1934) . > The c a t a l y t i c deconpos i t ion of hydrogen peroxide by

i r o n s a l t s . Proc . R. Soc. London, 147, 332.

Hahon, N. , Booth, J . A . , Boehlecke, B.A. and Merchant. J .A. (1980) . Enhanced v i r a l i n t e r f e r o n i n d u c t i o n by the minera l w o l l a s t o n i t e . J . I n t e r f e r o n Res . , 1, 49 .

H a l l i w e l l , B. and Gu t t e r i dge , J .M.C. (1984) . Oxygen t o x i c i t y , oxygen r a d i c a l s , t r a n s i t i o n meta ls and d i s e a s e . Biochem. J . , 219, 1 .

H a l l i w e l l , B. and Gu t t e r idge , J .M.C. (1985) . In : Free r a d i c a l s i n Biology and Medicine, Oxford Sc ience P u b l i c a t i o n s , Clarendon P r e s s , B e l f a s t .

H a l l i w e l l , B. and Gu t t e r idge , J .M.C. (1986) . Oxygen f r e e r a d i c a l s and i r o n in r e l a t i o n t o b io logy and medic ine: Some problems and concep t s . Arch. Bioch em. Biophys. , 246, 501 .

Har ing ton , J . S . , A l l i s o n , A.C. and Badami, D.V. (1975) . Mineral f i b r e s : Chemical, Phys ico-chemica l and b i o l o g i c a l p r o p e r t i e s . Adv. Pharmacol. Chemotherapy, 12, 2 9 1 .

Har ington , J . S . , M i l l e r , K. , and Macnab, G. (19 71) Hemolysis by a s b e s t o s . Environ. Res. , 4_, 9 5 .

H a r r i s o n , P.T.C. and Heath, J . C . (1986) . Apparent synergy in lung c a r c i n o g e n e s i s i n t e r a c t i o n between N-n i t roschep ta rae thy lene imine , p a r t i c u l a t e cadmium and c r o c i d o l i t e a s b e s t o s f i b r e s in r a t s . Carc inogenes i s , 7 , 1903.

110

Hart , R.W., Danie l , F . B . , Kindid, O.R., Beach, C.A., Joseph, L.B. and Well, R.C. (1980) . Elemental modi f i ca t ions and p o l y c y c l i c a romat ic hydrocarbons metabolism in human f i b r o b l a s t s . Environ. H l t h . P e r s p e c t . , 34, 59.

Hefner, R.E. and Gehring, P . J . (1975) . A comparison of r e l a t i v e r a t e s of hemolys is induced by va r ious f i b r o g e n i c and non f i b r o g e n i c p a r t i a l s wi th washed r a t e r y t h r o c y t e s Jji v i t r o . Am. Ind . Hyg. Assoc. J . 36, 734.

Hes te rberg , T.W. and B a r r e t t , J . C . (1985) . Induc t ion of a sbes tos f i b r e s of anaphase , a b n o r m a l i t i e s : Mechanism of aneuplo idy i nduc t i on and p o s s i b l e c a r c i n o g e n e s i s . Ca rc inogenes i s , 6 , 473.

Hes te rbe rg , T.W., Oshimura, M., B a r r e t t , J . C . (1985) . I n : In v i t r o Effec ts of Mineral Dus t s . E a r n s t . G. Beck and J . Bignon, ( E d s . ) , p 185. Sp r inge r -Ver l ag^ Ber l in^Heide lberg , New York.

Hill KE. and Burk, R.F. (1984). Influence of vitamin E and selenium on glutathione-dependent protection against microsomal lipid peroxidation, Biochem. Pharmacol., 33, 106 5.

Hogberg, J. and Kristoferson, A. (1977) . A correlation between glutathione levels and cellular damage in isolated hepatocytes. Eur. J. Biochem., 74, 77.

Hogberg, J. Moldeus, P., Arborgh, B., O'Brien, P.J. and Orrenius, S. (1975a). The consequences of lipid peroxidation in isolated hepatocytes. Eur. J. Biochem., 59, 457.

Hogberg, J., Orrenius, S. and Larson, R.E. (1975b). Lipid peroxidation in isolated hepatocytes Eur. J. Biochem. , 50, 595.

Hogberg, J., Orrenius, S. , and O'Brein, P.J. (1975c). Further studies on lipid peroxidase formation in isolated hepatocytes. Eur. J. Biochem., 59, 449.

I l l

Huang, S .L. , (19 79) . Amosite, Chrysotile and crocidolite asbestos are mutagenic to Chinese hamster lung cells. Mutat. Res., 68, 265.

Huang, S.L. Saggioro, D., Michelmann, H., and Mailing, H.V. (1978). Genetic effects of crocidolite asbestos in dhinese hamster lung cells. Mutat. Res. 57, 225.

Hv^nt, J., Pooley, F.D., and Richards, R.J. (1981). Biological reactivity of calcium silicate composites: In vitro studies. Environ. Res. 26, 51. •

H u n t e r , D. ( 1 9 6 9 ) . I n : D i s e a s e s of o c c u p a t i o n s , p . 1009 , 4 t h E d i t i o n , The E n g l i s h U n i v e r s i t i e s P r e s s . , London.

Huuskonen, M . S . , Ahlman, K. , M a t t s s o n , T . and T o s s a v a i n e n A. , (1980) . . A s b e s t o s d i s e a s e i n F i n l a n d . J_^ Occup. Med. , 22 , 7 5 1 .

Huuskonen, M . S . , J a r v i s a l o , J . , K o s h i n e n , H . , N i c k e l s , J . R a s a n e n , J . and A s p . , S . ( 1 9 8 3 a ) . P r e l i m i n a r y r e s u l t s from a ccS ior t of w o r k e r s exposed t o w o l l a s t o n i t e i n a F i n i s h l i m e s t o n e q u a r r y . Second . J . Work. E n v i r o n . H l t h . , 9_, 1 6 9 .

Huuskonen, M . S . , T o s s a v a i n e n , A . , K o s h i n e n . , H . , Z i t t i n g , A . , Khonen, O . , N i c k e l s , J . , Korhonen , K. , and V a a r a n e n , V. ( 1 9 8 3 b ) . W o l l a s t o n i t e e x p o s r u e and l u n g f i b r o s i s . E n v i r o n . Res . , 3 0 , 2 9 1 .

lARC Monographs on t h e E v a l u a t i o n of r i s k of c h e m i c a l s t o humans . H e a l t h O r g a n i s a t i o n , 1987 . •

t h e V o l . ,

c a r c i n o g e n i c 4 2 , World

I g u c h i , H. and Kojo , S . , (1989) P o s s i b l e g e n e r a t i o n of h y d r o g e n p e r o x i d e and l i p i d p e r o x i d a t i o n of e r y t h r o c y t e membrane by a s b e s t o s : C y t o t o x i c mechanism of a s b e s t o s . Biochem. I n t e r n a t . , 18 , 9 8 1 .

112

J a q u e s , L .M. , Brown, E . B . , B e r r e t t , J . M . , Bray, W.S. and W e l t e r , W. (1977) . S i a l i c a c i d : A c a l c i u m b i n d i n g c a r b o h y d r a t e . J . B i o l . Chem. 2 5 2 , 4 5 3 3 .

J a u r a n d , M.C. , Kheuang, L . , Magne, L. and Bignon, J . (1986) . Chromosomal changes i n d u c e d by c h r y s o t i l e f i b r e s o r b e n z o 3 , 4 - p y r e n e i n r a t p l e u r a l m e s o t h e l i a l c e l l s . M u t a t . R e s . , 169 , 1 4 1 .

J a u r a n d , M.C. , Magne. L. and Bignon, J . ( 1 9 7 9 ) . I n h i b i t i o n of p h o s p h o l i p i d s o f h e m o l y t i c a c t i o n by a s b e s t o s . Br . J . I n d . Med. , 3 6 , 1 1 3 .

J a u r e g u i , H . O . , Hayne r , N . T . , D r i s c o l l , J . L . , W i l l i a m s -H o l l a n d , R . , L i p s k y , M.H. , and G a l l e t t i , P.M. (1981) . T rypan b l u e dye u p t a k e and l a c t a t e d e h y d r o g e n a s e i n a d u l t r a t h e p a t o c y t e s - F r e s h l y i s o l a t e d c e l l s ^ c e l l s u s p e n s i o n s and p r i m a r y monolayer c u l t u r e s . I n v i t r o , 1 7 , 1 1 0 0 .

K a p l a n , H . , R e n i e r , A . , J a u r a n d , M.C. and Bignon, J . (1980) . I n : xn v i t r o E f f e c t s of M i n e r a l IXasts, R .C. Brown, I . P. Gormley> M. C h a m b e r l a i n and R. D a v i e s , ( E d s . ) , p . 2 5 1 . , Academic P r e s s , New York.

Kappus, H. (1985). In: Oxidative Stress, H. Sies, (Eds.) 273. Academic Press, London.

Kelsey, K.T., Christiani, D.C. and Little, J.B. (1986a) . Enhancement of benzo( a)pyrene-induced sister chrcsnatid exchanges in lymphocytes from cigarette smokers occupationally exposed to asbestos. J. Natl. Cancer Inst, 77, 321.

Kelsey, K.T., Yano, E., Liber, H.L. and Little, J.B. (1986b) . The in vitro genetic effects of fibrous erionite and crocidolite asbestos. Br. J. Cancer, 54, 107.

113

Kennedy, T . P . , Dodson, R. , Rao, N . V . , Henry , K. Y. , Hopk ins , C , B a s e r , M., T o l l e y , E. and H o i d a l , J . R . ( 1 9 8 9 ) . Dus t s c a u s i n g p n e u m o c o n i o s i s gen^erate OH* and p r o d u c e h e m o l y s i s . Arch . Bioch em. Biophys . , 269 , 3 59 .

K e n s l e r , T.W., and T r u s h , M.A. ( 1 9 8 4 ) . Role of oxygen r a d i c a l s i n tumor E n v i r o n . M u t a g e n . , ^ , 5 9 3 .

p r o m o t i o n .

Kimizuka , G. , Ohwada, H . , H a y a s h i , Y. , C o - c a r c i n o g e n i c e f f e c t of benzo( a ) p y r e n e i n t h e lung of P a t h o l . , 37 , 4 4 8 .

( 1 9 8 7 ) . a s b e s t o s and

h a m s t e r . A c t a

K i t s u g i , T . , Yamamura, T. and Kokubo, T. ( 1 9 8 9 ) . Bonding b e h a v i o u r of a g l a s s - c e r a m i c c o n t a i n i n g a p a t i t e and w o l l a s t o n i t e i n s e g m e n t a l r e p l a c e m e n t of t h e r a b b i t t i b i a u n d e r l o a d - b e a r i n g c o n d i t i o n s . J . Bone J o i n t S u r g . , 71A, 264.

K l a a s s e n , C D , and S t a c e y , N.H. ( 1 9 8 2 ) . I n : T o x i c o l o g y of t h e L i v e r , G. P l a a , W.R. ( E d s . ) , p . 1 4 7 . Raven P r e s s , New York.

Hewitt.

Koppenol, W.H. and Butler, J. (1977). Mechanism of reaction involving singlet oxygen and the superoxide anion. FEBS Lett., 83, 1.

Kosower, N.S. and Kosower, E.M. (1983). In: Functions of Glutathione, Biochemical Physiological, Toxicological, and Clinical Aspects, A Larson, S. Orrenius and A. Holmgren, (Eds.), p. 307. Raven Press, New York.

Krebs, H.A., Lund, P. and Edwards, M. (1979). In: Cell Populations, E. Reid, (Ed.), p. Horwool, Chichester.

1, Ellis

Krishna, G., Nath, J. and Ong, T. (1986). Murine bone marrow culture system for analysis. Mutat. Res. , 164, 91.

cytogenetic

114

Latt, S.A. (1973) . Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. Natl. Acad. Sci. (USA) , 70, 3395.

L a t t , S .A. ( 1 9 8 1 ) . S i s t e r c h r o m a t i d exchange f o r m a t i o n . Annu. Rev. G e n e t . 1 5 , 1 1 .

L a v a p p a , K . S . , Fu, M.M. and E p s t e i n , S . S . ( 1 9 7 5 ) . C y t o g e n e t i c s t u d i e s on c h r y s o t i l e a s b e s t o s E n v i r o n . R e s . , 1 0 , 1 6 5 .

L e c h n e r , J . F . , Haugen, A . , Tokiwa , T . , Trump, B . F . , a n d H a r r i s , C.C. ( 1 9 8 3 ) . I n : Human C a r c i n o g e n e s i s , C .C . H a r r i s and H. A u t r u p . ( E d s . ) , p . 5 6 1 , Academic P r e s s , New York.

L e m a i r e , I . , G i n g r a s , D. and L e m a i r e S . , ( 1 9 8 2 ) . Thymid ine i n c o r p o r a t i o n by l u n g f i b r o b l a s t s a s s e n s i t i v e a s s a y f o r b i o l o g i c a l a c t i v i t y of a s b e s t o s . E n v i r o n . R e s . , 2 8 , 3 9 9 .

L e m a i r e , I . , G i n g r a s , D . , and L e m a i r e , S . ( 1 9 8 6 ) . E f f e c t s of c h r y s o t i l e a s b e s t o s on ENA s y n t h e s i s and g rowth of human embryon ic l u n g f i b r o b l a s t s . J . E n v i r o n . P a t h o l . T o x i c o l . , 6 , 1 6 9 .

L i g h t , W.G. and Wei, E . T . ( 1 9 8 0 ) . I n : The i n v i t r o E f f e c t s of M i n e r a l D u s t s , R .C. Brown, I . P . Gormley, M. C h a m b e r l a i n and R . , D a v i e s , ( E d s . ) , p . 139 , Academic P r e s s , New York.

L i v i n g s t o n , G.K. , Rom, W.N. and M o r r i s , M.V. ( 1 9 8 0 ) . A s b e s t o s i nduced s i s t e r c h r o m a t i d exchange i n c u l t u r e d C h i n e s e h a m s t e r o v a r i a n f i b r o b l a s t s c e l l s . J . E n v i r o n . P a t h o l . T o x i c o l . , 4 , 3 7 3 .

Lowry, O . H . , Rosenbrough , N . J . , F a r r , A . L . and R a n d a l l , R . J . , ( 1 9 5 1 ) . P r o t e i n measuremen t w i t h t h e f o l i n p h e n o l r e a g e n t . J . B i o l . Chem., 19 3 , 2 6 5 .

115

Lutz, W.K. (19 79) . In v i t r o binding of organic chemicals to ENA as a quan t i t a t ive indica tor in the processes of chemical carcinogenesis . Mutat. Res. , 65, 289.

Mailing, H.V. (1971) . Dimethylnitrosamine: Formation of mutagenic compounds by interaction with mouse liver microsomes. Mutat. Res., 13, 825.

Mason, R.P. and Chignell, C.F. (1982). Free radicals in pharmacology and toxicology-Selected topics. Pharmacol. Res., 33, 189.

McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella microsome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. (USA), 72, 5135.

McCay, P.B. (1985). Vitamin. E: Interactions with free radicals and ascorbate. Ann. Rev. Nutr., 5, 32 3.

McCay, P.B. and Poyer, J.L. (19 76). In: The Enzymes of Biological Membranes, Vol. 1 p. 239., Plenum Press, New York.

McQueen, C.A. and Williams, G.M. (1982). Cytotoxicity of ^ xenobiotics in adult rat hepatocytes in primary culture. Fund. Appl. Toxicol., 2, 139.

Mengel, C.E. and Kann, Jr. H.E., (1966). Effects of in vitro hyperxia on erythrocytes. III. In vitro peroxidation of erythrocytes lipids. J. Clin. Invest. , 45, 1150.

M i l l e r , K. , ( 1 9 8 0 ) . In : The i n v i t r o E f f e c t s of Mineral D u s t s . R.C. Brown, I . P . Gormley, M., Chamberlain and R. D a v i e s , ( E d s . ) , p . 9 1 , Academic P r e s s , New York.

116

Mitchell, A.D., Casciano, D.A. , Meltz, M.L., Robinson, Williams, G.M. and Von Halle, D.E., San, R.H.C.,

E.S. (1983). Unscheduled DNA synthesis test: U.S. Environmental Protection Program. Mutat. Res., 123, 363.

report of the Agency-Genotox

Mitchell, D.B. and Acosta, D. (1981). Evaluation of the cytotoxicity of tricyclic antidepressants in primary cultures of rat hepatocytes. J. Toxicol. Environ. Health, 1_, 83.

Moldeus, P., Hogberg, J. and Orrenius, S. (1978). In: Methods in Enzymology S» Fleischer and L. Packer, (Eds.)., Vol, 52, p. 60. Academic Press, New York.

Moldeus, P. and Quanguan, J. (1987). Importance of the glutathione cycle metabolism. Pharmacol. Ther., 33, 37.

xn drug

Moorhead, P.S., Nowell, P.C., Mellman, W.J., Battips, D.M. and Hungeford, P.A. (1960). Chromosome preparations: Leukocyte cultures from human peripheral blood. Exp. Cell. Res., 20, 613.

Mossman, B.T., Eastman, A., Landesman, J.M. and Bresnick, E. (1983) . Effects of crocidolite and chrysotile asbestos on cellular uptake and metabolism of benzo(a)pyrene in Hamster tracheal epithelial cells. Environ. Hlth. Perspect., 51, 331.

Muller, H . J . , (1927) . A r t i f i c i a l transmutation of the gene, 84.

Science, 66,

Nakaraura, T. , Fuj i , T. and Ichxhara, A. (1985). Enzyme leakage due to the change of membrane permeabil i ty of primary cul tured r a t hepatocytes t rea ted with various hepatotoxins and i t s prevention by g lycyr rh iz in . Cell Biol. Toxicol. , 1 , 286.

117

Natarajan, A.T., Csukas, I. and Van Zeeland, A.A. (1981) . Contribution of incorporated 5-bromodeoxyuridine in DNA to the frequencies of sister chromatid exchanges induced by inhibitors by poly (ADP-ribose) - polymerase. Mutat. Res., 84, 125.

Nitowsky, H.M., Hsu, K.S. and Gordon, H.H. (1962). Vitamin E. Requirements of human infants. Vitamins Hormones, 20, 559.

Ono, K., Yamamura, T., Nakamura, T., Kakutani, Y., Kitsugi, T., Hyakuna, K., Kobubq, T., Oka, M. and Kotoura, Y. (1988) . Apatite- wollastonite containing glass ceramic fibrin mixture as a bone defect filler. J_j_ Biomed. Mater. Res. , 22, 869.

Oshimura, M. , Hesterberg, T.W., Tsutsui, T. and Barrett, J.C. (1984) . Correlation of asbestos induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture. Cancer Res., 44, 5017.

Pailes, W.H., Judy, D.J., Resnick, H. and Castranova, V. (1984). Relative effects of asbestos and wollastonite on alveolar macrophages. J_ Toxicol. Environ. Health, 14, 497.

Palekar, L.D., Eyre, J.F., Most, B.M. and Coffin, D.L. (1987). Metaphase and anaphase analysis of V-79 cells exposed to erionite, UICC chrysotile and UICC crocidolite. Carcinogenesis, 8 , 553.

Pascoe, G.A., Fariss, M.W., Olafsdottir, K. and Reed, D.J., (1987). A role of vitamin E in protection against cell injury: Maintenance of intracellular glutathione precursors and biosynthesis. Eur. J. Biochem., 166, 241.

Perderiset, M., Baillif, P. and Jaurand, M.C. (1988). Chemical analysis and photoelectron spectroscopy of macromolecule adsorption on the surface of attapulgite particles. J_>_ Colloid Interface Sci. . 121, 381.

118

Perderiset, M. , Saint Etienne, L. , Bignon, J. and Jaurand, M.C. (1989). Interactions of attapulgite (fibrous clay) Toxicol. Lett., 47, 303.

P e r r y , P . (1980) . I n : Chemica l Mutagens : P r i n c i p l e s and T h e i r D e t e c t i o n , F . J . d e S e r r e s and A. ( E d s . ) , V o l . 6 , p . 1 , Plenum P r e s s , New

Methods f o r H o l l a n e n d e r York.

P e r r y , P . and E v a n s , H . J . , (1975) . * C y t o l o g i c a l d e t e c t i o n of m u t a g e n - c a r c i n o g e n

e x p o s u r e by s i s t e r c h r o m a t i d e x c h a n g e . N a t u r e , 2 5 8 , 1 2 1 .

P e r r y , P and Wolff , F . (19 7 4 ) . New giemsa method f o r t h e d i f f e r e n t i a l s t a i n i n g of s i s t e r c h r o m a t i d s . N a t u r e , 2 5 1 , 156 .

P o t t , F . , Ziem, U. , R e i f f e r , F . J . , Huth , F . , E r n s t , H . , and Mohr, U. (198 7) . C a r c i n o g e n i c i t y s t u d i e s on f i b r e s m e t a l ccxnpounds and some o t h e r d u s t s i n r a t s . Exp. P a t h o l . , 3 2 , 1 2 9 .

P o t t s , W . J . , L e d e r e r , T . S . and G e h r i n g , P . T . ( 1 9 7 8 ) . Hemolys i s of washed r a t e r y t h r o c y t e s i n v i t r o b y d u s t p a r t i c l e s . Ann. I n d . Hyd. A s s o c . J . , 3 9 , 49 7 .

P r i c e - J o n e s , M . J . , Cubb ings , G. , and C h a m b e r l a i n , M (1980) . The g e n e t i c e f f e c t s of c r o c i d o l i t e Compar ison of chromosome a b n o r m a l i t i e s c h r o m a t i d e x c h a n g e s . M u t a t . R e s . , 79

a s b e s t o s : and s i s t e r

3 3 1 .

P r y o r , W . A . , S t a n l e y , J . P . and B l a i r , E . ( 1 9 7 6 ) . A u t o x i d a t i o n of p o l y u n s a t u r a t e d f a t t y a c i d s : I I s u g g e s t e d mechanism f o r t h e r e a c t i v e m a t e r i a l s from e n d o p e r o x i d e s . L i p i d s , 1 1 , 3 7 0 .

A f o r m a t i o n of TBA-

prOS t a g l a nd i n - 1 i k e

Rahman, Q. and C a s c i a n o , D.A. ( 1 9 8 5 ) . I n : Iji v i t r o E f f e c t s of M i n e r a l D u s t s ,

( E d s . ) , p . 4 8 3 . and J . Bignon, B e r l i n . H e i d e l b e r g .

E.G. Be ck S p r i n g e r - V e r l a g ^

119

Rahman, Q. , N a h i d , F . , J a i n , A.K. and A g a r w a l , S . S . ( 1 9 8 8 a ) . S i s t e r c h r o m a t i d exchange f r e q u e n c y and chromosomal a b e r r a t i o n s i n a s b e s t o s f a c t o r y w o r k e r s . V l l t h I n t e r n a t i o n a l P n e u m o c o n i o s i s C o n f e r e n c e , A u g u s t , 2 3 - 2 6 , P i t t s b u r g h , U.S.A.

Rahman, Q. (1988b) I n v i t r o

Nah id , F . , J a i n , A .K . , and Agarwa l , S.S

a s s e s s m e n t of t h e c y t o g e n e t i c e f f e c t s of c h r y s o t i l e f i b r e s u s i n g human b l o o d l y m p h o c y t e s . IVth I n t e r n a t i o n a l Workshop on E f f e c t s of M i n e r a l Dus ts on C e l l s , S e p t . 2 1 - 2 3 , Aube rge , Canada .

Rahman, Q . , ( 1 9 7 4 ) . A s b e s t o s i n d u c e d s i l i c a s o l u b i l i t y . 284 .

N a r a n g , S . , k a w , J . L . and Z a i d i , S .H .

h e m o l y s i s E n v i r o n .

i n r e l a t i o n t o i t s P h y s i o l . B iochem. , 4 ,

Rahman, Q. , V i s w a n a t h a n , P .N . and Z a i d i , S.H. (19 7 7 ) . Some new p e r s p e c t i v e s on t h e b i o l o g i c a l e f f e c t s of a s b e s t o s . E n v i r o n . R e s . , 14 , 4 8 7 .

Reddy, E . P . , R e y n o l d s , P . K . , S a n t o s , E . and B a r b a c i d , M. ( 1 9 8 2 ) . A p o i n t m u t a t i o n i s r e s p o n s i b l e f o r t h e a c q u i s i t i o n o f t r a n s f o r m i n g p r o p e r t i e s by t h e T24 human b l a d d e r c a r c i n o m a o n c o g e n e . N a t u r e , 300 , 1 4 9 .

Reed, D . J . and F a r i s s , M.W. ( 1 9 8 4 ) . G l u t a t h i o n e d e p l e t i o n and P h a r m a c o l . R e v . , 36 , 2 5 S .

s u s c e p t i b i l i t y .

Reed, D . J . , P a s c o e , G.A. and O l a f s d o t t i r , K. ( 1 9 8 7 ) . Some a s p e c t s of c e l l d e f e n s e mechanisms of g l u t a t h i o n e and v i t a m i n E d u r i n g c e l l i n j u r y . A r c h . T o x i c o l . 11 ( S u p p l . ) 3 4 .

R e i s s , B . , Tong, C . , T e l a n g , S. and W i l l i a m s , G . M . (1983) Enhancement of benzo( a ) p y r e n e m u t a g e n i c i t y bny c h r y s o t i l e a s b e s t o s i n r a t l i v e r e p i t h e l i a l c e l l s . E n v i r o n . R e s . , 3 1 , 1 0 0 .

R i c h a r d s , R . J . , B i o l o g i c a l c o m p o s i t e s :

T e t l e y , T .D. and Hunt , J . ( 1 9 8 1 ) . r e a c t i v i t y of c a l c i u m s i l c i a t e

I n v i v o s t u d i e s . E n v i r o n . R e s . 2 6 , 2 4 3 .

120

R i g o , A. j S t e v a n a t o , R-. , F i n a z z i - A g r o , A. and R o t i l i o , G. ( 1 9 7 7 ) . An a t t e m p t t o e v a l u a t e t h e r a t e of Habe r -Wei s s r e a c t i o n by u s i n g OH r a d i c a l s c a v e n g e r s . FEBS L e t t . , 8 0 , 130 .

R i t a , P . , Reddy, P . P . ( 1 9 8 6 ) . E f f e c t of c h r y s o t i l e a s b e s t o s f i b r e s on germ c e l l of m i c e . E n v i r o n . R e s . , 4 1 , 139 .

Rom, W.N., L i v i n g s t o n , G. K . , Casey , K . P . , Wood, S . D . , * E g g e r , M . J . , C h i u , G.L. and J e r c j m i n s k i , »L. ( 1 9 8 3 ) .

S i s t e r c h r o m a t i d e x c h a n g e f r e q u e n c y i n a s b e s t o s w o r k e r s . Jj_ N a t l . Cancer I n s t . , 7 0 , 4 5 .

R u s s e l l , W.L. ( 1 9 5 1 ) . X - r a y i n d u c e d m u t a t i o n s i n m i c e . Cold S p r i n g H a r b o r Symp. Quan t . B i o l . , 1 6 , 3 2 7 .

Russell, W.L. (1977). The role of mammalian in the future of chemical mutagenesis research. Arch. Toxicol., 38, 141.

Sakabe, H., Koshi, K. and Hayashi, H. (1970). In: Inhaled Particles. III.W.H. Walton (Ed.), Vol. I, p. 423, Unwin Bros, Old Working, Surrey,

Sandy, M.S., DiMonte, D. and Smith, M.T. (1988). Relationship between intracellular vitamin E, lipid peroxidation and chemical toxicity in hepatocytes. Toxicol. Appl. Pharmacol. , 93, 288.

Schepers, G.W.H., Durkan, T.M. and Delahant, A.B. (19 55). Effect of inhaled commercially hydrous calcium silicate dusts on animal tissues. Arch. Ind. Health, 12, 348.

Sedlack, V. and Ludsay, R.H. (1968). Estimation of total, protein bound and non-protein-SH group in tissue with Ellmanoic reagent. Anal. Biochem., 25, 192.

Sharma, O.P. and KrishnaMurti, C.R. (1980). Lipid peroxidation in animal tissues. J_ Sci. Indust. Res., 39, 110.

121

Shasby, D.M. , Petersen, M., Hodous, T., Boehlecke, B. and Merchant, J. (1979). In: Dust and Disease, R. Lemen and J.M. Dement, (Eds.), p. 251, Pathodox, Park Forest South.

S h i r a i s h i , Y. ( 1 9 8 2 ) . I n : S i s t e r Chromat id Exchange , A.A. Sandberg ( E d . ) , p . 39 5 , A l a n , R. L i s s , New York.

S i e s , H. and C a d e n a s , E . ( 1 9 8 3 ) . I n : B i o l o g i c a l B a s i s o f D e t o x i f i c a t i o n , J . C a l d w e l l

« and W.B. J a k b y ( E d s . ) , p . 1 8 1 , Academic P r e s s , London.

Sincock, A.M. (1977). In: Origin of Human Cancer, H.H. Hiatt, J.D. Watson, J.A. Winsten, (Eds.), p. 941, Cold Spring Harbor Laboratory.

Sincock, A.M., Delhonty, J.D.A. and Casey, G. (1982). A comparison of the cytogenetic response to asbestos and glass fibre in Chinese hamster and human cell lines. Mutat. Res., 101, 257.

Sincock, A.M. and Seabright, M. (1975). Induction of chromosome changes in Chinese hamster cells by exposure to asbestos firbes. Nature, 257, 56.

Singh, S.V. and Rahman, Q. (1987). Interrelationship between hemolysis and lipid peroxidation of human erythrocytes induced by silicic acid and silciate dust. J_ Appl. Toxicol., 7, 91.

Singh, S.V., Das, B., Rahman, Q. (198 3a). Relationship between solubility and hemolytic effects of toxic dusts. J^ Appl. Toxicol. , 3[, 14.

Singh, S.V., Viswanathan, P.N. and Rahman, Q. (1983b). Interaction between erythrocyte plasma membrane and silicate dusts. Environ. Hlth. Perspect. , 51, 55.

Skaug, v., Davies, R. and Gylseth, B. (1984). In vitro macrophage cytotoxicity of five calcium silicates. Br. J. Ind. Med., 41, 116.

122

Skaug, V. and Gylseth, B. (1983) . Hemolytic a c t i v i t y of f i v e d i f f e r e n t ca lc ium s i l i c a t e s . Environ. Hl th . P e r s p e c t . , 51, 19 5 .

Skaug, V. and Haugen, A. (1989) . In : E f f e c t s of Mineral Dusts on C e l l s , B.T. Mossman and R.O. Begin. ( E d s . ) , p . 7, Spr inger-Ver lag^ Ber l in^Heide lberg , New York.

Slater, T.F. (1984) . Free radical mechanisms in tissue injury. Biochem. J»I 222 I 1•

Stanton, M.F., Layard, M., Tegeris, A., Miller, E., May, M. and Kent, E. (1977). Carcinogenicity of fibrous glass: Pleural response in the rat in relation to fibre dimension. J_ Natl. Cancer Inst., 58, 587.

Stephens, R.F., Joseph, L.B.,Daniel, F.B., Schenck, K.M., Newman, H.A.I., Lipetz, P.D. and Millette, J.R. (1983). Environ. Hlth. Perspect., 51, 257.

Sugimura, T., Sato, S., Nagao, M., Yahagi, T., Matsushima, T. , Seino, Y., Takeuchi, M. and Kawachi, T. (19 76). In: Fundamentals in Cancer Prevention, P.N. Magee, T. Matsushima, T. Sugimura and S. Takayama, (Eds.), p. 191, University of Tokyo Press, Tokyo, and University Park Press, Baltimore, Maryland.

Tappel, A.L. (19 73). . Lipid peroxidation damage to cell components. Fed. Proc., 32, 1870.

Tice, R., Schneider, E.L. and Rary, J.M. (19 76). The utilization of bromodeoxyuridine incorporation into TMK for the analysis of cellular kinetics. Expt. Cell Res., 102, 232.

Trosic, I., Horvat, D. and Racic, J. (1986). The effect of asbestos on cells in culture. Periodicum. Biologerium., 88 , 269.

123

Trump, B . F . , B e r e z e s k y , I . K . and O s o r n i o V a r g a s , A.R. (1981) . I n : C e l l Death i n B io logy and P a t h o l o g y , I . D . Bowen and" R.A. L a c k s h i n , ( E d s . ) , p . 2 0 9 , Chapman and H a l l , New York.

Tyson, C .A . , H a w k - P r a t h e r , K . , S t o r y , D . I . and Gould , D.H. (1983) . C o r r e l a t i o n s of _in v i t r o and Jji v i v o h e p a t o t o x i c i t y f o r f i v e h a l o a l k a n e s . T o x i c o l . A p p l . P h a r m a c o l . , 70 , 289.

Ueda, N . , Ueneka, H . , Akematsu , T . and Sugiyama, T . (19 7 6 ) . P a r a l l e l d i s t r i b u t i o n of s i s t e r c h r o m a t i d e x c h a n g e s and chromosomal a b e r r a t i o n s . N a t u r e , 2 6 2 , 5 8 1 .

Vaca , C . E . , Wi lhe lm, J . and H a r m s - R i n g d a h l , M. ( 1 9 8 8 ) . I n t e r a c t i o n of l i p i d p e r o x i d a t i o n p r o d u c t s w i t h DNA. M u t a t . R e s . , 195 , 1 3 7 .

V a l e r i o , F . , d e F e r r a r i , M., O t a g i o , L . , R e p t t o , E . and S a n t i , L . ( 1 9 8 0 ) . I n : lARC S c i e n t i f i c P u b l i c a t i o n No. 30 , p . 4 8 5 , Lyon, F r a n c e .

V a l l y a t h a n , V . , Rob inson , V . , R e a s o r , M. J . and S t e t t e r , F . ( 1 9 8 4 ) . W o l l a s t o n i t e and a s b e s t o s c y t o t o x i c i t y . Am. Rev. R e s p i r . P i s . 1 2 9 , A 1 7 8 .

Vladimirov, Y.A. (1986). In: Free Radicals, Aging and Degenerative Diseases, J.E. Johnson, Jr., R. Walford, D. Harman and J. Miquel, (Eds.), p. 141, Liss, New York.

Wagner, J.C. and Berry, G. (1969). Mesotheliomas in rats following inoculation with asbestos. Br. J. Cancer, 2 3, 567.

Wang, N.S., Jaurand, M.C. Magne, L., Kheuang, L., Pinchon, M.C. and Bignon, J. (1987). Interactions between asbestos fibres and metaphase chromosomes of rat pleural mesothelial cells in culture: A scanning and transmission electron microscopic study. Am. J. Pathol. , 126, 343.

124

Warhei t , D. B. , H i l l , L.H. and- Brody, A.R. (1984) . In v i t r o e f f e c t s of c r o c i d o l i t e a sbes tos and w o l l a s t o n i t e on pulmonary macrophages and serum complement. Scan. E l e c t . Microscop. , 2 , 919 .

Wil l iams, G.M., Laspia , M.F. and Dunkel, V.C. (1982) . R e a l i a b i l i t y of t h e h e p a t o c y t e culture/DNA r e p a i r t e s t in t e s t i n g coded carc inogens and noncarcinogens . Mutat . R e s . , 97, 3 59.

Wi l l s , E.D. (1969) . L ip id peroxide format ion i n microsomes: General c o n s i d e r a i t o n . Biochem. J . , 113, 315.

Wolff, S. , (198 2) . In : S i s t e r Chromatid Exchange, S. Wolff, ( E d . ) , p . 4 1 , John Wiley, New York.

Wolff, S., Rodin, B. and Cleaver, J.E. (1977). Sister chromatid exchanges induced by mutagenic carcinogens in normal and xeroderma pigmentosum cells. Nature, 265, 347.

Wootton, I .D .P . (1964) . In : Mic ro -ana lys i s i n Medical Biochemistry, p . 101, J . and A. Church i l l L t d . , London.

Wright, A. , Gormley, I . P . , C o l l i n g s , P .L. and Davis , J.M.G. (1980) . In : The in v i t r o Ef fec t s of Mineral Dusts, R.C. Brown, I . P . Gormley, M. Chamberlain and R.Davies , ( E d s . ) , p . 25, Academic P r e s s , New York, London.

Wright, J . R . , Colby, H.D. , Mi le s ,P .R . (1981) . Cy toso l i c f a c t o r s v*iich a f f e c t microsomal l i p i d p e r o x i d a t i o n in lung and l i v e r . Arch. Biochem. Biophys. , 206, 296.

Z a i d i , S.H. (1969) . In : Experimental Pneumoconiosis, p . 35, Johns Hopkins P r e s s , Bal t imore .