Toxicology, 42 (1986) 195--204 Elsevier Scientific Publishers Ireland Ltd.

A COMPARATIVE STUDY OF THE REPRODUCTIVE EFFECTS OF METHADONE AND BENZO[a]PYRENE IN THE PREGNANT AND PSEUDOPREGNANT RAT

QUANG Q. BUI*, MINHTAM B. TRAN and WILLIAM L. WEST

Department of Pharmacology, College of Medicine Howard University, Washington D.C. 20059 (U.S.A.)

(Received April 4th, 1986) {Accepted June 12th, 1986)

SUMMARY

Benzo[a ]pyrene (BP; 50 mg/kg) or methadone (5 mg/kg) was given subcutaneously to pregnant rats at different stages of gestation. Both BP and methadone affected the reproductive performance of pregnant rats by significantly increasing the number of resorptions and fetal wastage, and by decreasing the fetal weight. The same dosage levels of BP and methadone were also given to pseudopregnant rats (PSP) with an induced decidual cell react ion (DCR) in an a t tempt to distinguish whether adverse effects occur in the maternal or fetal compar tment or both. Since the hormonal requirements for DCR and implantation are similar and the anatomical, histological, cytological, time sequential changes as well as appearance of the vasculature system for DCR and decidua are indistinguishable, PSP with DCR is similar to pregnancy except for the lack of a fetal compar tment . BP, in this PSP model, significantly reduced the uterine wet weight and cyclic nucleotide (cAMP) and cGMP) levels whereas methadone was wi thout a detectable effect. Our findings then suggest that BP may exert its effects adversely on both the maternal and fetal compartments , whereas methadone may act primarily in the fetal compar tment .

K e y words: Pseudopregnancy; Benzo[a ]pyrene ; Methadone; Reproductive effects

*Present address: To whom correspondence should be sent: Toxicology Branch (TS- 769C), Hazard Evaluation Division/OPP U.S. Environmental Protection Agency, 401 "M" St., S.W., Washington D.C. 20460, U.S.A. Abbreviations: BP, benzo[a ] pyrene; DCR, decidual cell reaction; PSP, pseudopregnant.

0300-483X/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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INTRODUCTION

Numerous chemicals have been reported to interfere with the reproductive capability of the mothers or to produce embryotoxici ty and/or fetotoxic effects. Methadone and benzo[a]pyrene (BP) can be cited as examples of such chemicals. The biological properties of methadone have been studied extensively in both humans and animals owing to its unique application in the maintenance of heroin addicts. In pregnant rats, administration of methadone is associated with an increase in maternal mortal i ty [1--4], resorption sites [ 2,3], stillbirths [1 ], infant mortal i ty [ 1,2,4], and a decrease in fetal birth weight [3,4]. Subcutaneous injection of methadone to pregnant mice is also associated with increased resorption sites, decreased fetal weight, and decreased in the number of live offspring [ 5]. BP is an ubiquitous urban pollutant and numerous studies have indicated that BP and/or its metabolites are potent carcinogens and mutagens [6]. In mice, BP has been shown to be teratogenic and embryotoxic [7]. Infertility associated with dramatic alterations in gonadal morphology and germ cell development has also been described in mice treated with BP [8]. Therefore, both methadone and BP may adversely affect reproduction. However, pregnancy consists of 2 inter- related compartments, maternal and fetal, hence, identifying the site of action of these 2 agents is worthy of investigation. We had previously proposed the use of the pseudopregnant (PSP) rat with a decidual cell reaction (DCI~) as a model to distinguish adverse effects between the maternal and fetal com- partments [9]. It is now well established that animals may be made PSP by a variety of agents or stimuli [10] which lead to cessation of cycling, persistent and functional corpora lutea, and the capability of developing decidual reactions characteristic of pregnancy. DCR can be artificially induced by different ways [11--16] and is referred to as a deciduoma [11] in order to differentiate it from the normal endometrial induced decidua arising from the implanting embryo stimulus. The hormonal requirements for the sensitization of the endometrium to respond to either an artificial stimulus (PSP) or an implantation (pregnancy) are identical [14,17]. The overall histological, cytological, time sequence changes and appearance of the vascular system in deciduoma formation are also similar to those of the decidua [18--20]. It has also been suggested that the PSP uterus with a DCR undergoes vascular changes similar to those of the pregnant uterus [21]. In addition, the hormonal levels monitored throughout PSP rat with a DCR are likewise similar but not identical to those of normal pregnancy owing to the lack of hormonal contributions from the fetal unit in PSP [9]. There- fore, the most noticeable difference between PSP with a DCR and pregnancy is the absence of a fetal compartment in this PSP model. PSP with an in- duced deciduoma animal model should provide useful information on events on the maternal side.

We wish first to confirm the reproductive effects of methadone and BP in pregnant rats and then localize the site of action of these agents by using the

196

proposed PSP animal model in an a t tempt to distinguish whether the adverse effects are in the maternal or fetal compartment or both.

MATERIALS AND METHODS

Animals Sexually mature male and female Sprague--Dawley rats (Charles River

Laboratories, Wilmington, MA) weighing between 225 and 250 g were acclimated for 2 weeks. Rats were individually housed in hanging cages under a controlled photoperiod of 12 h light/day, temperature {25°C), and humidi ty (60--80%) with food (Purina Laboratory Rodent Chow) and tap water ad libitum. All females were subjected to daily vaginal lavage 7 days/ week to determine the stage of their estrous cycle. Only rats that exhibited at least 2 normal, 4- or 5-day estrous cycles were selected.

Pregnancy Following the initial adjustment period, males were housed overnight with

randomly selected females at a ratio of 1 male: 2 females/cage. Day 1 of pregnancy was defined as the day on which a sperm plug and motile sperm in a vaginal smear were detected. Once mating was determined, each female was separated, individually housed in clear polypropylene cage with stainless steel top, and identified.

Pseudopregnancy Pseudopregnancy was induced by 2 electric stimulations of the uterine

cervix [16] using a Yeda Cervical Stimulator (Rohobot, Israel). The first stimulus was performed on the afternoon of the proestrus day (Day 0) followed by a second stimulus 24 h later, on estrus day [10]. The intensity of the electric stimulus was 15 V, 30 ms/pulse at regular intervals of 6 ms. The day when the microscopic examination of the vaginal lavage showed cornification followed by leukocytic infiltration was designated as Day 1 of PSP. Daily smears were then made to monitor the persistence of the PSP status of each animal. On Day 5 of PSP [15], induction of decidual tissue was performed via a midventral laparotomy in lightly etherized animals and experimental decidualization was initiated by scratching the antimesometrial surface of the endometrium [13] with a blunt needle and inserting of a silk thread (silicone-treated black, Davis Geck silk No. 0-1081-61) into the left uterine horn [9]. The decidual cell reaction was confirmed at autopsy by gross appearance of the uterine horn and by removing and weighing the deciduoma.

Treatment Methadone (Dolophine HC1, Eli Lilly Co., Indianapolis, IN) was given

daily, subcutaneously (5 mg/kg), to both pregnant and PSP rats in a constant volume of physiological saline (10 ml/kg). The dose of methadone was adjusted daily according to the body weight variations of the animals. The

197

daily dose of 5 mg/kg of methadone was considered since this dose did not produce catatonia and would be least likely to produce dependence on the narcotic [9] . BP (Aldrich Chemical Co., Milwaukee, WI) was also given daily, subcutaneously (50 mg/kg), to both pregnant and PSP rats in a constant volume of corn oil (2 ml/kg). This dosage level of BP was selected based upon previous work in this laboratory [22].

Pregnant animals were treated with methadone or BP from Days 6--8 or 6--11 of gestation corresponding to the day of deciduoma induction in PSP rats. On Day 20 of gestation, estimated at 24 h preparturition, the dams were sacrificed by cervical dislocation. The abdominal wall was incised longitudinally and both uterine horns were carefully exposed. The metrial glands were counted identifying original implantation sites. An implantation site not occupied by a fetus was designated as a resorption site. The viability and body weight of each fetus was recorded.

Methadone or BP were administered to PSP rats from Days 6--8 or 6 - 11 of PSP. PSP animals were sacrificed on Days 9 and 12 of PSP after receiving, respectively, 3 or 6 consecutive daily injections. The uterus with deciduoma was removed and weighed to ascertain the presence of development of deciduoma. Sacrifice of PSP animals could not be delayed beyond Day 12 of PSP, since the deciduoma will undergo rapid regression [ 19,23].

Tissue sampling and assays of cyclic nucleotides Upon sacrifice, uterine tissues of PSP rats were removed, immediately

placed on dry-ice, and stored at -20°C until assayed. Uterine tissues were homogenized in ethanol (50% w/v), centrifuged, and the supernatant was collected. The precipitate was washed with 1 ml of ethanol in water (2:1 v/v), centrifuged, and the supernatants were combined and evaporated to dryness at 55°C under a stream of air. The residues were dissolved in Tris- EDTA (0.05 M Tris, pH 7.5 containing 4 mM EDTA). The mixtures were centrifuged and the supernatant was used in the detection of cyclic nucleo- tides using radioimmunoassay techniques (Amersham Corp., Illinois Heights, IL). All determinations were made in triplicate.

Statistics Intergroup differences were assessed by the Student 's t-test or analysis of

variance followed by Newman-Keuls exam where appropriate. The number of fetal resorptions and fetal wastage were compared by Chi-Square analysis. For all statistical analyses, a level of probability of P ~ 0.05 was used for significance.

RESULTS

There were no maternal deaths in either the treated or control groups. All dams appeared normal upon gross observation throughout the study. The reproductive status of pregnant rats treated with BP or methadone is shown in Tables I and II, respectively.

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TABLE I

REPRODUCTIVE STATUS OF PREGNANT RATS TREATED WITH BP (50 mg/kg/day) FROM DAYS 6 TO 8 AND 6 TO 11 OF GESTATION

Control BP BP (corn oil) 3 inject. 6 inject.

Number of dams 15 10 10 Implantations/litter a 13.3 + 0.7 12.3 + 1.7 13.4 + 0.4 Number of dead fetuses 2 1 1 Number of resorptions 6 48" 65 * Fetal wastage/dam a 0.5 _+ 0.2 6.1 + 2.5* 6.6 + 2.0* Number of live fetuses 192 49* 68* Fetal weight (g)a 3.6 -+ 0.4 1.3 -+ 0.1" 1.4 + 0.1" Uterine weight (g)a 4.5 + 0.4 1.7 _+ 0.1" 1.8 + 0.4*

aMean + S . E . M . *Significantly different from control values at P < 0.05.

T h e r e were n o s i g n i f i c a n t d i f f e r e n c e s in t he n u m b e r of i m p l a n t a t i o n s

b e t w e e n the t r e a t e d a n d c o n t r o l an ima l s . H o w e v e r , ra ts t r e a t e d w i t h BP e x h i b i t e d a s t a t i s t i ca l ly s i g n i f i c a n t inc rease in t he n u m b e r of r e s o r p t i o n s a n d fe ta l was t age (dead fe tuses p lus r e s o r p t i o n s ) . T h e fe ta l we igh t s f r o m the BP- t r e a t e d g r o u p s were also s i g n i f i c a n t l y less t h a n c o n t r o l values . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s in u t e r i n e w e i g h t were o b s e r v e d b e t w e e n t he t r e a t e d a n d c o n t r o l rats .

No s i g n i f i c a n t d i f f e r e n c e s in t he n u m b e r o f i m p l a n t a t i o n s a n d d e a d

fe tuses we re o b s e r v e d b e t w e e n the m e t h a d o n e - t r e a t e d a n d c o n t r o l g roups .

H o w e v e r , a d m i n i s t r a t i o n of m e t h a d o n e a t va r ious stages of p r e g n a n c y

TABLE II

REPRODUCTIVE STATUS OF PREGNANT RATS TREATED WITH METHADONE (55 mg/kg) FROM DAYS 6 TO 8 AND 6 TO 11 OF GESTATION

Control Methadone Methadone (saline) 3 inject. 6 inject.

Number of dams 9 8 9 Implantations/dam a 12.9 _+ 2.0 12.7 _+ 1.3 13.3 + 1.3 Number of dead fetuses 1 2 5 Number of resorptions 2 4 19" Fetal wastage a 0.3 _+ 0.1 0.7 _+ 0.2 2.7 _+ 0.7* Number of live fetuses 113 96 96 Fetal weight (g)a 3.8 _+ 0.3 2.9 + 0.1" 2.7 _+ 0.3* Uterine weight (g)a 4.7 -+ 0.6 4.1 _+ 0.3 4.2 _+ 0.5

aMean _+ S .E .M. *Significantly different from control values at P < 0.05.

1 9 9

1,200

~ ) 1,000 *m l

800

=

600

mg

LlJ_I r._ E

O i

BB control, saline [:]control, corn oil i mmethadone i

- -BP i

9 12 Days of PSP Fig. 1. U t e r i n e w e t w e i g h t o f c o n t r o l ( sa l ine or c o r n oi l ) PSP r a t s w i t h d e c i d u a l cell r e a c t i o n o r t r e a t e d w i t h e i t h e r BP or m e t h a d o n e f r o m d a y s 6 to 8 o r 6 t o 11 o f PSP. E a c h ba r r e p r e s e n t s t h e m e a n ± S .E .M. o f 6 a n i m a l s . * D e n o t e s s t a t i s t i c a l l y d i f f e r e n t f r o m c o n - t ro l va lues .

significantly increased the mean fetal wastage and decreased the mean fetal weight.

Changes in weight of the uterine horns with DCR as a funct ion of time and after BP and methadone administration are shown in Fig. 1. No statisti- cally significant differences were observed on Day 9 of PSP between the methadone , BP, and control groups. However, t rea tment of PSP animals with BP for 6 consecutive days resulted in a significant decrease in uterine weight as evidenced on Day 12 of PSP.

The levels of uter ine cAMP in PSP rats were significantly depressed by BP t rea tment on both Days 9 and 12 of PSP but not by methadone (Fig. 2). Similarly, the uterine cGMP levels were significantly decreased by BP but unaffected by methadone (Fig. 3).

D I S C U S S I O N

Methadone, administered at various stages during pregnancy, significantly increased the number of resorptions and fetal wastage, and decreased the

200

Q

D,

.<

60

40

20

1 m a n

inn " g g Im

l I gg am BB am

• control, saline I-Icontrol, corn oil

methadone -----~BF

o ~ 9 12 Days 0I PSP

Fig. 2. PSP rats with decidual cell reaction were treated with saline, corn oil, BP, or methadone from days 6 to 8 or 6 to 11 of PSP. Uterine levels of cAMP were assayed by radioimmunoassay techniques on days 9 and 12 of PSP. Each bar represents the mean -+ S.E.M. of 6 animals. *Denotes statistically different from the corresponding control values.

fetal weight (Table II). Similar findings had also been r epo r t ed by m a n y invest igators in the rat [2- -4] as well as in the mouse [5 ] . Our da ta reveal t ha t BP, adminis te red to rats beginning on Day 6 of gestat ion, m ay be b o th e m b r y o t o x i c and f e t o t o x i c as evidenced by an increase in resorp t ion sites and a decrease in the n u m b e r of live offspr ing and fetal weight (Table I). In mice, BP has been d o c u m e n t e d to be e m b r y o t o x i c and tera togenic [7] and it m a y also adversely a f fec t fer t i l i ty by altering gonadal m o r p h o l o g y and germ cell d e v e l o p m e n t [8 ] . These findings tha t BP and m e t h a d o n e b o th a f fec t r e p r o d u c t i o n satisfied our cri ter ia fo r thei r selection.

In the PSP rat with DCR, t r e a t m e n t with BP signif icantly reduced the u ter ine we t weight and cyclic nuc leo t ide levels whereas m e t h a d o n e had no observable e f fec t s on these paramete rs (Fig. 1, 2 and 3). I t is well establ ished tha t dur ing pregnancy , estrogens are i m p o r t a n t fo r the h y p e r t r o p h y , hyper - plasia, and h y p e r e m i a of the u terus to ef fec t ive ly assure its d e v e l o p m e n t [ 2 4 - 2 6 ] . BP has been shown to c o m p e t e wi th the es t rogen for the u te r ine estrogen recep to r s [ 2 2 ] , to in ter fere with the m o n o o x y g e n a s e en zy m es necessary fo r the biosynthesis of active ca techol estrogens f rom est rogen precursors [ 2 7 ] , and to exe r t proges terone- l ike e f fec ts [ 2 2 ] . The anti- es t rogenic e f fec ts of BP may resul t in inadequa te s t imula t ion of u te r ine

201

2.5

2

1.5

1

0.5

• control, saline

[ ] control, corn oil ~ B m methadone -------BP

m m m m

mm m m m m m

,A-

9 12 Days of PSP Fig. 3. PSP rats wi th decidual cell react ion were t rea ted wi th saline, corn oil, BP, or m e t h a d o n e f rom days 6 to 8 or 6 to 11 of PSP. Uter ine levels of cGMP were assayed by rad io immunoassay techniques on days 9 and 12 of PSP. Each bar represents the mean -+ S.E.M. of 6 animals. *Denotes stat ist ical ly d i f fe rent f rom the cor responding con t ro l values.

tissues [28] leading to the decrease in uterine weight observed (Fig. 1). Since estrogens are also known to increase cGMP levels in the rat uterus by enhancing the activity of guanylate cyclase [29--31] and/or by decreasing the specific activities cGMP phosphodiesterase [38] , BP, by possessing antiestrogenic activities [22 ,28] , may suppressed cGMP levels (Fig. 3). Furthermore, progesterone [32] as well as progesterone-like agonists such as BP [22] may stimulate cyclic nucleotide phosphodiesterases resulting in a decrease of both cAMP (Fig. 2) and cGMP {Fig. 3) levels. The exact mecha- nism by which BP produces its effects on the uterus remains problematical and requires further investigation. Methadone, on the other hand, exerts no noticeable effects in the PSP rats with a DCR.

BP, in this PSP model, significantly reduced the uterine wet weight and cyclic nucleotide (cAMP and cGMP) levels whereas methadone was without a detectable effect. These findings suggest that BP may exert its effects

202

a d v e r s e l y o n b o t h t h e m a t e r n a l a n d f e t a l c o m p a r t m e n t s , w h e r e a s m e t h a d o n e m a y a c t p r i m a r i l y in t h e f e t a l c o m p a r t m e n t .

B a s e d o n t h e a s s u m p t i o n t h a t t h e i n d u c e d D C R in PSP a n i m a l s u n d e r g o a c y c l e o f m o r p h o l o g i c , h o r m o n a l a n d b i o c h e m i c a l g r o w t h , a n d r e g r e s s i o n w h i c h p a r a l l e l c h a n g e s o c c u r r i n g a f t e r u t e r i n e i m p l a n t a t i o n [ 1 4 , 1 5 , 2 0 ] d u r i n g p r e g n a n c y , t h e use o f t h e PSP r a t w i t h a D C R m a y p r o v i d e a m e a n s t o l o c a l i z e adve r se d r u g e f f e c t s b e t w e e n t h e m a t e r n a l a n d f e t a l c o m p a r t m e n t s .

ACKNOWLEDGEMENTS

Th i s w o r k was s u p p o r t e d in p a r t b y g e n e r o u s g i f t s f r o m H o f f m a n L a R o c h e Inc . , A m e r i c a n C y a n a m i d Co . , a n d P e n w a l t F o u n d a t i o n .

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