TERATOLOGY 42~611-618 (1990)
Teratogenic Effect of the Cholesterol Synthesis Inhibitor AY 9944 on Rat Embryos In Vitro M. REPETTO, J.C. MAZIERE, D. CITADELLE, R. DUPUIS, M. MEIER, S.BIADE, D. QUIEC, AND C. ROUX Laboratoire ZEmbryologie, CHU Saint-Antoine, 27, rue Chaligny, 75012 Paris, France (M.R.,D.C., R.D., M.M., S.B., D.Q., C.R.) and Laboratoire de Biochimie, CHU Saint-Antoine, 27, rue Chaligny, 75012, Paris, France (J.C.M.)
ABSTRACT
AY 9944 [trans-l,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; ( C ) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 pg/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dosedependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.
AY 9944 ([trans-1,4-bis(2-chlorobenzyl-'80; Barbu et al., '84).The sterol perturbaaminomethyl) cyclohexane dihydrochlo- tions included both a decrease in cholesterride]) is an amphiphilic cationic substance olemia and an accumulation of 7-dehydrowhich has multiple cellular actions, the best cholesterol (Barbu et al., '88). However, known of which is the inhibition of choles- cholesterol supplementation of the diet of terol synthesis. We have previously re- pregnant dams can prevent specific malforported that AY 9944 induced fetal abnor- mations (Roux et al., '79b). The effectiveness malities in pregnant Wistar rats (Roux and of the supplementation varies according to Aubry, '66).The most characteristic malfor- the timing of cholesterol administration, bemations in vivo are of the holoprosencepha- ing complete only when the supplementalic type; this malformation is externally vis- tion starts on the day of AY 9944 treatment ible in 25% of fetuses but 80% of the (Barbu et al., '88). It therefore seems that apparently normal fetuses were found to sterol perturbations are an important caushave isolated pituitary agenesis (Roux et ative factor in embryonic malformations. But it remains questionable whether materal., '79a). We have also shown that administration of nal sterols are the most important factor or AY 9944 on day 4 of gestation produces a significant decrease of maternal plasma sterol levels on day 10 of gestation, when cranReceived January 30, 1990; accepted July 18, 1990. iofacial malformations occur, and that the Address reprint requests to Dr. C. Roux, Laboratoire dEmnumber of malformed fetuses is related to bryologie, CHU Saint-Antoine, 27, rue Chaligny, 75012 Paris, the level of maternal sterolemia (Roux et al., France.
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whether AY 9944 can directly modify the sterols in the embryo. Steele et al. ('83) have used hypolipidemic agents and embryo culture to demonstrate that the agents themselves are teratogenic when introduced into the culture medium but that treated maternal serum is not teratogenic. These experiments on whole embryo culture were therefore designed to show whether AY 9944 has a direct or indirect action on the embryo by changing the maternal milieu. MATERIAL AND METHODS
Embryo culture conditions Wistar rats (Charles River France), weighing approximately 200 g, were housed under standard conditions and a 12 h light/ dark cycle (darkness from midnight to midday). Females were mated with males of the same strain between 8 and 10 h. The day when sperm was found in vaginal smears was designated as day 1 of gestation. Wistar rat embryos (early somite stage) within the intact visceral yolk sac and amnion were explanted from the uteri of pregnant dams on day 11 of gestation between 10.00 and 12.00 h. The age of embryos was 10 days + 0 to 4 h. They were cultured for 48 h according to the method used by New ('78). Each intact embryo was placed in a sterile round-bottom flask (25 ml) containing 2 ml of culture medium and a gas mixture of 5% O,, 5% C02, and 90% N,. The flask was placed on a rotor in a n incubator a t 37°C; the gas mixture was changed with mixtures of increasing 0, concentrations and 5% CO, in N, at regular intervals. The culture medium utilized was fresh sterile serum collected from adult Wistar rats. Sera from several rats were immediately centrifuged, pooled, and heat inactivated (Steele and New, '74). No antibiotics were added. The embryos were cultured by series of 8 to 15 individuals on the same day. Each series included 2 or 3 controls. As far as possible, controls and treated embryos were taken from the same dam. Experimental protocol Four experimental conditions were examined: Controls (group A): 21 embryos from 8 normal mothers were cultured in normal rat serum.
Culture in serum from AE' 9944-treated rats (group B): 26 normal embryos, from 4 rats, were cultured in serum from rats treated with AY 9944. Nonpregnant rats were given a single dose of 50 mg/kg of AY 9944 in 5 ml/kg of distilled water by intubation. Blood was withdrawn 6 days later for serum preparation. Six days is the time necessary to produce the greatest hypocholesterolemia (Barbu et al., '84). Culture with AY 9944 in the culture medium (group C): 20 normal embryos, from 3 mothers, were cultured in serum of normal rats containing AY 9944. The drug was added directly to the serum. AY 9944 was added over a range of 0-1000 pg/ml serum in a n initial survey experiment. The drug concentrations in a second experiment were 0, 1, and 2 pg/ml. Eight embryos were cultured in serum to which was added l pg/ml (group C1) and 12 in serum to which was added 2 pgiml (group C 2 ) . Culture of embryos from AY 9944-treated dams in normal rat serum (group D): 33 embryos, from 4 females treated with AY 9944, were cultured in normal rat serum. Pregnant rats were dosed by intubation with 50 mg/kg of AY 9944 in 5 mlikg of distilled water on day 4 of gestation. The embryos were explanted from dam uteri on day 11of gestation (their age was 10 days + 0 to 4 h) and cultured 48 h in normal serum.
Morphological assessment of development The conceptuses were removed after 48 h of culture, washed in phosphate-buffered saline (PBS), and examined under a stereomicroscope for assessment of growth and differentiation. The diameter and vascularisation of the yolk sac, the head, and crownrump lengths of all the embryos were used as indicators of embryonic growth. The degrees of differentiation were evaluated using the morphological scoring system developed by Brown and Fabro ('81) and any anomalies were recorded. Some embryos were recently dead a t the end of the culture period. Although their gross morphology could be examined, they were not included in scoring or growth measurements. The macroscopically recognizable developmental steps of the embryos a t the end of the culture period were taken as criteria for a first macroscopic estimation. However, histological investigations were also performed to obtain further information. The
613
TERATOGENESIS OF AY 9944 IN VITRO TABLE 1 . Score and growth measurements (means f SO) of the four groups of embryos cultured for 48 h Crown-rump Head Yolk sac Score length length diameter 2.99 2 0.20 6.40 ? 0.41 49.70 t 0.9 5.3 k 0.32 Controls ( n = 21) 5.97 f 0.50** 2.86 i 0.25n.s. 47.02 5 1.58*** 5.08 2 0.48n.s. Group B ( n = 30) 5.18 I 0.44n.s. 2.96 t 0.lln.s. 6.41 k 0.28n.s. Group C1 ( n = 8) 47.75 ? 2.0** 5.27 0.72*** 2.15 5 0.44*** Group C2 ( n = 12) 41.95 f 2.86*** 4.02 t 0.53*** 45.66 t 3.80*** 4.80 t 0.40*** 2.53 k 0.26*** 6.09 i 0.39** Group D (n = 33)
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Teratogenic Effect of the Cholesterol Synthesis Inhibitor AY 9944 on Rat Embryos In Vitro M. REPETTO, J.C. MAZIERE, D. CITADELLE, R. DUPUIS, M. MEIER, S.BIADE, D. QUIEC, AND C. ROUX Laboratoire ZEmbryologie, CHU Saint-Antoine, 27, rue Chaligny, 75012 Paris, France (M.R.,D.C., R.D., M.M., S.B., D.Q., C.R.) and Laboratoire de Biochimie, CHU Saint-Antoine, 27, rue Chaligny, 75012, Paris, France (J.C.M.)
ABSTRACT
AY 9944 [trans-l,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; ( C ) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 pg/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dosedependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.
AY 9944 ([trans-1,4-bis(2-chlorobenzyl-'80; Barbu et al., '84).The sterol perturbaaminomethyl) cyclohexane dihydrochlo- tions included both a decrease in cholesterride]) is an amphiphilic cationic substance olemia and an accumulation of 7-dehydrowhich has multiple cellular actions, the best cholesterol (Barbu et al., '88). However, known of which is the inhibition of choles- cholesterol supplementation of the diet of terol synthesis. We have previously re- pregnant dams can prevent specific malforported that AY 9944 induced fetal abnor- mations (Roux et al., '79b). The effectiveness malities in pregnant Wistar rats (Roux and of the supplementation varies according to Aubry, '66).The most characteristic malfor- the timing of cholesterol administration, bemations in vivo are of the holoprosencepha- ing complete only when the supplementalic type; this malformation is externally vis- tion starts on the day of AY 9944 treatment ible in 25% of fetuses but 80% of the (Barbu et al., '88). It therefore seems that apparently normal fetuses were found to sterol perturbations are an important caushave isolated pituitary agenesis (Roux et ative factor in embryonic malformations. But it remains questionable whether materal., '79a). We have also shown that administration of nal sterols are the most important factor or AY 9944 on day 4 of gestation produces a significant decrease of maternal plasma sterol levels on day 10 of gestation, when cranReceived January 30, 1990; accepted July 18, 1990. iofacial malformations occur, and that the Address reprint requests to Dr. C. Roux, Laboratoire dEmnumber of malformed fetuses is related to bryologie, CHU Saint-Antoine, 27, rue Chaligny, 75012 Paris, the level of maternal sterolemia (Roux et al., France.
0 1990 WILEY-LISS, INC.
612
M. REPETTO E T AL.
whether AY 9944 can directly modify the sterols in the embryo. Steele et al. ('83) have used hypolipidemic agents and embryo culture to demonstrate that the agents themselves are teratogenic when introduced into the culture medium but that treated maternal serum is not teratogenic. These experiments on whole embryo culture were therefore designed to show whether AY 9944 has a direct or indirect action on the embryo by changing the maternal milieu. MATERIAL AND METHODS
Embryo culture conditions Wistar rats (Charles River France), weighing approximately 200 g, were housed under standard conditions and a 12 h light/ dark cycle (darkness from midnight to midday). Females were mated with males of the same strain between 8 and 10 h. The day when sperm was found in vaginal smears was designated as day 1 of gestation. Wistar rat embryos (early somite stage) within the intact visceral yolk sac and amnion were explanted from the uteri of pregnant dams on day 11 of gestation between 10.00 and 12.00 h. The age of embryos was 10 days + 0 to 4 h. They were cultured for 48 h according to the method used by New ('78). Each intact embryo was placed in a sterile round-bottom flask (25 ml) containing 2 ml of culture medium and a gas mixture of 5% O,, 5% C02, and 90% N,. The flask was placed on a rotor in a n incubator a t 37°C; the gas mixture was changed with mixtures of increasing 0, concentrations and 5% CO, in N, at regular intervals. The culture medium utilized was fresh sterile serum collected from adult Wistar rats. Sera from several rats were immediately centrifuged, pooled, and heat inactivated (Steele and New, '74). No antibiotics were added. The embryos were cultured by series of 8 to 15 individuals on the same day. Each series included 2 or 3 controls. As far as possible, controls and treated embryos were taken from the same dam. Experimental protocol Four experimental conditions were examined: Controls (group A): 21 embryos from 8 normal mothers were cultured in normal rat serum.
Culture in serum from AE' 9944-treated rats (group B): 26 normal embryos, from 4 rats, were cultured in serum from rats treated with AY 9944. Nonpregnant rats were given a single dose of 50 mg/kg of AY 9944 in 5 ml/kg of distilled water by intubation. Blood was withdrawn 6 days later for serum preparation. Six days is the time necessary to produce the greatest hypocholesterolemia (Barbu et al., '84). Culture with AY 9944 in the culture medium (group C): 20 normal embryos, from 3 mothers, were cultured in serum of normal rats containing AY 9944. The drug was added directly to the serum. AY 9944 was added over a range of 0-1000 pg/ml serum in a n initial survey experiment. The drug concentrations in a second experiment were 0, 1, and 2 pg/ml. Eight embryos were cultured in serum to which was added l pg/ml (group C1) and 12 in serum to which was added 2 pgiml (group C 2 ) . Culture of embryos from AY 9944-treated dams in normal rat serum (group D): 33 embryos, from 4 females treated with AY 9944, were cultured in normal rat serum. Pregnant rats were dosed by intubation with 50 mg/kg of AY 9944 in 5 mlikg of distilled water on day 4 of gestation. The embryos were explanted from dam uteri on day 11of gestation (their age was 10 days + 0 to 4 h) and cultured 48 h in normal serum.
Morphological assessment of development The conceptuses were removed after 48 h of culture, washed in phosphate-buffered saline (PBS), and examined under a stereomicroscope for assessment of growth and differentiation. The diameter and vascularisation of the yolk sac, the head, and crownrump lengths of all the embryos were used as indicators of embryonic growth. The degrees of differentiation were evaluated using the morphological scoring system developed by Brown and Fabro ('81) and any anomalies were recorded. Some embryos were recently dead a t the end of the culture period. Although their gross morphology could be examined, they were not included in scoring or growth measurements. The macroscopically recognizable developmental steps of the embryos a t the end of the culture period were taken as criteria for a first macroscopic estimation. However, histological investigations were also performed to obtain further information. The
613
TERATOGENESIS OF AY 9944 IN VITRO TABLE 1 . Score and growth measurements (means f SO) of the four groups of embryos cultured for 48 h Crown-rump Head Yolk sac Score length length diameter 2.99 2 0.20 6.40 ? 0.41 49.70 t 0.9 5.3 k 0.32 Controls ( n = 21) 5.97 f 0.50** 2.86 i 0.25n.s. 47.02 5 1.58*** 5.08 2 0.48n.s. Group B ( n = 30) 5.18 I 0.44n.s. 2.96 t 0.lln.s. 6.41 k 0.28n.s. Group C1 ( n = 8) 47.75 ? 2.0** 5.27 0.72*** 2.15 5 0.44*** Group C2 ( n = 12) 41.95 f 2.86*** 4.02 t 0.53*** 45.66 t 3.80*** 4.80 t 0.40*** 2.53 k 0.26*** 6.09 i 0.39** Group D (n = 33)
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***P
