PHENYTOIN COVALENT BINDING AND EMBRYOPATHY IN MOUSE EMBRYOS COCULTURED WITH MATERNAL HEPATOCYTES FROM MOUSE, RAT, AND RABBIT

The anticonvulsant drug phenytoin is teratogenic in a variety of speci es including humans. Traditional embryo culture studies have employed the addition of 9000 g supernatant (S-9) or microsomal fractions from induced rat or mouse liver as an exogenous bioactivating system to app roximate a maternal contribution, However, cellular fractions, unlike cultured intact hepatocytes, may themselves be embryotoxic, and do not reflect the in vivo balance of bioactivation and detoxification. To e valuate in vitro the known in vivo differential species susceptibility to phenytoin teratogenesis day 9.5 (day of plug = day 1) mouse embryo s either were cultured alone for 24 hr or were co-cultured with hepato cytes from maternal mice, rats or male rabbits, thereby exposing the e mbryos to the effects of potential species-specific phenytoin metaboli sm. In the absence of hepatocytes, phenytoin embryotoxicity was concen tration dependent (0, 10, 20 and 60 mu g/mL), with decreases in embryo nic growth, reflected by reduced yolk sac diameter and crown rump leng th, apparent within the maternal therapeutic range (20 mu g/mL). Coval ent binding of the radiolabeled drug to live embryonic tissue was sign ificantly higher than in control embryos previously killed by fixation , suggesting that the embryo can bioactivate phenytoin to a toxic reac tive intermediate, Mouse embryos grew equally well with hepatocytes fr om all three species, indicating interspecies tissue compatibility. Th e addition of rat and rabbit hepatocytes, but not mouse hepatocytes, s ignificantly enhanced the phenytoin-induced impairment of mouse embryo nic development, as demonstrated by reductions in somite number. The p henytoin-induced impairment of mouse embryonic growth was not enhanced by the addition of rat or rabbit hepatocytes, while mouse hepatocytes conferred protection. The covalent binding of phenytoin to extracellu lar proteins in the culture medium was not enhanced by the addition of mouse hepatocytes. These results suggest that mouse embryos intrinsic ally can bioactivate phenytoin to a toxic reactive intermediate, with embryopathic consequences. The protection conferred by maternal mouse hepatocytes suggests a species-specific maternal biochemical balance f avouring detoxification that is not shared by rat and rabbit hepatocyt es, which enhanced phenytoin embryopathy. Thus, while phenytoin terato genicity likely involves embryonic bioactivation, maternal determinant s may contribute variably to teratologic susceptibility in a species-s pecific manner.