Phenytoin covalent binding and embryopathy in mouse embryos co-cultured with maternal hepatocytes from mouse, rat,...

The anticonvulsant drug phenytoin is teratogenic in a variety of species 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 approximate 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 evaluate in vitro the known in vivo differential species susceptibility to phenytoin teratogenesis, day 9.5 (day of plug = day 1) mouse embryos either were cultured alone for 24 hr or were co-cultured with hepatocytes from maternal mice, rats or male rabbits, thereby exposing the embryos to the effects of potential species-specific phenytoin metabolism. In the absence of hepatocytes, phenytoin embryotoxicity was concentration dependent (0, 10, 20 and 60 micrograms/mL), with decreases in embryonic growth, reflected by reduced yolk sac diameter and crown rump length, apparent within the maternal therapeutic range (20 micrograms/mL). Covalent binding of the radiolabeled drug to live embryonic tissue was significantly higher than in control embryos previously killed by fixation, suggesting that the embryo can bioactivate phenytoin to a toxic reactive intermediate. Mouse embryos grew equally well with hepatocytes from all three species, indicating interspecies tissue compatibility. The addition of rat and rabbit hepatocytes, but not mouse hepatocytes, significantly enhanced the phenytoin-induced impairment of mouse embryonic development, as demonstrated by reductions in somite number. The phenytoin-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 extracellular proteins in the culture medium was not enhanced by the addition of mouse hepatocytes. These results suggest that mouse embryos intrinsically 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 favouring detoxification that is not shared by rat and rabbit hepatocytes, which enhanced phenytoin embryopathy. Thus, while phenytoin teratogenicity likely involves embryonic bioactivation, maternal determinants may contribute variably to teratologic susceptibility in a species-specific manner.