Ozolins T R, Wiley M J, Wells P G
Faculty of Pharmacy, University of Toronto, Canada.
Biochem Pharmacol. 1995 Nov 27;50(11):1831-40. doi: 10.1016/0006-2952(95)02076-4.
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.
抗惊厥药物苯妥英在包括人类在内的多种物种中具有致畸性。传统的胚胎培养研究采用添加来自诱导大鼠或小鼠肝脏的9000g上清液（S-9）或微粒体部分作为外源性生物激活系统，以模拟母体的作用。然而，与培养的完整肝细胞不同，细胞部分本身可能具有胚胎毒性，并且不能反映生物激活和解毒的体内平衡。为了在体外评估已知的体内对苯妥英致畸作用的不同物种易感性，将第9.5天（交配日=第1天）的小鼠胚胎单独培养24小时，或与母鼠、大鼠或雄性兔的肝细胞共培养，从而使胚胎暴露于潜在的物种特异性苯妥英代谢的影响之下。在没有肝细胞的情况下，苯妥英的胚胎毒性呈浓度依赖性（0、10、20和60微克/毫升），胚胎生长减少，表现为卵黄囊直径和头臀长度减小，在母体治疗范围内（20微克/毫升）即可明显观察到。放射性标记药物与活胚胎组织的共价结合明显高于先前通过固定杀死的对照胚胎，这表明胚胎可以将苯妥英生物激活为有毒的反应性中间体。小鼠胚胎与所有三种物种的肝细胞生长情况相同，表明种间组织相容性良好。添加大鼠和兔肝细胞，但不添加小鼠肝细胞，显著增强了苯妥英诱导的小鼠胚胎发育损伤，表现为体节数量减少。添加大鼠或兔肝细胞并没有增强苯妥英诱导的小鼠胚胎生长损伤，而小鼠肝细胞则提供了保护作用。添加小鼠肝细胞并没有增强苯妥英与培养基中细胞外蛋白质的共价结合。这些结果表明，小鼠胚胎本身可以将苯妥英生物激活为有毒的反应性中间体，并产生胚胎病变后果。母鼠肝细胞提供的保护作用表明存在一种物种特异性的母体生化平衡，有利于解毒，而大鼠和兔肝细胞则不具备这种平衡，它们会增强苯妥英胚胎病。因此，虽然苯妥英致畸性可能涉及胚胎生物激活，但母体因素可能以物种特异性方式对致畸易感性产生不同的影响。
