Analysis of mechanism for thalidomide-induced teratogenicity using knock-out mice

为了评价胚胎成纤维细胞是否是分析致畸性机制的有用工具，用7种致畸和/或致癌化学品（沙利度胺、苯妥英、DMBA、苯并[a]芘、萘、PhIP和MeIQx）检测胚胎成纤维细胞的细胞毒性。除DMBA和PhIP外，其他5种化学物质对胚胎成纤维细胞无毒性，提示胚胎成纤维细胞可能缺乏代谢活化能力。因此，为了补偿胚胎成纤维细胞中代谢活化的效力，采用肝微粒体预孵育方法进行胚胎成纤维细胞的细胞毒性试验。通过使用孕兔肝微粒体的预孵育方法，沙利度胺对胚胎成纤维细胞产生剂量依赖性细胞毒性，但对孕小鼠则没有。这些结果与沙利度胺致畸性的种属差异一致。此外，细胞色素P450（P450）抑制剂1-氨基苯并三唑或α-萘甲酮（α-NF）的加入抑制了沙利度胺对胚胎成纤维细胞的细胞毒性。沙利度胺处理还通过使用孕兔肝微粒体预孵育抑制了胚胎成纤维细胞的细胞增殖。通过加入1-氨基苯并三唑或α-NF来消除沙利度胺的这种抑制作用。为了确定微粒体环氧化物水解酶（mEH）是否参与沙利度胺诱导的致畸性，分析了mEH缺失小鼠和野生型小鼠之间沙利度胺处理对胚胎成纤维细胞细胞毒性的影响。沙利度胺对小鼠胚胎成纤维细胞增殖的抑制作用与野生型小鼠胚胎成纤维细胞无明显差异，提示P450参与了沙利度胺对胚胎成纤维细胞增殖的抑制作用

To evaluate whether embryo fibroblasts are useful tool to analyze the mechanism of teratogenicity, cytotoxicity of embryo fibroblasts was examined with seven chemicals of teratogens and/or carcinogens (thalidmide, phenytoin, DMBA, benzo[a]pyrene, naphthalene, PhIP and MeIQx). Five chemicals except DMBA and PhIP demonstrated no cytotoxicity in embryo fibroblasts suggesting the possibility that embryo fibroblasts lack the ability of metabolic activation. Thus, to compensate the potency of metabolic activation in embryo fibroblasts, pre-incubation method using liver microsomes was applied for cytotoxicity assay in embryo fibroblasts. Thalidomide produced dose-dependent cytotoxicity to embryo fibroblasts by the pre-incubation method using liver microsomes from pregnant rabbits, but not from pregnant mice. These results are consistent with the species difference on teratogenicity of thalidomide. Furthermore, the addition of cytochrome P450 (P450) inhibitor, 1-aminobenzotriazole or α- naphthoflavone (α-NF) suppressed thalidomide-induced cytotoxicity to embryo fibroblasts. The treatment of thalidomide also suppressed cell proliferation of embryo fibroblasts by the pre-incubation using liver microsomes from pregnant rabbits. This suppressive effect of thalidomide was removed by the addition of 1-aminobenzotriazole or α-NF.To identify whether microsomal epoxide hydrolase (mEH) is involved in thalidomide-induced teratoge-nicity, the effect of thalidomide treatment on cytotoxicity of embryo fibroblasts was analyzed between mEH-null and the wild-type mice. No significant difference was observed in thalidomide-induced cytotoxicity of embryo fibroblasts from mEH-null and the wild-type mice.These results suggest that P450 is involved in thalidomide-induced cell proliferation inhibition to embryo fibro-blasts