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

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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