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

沙利度胺基因敲除小鼠致畸机制分析

• 批准号: 11672207
• 负责人: MIYATA Masaaki
• 金额: $ 2.56万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672207/
• 关键词: Thalidomide; Embryo fibroblast; Teratogenicity; Epoxide hydrolase; Knock-out mice; ノックアウトマウス; 遺伝子欠損マウス; 細胞増殖

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

为了评价胚胎成纤维细胞能否作为分析致畸机制的有用工具，用7种致畸或致癌物质(苯妥英钠、苯妥英钠、DMBA、苯并[a]芘、萘、PhIP和MeIQx)检测了胚胎成纤维细胞的细胞毒性。除DMBA和PhIP外，其余5种化合物对胚胎成纤维细胞均无细胞毒性，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代谢激活的效力，采用肝微粒体预孵育法测定胚胎成纤维细胞的细胞毒作用。沙利度胺对胚胎成纤维细胞有剂量依赖性的细胞毒作用，用孕兔肝微粒体预孵育的方法，而不是孕鼠的。这些结果与沙利度胺致畸作用的物种差异是一致的。此外，加入细胞色素P450(P450)抑制剂1-氨基苯并三氮唑或α-萘黄酮(α-NF)可抑制沙利度胺对胚胎成纤维细胞的细胞毒作用。沙利度胺治疗也抑制胚胎成纤维细胞的细胞增殖，通过预先孵育妊娠兔的肝微粒体。加入1-氨基苯并三氮唑或α-NF可消除反应停对胚胎成纤维细胞的抑制作用。为确定微粒体环氧化物水解酶(Meh)是否参与反应停的致畸作用，我们分析了反应停对mh阴性和野生型小鼠胚胎成纤维细胞细胞毒作用的影响。沙利度胺对胚胎成纤维细胞的细胞毒作用与野生型小鼠无明显差异，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。