DRUG METABOLIZING ENZYMES IN ANIMAL MODELS AND HUMAN TISSUE

动物模型和人体组织中的药物代谢酶

• 批准号: 3840984
• 负责人: J A GOLDSTEIN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3840984
• 关键词: alleles; benzopyrenes; complementary DNA; cytochrome P450; drug metabolism; enzyme mechanism; gene expression; genetic polymorphism; genetic regulatory element; human genetic material tag; human tissue; isozymes; liver metabolism; messenger RNA; mutagens; phenytoin; site directed mutagenesis; tissue /cell culture; tolbutamide; toxin metabolism; warfarin; yeasts

The cytochrome P450s comprise the principal monooxygenase system which metabolizes foreign chemicals. There are genetic polymorphisms in many of these enzymes including the CYP2C subfamily (S-mephenytoin metabolism) in man. We have isolated and characterized cDNAs for all of the known members of the CYP2C subfamily including cDNAs for two new genes (2C18 and 2C19). S-mephenytoin metabolism in human liver correlated highly with 2C18 mRNA (r = 0.95) and to a lesser extent with 2C9 (r = 0.65), suggesting that one or both of these isozymes are involved in this polymorphism. Six members of the 2C subfamily (2C8, 2C9(2 alleles), 2C18(2 alleles), and 2C19)) were expressed in a yeast cDNA expression system and the recombinant P450s purified from yeast. 2C18 stereospecifically 4'-hydroxylated S- mephenytoin, while neither 2C8 nor 2C9 metabolized this substrate. In contrast, the Ile359 allele of 2C9 metabolized tolbutamide preferentially, while the substitution of one amino acid (Leu359) essentially abolished activity. These results suggest that 2C18 is important in S-mephenytoin metabolism and 2C9 in tolbutamide metabolism, but that allelic variations may also affect metabolism. The anti-coagulant warfarin was also region and stereospecifically metabolized by this subfamily. 2C9 7-hydroxylated S-warfarin, while 2C18 4-hydroxylated S warfarin. The 2C cDNAs have been inserted into the AHH/TK- cell line, and future studies will characterize their ability to metabolize premutagens. Site-specific mutagenesis was used to produce know allelic variations of human 2C9. These have been tested for their effects on warfarin metabolism and will be tested for their ability to alter metabolism of S-mephenytoin and tolbutamide. The contribution of these enzymes to benzpyrene metabolism will be addressed in future studies. Upstream regions of 2C18 and 2C9 have been isolated. Future studies will address the role of allelic variations in coding and regulatory regions on the ability of humans to metabolize selected drugs. Other studies are addressing the variability of human 1A1 and 1A2 in human liver.

细胞色素P450包括主要的单加氧酶系统，该系统 代谢外来化学物质。许多人都存在遗传多态 这些酶包括CYP2C亚家族(S-美苯妥因代谢)在 天哪。我们已经分离并鉴定了所有已知成员的cDNA 包括两个新基因(2C18和2C19)的cDNA。 人肝组织中S-美苯妥因代谢与2C18mR-NA高度相关(r =0.95)，与2C9的相关性较小(r=0.65)，表明一个或 这两种同工酶都参与了这种多态。六名成员 2C亚家族(2C8、2C9(2个等位基因)、2C18(2个等位基因)和2C19) 酵母表达系统的构建及重组P450蛋白的构建 从酵母中提纯。2C18立体专一性4‘-羟化S- 而2C8和2C9都不代谢该底物。在……里面 相比之下，2C9的Ile359等位基因优先代谢甲苯丁胺， 而一种氨基酸(Leu359)的替代基本上取消了 活动。这些结果表明，2C18在S-美苯妥因中起重要作用 代谢和2C9在甲苯磺丁胺代谢中，但等位基因变异 也可能影响新陈代谢。抗凝血剂华法林也是区域 并由这个亚家族进行立体特异性代谢。2C9 7-羟基化 S-华法林，2C18 4-羟化S华法林。2C的cDNA已经被 插入到AHH/TK细胞系中，未来的研究将表征 它们代谢前诱变剂的能力。定点突变是 用于产生已知的人类2C9等位基因变异。这些都是 测试它们对华法林代谢的影响，并将测试 它们改变S-美苯妥因和甲苯丁胺代谢的能力。这个 这些酶对苯并芘新陈代谢的贡献将在 未来的研究。2C18和2C9的上游区域已被分离。 未来的研究将解决等位基因变异在编码和编码中的作用 人类代谢选定药物的能力的调控区域。 其他研究正在解决人类1A1和1A2在人类中的变异性 肝脏。