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: 3855814
• 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/3855814
• 关键词: aflatoxins; alleles; complementary DNA; cytochrome P450; drug metabolism; environmental toxicology; enzyme mechanism; gene expression; genetic polymorphism; human genetic material tag; human tissue; isozymes; laboratory rat; liver metabolism; messenger RNA; molecular cloning; mutagens; phenytoin; point mutation; site directed mutagenesis; tobacco abuse; tolbutamide; toxin metabolism; transfection; yeasts

The cytochrome P450s comprise the principle monooxygenase system which metabolizes foreign chemicals. Some of these enzymes are polymorphic in man. We have isolate and characterized cDNAs for 8 members of the CYP2C subfamily including cDNA representing 2C8, 2C9 and three new genes (2C18, 2C17, and 2C19). A number of allelic variants were isolated. Five full length clones (representing allelic variants of 2C9, 2C18, 2Cl9) were expressed in a Cos cell expression system. Differences in metabolism of tolbutamide (primarily 2C9) and mephenytoin (2C18) were noted. Livers from humans which varied dramatically in their ability to metabolize the drug S- mephenytoin have been analyzed for 2C8, 2C9, 2C18, and 2C19 mRNAs. S-mephenytoin 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. Similar methods are being established to examine the variability of human live P4501A2. A phenotypic difference in a rat P450 (2Cl3) was determined by site-directed mutagenesis to result from a single amino acid substitution in a semi-conserved region (180) which altered the half-life. Alteration of this amino acid in a human P450 by site-directed mutagenesis also decreased its half-life showing the importance of this residue. All of the full length human CYP2C cDNAs were the expressed in a yeast cDNA expression system. Metabolism was poorer than anticipated therefore, the human P450s are being purified from yeast microsomes in order to characterize their metabolism further. They have also been inserted into the AHH/TK cell line in order to characterize their ability to metabolize premutagens Preliminary data suggests that one cytochrome may metabolize aflatoxin. We ar attempting to address the cytochrome responsible for the phenotypic variation of mephenytoin metabolism in humans as well as the consequences of other phenotypic variations in humans. We are also addressing the effects of cigarette smoking an environmental chemicals on hepatic levels of another variable enzyme P4501A2.

细胞色素 P450 包含主要的单加氧酶系统， 代谢外来化学物质。 其中一些酶具有多态性 男人。 我们分离并表征了 CYP2C 8 名成员的 cDNA 亚家族包括代表 2C8、2C9 的 cDNA 和三个新基因（2C18、 2C17和2C19)。 分离出许多等位基因变体。 五满 长度克隆（代表 2C9、2C18、2C19 的等位基因变体） 在 Cos 细胞表达系统中表达。 新陈代谢的差异 注意到甲苯磺丁脲（主要是 2C9）和美芬妥英（2C18）。 肝脏来自 人类代谢药物 S 的能力差异很大 已对美芬妥英的 2C8、2C9、2C18 和 2C19 mRNA 进行了分析。 S-美芬妥英与 2C18 mRNA 高度相关 (r = 0.95)，与较小的相关 2C9 的程度（r = 0.65）表明这些同工酶中的一种或两种是 参与这种多态性。 类似的方法正在建立 检查人类活体 P4501A2 的变异性。 表型差异 通过定点诱变测定大鼠 P450 (2Cl3)，结果 来自半保守区域中的单个氨基酸取代 (180) 这改变了半衰期。 人 P450 中该氨基酸的改变 通过定点诱变也缩短了其半衰期 该残留物的重要性。 所有全长人类 CYP2C cDNA 在酵母cDNA表达系统中表达。 新陈代谢较差 因此，人类 P450 正在从酵母中纯化出来，超出预期 微粒体以进一步表征其代谢。 他们有 也被插入 AHH/TK 细胞系中，以表征其 代谢前诱变剂的能力初步数据表明， 细胞色素可以代谢黄曲霉毒素。 我们正试图解决 细胞色素负责美芬妥英的表型变异 人类的新陈代谢以及其他表型的后果 人类的变异。 我们也在解决香烟的影响 吸烟环境化学物质对另一个变量的肝脏水平的影响 酶 P4501A2。