DRUG METABOLIZING ENZYMES IN ANIMAL MODELS AND HUMAN TISSUE

酶包括单加氧酶系统，其代谢 外国化学品 我们正在研究CYP 2C亚家族，其中包括 P450，其在人中是多态性的并且代谢药物S- 美芬妥英我们使用酵母cDNA表达系统表达CYP 2C 8， 2C 9、2C 18和2C 19以及几种等位基因变体，并比较了它们的 这些重组蛋白代谢华法林的能力，以及 甲苯磺丁脲 2C 9是甲苯磺丁脲的主要代谢酶， 迄今为止，仅发现2C 18代谢S-美芬妥英， 美芬妥英的周转率较低。 我们正在扩展 对S-美芬妥英代谢进行了更严格的优化研究 孵育条件 为了确定这些酶中的哪一种 在多态性方面，我们正试图开发特异性抗体， 所有四种基因产物来比较代谢和蛋白质水平。 的 重组蛋白在细菌中表达， 生产 2C 18的完整基因和除外显子外的所有2C 9 6个已从人肝中分离到，upstram区> 1 kb， 外显子和内含子连接测序。 我们测序了上游区域 2C 18来自高代谢者和低代谢者，但未发现序列 差异 2C 18和2C 9的上游区域已经表达 使用荧光素酶报告基因构建体。 相对启动子 2C 9和2C 18的能力似乎与更高的表达一致， 2C 9在人体肝脏中的作用 潜在的肝脏特异性位点，糖皮质激素 反应元件、可能的抑制区和 可以推断苯巴比妥诱导的药物已被鉴定。 未来 研究将解决影响S-代谢的遗传缺陷， 美芬妥英和其他2C底物。我们也在检查 人肝脏中CYP 1A 2和CYP 1A 1表达的变异性。 PCR 分析将把这些基因的表达与吸烟史联系起来 和接触二恶英 诱导CYP 1A 1的剂量反应数据 和CYP 1A 2正在人类和大鼠肝脏切片中进行。

The CYP enzymes comprise the monooxygenase system which metabolizes foreign chemicals. We are studying the CYP2C subfamily which includes a P450 which is polymorphic in man and metabolizes the drug S- mephenytoin. We used a yeast cDNA expression system to express CYP2C8, 2C9, 2C18, and 2C19 and several allelic variants and compared their ability of these recombinant proteins to metabolize warfarin, and tolbutamide. 2C9 was the principal enzyme metabolizing tolbutamide, but only 2C18 has thus far been found to metabolize S-mephenytoin but turnover numbers for mephenytoin were somewhat low. We are extending the S-mephenytoin metabolism with a more rigorous study of optimum incubation conditions. To identify which of these enzymes is involved in the polymorphism, we are attempting to develop specific antibodies to all four gene products to compare metabolism with protein levels. The recombinant proteins are being expressed in bacteria prior to antibody production. The complete gene for 2C18 and all of 2C9 except for exon 6 have been isolated from human liver and >1kb of upstram region and all exons and intron junctions sequenced. We sequenced the upstream region of 2C18 from a high and poor metabolizer but found no sequence differences. Upstream regions of 2C18 and 2C9 have been expressed using a luciferase reporter gene construct. The relative promotor abilities of 2C9 and 2C18 appear consistent with the greater expression of 2C9 in human liver. Potential liver-specific sites, glucocorticoid responsive elements, a possible inhibitory region, and an element which may infer phenobarbital inducibility have been identified. Future studies will address the genetic defect(s) which affect metabolism of S- mephenytoin and other 2C substrates in humans. We are also examing variabilities in expression of CYP1A2 and CYP1A1 in human liver. PCR analysis will correlate expression of these genes with smoking history and exposure to dioxins. Dose response data for the induction of CYP1A1 AND CYP1A2 are being performed in human and rat liver slices.