DRUG METABOLIZING ENZYMES IN ANIMAL MODELS AND HUMAN TISSUE

CYP酶组成代谢的单加氧酶系统 外国化学品。我们正在研究CYP2C亚家族，包括 一种在人类中具有多态并代谢药物S的P450- 甲苯妥因。我们利用酵母cDNAs表达系统表达了细胞色素P450 2 C8， 2C9、2C18、2C19和几种等位基因变异体的比较 这些重组蛋白代谢华法林的能力，以及 甲苯丁酰胺。2C9是甲苯丁胺的主要代谢酶，但 到目前为止，只有2C18被发现能代谢S-美苯妥因，但 美芬妥因的营业额较低。我们正在延长 用更严谨的方法研究S-美苯妥因代谢的最佳条件 孵化条件。以确定这些酶中的哪一种涉及 在这种多态中，我们试图开发特定的抗体来 所有四种基因产品都用来比较新陈代谢和蛋白质水平。这个 重组蛋白在细菌中的表达先于抗体 制作。2C18和2C9除外显子外的全部基因 已从人的肝脏和1kb的上游区分离到6个 外显子和内含子连接被测序。我们对上游地区进行了测序 2C18来自一种高和差的代谢物，但没有发现序列 不同之处。表达了2C18和2C9的上游区域 使用荧光素酶报告基因构建。相对推动者 2C9和2C18的能力似乎与更大的表达一致 2C9在人体肝脏中的表达。潜在的肝脏特异性部位，糖皮质激素 响应元件、可能的抑制区域以及 可能推断苯巴比妥的诱导性已被确定。未来 研究将解决影响S新陈代谢的遗传缺陷(S)- 人体内的美苯妥因和其他2C底物。我们还在检查 人肝脏细胞色素P450 1A2和细胞色素P1A1表达的变化聚合酶链反应 分析将把这些基因的表达与吸烟史联系起来 以及接触到二恶英。细胞色素P1A1诱导的剂量效应数据 和CYP1A2正在人和大鼠的肝脏切片上进行试验。

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.