DRUG METABOLIZING ENZYMES IN ANIMAL MODELS AND HUMAN TISSUE

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。这些酶中的一些是多态的 天哪。我们已经分离并鉴定了8个CYP2C成员的cDNA 亚家族包括代表2C8、2C9和三个新基因(2C18、 2C17和2C19)。分离到大量的等位基因变异。五个满额 长度克隆(代表2C9、2C18、2Cl9的等位变异)为 在Cos细胞表达系统中表达。新陈代谢的差异 甲苯丁胺(主要是2C9)和美芬妥因(2C18)被发现。肝脏来自 人类代谢药物S的能力有很大的不同- 已经分析了美芬妥因的2C8、2C9、2C18和2C19 mRNAs。 S-美苯妥因与2C18mRNA高度相关(r=0.95m RNA)，与2C18m RNA相关性较小 与2C9的遗传距离(r=0.65)表明这两种同工酶中的一种或两种都是 与这种多态有关。正在建立类似的方法来 检测人类活体P4501A2的变异性。表型上的差异 用定点突变的方法检测了大鼠P450(2Cl3)。 从半保守区中的单一氨基酸取代(180) 这改变了半衰期。人P450中这一氨基酸的变化 通过定点突变也减少了它的半衰期，显示出 这种残留物的重要性。所有全长的人类CYP2C cDNA都是 在酵母基因表达系统中进行了表达。新陈代谢较差 因此，正在从酵母中提纯人类P450 以进一步研究它们的新陈代谢。他们有 也被插入到AHH/TK细胞系中，以鉴定其 代谢前诱变剂的能力初步数据表明 细胞色素可代谢黄曲霉毒素。我们正在尝试解决 美苯妥因表型变异的细胞色素 人类的新陈代谢以及其他表型的后果 人类的变异。我们还在解决香烟的影响 吸烟一种环境化学物质对肝脏水平的另一变量 酶P4501A2。

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.