DRUG METABOLIZING ENZYMES IN ANIMAL MODELS AND HUMAN TISSUE

细胞色素P450包含主要的单加氧酶系统， 代谢外来化学物质这些酶中的一些是多态的。我们 正在克隆和测序这些P450基因的cDNA， 变化.然后，我们使用cDNA表达系统，如酵母和 COS-1细胞，以评估单个P450在药物代谢中的作用， 诱变剂我们已经从两个人的肝脏中制备了文库。无cDNA克隆 对于P450 IIC 8，在IIC 8表型低的人肝脏中鉴定出 蛋白北方分析和PCR分析也表明， 这种酶的mRNA水平。然而，一个新人类的cDNA（254 c） IIC P-450被识别。我们正在寻找一个全长克隆， 鉴定并表达这种酶。第二个图书馆是由一个 人肝高IIC 8，和90个基本上全长cDNA克隆， 鉴定了IIC亚家族。杂交研究表明，IIC 8 代表32%的克隆，约60%与 IIC 9（MP4或MP8）。两个变体全长IIC 9克隆被克隆。 鉴定两个额外的cDNA克隆代表了一个新的 已对全长IIC P-450（s）进行测序。另外的cDNA正在 表征了在酵母中的表达研究比较了大鼠 肝P450 IIC 13及其表型变异体。P450 IIC 13在大肠杆菌中表达， 酵母细胞的水平比其表型变体高5-7倍 含有9个碱基取代，表明基因中的点突变 P450 IIC 13 mRNA表达缺失。我们目前正在 使用cDNA表达系统表达人P450 IIC的成员， 亚科这些人类P450代谢重要药物的能力 和环境化学品（美芬妥英，甲苯磺丁脲，和各种类别 将分析前诱变剂）。肝脏组织约25-50 将分析人类P450酶可能的表型变异性 使用北方分析。这些研究将评估个人的作用 化学诱变剂代谢活化/失活中的P450， 致癌物质。

The cytochrome P450s comprise the principle monooxygenase system which metabolizes foreign chemicals. Some of these enzymes are polymorphic. We are cloning and sequencing cDNAs for these P450s from individuals showing variations. We are then using cDNA expression systems such as yeast and COS-1 cells to assess the role of individual P450s to metabolize drugs and mutagens. We have prepared libraries from two human livers. No cDNA clones for P450IIC8 were identified in a human liver phenotypically low in IIC8 protein. Northern analysis and PCR analysis also indicate extremely low levels of the mRNA for this enzyme. However, a cDNA(254c) for a new human IIC P-450 was identified. We are searching for a full length clone to identify and express this enzyme. A second library was constructed from a human liver high in IIC8, and 90 essentially full length cDNA clones in the IIC subfamily were identified. Hybridization studies indicate that IIC8 represents 32% of the clones, and about 60% are identical or similar to IIC9 (either MP4 or MP8). Two variant full-length IIC9 clones were identified. Two additional cDNA clones representing variants of a new full-length IIC P-450(s) have been sequenced. Additional cDNAs are being characterized. Expression studies in yeast have compared expression of rat liver P450IIC13 and its phenotypic variant. P450IIC13 was expressed in yeast cells at a level 5-7-fold higher than its phenotypic variant containing 9 base substitutions, demonstrating that point mutations in the mRNA result in the defective expression of P450IIC13. We are presently using cDNA expression systems to express members of the human P450IIC subfamily. The ability of these human P450s to metabolize important drugs and environmental chemicals (mephenytoin, tolbutamide, and various classes of promutagens) will be analyzed. Liver tissue from approximately 25-50 humans will be analyzed for possible phenotypic variability of P450 enzymes using Northern analysis. These studies will assess the role of individual P450s in metabolic activation/deactivation of chemical mutagens and carcinogens.