Chun Y J
College of Pharmacy, Chung Ang University, Seoul, Korea.
Arch Pharm Res. 1998 Jun;21(3):305-9. doi: 10.1007/BF02975292.
Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver. It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines. In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expression was achieved at 48 hrs after incubating at 30 degrees C in terrific broth containing ampicillin, IPTG and other supplements. High level expression of P 450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PAGE. The well-known mutagens 2-aminoanthracene and MelQ increased the revertant colonies of E. coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner. The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals.
人细胞色素P450 1A2是人类肝脏中主要的细胞色素P450之一。已知它能够激活多种致癌物，如芳胺和杂环胺。为了开发具有人P450的新型细菌致突变性测试系统，将插入到pCW细菌表达载体中的人P450 1A2全长cDNA导入大肠杆菌WP2 uvrA菌株，该菌株是用于细菌反向致突变性测定的著名大肠杆菌菌株。表达的人细胞色素P450 1A2显示出典型的P450血红蛋白光谱。在含有氨苄青霉素、异丙基-β-D-硫代半乳糖苷（IPTG）和其他补充剂的 terrific肉汤中于30℃孵育48小时后达到最大表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定了大肠杆菌WP2 uvrA膜中人细胞色素P450 1A2的高水平表达。著名的诱变剂2-氨基蒽和MelQ以剂量依赖性方式增加了表达人细胞色素P450 1A2的大肠杆菌WP2 uvrA的回复菌落数，且无需外源性大鼠肝线粒体后上清液（S9组分）。结果表明，人细胞色素P450在细菌致突变性测试菌株中的功能性表达将为研究新药和环境化学物质的诱变和致癌机制提供有用的工具。
