EXPRESSION OF CYTOCHROME P-450S AND THEIR ROLE IN MUTAGENESIS & CARCINOGENESIS

细胞色素 P-450S 的表达及其在诱变中的作用

• 批准号: 3874714
• 负责人: N BATTULA
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874714
• 关键词: DNA; Retroviridae; acetylaminofluorene; adduct; biotransformation; carbopolycyclic compound; chemical addition; chemical carcinogen; chemical carcinogenesis; cyclic amine; cytochrome P450; drug carcinogenesis; drug interactions; enzyme biosynthesis; enzyme substrate complex; gene expression; genetic manipulation; heterocyclic compounds; hypoxanthine phosphoribosyltransferase; laboratory mouse; molecular cloning; mutagens; nucleic acid sequence; nutrition related neoplasm /cancer; nutrition related tag; toxicant interaction; toxin metabolism; vaccinia virus

The cytochrome P450 superfamily of genes code for a number of enzymes which metabolize a wide array of compounds including endobiotics and xenobiotics. The ability of the cytochrome P450s to carry out such diversity of functions on a variety of substrates results from multiple forms of distinct P450s. We are primarily interested in the biochemical and molecular mechanisms of chemical carcinogenesis. To elucidate the role of P450s involved in the biotransformation of xenobiotics to toxic, mutagenic and carcinogenic forms, it is essential that the individual P450s are expressed in cells which lack endogenous activity for these enzymes. Toward this goal we have developed expression systems by which DNAs coding for individual cytochrome P450s can be delivered into mammalian cells and these engineered cell lines can be analyzed for the consequences of functional expression of cytochromes P450. Rodent cells were transduced with recombinant retroviruses containing the DNA for cytochrome P4501A2. Analysis of the clones for P4501A2 showed colinear integration of a single copy of the DNA into the chromosomal DNA of host cells and the expected P4501A2 activities. Clones constitutively expressing cytochrome P4501A2 when exposed to the food-derived carcinogenic heterocyclic amines IQ, MeIQx, and PhIP, and the aromatic amines AF and AAF each formed specific DNA adducts with the host cell DNA. The adduct formation was dependent on the duration of exposure and the dose of the carcinogen. 7,8-Benzoflavone, a known inhibitor of P4501A2 activity, blocked the formation of these adducts, indicating that metabolic activation by P4501A2 is required for the adduct formation. Analysis of clones containing DNA-adducts for possible adduct-directed mutations showed that the cells indeed suffered mutations in the hypoxanthine-guanine phosphoribosyl transferase gene and the mutant clones lacked HGPRT activity. Thus, we established a well-defined prototype mammalian cell system expressing cytochrome P4501A2 for biochemical and molecular analysis of mutagenesis induced by xenobiotics.

细胞色素P450基因超家族编码许多酶， 代谢包括内源性和外源性物质在内的多种化合物。 细胞色素P450进行这种多样性的能力， 在各种基板上的功能是由多种形式的 不同的P450我们主要对生物化学和 化学致癌的分子机制。阐明的作用 P450参与外源性物质生物转化为毒性、致突变性 和致癌形式，这是必不可少的，个别P450是 在缺乏这些酶的内源活性的细胞中表达。朝向 为了实现这一目标，我们开发了表达系统， 单个细胞色素P450可以被递送到哺乳动物细胞中， 可以分析工程化细胞系的功能性改变的后果， 细胞色素P450的表达。啮齿类动物细胞用 含有细胞色素P4501 A2 DNA的重组逆转录病毒。 对P4501 A2的克隆的分析显示了单个P4501 A2的共线性整合。 将DNA拷贝到宿主细胞的染色体DNA中，并将预期的 P4501 A2活性。组成型表达细胞色素P4501 A2的克隆 当暴露于食物来源的致癌杂环胺IQ时， MeIQx和PhIP，以及芳族胺AF和AAF各自形成特定的 DNA与宿主细胞DNA加合。加合物的形成取决于 暴露的持续时间和致癌物的剂量。7，8-苯并异丁酮， 一种已知的P4501 A2活性抑制剂， 加合物，表明P4501 A2的代谢活化是 加合物的形成。含有DNA加合物的克隆的分析 可能的内收定向突变表明，细胞确实遭受了 次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因中的突变， 突变克隆缺乏HGPRT活性。于是，我们建立了一个 表达细胞色素P4501 A2的明确定义的原型哺乳动物细胞系统 用于生物化学和分子生物学分析 异生物质