AMES MUTAGENICITY TESTING WITH RECOMBINANT HUMAN P450S

Ames致突变性测试是一种广泛使用的评估 药物和化学品的遗传毒性和潜在致癌性。这 测试测量组氨酸营养缺陷症逆转的频率 鼠伤寒沙门氏菌与 测试化学物质和代谢激活系统。这个激活系统 最常见的是组织的9000 x g上清液(S9部分) 匀浆，最常见的来源于啮齿动物的肝脏。.的存在 激活组织中的酶，特别是细胞色素P450 匀浆促进诱变剂和致癌物转化为 它们的活性代谢物，然后能够与细胞结合 包括DNA在内的大分子。目前的建议将消除 利用实验动物作为活化酶的来源 在试验菌中直接表达这些酶的制备 (小鼠沙门氏菌)。此外，Ames测试的几个缺点是 目前使用的将被消除：1)组织的未定义性质 匀浆将被已知结构和结构的酶取代 2)有可能使用克隆人，而不是 啮齿动物，酶，从而更准确地估计对人类的风险 暴露在测试化学品中；以及3)可以发生激活 在细菌细胞内，而不是在细胞外，促进 潜在的短命诱变剂与靶DNA的相互作用 从而提高了检测的灵敏度。的具体目标 这项建议包括： 1)人细胞色素P450与细胞色素P450还原酶共表达 在鼠伤寒沙门氏菌中并测定这些酶的胞内水平 通过功能和免疫化学分析；如有必要，修改 表达载体(含有一个或多个P450拷贝的质粒 和还原酶cDNA以及一个或多个启动子)，以优化 最大活性的两种表达酶的比例； 2)对优化后的体系的酶参数进行了表征 适当的底物(S)，重要的是要证明 活体中存在特定的P450催化的单加氧酶活性 细菌培养； 3)建立转化鼠伤寒沙门氏菌细胞在 通过添加一种已知会导致基因突变的测试物质进行的Ames测试 并与细菌细胞比较，对组氨酸逆转株进行评分 不包含P450表达系统，但在存在下孵化 或不含人肝S9组分；以及 4)扩大“重组P450 Ames试验”，成立一个专家组 含有一系列人细胞色素的鼠伤寒沙门氏菌重组株 P450，集中在那些最常见的与激活 致癌和致突变化学品，并验证这一用途 系统。

The Ames test for mutagenicity is a widely used protocol to estimate the genotoxicity and potential carcinogenicity of drugs and chemicals. This test measures the frequency of reversion of histidine auxotrophs of Salmonella typhimurium following their incubation in the presence of the test chemical and a metabolic activating system. This activating system is most often the 9000 x g supernatant (S9 fraction) of a tissue homogenate, most commonly derived from rodent liver. The presence of activating enzymes, notably the cytochromes P450, in this tissue homogenate promotes the conversion of promutagens and procarcinogens to their active metabolites, which are then capable of binding to cellular macromolecules, including DNA. The present proposal would eliminate the use of laboratory animals as a source of the activating enzyme preparation by directly expressing these enzymes in the test bacterium (S. typhimurium). Moreover, several shortcomings of the Ames test as presently used would be eliminated: 1) the undefined nature of the tissue homogenate would be replaced with enzymes of known structure and activity; and 2) it would be possible to use cloned human, rather than rodent, enzymes, and thereby more accurately estimate the risk to man of exposure to the test chemical; and 3) the activation can take place inside the bacterial cell, rather than outside, facilitating the interaction of potentially short-lived mutagens with the target DNA and thereby enhancing the sensitivity of the assay. The specific aims of this proposal are: 1) To coexpress a human cytochrome P450 with cytochrome P450 reductase in S. typhimurium and determine the intracellular levels of these enzymes by functional and immunochemical assays; if necessary, to modify the expression vector (a plasmid containing one or more copies of the P450 and reductase cDNAs, and one or more promoters) so as to optimize the ratio of the two expressed enzymes for maximum activity; 2) To characterize the enzymatic parameters of the optimized system with appropriate substrate(s), and, importantly, to demonstrate that the specific P450-catalyzed monooxygenase activity is present in live bacterial cultures; 3) To establish the utility of the transformed S. typhimurium cells in an Ames test by adding a test substance known to cause genetic mutations and scoring for histidine revertants, with comparison to bacterial cells not containing a P450 expression system, but incubated in the presence or absence of a human liver S9 fraction; and 4) To expand the "recombinant P450 Ames test" by establishing a panel of S. typhimurium recombinants containing a series of human cytochromes P450, focussing on those most often associated with the activation of carcinogenic and mutagenic chemicals, and verifying the utility of this system.