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AMES MUTAGENICITY TESTING WITH RECOMBINANT HUMAN P450S

使用重组人 P450S 进行 AMES 突变性测试

基本信息

批准号：
2391600
负责人：
TODD D PORTER
金额：
$ 14.28万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-29 至 1999-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2391600
关键词：
Salmonella typhimurium alternatives to animals in research carcinogen testing chemical carcinogen clone cells cytochrome P450 drug carcinogenesis enzyme activity human tissue immunochemistry mutagen testing recombinant DNA ultraviolet spectrometry

项目摘要

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.

致突变性的艾姆斯试验是一种广泛使用的方案，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型的回复频率，鼠伤寒沙门氏菌在存在以下物质的情况下孵育后测试化学品和代谢激活系统。这个激活系统最常见的是9000 x g组织上清液（S9组分）匀浆，最常来自啮齿动物肝脏。的存在激活酶，特别是细胞色素P450，在这个组织中匀浆促进前诱变剂和前致癌剂转化为它们的活性代谢物，然后能够结合到细胞大分子，包括DNA。本提案将取消使用实验室动物作为活化酶的来源通过在测试细菌中直接表达这些酶的制备（S.鼠伤寒沙门氏菌）。此外，艾姆斯试验的几个缺点， 1）组织的不确定性质匀浆可以用已知结构酶代替，（2）可以利用克隆人，而不是啮齿动物，酶，从而更准确地估计风险的人暴露于测试化学品;以及3）活化可以发生在细菌细胞内部，而不是外部，潜在的短寿命诱变剂与靶DNA的相互作用，从而提高测定的灵敏度。的具体目标这些建议是： 1)共表达人细胞色素P450和细胞色素P450还原酶 in S.并确定这些酶的细胞内水平通过功能和免疫化学测定;如有必要，表达载体（含有P450的一个或多个拷贝的质粒和还原酶cDNA，以及一个或多个启动子），从而优化所述酶的表达。最大活性的两种表达酶的比例; 2)为了表征优化系统的酶促参数，适当的基质，重要的是，要证明肝脏中存在特异性P450催化单加氧酶活性细菌培养物; 3)为了建立变换后的S.鼠伤寒杆菌细胞通过添加已知会导致基因突变的测试物质进行艾姆斯测试与细菌细胞相比，不含P450表达系统，但在或不存在人肝S9组分;和 4)通过建立一个panel，扩展“重组P450艾姆斯试验”， S.含有一系列人细胞色素的鼠伤寒杆菌重组体 P450，关注那些最常与激活致癌和致突变化学品，并验证这一效用系统