AMES MUTAGENICITY TESTING WITH RECOMBINANT HUMAN P450S

使用重组人 P450S 进行 AMES 突变性测试

• 批准号: 2155287
• 负责人: TODD D PORTER
• 金额: $ 11.84万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-29 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2155287
• 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

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.

艾姆斯致突变性试验是一种广泛使用的方法来估计致突变性 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细胞的逆转频率 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 最常见的是组织的 9000 x g 上清液（S9 部分） 匀浆，最常见来自啮齿动物肝脏。 的存在 激活该组织中的酶，特别是细胞色素 P450 匀浆促进促诱变剂和促癌剂转化为 它们的活性代谢物，然后能够与细胞结合 大分子，包括DNA。 目前的提案将消除 使用实验动物作为激活酶的来源 通过在测试细菌中直接表达这些酶来制备 （鼠伤寒沙门氏菌）。 此外，艾姆斯测试的几个缺点如下： 目前使用的将被消除：1）组织的不确定性质 匀浆将被已知结构的酶替代并且 活动; 2）有可能使用克隆人，而不是 啮齿动物、酶，从而更准确地估计人类的风险 暴露于测试化学品； 3) 可以进行激活 在细菌细胞内部，而不是外部，促进 潜在的短寿命诱变剂与目标 DNA 的相互作用 从而提高测定的灵敏度。 具体目标 该提案是： 1) 与细胞色素 P450 还原酶共表达人细胞色素 P450 鼠伤寒沙门氏菌并测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，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 两种表达的酶的最大活性之比； 2) 表征优化系统的酶参数 适当的底物，并且重要的是，证明 活体中存在特定的 P450 催化单加氧酶活性 细菌培养； 3) 建立转化的鼠伤寒沙门氏菌细胞在 艾姆斯测试，添加已知会导致基因突变的测试物质 和组氨酸回复突变体的评分，与细菌细胞的比较 不含 P450 表达系统，但在存在下孵育 或缺乏人肝脏 S9 组分；和 4) 通过建立一组来扩大“重组P450 Ames测试” 含有一系列人类细胞色素的鼠伤寒沙门氏菌重组体 P450，重点关注那些最常与激活相关的 致癌和致突变化学品，并验证其效用 系统。