DNA DAMAGE BY BIOACTIVATED XENOBIOTICS

生物活性异生物质造成的 DNA 损伤

• 批准号: 2882830
• 负责人: J CHRISTOPHER STATES
• 金额: $ 3.95万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2882830
• 关键词: DNA damage; adduct; benzopyrenes; chemical carcinogenesis; chromatin; dosage; environmental toxicology; gene mutation; mutagens; polymerase chain reaction; tissue /cell culture; toxin metabolism

DESCRIPTION (Adapted from the Investigator's Abstract): Mutagenesis by environmental agents is believed to be an initiating event in carcinogenesis. Many environmental chemicals are metabolically converted into mutagens. Mutagenesis in cultured cells treated with exogenously supplied preformed mutagens has been studied extensively but there is little information available on mutagenesis by mutagens produced within cells at physiologically relevant levels. The intrinsic differences between cells treated with exogenous reactive metabolites and metabolically competent cells treated with metabolic precursors include both the subcellular localization and the rate of exposure to reactive metabolites. Quantitative differences in the macromolecules which are covalently modified by the reactive metabolites cause alterations in cytotoxicity and genotoxicity. The few investigations comparing genotoxicity of reactive metabolites supplied exogenously and produced endogenously demonstrated dramatic differences. Clearly, more work using metabolically competent cells which more closely resemble the in vivo exposure condition is needed. The long term goal of the current proposal is to understand the mechanisms of mutagenesis by metabolically activated environmental agents considered prime candidates for initiation of carcinogenesis in exposed human populations. The working hypothesis that the gene specific distribution of DNA adducts is enhanced in cells metabolically producing mutagens and it is the distribution of adducts that determines the toxicological response. The specific aims are: 1. To determine the aspects of chromatin structure contributing to preferential DNA damage in expressed genes, and 2. to investigate the role of dose rate in mutagenesis and gene specific DNA damage. The proposed studies will use CYP1Al -expressing human cells and benzo[a]pyrene metabolites as a model system. Gene specific DNA damage levels determined by quantitative PCR will be compared between genes and correlated with transcription rates determined by quantitative reverse transcription PCR. Mutagenesis will be followed by HPRT mutant induction and the effect of altering dose rates will be determined. The distribution of DNA damage within the HPRT and p53 genes will be monitored by ligation mediated - PCR (LM-PCR). Mutation spectra will be determined by sequencing RT-PCR produced mutant HPRT cDNAs and correlated with DNA damage spectra. Adducted proteins will be analyzed to determine potential contributions of protein modification to the toxicological response. These studies will provide needed information elucidating the interplay between chromatin structure in active genes and dose rate in determining gene specific DNA damage by a model carcinogen. The data obtained will provide a model on which to base investigations of other bioactivated chemical carcinogens.

描述(改编自《调查者摘要》)：诱变 环境代理人被认为是一个启蒙事件 致癌。许多环境化学物质被新陈代谢转化 变成了诱变剂。外源性辐射对培养细胞的致突变作用 提供的预成型诱变剂已被广泛研究，但很少有 有关细胞内产生的诱变剂的诱变作用的信息，请访问 生理上相关的水平。细胞之间的内在差异 用外源反应性代谢物处理并具有代谢能力 用代谢前体处理的细胞包括亚细胞 定位和暴露于反应性代谢物的比率。量化 被共价修饰的大分子的差异 反应性代谢物引起细胞毒性和遗传毒性的改变。 比较反应性代谢物遗传毒性的少数研究 外源供应和内源生产展示了戏剧性 不同之处。显然，更多的工作是利用具有代谢能力的细胞 更接近体内暴露的条件是必要的。《长河》 当前提案的长期目标是了解 被认为是最重要的代谢激活的环境物质的诱变 在暴露的人群中启动致癌的候选对象。 工作假说是DNA加合物的基因特异性分布是 增强了细胞代谢产生诱变剂的能力，它是 决定毒理学反应的加合物的分布。这个 具体目标是：1.确定染色质结构的各个方面 促进表达基因的优先DNA损伤，以及2.对 剂量率在诱变和基因特异性DNA中的作用 损坏。拟议的研究将使用表达CYP1Al的人类细胞和 以苯并[a]芘代谢物为模型体系。基因特异性DNA损伤 通过定量聚合酶链式反应确定的水平将在基因和 与定量反向测定的转录速率相关 转录聚合酶链式反应。诱变之后将进行HPRT突变诱导 改变剂量率的效果将得到确定。分布情况 HPRT和P53基因的DNA损伤将通过连接进行监测 中介-聚合酶链式反应(Lm-PCR)。突变谱将通过测序确定 RT-PCR产生突变的HPRT cDNAs，并与DNA损伤谱相关。 将对加合物蛋白质进行分析，以确定其潜在贡献 蛋白质修饰对毒理学的反应。这些研究将 提供必要的信息，阐明染色质之间的相互作用 活性基因的结构与基因特异性DNA测定中的剂量率 一种模型致癌物质造成的损害。所获得的数据将提供一个关于 这为其他生物活性化学致癌物的研究奠定了基础。