DNA DAMAGE BY BIOACTIVATED XENOBIOTICS

生物活性异生物质造成的 DNA 损伤

• 批准号: 2668338
• 负责人: J CHRISTOPHER STATES
• 金额: $ 20.88万
• 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 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2668338
• 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（改编自研究者摘要）：诱变 环境因素被认为是一个启动事件， 致癌作用 许多环境中的化学物质在代谢过程中 转化为诱变剂 外源性药物处理培养细胞的致突变性 提供的预成型诱变剂已被广泛研究，但很少有 关于细胞内产生的诱变剂引起的诱变的现有资料， 生理相关水平。 细胞之间的内在差异 用外源性反应性代谢物处理， 用代谢前体处理的细胞包括亚细胞的 定位和暴露于活性代谢物的速率。 定量 通过共价修饰的大分子的差异 活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 外源供给和内源生产的结果显示， 差异 显然，更多的工作是使用代谢活性细胞， 需要更接近于体内暴露条件。 长 本提案的长期目标是了解 被认为是主要的代谢活化环境因子的诱变 在接触人群中引发致癌作用的候选者。 DNA加合物的基因特异性分布的工作假设是 在代谢产生诱变剂的细胞中增强， 决定毒理学反应的加合物分布。 的 具体目标是：1. 确定染色质结构的各个方面 有助于表达基因中的优先DNA损伤，和2. 到 研究剂量率在诱变和基因特异性DNA中的作用 损害 拟议的研究将使用表达CYP 1A 1的人细胞， 苯并[a]芘代谢物作为模型系统。 基因特异性DNA损伤 通过定量PCR测定的水平将在基因和 与通过定量逆转录确定的转录速率相关 转录PCR。 诱变后将进行HPRT突变体诱导 并且将确定改变剂量率的效果。 分布 HPRT和p53基因内的DNA损伤将通过连接来监测 介导PCR（LM-PCR）。 将通过测序确定突变谱 RT-PCR产生突变HPRT cDNA，并与DNA损伤谱相关。 将分析加合蛋白以确定以下物质的潜在贡献： 蛋白质修饰的毒理学反应。 这些研究将 提供所需的信息，阐明染色质之间的相互作用 活性基因结构与剂量率在基因特异性DNA测定中的应用 模型致癌物造成的损害。 获得的数据将提供一个模型， 这是研究其他生物活性化学致癌物的基础。