MECHANISM OF CHROMIUM CARCINOGENICITY

铬致癌机制

• 批准号: 2156355
• 负责人: KAREN E WETTERHAHN
• 金额: $ 23.51万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2156355
• 关键词: DNA replication; adduct; cellular oncology; chemical carcinogenesis; chick embryo; chromium; crosslink; electron spin resonance spectroscopy; free radical oxygen; genetic promoter element; glutathione; hydroxyl radical; laboratory rat; metal metabolism; metallothionein; mitochondrial DNA; nucleic acid denaturation; nucleic acid repetitive sequence; operon; restriction mapping; singlet oxygen; toxin metabolism; transcription factor

The overall objective of this research project is to understand the mechanism by which chromium(VI) compounds act as carcinogens. We plan to test the following hypotheses: (1) that metabolic activation of chromium(VI) results in "reactive intermediates" which give rise to specific DNA lesions; (2) that the specific DNA lesions occur at defined DNA sequences which are preferentially attacked because of their DNA structure; and (3) that the specific chromium(VI)-induced DNA lesions affect the normal template activity of DNA by altering DNA-protein interactions. The following approaches which include both in vitro and in vivo experimental systems will be used in attacking this problem: (1) The chromium(V) and radical intermediates generated during the metabolic activation of chromium(VI) by redox-active cellular components will be determined. EPR spectroscopy will be used to detect chromium(V) species and spin traps will be used to detect singlet oxygen and hydroxyl and thiyl radical species formed upon reaction of chromium(VI) be pretreated with xenobiotics which specifically affect the levels of the various cellular redox components and change in the production of the chromium(V) and radical species in various tissues will be determined. (2) Chromium (VI)-induced DNA lesions, in the form of DNA strand breaks, DNA cross-links, chromium-DNA adducts and radical-DNA adducts, which result from the attack f "active intermediates" formed during the metabolism of chromium(VI) will be determined in plasmid DNA and restriction fragments in vitro and in mitochondrial and nuclear DNA in vivo. These DNAs differ in size, conformation and associated proteins, and therefore, may differ as targets for chromium(VI)-induced DNA lesions. The sequence/conformation specificity of the chromium-DNA adducts will be examined, and the chromium-DNA adducts will be isolated, characterized and compared with synthetic chromium-nucleotide complexes. Antibodies will be raised to chromium-DNA adducts isolated from the in vitro reactions, and will be used to analyze DNA isolated from tissues of rats and chick embryos treated with chromium(VI) in vivo. (3) The effect of chromium-induced DNA lesions on protein-DNA interactions will be examined. The ability of DNA polymerase to synthesize daughter DNA strands in the presence of chromium-DNA adducts on the DNA template will be determined. The ability of gene regulatory proteins to bind to their specific DNA recognition elements after formation of chromium-DNA adducts within these sequences will also be determined. The interaction of lac repressor CAP and RNA polymerase with a DNA restriction fragment containing the promoter and operator sequences of the E. coli lactose operon, and interaction of glucocorticoid receptor and metal regulatory proteins to a restriction fragment containing the matallothionein promoter will be examined. These studies should elucidate critical cellular pathways which lead to the carcinogenic activity of chromium(VI) compounds.

本研究项目的总体目标是了解 铬（VI）化合物作为致癌物的机制。 我们计划 为了验证以下假设：（1）代谢活化 铬（VI）产生“反应性中间体”， 特异性DNA损伤;（2）特异性DNA损伤发生在定义的 DNA序列，由于其DNA 结构;（3）特定的铬（VI）诱导的DNA损伤 通过改变DNA-蛋白质影响DNA的正常模板活性 交互. 以下方法包括体外和 体内实验系统将用于解决这个问题： (1)铬（V）和自由基中间体在反应过程中产生， 氧化还原活性细胞组分对铬（VI）的代谢活化 将被确定。 EPR光谱将用于检测铬（V） 物种和自旋陷阱将用于检测单线态氧， 铬（VI）反应时形成的羟基和硫代自由基物质 用异生物素进行预处理， 各种细胞氧化还原组分和生产中的变化 铬（V）和各种组织中的自由基物质将被 测定 (2)铬（VI）诱导的DNA损伤，以DNA的形式 链断裂、DNA交联、铬-DNA加合物和自由基-DNA 加合物，这是由于攻击f“活性中间体”形成 在铬（VI）的代谢过程中，将在质粒DNA中测定 和限制性片段在体外和线粒体和核DNA in vivo. 这些DNA在大小、构象和相关的 蛋白质，因此，可能不同的目标铬（VI）诱导 DNA损伤 铬-DNA的序列/构象特异性 将检查加合物，并分离铬-DNA加合物， 其特征在于，并与合成的铬-核苷酸络合物进行比较。 将针对从体内分离的铬-DNA加合物产生抗体 体外反应，并将用于分析从组织中分离的DNA 铬（VI）处理的大鼠和鸡胚在体内。 (3)的 铬诱导的DNA损伤对蛋白质-DNA相互作用的影响将 接受检查。 DNA聚合酶合成子代DNA的能力 在DNA模板上存在铬-DNA加合物的情况下， 被确定。 基因调节蛋白结合其 铬-DNA形成后的特异性DNA识别元件 还将测定这些序列内的加合物。 的相互作用 lac阻遏物CAP和RNA聚合酶与DNA限制性片段 含有E.大肠杆菌乳糖 糖皮质激素受体与金属调控因子的相互作用 将蛋白质与含有金属硫蛋白的限制性片段连接 将对发起人进行审查。 这些研究应该阐明关键的 导致致癌活性的细胞途径 铬（VI）化合物。