MECHANISM OF CHROMIUM CARCINOGENICITY

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 为了检验以下假设：(1)新陈代谢激活 铬(VI)会产生“活性中间体”，从而引起 特定DNA损伤；(2)特定DNA损伤发生在定义的 由于其DNA而优先受到攻击的DNA序列 结构；以及(3)特异性铬(VI)引起的DNA损伤 DNA-蛋白质改变对DNA正常模板活性的影响 互动。以下方法既包括体外培养，也包括 体内实验系统将被用来解决这个问题： (1)在反应过程中产生的铬(V)和自由基中间体 氧化还原活性细胞组分对铬(VI)的代谢活化 将会被确定。电子顺磁共振波谱将用于铬(V)的检测 物种和自旋陷阱将被用来检测单线态氧和 铬(VI)反应生成的羟基和硫基自由基物种 用外源物质进行预处理，这些物质会特别影响 不同的细胞氧化还原成分及其在生产中的变化 各种组织中的铬(V)和自由基物种将是 下定决心。(2)铬(VI)诱导的DNA损伤，以DNA形式存在 链断裂、DNA交联、铬-DNA加合物和自由基DNA 加合物，由“活性中间体”的攻击而形成 在铬的代谢过程中，六价铬会被测定在质粒DNA中 以及在线粒体和核DNA中的限制性片段 在活体内。这些DNA在大小、构象和相关的 因此，蛋白质作为铬(VI)诱导的靶标可能有所不同 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， 表征并与合成的铬-核苷酸络合物进行比较。 抗体将被提高到从In分离的铬-DNA加合物 体外反应，并将用于分析从组织中分离的DNA 在体内用铬(VI)处理的大鼠和鸡胚胎。(3) 铬致DNA损伤对蛋白质-DNA相互作用的影响 接受检查。DNA聚合酶合成子DNA的能力 DNA模板上存在铬-DNA加合物的链将 要下定决心。基因调控蛋白与其基因结合的能力 铬-DNA形成后的特异性DNA识别元件 还将确定这些序列中的加合物。互动 带有DNA限制性片段的乳糖抑制物CAP和RNA聚合酶 含有大肠杆菌乳糖的启动子和操纵子序列 操纵子、糖皮质激素受体与金属调节的相互作用 蛋白质到含有金属硫蛋白的限制性片段 发起人将接受检查。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。

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.