MECHANISM OF CHROMIUM CARCINOGENICITY

铬致癌机制

• 批准号: 2156356
• 负责人: KAREN E WETTERHAHN
• 金额: $ 24.5万
• 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/2156356
• 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.

该研究项目的总体目的是了解 铬（VI）化合物作为致癌物的机制。 我们计划 测试以下假设：（1）代谢激活 铬（VI）导致“反应性中间体”产生 特定的DNA病变； （2）特定的DNA病变发生在定义 由于其DNA而优先攻击的DNA序列 结构; （3）特定铬（VI）诱导的DNA病变 通过改变DNA蛋白来影响DNA的正常模板活性 互动。 以下方法，包括体外和 体内实验系统将用于攻击此问题： （1）在该期间产生的铬（V）和自由基中间体 氧化还原活性细胞成分对铬（VI）的代谢激活 将确定。 EPR光谱法将用于检测铬（V） 物种和自旋陷阱将用于检测单线氧和 铬反应（VI）形成的羟基和硫基自由基物种 用异种生物预处理，特别影响 各种细胞氧化还原成分和生产的变化 各种组织中的铬（V）和自由基物种将是 决定。 （2）铬（VI）诱导的DNA病变，以DNA的形式 链断裂，DNA交联，铬-DNA加合物和自由基-DNA 加合物，是由攻击f“主动中间体”形成的。 铬（VI）的代谢期间将在质粒DNA中确定 以及在体外和线粒体和核DNA中的限制片段 体内。 这些DNA的大小，构象和相关的不同 蛋白质，因此，作为铬（VI）诱导的靶标可能会有所不同 DNA病变。 铬-DNA的序列/构象特异性 将检查加合物，并将铬-DNA加合物分离出来， 表征并与核苷酸合成铬合物络合物进行了比较。 将从IN分离到铬-DNA加合物升高抗体 体外反应，将用于分析从组织中分离的DNA 在体内用铬（VI）处理的大鼠和雏鸡的胚胎。 （3） 铬诱导的DNA病变对蛋白DNA相互作用的影响将 被检查。 DNA聚合酶合成子DNA的能力 在DNA模板上存在铬-DNA加合物存在的情况下将 可以确定。 基因调节蛋白与其结合的能力 铬-DNA形成后的特定DNA识别元件 这些序列中的加合物也将被确定。 相互作用 具有DNA限制片段的LAC阻遏帽和RNA聚合酶的 包含大肠杆菌乳糖的启动子和算子序列 操纵子，以及糖皮质激素受体和金属调节的相互作用 蛋白质到包含Matallothionein的限制片段 将检查发起人。 这些研究应阐明关键 导致致癌活性的细胞途径 铬（VI）化合物。