Arsenic co-carcinogenesis with UVR: nitrosation and oxidation of target proteins

描述（由申请人提供）：人类通过饮用水广泛接触砷，其水平超过环境保护署和世界卫生组织的最低污染物水平10微克/升，这是一个国家和国际问题。人们越来越认识到，低浓度和无细胞毒性的砷可以放大其他遗传毒性物质（如紫外线辐射）的DNA损伤和致癌潜力，至少部分是通过抑制DNA修复过程。砷抑制DNA修复靶蛋白的机制对于理解砷的致癌和共致癌潜力以及确定逆转或预防人群中砷暴露的不良影响的途径至关重要。目前的项目将测试砷产生的活性氧和氮物质通过与锌指结构域的氧化还原敏感的半胱氨酸残基反应来抑制锌指DNA修复蛋白的活性的假设。在目的1中，我们将研究砷介导的诱导型一氧化氮合酶和NADPH氧化酶（NOX）的诱导和随后的一氧化氮和超氧化物的产生对两种DNA修复蛋白（XPA和PARP-1）的活性，DNA修复和角质形成细胞的遗传毒性的影响。iNOS和NOX的遗传和药理学破坏将确定砷介导的DNA修复抑制中涉及的途径。目标2将研究砷产生的活性氧和氮物质与XPA和PARP-1的锌指的相互作用，并应用分析技术来定义锌指结构域的特定修饰和锌结合的后果。我们的实验室和其他实验室产生的数据表明砷与锌指结构结合的选择性，我们发现砷结合而不是锌结合的锌指肽非常容易氧化。我们将测试砷与锌指的结合是否转化为靶向氧化和亚硝化修饰以及锌指蛋白功能的丧失。在目标3中，我们将使用遗传模型测试砷和紫外线辐射在体内DNA损伤和皮肤肿瘤发生中的协同作用对iNOS和NOX的依赖性。因此，本项目使用多方面的方法严格测试砷抑制关键DNA修复靶蛋白的机制。这些研究的结果将提高我们对砷破坏锌指DNA修复蛋白功能的机制的理解，并可能对砷暴露人群的治疗或预防干预产生重大影响。此外，这些研究可能会导致关于癌症和其他砷相关疾病的潜在砷靶点的可验证假设。

DESCRIPTION (provided by applicant): Widespread human exposure to arsenic through drinking water at levels in excess of the Environmental Protection Agency and World Health Organization minimum contaminant level of 10 �g/L is a national and international concern. It is becoming increasingly appreciated that low and non-cytotoxic concentrations of arsenic can amplify the DNA damaging and carcinogenic potential of other genotoxic agents such as ultraviolet radiation, at least in part, through inhibition of DNA repair processes. The mechanisms by which arsenic inhibits DNA repair target proteins is central to understanding the carcinogenic and co-carcinogenic potential of arsenic and to identify avenues to reverse or prevent the adverse effects of arsenic exposure in human populations. The current project will test the hypothesis that arsenic-generated reactive oxygen and nitrogen species inhibits the activity of zinc finger DNA repair proteins through reaction with redox-sensitive cysteine residues of the zinc finger domains. In Aim 1 we will investigate the impact of arsenic-mediated iNOS and NADPH oxidase (NOX) induction and subsequent nitric oxide and superoxide generation on the activity of two DNA repair proteins (XPA and PARP-1), DNA repair and genotoxicity in keratinocytes. Genetic and pharmacologic disruption of iNOS and NOX will define which pathway(s) are involved in arsenic-mediated inhibition of DNA repair. Aim 2 will investigate the interaction of arsenic-generated reactive oxygen and nitrogen species with the zinc fingers of XPA and PARP-1 and apply analytical techniques to define specific modifications of the zinc finger domain and consequences with regard to zinc binding. Data generated by our laboratories and others indicate selectivity for arsenic binding to zinc finger structures and we find that arsenic- bound, but not zinc bound, zinc finger peptide is highly vulnerable to oxidation We will test whether arsenic binding to a zinc finger translates to targeted oxidative and nitrosative modification and loss of zinc finger protein function. In Aim 3, we will test the iNOS and NOX dependence for the reported synergism between arsenic and ultraviolet radiation in DNA damage and skin tumorigenesis in vivo using genetic models. Thus, this project rigorously tests mechanisms of arsenic inhibition of key DNA repair target proteins using a multi- faceted approach. The outcomes from these studies will improve our understanding of mechanisms underlying arsenic disruption of zinc finger DNA repair protein function and may have significant impact on treatments or preventative interventions for arsenic exposed populations. Additionally, these studies may lead to testable hypotheses regarding potential arsenic targets in cancer and other arsenic-associated diseases.