Zinc Chemoprevention of Arsenic Co-Carcinogenesis

描述（由申请人提供）：有强有力的证据表明，除了已知的高砷浓度的直接致癌作用外，低砷、非致癌水平的砷与其他dna损伤剂（如苯并[a]芘和紫外线辐射（UVR））协同作用，增强致癌作用。这一观察结果导致砷被确定为一种共同致癌物。砷共同致癌的一种作用模式是抑制含有锌指DNA结合基序的DNA修复蛋白。本实验室的研究表明：1)具有e3半胱氨酸残基的锌指蛋白是亚砷酸盐结合的直接分子靶点，包括着色性干皮病A组（XPA）和聚（adp -核糖）聚合酶(PARP)-1，它们分别是核苷酸切除修复（NER）和碱基切除修复（BER）的关键蛋白；ii)低水平的亚砷酸盐会增强紫外线辐射引起的光产物和氧化性DNA损伤；iii)补充锌可以防止亚砷酸盐对PARP-1的抑制作用，增强uvr诱导的DNA损伤和突变。我们假设补充锌可以通过恢复BER和NER途径的DNA修复能力来防止亚砷酸盐增强uvr诱导的皮肤肿瘤。该研究将探讨一种新的机制，即锌可能通过与DNA修复途径中不同的分子靶点相互作用来预防砷增强性致癌。在目标1中，我们将使用多种分析方法来确定亚砷酸盐和锌与代表PARP-1和XPA锌指结构域的合成肽以及纯化蛋白的竞争相互作用。这些发现将与功能分析相结合，以确定亚砷酸盐对靶蛋白的亲和力和锌相互作用的任何已确定差异的后果。目的2将分析从处理细胞中分离的PARP-1和XPA，以测量砷和锌与蛋白质的结合及其相应的功能后果，以揭示锌逆转砷效应的具体机制。在Aim 3中，我们将通过生化方法验证PARP-1和XPA是体内砷的靶点，并验证锌可以防止砷增强uvr诱导的DNA损伤和皮肤肿瘤的假设。这些研究的结果有望证明锌恢复PARP-1和XPA活性的能力，从而提高基因组完整性，减少砷对共致癌的贡献。虽然锌主要通过其报道的一般抗氧化活性而被认为具有化学保护作用，但该建议的创新之处是关注锌阻止亚砷酸盐与特定锌指DNA修复蛋白靶点结合的新机制，从而逆转亚砷酸盐对DNA修复的抑制作用。本研究将为明确锌补充剂减少砷共致癌的机制提供坚实的基础，并为锌补充剂预防砷共致癌的潜力提供原理证明。如果得到验证，补充锌可能是砷暴露人群中成本低且易于实施的化学预防策略。

DESCRIPTION (provided by applicant): There is strong evidence that in addition to the known direct carcinogenic actions of high arsenic concentrations, low, non-carcinogenic levels of arsenic synergize with other DNA-damaging agents such as benzo[a]pyrene and ultraviolet radiation (UVR) to enhance carcinogenesis. This observation has led to the identification of arsenic as a co-carcinogen. One mode of action to account for arsenic co-carcinogenesis is inhibition of DNA repair proteins containing zinc finger DNA binding motifs. Research from our lab demonstrates that: i) zinc finger proteins with e3 cysteine residues are direct molecular targets for arsenite binding, including xeroderma pigmentosum group A (XPA) and poly(ADP-ribose)polymerase (PARP)-1, which are the critical proteins for nucleotide excision repair (NER) and base excision repair (BER) respectively; ii) low levels of arsenite enhance UVR-induced photoproducts and oxidative DNA damage; and iii) supplemental zinc prevents arsenite effects on PARP-1 inhibition, enhancement of UVR-induced DNA damage and mutations. We hypothesize that zinc supplement will prevent arsenite enhancement of UVR-induced skin tumors by restoring the DNA repair capabilities in both BER and NER pathways. The proposed study will address a novel mechanism by which zinc may act to prevent arsenic-augmented carcinogenesis through interaction with distinct molecular targets in DNA repair pathways. In Aim 1, we will use multiple analytical approaches to definitively examine the competitive interaction of arsenite and zinc with synthetic peptides representing the PARP-1 and XPA zinc finger domains and with purified protein. These findings will be coupled with functional assays to establish the consequences of any identified differences in arsenite affinity for target proteins and zinc interactions. Aim 2 will analyze PARP-1 and XPA isolated from treated cells to measure arsenic and zinc binding to protein and the corresponding functional consequences to reveal the specific mechanism by which zinc reverses the arsenic effects. In Aim 3, we will validate PARP-1 and XPA as arsenic targets in vivo using biochemical methods, and test the hypothesis that zinc will prevent arsenic enhancement of UVR-induced DNA damage and skin tumors. The outcomes from the proposed studies are expected to demonstrate the capability of zinc to restore PARP-1 and XPA activity leading to improved genomic integrity and a decrease in the arsenic contribution to co-carcinogenesis. Although zinc has been considered chemoprotective largely through its reported general antioxidant activities, this proposal is innovative by focusing on a novel mechanism by which zinc prevents arsenite binding with specific zinc finger DNA repair protein targets, thus reversing the arsenite inhibition of DNA repair. This study will provide the solid foundations for a clear mechanistic understanding of how supplemental zinc reduces arsenic co-carcinogenesis, and provide the proof of principle for the potential of zinc supplement to prevent arsenic co-carcinogenesis. If validated, supplemental zinc could represent a low cost and easily implemented strategy for chemoprevention in arsenic exposed populations.