Epigenetic effects on susceptibility to heavy metal & PAH induced DNA damage

重金属如砷、镍、铬以及多环芳烃(PAHs)是常见的环境污染物。人类接触这种毒物会极大地增加患癌症的风险。多环芳烃的致癌作用被归因于它们与DNA的共价结合，这可能导致突变，最终导致致癌。金属致癌的机制还不是很清楚。研究发现，镍和砷暴露可引起DNA甲基化。这种非突变的表观遗传学变化也可能导致肿瘤抑制基因的抑制，如p53基因，从而触发肿瘤发生。最近，我们发现C5胞嘧啶甲基化CpG位上的鸟嘌呤是许多多环芳烃的主要DNA靶标，包括苯并(A)芘二醇环氧化物(BPDE)。事实上，我们已经发现抑癌基因p53中的BPDE结合谱与该基因在肺癌中的突变谱相吻合。已经发现，突变的P53基因足以在动物模型中引发癌症发生，更重要的是，50%的人类癌症存在P53基因突变。这些发现使我们假设，靶向DNA损伤，而不是选择，是癌症中p53突变谱的主要决定因素，CpG选择是人类癌症中p53突变谱的主要决定因素，单个p53基因的CpG甲基化状态可能决定该基因对DNA损伤和突变的易感性。这些发现使我们假设，靶向DNA损伤，而不是选择，是人类癌症P53突变谱的主要决定因素，单个P53基因的CpG甲基化状态可能决定该基因对DNA损伤和突变的易感性。根据这些发现，我们认为镍、砷和铬的致癌作用可能部分归因于它们诱导DNA高甲基化的能力，这反过来又增强了甲基化的抑癌基因和原癌基因对大量致癌物引起的DNA损伤和突变的易感性。这项研究的目的是使用两项最先进的技术来验证这一假设：UvrABC连接介导的聚合酶链式反应在单核苷酸水平绘制DNA加合物的图谱，以及p53基因芯片检测突变和胞嘧啶甲基化。我们将确定：1)不同个体淋巴细胞中P53基因的甲基化状态及其与BDE损伤易感性的关系；2)镍、砷和铬是否引起P53基因甲基化状态的改变，以及镍、砷和铬是否引起P53基因甲基化状态的改变，从而改变该基因对BPDE诱导的DNA损伤的易感性；以及3)镍、砷和铬处理对P53、β-肌动蛋白和HPRT基因中BPDE-DNA加合物修复的影响。

Heavy metals such as arsenic, nickel, and chromium as well as polycyclic aromatic hydrocarbons (PAHs) as benzo(a)pyrene are common environmental contaminants. Human exposure to such toxicants greatly increases cancer risk. The carcinogenicity of PAHs has been attributed to their covalent binding to DNA, which can result in mutations that ultimately lead to carcinogenesis. The mechanisms of metal-induced carcinogenesis are less clear. It has been found that nickel and arsenic exposure can induce DNA hypermethylation. Such non-mutational epigenetic changes could also result in suppression of tumor suppressor genes, such as the p53 gene, triggering tumorigenesis. Recently, we have found that the guanines at C5 cytosine methylated CpG sites are the major DNA targets of many PAHs, including benzo(a)pyrene diol epoxide (BPDE). In fact, we have found that the BPDE binding spectrum in the tumor suppressor p53 gene coincides with the mutation spectrum of this gene in lung cancer. It has been found that a mutated p53 gene is sufficient to trigger carcinogenesis in animal models, and that more importantly, 50% of human cancers have a mutation in the p53 gene. These findings have lead us to hypothesize that targeted DNA damage, rather than selection, is the major determinant of the p53 mutation spectrum in cancer, and that the CpG selection, is the major determinant of the p53 mutation spectrum in human cancer, and that the CpG methylation status of an individual p53 gene may determine the susceptibility of this gene to DNA damage and mutation. These findings have led us to hypothesize that targeted DNA damage, rather than selection, is the major determinant of the p53 mutation spectrum in human cancer, and that the CpG methylation status of an individual p53 gene may determine the susceptibility of this gene to DNA damage and mutation. In light of these findings, we propose that the carcinogenicity of nickel, arsenic and chromium may be partly due to their ability to induce DNA hypermethylation, which in turn enhances the susceptibility of methylated tumor suppressor genes and protooncogenes to bulky carcinogen-induced DNA damage and mutations. The objective of this research is to test the hypothesis using two state-of-the-art technologies; UvrABC-ligation-mediated-PCR to map DNA adducts at the single nucleotide level, and the p53 GeneChip to detect mutations and cytosine methylation. We will determine: 1) the p53 gene methylation status in lymphocytes of different individuals and assess its relationship with the susceptibility to BDE damage, 2) whether nickel, arsenic, and chromium induce changes in the methylation status of the p53 gene and whether nickel, arsenic, and chromium induce changes in the methylation status of the p53 gene and, consequently, in the susceptibility of this gene to BPDE induced-DNA damage, and 3) the effect of nickel, arsenic and chromium treatment on the repair of BPDE-DNA adducts in the p53, beta-actin and HPRT gene.