Particulate Cr(VI) Toxicology in Human Lung Epithelial Cells and Fibroblasts

描述(由申请人提供)：六价铬(Cr(VI))，也称为铬酸盐，是一个主要的公共卫生问题。铬酸盐，特别是不溶性化合物，是公认的人类肺癌致癌物。我们的研究重点是探讨目前尚不清楚的铬(VI)致癌机制。最近的研究表明，微粒铬(VI)会导致染色体不稳定，这是人类肺癌的一个特征。然而，铬(VI)是如何诱导染色体不稳定的，目前还知之甚少。我们的初步数据显示，长期暴露在微粒铬(VI)中会导致DNA修复缺陷的表型，这是染色体不稳定的基础，因此，本研究的目标是表征这种修复缺陷及其影响，并了解导致它发生的机制。我们将检验这一假设，即颗粒铬(VI)诱导表观遗传变化，使铬(VI)诱导的DNA双链断裂的同源重组修复失活，从而增加染色体不稳定性和致癌作用。我们将通过四个相互关联的具体目标来检验这一假设。目的1利用免疫印迹、免疫荧光和修复试验，鉴定同源重组修复缺陷，并显示颗粒铬(VI)导致细胞失活RAD51介导的反应，并切换到低保真的非同源末端连接(NHEJ)修复。目的2利用细胞遗传学技术和接触非抑制生长和贴壁非抑制生长分析，显示颗粒铬(VI)失活RAD51反应的细胞获得染色体不稳定性，并经历肿瘤转化。目的3将通过乙酰化状态、基因表达以及蛋白质相互作用和定位的分析，通过影响E2F1、ATM、Rad51C和RAD51来识别导致RAD51反应丧失的表观遗传变化(乙酰化增加)。最后， 目的4将通过乙酰化和组蛋白脱乙酰酶活性的测定来研究颗粒铬(VI)与乙酰基的结合来表征微粒铬(VI)引起的乙酰化变化的机制。每个目标都将集中在人类肺细胞上，并确认在人类铬(VI)肿瘤中的关键发现。这些结果将导致有关铬(VI)与DNA双链断裂机制相互作用、乙酰化状态的详细信息的首次报道，以及暴露于铬(VI)工人的肿瘤中这些方面的首次特征。这项研究具有重要意义，因为它将提供：1)对微粒铬(VI)S致癌机制的了解；2)更好地评估接触微粒风险的基本信息；以及3)进一步研究铬(VI)、其他金属和一般肺癌的机制方法。

DESCRIPTION (provided by applicant): Hexavalent chromium (Cr(VI)), also known as chromate, is a major public health concern. Chromates, particularly the insoluble compounds, are well-established human lung carcinogens. Our study focuses on investigating the mechanisms of Cr(VI)-induced carcinogenesis, which are currently unknown. Recent studies indicate that particulate Cr(VI) induces chromosome instability, which is a hallmark of human lung tumors. However, how Cr(VI) induces chromosome instability is poorly understood. Our preliminary data show that chronic exposure to particulate Cr(VI) induces a DNA repair deficient phenotype that underlies the chromosome instability, and thus, the goal of this research is to characterize this repair deficiency and its impact and understand the mechanisms that cause it to occur. We will test the hypothesis that particulate Cr(VI) induces epigenetic changes inactivating homologous recombination repair of Cr(VI)-induced DNA double strand breaks resulting in increased chromosome instability and carcinogenesis. We will test this hypothesis through four interrelated specific aims. Aim 1 will characterize the homologous recombination repair defect and show that particulate Cr(VI) causes cells to inactivate Rad51-mediated response and switch to lower fidelity non-homologous end joining (NHEJ) repair using immunoblotting, immunofluorescence and repair assays. Aim 2 will show that the cells with particulate Cr(VI)-inactivated Rad51 response acquire chromosome instability and undergo neoplastic transformation using cytogenetic techniques and assays for contact- uninhibited and anchorage independent growth. Aim 3 will identify an epigenetic change (increased acetylation) that causes the loss of Rad51 response by affecting E2F1, ATM, Rad51C and Rad51 with assays for acetylation status, gene expression, and protein interactions and localization. Finally, Aim 4 will characterize the mechanism for particulate Cr(VI)-induced acetylation changes studying Cr(VI) binding to acetyl groups using assays for acetylation and histone deacetylase activity. Each aim will focus on human lung cells and confirm key findings in human Cr(VI) tumors. Results will lead to the first reports of detailed information of the interactions of Cr(VI with DNA double strand break machinery, acetylation status and the first characterizations of these aspects in tumors from Cr(VI)-exposed workers. This research is significant because it will provide: 1) An understanding of particulate Cr(VI)'s carcinogenic mechanism; 2) Essential information to better assess the risk of exposure to particulates; and 3) A mechanistic approach for further study of Cr(VI), other metals, and lung cancer in general.