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

人肺上皮细胞和成纤维细胞中的颗粒 Cr(VI) 毒理学

• 批准号: 8578064
• 负责人: John Pierce Wise
• 金额: $ 33.33万
• 依托单位: UNIVERSITY OF SOUTHERN MAINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-06 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8578064
• 关键词: Acetylation; Affect; Anchorage-Independent Growth; Binding; Biological Assay; Cancer Etiology; Carcinogen exposure; Carcinogens; Cells; Chromates; Chromium Compounds; Chromosomal Instability; Chronic; Cytogenetic Analysis; Cytoplasm; DNA Double Strand Break; DNA Repair; Data; Deacetylation; Defect; Double Strand Break Repair; E2F1 gene; Environmental and Occupational Exposure; Epigenetic Process; Epithelial Cells; Event; Exposure to; Fibroblasts; Frequencies; Gene Expression; Genomic Instability; Goals; Histone Deacetylase; Human; Immunoblotting; Immunofluorescence Immunologic; In Vitro; Ions; Lead; Lung; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Mediating; Metals; Modeling; Molecular; Mutation; Neoplastic Cell Transformation; Nonhomologous DNA End Joining; Nuclear; Particulate; Peptide Mapping; Phenotype; Proteins; Public Health; Publishing; Reporting; Research; Risk; Salts; Solubility; Spectral Karyotyping; System; Techniques; Testing; Toxicology; Zinc; carcinogenesis; chromium hexavalent ion; homologous recombination; interest; prevent; public health relevance; recombinational repair; repaired; response; tandem mass spectrometry; tumor; zinc chromate

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.

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，是公认的人类肺部致癌物。我们的研究重点是调查铬（VI）诱导的致癌机制，这是目前未知的。最近的研究表明，颗粒铬（VI）诱导染色体不稳定性，这是人类肺肿瘤的标志。然而，铬（VI）如何诱导染色体不稳定性知之甚少。我们的初步数据表明，慢性暴露于颗粒铬（VI）诱导的DNA修复缺陷的表型，染色体不稳定性的基础，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。我们将测试的假设，颗粒铬（VI）诱导表观遗传变化失活同源重组修复铬（VI）诱导的DNA双链断裂，导致染色体不稳定性增加和致癌作用。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将表征同源重组修复缺陷，并使用免疫印迹、免疫荧光和修复测定显示颗粒Cr（VI）导致细胞抑制Rad 51介导的反应并切换到较低保真度的非同源末端连接（NHEJ）修复。目的2将显示具有颗粒状Cr（VI）失活的Rad 51响应的细胞获得染色体不稳定性，并使用细胞遗传学技术和接触非抑制和锚定非依赖性生长的测定进行肿瘤转化。目的3将通过乙酰化状态、基因表达和蛋白质相互作用和定位的测定来鉴定通过影响E2 F1、ATM、Rad 51 C和Rad 51而导致Rad 51应答丧失的表观遗传变化（乙酰化增加）。最后， 目的4将表征颗粒铬（VI）诱导的乙酰化变化的机制，研究铬（VI）结合乙酰基使用乙酰化和组蛋白脱乙酰酶活性测定。每个目标都将集中在人类肺细胞上，并确认人类Cr（VI）肿瘤的关键发现。结果将导致第一份关于Cr（VI）与DNA双链断裂机制相互作用、乙酰化状态的详细信息的报告，以及暴露于Cr（VI）的工人的肿瘤中这些方面的首次表征。这项研究是重要的，因为它将提供：1）颗粒铬（VI）的致癌机制的理解; 2）基本信息，以更好地评估暴露于颗粒的风险;和3）的机制方法，为进一步研究铬（VI），其他金属，和肺癌的一般。