Determining DNA Repair Capacities for Correlations with DNA Adductomes

确定 DNA 修复能力与 DNA 加合物的相关性

• 批准号: 9390162
• 负责人: Nicholas E Geacintov
• 金额: $ 27.74万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9390162
• 关键词: Air; Base Excision Repairs; Biological; Biological Assay; Biological Markers; Biological Models; Bypass; Cancer Etiology; Cancer Family; Cell Extracts; Cell Survival; Cells; Chemicals; Coupled; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA Sequence; DNA biosynthesis; DNA lesion; Data; Detection; Development; Diet; Disease; Environment; Environmental Carcinogens; Environmental Risk Factor; Excision; Excision Repair; Experimental Designs; Exposure to; Fibroblasts; Genetic Materials; Genetic Transcription; Genome; Health; Human; Human Genome; In Vitro; Individual; Industrialization; Knowledge; Label; Laboratories; Lead; Lesion; Libraries; Liquid Chromatography; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Methodology; Methods; Monitor; Mutation; Nature; Nucleotide Excision Repair; Nucleotides; Oligonucleotides; Oncogenes; Pathway interactions; Pattern; Plasmid Cloning Vector; Play; Poison; Polymerase; Reproducibility; Research; Resistance; Risk; Risk Assessment; Role; Series; Site; Societies; Structure; System; Techniques; Technology; Testing; Time; Tissues; Transcription-Coupled Repair; Transfection; Tumor Suppressor Genes; Water; Workplace; adduct; cancer initiation; carcinogenesis; chemical binding; epidemiology study; experimental study; exposed human population; genotoxicity; homologous recombination; human DNA; improved; in vivo; insight; method development; novel; repaired; tandem mass spectrometry

Epidemiological studies suggest that a significant fraction of human cancers in industrialized societies are induced by environmental factors such as genotoxic chemicals that are ubiquitous in urban environments. Rapid developments in the field of mass spectrometry promise to provide more and more extensive human DNA adductomics maps. However, for a deeper appreciation of the significance of the DNA adductomics results, it is important to establish which of these DNA adducts are a greater threat to human health than others. The first and critically important cellular defense against genotoxic DNA adducts is cellular DNA repair. Extensive experiments in human cell extracts in vitro have shown that there is a wide range of DNA adducts that persist in human cells and tissues for long periods of time, while others disappear more quickly. Research on the DNA repair capacities (DRC) of different isomeric and other forms of the same family of cancer-causing DNA adducts has shown that there are significant differences in DNA repair capacities in human cell extract model systems in vitro. There are classes of adducts that resist DNA repair entirely, and are therefore considered to be the most genotoxic ones. However, it is not known whether the results obtained in cell extract experiments in vitro carry over to the cellular environment. Therefore, In this project, the development of methods are proposed for measuring the repair of chemically defined DNA adducts in cellular environments in intact cells. Methods will be developed that will differentiate the repair of different DNA adducts by the three major human DNA repair systems: Nucleotide Excision Repair (NER), Transcription Coupled Repair (TCR) and Base Excision Repair (BER). Previous efforts to monitor these pathways in a unified manner using the same DNA substrates, were hindered by difficulties in recovering the products of the different DNA repair mechanisms from intact cells. Preliminary results show that this approach is now feasible using 32P-labeling techniques. In this project, plasmid vectors containing single chemically and structurally defined DNA lesions belonging to different classes of DNA adducts will be transfected into single cells, and the repair of these lesions will be monitored as a function of time. The results from the proposed DNA repair capacity experiments will ultimately provide new information for assessing the significance of DNA adductomic patterns. Another objective is to improve the present biomarker technologies that simply document the presence of DNA lesions, but do not provide critical information about an individual’s capacity to remove that damage from the genome. An accurate knowledge of individual DNA repair capacities will be useful for assessing the risk of such individuals for developing cancers associated with exposure to environmental carcinogens. In summary, the biological significance of DNA adductomics data for evaluating the risk to human health due to exposure to toxic chemicals in the environment or work place, would be markedly enhanced by a knowledge of the cellular DRC of the different DNA adducts.

流行病学研究表明，在工业化社会中，相当一部分人类癌症是 由环境因素引起的，例如在城市环境中普遍存在的遗传毒性化学物质。 质谱学领域的快速发展有望为人类提供越来越广泛的 DNA内收分离图谱。然而，为了更深入地了解DNA加合物的意义 结果，重要的是要确定这些DNA加合物中哪些对人类健康的威胁比 其他。针对遗传毒性DNA加合物的第一个也是至关重要的细胞防御是细胞DNA修复。 在体外对人类细胞提取物进行的广泛实验表明，存在着广泛的DNA加合物。 它们在人体细胞和组织中持续很长时间，而其他的则消失得更快。研究 同一致癌家族不同异构体及其他形式DNA修复能力的研究 DNA加合物表明，人类细胞提取物中的DNA修复能力存在显著差异 体外模型系统。有几类加合物完全抵抗DNA修复，因此 被认为是基因毒性最强的一种。然而，目前尚不清楚在细胞中获得的结果是否 体外提取实验延续到细胞环境中。因此，在这个项目中，开发 提出了在细胞环境中测量化学定义的DNA加合物修复的方法 在完好无损的细胞中。将开发出区分三种不同DNA加合物修复的方法 人类主要DNA修复系统：核苷酸切除修复(NER)、转录偶联修复(TCR) 和碱基切除修复(BER)。以统一的方式使用 相同的DNA底物，由于难以回收不同DNA修复的产物而受到阻碍 来自完整细胞的机制。初步结果表明，这种方法现在使用32P标记是可行的。 技巧。在这个项目中，含有单个化学和结构定义的DNA损伤的质粒载体 属于不同类别的DNA加合物将被转染到单个细胞中，并修复这些 损伤将作为时间的函数进行监测。根据拟议的DNA修复能力得出的结果 实验最终将为评估DNA内收切除法的意义提供新的信息 模式。另一个目标是改进目前的生物标记技术，这些技术简单地记录 DNA损伤的存在，但不提供关于个人移除该损伤的能力的关键信息 基因组造成的损害。对个体DNA修复能力的准确了解将有助于 评估这些人患与环境暴露有关的癌症的风险 致癌物质。综上所述，DNA加合物数据对于评估发病风险的生物学意义。 由于暴露在环境或工作场所的有毒化学物质中，人体健康将显著 通过了解不同DNA加合物的细胞DRC而得到加强。