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，人类癌症的很大一部分是由癌症引起的。 由环境因素引起的，如在城市环境中普遍存在的遗传毒性化学品。 质谱领域的快速发展有望为人类提供越来越广泛的信息。 DNA内收酶图谱。然而，为了更深入地理解DNA内收体学的重要性， 因此，重要的是要确定这些DNA加合物中哪一种对人类健康的威胁比 他人第一个和至关重要的细胞防御基因毒性DNA加合物是细胞DNA修复。 在体外对人类细胞提取物进行的大量实验表明，DNA加合物的范围很广 在人体细胞和组织中存在很长一段时间，而其他人消失得更快。研究 关于同一致癌家族的不同同分异构体和其他形式的DNA修复能力（DRC） DNA加合物已经表明，在人类细胞提取物中，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， 被认为是最具遗传毒性的。然而，尚不清楚细胞中获得的结果是否 体外提取物实验中残留到细胞环境中。因此，在本项目中，开发 提出了一种方法来测量细胞环境中化学定义的DNA加合物的修复 在完整的细胞中。方法将被开发，将区分不同的DNA加合物的修复由三个 主要的人类DNA修复系统：核苷酸切除修复（NER），转录偶联修复（TCR） 基底切除修复术（BER）以前的努力，以统一的方式监测这些途径，使用 相同的DNA底物，在回收不同的DNA修复产物的困难， 完整细胞的机制。初步结果表明，这种方法现在是可行的，使用32P标记 技术.在这个项目中，质粒载体含有单一的化学和结构定义的DNA损伤， 属于不同类别的DNA加合物将被转染到单细胞中，并且这些加合物的修复将在细胞中进行。 病变将作为时间的函数进行监测。DNA修复能力的结果 实验将最终为评估DNA内收体的重要性提供新的信息， 模式.另一个目的是改进目前的生物标志物技术，该技术简单地记录了生物标志物。 DNA损伤的存在，但不能提供关于个体去除DNA损伤的能力的关键信息。 来自基因组的伤害对个体DNA修复能力的准确了解将有助于 评估这些人患上与暴露于环境污染有关的癌症的风险， 致癌物质。总而言之，DNA内收体数据对于评估以下风险的生物学意义是： 由于暴露于环境或工作场所的有毒化学品， 通过对不同DNA加合物的细胞DRC的了解来增强。