DNA甲基化及其关联基因突变对苯作业工人遗传损伤的调控

Benzene (BZ) is a well-known established human carcinogen. With huge crowd of occupationally BZ-exposed workers in China, it is of great theoretical and practical significance to seek for genetic damage and DNA repair capacity indicators for the prevention and control of BZ-induced occupational health hazard. Our previous studies have shown that BZ-exposed workers have significantly elevated MN frequencies compared with the controls under the current occupational health standard. Recent studies have confirmed that genetic damage, DNA methylation in promotor region and mutations in DNA methylation related genes are both tightly associated with leukemia. In this proposal, the cytokinesis-block micronuclei (CBMN) assay will be used to measure the genetic damage and the CBMN-based 3AB method is developed to detect the DNA repair capacity of BZ-exposed workers. Additionally, we project to perform genome-wide methylation profiling using chip technology, and detect the mutaion in DNA methylation related genes by second-generation sequencing and PCR-RFLP, seeking for the potential biomarkers. Based on exposure assesment among benzene-exposed workers, we explore the potential relationship between DNA methylation, mutaion in DNA methylation related genes and genetic damage as well as its repair capacity. This study will help to reveal the molecular mechanisms of BZ-induced genetic damage. The results from our study can be used as scientfic references in revising the BZ occupational health standard and improving occupational health surveillance program for BZ-exposed workers and other carcinogen-exposed population.

苯是确定的人类致癌物。我国纯苯产量大,接触工人众多。本课题组近年研究表明，在当前职业卫生标准下，苯作业工人的微核率较对照组显著增加。最近研究证实DNA甲基化与肿瘤发生密切相关，同时DNA甲基化关联基因调控着机体DNA甲基化的发生和清除，甲基化关联基因存在大量突变，某些突变导致酶活性改变，继而引起DNA甲基化改变和影响蛋白表达，进而影响机体生命活动。本项目通过苯作业工人的接触评估,应用胞质分裂阻滞微核（CB微核)和基于CB微核的3AB指数分别检测苯作业工人外周血淋巴细胞的遗传损伤及其修复能力，确定苯致血液损害和遗传损伤的基准剂量；应用甲基化芯片绘制全基因组启动子DNA甲基化谱；应用二代测序和PCR-RFLP检测甲基化关联基因的突变位点。探讨DNA甲基化关联基因突变与甲基化的关联，并研究其对苯致遗传损伤及其修复能力的内在联系，为修订苯职业卫生标准和完善苯作业工人健康监护方案提供科学依据。

苯是常见的工业化学物，能导致苯中毒和白血病。本项目通过职业流行病学调研和接触评估，评价苯作业工人遗传损伤和外周血DNA 总体甲基化改变的风险；采用芯片技术筛选苯中毒病例组和对照组DNA差异甲基化位点，并用焦磷酸测序验证 DNA 甲基化水平，进一步用实时荧光定量PCR对差异甲基化基因的表达状态进行验证，阐明苯接触工人的遗传损伤和甲基化水平改变，为探索慢性苯中毒的发病机制提供科学依据。.通过职业健康体检和遗传损伤检测，收集温州鞋厂工人生物样本410余例；安徽某汽车喷漆车间工人样本510余例。课题组分别采样BMDS软件和PROAST 38.9软件进行基于微核的苯的基准剂量评估，发现以双核微核为指标的遗传损伤的基准剂量远低于以白细胞计数作为效应终点，并且低于我国现行职业卫生标准。.通过对DNMT3A/3B常见突变位点检测，分析其对苯作业工人外周血DNA总体甲基化水平和遗传损伤影响，发现DNMT3A R882突变等位基因（R882H和R882C）和DNMT3B rs24909 GG等位基因与总体DNA低甲基化显著相关。其中R822突变等位基因（R882H和R882C）中的微核率高于野生型。DNA甲基化关联基因突变能够影响总体DNA甲基化水平，也与遗传损伤存在关联。.选择6例慢性苯中毒患者和6名匹配的健康对照工人，经芯片技术对全基因组甲基化位点和表达谱分析发现：病例组与对照组差异甲基化位点总数目为679个，其对应的基因共383个。将将差异甲基化基因做pathway富集分析并建立network，结果显示与苯中毒相关的pathway有PI3K-Akt信号通路，cAMP信号通路，细胞因子受体相互作用等。在PI3K-Akt信号通路中包括两个发生高甲基化差异且低表达的差异基因：CSF3R基因和CREB5基因；发生低甲基化且高表达的差异基因F2R基因，并经焦磷酸测序验证DNA 甲基化水平和扩大样本进一步用实时荧光定量PCR对差异甲基化基因的表达状态进行验证。.综上，在我国现行职业卫生标准下，苯仍能导致遗传损伤和DNA甲基化改变，DNA甲基化关联基因突变能够影响DNA甲基化水平。