1,3-丁二烯致细胞核质桥形成的DNA甲基化作用机制研究

1,3-butadiene(BD) is an important environmental pollutant and industrial chemical with occupationally exposed subjects worldwidely. It was classified as a human carcinogen in 2007 by the International Agency for Research on Cancer. So it is critical to verify the biomarkers which can reflect the harmful effects of BD in exposed populations to prevent the BD-related cancer. Our previous study in China showed that the frequency of nucleoplasmic bridge (NPB) increased significantly in BD-exposed workers when compared with the subjects in control group, suggesting that NPB may be a sensitive indicator for the genetic damage induced by BD. However the progress and molecular mechanisms underlying the formation of NPB induced by BD has not been elucidated. Our previous results also displayed that the frequcies of micronucleus and NPB increased simultaneously in BD-exposed group, and there were positive relationship between these genotoxic indexes. Combined with the theory of NPB origin, it can be concluded that BD induces NPB by causing the DNA double strands breaks and DNA recombination repair system should play an important role in the progress of NPB formation. On the other hand,the latest report showed that BD was also an epigenetic toxicant with extensive demethylation on global DNA and DNA repetitive elements in mice after short-term inhalational exposure to low dose of BD. Since DNA methylation modification is one of the important mechanisms of expressional regulation for genes in DNA recombination systems, the following scientific hypothesis can be acquired: BD may cause the dysregulation of genes in DNA recombination systems by inducing DNA methylation modification, then DNA double strands breaks indcued by BD is prone to be repaired by the way of non-homologouse end-joining, which will lead to the extra formation of NPB. Based on the hypothesis, this research is planned to verify the dynamic progress from DNA double strands breaks to formation of NPB induced by BD firstly. Then in vitro and in vivo tests will be conducted to screen the target genes in DNA recombination system which are dysregulated by the way of DNA methylation modification, and to verify the effects on the formation of NPB induced by BD. Finally epidemiologic study will conducted to clarify the relationship between BD exposure with genetic damage and epigenetic damage in BD occupationally exposed workers in China. This research will be helpful to explain the molecular mechanisms of genotoxicity induced by BD, and to provide the evidence of NPB as a sensitive indicator of reflecting the genetic damage in BD-exposed populations.

1,3-丁二烯（BD）是重要环境污染物和确定人类致癌物。前期研究发现BD职业接触人群遗传损伤指标核质桥（NPB）率显著升高，但BD诱导NPB形成的机制不明。前期研究及NPB形成方式提示，BD诱导的NPB来源于DNA双链断裂后的DNA重组修复过程。最新报道表明BD可引起显著的DNA甲基化改变，由于甲基化修饰是DNA重组修复系统基因表达调控的重要机制，由此提出：BD通过对DNA重组修复系统关键基因的甲基化调控，引起该系统功能异常，使BD所致的DNA双链断裂倾向于发生易错修复，导致NPB过多生成。项目拟在确定BD诱导NPB形成的动力学过程基础上，揭示DNA重组修复系统关键基因在BD致NPB形成中的甲基化调控机制，进一步在职业接触人群中验证BD接触与遗传损伤和表观遗传损伤的反应关系。综合分析阐明BD诱导NPB形成的DNA甲基化作用机制。为深入阐释BD遗传毒性机理提供科学依据。

1,3-丁二烯（1, 3-butadiene, BD）是重要的环境污染物和人类致癌物。既往研究发现BD职业暴露人群中染色体损伤标志核质桥（nucleoplasmic bridge, NPB）比率显著升高，但BD诱导NPB形成的机制不明。本项目提出BD通过影响DNA重组修复系统的基因调控，导致BD诱导的DNA双链断裂倾向于非同源末端连接的易错修复方式，诱导NPB形成。同时BD可能通过DNA甲基化修饰影响染色体损伤的发生。基于这一科学假设，本项目围绕BD及其活性代谢产物1,2:3,4-二环氧丁烯（DEB）的遗传损伤及其分子机制，从以下三个方面开展了研究：1、建立DEB染毒的GC-2和TK6细胞模型，研究DEB致NPB形成的细胞动力学过程并分析其关键环节。发现DEB诱导细胞DNA双链断裂，同时诱导微核、核质桥以及核芽突等染色体损伤。证实DEB通过Chk1通路诱导细胞周期G2期阻滞，同时DEB诱导的ROS升高介导了细胞周期阻滞和DNA损伤。2、研究DNA 双链断裂（DSB）修复机制在DEB所致染色体损伤中的作用。发现DEB诱导细胞DSB重组修复失衡，包括提高非同源末端连接（NHEJ）修复效率，降低同源重组（HR）修复效率。影响DSB修复相关基因表达，包括降低HR相关基因BRCA1表达，提高NHEJ相关基因DNA-PKcs, XLF等表达，提高NHEJ相关基因Ku80与DNA的结合。发现抑制NHEJ修复可降低DEB诱导的NPB形成。3、研究BD对基因组甲基化的影响以及甲基化改变在BD所致染色体损伤中的作用。发现DEB和BD可导致TK6细胞和小鼠基因组低甲基化并抑制DNA甲基转移酶表达；发现提高DNA甲基化水平可降低DEB 和BD诱导的细胞染色体损伤。BD职业接触人群中，亚甲基四氢叶酸还原酶基因（MTHFR）多态性与染色体损伤相关，提示催化甲基供体形成的关键酶基因的突变可影响BD所致的染色体损伤。通过研究，证实了BD诱导DNA双链断裂致NPB形成的动力学过程，揭示了DNA重组修复系统关键基因在BD致NPB形成中的作用，以及DNA甲基化调控在BD遗传损伤中的作用。这些研究结果为阐明NPB的形成机制，以及将其用于BD接触人群遗传损伤危险度评估的生物标志物提供了科学依据。