组蛋白修饰介导职业暴露多环芳烃早期损伤效应机制研究

The occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is one of the most important causes of lung cancer. Elucidating the underlying mechanism would be meaningful for selection of effective early monitoring biomarkers. Our previous study indicated that histone modifications were changed when the cells were exposed to PAHs, and the alternations may play important roles in early biological effects. In order to further explore the relationship between them, we intend to study the function and action mechanism of histone modification in the early health effect of PAHs exposure in occupational population and in vitro cells. By measuring the levels of PAHs in working environment and biomarkers of PAHs exposure in human biological samples, we will obtain the internal and external exposure levels of PAHs, and the genotoxic and oxidative damage effect of PAHs, which were identified as early damages induced by PAHs exposure. We intend to detect the acetylation and methylation of histone H3 lysine sites which are screened from in vitro assays, then detect the alterations in lymphocytes of occupational population by means of ELISA, finding the correlation between histone modifications and the PAHs exposure or early biological effects. Then we will study the function of histone modification in DNA damage repair and oxidative stress induced by PAHs exposure using the histone modification defect cell lines. Also we will study the action mechanism of hisone modification by ChIP-seq and mRNA sequencing, which will find the critical genes that be regulated in the process. This study will shed light on the mechanism of PAHs toxicity from a new angle, and provide important information for using critical histone modifications as new biomarkers of PAHs exposure and/or early damage effects.

职业环境多环芳烃（polycyclic aromatic hydrocarbons, PAHs）暴露是导致肺癌的重要因素之一，阐明其损伤效应机制有助于筛选早期敏感生物标志物。我们前期研究发现PAHs暴露可引起组蛋白修饰改变并具有生物学意义。为进一步探明两者的关联，拟采用PAHs职业暴露人群分子流行病学和体外细胞机制研究，探讨组蛋白修饰在PAHs早期损伤效应中的功能和机制。用体外细胞实验筛选PAHs暴露时组蛋白H3的修饰改变，并检测其在人群淋巴细胞中的状态，结合PAHs暴露水平，以及PAHs暴露早期效应DNA损伤和氧化损伤指标，明确组蛋白修饰改变与PAHs暴露及早期效应的关联。构建特异组蛋白修饰缺陷细胞模型，探讨其在早期效应中的功能；通过ChIP-seq、mRNA测序及功能分析获得关键性调控基因。本研究从新的视角揭示PAHs暴露的毒性机制，为寻找新型早期损伤生物标志物提供科学依据。

职业环境多环芳烃(polycyclic aromatic hydrocarbons,PAHs) 暴露是导致肺癌的重要因素之一，阐明其损伤作用机制有助于筛选早期敏感生物标志。本项目以本溪钢铁公司焦炉车间为研究现场，募集了190例焦化厂男性工人和100例对照男性工人并进行了PAHs内暴露(尿1-OHP)、DNA损伤和特异组蛋白修饰检测。发现尿1-OHP水平与空气PAHs浓度趋势一致（炉顶>炉侧>炉底>对照环境），且1-OHP与DNA损伤、外周血淋巴细胞H3K36me3水平均存在正相关关系，提示H3K36me3可作为PAHs暴露标志。根据体外实验结果，检测了人群外周血淋巴细胞特异组蛋白修饰，发现暴露组H3K27/36me3升高，H3K4me3则降低，其中H3K4me3和H3K36me3与DNA损伤呈负相关关系。ChIP-qPCR结果显示MGMT和MLH1基因编码区序列富集在H3K36me3区域，且基因调控有时间—效应关系；通过DSB修复模型发现36位点突变可导致DSB修复延迟，且MGMT和MLH1的表达水平下降，说明H3K36me3通过参与调控MGMT和MLH1基因表达而介导PAHs引起的DNA损伤。此外，我们构建了上述特异位点突变细胞株，发现染色体不稳定性增加、本底微核率升高、36位点突变细胞生长加速，并且H3K4/9/36me3在恶性转化过程中明显改变，其中H3.1K36、H3.3K4/36位点突变都可以促进软琼脂克隆形成。上述结果说明特异组蛋白位点突变可影响染色质疏松状态、增加染色体不稳定性、改变细胞生长特征、影响其锚着独立生长特性，证实了组蛋白修饰在DNA损伤修复和PAHs致癌中发挥作用。此外，我们还发现低剂量BaP暴露可通过H3K18ac和H3K27ac对AhR介导的主要代谢通路进行调控，影响BaP暴露引起的DNA损伤和细胞增殖等早期毒性效应；p-H3S10和p-H3S28则通过调控DNA损伤修复基因表达在致癌过程中发挥重要作用，该部分结果从代谢和磷酸化修饰角度更加全面的对组蛋白修饰在PAHs诱导的早期损伤中的作用和机制进行了阐释。本课题明确了H3K36me3、H3K18ac、H3K27ac、p-H3S10和p-H3S28在PAHs致早期损伤中的作用及机制，从新的视角揭示了PAHs暴露的毒性机理，为组蛋白修在职业暴露人群健康监测中的应用提供了理论依据。