【作者】 王艳华；

【导师】 段化伟； 郑玉新； 叶萌；

【作者基本信息】 中国疾病预防控制中心 ， 卫生毒理学， 2017， 硕士

【摘要】 焦炉逸散物(Coke oven emissions,COE)是焦化生产过程中的主要排放污染物,其中含有大量的颗粒物(Particulate matter,PM)和多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)。流行病学研究表明,高浓度的颗粒物或多环芳烃暴露是呼吸系统疾病发生发展的主要原因之一,其中COE所致肺癌被列为明确的职业性肿瘤。细颗粒物(Fine particulate matter,PM2.5)由于具有小于2.5μμm的空气动力学直径和易于富集有毒有害物质的表面,对呼吸系统的损害效应也更加明确。但在COE暴露与效应标志物的剂量-反应关系研究中,以致癌性比较明确的PAHs为主要目标污染物开展的较多,还缺少对于其中PM2.5健康效应的暴露-反应关系的分析。虽然,流行病学研究已经证实职业COE暴露与焦炉工人罹患肺癌间的因果联系,但从暴露到肺癌发生过程中的重要生物学改变和早期不良健康效应的标志物研究还很有限。研究发现,循环系统中多种蛋白质表达的变化与维持呼吸系统稳态密切相关,包括肺表面活性蛋白 A(Surfactant protein A,SP-A)、肺表面活性蛋白 D(Surfactant protein D,SP-D)和克拉拉细胞蛋白(Club cell protein,CC16)等。研究人员已经在临床科研工作中开展了多项肺损伤蛋白与慢性阻塞性肺病等呼吸疾病发生和进展的研究。但是,关于COE致血液中肺损伤相关标志物改变的研究还未见报道。颗粒物和PAHs暴露可导致多种疾病的发病风险增加,尤其是在致癌过程中,表观遗传学改变可能发挥了重要作用。表观遗传不涉及DNA序列的改变,具有可逆性等特点。DNA甲基化是重要的表观遗传修饰之一。我们以及其他研究团队在早期研究中发现PAHs暴露可以导致多个DNA修复基因和抑癌基因的甲基化异常改变。但对基因组整体水平的甲基化研究报道较少,目前也缺乏PM2.5致机体基因组DNA甲基化与DNA甲基转移酶(DNA methyltransferase,DNMTs)关系的研究。为了解释上述问题,我们在北方某焦化厂人群中开展了 COE暴露与早期损害标志物的暴露-反应关系的分子流行病学研究。首先对工作场所多个工种进行颗粒物和多环芳烃等污染物的暴露评价;然后招募558名焦炉作业工人作为暴露人群,210名氧气厂和冷轧厂工人作为对照。检测血清中机体炎症水平(hs-CRP)、肺损伤标志物(CC16,SP-A和SP-D)水平,结合肺功能变化,探讨焦炉逸散物中PM2.5和PAHs对肺部的损伤效应。观察研究对象白细胞基因组DNA甲基化水平和DNA甲基化转移酶的表达水平改变,并结合中介效应探讨DNA甲基化转移酶在COE主要成分致基因组DNA甲基化和肺损伤改变中的作用。一、焦炉逸散物致血清呼吸系统损伤标志物的改变通过称重法计算COE中的PM2.5含量,超高效液相色谱法检测颗粒相中多环芳烃的浓度。超高效液相色谱串联质谱法检测尿中1-羟基芘(1-hydroxyrene,1-OHP)的浓度,ELISA法检测血清中SP-A、SP-D和CC16的浓度,同时检测研究对象的肺功能。研究发现随着COE中PM2.5和PAHs暴露程度的增高,血清CC16呈降低趋势(P=0.046)。在全人群为基础的暴露-反应关系分析中发现,COE中PM2.5增加1个IQR(121.98 μg/m3)导致血清 CC16 降低 5.24%(P= 0.004);COE 中总 PAHs 增加 1 个IQR(3.81μg/μm3),会导致血清 CC16 降低 5.69%(P = 0.027);尿 1-OHP 每增加 1个IQR(1.06μmol/mol肌酐),会导致血清CC16降低4.67%(P= 0.041)。在男性工人中,还发现血清 CC16 每降低 1 个 IQR(3.95 ng/mL),FEVl/FVC 降低 0.86%(P=0.045)。二、焦炉逸散物致基因组DNA甲基化改变采用ELISA法检测基因组DNA甲基化(5-mC%)的含量,实时荧光定量PCR(RT-qPCR)法进行DNMTs的mRNA定量分析,同时检测研究对象血清中的叶酸和维生素B12水平。研究发现,全人群中,随着COE暴露水平的增加,基因组DNA甲基化(5-mC%)水平呈显著的下降趋势(P = 0.001)。暴露-反应关系分析发现,COE中PM2.5增加一个IQR(121.98 μg/m3),会引起基因组 DNA 甲基化(5-mC%)降低 5.78%(P<0.001);总PAHs每增加一个IQR(3.81 μg/m3),导致基因组DNA甲基化降低6.09%(P=0.007)。研究发现尿1-OHP与基因组DNA的5-mC含量呈显著的负相关(β=-0.025,P<0.001)。DNA甲基化转移酶的定量研究发现,随着COE暴露程度的增加,DNMT3A基因的mRNA表达水平呈下降趋势(P = 0.047);其中总PAHs每增加一个IQR(3.81 μg/m3),DNMT3A表达量降低13.25%(P= 0.027),没有发现DNMT1表达量的显著改变。回归分析显示基因组DNA甲基化(5-mC%)与DNMT1和DNMT3A均存在显著的正相关。中介效应分析发现PAHs暴露对基因组DNA甲基化效应的5.95%可以被DNMT3A所介导。三、焦炉逸散物致肺损伤与DNA甲基化标志物的关系研究采用相关分析和多元线性回归分析探讨了肺损伤标志物(血清CC16、SP-A、SP-D)、肺功能与DNA甲基化指标(5-mC%、DNMT1、DNMT3A)间的相互关系。发现血清CC16的浓度与DNMT1表达量呈显著的正相关(r = 0.075,P=0.040)。经年龄分层后发现,在小于45岁年龄组中血清CC16与DNMT1和DNMT3A均存在显著的正相关。中介效应分析没有发现DNA甲基化在COE致肺损伤中的中介作用。研究结论1.研究发现焦炉逸散物中PM2.5和PAHs都可以导致呼吸系统损伤和DNA甲基化改变,且其作用模式不同。2.焦炉逸散物暴露与血清CC16存在明确的暴露-反应关系,血清CC16降低可作为焦炉逸散物致肺损伤的早期效应标志物。3.焦炉逸散物暴露可引起基因组DNA低甲基化改变,DNMT3A是介导基因组DNA甲基化降低的重要因素。更多还原

【Abstract】 Coke oven emissions(COE)are the main pollutants in the coking production,which contains a large number of Particulate matter(PM)and Polycyclic aromatic hydrocarbons(PAHs).Epidemiological studies showed that exposure to high concentrations of particulate or PAHs is one of the major causes of the respiratory diseases,and cancer caused by COE is classified as a clear occupational tumor.Fine particulate matter(PM2 5)has aerodynamic diameter less than 2.5μm and is easy to enrich toxic and harmful substances on the surfaces,and the adverse effect of PM2.5 on respiratory system is more clearly.PAHs is the main target pollutant in the studies of adverse effect induced by COE exposure,but there is still lack of the analysis of exposure-response relationship between PM2.5 exposure and adverse effect.Occupational COE exposure leads to lung cancer,but there are few studies on the changes of important biological molecules and the early health effect biomarkers in this process.Pulmonary proteins are involved in maintaining the homeostasis of respiratory system including Clara cell protein(CC16),surfactant protein A(SP-A),and surfactant protein D(SP-D).Researchers have been conducting a number of studies on the association of lung injury proteins and pulmonary disease in clinical research.However,there are few studies on the changes of lung injury proteins induced by COE.Exposure to PM and PAHs can lead to an increased risk of multiple diseases,and epigenetic changes may play an important role in the process of carcinogenesis.Genetic damage was hard to reverse,but epigenetic inheritance does not involve changes in DNA sequences which was reversible.DNA methylation is the most widely studied epigenetic modification so far.Researches and our previous studies found that PAHs exposure can cause the aberrant methylation of DNA repair genes and tumor suppressor genes.There are lack of studies on the global DNA methylation induced by PM2.5 and PAHs and studies on the association of global DNA methylation and DNA methyltransferase(DNMTs).We conducted a molecular epidemiological study.Exposure assessment of PM2.5 and PAHs were carried out in several workplaces.We enrolled 558 coke plant workers and 210 control workers in oxygen plant and cold-rolling mill in China.We measured serum high-sensitive C-reactive protein in all subjects.Pulmonary injury was measured by lung function and serum Club cell protein(CC16),surfactant protein A(SP-A),and surfactant protein D(SP-D).Epigenetic modification effects were detected by global DNA methylation level and DNMTs in human white blood cells.1.Respiratory system injury marker with coke oven emissions exposureThe concentrations of PM2.5 were analyzed by weighing method.Ultra-high performance liquid chromatography(UPLC)was used to determine the concentration of 16 PAHs.An ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS/MS)method was used to measure the urinary 1-OHP.Serum high-sensitivity C-reactive protein(hs-CRP)was measured by an automatic biochemistry analyzer.Serum CC16,SP-A and SP-D were measured by sandwich ELISA.The lung function tests were conducted by a pulmonologist using a fixed electronic spirometer.Serum CC16 levels reduced with the increasing occupational exposure history of COE.An IQR(121.98 μg/m3)increased of PM2.5 was associated with 5.24%decrease(P=0.004)in serum CC16.An IQR(3.81 μg/m3)increased of ∑PAHs was associated with 5.69%decrease(P=0.027)in serum CC16.Serum CC16 was also identified with each IQR increase in urinary 1-OHP concentration(1.06 μmol/mol creatinine)associated with 4.67%decrease in serum CC16(P=0.041).Furthermore an IQR decrease in serum CC16(3.95 ng/mL)was associated with 0.87%decrease in FEV1/FVC in the male participants(P = 0.045).2.DNA methylation induced by coke oven emissions exposureGlobal DNA methylation(5-mC%)was measured by methylated DNA quantification kit.Quantitative analysis of DNMTs was measured by real-time quantitative PCR(RT-qPCR)method.Serum folic acid and vitamin B12 levels were also examined.Global DNA methylation(5-mC%)reduced with the increasing occupational exposure history of COE.An IQR(121.98 μg/m3)increased of PM2.5 was associated with 5.78%decrease(P<0.001)in Global DNA methylation(5-mC%).An IQR(3.81 μg/m3)increased of ∑PAHs was associated with 6.09%decrease(P=0.007)in Global DNA methylation(5-mC%).Urinary 1-OHP was negatively correlated with Global DNA methylation(5-mC%)(β=-0.025,P<0.001).The association of ambient environmental PM2.5 with DNMT1 and DNMT3A were not found.An IQR(3.81 μg/m3)increased of ∑PAHs was associated with 13.25%decrease(P=0.027)in DNMT3A.No significant correlation was found between urinary 1-OHP with DNMT1 and DNMT3A.Regression analysis showed that global DNA methylation(5-mC%)was positively correlated with DNMT1 and DNMT3A.Mediation model analyzed 5.95%of the effects of PAHs exposure on global DNA methylation mediated through changes in DNMT3 A.3.Association of lung injury and DNA methylation markers induced by coke oven exposureThe correlation between lung injury markers(serum CC16,SP-A,SP-D),lung function and DNA methylation(5-mC%,DNMT1,DNMT3A)was analyzed by correlation analysis and multiple linear regression analysis.Serum CC16 concentration was found to be positively correlated with DNMT1(r = 0.075,P = 0.040).After layered by age,there was a significant positive correlation between serum CC16 and DNMT1 and DNMT3A in patients younger than 45 years.Mediation of DNA methylation in lung injury induced by COE was not found.Conclusions1.Reduced serum Club cell protein as an early pulmonary injury marker for COE exposure.2.COE exposure can cause the reduction of DNA methylation.And the reduction of DNMT3 A is an important factor in the decrease of global DNA methylation.DNMT3 A is not the key factor mediating lung injury caused by COE.更多还原