焦炉逸散物致呼吸系统损伤与基因组DNA甲基化改变的生物标志物研究

发布时间：2018-09-09 16:08

【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%(P0.001);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,P0.001)。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。
[Abstract]:Coke oven emissions (COE) are the main pollutants discharged during the coking process, which contain a large number of particulate matter (PM) and polycyclic aromatic hydrocarbons (PAHs). Epidemiological studies have shown that exposure to high concentrations of particulate matter or polycyclic aromatic hydrocarbons (PAHs) is the occurrence and development of respiratory diseases. Fine particulate matter (PM2.5) has an aerodynamic diameter of less than 2.5 microns and is prone to enrich toxic and harmful substances on the surface. The damage effect on respiratory system is also more clear. However, the exposure and effect markers of COE are agents. Among the dose-response relationships, PAHs with more definite carcinogenicity are the main target pollutants, and there is a lack of analysis on the exposure-response relationship of PM2.5 health effects. Although epidemiological studies have confirmed the causal link between occupational COE exposure and lung cancer in coke oven workers, the process from exposure to lung cancer is still lacking. Significant biological changes and biomarkers of early adverse health effects are still limited. Studies have found that changes in the expression of various proteins in the circulatory system are closely related to the maintenance of respiratory homeostasis, including lung surfactant protein A (SP-A), lung surfactant protein D (SP-D) and clara. Club cell protein (CC16) and so on. Researchers have carried out a number of clinical research work in lung injury protein and chronic obstructive pulmonary disease and other respiratory disease occurrence and progress of research. However, the study on COE-induced lung injury related markers in the blood has not been reported. Particulate matter and PAHs exposure can lead to a variety of. Epigenetic changes may play an important role in increasing the risk of disease, especially in carcinogenesis. Epigenetics does not involve changes in DNA sequences and is reversible. DNA methylation is one of the most important epigenetic modifications. We and other research teams found that PAHs exposure can lead to more in early studies. The methylation of DNA repair genes and tumor suppressor genes was abnormal. However, there were few reports on the methylation at the whole genome level. At present, there was no study on the relationship between DNA methyltransferase (DNMTs) and DNA methyltransferase (PM2.5). In order to explain the above problems, we carried out a study in a northern coking plant population. Molecular epidemiological study on the relationship between COE exposure and exposure-response of early damage markers was carried out. Firstly, exposure to particulate matter and polycyclic aromatic hydrocarbons (PAHs) was assessed in several workplaces. Then 558 coke oven workers were recruited as exposed population and 210 workers in oxygen plant and cold rolling plant as controls. The levels of somatic inflammation (hs-CRP), lung injury markers (CC16, SP-A and SP-D) and the effects of PM2.5 and PAHs in coke oven effluents on lung function were investigated. The changes of genomic DNA methylation and expression of DNA methylation transferase in leukocytes were observed, and the mediating effects of DNA methylation transferase were discussed. The role of coke oven fugitives in the alteration of genomic DNA methylation and lung injury induced by COE. 1. Changes of biomarkers in serum respiratory system injury induced by coke oven fugitives. PM2.5 content in COE was calculated by weighing method. Polycyclic aromatic hydrocarbons (PAHs) concentration in granular phase was detected by ultrahigh performance liquid chromatography-tandem mass spectrometry. Urine 1-Hydroxyl group was detected by ultrahigh performance liquid chromatography-tandem mass spectrometry. The concentration of pyrene (1-hydroxyrene, 1-OHP) in serum was detected by ELISA. The pulmonary function of the subjects was also measured. The study found that the serum CC16 decreased with the increase of exposure to PM2.5 and P AHs in COE (P = 0.046). In a population-based exposure-response relationship analysis, one IQR (12) was increased by PM2.5 in COE. 1.98 ug/m3 decreased serum CC16 by 5.24% (P = 0.004); total PAHs increased by 1 IQR (3.81 ug/m3) in COE, decreased serum CC16 by 5.69% (P = 0.027); urinary 1-OHP increased by 1 IQR (1.06 ugol/mol creatinine) and decreased serum CC16 by 4.67% (P = 0.041) in male workers. Ng / mL, FEVl / FVC decreased by 0.86% (P = 0.045). 2. Genomic DNA methylation induced by coke oven fugitives was detected by ELISA. Genomic DNA methylation (5-mC%) was detected by real-time fluorescence quantitative PCR (RT-qPCR) and the levels of folic acid and vitamin B12 in serum were detected by real-time fluorescence quantitative PCR. Genomic DNA methylation (5-mC%) decreased significantly with the increase of COE exposure (P = 0.001). Exposure-response relationship analysis showed that an IQR (121.98 ug/m3) was added to PM2.5 in COE, resulting in a 5.78% (P 0.001) decrease in genomic DNA methylation (5-mC%) and an IQR (3.81 ug/m3) increase in total PAHs, resulting in genomic DNA methylation. A significant negative correlation was found between urinary 1-OHP and genomic DNA 5-mC content (beta = - 0.025, P 0.001). Quantitative study of DNA methylation transferase showed that the mRNA expression level of DNMT3A gene decreased with the increase of COE exposure (P = 0.047), and the total PAHs increased by an IQR (3.81 ug/m3) and DNMT3A table. Regression analysis showed that genomic DNA methylation (5-mC%) was positively correlated with both DNMT1 and DNMT3A. Intermediate effect analysis showed that 5.95% of genomic DNA methylation induced by PAHs exposure could be mediated by DNMT3A. 3. lung injury induced by coke oven fugitives and DNA A Correlation analysis and multiple linear regression analysis were used to explore the relationship between lung injury markers (serum CC16, SP-A, SP-D), lung function and DNA methylation markers (5-mC, DNMT1, DNMT3A). There was no mediating role of DNA methylation in COE-induced lung injury. Conclusion 1. Both PM2.5 and PAHs in coke oven effluents can cause respiratory system damage and DNA methylation changes, and their modes of action are different. 2. There is a clear exposure-response relationship between coke oven fugitive exposure and serum CC16. Reduction of serum CC16 can be used as an early marker of lung injury induced by coke oven fugitive exposure. 3. Coke oven fugitive exposure can cause hypomethylation of genomic DNA. DNMT3A is an important factor mediating the reduction of genomic DNA methylation.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R13

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