柴油机尾气暴露人群乙烯基DNA加合物与DNA甲基化水平的关系研究

Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust

目的 探讨柴油机尾气（DEE）暴露人群乙烯基DNA加合物与细胞周期蛋白依赖性激酶抑制剂2A基因（P16）、Ras相关结构域家族1A基因（RASSF1A）和O-6-甲基鸟嘌呤-DNA甲基转移酶基因（MGMT）启动子区DNA甲基化水平的关联性.方法 采用整群抽样法于2012年选取河南省某柴油机厂124名车间试车工人为DEE暴露组,选取同地区自来水厂112名非DEE暴露工人为对照组.收集研究对象的人口学信息,并采集静脉血和班后尿液.应用高效液相色谱-质谱联用方法检测研究对象尿液中乙烯基DNA加合物的生成情况,包括N6-亚乙烯基-2＆#39;-脱氧腺苷（εdA）和3,N4-亚乙烯基-2＆#39;-脱氧胞苷（εdC）.运用焦磷酸测序方法分析P16、RASSF1A和MGMT启动子区DNA甲基化水平,以5-甲基胞嘧啶（5mC）占胞嘧啶的百分比表示（%5mC）.采用Spearman相关分析和多重线性回归分析乙烯基DNA加合物和DNA甲基化水平的关系.结果 暴露组和对照组工人尿液中 εdA浓度的P50（P25~P75）分别为230.00（98.04~470.91）、102.10（49.95~194.48）pmol/g肌酐,暴露组高于对照组（P〈0.001）.暴露组工人P16、RASSF1A和MGMT启动子区DNA甲基化水平分别为2.04＆#177;0.41、2.19（1.94~2.51）和2.22（1.94~2.46）%5mC,对照组工人分别为2.19＆#177;0.40、2.41（2.11~2.67）和2.44（2.15~2.91）%5mC,暴露组低于对照组（P值分别为0.005,0.002和〈0.001）.Spearman相关分析显示,P16、RASSF1A和MGMT启动子区DNA甲基化水平与尿液中εdA浓度呈负相关（r值分别为-0.155、-0.137和-0.198,P值均〈0.05）,与εdC浓度相关无统计学意义（P〉0.05）.多重线性回归分析显示,在非吸烟人群中,εdA与P16、RASSF1A和MGMT启动子区DNA甲基化负相关[β（95%CI）值分别为-0.068（-0.132^-0.003）、-0.082（-0.159^-0.004）和-0.048（-0.090^-0.007）,P值分别为0.039,0.039和0.024],εdC与MGMT启动子区DNA甲基化负相关[β（95%CI）值为-0.094（-0.179^-0.008）,P=0.032].结论 DEE暴露可引起εdA升高,P16、RASSF1A和MGMT启动子区DNA甲基化水平降低.

Objective To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A （P16）, Ras association domain family 1 （RASSF1A） and O-6-methylguanine-DNA methyltransferase （MGMT） in workers with occupational exposure to diesel engine exhaust （DEE）. Methods We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2＆#39;-deoxyadenosine （εdA） and 3,N4-etheno-2′-deoxycytidine （εdC）. The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine （5mC） over the sum of cytosines （% 5mC）. Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results The median （P25-P75） of urinary εdA level was 230.00 （98.04-470.91） pmol/g creatinine in DEE exposure group, and 102.10 （49.95-194.48） creatinine in control group. The level of εdA was higher in DEE exposure group than control group （P〈0.001）. DNA methylation levels of P16, RASSF1A and MGMT were 2.04＆#177;0.41, 2.19 （1.94-2.51）, 2.22 （1.94-2.46） %5mC in exposure group, and 2.19＆#177;0.40, 2.41 （2.11-2.67）, 2.44 （2.15-2.91） %5mC in control group. DNA methylation levels were lower in exposure group （P values were 0.005, 0.002 and 0.001, respectively）. Spearman correlation analysis showed that DNA methylation levels of P16, RASSF1A, and MGMT were negative associated with urinary ε dA level （r values were -0.155, -0.137, and -0.198, respectively, P〈0.05）. No significant correlation was observed between the εdC level and any measured DNA methylation levels （P〉0.05）. Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group （ β （95%CI） was -0.068 （-0.132--0.003）, -0.082 （-0.159--0.004） and -0.048 （- 0.090-- 0.007）,P values were 0.039, 0.039 and 0.024, respectively）. Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group （ β （95%CI） was -0.094 （-0.179--0.008）, P=0.032）. Conclusion DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A and MGMT.