【作者】 王君；

【导师】 金银龙；

【作者基本信息】 中国疾病预防控制中心 ， 劳动卫生与环境卫生学， 2014， 博士

【摘要】 目的：研究北方某市母婴多环芳烃(PAHs)负荷水平及其影响因素以及宫内多环芳烃暴露与脐带血生成BPDE-DNA加合物关系的研究；探讨宫内暴露PAHs对胎儿基因组甲基化的影响；研究宫内PAHs暴露水平对胎儿特异基因甲基化的影响；了解宫内暴露对基因甲基化与生成DNA加合物的关系。方法：1.应用现场流行病学的调查方法,对所选择的待产孕妇先进行流行病学调查,并采集孕妇的外周静脉血和新生儿脐带血：2.用高效液相色谱-荧光色谱法测定孕妇静脉血和脐带血血清AnT、FluA、Pyr、 BaA、BbF、BkF、BaP、DBahA和BghiP共9种PAHs的浓度；3.采用毛细管电泳-激光诱导荧光检测部分脐血中的BPDE-DNA加合物的浓度；4.利用LC-MS/MS建立的全基因组甲基化水平分析方法测定相应的脐带血DNA基因组甲基化水平,利用单因素及多元回归的统计方法分析评价宫内暴露对胎儿全基因组甲基化水平的影响；5.用焦磷酸测序法测定P53、P16特定基因位点甲基化,先后用单因素分析和多元逐步回归的方法分析影响基因甲基化的主要因素；6.分别用单因素及多元逐步回归模型分析BPDE-DNA加合物与基因组甲基化水平及特异基因P16、P53位点甲基化的关系。结果：1.母、脐血清中Pyr的检出率在9种多环芳烃中是最高,其检出率分别是98.9%和95.2%。其余几种PAHs的检出率也多在60%以上,其中BaP的血清负荷水平在母、脐中分别为47.3%和49.7%,检出浓度中位数分别为0.035(P25：0.020,P75:0.072) ng/ml和0.035(P25:0.020,P75:0.095ng/ml)平均浓度分别约为(0.053±0.044) ng/ml和(0.062±0.053) ng/ml.经秩和配对检验,显示脐带血中AnT、Pyr、BaA、BkF、BaP和BghiP的含量均高于相应母血,差异有统计学意义。2.家庭住宅距离交通主干道比较近的孕妇血清BaP含量比较高(P<0.05)。3.胎儿脐带血中的BPDE-DNA加合物的检出率是86.1%,但脐带血中的BPDE-DNA加合物与孕妇、脐带血血清BaP水平无明显相关。4.单因素分析结果显示,不同PAHs的种类及不同负荷水平对基因组甲基化的影响各不相同。对于血清BaP和BaA来说,随着血清负荷水平增加,结果表明相应的全基因组甲基化水平受到了影响,呈现减低趋势。对于血清BkF和血清FluA,结果显示其负荷水平与相应的全基因组甲基化呈正相关。另外其他三种PAHs没有发现其负荷水平对甲基化有明显的影响。在排除了其他干扰后的多元回归分析表明,血清BaP和血清BkF仍然能进入回归模型,说明PAHs暴露是基因组甲基化水平发生改变的主要影响因素。5.单因素分析,发现新生儿的性别和母亲孕期服用叶酸可能会对两种基因的甲基化水平有影响。经对脐带血清AnT、FluA、Pyr、BaA、BbF、BkF、BaP、DBahA和BghiP对基因甲基化水平影响的单因素分析,发现血清BaP水平可能影响P53和P16基因的甲基化水平,负荷组显示的甲基化水平比低负荷组要低,差别有统计学意义。血清BbF和BkF的水平则正好相反,结果显示有增加基因甲基化水平的可能。而血清BghiP、BaA、FluA则在不同的基因、相同基因的不同位点都影响不同。通过多元回归模型的分析,在排除了其他干扰因素后,仍有不同种类的PAHs进入模型。说明PAHs的暴露是影响P53和P16基因甲基化水平改变的主要影响因素。6.经单因素分析可知,BPDE-DNA加合物与基因组甲基化及P53、P16的位点甲基化水平均有关系。多元逐步回归分析结果显示,基因组甲基化水平与可检测到的BPDE-DNA加合物呈正相关,而P16的位点基因甲基化水平与BPDE-DNA加合物水平呈负相关关系。7.结果显示,PAHs暴露后,DNA甲基化水平比BPDE-DNA加合物更容易检测的到。结论：1.本研究中的孕期妇女体内有一定的PAHs负荷水平,家庭离交通主干道的距离是体内PAHs负荷水平的主要影响因素。室外空气污染可能在PAHs的暴露中起主要作用。2.母婴体内PAHs负荷存在着相关性,血清BaP可以作为评价PAHs暴露水平的代表物质。3.证实了PAHs的跨胎盘暴露已造成发育中的胎儿DNA的早期损伤。4.宫内PAHs的暴露影响了胎儿基因组甲基化状态的改变,不同PAHs对胎儿基因组甲基化状态的影响方式不同。5.环境低水平的的宫内多环芳烃暴露也会对影响到胎儿P53、P16抑癌基因的甲基化。这种改变有可能会促进基因的其他损伤行为发生而增加胎儿出生以后的患病风险。6.基因组甲基化与特异位点的甲基化水平均与可检测到的加合物有关。DNA的甲基化与BPDE-DNA加合物的形成之间存在相互促进或抑制作用的可能。更多还原

【Abstract】 Objective:To investigate the body burdens of polycyclic aromatic hydrocarbons (PAHs) of pregnant women and to explore the influencing factors related to PAHs exposures. To discuss the relationship between BaP levels and BPDE-DNA adducts in cord blood. To explore the effect on the global DNA methylation and specific gene methylation in cord blood caused by prenatal exposure. To research the relationship between prenatal PAHs exposure on BPDE-DNA adducts and DNA methylation in cord blood.Methods:1. Questionnaire investigation on random selected and voluntarily participating pregnant women was conducted and maternal blood and cord blood was collected.2.216pregnant women from one hospital were investigated and questionnaire survey was conducted to obtain individual behavior and life style during the period of July to December2012. PAHs levels in205pregnant women serum samples was detected by high-performance liquid chromatography(HPLC).3. Capillary electrophoresis-laser induced nuorescence immunoassay method was used to determine the concentrations of BPDE-DNA adducts.4. The globle DNA methylation was scanning by LC-MS/MS. And simple univariate statistics analysis and multivariate linear stepwise regression was also conducted to select the important factors of the change on globle DNA methylation.5. The gene-specific methylation level was quantified by pyrosequencing in cord blood. And T-test and multivariate linear stepwise regression was also carried to select the important factors of the change on gene-specific methylation.6. Relationship between prenatal PAHs exposure on BPDE-DNA adducts and DNA methylation in cord blood was analyzed by T-test and multivariate linear stepwise regression.Results:1. The detection rate of Pyr in205pregnant women serum samples was still the highest which was98.9%and95.2%. and the detection rate of BaP in205pregnant women serum samples was47.3%, and the mean BaP concentration was0.035ng/ml (P25:0.02,P75:0.07) ng/ml. The concentration of AnT、Pyr、BaA、BkF、BaP and BghiP in cord serum is statistically higher than those in paired maternal (p=0.002)2. There was much higher BaP concentration in pregnant women who lived nearby the traffic artery.3. The detection rate of BPDE-DNA adducts in cord blood was86.1%. But the level of PAH-DNA adducts detected were not significantly associated with BaP either in cord or maternal serum.4. According to the results of LC-MS/MS analysis and T-test, various kinds and levels of PAHs has individual effect on global DNA methylation. Statistics of multivariate linear stepwise regression reveals that the concentration of BaP and BkF in cord blood serum was an important factor on the global methylation.5. According to the results of pyrosequencing in cord blood analysis and statistics of T-test, both the gender of newborns and folic acid supplement during pregnancy can affect the global methylation changes. The concentration of BaP could negatively influence the gene-specific methylation of CpG site, but BbF and BkF has the opposite result. Multivariate linear stepwise regression reveals that several PAHs can be fitted the model, that is, PAHs exposure in gestion period is the most important factor for the gene-specific site methylation of P16and P53.6. T-test shows that the levels of DNA methylation are associated with the presence of detectable BPDE-DNA adducts. Both the positive association relationship between global DNA methylation and the presence of BPDE-DNA adducts and the negative association between site-specific methylation and adducts are revealed after multivariate linear stepwise regression.7. The level of DNA methylation was more easily detected than BPDE-DNA adducts for PAHs exposure.Conclusions:1. Several kinds of PAHs can be detected in general pregnant woman of the study. Living nearby the traffic artery is the main factor among all of the surveying factors and outdoor air pollution maybe play an important role in prenatal PAHs exposure.2. PAHs can be detected in maternal and umbilical serum, and body levels of PAHs in maternal serum are related with those in body of fetus. BaP can be regarded as the representative of the total PAHs in evaluating the level of PAHs exposure.3. DNA damage in the developing through the transplacental exposure to PAHs from maternal fetus was also confirmed.4. Prenatal exposure to PAHs can change the global DNA methylation and different kinds of PAHs have different effects on global DNA methylation which means they may have their individual mechanisms of adverse effects.5. Prenatal exposure to PAHs can also ater the site-specific methylation of P53and P16, which maybe result in other gene damage and increase the risk of future diseases for the baby.6. Global DNA methylation and site-specific methylation of P16are related with the presenceof detectable BPDE-DNA adducts and there maybe interactions either inhibiting or accelerating between them.更多还原