【作者】 董少霞；

【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， 2008， 博士

【摘要】 研究背景研究表明,胎儿和新生儿比成人对环境有害物质的危害更敏感,可能源于不同的暴露途径、生理上的不完善和更长的生命周期使疾病在早期生命阶段启动发展。多环芳烃是最具特征的环境毒物之一,一些PAHs是致突变、致癌和生殖发育毒物,还可与DNA结合引起DNA损伤,通过干扰激素代谢途径或干扰激素活性而使内分泌系统紊乱。实验研究已发现经胎盘的PAHs暴露与不良出生结局有关。有证据表明,目前严重的环境污染,尤其是空气污染可能与不良出生结局有关,其中PAHs起着重要的作用。目前的研究大多由于缺乏有效的定量暴露资料来将普遍的环境污染因素和婴幼儿健康影响研究在因果上联系起来。分子流行病学运用生物标志可以作为确定环境-易感性关系的有效工具。同时,由于人类健康或疾病状态是由遗传因素与环境因素相互复杂作用的结果,机体损伤反应不仅与暴露于环境有害因素的程度有关,同时还与遗传易感性或耐受性有着密切联系,即存在环境与基因交互作用,探讨基因与暴露的相互关系已经成为目前环境与健康领域发展的方向之一。宫内胎儿通过母体间接暴露于多环芳烃,由于受到胎盘屏障的选择性和通透性的作用、胎盘酶活性的影响、以及母体和自身代谢酶基因的表达、酶活性的综合影响,使得胎儿的暴露及其效应的影响因素更为复杂。而目前尚缺乏对宫内胎儿PAHs暴露的有效生物标志及其效应的探讨,不多的几项针对基因对代谢和胎儿发育交互作用的研究结论也不统一。太原市是我国环境空气污染甚为严重的城市,近年来一直处于污染的前列。其中尤以颗粒物及PAHs污染为甚。此外,该城市的出生缺陷及其他不良出生结局水平很高。因此,在此环境中探讨宫内PAHs暴露的相关生物标志及其健康影响具有重要的现实意义,可为进一步开展筛检易感人群、进一步开展健康危险度评价提供科学的理论依据。研究目的本研究通过流行病学调查和生物样品检测,了解太原市母亲及新生儿多环芳烃暴露现状,明确母婴暴露的相关关系及其影响因素,探讨有效表征宫内PAHs暴露的生物标志;阐明代谢酶基因多态性(CYP1A1 MspI、GSTM1、GSTT1、GSTP1)与PAHs代谢的关系及其修饰作用;探讨PAHs暴露同代谢酶基因多态性对于胎儿生长发育的交互作用,从而为有效的进行易感人群筛检和干预、开展危险性评价提供科学依据。研究方法1研究现场污染状况分析采用常规大气污染物监测方法,在研究现场定点采样,送回实验室采用高效液相色谱-荧光检测法测定PM2.5上9种PAHs含量,用于评价研究现场的污染状况,提供本研究的背景资料。2流行病学调查采用横断面调查的方法,对研究对象进行问卷调查,了解母亲的一般情况、生活环境情况,吸烟饮酒情况,家族及生育史,新生儿发育状况等。3生物样品的收集和测定3.1采集母亲产前中段尿样,采用酶水解—高效液相色谱法测定尿样中1-OHPy;3.2采集产前及产后当天母亲静脉血及产时新生儿脐带血,采用高效液相色谱-荧光检测法测定母婴血清中7种PAHs水平;3.3采血即刻采用培养法检测母婴血白细胞微核率;3.4提取母婴全血基因组DNA,采用PCR-RFLP或多重PCR法检测CYP1A1、GSTP1、GSTM1、GSTT1基因多态性。研究结果第一部分太原市大气中多环芳烃水平太原市大气中可检出多种致癌性PAHs,其中B(a)P含量为0.0303μg/m~3,超出国家居民区大气标准(0.01μg/m~3)2倍以上.第二部分母婴PAHs暴露评价及其相关因素1母亲尿样1-OHPy检出率为93.8%,检出浓度为:0.794μmol/mol Cr(IQR0.519～1.369μmol/mol Cr)。有92.5%的母亲尿样1-OHPy含量高于以往推荐的参考值(一般居民尿中1-OHPy的生物暴露限值0.1μmol/mol Cr),同时,13.3%的母亲尿样1-OHPy含量高于焦炉工人生物暴露限值(1.90μmol/mol Cr),表明研究人群PAHs暴露水平普遍较高;2影响母亲尿样1-OHPy含量的单因素分析显示,厨房和客厅或卧室未分开、做饭时未使用抽油烟机以及教育程度低的母亲其尿样中1-OHPy含量偏高(P＜0.05):3多元线性回归结果显示,母亲每周烹调时间、被动吸烟和取暖方式是影响母亲尿样1-OHPy水平的主要因素;当调整了其它因素后,母亲每周烹调时间每增加1h,经对数转换后的母亲尿样1-OHPy含量相应增加0.004个单位;有被动吸烟者母亲尿样1-OHPy含量相应增加0.064个单位;燃煤取暖的母亲比使用集中供暖的母亲经对数转换后的母亲尿样1-OHPy含量高0.063个单位:并且被动吸烟和烹调时间、取暖方式和烹调均有交互作用。4母婴血清中均可检出多种致癌性PAHs,脐带血B(k)F(P=0.002)、B(a)P(P=0.002)、DB(a,h)A(P＜0.001)及T(P=0.034)水平均明显高于母亲血清中的含量;5单个PAHs及总PAHs以及T水平在母婴之间均有相关关系存在(P＜0.05):6母亲尿样1-OHPy同母血清及新生儿脐带血中PAHs之间均没有相关性。第三部分代谢酶基因多态性与母婴PAHs暴露关系1母亲GSTT1缺失型、GSTM1缺失型、GSTP1杂合/纯合突变型及CYP1A1MspI杂合/纯合突变型携带者母婴血清PAHs水平均高于正常型或野生纯合型,但通过Mann-Whitney Test检验,没有统计学差异(P＞0.05);2新生儿GSTM1缺失型携带者脐带血中7种PAHs及TPAHs、T均高于GSTM1正常型,其中B(b)F、B(a)P、B(ghi)P、TPAHs水平两组之间的差异具有统计学意义(P＜0.05):GSTM1、GSTT1联合缺失型新生儿脐带血中7种PAHs及TPAHs、T值也均高于正常型,其中B(k)F、B(ghi)P及TPAHs明显较高(P＜0.05)。而GSTP1杂合/纯合突变型及CYP1A1MspI杂合/纯合突变型的血清PAHs水平虽然高于野生型,但经Mann-Whitney Test检验,差异没有统计学意义(P＞0.05)。3校正年龄、教育水平、取暖方式、通风、每周做饭时间分组、被动吸烟等因素后,CYP1A1纯合突变型母亲的尿样1-OHPy水平明显高于野生型(1.6530vs 1.1624μmol/molCr,P=0.047):GSTP1纯合突变型1-OHPy水平明显高于野生型(1.0076 vs 0.9622μmol/molCr,P=0.009);4多基因模型中,CYP1A1和GSTP1对母亲尿样1-OHPy浓度具有修饰作用(P=0.046及P=0.029)。多基因模型的决定系数为0.262:5基因一基因交互作用分析显示对尿中1-OHPy浓度有修饰作用的多态位点主要有CYP1A1和GSTM1。第四部分母婴遗传损伤及宫内PAHs暴露与代谢酶基因多态性对生长发育的交互作用1母婴微核水平具有相关性(spearman correlation,R=0.212,P=0.037),且母亲高于新生儿(Wilconxon signed ranks test,Z=-3.325,P＜0.05);2单因素分析发现,不使用抽油烟机的母亲外周血淋巴细胞微核率要高于使用抽油烟机者(P=0.038);随年龄的增长,母亲外周血淋巴细胞微核率有增加趋势(P=0.024);3母亲常吃油炸食品对新生儿微核率有显著性影响,经常吃油炸食品的母亲其新生儿脐带血微核率明显偏高(P＜0.05);新生儿微核率水平没有性别上的差异(P＞0.05);4不同PAHs暴露水平下微核率没有明显差异(P＞0.05);5母亲四种代谢酶基因多态性对母亲外周血MN以及新生儿脐带血MN均没有显著性影响(P＞0.05)。6新生儿四种代谢酶基因多态性对脐带血MN没有显著性影响(P＞0.05)。7没有发现PAHs暴露和代谢酶基因多态性对微核形成的交互作用。8 PAHs高暴露时,GSTP1杂合/突变型基因携带者其新生儿出生体重较低,而在低PAHs暴露组偏高,但差异没有达到显著性水平(P=0.086)。9采用多元线性回归模型将母亲年龄、文化程度、被动吸烟、取暖方式、每周做饭时间分组、婴儿性别、孕周、母亲BMI等混杂因素调整后发现,GSTP1 AG/GG基因型携带者其新生儿头围有减小趋势(P=0.095)。10控制了相关混杂因素后,母亲GSTP1基因多态性同PAHs暴露对新生儿出生头围的影响可能有交互作用存在(F=3.397,P=0.069);11多元线性回归模型并将母亲年龄、文化程度、被动吸烟、取暖方式、每周做饭时间分组、新生儿性别、孕周、母亲BMI等混杂因素调整后,PAHs暴露同CYP1A1及GSTM1基因多态性对出生体重有交互作用(分别P=0.034和P=0.062)。研究结论1.太原市大气PAHs污染较为严重;2.太原市母婴体内均可检出多种PAHs,新生儿脐带血中多种PAHs高于母体水平,且母婴PAHs水平之间具有相关性,可以用母亲外周血PAHs水平尤其是芘水平来表征胎儿的宫内暴露水平;3.大气污染、居室取暖方式、烹调频次、被动吸烟是太原市孕期妇女PAHs暴露的相关因素,并且被动吸烟及烹调习惯、取暖方式和烹调频次之间存在交互作用;4.代谢酶基因多态性对PAHs体内代谢具有修饰作用,母亲CYP1A1、GSTP1基因多态性对芘的代谢中可能起着重要得作用,CYP1A1与GSTM1对芘的代谢具有交互作用。新生儿GSTM1基因多态性对PAHs代谢起着重要作用。5.宫内PAHs暴露和代谢酶基因多态性对新生儿生长发育具有交互作用。母亲GSTP1基因多态性与PAHs暴露对新生儿出生体重、头围的影响具有交互作用;新生儿CYP1A1、GSTM1与PAHs暴露对新生儿出生体重具有交互作用。综上研究结果,本研究人群(孕期妇女)具有较高的PAHs暴露水平,并且通过体内代谢及胎盘的转运影响到了发育中的胎儿。本研究结果首次利用较大的样本量关注了母婴体内PAHs暴露及代谢的关系,初步提出可以用母体血清芘水平来表征胎儿体内的代谢;并且发现代谢酶基因多态性在暴露及效应中均起到一定的修饰作用。鉴于本研究是一次横断面调查,并且初步性的结论尚需在不同人群及不同污染程度的地区来开展相关验证工作,建议开展大样本的对列研究以及同时考虑多种代谢酶基因多态性以进一步明确宫内胎儿PAHs暴露健康影响的关键时期,具体机制以及定量的关联也有待于今后进一步深入研究。更多还原

【Abstract】 BackgroundEvidence has shown that fetuses and infants are more vulnerable than adults to a variety of environmental toxicants because of their differential exposure patterns, physical immaturity and a longer lifetime over which disease initiated in early life can develop.Polycyclic aromatic hydrocarbons(PAHs) are among the best-characterized environmental toxicants.A number of PAHs are reproductive and developmental toxicants,as well as mutagens and carcinogens.In addition to their ability to bind to and damage DNA,PAHs such as benzo[a]pyrene are capable of disrupting the endocrine system by altering metabolic pathways of natural hormones or interfering with their activity.Laboratory studies have found an association between transplacental exposure to certain PAHs and adverse reproductive outcomes.And more and more evidences have appeared to indicate that there is an association between air pollution and adverse birth outcomes in human.Furthermore,PAHs may be the most prominent agents in polluted urban air.Birth outcomes are important because they are indicators of the health of the newborns and infants.In addition,low birth weight and impaired growth in the first year of life are known to influence the subsequent health status of individuals,including increased mortality and morbidity in childhood and an elevated risk of hypertension,coronary heart disease,and non-insulin-dependent diabetes in adulthood.Most of the research to date has lacked adequate exposure and dosimeter data to forge causal links between common environmental factors and health effects in the young.In this regard,molecular epidemiology using biomarkers can be a valuable tool in defining environment-susceptibility relationships when used in conjunction with sound monitoring and epidemiologic methodologies.Meanwhile,health and disease are influenced by genetic and environmental factors simultaneously,and human’s response to the environmental toxicants or other deleterious factors is strongly linked to not only the dosage or degree of the environmental factors but also the genetic susceptibility.To explore the interactions between exposure and genetic polymorphisms has been the focus in environmental health fields.Fetus exposed to PAHs through the placenta and exposure evaluation is very complicated due to the highly selectivity,enzyme activity in placenta and the genetic polymorphisms both of maternal and newborns.To date,few studies have focused the effective biomarkers to evaluate the extent and impacts of prenatal exposure to PAHs. Though several studies have explored the modification of the genetic polymorphisms to the metabolism of PAHs and the interactions on fetal growth and development,the results are inconsistent.So it is urgent to explore the association.Environmental surveillance data shows that air in Taiyuan City is always heavily polluted and especially by the particulate matter(PM) and PAHs.Further,Birth defects incidence is high and used to be called the "Everest".Beside,adverse birth outcomes are also high.Therefore it is very important to discuss the prenatal exposure to PAHs for the specific biomarkers and the health impacts,further to help to identify high-risk population and improve the health risk evaluation.ObjectivesTo study the present PAHs exposure levels of pregnant and fetus in Taiyuan city by epidemiology investigation and biological surveillance;To explore the relationship between maternal and infant exposure and the related factors;To facilitate more effective discussions on the specific biomarkers which can be used to indicate the prenatal exposure;To estimate the modification role of metabolic genetic polymorphisms on the PAHs metabolism;To investigate the interactions of prenatal exposure to PAHs and the metabolic genetic polymorphisms;In sum,to provide scientific data for more effectively identification of high risk population,for more effective prevention and protection and for more pr(?)cised risk evaluation.Methods1 Air monitoringHigh-performance liquid chromatography(HPLC) with subsequent fluorescence detection was used to determine the PAHs(nine kinds) levels adhered to PM2.5.2 Epidemiology investigationsField study was conducted in Taiyuan city.A detailed,validated questionnaire administered to the mother within 2 days postpartum included information on the demography,smoking and drinking,residential and environmental exposures,disease and pregnant history,newborn development indexes.3 Sampling and measuringMaternal blood(10ml) was collected within 1 day postpartum,and umbilical cord blood(20ml) was collected at delivery.Samples were transported to the laboratory immediately after collection.About 0.5ml whole blood(both maternal and newborn) were cultured in culture medium to analyze the micronuclei frequency;the other blood were centrifuged to gain the serum for the PAHs measuring;and genomic DNA was extracted from lymphocytes by standard techniques.The genotyping of CYP1A1 MspI、GSTM1、GSTT1 and GSTP1 was performed in duplicate,using techniques based on PCR or PCR-RFLP.Each participant was asked to provide a 50 ml urine sample.Urine 1-OHPy concentration was measured using a modification of the HPLC method incubated for 4h in 37℃withβ- glucuronidase.ResultsPartⅠAir monitoringNine kinds of carcinogenic PAHs were detected in Taiyuan.B(a)P concentration is 0.0303μg/m~3,which is two times higher than the air standard(0.01μg/m~3).It shows that PAHs pollution is heavy in the study field.PartⅡPAHs exposure and the related factors1 In our study,the percentage of detectable samples in urine was 93.8%.1-OHPy concentration(μmol/mol Cr) was 0.794(median) and IQR was 0.519～1.369 (μmol/mol Cr).92.5%of the maternal urine 1-OHPy level were higher than that in the common residents(0.11μmol/mol Cr) and 13.3%of the maternal urine 1-OHPy level were higher than the exposure limit of Coke-oven workers (1.90μmol/mol Cr).2 There were several factors related to the 1-OHPy concentration,such as education, usage of cooking fume extractor and the kitchen separated from the sitting room or living room.Cooking frequencies,passive smoking and residential heating are the independent factors to the 1-OHPy level after controlling the other confounders in multiple linear regression models.3 Several kinds of carcinogenic PAHs were detected in nearly all the serum of both the mothers and newborns.The serum concentration of B(k)F,B(a)P,DB(a,h)A and T value in fetal were significantly higher than paired mother tissues.4 Every PAHs in serum was positively related to the other PAHs both in mother and newborn.And the PAHs in umbilical cord blood were also related to the same PAHs in paired mother tissues.5 1-OHPy concentration was positively correlated to the total PAHs,but no relationship were observed between the 1-OHPy and the PAHs level in umbilical cord blood.PartⅢMetabolic genetic polymorphisms and PAHs exposure1 The PAHs levels in serum both of mothers and newborns were higher in mothers with genotype TC/CC at CYP1A1 MspI,AG/GG at GSTP1,GSTM1 null or GSTT1 null genotype than that with the wild genotype or non-null genotype.But no significant difference was observed by Mann-Whitney Test.2 All the seven kinds of PAHs,the total PAHs and the T value in umbilical cord blood were higher in newborn with GSTM1 null genotype than that with GSTM1 non-null genotype.And significant difference were found only for the B(b)F、B(a)P、B(ghi)P and TPAHs level between the GST null and non-null genotypes.3 The same results were evident in newborns with the GSTM1 null and GSTT1 null combination than that with present genotype.4 1-OHPy level is significantly higher in urine of mothers with CYP1A1 homozygous at CC than that in wild genotype(1.6530 vs 1.1624μmol/molCr, P=0.047).And the mothers with homozygous GG at GSTP1 had a significantly higher level of 1-OHPy than the wild genotype group(1.0076 vs 0.9622μmol/molCr,P=0.009.)5 CYP1A1 and GSTP1 could modify the 1-OHPy concentration in the multi-gene model.R~2=0.262.6 Urinary 1-OHPy concentration could be influenced by CYP1A1 and GSTM1 polymorphisms when taking account into the some two way gene-gene interaction.PartⅣGenetic damage and the gene-exposure interaction to the fetal development1 MN frequency in blood was correlated between the paired mothers and newborns (R=0.212,P=0.037 )and MN frequency was higher in maternal blood than that in umbilical cord blood(Wilconxon signed ranks test,Z=-3.325,P＜0.05 ).2 A significant(P=0.038) higher MN frequency was found in lymphocytes of mothers with no usage of cooking fume extractor when cooking.MN frequency increased with age(P=0.024) in this adult population aged 18-42 years.3 MN frequency was higher in umbilical cord blood when the paired mother used to eat more fried or fumed food during pregnancy(P＜0.05 ).4 Newborns showed the same MN frequency regardless of sex(P＞0.05).5 MN frequency showed no significant difference among the different PAHs exposure levels(P＞0.05).6 There was no significant correlation between the four metabolic genetic polymorphisms and the MN frequency in the blood both of mother and newborn (P＞0.05).7 No interactive effect of exposure to PAHs and metabolic genetic polymorphisms was found on MN frequency in umbilical cord blood.8 Birth weight were lower in newborns with genotype AG/GG at GSTP1 within the high PAHs exposure group but were higher within the low PAHs group,whereas the interactive effect was not statistically significant(F=3.383,P=0.086)9 The birth head circumference reduced in the newborns with AG/GG genotype at GSTP1 after adjusting for age,education,passive smoking,history of pregnancy, residential heating,cooking frequency,pre-pregnancy height and weight,infant sex and gestational age(P=0.095). 10 An interactive effects were found between GSTP1 gene polymorphism and prenatal exposure to PAHs(F=3.397,P=0.069)11 There were interactive effects between PAHs exposure and CYP1A1 or GSTM1 gene polymorphism after adjusting the other confounders such as mother age, education,passive smoking,history of pregnancy,residential heating,air ventilating,cooking frequency,pre-pregnancy height and weight,infant sex and gestational age(P=0.034 and P=0.062,respectively).Conclusions1 Taiyuan is heavily polluted by PAHs.2 Several carcinogenic PAHs were detected in almost all the paired mother and newborns.The levels in umbilical cord blood were similar or higher compared to the paired mothers and a correlation could be found between the mothers and newborns.So we suggested that the PAHs levels(especially Pyr) in materal serum may be used to as biomarkers of the level of fetus in utero.3 Residential heating style,cooking frequency and passive smoking were the important factors associated to the PAHs exposure during the pregnancy period. Not only passive smoking and cooking frequency,but also residential heating style and cooking frequency have a joint effect on the 1-OHPy levels.The highest 1-OHPy level exists in the subjects with passive smoking and more cooking frequency or the subjects with coal-stove heating and more cooking frequency.4 Genetic polymorphisms play an important role in modulating the PAHs -metabolizing.5 It demonstrated that there are interactive effects on the fetus growth and development between the prenatal PAHs exposure and metabolic genetic polymorphisms.In summary,the study subjects showed higher exposure levels to PAHs and the fetus may be damaged through the placenta.Metabolic genetic polymorphisms may play a modulating role in the exposure and effective evaluation.Due to the limitation of the cross-section study,we suggest that additional cohort studies with larger samples and more metabolic genetic polymorphisms will be needed to confirm the findings and to explore the mechanism or the relationship.更多还原