【作者】 周文珊；

【导师】 鲁文清；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2010， 博士

【摘要】 饮用水加氯消毒可杀灭水中病原微生物,有效防止介水疾病的发生和传播,是迄今为止世界范围内最广泛采用的饮水净化消毒方法。然而,在消毒过程中,氯制剂与原水中的有机物质反应可产生大量氯化消毒副产物(chlorination disinfection by-products, CDBPs).毒理学研究显示,一些CDBPs在实验动物中可引起出生动物体重和活产率的降低,心血管系统和神经系统畸形等不良妊娠结局,提示CDBPs可能存在一定的生殖毒性。然而关于CDBPs暴露与低体重、流产、死胎和出生缺陷等的人群流行病学研究并未得到一致性论断。对CDBPs暴露评估的不准确性可能是导致研究结果不一致的主要原因,如何在CDBPs与人类生殖健康关系的研究中更准确评估人群CDBPs的暴露水平成为在开展相关研究时重点需要解决的问题。目的：本研究以新生儿体重这一生殖健康结局作为观察指标,通过对产妇所在区域管网水中多种CDBPs实行按月定点、定时监测、并通过对产妇孕晚期尿三氯乙酸水平的分析,分别从外暴露与内暴露两个方面对产妇怀孕期间CDBPs的暴露水平进行评估,研究其暴露水平对新生儿体重的影响。其中,采用尿三氯乙酸作为内暴露标志物,在CDBPs健康危害的人群流行病学研究中尚属首次。此外,鉴于CDBPs对人类生殖健康影响的分子机制尚不清楚,本文拟对新生儿代谢基因和DNA修复基因的多态性进行筛查,初步探讨CDBPs对人类生殖健康产生影响的分子机制。方法：研究对象均来自于2008年1月至2009年5月期间到武汉某医院妇产科就诊的产妇和新生儿。产妇与新生儿的基本信息均摘抄于医院分娩记录本。研究分三部分展开。第一部分：选择某水厂供水区域为研究范围,根据与水厂距离由近至远分别设置距水厂1km、4km、8km三个采样点,在2008年1月至2009年5月期间对该水厂末梢水中四种三卤甲烷(三氯甲烷、一氯二溴甲烷、二氯一溴甲烷和三溴甲烷)和两种氯代乙酸(二氯乙酸和三氯乙酸)进行按月、定时、定点监测,充分考虑了由时间和空间变化引起的管网水中CDBPs浓度的波动对暴露评估带来的影响。同时选取怀孕期间一直居住在该供水区域的1385名产妇为研究对象,以此外暴露数据为依据,评估产妇孕早期、孕中期、孕晚期、怀孕全程平均暴露水平以及孕早、中期联合暴露水平与新生儿体重的关系。第二部分：采用高效液相色谱方法对398名产妇孕晚期尿中三氯乙酸水平进行检测,并以此作为评价CDBPs暴露的内暴露标志,对产妇孕晚期CDBPs暴露水平与新生儿体重之间的关系进行分析。该方法较以往流行病学研究中仅采用CDBPs外暴露的评估方法更为精确。此外,通过问卷调查,以获得更多的关于产妇用水习惯、经济收入、病史等资料,在充分控制影响新生儿体重混杂因素的同时,对不同途径CDBPs暴露与新生儿体重的关系进行研究。第三部分：采集158名新生儿脐带血并提取其DNA样本,检测其代谢酶CYP2E1(G-1259C)和DNA修复酶hOGG1(Ser326Cys)两种基因多态的分布情况。同时以其母亲尿三氯乙酸水平作为暴露评估依据,分析环境-基因交互作用对新生儿体重的影响,初步探讨CDBPs对人类生殖健康影响的分子机制。结果：本研究第一部分中17个月水样监测结果显示,管网水中CDBPs浓度随末梢水至水厂距离以及时间而波动。在被检测的51份水样中,三氯甲烷范围为3.15-85.75μg/L,二氯一溴甲烷为0.77-12.44μg/L,一氯二溴甲烷为未检出～4.21μg/L,三溴甲烷为未检出～18.15μg/L,二氯乙酸为6.05～28.76μg/L,三氯乙酸为1.93～25.51μg/L。除7月份一份水样的三氯甲烷高于我国生活饮用水卫生标准限值外,其余水样CDBPs含量均在限值以下。以此环境监测数据为依据,我们对产妇怀孕期间CDBPs水平进行了分析评估,结果显示,产妇孕晚期高暴露三氯甲烷和溴代三卤甲烷可能与新生儿体重降低有关,OR值分别为1.82和1.51,95%C1分别为1.10～3.02和1.05-2.17。对产妇孕早期与孕中期联合暴露CDBPs的分析显示,高浓度的二氯乙酸暴露可能导致新生儿体重的减少(OR=1.60,95%CI：1.02～2.47)。本文第二部份中385名产妇尿三氯乙酸水平分布范围为0.9μg/gCr-123.3μg／gCr。产妇尿三氯乙酸水平与新生儿体重的线性模型结果显示,产妇孕晚期尿三氯乙酸水平的增高可能与新生体重降低有关(p=-0.15,95%CI：-0.31--0.01,P=0.04)。同时,研究结果还显示产妇尿三氯乙酸水平与每日平均饮水量呈正相关,且具有统计学意义(β=0.23,95%CI：0.38-14.06,P=0.04)。在本文第三部分环境与基因交互作用的研究中,158名新生儿CYP2E1G-1259C基因多态分布情况为CC=7(4.4%),CG=56(35.4%),GG=95(60.2%)：hOGG1Ser326Cys基因多态分布情况为Cys／Cys=23(14.6%),Ser／Cys=74(46.8%),Ser／Ser=61(38.6%)。在这两种基因多态中,突变型纯合子(CC和Cys／Cys)与其余两种基因型相比新生儿平均体重较低。我们在具有这两种基因不同基因型的人群中,未观察到CDBPs高暴露组与低暴露组之间新生儿体重存在显著性差异,即产妇CDBPs暴露水平与新生儿体重的关系可能不受该两种基因多态的影响。但由于该实验研究对象数量较少,其结果需要在更大人群样本中加以验证。结论：(1)怀孕期间产妇CDBPs暴露水平可能与新生儿出生体重降低有关。孕晚期是胎儿生长的关键时期,本文发现孕晚期产妇高暴露三氯甲烷和溴代三卤甲烷可能会导致新生儿出生体重的减少。产妇孕早、中期联合暴露高水平的二氯乙酸可能引起新生儿体重的减少,提示孕早期和孕中期可能是二氯乙酸对胎儿生长产生作用的敏感时期。(2)产妇尿三氯乙酸水平的增高可能导致新生儿体重减少。尿三氯乙酸可作为内暴露标志评价人群CDBPs的暴露水平。(3)本文未发现代谢酶CYP2E1(G-1259C)和DNA修复酶hOGG1(Ser326Cys)两种基因多态与CDBPs暴露的交互作用对新生儿体重的影响,CDBPs对人类健康产生影响的分子机制尚有待进一步探讨。创新点：(1)对管网水中多种CDBPs的浓度进行监测,历时17个月,监测过程中同时考虑了时间和空间变化对外暴露评估的影响。(2)在人群流行病学领域中,首次采用尿三氯乙酸为内暴露标志评价人群CDBPs暴露水平。(3)CDBPs对人类健康影响的分子机制尚未阐明,本研究从一相代谢酶(CYP2E1)和DNA修复酶(hOGG1)两种基因多态性入手,初步探讨CDBPs对生殖健康影响的分子机制。更多还原

【Abstract】 Chlorination is applied to kill pathogenic microorganisms in drinking water and has been widely used over the world to reduce the incidence of waterborne diseases. However, chlorination disinfection by-products (CDBPs) are formed when disinfectants used in water treatment plants react with natural organic matter present in the source water. Several of the halogenated by-products have been evaluated in animal studies and showed evidences of their potential to induce adverse reproductive health outcomes, such as reductions in birth weight and live birth, and congenital malformations of the cardiovascular and neurological systems. In epidemiological studies, the relationship between CDBPs exposure and reproductive health outcomes such as low birth weight, pregnancy loss, still birth and birth defects remains unclear, mainly owing to limitations in the exposure assessment in most studies. How to make CDBP exposure measurement more accurate has become a major problem when conducting CDBPs health effects studies.ObjectivesWe conducted this study to investigate the relationship between CDBPs exposure and reduced birth weight. Two means of exposure assessment, external and internal exposure measurement, were adopted_in our study. Several of CDBPs levels in the pregnant women living_in a same area were monitored at three locations in the middle of each month to collect the data for external exposure assessment and we also examined the trichloracetic acid (TCAA) levels in the first morning urine obtained from the pregnant women, and these levels were used as biomarker of CDBPs internal exposure. Besides, our study is the first one to use urinary TCAA as biomarker for CDBPs exposure in epidemiological studies. The molecular mechanism by which CDBPs may influence human reproductive health is not well understood and in order to investigate the mechanisms, we analyzed the association between CYP2E1G1259C and hOGG1Ser326Cys genotype in newborns and CDBPs’health effects.MethodsPregnant women and newborns in our study were recruited form one hospital in Wuhan during the period from Jan.2008 to May 2009. Their basic information such as maternal age, education, disease history, gestational duration, and newborn’s birth weight were obtained from the hospital birth records.In our first part, the pregnant women were recruited from one area where the water supply was from a single water plant. Water samples were collected from 3 locations:point 1 (about lkm from the plant), point 2 (about 4 km from the plant), and point 3 (about 8 km from the plant). During the period from Jan 2008 to May 2009, water samples were monthly collected from the these 3 points at fixed intervals, and the parameters including four halomethanes (chloroform, chlorodibromomethane, bromodicloromethane and bromoform) and two chloro-haloacetic acids (trichloracetic acid and dichloroacetic acid) were obtained from these samples. Both time and distance had taken into account when we collected this data. We selected 1385 pregnant women who were living in this area during their pregnancy and their trimester-specific CDBPs exposure assessment was based on this environmental data. Then we analyzed the relationship between CDBPs exposure during the pregnancy and their newborns’birth weight among these 1385 women.A number of 398 pregnant women were participated in our second part study and we had examined the urinary trichloracetic acid levels in their first morning urine, and this was used as internal exposure biomarker. This exposure assessment is more accurate than that used in the previous epidemiological studies. The association between maternal urinary trichloracetic acid levels and fetal birth weight was investigated in this part. In addition, we conducted a questionnaire survey to collect the vital information such as maternal water-use behaviors, family incomes, and reproductive disease history to control the birth weight confounders and we also investigate the effects of multi-route exposure to CDBPs.In the third part, we collected the cord blood of 158 newborns for DNA isolation. Taqman technique was adopted to genotype the CYP2E1 (G-1259C) and hOGGl (Ser326Cys) polymorphisms and maternal urinary trichloracetic acid levels were used for CDBPs exposure assessment. With these results, we conducted the gene-environment interaction analysis and try to find out the molecular mechanism of CDBPs.ResultsOur drinking water monitor lasted for 17 months and the CDBPs levels from month to month and from one point to another. Among the 51 water samples, the concentration ranges are:3.15～85.75 mg/L for chloroform,0.77～12.44 mg/L for chlorodibromoethane, LOD (limit of detection)-4.21 mg/L for bromodicloromethane, LOD～18.15 mg/L for bromoform,6.05-28.76 mg/L for dichloroacetic acid and 1.93～25.51mg/L for trichloracetic acid. The levels of CDBPs met the national standards for drinking water quality_except for the chloroform in one sample in July 2008. Based on this environmental data, we estimated maternal exposure levels and we found that high exposure to chloroform and bromo-trihalomethanes during the last trimester may result in increased risk in reduced birth weight (OR=1.82,95CI: 1.10～3.02; OR=1.51,95CI:1.05～2.17, respectively). Besides, exposure to high level of dichloroacetic acid during the first two trimesters could also lead to reduction of birth weight, with an OR of 1.60 (95CI:1.02～2.47).The urinary trichloroacetic acid levels among 398 women ranges from 0.9～123.3μg/gCr. Our results showed that high level of urinary trichloroacetic acid in the late pregnancy may be associated with reduced birth weight (β=-0.15,95%CI: 0.31～-0.01, P=0.04). In addition, we also observed that there was a positive correlation between urinary trichloroacetic acid levels and water consumption(β=0.23, 95%CI:0.38-14.06, P=0.04).In our gene-environment interaction study,158 newborns were genotyped for the CYP2E1 G1259C and hOGG1 Ser326Cys polymorphisms and the distribution of the polymorphism is:CC=7 (4.4%), CG=56 (35.4%), GG=95 (60.2%) forCYP2E1 and Cys/Cys=23(14.6%), Ser/Cys=74(46.8%), Ser/Ser=61(38.6%) for hOGGl. Compared with the newborns with heterozygous and wild type homozygous, the mean birth weight of newborns with the CC genotype for CYP2E1 or Cys/Cys genotype for hOGG1 were lower than others. However, when we divided newborns into two groups, wild type homozygous and other genotypes, we did not find any differences in birth weight between high and normal exposure groups, which indicated that these two polymorphisms may not relate to CDBPs effects on birth weight. But the sample size in our study is a bit small and our results need to be confirmed in large scale studies.ConclusionIn conclusion, our results showed evidences that exposure to CDBPs during pregnancy may relate to reduced birth weight. Many scientists consider the last trimester as an important period for fetal growth, and our study found that high exposure to chloroform and bromo-trihalomethanes may lead to reductions in birth weight. In addition, exposure to high levels of dichloroacetic acid during the first two trimesters may also result in reduced birth weight, which indicated that fetal growth during the first and second trimester may be more sensitive to dichloroacetic acid. Meanwhile, by using urinary trichlroacetic acid as the internal biomarker, we observed that high exposure to CDBPs can lead to reductions in birth weight. In our third part, we did not found the gene-environment interaction effects of CYP2E1 (G-1259C) andhOGG1 (Ser326Cys) polymorphism and the molecular mechanism of CDBPs still need to be studies in the future.The new ideas(1) We conducted a 17 months study to monitor several CDBPs concentrations distribution systems and during our study both the time and geographic variations were taken into account.(2) Our study is the first one to use urinary trichloroacetic acid as the biomarker for CDBPs exposure in epidemiological studies.(3) The molecular mechanism through which CDBPs may influence human reproductive health is still unclear. We studied the polymorphism of phaseⅠmetabolism enzymes (CYP2E1) and DNA repair enzymes (hOGGl) to investigate the molecular mechanism of CDBPs.更多还原