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1、附件2论文中英文摘要作者姓名：徐苑苑论文题目：饮水型砷暴露人群砷甲基化模式及其与机体氧化应激状态关系的研究作者简介：徐苑苑，女，1980年11月出生，2006年9月师从于中国医科大学公共卫生学院孙贵范教授，于2009年6月获博士学位。中 文 摘 要目的砷及其化合物是国际癌症研究机构（IARC）确认的人类致癌物。长期饮用高砷水可导致砷性皮肤损伤及皮肤癌，并与全身其他多组织器官的肿瘤、心血管疾病、糖尿病及儿童智力损伤的发生有关。目前，全球约有2亿人因长期饮用高砷水而面临健康威胁。然而，高砷暴露人群的砷中毒易感性有所不同。作为一种外来化合物，砷在体内的代谢转化与其毒性的发生发展关系密切。饮用水中的砷
2、主要是无机砷（iAs），iAs在人体内发生以甲基化为主的生物转化，可产生多种具有不同毒性和靶器官的代谢产物。流行病学调查发现，对砷进行二甲基化能力较强或尿中一甲基砷（MMA）相对含量较少的人群患砷暴露相关疾病的风险较低，这提示砷中毒易感性与砷甲基化模式有关。因此，砷甲基化模式的研究对于进一步探索砷中毒的发病机制和预防砷中毒的发生都具有重要意义。氧化应激学说是砷中毒发病机制的重要理论之一。砷甲基化是否会影响机体的氧化应激状态，是否通过对氧化应激状态的影响而导致人群砷中毒易感性差异，尚缺乏相关报道。本研究通过对我国亚急性和慢性饮水型砷暴露人群的调查研究，分析不同特征人群砷甲基化特点，探讨谷胱甘肽硫
3、转移酶-1（GSTO1）和谷胱甘肽硫转移酶-2（GSTO2）基因多态性对砷甲基化的影响。同时，以人群血抗氧化物和尿DNA氧化损伤标记物反映砷暴露对机体氧化应激状态的影响，并分析机体氧化应激状态与砷甲基化模式和GSTO1、GSTO2基因多态性之间的联系。方 法1、研究对象（1）亚急性砷暴露人群：76名研究对象来自2004年12月辽宁省阜新市某铜冶炼厂因排污管破裂污染饮用水 水砷含量为（48.5 4.3）mg/L 引发亚急性砷中毒事件时，阜新市中心医院收治的患者。（2）慢性高砷暴露人群：研究对象来自内蒙古呼和浩特市周边3个不同饮用水砷浓度的高砷暴露村（乃莫板、口肯板和什力格图）。3村均采用集中供水
4、方式为本村居民提供生活用水，供水水源为各自村中的深井，井水砷浓度分别为0.09 mg/L、0.16 mg/L和0.24 mg/L。已知高砷暴露时间分别为7年、7年、6年。参与研究的调查对象为：0.16 mg/L砷暴露组108人，0.09 mg/L砷暴露组100人，0.24 mg/L砷暴露组72人。（3）慢性低砷暴露（对照）人群：来自内蒙古呼和浩特市土默特左旗田家营村，该村亦为集中供水，水砷浓度0.02 mg/L，低于中国农村饮用水砷最高允许浓度0.05 mg/L。2、流行病学调查亚急性与慢性砷暴露比较分析人群：亚急性砷中毒病人资料从阜新市中心医院获得。对与之比较的慢性砷暴露人群采用横断面调查，
5、收集人群饮水方式、饮水量、疾病史等信息。慢性饮水型砷暴露人群砷甲基化模式与氧化应激状态分析研究对象：通过调查问卷获得研究对象的吸烟、饮酒、饮食习惯、饮水方式与每日饮水量、疾病史等信息。由专业医师对调查对象进行体检，并依据中国砷中毒诊断标准（WS/T211-2001）进行砷中毒症状检查。3、样品采集（1）尿液样品：采集亚急性砷中毒患者在收治入院未进行治疗时的尿样；采集慢性砷暴露人群的即时尿样。上述样品均放置于0-4 冰盒中运送回实验室，-80 保存待测。（2）血液样品：采集人群空腹静脉血5 ml，加入肝素包被的抗凝管，根据不同实验目的现场分装，于液氮中保存运送回实验室，-80 保存待测。4、尿形
6、态砷分析尿液样品与2 mol/LNaOH等体积比混合，于100 恒温消化3 h，采用冷井捕集-氢化物发生-原子吸收分光光度法测定样品中iAs、MMA、二甲基砷（DMA）和三甲基砷（TMA）含量，并以各形态砷含量之和作为总砷（TAs）含量。以苦味酸法检测尿中的肌酐（Cr）水平以校正尿砷含量。用尿中不同形态砷所占总砷百分比（iAs%、MMA%和DMA%）和2个砷甲基化率指标 一甲基化率（FMR）和二甲基化率（SMR） 反映机体砷甲基化模式。5、基因型分析采用聚合酶链式反应-限制性片断长度多态性（PCR-RFLP）方法检测GSTO1基因A140D和GSTO2基因N142D的基因多态性。6、全血还原型
7、谷胱甘肽（GSH）含量与超氧化物歧化酶（SOD）活性测定以5,5-二硫代-2-硝基苯甲酸（DTNB）法测定全血GSH含量；采用亚硝酸盐形成法测定SOD活性；以氰化物高铁血红蛋白形成法测定血红蛋白水平，对血GSH含量和SOD活性进行校正。7、尿8-羟基-2-脱氧鸟苷（8-OHdG）水平测定采用酶联免疫吸附法测定尿8-OHdG含量，以尿Cr水平对其进行校正。8、统计分析用SPSS 11.0对数据进行统计分析。对自身不服从正态分布并且转化后依然不服从或近似正态分布的数据采用非参数检验：2组间比较采用Mann-Whitney U 检验；3组间比较采用Kruskal-Wallis H检验。对转化后服从或
8、近似正态分布的数据进行参数检验：采用单因素方差分析和LSD法比较2组以上数值变量间是否具有统计学差异；采用t检验比较2组间数值变量是否具有统计学差异。应用多元线性回归模型，分析机体氧化应激状态与砷甲基化能力的联系强度和相关性。结 果1、亚急性砷暴露与慢性高砷暴露和慢性低砷暴露（对照）人群砷甲基化模式比较不同类型砷暴露人群中均发现尿形态砷构成比均存在明显的个体差异；尿中各种形态砷和TAs含量在亚急性砷中毒人群中最高，在慢性低砷暴露人群中最低，且各组间差异均具有统计学意义（P 慢性高砷暴露人群 慢性低砷暴露人群；而DMA%、FMR和SMR在3组人群中从高到低为：慢性低砷暴露人群 慢性高砷暴露人群
9、亚急性砷暴露人群。上述指标在各组间差异均有统计学意义（P 0.05）。2、亚急性与慢性高、低砷暴露人群尿8-OHdG水平比较亚急性砷暴露人群尿8-OHdG水平显著高于慢性高砷和低砷暴露人群（P 0.05），慢性高砷暴露人群尿8-OHdG水平显著高于慢性低砷暴露人群（P 0.09 mg/L砷暴露组 0.02 mg/L砷暴露组，且各组间差异均有统计学意义（P 0.05）。0.16 mg/L和0.09 mg/L2个高砷暴露组人群尿iAs%和MMA%均显著高于0.02 mg/L低砷暴露组（P 0.05），而DMA%、FMR和SMR均显著低于0.02 mg/L砷暴露组（P 0.05）；0.16 mg/L
10、砷暴露组人群仅尿DMA%和SMR显著高于0.09 mg/L砷暴露组（P 0.05），儿童尿MMA%均显著低于同组成人（P 0.05），而DMA%和SMR均显著高于同组成人（P 0.05）。0.02 mg/L低砷暴露组儿童尿TAs含量显著高于成人（P 0.05）。5、男性与女性尿砷含量与砷甲基化模式的比较同一砷浓度暴露条件下，未见男性与女性在尿形态砷含量和砷甲基化模式上存在统计学差异（P 0.05）。6、砷暴露时间对尿砷含量和砷甲基化模式的影响0.16 mg/L砷暴露组25名儿童和39名成人在砷暴露7年和9年尿TAs含量均无显著差异（P 0.05）。儿童与成人砷暴露9年时尿iAs%和MMA%均显
11、著高于砷暴露7年（P 0.05），而DMA%、FMR和SMR均显著低于砷暴露7年（P 0.05）。砷暴露7年时，尿iAs%具有显著差异（P 0.05）的3组人群在砷暴露9年时尿iAs%差异无统计学意义；砷暴露2年前后，机体相对砷甲基化模式发生改变。7、砷甲基化模式的家庭相关性各形态砷百分比和2次砷甲基化率在兄弟姐妹和父母子女间均呈现显著相关性（P 0.05）。9、砷暴露对血GSH含量和SOD活性的影响0.16 mg/L砷暴露组儿童与成人血GSH含量均显著低于0.09mg/L和0.02 mg/L砷暴露组儿童与成人（P 0.05）；同一砷暴露组内，仅0.09 mg/L砷暴露儿童与成人血GSH含量差
12、异具有统计学意义（儿童 成人，P 0.05）。0.16 mg/L砷暴露组儿童与成人血SOD活性均显著低于0.09 mg/L砷暴露组（P 0.05），0.09 mg/L砷暴露组儿童和成人SOD活性均高于0.02 mg/L砷暴露组（P值分别为 0.05）。10、砷暴露对人群尿8-OHdG水平的影响不同砷浓度暴露组成人尿8-OHdG水平比较：0.16 mg/L砷暴露组 0.09 mg/L砷暴露组 0.02 mg/L砷暴露组，且各组间差异具有统计学意义（P 0.05）；0.16 mg/L和0.09 mg/L砷暴露组间儿童尿8-OHdG水平无显著差异，但均显著高于0.02 mg/L砷暴露组（P 0.05
13、）。高砷暴露9年时，儿童与成人尿8-OHdG水平均显著高于砷暴露7年（P 0.05）。结论1、人体的砷甲基化能力随砷暴露剂量率升高或暴露时间延长而降低；同样砷浓度暴露水平下，砷甲基化模式无显著性别差异，儿童的砷甲基化能力高于成人，机体的砷甲基化模式与GSTO1 A140D或GSTO2 N142D的基因多态性无关。除遗传因素外，外源性因素对砷甲基化模式的影响不容忽视。2、高砷暴露可使人体抗氧化能力下降，DNA氧化损伤加重，且砷所致DNA氧化损伤可随砷暴露剂量率的升高或暴露时间的延长而加重。3、砷暴露人群的氧化应激状态与砷甲基化模式关系密切，这可能是砷甲基化模式多样性影响砷中毒易感性的重要原因。关
14、键词： 砷中毒；砷甲基化；谷胱甘肽硫转移酶-1；谷胱甘肽硫转移酶-2；氧化应激；还原型谷胱甘肽；超氧化物歧化酶；8-羟基-2-脱氧鸟苷Study on Arsenic Methylation Pattern and Its Association with Oxidative Stress Status in Populations Exposed to Arsenic in Drinking WaterXu YuanyuanABSTRACTObjectiveArsenic has been identified as a human carcinogen by the Internation
15、al Agency for Research on Cancer (IARC). The long exposure to arsenic in drinking water can cause the classical dermal stigmata and even skin cancers, and also is related to the development of cancers of several organs, cardiovascular diseases, diabetes and the impairment of intelligence in children
16、. It is estimated that 200 million people are being under the threat of high arsenic in drinking water in the world. However, the susceptibility to arsenicosis is different in subjects exposed to high arsenic. Arsenic is a xenobiotic, and the metabolism of arsenic in the body may be closely related
17、to the development of its toxicity. The prime biotransformation of inorganic arsenic (iAs) in human bodies is methylation, which results in arsenic metabolites different in toxicity and target organs. Epidemiological studies have suggested that subjects with higher secondary arsenic methylation capa
18、city or lower relative content of monomethylated arsenic (MMA) in the urine have lower risk of arsenic-related diseases. The susceptibility to arsenicosis is at least partly related to the pattern of arsenic methylation. Thus, the study on factors influencing arsenic methylation is essential to the
19、further study on the mechanism and the prevention of arsenicosis. Oxidative stress is one of the most important theories in the mechanism of arsenicosis. Could arsenic methylation pattern affect the status of oxidative stress? Is it possible that arsenic methylation pattern related to the susceptibi
20、lity to arsenicosis through oxidative stress? These answers remain unclear. The present study was conducted in subjects exposed to arsenic through drinking water subacutely and chronically in China. Arsenic methylation pattern among different kinds of populations, as well as effects of polymorphisms
21、 of glutathione S-transferases omega 1 (GSTO1) and glutathione S-transferases omega 2 (GSTO2) genes on arsenic methylation pattern, were studied. In addition, the status of oxidative stress status for study subjects were assessed on the bases of antioxidants in blood and oxidants DNA lesions in urin
22、e. Associations of oxidative stress with arsenic methylation pattern and polymorphisms of GSTO1 and GSTO2 were also analyzed. Methods1. Study subjects(1) Subacute arsenic-exposed subjects: 76 subjects were from patients admitted to the Centre Hospital of Fuxin in accidental subacute arsenic poisonin
23、g of Fuxin in December, 2004. In the accident, pollution of drinking water was caused by the leakage of arsenic-containing waste from the drain pipe in a copper-smelting factory. The concentration of arsenic in the polluted well water was (48.5 4.3) mg/L. (2) Chronic high arsenic-exposed subjects: T
24、he subjects were from 3 high arsenic-exposed villages (Naimoban, Koukenban and Shiligetu) near hohhot in Inner Mongolia, China. In these villages, centralized tap-water systems were established and supplied water to all villagers for daily life for at least 6 years. Unfortunately, the tap-water cont
25、ained high concentrations of arsenic, as much as 0.09 mg/L, 0.16 mg/L and 0.24 mg/L, respectively. 100 subjects in 0.09 mg/L arsenic-exposed group, 108 subjects in 0.16 mg/L arsenic-exposed group, and 72 subjects in 0.24 mg/L arsenic-exposed group were recruited in this study. (3) Chronic low arseni
26、c-exposed (control) subjects: The subjects were from Tianjiaying village around Hohhot in Inner Mongolia, China. Centralized tap-water with 0.02 mg/L arsenic, less than the maximum allowable concentration of arsenic in drinking water (0.05 mg/L) in rural of China, was provided in this village.2. Epi
27、demiological investigationStudy on the subacute and chronic arsenic-exposed subjects: data on subacute arsenic-exposed subjects were obtained from the Central Hospital of Fuxin. Cross sectional studies were conducted in the chronic arsenic-exposed subjects compared with the subacute exposed, in whic
28、h data on drinking method, daily ingestion of water, disease history were collected. Study on arsenic methylation and oxidative stress of subjects chronically exposed to high arsenic in drinking water: Data on age, sex, smoking, drinking, dietary habits, daily water ingestion, medical history and ot
29、hers were obtained by questionnaire. Trained doctors conducted detailed physical examinations and arsenicosis identification according to the Diagnosis Standards on Arsenicosis of China (WS/T211-2001). 3. Sample collection(1) Urine samples: Urine samples of subacute arsenic-exposed subjects were col
30、lected prior to any therapeutic intervention after they were admitted to the hospital. Spot urine samples of chronic arsenic-exposed subjects were collected. All urine samples were shipped to lab in 0-4 ice box and kept in -80 before analysis.(2) Blood samples: 15 ml of fasting venous blood was take
31、n from the study subjects, inoculated into anticoagulated tubes (containing heparin) and aliquoted. Then the samples were shipped to lab in liquid nitrogen and kept in -80 before analysis.4. Determination of arsenic metabolitesUrine samples were mix with 2 mol/L NaOH (1/1, v/v) and digested at 100 f
32、or 3 h. Then cold trap hydride generation-atomic absorption spectrometry were applied to determine the content of iAs, MMA, dimethylated arsenic (DMA) and trimethylated arsenic (TMA). The total arsenic (TAs) content was calculated by summing up the content of all above arsenicals. The levels of urin
33、ary creatinine (Cr) determined with jaffe assay were used to correct the concentrations of arsenic in the urine. Proportions of urinary arsenicals (iAs%, MMA% and DMA %) and the 2 arsenic methylation ratios first methylation ratio (FMR) and secondary methylation ration (SMR) were used to assess arse
34、nic methylation capacity of the body. 5. Genotype analysisPolymerase Chain Reaction/Restriction fragment length polymorphism (PCR/RFLP) was used to detect polymorphisms of GSTO1 and GSTO2 genotypes. 6. Determination of redued glutashione (GSH) content and superoxide dismutase (SOD) activity in blood
35、5,5-dithiobis-2-nitrobenzoic acid (DTNB) method and nitrite-generating method were used to determine GSH content and SOD activity in blood, respectively. levels of haemoglobin were assayed to correct GSH content and SOD activity。7. Determination of urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) levelsE
36、nzyme linked immunobsorbentassy (ELISA) Kit was applied for the determination of urinary 8-OHdG levels. Urinary Cr levels determined with jaffe assay were used to correct urinary 8-OHdG levels.8. Statistical analysisStatistical analysis was conducted by using the SPSS software (version 11.0). Nonpar
37、ametric tests were applied to data which were not fit or approximately fit normal distribution even after transformation. Mann-Whitney U test and Kruskal-Wallis H test were used to determine the statistical significance for the differences between 2 and 3 groups, respectively. Parametric tests were
38、applied to data which were fit or approximately fit normal distribution after transformation. One-way ANOVA and LSD test were performed to determine the statistical significance for the differences among 3 groups. T-test was used to determine the statistical significance for the differences between
39、2 groups. Multiple linear regression analyses were applied to assess the associations of oxidative stress status with arsenic methylation pattern.Results1. Comparison of arsenic methylation pattern among subacute, chronic high and low arsenic-exposed subjectsIndividuals in subacute and chronic arsen
40、ic-exposed groups were all different in concentrations and proportions of urinary arsenic metabolites. Concentrations of urinary arsenicals and TAs were highest for subacute arsenic-exposed subjects and lowest for chronic low arsenic-exposed subjects, and the differences between groups were statisti
41、cally significant (P chronic high arsenic exposed subjects chronic low arsenic-exposed subjects; The order of DMA%, FMR and SMR values, from highest to lowest, was as follows: chronic low arsenic exposed subjects chronic high arsenic exposed subjects subacute arsenic-exposed subjects. All above diff
42、erences between groups were statistically significant (P 0.05).2. Comparison of urinary 8-OHdG levels among subacute, chronic high and low arsenic-exposed subjectsThe levels of urinary 8-OHdG were highest for subacute arsenic-exposed subjects and lowest for chronic low arsenic-exposed subjects, and
43、the differences were statistically significant (P 0.09 mg/L-arsenic-exposed subjects 0.02 mg/L-arsenic-exposed subjects, and the differences were statistically significant (P 0.05). The values of urinary iAs% and MMA% were significantly higher, whereas the values of DMA%, FMR and SMR were significan
44、tly lower (P 0.05) in 0.16 mg/L and 0.09 mg/L high arsenic-exposed subjects, compared with 0.02 mg/L low arsenic-exposed subjects. Only the values of urinary DMA% and SMR were significantly different between 0.16 mg/L and 0.09 mg/L arsenic exposed groups, both of which were higher for the 0.16 mg/L-exposed (P 0.05); however, children were significantly lower