【作者】 张黎；

【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， 2011， 博士

【摘要】 饮用水消毒是保证城市供水安全的的重要步骤,但饮用水在消毒过程中会产生大量消毒副产物(disinfection by-products, DBPs)。研究表明,一些DBPs具有明显的致突变性和遗传毒性,或者致癌性。至今已发现的DBPs超过600种,除了氯消毒会生成DBPs,采用臭氧、氯胺、二氧化氯等消毒方式也会产生大量DBPs。在日常生活中,人群对DBPs的暴露呈现长期性、暴露方式多样性、多种DBPs混合暴露的特点。运用流行病学或者慢性哺乳动物致癌试验等方法,评价众多DBPs昆合暴露对人群健康的影响,在现有的技术条件下还难以实现。因此,构建一套短期遗传毒理学试验检测体系,对饮用水中DBPs昆合暴露的遗传毒性进行快速、准确的评价具有重要的现实意义。目的：以单细胞凝胶电泳试验(single cell gel electrophoresis assay, SCGE)为核心技术,以人类来源的肝肿瘤细胞(human-derived hepatoma line, HepG2)为靶细胞,组成SCGE/HepG2测试系统,对饮用水中普遍存在且对人类健康威胁较大的5类共15种DBPs的遗传毒性进行分析和比较,评估该测试系统的检验效能和敏感性;运用SCGE/HepG2测试系统对氯化消毒前后水样提取物的遗传毒性特征及影响因素进行评价分析；探讨该测试系统在饮用水安全性评价体系中的可行性,为其纳入饮用水安全评价体系提供实验依据；应用SCGE/HepG2测试系统结合氧化损伤标志,探讨DBPs引起靶细胞遗传毒性损伤的机制。方法：1、应用SCGE/HepG2测试系统检测15种DBPs对HepG2细胞DNA损伤的作用。15种DPBs包括4种三卤甲烷、6种卤乙酸、3种卤乙腈、1种呋喃酮和1种醛类。每种DBP均设计5个染毒剂量,染毒剂量范围为0.001-10000μmol/l,同时设溶剂对照、阳性对照和空白对照。染毒时间均为4小时。采集细胞DNA迁移图,应用CASP软件分析采集的图像,尾部DNA含量、尾长和Oliver尾矩值作为判断细胞DNA损伤强度的指标。2、应用SCGE/HepG2测试系统评价水体样本的遗传毒性效应。分别于2009年的枯水期和丰水期,采集武汉市以长江和汉江为水源的两个水厂的原水、经氯化消毒处理后的出厂水以及管网末梢水。采用XAD-2大孔树脂对水样进行浓缩提取。应用SCGE/HepG2测试系统分析水样提取物的遗传毒性效应,同时分析水中DBPs的含量。水样提取物设为4个染毒浓度,分别相当于1.2、6、30及150ml水样/ml培养液,染毒时间为24小时。水中DBPs含量分析采用顶空毛细管柱气相色谱法。3、SCGE/HepG2测试系统结合氧化损伤标志探讨DBPs的遗传学损伤机制。选择消毒副产物水合氯醛(chloral hydrate, CH)为目标物,采用慢性细胞毒性试验,检测CH致HepG2的细胞毒性,计算细胞毒性潜力值。在含有或者不含抗氧化剂过氧化氢酶(Catalase)/或丁基羟胺茴香醚(Butyl hydroxylamine Anisole,BHA)的条件下,用SCGE/HepG2测试系统检测CH对HepG2细胞的遗传学损伤效应。测定CH染毒后的细胞内活性氧(Reactive Oxygen Species,ROS)、丙二醛(Malondialdehyde, MDA)、超氧化物歧化酶(Superoxide Dismutase, SOD)和还原型谷胱甘肽(Glutathione, GSH)的含量；CH染毒系列分别为7.8125、15.625、31.25、62.5、125、250、500和1000μmol/l;染毒时间分别为4和24小时。结果：第一部分结果：根据引起HepG2细胞DNA明显损伤的最低浓度,将DBPs遗传毒性的强弱按类别排序如下：①4种三卤甲烷：一溴二氯甲烷(bromodichloromethane, BDCM)>一氯二溴甲烷(dibromochloromethane, DBCM)>溴仿(tribromomethane,TBM)>氯仿(trichloromethane, TCM);②6种卤乙酸：碘乙酸(iodoacetic acid, IA)>溴乙酸(bromoacetic acid, BA)>二溴乙酸(dibromoacetic acid, DBA)>二氯乙酸(dichloracetic acid, DC A)>三氯乙酸(trichloroacetic acid, TCA),氯乙酸(chloroacetic acid, CA)无明显的DNA损伤作用；③3种卤乙腈：二溴乙腈(dibromoacetonitrile, DBN)≈二氯乙腈(dichloroacetonitrile, DCN)>三氯乙腈(trichloroacetonitrile, TCN);④属于呋喃类的3-氯-4-二氯甲基-5-羟基-2(5氢)-呋喃酮(3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, MX)≈TCA;⑤而属于醛类的CH≈DCA。在15种DBPs中,卤乙酸类的IA对细胞DNA的损伤作用最强,BA次之。SCGE/HepG2测试系统可检测出14/15种DBPs的遗传毒性,仅卤乙酸类的CA在测试系统中未显示出遗传毒性。第二部分结果：①汉江、长江水样中均检出TCM和BDCM,二者在消毒后水样中的含量均高于原水,且在枯水期汉江水样中的含量高于同期长江水样；②与溶剂对照相比,汉江水源原水、出厂水、管网末梢水提取物主要在中、高浓度时(30、150ml/ml)诱导HepG2细胞DNA损伤明显增加(P<0.05或P<0.01)；与同浓度原水相比,丰水期汉江水源出厂水提取物在低、中、高浓度(6、30、150ml/ml),以及管网末梢水提取物在低浓度(1.2、6ml/ml),引起DNA损伤明显增加；③与溶剂对照相比,长江水源原水、出厂水和管网末梢水提取物主要在中、高浓度(30、150ml/m1)诱导HepG2细胞DNA损伤明显增加(P<0.05或P<0.01)；与同浓度原水相比,枯水期长江水源出厂水提取物和丰水期管网末梢水提取物在高浓度(150ml/ml),引起DNA损伤明显增加(P<0.05或P<0.01)；④不论是汉江水源还是长江水源,枯水期水样提取物诱导的HepG2细胞OTM值均高于丰水期(P<0.05或P<0.01)；⑤不论是枯水期还是丰水期,汉江水源水样提取物诱导的HepG2细胞OTM值均高于长江水源(P<0.01)。第三部分结果：①消毒副产物CH引起HepG2细胞的％C1/2值为2.36×10-3mol/l;②BHA或者Catalase单独染毒均未引起HepG2细胞的DNA损伤,40μmol/l的CH单独染毒可使HepG2细胞的DNA损伤明显增加。不同浓度的BHA或Catalase与40μmol/1的CH联合染毒时,DNA损伤较之CH单独染毒明显降低(P<0.001)；③在染毒4小时的条件下,CH浓度达500μmol/l以上时,HepG2细胞内ROS生成量明显增加、GSH含量明显下降(P<0.05或P<0.01),但MDA和SOD含量没有明显的影响;④在染毒24小时的条件下,CH浓度达125μmol/l以上时,HepG2细胞内ROS生成量明显增加、GSH含量显著下降(P<0.05或P<0.01)；高浓度的CH(1000μmol/l)可引起细胞内MDA生成量上升和SOD含量下降(P<0.05)；⑤相关分析表明,CH诱导的ROS含量的变化与GSH和SOD含量的变化呈负相关(P<0.05或P<0.01)、与MDA含量的变化呈正相关(P<0.01)；GSH含量的变化与MDA含量变化呈负相关(P<0.01)、与SOD含量变化呈正相关(P<0.01)；MDA含量的变化与SOD含量变化呈负相关(P<0.01)。结论：(1)5类共15种DBPs中,卤乙酸类对HepG2细胞的DNA损伤作用最强。DBPs对细胞DNA的损伤作用还因取代基而不同,碘代DBP的DNA损伤作用高于溴代DBP,溴代DBP对DNA的损伤作用则高于氯代DBP。(2)氯化消毒增加了地表水的遗传毒性；水样提取物的遗传毒性受到水文期和水源因素的影响,枯水期水样提取物遗传毒性高于丰水期,汉江水源水样提取物遗传毒性高于长江水源。(3) SCGE/HepG2测试系统能够快速、有效地检测15种DBPs和水样提取物的遗传毒性,结合氧化损伤标志还可用于DBPs致DNA损伤及氧化损伤机制的探讨。(4)建议将SCGE/HepG2测试系统纳入饮用水的安全评价体系中更多还原

【Abstract】 Disinfection is of unquestionable importance in the supply of safe drinking-water. However, disinfection of drinking water can result in formation of an array of disinfection by-products (DBPs). Many studies indicated that some of DBPs were mutagenic and genotoxic or carcinogenic. So far, more than 600 DBPs have been reported in the literature. DBPs are formed when chemical disinfectants such as chlorine, ozone, chlorine dioxide and chloramines are used during drinking water treatment. Human exposure to DBPs presents the characteristics of long-term, multiple routes and mixed exposure. It is difficult to evaluate the health effects of mixed exposure to DBPs by chronic mammalian carcinogenicity or epidemiological studies now. Therefore, a fast and accurate evaluation of genotoxicity risk to DBPs mixture exposure in drinking water by a set of short-term genotoxicity test systems is needed.Objectives:The aim of this study was:(i) to build SCGE/HepG2 test system, which is composed of single cell gel electrophoresis (SCGE) assay as a core technique and the human-derived hepatoma line (HepG2) as target cells; and to evaluate the performance and sensitivity of SCGE/HepG2 test system through the genotoxicity test of fifteen DBPs which are common in drinking water and have adverse effect to health; (ii) to evaluate the genotoxicity of the extracts of chlorinated drinking water using SCGE/HepG2 test system; and to explore the feasibility of introducing SCGE/HepG2 test system into evaluation system for drinking water safety; (ⅲ) to study the genotoxic mechanism of disinfection by-products CH using SCGE/HepG2 test system and oxidative stress experiments.Method:(ⅰ) The effect of fifteen DBPs on DNA damage in HepG2 was investigated by SCGE/HepG2 test system. These fifteen DBPs include four trihalomethanes (THMs), six haloacetic acides (HAAs), three haloacetonitriles (HANs), one furanone and one aldehyde. HepG2 cells were exposed to each DBP at five concentrations ranged from 0.001 to 10000μmol/l for 4 hours with blank control, solvent control and positive control. The images of DNA migration were analyzed by CASP software. As indicators of DNA damage, tail DNA content, tail length and Olive tail moment (OTM) were analyzed by software.(ⅱ) The genotoxicity of water samples was investigated by SCGE/HepG2 test system. Water samples including raw water, finished water after chlorination disinfection and tap water were collected in winter and summer in 2009 from two water plants which used Han River and Yangtze River as water source. Water samples were extracted by XAD-2 resin. The genotoxicity of the water extracts was investigated by SCGE/HepG2 test system. Meanwhile, the content of DBPs was analyzed. HepG2 cells were exposed to the water extracts at the concentrations of 1.2,6,30 and 150 ml water/ml medium for 24 hours. DBPs analysis was conducted by gas chromatography with headspace capillary column.(ⅲ) The genotoxic mechanism of DBPs was investigated by SCGE/HepG2 test system and oxidative stress experiments. Chloral hydrate (CH) was selected as the target DBP. The cytotoxicity of CH was detected using microplate cytotoxicity assay. The genotoxicity of CH was measured with and without antioxidants (catalase and butylated hydroxyanisole (BHA)) using SCGE/HepG2 test system. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) were measured after HepG2 cells were exposed to CH at the concentrations of 7.8125,15.625, 31.25,62.5,125,250,500 and 1000μmol/l for 4 or 24 hours.Results:Part 1:Based on the minimal DBPs concentration inducing significant DNA damage, the rank order of the DNA damage potency is:(ⅰ) bromodichloromethane (BDCM)> dibromochloromethane (DBCM)> tribromomethane (TBM)> trichloromethane (TCM) of the four THMs; (ii) iodoacetic acid (IA)> bromoacetic acid (BA)> dibromoacetic acid (DBA)> dichloracetic acid (DCA)> trichloroacetic acid (TCA) of the five HAAs; (iii) dibromoacetonitrile (DBN)≈dichloroacetonitrile (DCN)> trichloroacetonitrile (TCN) of the three HANs; (iv) MX and CH showed DNA damage potency similar to TCA and DCA, respectively. IA is the most genotoxic DBP in the fifteen DBPs, followed by BA. Fourteen of fifteen DBPs were shown to be genotoxic using SCGE/HepG2 test system. Chloroacetic acid (CA) was not genotoxic in this test system.Part 2:(ⅰ) TCM and BDCM were detected in water samples of Han River and Yangtze River. The contents of TCM and BDCM in disinfected water and in winter from Han River were higher than that in raw water and in the same season from Yangtze River, respectively. (ii) The genotoxicity results of water samples from Han River are represented as folloews: when compared with the solvent control, the extracts of raw water, finished water and tap water led to a significant increase in DNA damage in HepG2 cells at the concentrations of 30,150 ml water/ml medium (P< 0.05 or P< 0.01); when compared with raw water, a significant increase in DNA damage was caused by the extracts of finished water at the concentrations of 6,30,150 ml water/ml medium, and the extracts of tap water at the concentrations of 1.2,6 ml water/ml medium in summer (P< 0.05 or P< 0.01). (iii) The genotoxicity results of water samples from Yangtze River are represented as folloews: when compared with the solvent control, the extracts of raw water, finished water and tap water led to a significant increase in DNA damage in HepG2 cells at the concentrations of 30,150 ml water/ml medium (P< 0.05 or P< 0.01); when compared with raw water, a significant increase in DNA damage was caused by the extracts of finished water in winter and tap water in summer at the concentration of 150 ml water/ml medium (P< 0.05 or P< 0.01). (iv) The value of OTM caused by water extracts in winter was higher than that in summer (P<0.05 or P<0.01), whatever water samples were from Han River or Yangtze River.(v) The value of OTM e caused by water extracts from Han River was higher than that from Yangtze River (P<0.05 or P<0.01), whatever water sample was collected in winter or summer. Part 3:(ⅰ) The value of %C1/2 of CH for HepG2 cell was 2.36×10-3 mol/1. (ii) Neither catalase nor BHA caused DNA damage in HepG2 cells. When HepG2 cells were exposed to CH at the concentration of 40μmol/l, a significant increase in DNA damage was caused. DNA damage caused by CH was significantly decreased by addition of catalase or BHA (P <0.01). (iii) The content of ROS was increased and GSH was decreased when HepG2 cells were exposed to CH at the high concentrations (500,1000μmol/l) of CH for 4 h (P< 0.05 or P< 0.01); there were no changes for MDA and SOD. (iv) When the cells were exposed to CH at the concentration of 125,250,500,1000μmol/l for 24 h, the content of ROS was increased and GSH was decreased (P< 0.05 or P< 0.01); the content of MDA was increased and SOD was decreased at the highest concentration (1000μmol/l) of CH (P< 0.05). (v) The content of ROS was negatively correlated with that of GSH or SOD (P< 0.05 or P< 0.01), and positively correlated with that of MDA (P< 0.01). The content of GSH was positively correlated with that of SOD (P< 0.05), and negatively correlated with that of MDA (P< 0.01). The content of MDA was negatively correlated with hat of SOD (P<0.01).Conclusion:1. HAAs are the most genotoxic DBPs in the fifteen DBPs. The rank order of the DNA damage potency of DBPs is:iodo-> bromo-> chloro-DBPs across different structural DBP classes.2. Chlorination disinfection may enhance the genotoxicity of surface water. The genotoxicity of water extract may vary according to seasons and water sources. The genotoxicity of the water extract in winter or from Han River is higher than that in summer or from Yangtze River, respectively.3. SCGE/HepG2 test system is a rapid and sensitive tool for evaluating the genotoxicity of DBPs and the water extracts. SCGE/HepG2 test system combined with oxidative stress may be used to investigate the mechanism of genotoxicity of DBPs.4. SCGE/HepG2 test system is recommended as a tool for the assessment of drinking water safety.更多还原