| 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.
|
PDF Full Text Request