Use Of HGPRT Gene Mutation Assay And SOS/umu Test In The Genotoxicity Risk Assessment Of Drinking Water Disinfection Byproducts

Disinfection of drinking water can result in formation of an array of DBPs (disinfection by-products). Toxicological studies have shown that some of DBPs was mutagenic and induced genotoxic and carcinogenic effect on mammalian. The presence of DBPs, therefore, is a major issue for safe drinking water. Due to the large number of DBPs identified in drinking waters, the full biological effects of the mixtures of DBPs were not reflected in any of the toxicology studies of individual DBPs. Moreover, human health risk evaluation of DBPs mixture exposure using long term carcinogenicity tests and epidemiological studies, still had technical difficulties. Therefore, selecting a battery of short-term genotoxicity tests is of very important meaning for the safty assessment of DBPs mxitrue exposure in drinking water.Objectives:The aim of this study was:(â…°) to evaluate the genotoxicity of 13 DBPs which have been identified to be mutagenic and/or cancinoginic using HGPRT (hypoxanthine-guanine phosphoribosyl transferase) gene mutation assay and SOS/umu test and. to investigate the relationship between the genotoxicity and cytotoxicity induced by DBPs using assays mentined above and microplate-based assay. (â…±) to identify potential genotoxity of the pre-disinfection and post-disinfection water samples by using HGPRT gene mutation assay and SOS/umu test. (â…²) to obtain more information about toxicological features of 13 DBPs and to explore the feasibility of introducing these assays into evaluation system for drinking water safty through the studies above.Methods:1. The cytotoxicity in CHO (chinese hamster ovary) cell and Salmonella typhimurium were measured by using micoplate-based assay. The cells were treated with multiple levels of DBPs for 72hr in CHO and 2hr in Salmonella typhimurium. We measured the absorbances and counted the cell density. The cytotoxic potency was calculated by an equation obtained after the curve fitting. The mutagenic potency of 13 DBPs was measured using CHO/HGPRT assay. In the assay, CHO cells were treated with four different concentrations of DBPs for 4hr and a negative control and a positive control were used at same time for the quality control of the results. Genotoxic potency was shown as the slope of the concentration-response curve. The effect of 13 DBPs on DNA damage was measured using SOS/umu test in Salmonella typhimurium. In this test, a multiple concentrations of DBPs were designed to treat the cells for 2hr. The genotoxicity was considered to be positive if a DPB induced an induction ratio of 2. The genotoxic potency was calculated by an equation obtained after the curve fitting and used to compare genotoxic intencity of 13 DBPs.2. Water samples including the raw water, finished water and tap water were collected from a water plant in January, May and July. Hanjiang River is the water source for this plant. These water samples were acidified (pH=2) with HCl and extrated by XAD-2 resin. In HGPRT assay, the cells were treated with the water extracts at four different concentrations (corresponding to 1.2,6,30 and 150 ml water/ml medium) for 4hr. While in SOS/umu test, the cells were treated with a series of concentrations corresponding to 1 to 2000 ml water/ml medium for 2 hr.Results:1. According to the cytotoxic potency values, the rank order in decreasing cytotoxic activity of 13 DBPs to CHO cells was DBN> IA> BCN> BA> MX> DCN> CN> TCN> DBA> CA> CH> DCA> TCA. The tested DBPs were shown to be mutagenic to CHO cells in HGPRT assay, except for BCN, CN, CH and TCA. We compared the genotoxic potency of 13 DBPs and listed them as follows:MX> DBN> IA> DBA> DCN> TCN> BA> CA> DCA. 2. We found that 13 DBPs cytotoxicity to Salmonella typhimurium were different and the order from the highest toxicity to the lowest toxicity is:MX> IA> DBN> BCN> BA > TCN> DCN> CA> DCA> DBA> CN> TCA> CH. The induction of DNA damage by the 13 DBPs was measured by SOS/umu test and the rank order in decreasing genotoxicity was:MX> DBN> IA> BCN> BA> TCN> DBA> DCA, while CN, DCN, CA, TCA and CH were refractory.3. The CHO/HGPRT assay shown that the extracts from finished water (Jan) and tap water (Jan) increased the HGPRT gene mutant frequency at the highest concentration (150 ml water/ml medium). When compared with the negative control, the lowest concentration resulting in a significant increase of gene mutation was 30 ml water/ml medium for tap water (May). Meanwhile, the extracts from finished water (Jan) and tap water (Jan and May) caused significantly higher levels of mutation than the raw water at the highest concentration (150 ml water/ml medium). However, all extracts from July did not show any genotoxicity in CHO/HGPRT assay. The results presented in SOS/umu test showed that all extracts were genotoxic to Salmonella typhimurium except one sample (raw water, May). The genotoxic potency ranking for the water extracts was as follows:finished water > tap water> raw water (the samples from Jan and May); raw water> tap water> finished water (the samples from July).Conclusions:In sumary, CHO/HGPRT assay and SOS/umu test was well suit for the genotoxiciy analysis of DBPs and water samples. In addition, the two assays can be used to compare the genotoxic intencity quantityly. The cytotoxic potency of 13 DBPs was significantly and highly correlated with their genotoxic potency. By validation for the two assays using high priority DBPs and genotoxicity evaluation of the water samples before and after chlorinated disinfection using these two assays, the results were confirmed to be reliable. Therefore, these two rapid genotoxicity assays were recommended as a tool for the assessment of drinking water safty.