Cytotoxicity And Genotoxicity Analysis For Chlorinated Disinfection By-products Of Drinking Water In HepG2 Cells

Objective:Chlorinated disinfection by-products (CDBPs) commonly exist in chlorinated drinking water. Many kinds of CDBPs have been proved to process mutagenic, teratogenic and carcinogenic effect in both toxicological and epidemiological studies, which pose a potential threat on public health. It's urgent to establish the rational and effective integrated test facility to monitor the genetoxicity of the drinking water. In the present study, we used an integrated approach of cytotoxicity assay and cytokinnesis-block micronucleus method (CBMNT) to evaluate the cytotoxicity and genetoxicity of a selected group of CDBPs in HepG2 cells, and thus to explore the feasibility of such an integrated approach in drinking water safety evaluation.Methods:(1) Five CDBPs with high priority (dichloroacetic acid, DCA; trichloroacetic acid, TCA; dibromoacetic acid, DBA; dichloroacetonitrile, DCAN; 3 - chloro -4 - (dichloromethyl) -5 hydroxy -2 (5 H) - furanone, MX) were selected for toxicity evaluation. We used an integrated approach of crystal violet staining assay and CBMNT to evaluate and rank their cytotoxicity and genetoxicity, and analyze the relationship between their cytotoxicity and genotoxicity.(2) The genotoxicity of organic extracts of surface water before and after chlorination disinfection in a waterworks in Wuhan was detected using CBMNT in HepG2 cells. Results:(1) Cytotoxicity of CDBPs in HepG2 cells within 72 hours The rank order in declining of cytotoxicity of five CDBPs was MX> DCAN> DBA> DCA> TCA, the % C ? values (mmol/L) were as follows respectively: 0.090, 0.284, 1.217, 5.067 and 9.335.(2) Genotoxicity of CDBPs in HepG2 cells Compared with solvent control, the lowest concentration inducing a significant genotoxic effect (LCG) of five CDBPs were as follows: MX, 100μmol/L; DCAN, 200μmol/L; DBA, 500μmol/L; DCA, 1000μmol/L; TCA, 5000μmol/L (P <0.05 for all). Based on LCG and C factor (C factor = MN max group / MN solvent control group), the rank order in decreasing of genotoxic activity of five CDBPs was MX> DCAN> DBA> DCA> TCA. This order was the same with cytotoxicity order.(3) Genotoxicity of the water extracts in HepG2 cells Compared with solvent control, the extract of raw water didn't cause significant increase of micronuclei frequencies in HepG2 cells at any concentration (P> 0.05 for all), while the micronuclei frequencies were markedly increased at the highest concentration ( 150ml/ml culture medium) of finished water and tap water (P <0.05 for both). The results suggested that the chlorination disinfection could increase the genotoxicity of surface water.Conclusion:The crystal violet staining assay was proved to be a good method to detect the cytotoxicity of CDBPs. For the stable and accurate results, CBMNT/HepG2 could effectively detect the damage of chromosome caused by exposure to CDBPs and water extract mixtures. So, it's considered to be feasible to use the integrated approach of crystal violet staining cytotoxicity assay and CBMNT in HepG2 cells to evaluate the safety of drinking water.