Cytotoxicity And Genotoxicity Of Halobenzoquinones Disinfection By-products

Chlorinated disinfection by-products?DBPs?inevitably form during the disinfection of drinking water.Halobenzoquinones?HBQs?are a new class of unregulated DBPs in recent years.Previous studies demonstrated that HBQs are more toxic than some regulated haloacetic acids and trihalomethanes.HBQs can produce intracellular reactive oxygen species and induce DNA damage,and it may have potential genotoxicity.Genotoxic studies on mammalian cells with gene mutation,DNA and chromosomal damage as the end point are still insufficient.Thus,we conducted a battery of in vitro assays to determine the cytotoxicity and genotoxicity of six HBQs.This study will provide scientific evidence for health risk assessment and establishment of standard for drinking water.1 Cytotoxicity of six HBQsObjective:The objective of this study is to evaluate the 72 h cytotoxicity of six HBQs.Methods:The cells were seeded into 96-well plates with 1,000 for 24 h,then exposed to six HBQs?2,6-dichloro-1,4-benzoquinone?2,6-DCBQ?,2,5-dichloro-1,4-benzoquinone?2,5-DCBQ?,2,6-dibromo-1,4-benzoquinone?2,6-DBBQ?,2,5-dibromo-1,4-benzoquinone?2,5-DBBQ?,2,6-diiodo-1,4-benzoquinone?2,6-DIBQ?,2,3-diiodo-1,4-benzoquinone?2,3-DIBQ??at different concentrations for 72 h,respectively,with the solvent control?0.5%DMSO?.Then the absorbance values were measured by the microplate reader at 450 nm,and the relative cell viability?%?were calculated.Results:The rank order of cytotoxicity to CHO-K1 cells was as follows:2,3-DIBQ>2,6-DIBQ>2,6-DBBQ>2,5-DBBQ>2,5-DCBQ>2,6-DCBQ.The half lethal doses(IC₅₀)of six HBQs were at 10^-55 M level.And the toxicity of HBQs were more toxic than almost trihalomethanes and most of the haloacetic acids,and were less toxic than most of the haloacetonitriles.IC₅₀0 had a significant correlation with the molar surface area?S??P<0.05?,but had no correlation with the logarithm of the octanol-water partition coefficient?logP?,the electronic properties and quantum mechanical descriptors(E_HOMO,E_LUMO),dipole moment???,molar refractivity?R??P>0.05?.Conclusion:The IC₅₀ of HBQs to CHO-K1 cells ranged from 14.61?M to31.90?M.The cytotoxicity of HBQs was closely related to the physicochemical properties.2 Genotoxicity of six HBQs in CHO-K1 cellsObjective:The aim of this study is to evaluate the genotoxicity of six HBQs by hypoxanthine phosphoribosyltransferase?HPRT?gene mutation assay,single cell gel electrophoresis?SCGE?Assay and cytokinesis-block micronucleus?CBMN?assay.Methods:The CHO-K1 cells were exposed to four different concentrations of six HBQs for 4 h,respectively.The solvent and positive controls were 0.5%DMSO and ethylmethyl sulfone?EMS?,respectively.Then the cells were collected and tested mutagenic effects by HPRT gene mutation assay with 5?g/mL 6-TG.The CHO-K1 cells were exposed to six different concentrations of HBQs.The solvent and positive controls were DMSO and EMS,respectively.After 24h,Tail DNA%,Tail length and Olive tail moment were detected by SCGE assay.The CHO-K1 cells simultaneously treated with 5?g/mL cytochalasin B and different four concentrations of HBQs for 1.5-2 cells cycles.The solvent and positive controls were DMSO and mitomycin C,respectively.Then the number of binucleated cells containing the micronucleus was counted,and the rate of micronucleus,nuclear buds and nuclear bridges were calculated.And analyze the relationship of genotoxicity with cytotoxicity and physicochemical structure parameters.Results:The mutation rates of the six HBQs showed no statistically different with negative control groups?P>0.05?.With the increase of2,6-DCBQ exposure dose,10?M could induced significantly increase in Tail DNA%and Tail length?P<0.05?,and 15?M 2,6-DCBQ could increase Olive tail moment?P<0.05?.1?M 2,5-DCBQ could produce in a significant increase in Tail DNA%,Tail length and Olive tail moment?P<0.05?.However,with the increased doses of 2,6-DCBQ and 2,5-DCBQ,Tail DNA%,Tail length,and Olive tail moment decreased again.The results of remaining 4 HBQs were negative in SCGE assay.2,6-DCBQ,2,6-DBBQ,2,6-DIBQ and 2,5-DCBQ could induced a significant increase in micronucleus rate,nuclear buds rate,and nuclear bridges rate?P<0.05?,and a significant dose-dependent increase was observed.The results of remaining two HBQs by CBMN assay were negative.The rank of genotoxicity was 2,5-DCBQ>2,6-DBBQ>2,6-DIBQ>2,6-DCBQ.The genotoxicity of HBQs had a significant correlation with IC₅₀?P<0.05?,but had no correlations with molar refractivity?R?,the octanol-water partition,the molar surface area?S?,the logarithm of the electronic properties,quantum mechanical descriptors(E_HOMO,E_LUMO)and dipole moment????P>0.05?.Conclusion:The six HBQs did not have mutagenicity in the CHO-K1/HPRT assay system.The CHO-K1/SCGE system showed that2,6-DCBQ and 2,5-DCBQ may induce DNA damage.However,the other 4HBQs have not observed DNA toxicity.CHO-K1/CBMN assay showed that2,6-DCBQ,2,5-DCBQ,2,6-DBBQ and 2,6-DIBQ exhibited chromosomal damage in CHO-K1 cells.The chromosomal toxicity of other 2 HBQs had not been observed.The rank of genotoxicity was 2,5-DCBQ>2,6-DBBQ>2,6-DIBQ>2,6-DCBQ.It was related to the cytotoxicity of HBQs.In general,three genotoxicity assay showed 6 HBQs are suspicious genotoxic agent.