The Genomic Study Of DNA Damage And Repair Induced By Halobenzoquinone Disinfection Byproduct In Human Uroepithelial Cells

Objective:Disinfection of drinking water is one of the most significant achievements of public health since the 20th century,because it has effectively reduced the risk of waterborne diseases.However,the disinfection process has produced unnecessary,even harmful substances,named water disinfection byproducts（DBPs）.People are exposed to DBPs through a variety of ways,including oral,respiratory and skin contact absorption.Epidemiological studies have shown that DBPs are associated with a variety of health risks,such as stillbirth,spontaneous abortion,birth defects,low birth weight,bladder cancer and colorectal cancer.Halobenzoquinones（HBQs）are an emerging class of DBPs that have been found to occur widely in treated tap water and recreational waters at levels up to 300 ng/L.Studies have shown that HBQs have greater cytotoxicity in Chinese hamster ovary（CHO）cells than other regulated DBPs.In addition,HBQ’s cytotoxicity is related to their structures,and the rank orders of cytotoxicity were followed as2,6-diiodo-1,4-benzoquinone（2,6-DIBQ）>2,6-dichloro-1,4-benzoquinone（2,6-DCBQ）>2,6-dibromo-1,4-benzoquinone（2,6-DBBQ）.HBQs have also been predicted as potential bladder carcinogens by quantitative structure-toxicity relationship（QSTR）analysis,but there is still a lack of direct experimental evidence of mutagenicity and carcinogenicity of HBQs.The purpose of this study was to investigate the effects of HBQs on gene expression of DNA damage repair related pathway in human uroepithelial cells（SV-HUC-1）.In addition,the aim of our study is to compare the effects of different halogen substituents on toxicity of HBQs,so as to provide reference for health risk assessment of HBQs,prediction of carcinogenicity of HBQs and formulation of drinking water sanitation standards in the future.Methods:SV-HUC-1 cells were selected as experimental objects,and three kinds of HBQs（2,6-DCBQ、2,6-DBBQ和2,6-DIBQ）were used as the testing compounds.The effect of HBQs on survival rate of SV-HUC-1 cells was detected by CCK-8 kit.The morphological changes of HBQs were observed by living cell imaging system.Flow cytometry was used to detect the effects of HBQs on intracellularγH2AX levels at different concentration and different exposure time.The effect of HBQs on DNA damage was detected by alkaline comet assay.Finally,PCR Array was used to detect the expression levels of 84 target genes related to DNA repair pathway.Results:1.Effects of HBQs on survival rate and morphology of SV-HUC-1 cells.The results of CCK-8 assay showed that the survival rate of SV-HUC-1 cells exposed to HBQs with different concentrations and substituents for 24 h generally decreased with the increase of HBQs dose,indicating a concentration-dependent relationship.The IC₅₀of the three HBQs on SV-HUC-1 cells were 2,6-DCBQ at 137μM,2,6-DBBQ at139μM,and 2,6-DIBQ at 81μM,respectively.After exposure to HBQs for 24 h,the cells in the control group grew well,with long spindle shape,adherent growth and good refraction.With the increase of the concentration of 3 HBQs,the cells gradually shrinked,became round and smaller volume,and refracting power of the cells declined.And there were even floating cells,and cell fragments appeared in the intercellular space.2.Effect of HBQs on cell survival rate of SV-HUC-1 cells treated with IC₁₀,IC₂₀and IC₃₀for 3 h.The results showed that compared with the negative control,there was no significant statistical difference of cell viability in all groups（P≥0.05）.3.Effects of HBQs on DNA damage in SV-HUC-1 cells（Flow cytometry）.The expression ofγH2AX in the nucleus was detected by flow cytometry.The expression ofγH2AX increased with the increase of the concentration of the three HBQs,presenting a concentration-dependent relationship.Compared with the negative control,the difference in their equivalent biological concentration IC₂₀was statistically significant（P<0.05）,therefore,their IC₂₀was used as the concentration of exposure.3 h,6 h,12 h and 24 h were used as the exposure time of HBQs.The results showed that the DNA damage was most obvious when the exposure time of HBQs was 3 h,and with the increase of exposure time,the level ofγH2AX decreased,but was still higher than that of the negative control.Therefore,3 h was chosen as the exposure time of the following experiments.4.Effects of HBQs on DNA damage in SV-HUC-1 cells（Alkaline Comet Assay）.The three HBQs were exposed to SV-HUC-1 cells for 3 h with their respective IC₂₀.The comet assay was used to observe the DNA trailing of SV-HUC-1cells.The cells in three HBQs group had obvious trailing phenomenon compared with that of negative control.The contents of tail DNA,tail length and Olive tail moment were significantly different from those of the negative control,indicating that HBQs have caused double-strand DNA break,but the genotoxicity of HBQs with three different substituents was not significantly different.5.The genomic study of DNA repair in SV-HUC-1 cells.The genotoxic effects of three HBQs on SV-HUC-1 cells were studied based on toxicological genomics.The IC₂₀of each of the three HBQs was selected as the exposure concentration and the cells were exposed to HBQs for 3 h,the significantly differentially expressed genes were obtained through transcriptome analysis,and functional cluster analysis and pathway enrichment analysis were conducted for these genes.The results showed that there were many differentially expressed genes clustered in the pathways related to base excision repair,nucleotide excision repair and homologous recombination repair.In addition,differentially expressed genes were also clustered in the pathways related to mismatch repair,non-homologous terminal junction repair,cell cycle regulation,p53 signaling pathway and DNA splicing.Conclusions:1.HBQs can cause cytotoxicity of human uoepithelial cells,and the rank orders of cytotoxicity were 2,6-DIBQ>2,6-DCBQ≈2,6-DBBQ;2.HBQs can lead to DNA damage in human uroepithelial cells,resulting in genotoxicity,and there was little difference in genotoxicity among the three HBQs;3.HBQs can disturb DNA repair pathways,which mainly affect base excision repair,nucleotide excision repair and homologous recombination repair pathways.