Potassium Bramate-induced Genotoxicity And Possible Involvement Of Oxidative Stress In Human HepG2 Cells

Introduction:Potassium bromate (PB) is a by-product of the use of ozone in drinking water disinfection, and also as a widely used food additive that is employed to improve the quality of flour, by acting as a bleaching agent. Considering the genotoxicity and caicinogenesis of it, PB was banned to be the flour additive in many countries. In 2007, The Chinese government proposed the provisional guideline value of PB 0.01 mg/1 in drinking water what was in accord with the one proposed by WHO in 2004.In 1987 the International Agency for Research on Cancer (IARC) assessed PB as a Group 2B carcinogen. It proved that PB was a kind of compound which could increase the risk of human cancer. PB has been evaluated for its genotoxic effects. However, the mechanisms of the effects are not well understood. PB was able to induce the generation of reactive oxygen species (ROS) and a significant reduction of intracellular GSH. In addition, it reported that the changes of lysosomal membrane stability and mitochondrial membrane potential may be involved in the mechanisms of PB-induced genotoxicity. Therefore, the aim of this study was to assess the genotoxic effects of PB in vitro and to elucidate the mechanism of PB-induced genotoxicity. Since oral exposure is the route of contamination by PB and the liver is the initial site of PB metabolism, HepG2 cell line is considered to be a more suitable system in our study. The HepG2 cell line retains many of the functions of normal liver cells and expresses the activities of several phases I and II xenobiotic metabolizing enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.Methods:HepG2 cells were selected as test system. We used the single cell gel electrophoresis assay (SCGE) and the micronucleus test (MNT) to study the genotoxic effects of PB. To elucidate the oxidative DNA damage mechanism in HepG2 cells, we used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) to monitor the levels of ROS and glutathione (GSH). DL-buthionine sulphoximine (BSO) and hydroxytyrosol (HT) were used to modulate the level of GSH in HepG2 cells, and the effects of GSH on PB-induced genotoxicity were determined by the SCGE and MNT. To further investigate the mechanism of genotoxicity of PB in HepG2 cells, we used Acridine orange (AO) and Rhodamine 123 to measure the changes of lysosomal membrane stability and mitochondrial membrane potential. In addition,8-hydroxyderoxyguanosine (8-OHdG), which is a reliable marker for oxidative DNA damage, was also measured by immunocytochemistry staining analysis. The data were statistically analyzed by SPSS v 11.5 software.Results:In the SCGE and MNT, PB increased the DNA migration and the MN frequencies at the concentrations of 1.56 mM—12.5 mM and 0.12 mM—1 mM, respectively. A statistical significance was not observed in HepG2 cells when the cells exposed with PB (0 mM—12.5 mM) for 40 min. DNA damage induced by PB was significantly reduced a dose-dependent manner in cells pre-treated with HT (12.5μM—50μM) for 30 min in the SCGE assay. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the PB-induced genotoxic effects and that when the intracellular GSH content was elevated by HT, the chromosome damage induced by PB was significantly prevented in the concentrations (0.12 mM—1 mM). Moreover, PB significantly caused 8-OHdG formation and the change of lysosomal membrane stability in HepG2 cells at concentrations from 6.25 mM to 12.5 mM. In addition, the change of mitochondrial membrane potential induced by PB (0 mM—12.5 mM) in the assay.Conclusion:The results suggest that PB caused DNA strand breaks and chromosome damage, which indicate that PB induced genotoxic effects in HepG2 cells. PB- induced increase of 8-OHdG formation was considered as oxidative damage of DNA. PB exerts genotoxic effects in HepG2 cells, probably through the formation of ROS, depletion of GSH, increase of 8-OHdG formation and the change of lysosomal membrane stability, which cause oxidative DNA damage.