| Objective:Copper 8-quinolinolate(Copper 8-quinolinolate,CuQ) is ex- tensively used as a fungicide in many countries and was used as a fungicide for controlling the diseases of freckle, scab, and black spot in vegetables and fruits.In addition,CuQ is approved for use as a wood preservative for control of sapstain, mold and decay in unfinished wood and wood products. Treated wood materials may be used in contact with fruit, vegetables and other foodstuffs in an area such as greenhouses and greenhouse items, produce picking boxes, mushroom trays and vegetables stakes.The carcinogenic risk of PAT to humans is classified in Group3 by the International Agency for Research on Cancer (IARC), since the evidence of carcinogenicity is inadequate in humans and in experimental animals. CuQ has been reported to dose-dependently increased the reverse mutation of S. typhimurium TA97, TA100, TA102 in assay with rat liver S9, and positive response of S9-activated in S. typhimurium TA100 with Ames test and in Bacillus subtilis with rec assay CuQ-induced hepatotoxicity has been ob- served in experimental animals. Therefore, we explore the DNA damage effects of CuQ in Human hepatoma cell line (HepG2) cells and the reaction mechanisms. HepG2 cells were selected as test system, which was orig- inally derived from a human hepatoblastoma. 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. It was considered to be more suitable for reflecting the metabolism of xenobiotics in the human body better than other mammalian cell lines.The aim of this study was to assess the DNA damage effects of CuQ and to elucidate the reaction mechanisms by use of human hepatoma HepG2 cells. Thus it may provide some information for the safety assessment to humans on CuQ.Methods: HepG2 cells were selected as test system. DNA-strand breaks in HepG2 cells were evaluated by single cell gel electrophoresis assay (SCGE). To elucidate the DNA damage mechanism in HepG2 cells, Catalase (CAT) activity was determined and the level of lipid peroxidation was determined by measurement of thiobarbituric acid-reactive substances (TBARS). We measured the effect of CuQ on NF-κB by western blotting analysis. 8-hydroxyderoxyguanosine (8-OHdG), which is a reliable marker for oxidative DNA damage, was also measured by immunoperoxidase stain- ing analysis. We used the O-phthalaldehyde (OPT) to monitor the levels of GSH. DL-buthionine sulphoximine (BSO) were used to modulate the level of GSH in HepG2 cells, and evaluate the effects of GSH on CuQ-indudced DNA damage.Results:Exposure to CuQ caused significant increase of DNA migra- tion in comet assay at all tested concentrations (0.5μM-4μM) in HepG2 cells,suggesting that CuQ caused DNA damage in HepG2 cells. CuQ signi- ficantly decreased levels of glutathione (GSH) and the activity of catalase (CAT) in HepG2 cells and increased levels of lipid peroxidation product thiob-arbituric acid reactive substances (TBARS). Moreover, CuQ signi- ficantly increased accumulation of the p65 subunit of NF-κB into nucleas, the formation of 8-hydroxydeoxyguanosine (8-OHdG). The intracellular GSH level was modulated by pre-treatment with buthionine(S,R) sulf- oximine (BSO),a specific GSH synthesis inhibitor, it was found that depletion of GSH in HepG2 cells treated with BSO dramatically increased the susceptibility of HepG2 cells to CuQ-induced DNA damage.Conclusion: CuQ exerts DNA damage by oxidative stress in HepG2 cells; GSH, as a main intracellular antioxidant, is responsible for cellular defense against CuQ-induced DNA damage. |
