Possible Involvement Of Oxidative Stress In Patulin-induced Genotoxicity In Human HepG2 Cells

Introduction: Patulin (PAT),is a mycotoxin produced by several species of Penicillium, Aspergillus and Byssochlamys, with the most com- monly encountered species being Penicillium expansum. PAT is a frequent contaminant of ripe apples and apple products such as apple juice and cider. Concerns about the toxin properties of patulin have led various countries and the World Health Organization to establish 50μg/L as the recommended limit in apple juice.The carcinogenic risk of PAT to humans is classified in Group 3 by the International Agency for Research on Cancer (IARC), since the evidence of carcinogenicity is inadequate in humans and in experimental animals. PAT has been evaluated for its genotoxic effects. However, the mechanisms of the effects are not well understood. PAT caused the increase or decrease of intracellular glutathione (GSH) level. GSH is a main intracellular antioxi- dant. So we explore the role of oxidative stress in PAT-induced genotoxicity. Since oral exposure is the route of contamination by PAT, HepG2 cell line is considered to be a more suitable system for detecting the PAT–induced DNA damage in vitro.The HepG2 cell line was originally 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 genotoxic effects of PAT and to elucidate the underlying mechanism of PAT-induced DNA damage. We hope the results could give some useful information for the safety asse- ssment to humans on PAT.Methods: HepG2 cells were selected as test system. We used the single cell gel electrophoresis assay (SCGE) in addition to the micronucleus test (MNT) to study the genotoxic effects of PAT. To elucidate the underlying mechanism of PAT-induced genotoxicity, DL-buthionine sulphoximine (BSO) and N-acetylcysteine (NAC) were used to modulate the level of GSH in HepG2 cells, and the effects of GSH on PAT-indudced genotoxicity were detrmined by the SCGE and MNT. We used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and 0-phthalaldehyde (OPT) to monitor the levels of reactive oxygen species (ROS) and GSH. We evaluated the level of lipid peroxidation by measurement of thiobarbituric acid-reactive substances (TBARS). In addition, 8-hydroxyderoxyguanosine (8-OHdG), which is a reliable marker for oxidative DNA damage, was also measured by immuno- peroxidase staining analysis. We measured the level of p53 by western blotting analysis. The data were statistically analyzed by SPSS v 11.5 software.Results: In the SCGE and MNT, PAT increased the DNA migration and the MN frequencies at the concentrations of 20μM—40μM and 0.38μM—0.75μM, respectively. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the PAT-induced genotoxic effects and that when the intracellular GSH content was elevated by NAC, the chromosome damage induced by PAT was significantly prevented in our test concentrations (0.19μM—0.75μM). The formation of TBARS was observed in HepG2 cells exposed to PAT. With the increasing of PAT concentration, the staining intensity of 8-OHdG increased obviously. PAT- induced the accumulation of p53 protein was observed at the concentration of 0.75μM by western blotting in HepG2 cells.Conclusion: PAT could be genotoxic to HepG2 cell. PAT exerts genotoxic effects in HepG2 cells, probably through the formation of ROS and depletion of GSH, which cause oxidative DNA damage. P53 protein plays important roles in the defense against PAT-induced DNA damage.