The Role Of Oxidative Stress In Deoxynivalenol-induced DNA Damage In HepG2 Cells

Objective: Deoxynivalenol (DON) is a trichothecene mycotoxin and a cereals contamination, which is found widely in the moldy wheat, barley, maize and flour. It is considered one of the most natural toxins of Fusarium and it is highly contaminated in the feedstuff around the whole world. It is one of the most important Fusarium toxins that may potentially affect human and animal health and productivity, confirmed by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO).The International Agency for Research on Cancer (IARC), considered DON to be a Group 3, which is"not classifiable according to its carcino- genicity to humans". DON was mutagenic in many mammalian cells in vitro using chromosome aberration. The production of lipid peroxidation, malondialdehyde (MDA), was found in vitro. Liver is the major site of DON metabolism. In vitro studies showed genotoxicity of DON and this was partly related to the production of free radicals and of reactive oxygen species (ROS). This study was to assess the role of oxidative stress in deoxynivalenol-induced DNA damage. In this study, we selected a meta- bolically competent human hepatoma line (HepG2), which retains many of the functions of normal liver cells and expresses the activities of several phase I and phase II xenobiotic metabolizing enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.The object of present study is to explore whether DON causes DNA damage in HepG2 cells and to elucidate the underlying mechanism of DON- induced DNA damage. Thus it may provide some information for safety assessment to humans on DON. Method:HepG2 cells were selected as test system. DNA damage induced by DON was assessed by single cell gel electrophoresis (SCGE) assays. Hydroxytyrosol (HT) was used as an antioxidant to protect DNA from damage in the comet assays. To further investigate the mechanisms, we used the 2, 7-dichlorofluorescein diacetate (DCFH-DA) to monitor the levels of reactive oxygen species (ROS). 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.Results: In the comet assay, a significant dose-dependent increase of DNA migration was found after treatment with DON at concentrations ranging from 3.75μM to 30μM. DNA migration was monitored by pre- treatment with hydroxytyrosol (HT) as an antioxidant in comet assay. It was found that DNA migration with pre-treatment of HT was dramatically decreased. The DNA damage induced by DON was almost completely prevented. DON at concentrations from 15μM to 60μM induced the increa- sed levels of ROS. The formation of TBARS was observed in HepG2 cells exposed to DON and with the increasing of DON concentration, the staining intensity of 8-OHdG increased obviously, the doses being 3.75μM~15μM and 7.5μM ~60μM.Conclusion: DON caused DNA damage in HepG2 cells, probably through oxidative stress; HT, as an antioxidant, plays a vital role in the defense against DON-induced DNA damage.