Genotoxic Effect And Oxidative DNA Damage In HepG2 Cells Exposed To Sudan IV

Introduction: Sudan IV (C24H20N4O), a synthetic lipid soluble azo pigment, is widely used in various industrial fields. They had also been adopted for coloring various food- stuffs, particularly in those containing chilli powders, because of their intense dark red color.In 1975, the international agency for research on cancer (IARC) assessed Sudan IV as group 3 carcinogen, for which the evidence of carcinogenicity was inadequate in humans and inadequate or limited in experimental animals. Sudan IV is mutagenic, but only after chemical reduction and metabolic activation. Reduction can take place by means of reductase of the gastrointestinal microflora and also through microsomal and cytosolic reductase of the liver and extra-hepatic tissues. The toxic and/or carcinogenic effects of Sudan IV in the gut or liver are possibly due to the products into which they are degrades. Sudan IV is a liver-specific toxin known as a carcinogen in experimental animals. but neither the relevance of these tumours to humans nor their mechanisms of induction is clear.In this study, we selected a metabolically 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 objective of the study was to assess the genotoxic effects of Sudan IV in vitro and to elucidate the mechanisms in these cells. Thus it may provide some information for safety assessment to humans on Sudan IV.Methods: HepG2 cells were selected as test system. DNA-strand breaks in HepG2 cells were evaluated by single cell gel electrophoresis assay (SCGE). Micronucleus test (MNT) which reflects chromosome breakage and/or chromosome loss was applied. The level of the oxidative production 8-Hydroxydexyguanosine (8-OHdG) was measured using immunoperoxidase staining. The level of intracellular ROS was monitored with 2', 7-dichlorofluorescein diacetate (DCFH-DA) assay. To explore the role of GSH in Sudan IV-induced DNA damage, the intracellular GSH level in HepG2 cells was modulated with buthionine-(S,R)-sulfoximine(BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor ,and the effects of GSH on Sudan IV-induced DNA damage were determined by the comet assay. Furthermore, the effect of GSH depletion on cytotoxicity of Sudan IV in HepG2 cells was examined by the cell viability assay.Results: In the SCGE and MNT, a dose-dependent increase of DNA migration and of the MN frequencies was found after treatment with the test compound. Sudan IV (25-100μM) causes a significant increase in DNA damage of HepG2 cells for 1h. Twenty-four hours exposure of the cells to Sudan IV (50-100μM) results in a significant increase in the MN frequencies. The formation of intracellular ROS was significantly increased in Sudan IV-treated cells exposed to concentration (50 and 100μM) for 1 h. Sudan IV at the dose (12.5-100μM) caused a significant oxidative damage through 8-OHdG formation in HepG2 cells for 3 h. It was also found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to Sudan IV-induced DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by Sudan IV was almost completely prevented.Conclusion:Based on these data we believe that Sudan IV exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.