| Introduction:Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I is not permitted for use at any level of food production in most of the countries.In 1975 the International Agency for Research on Cancer (IARC), assessed Sudan I as a Group 3 carcinogen. With respect to Sudan I genot -oxicity data, positive results were observed both in vivo and in vitro upon metabolic activation. The liver is the target site of Sudan I toxicity. Sudan I is metabolized by several routes, reactive oxygen species (ROS) are produced. The aim of this study was to assess the genotoxic effects of Sudan I in vitro and to elucidate the mechanism of oxidative DNA damage. 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 metabolising enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.The aim of present study is to explore whether Sudan I causes genotoxic effect in HepG2 cells and to elucidate the underlying mechanism whether it probably via ROS-induced oxidative DNA damage. Thus it may provide some information for safety assessment to humans on Sudan I.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 production8-Hydroxydeoxyguanosine (8-OHdG) was measured using immunoperoxidase staining. The level of intracellular ROS was monitored with 2, 7-dichlorofluorescein diacetate (DCFH-DA) assay. Lipid peroxidation as the measure of cell oxidative injury was determined by measurement of thiobarbituric acid– reactive substances (TBARS).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 I (25-100μM) causes a significant increase in DNA damage of HepG2 cells for 1 h. Twenty-four hours exposure of the cells to different concentrations of Sudan I (25-100μM) results in a significant increase in the MN frequencies. The formation of intracellular ROS was significantly increased in Sudan I-treated cells exposed to higher concentration (100μM) for 1 h. Sudan I at higher dose (50-100μM) caused a significant oxidative damage through 8-OHdG formation in HepG2 cells for 3 h. Sudan I at the higher concentrations (50-100μM) exposure caused increase in the levels of TBARS as an index of cellular lipid peroxidation in HepG2 cells for 24 h.Conclusion:The data suggest that Sudan I caused DNA strand breaks and chromosome breaks, which indicate that Sudan I induced genotoxic effects in HepG2 cells. Significantly increased levels of ROS, 8-OHdG, TBARS were observed in HepG2 cells at higher concentrations. It may be suggested that the genotoxic effects of Sudan I in HepG2 cells probably depend on the ROS -induced oxidative DNA damage occurring at higher doses. |
