Possible Involvement Of Oxidative Stressin Octachlorodipropyl Ether-induced Genotoxicity In Human HepG2 Cells

Introduction:Octachlorodipropyl ether (OCDPE) is an insecticidal synergist or an active ingredient of pyrethroid, organophosphorus pesticides and a bioavailable environmental contaminant. It's generally exists in the commercial agricultural and household insecticides.According to previous studies on OCDPE, the acute toxicity has been confirmed to be low, while the subacute or chronic toxicity has been demonstrated to be obvious, for example subacute hepatotoxicity. Furthermore, OCDPE has been reported to have carcinogenicity, contact allergenicity and mutagenicity. However, the OCDPE-induced genotoxicity, especially in humans, has not been well understood now. The aim of this study was to assess the genotoxic effects of OCDPE 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 OCDPE 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 OCDPE.Methods:HepG2 cells were selected as test system. We used the single cell gel electrophoresis assay (SCGE) and the micronucleus test (MNT) to study the genotoxic effects of OCDPE. To elucidate the oxidative DNA damage mechanism in HepG2 cells, we used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH); we used Acridine orange (AO) and Rhodamine 123 to measure the changes of lysosomal membrane stability and mitochondrial membrane potential. In addition, 8-hydroxyderoxyguanosine (8-OHdG), which is a reliable marker for oxidative DNA damage, was also measured by immunocytochemistry staining analysis. The data were statistically analyzed by SPSS v 11.5 software.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. OCDPE (20μM-80μM) causes a significant increase in DNA damage of HepG2 cells for 80 min. Twenty-four hours exposure of the cells to different concentrations of OCDPE (10μM-40μM) results in a significant increase in the MN frequencies; OCDPE at dose (20μM-80μM) caused a significant oxidative damage through 8-OHdG formation in HepG2 cells for 3 h. The formation of intracellular ROS was significantly increased and intracellular GSH was decreased in OCDPE-treated cells exposed to higher concentration (20μM-80μM) for 80 min; The effect on lysosomes observed in HepG2 cells was a statistically significant increase when the cells were exposed to (20μM-80μM) OCDPE for 50 min; mitochondrial membrane potential decreased after treatment with OCDPE (20μM-80μM) for 70 min.Conclusion:The data suggest that OCDPE caused DNA strand breaks and chromosome breaks, which indicate that OCDPE induced genotoxic effects in HepG2 cells. Significantly increased levels of 8-OhdG, ROS and decreased levels of GSH were observed in HepG2 cells at higher concentrations. Moreover, The effect on lysosomes observed in HepG2 cells was a statistically significant increase and the mitochondrial membrane potential was a statistically significant decrease. It may be suggested that the genotoxic effects of OCDPE in HepG2 cells probably depend on the ROS induced oxidative DNA damage occurring at higher doses and the lysosomes could be one of the main biochemical targets for OCDPE toxicity since the lysosomal damage was observed before any effect on mitochondria membrane potential, GSH levels, formation of reactive oxygen species or DNA damage.