Possible Involvement Of Oxidative Stress And Lysosomal Membrane Integratiy In Ortho-phenylphenol Induced-DNA Damage In Human HepG2 Cell

Objective:Ortho-phenylphenol (OPP) is a broad-spectrum fungicide and anti-bacterial agent used for the anti-fungal treatment of citrus fruits and vegetables before packaging and for impregnation of fruit wrappers during shipping. OPP can seep into pulp through fruit wall and not been cleaned as a result from its systemic, which caused toxicity of liver, kidney and nervous system.Several studies suggested that OPP have carcinogenicity and mutagenicity. Mariko Murata indicated that OPP-induced DNA damage involved generation of ROS. The aim of this study was to assess the mechanism of OPP-induced DNA damage involved with oxidative stress and lysosomal membrane integrity, providing some information for safety assessment to human on OPP.Methods:HepG2 cells were selected as test system. The single cell gel electrophoresis assay (SCGE) was used to detect the DNA damage induced by OPP and study the genotoxicity of OPP. To elucidate the possible mechanism of DNA damage caused by OPP in HepG2 cells, we used 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. The effects of oxidative stress and lysosomal membrane integrity on DNA damage were observed when HepG2 cells were pretreated with HT, NH4Cl, pepstain A and desipramine for 1h before. The date was analyzed by SPSS 11.5.Results:OPP (200-800μM), for 1h, caused a significant increase of the DNA migration, the levels of intracellular ROS, GSH and mitochondrial inactivation in a dose-dependent manner. OPP, for 40min, caused a significant decrease in lysosomal rupture. Cells pretreated HT, NH4Cl, pepstain A and desipramine, its DNA fragmentation induced by OPP were decreased as the same as the change of intracellular ROS, GSH, lysosomal stability and mitochondrial membrane potential, which indicated that they protect HepG2 cells DNA from fragmentation.Conclusion:The results suggest that OPP caused DNA strand breaks, which indicates that OPP induced genotoxic effects in HepG2 cells. Since, after HepG2 cells pretreated with HT, DNA strand breaks were significantly reduced, intracellular ROS and GSH deleption induced by OPP were decreased, we suggest that DNA damage induced by OPP involved oxidative stress. Meanwhile, lysosomal membrane and mitochondrial membrane were also protected when cells were pretreated with HT and NH4Cl, DNA strand breaks induced by OPP were reduced by pepstain A and desipramine, which tell us that lysosomal membrane integrity may be the mechanism of DNA damage induced by OPP.