Oxidative Stress Mediated Dna Damage and Apoptosis in Arsenic-treated Human Hepatocellular Carcinoma Cells

Recent studies in our laboratory indicated that oxidative stress plays a key role in arsenic trioxide (ATO)-induced cytotoxicity in human cancer cells. In the present investigation, we used human hepatocellular carcinoma (HepG2) cells as a model to determine whether arsenic induced DNA damage and apoptosis is mediated through oxidative stress. To achieve this goal, oxidative stress biomarkers were measured by lipid peroxidation, glutathione peroxidase, and catalase assays, respectively. The degree of DNA damage was estimated by the means of comet assay. Apoptosis was measured by flow cytometry analysis of phosphatidylserine externalization (Annexin V assay) and caspase 3 activities, and by DNA laddering assay. The results of the lipid peroxidation showed a significant increase (p <0.05) of malondialdehyde (MDA) levels with increasing ATO concentrations. Results from the glutathione peroxidase and catalase assays showed a gradual decrease in antioxidant enzyme activity in ATO-treated cells as compared the control. A significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage were observed in ATO-treated cells using the comet assay. In regard to the annexin V assay, annexin V positive cell expression was not detected at 24 hours of ATO exposure. A slight dosedependent increase (p< 0.05) was recorded in ATO-treated cells with regard to caspase 3 activation. These results were confirmed by data of the DNA laddering assay showing a clear evidence of nucleosomal DNA fragmentation in ATO-treated cells. Taken together, our research demonstrated that oxidative stress modulates ATO mediated DNA damage and apoptosis in HepG2 cells.