Genotoxic Effect of Inorganic Arsenic Compounds Using Comet Assay

Background: Arsenic is an environmental chemical of toxicological concern today since it has been shown to be a human genotoxin and carcinogen.

Aims: The aim of this study was to determine the genotoxic effect in HepG2 cells of the four arsenic compounds by measuring DNA damage, depletion of cellular glutathione (GSH) as a measure of oxidative stress as well as antioxidant agent and apoptotic and necrotic events.

Study Design: Arsenic is an environmental chemical of toxicological concern today since it has been shown to be a human genotoxin and carcinogen. This project investigated four forms of inorganic arsenic: arsenate (As+5), sodium arsenite (As+3), arsenic trioxide (As2O3) and dimethyl arsenic acid (DMA+5); a major metabolite of arsenate.

Methodology: HepG2 cells (1.5x105/ml) treated with the four arsenics (10 µM) for 24 hr. Harvested cells were analysed for GSH concentration by reverse phase HPLC with fluorescence detection. Cells were investigated for DNA damage using the Comet assay. Cells were analysed by flow cytometry to detect apoptosis and necrosis, all running in parallel.

Results: The DNA damage for cells dosed with DMA or As2O3 was not significantly different from control. However, significant DNA damage was seen for cells treated with arsenite and arsenate (p<0.001, p<0.05 respectively). Reduced glutathione was increased by arsenic compounds compared to control. However, this was only statistically significant for arsenite (p<0.001) and arsenate (p<0.05). Whereas, results not indicated apoptosis and necrosis in viable adherent cells although this was not statistically different.

Conclusions: All four arsenicals appeared to increase GSH content and DNA damage compared to control especially both arsenite and arsenate significantly are different. Further experiments are required to assess the mechanism(s) of genotoxicity of the arsenicals in GSH depletion.