Genotoxic Effect of Arsenate and Arsenite in Human HaCat Cells in Culture Using Comet Assay

Abstract

Arsenic is an environmental chemical of toxicological concern today since it is a human genotoxin and chronic exposure is associated with development of cancers, including skin. Inorganic arsenate is metabolically reduced to arsenite by glutathione (GSH) prior to methylation. The aim of this study was to determine the relative toxic effects of arsenate and arsenite in HaCat cells (immortalized human keratinocytes) in vitro by measuring cytotoxicity, DNA damage, depletion of glutathione and apoptotic and necrotic events. HaCat cells were treated with arsenate and arsenite (10 μM) for DNA damage detection using Comet assay and cytotoxicity (10, 60 and 100 μM) all measured at 24 hr. In some experiment arsenate or arsenite (10 μM) was added at the same time as BSO 10 μM for 24 hr, and GSH levels were measured by HPLC with fluorescence detection. Flow cytometry was used to investigate apoptotic and necrotic events following arsenate and arsenite (10 μM) treatment for 24 hr. Arsenate and arsenite at 60 and 100 μM, but not 10 μM, reduced the number of adherent viable cells with time. Therefore, DNA damage could only be measured at 10 μM as at higher concentrations the cells did not produce classical Comets but showed fragmentation. DNA damage was significantly (p < 0.001) increased in cells treated for 24 hr with 10 μM arsenate and arsenite compared to control. GSH levels were significantly increased in HaCat cells treated with10 μM arsenate and arsenite (p < 0.05, p < 0.001, respectively) compared to control. Cells treated with buthionine sulphoximine (BSO) at the same time as arsenate had increased GSH levels (p < 0.001), but arsenite and BSO did not increase cellular GSH. Arsenate and arsenite increased apoptosis, and arsenate increased necrosis, although none of the values reached statistical significance. Arsenite was more cytotoxic than arsenate. Arsenate and arsenite are known to produce oxidative stress involving ROS formation and depletion of glutathione. The increase in GSH levels at low doses of arsenate and arsenite, and by arsenate even in the presence of BSO.