Vitamins E and C exert protective roles in hydrogen peroxide-induced DNA damage in human peripheral blood mononuclear cells

Abstract

Hydrogen peroxide (H2O2) exerts strong oxidative, cytotoxic, and genotoxic effects, whereas vitamins C and E are potent non-enzymatic antioxidants. This study aimed to demonstrate the ameliorative effects of vitamins C and E, individually or in combination, on H2O2-induced DNA damage using the alkaline Comet Assay with silver nitrate staining and visual scoring. Trypan blue exclusion assay was used to determine the cytotoxicity of the treatments, whereas alkaline Comet Assay with silver nitrate staining was used to quantify DNA damage. DNA damage was assessed by the method of visual comet scoring and expressed in arbitrary units. Human peripheral blood mononuclear cells (PBMCs) were pretreated with 100 ?M vitamin C and E for 30 min, individually or in combination, followed by a treatment with 100 ?M H2O2 for 30 min. Untreated cells were used as a negative control, whereas cells treated with 100 ?M H2O2 only were used as a positive control. We observed a considerable H2O2-induced DNA damage in the positive control, which was reduced in vitamin-pretreated cells. The combination of vitamins C and E led to the greatest amelioration of DNA damage. In our hands, Comet Assay with silver nitrate staining and visual scoring represents a rapid and reliable method to investigate the protective effects of vitamins C and E on H2O2-induced DNA damage.