Effect of Ascorbic Acid (Vitamin C) on H2O2 Induced Oxidative DNA Damage in Human Lymphocytes Estimated by Comet Assay

Abstract

"Oxidative DNA damage induced by reactive oxygen species and free radicals. Reactive Oxygen Species induced oxidative damage plays a key role in DNA damage". Our study aimed to identify the protective effect of ascorbic acid (AA, vitamin C) against hydrogen peroxide induced oxidative damage in DNA using Comet assay. Lymphocytes pretreated with or without antioxidants, incubated at 37°C for 30 minutes, then H2O2 (100µM) was added, and incubated again at 37°C for 60 minutes. Viability of cells was detected by trypan blue stain exclusion method. The decrease in viability brought about by H2O2 when the cells incubated for 60 minutes and the viability was present to be 39±3% from 80±4% and it was highly developed by the found of AA at 100 μM which appeared 72 ± 1%. These results indicate that the activity of the ascorbic acid as antioxidantas evidenced by its ability to suppress the oxidative effect against H2O2 and protect the lymphocytes. Estimation of comet tail moment and tail length in human lymphocyte treated with 100µM of hydrogen peroxide as positive control showed that 13± 4.5% of the cells showed no DNA damage while the DNA damage from low to very high damage were 19± 3.5%, 12.2± 2.3%, 14± 3.2% and 37± 3.0%, respectively. In contrast, treatment of the cells with 100 µM H2O2in combination with 10, 25,75 and 100 µM of AA reduced the percentage of DNA damage according to the concentrations used and they were 9± 2.3%, 4.5± 1.6%, 6± 0.5% and 10± 1.4%, respectively. In addition, H2O2 induced DNA damageat percentage of 78% at concentration of 100 µmol/L withoutthe addition of ascorbic acid. while the treatment of human lymphocyte with ascorbic acid was able to reduce oxidative DNA damage by 17% in comparison with control group.