Evaluation of the level of DNA damage and repair in human lymphocytes cultured in the presence of Beta- Carotene using comet assay (single cell gel electrophoresis)

Abstract

Background: Reactive oxygen species (free radicals) can cause various damage to different parts of the body, including the blood. Oxidative DNA damage can be measured in lymphocytes by various techniques which is a useful way to evaluate the degree of oxidative stress. The Comet assay is important technique for assess the damage or repair of DNA in human cultured lymphocytes.Aim of the study: To assess the levels of DNA damage and to measure the proportion of the DNA cellular repair in human lymphocytes cultured in vitro conditions, the impact of the presence of Beta-Carotene by measuring the Comet tail moment.Subjects and Methods: The study included 50 individuals aged between 20-50 years, during the period from12 October 2014 to 19 November 2015 and from healthy individuals, non-smokers, and non-conception of any type of vitamins before 1-2 weeks of sampling. Ten milliliters of hole blood sample taken to hepernized container, 10 samples (5 males, 5 females) to study the toxicity of different concentrations of Beta-Carotene (100, 10000) µg/ml on cultured lymphocyte by trazoleom assay; then take another samples (40) to assess the level of DNA damage in cultured lymphocytes by Comet assay in presence of the two different concentrations of Beta-Carotene (100, 10000) µg/ml, (20 individuals every concentration). Results: There were a damage occur in DNA of the cultured lymphocytes by the effect of the presence of hydrogen peroxide, and there was repair occur by the presence of Beta-Carotene at the concentrations (100 and 10000) µg/ml, and also there were a significant change in the average of tail moment (in Comet assay) as an indicator of a positive effect for Beta-Carotene to protect DNA of the cultured lymphocyte cells.Conclusions: This study demonstrated the protective effects of in vitro applications of Beta Carotene in different concentrations (100, 10000) µg/ml on DNA damage induced by H2O2 in lymphocyte cultures of healthy individuals, via Comet assay (tail moment), which showed that the most effective concentration of Beta-Carotene as antioxidant was in the concentration of 10000 µg.