Evaluating the degree of oxidative DNA damage and apoptosis in human lymphocytes cultured in the presence of beta-carotene using comet assay, and FAS L (CD95)

Background: Reactive oxygen species can cause various damage to different parts of the body, including the blood. It can damage the proteins, lipids as well as the DNA components of the blood. Oxidative DNA damage can be measured in lymphocytes by various techniques which is a useful way to assess the overall degree of oxidative stress. This study measures the levels of DNA damage and assess the proportion of the DNA cellular repair in human lymphocytes cultured in vitro conditions, the impact of the presence of beta-carotene using comet assay (single cell gel electrophoresis), and to evaluate the apoptosis in these cells if it may occur.

Subjects and Methods: The study included 50 individuals aged between 20-50 years, during the period from October 2014 to November 2015. All participants were healthy, non-smokers with no family history of any disease. Also, they were not taking any type of vitamins or dietary supplements. Ten milliliters of total blood sample were collected in hepernized containers. Random blood samples of ten participants (5 males, 5 females) were used to study the effects of different concentrations of beta-carotene (100 and 10000) µg/ml on cultured lymphocytes by trazoleom assay. Samples from the remaining 40 participants were used to assess the levels of DNA damage in cultured lymphocytes using single cell gel electrophoresis in presence of the two different concentrations of beta-carotene (100 and 10000) µg /ml, and also to measure Fas L (CD95).

Results: Treating the lymphocytic cells with hydrogen peroxide caused a considerable damage to the DNA, however, lymphocytic cells treated with beta-carotene (at concentrations of 100 and 10000 µg/ml) showed less significant DNA damage. This was also associated with a significant change in the average tail lengths (in comet assay), indicating the positive effects of beta-carotene on the lymphocytes. Fas L (DC95) was not detected among the healthy lymphocytes, which was not associated with lymphocytic stimulation in response to beta-carotene.

Conclusions: This study proved the beneficial antioxidant capacity of beta-carotene in reducing the degree of the oxidative stress as manifested by the high levels of oxidative DNA damage measured via comet assay. A high concentration (10000 ug/ml) of beta-carotene proved to be highly Beneficial to the lymphocytes compared to a lower concentrations.