Antioxidants Prevent the Lymphocyte DNA Damage Induced by PMA-Stimulated Monocytes

Abstract

Oxidative stress has been related to several degenerative diseases such as cancer and coronary heart disease. Reactive oxygen species can damage different cellular macromolecules, including DNA, which is directly responsible for mutation and carcinogenesis. In this study, monocytes that were activated by phorbol 12-myristate 13-acetate (PMA) were coincubated with lymphocytes, and the DNA damage was measured by single-cell gel electrophoresis (comet) assay. Stimulation of monocytes with PMA activates the "respiratory burst," which evokes DNA damage in lymphocytes. The extent of the damage is related to the concentration of monocytes and the exposure time. Exogenous addition of superoxide dismutase did not prevent the DNA damage, which suggests that superoxide ions are not directly responsible for the damage. Partial protection was observed when catalase was included (60% protection), which indicates that other reactive species, in addition to H₂O₂, are responsible for the damage. In this system, the protective activity of natural antioxidants at different concentrations was also investigated. After coincubation of PMA-activated monocytes with lymphocytes in the presence of each antioxidant for one hour at 37°C, the lymphocyte DNA damage was determined. All the compounds protected the lymphocytes to a certain degree, with a maximum effect at different concentrations: 41% protection with 1 μM ascorbic acid, 55% protection with 40 μM α-tocopherol, 50% protection with 3 μM β-carotene, and 56% protection with 5 μM quercetin. On the basis of these results, we maintain that this "ex vivo model," more closely related to physiological conditions, could be used to test the antioxidant activity of different compounds.