Oxygen free radicals production and DNA damage/repair activity in lymphocytes of elderly subjects





J. Jajte, J. Grzegorczyk, J. Błasiak, A. Sapota

Department of Toxicology, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1 Str., 90.151 Lodz, Poland



Purpose: A growing body of experimental data indicates that increased levels of DNA damage is associated with aging process. It has been also hypothesized that reactive oxygen species play an important role in the aging and cellular senescence. The aim of our study was to examine the relationship between oxygen free radicals production and DNA damage/repair in lymphocytes with age and selective diagnostic parameters in elderly subjects.

Method: The population (aged 60-92) consisted of controls healthy elderly and ill patients with atherosclerosis. The healthy status was assessed through a medical examination and by routinely clinical laboratory analysis. We have used spectrofluorimetry and/or flow cytometry (FACS) measurements to study ROS production in cells. In these methods oxidation of DHR or DCF, as fluorescent probes, by ROS was recorded. Additionally, we used for lymphocytes two different stimulus: PMA (phorbol 12-myristate 13-acetate) as high stimulus and fMLP (N-formyl-MetLeuPhe) as low physiological stimulus.

The level of DNA damage and the kinetics of DNA repair was evaluated by the alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) for oxidized pyrimidines, and formamidopyrimidine-DNA glycosylase (Fpg) for oxidized purines, recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. To assess DNA repair activity we determined the sensitivity to DNA-damaging agent hydrogen peroxide and the efficacy of removing of DNA damage induced by this agent in peripheral blood lymphocytes.

Results: We have found significant correlation between ROS generation in lymphocytes and DNA damage/repair activity in healthy elderly and ill old patients with some parameters of clinical laboratory analysis (e.g. cholesterol) and good correlation with the pathological changes, mainly infections and atherosclerosis, in old patients.

Conclusions: Our data provide direct support that analyzing of ROS generation in lymphocytes with assessing the DNA damage/repair activity of controls healthy elderly and ill old patients might be helpful in clinical diagnosis of the age-related diseases.

This work was supported by the ZINCAGE project (FOOD-CT-2003-506850) of the EU 6th Framework Program.




Key words: ROS, DNA damage, DNA repair, lymphocytes, elderly subjects







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