EVALUATION OF OXIDATIVE STRESS IN CELLS - A CORRELATION BETWEEN AN EPR METHOD AND CONVENTIONAL TECHNIQUES

The oxidative modification of rat thymocytes induced by the thermolabi le azocompound 2,2'-azobis(2-amidinopropane) dihydrochloride was exami ned by different methods. The azocompound, being water-soluble, is abl e to generate the initiating radicals at constant rate outside the cel l. Oxygen consumption due to thymocyte oxidation was evaluated by an E PR method based on the effect of oxygen on the width of the EPR line o f fusinite. This derivative of coal is a new paramagnetic probe sensit ive to oxygen concentration, nontoxic and quite stable in biological s ystems. We obtained a constant rate of oxidation that increased with t he assay temperature and the number of thymocytes exposed to oxidative stress. Oxygen consumption was then correlated to the cell viability, to the loss of unsaturated fatty acids and to the depletion of sulphy dryl groups of proteins. The content of polyunsaturated fatty acids di d not change after one hour of treatment. During the second hour a par tial lipid peroxidation occurred with consequent decrease in cell viab ility. Protein thiols were depleted at a slow rate during the first tw o hours of incubation, after which period a higher rate of oxidation o ccurred. By using the above cited EPR method, we also determined very accurately the total lipid-soluble radical trapping antioxidant capabi lity and the oxidizability of the thymocyte lipid extract. This study suggests that, at present, a quantitative correlation among results ob tained by different methods that evaluate oxidative stress in cells is not feasible. However as the proposed EPR technique accurately and se nsitively measures oxygen concentration, it can be successfully used i ) to put in evidence differences in oxidizability of different cell ty pes, ii) to compare the efficiency of different systems in generating radical stress, iii) to perform measures when only a low number of cel ls is available, iv) to determine very precisely the total lipid-solub le radical trapping antioxidant capability and the oxidizability of ce ll lipid extracts.