Evaluation of oxidative stress in cells: A correlation between an EPR method and conventional techniques

The oxidative modification of rat thymocytes induced by the thermolabile azocompound 2,2′-azobis(2-amidinopropane) dihydrochloride was examined by different methods. The azocompound, being water-soluble, is able to generate the initiating radicals at constant rate outside the cell. Oxygen consumption due to thymocyte oxidation was evaluated by an EPR method based on the effect of oxygen on the width of the EPR line of fusinite. This derivative of coal is a new paramagnetic probe sensitive to oxygen concentration, nontoxic and quite stable in biological systems. We obtained a constant rate of oxidation that increased with the 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 sulphydryl groups of proteins. The content of polyunsaturated fatty acids did not change after one hour of treatment. During the second hour a partial lipid peroxidation occurred with consequent decrease in cell viability. Protein thiols were depleted at a slow rate during the first two hours of incubation, after which period a higher rate of oxidation occurred. By using the above cited EPR method, we also determined very accurately the total lipid-soluble radical trapping antioxidant capability and the oxidizability of the thymocyte lipid extract. This study suggests that, at present, a quantitative correlation among results obtained by different methods that evaluate oxidative stress in cells is not feasible. However as the proposed EPR technique accurately and sensitively measures oxygen concentration, it can be successfully used i) to put in evidence differences in oxidizability of different cell types, ii) to compare the efficiency of different systems in generating radical stress, iii) to perform measures when only a low number of cells is available, iv) to determine very precisely the total lipid-soluble radical trapping antioxidant capability and the oxidizability of cell lipid extracts.