Exposure of rat thymocytes to hydrogen peroxide increases annexin V binding to membranes: inhibitory actions of deferoxamine and quercetin

Effects of hydrogen peroxide (H2O2) on rat thymocytes were examined, using a flow cytometer and three fluorescent probes, annexin V-fluorescein isothiocyanate (annexin V-FITC) for detecting phosphatidylserine expressed on the membrane surface, ethidium bromide for estimating dead cells, and fluo-3-acetoxymethyl ester (fluo-3-AM) for monitoring changes in intracellular Ca2+ concentration ([Ca2+](i)), to characterize H2O2-induced cytotoxicity. Exposure to H2O2 (30 mu M or more) increased the number of annexin V-positive live cells dose- and time-dependently while the number of dead cells increased at concentrations of 1 mM or more. H2O2 (30 mu M or more) increased [Ca2+](i) in a dose-dependent manner. Threshold concentration of H2O2 to increase [Ca2+](i) was similar to that to increase annexin V binding to membranes. The H2O2-induced change in cell membranes was attenuated under Ca2+-free conditions. Therefore, it is likely that Ca2+ is involved in the H2O2-induced cytotoxicity. Deferoxamine was effective to protect the cells suffering from H2O2-induced oxidative stress, suggesting a contribution of hydroxyl radicals generated by the Fenton reaction. Quercetin also exerted a potent protective action on cells suffering from H2O2-induced oxidative stress. The results indicate that the exposure of rat thymocytes to H2O2 at micromolar concentrations increases annexin V binding to cell membranes in a Ca2+-dependent manner, suggesting the possibility that the oxidative stress caused by H2O2 (and/or hydroxyl radicals) induces apoptosis via increasing [Ca2+](i). (C) 1999 Elsevier Science B.V. All rights reserved.