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

Abstract Effects of hydrogen peroxide (H 2 O 2 ) 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 Ca 2+ concentration ([Ca 2+ ] i ), to characterize H 2 O 2 -induced cytotoxicity. Exposure to H 2 O 2 (30 μM 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. H 2 O 2 (30 μM or more) increased [Ca 2+ ] i in a dose-dependent manner. Threshold concentration of H 2 O 2 to increase [Ca 2+ ] i was similar to that to increase annexin V binding to membranes. The H 2 O 2 -induced change in cell membranes was attenuated under Ca 2+ -free conditions. Therefore, it is likely that Ca 2+ is involved in the H 2 O 2 -induced cytotoxicity. Deferoxamine was effective to protect the cells suffering from H 2 O 2 -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 H 2 O 2 -induced oxidative stress. The results indicate that the exposure of rat thymocytes to H 2 O 2 at micromolar concentrations increases annexin V binding to cell membranes in a Ca 2+ -dependent manner, suggesting the possibility that the oxidative stress caused by H 2 O 2 (and/or hydroxyl radicals) induces apoptosis via increasing [Ca 2+ ] i .