Exposure of rat thymocytes to hydrogen peroxide increases annexin V binding to membranes: inhibitory actions of deferoxamine and quercetin.
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 microM 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 microM 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).
Oyama
Y
Y
Laboratory of Cellular Signaling, Faculty of Integrated Arts and Sciences, The University of Tokushima, Minami-Jyosanjima 1-1, Tokushima, Japan. oyama@ias.tokushima-u.ac.jp
Noguchi
S
S
Nakata
M
M
Okada
Y
Y
Yamazaki
Y
Y
Funai
M
M
Chikahisa
L
L
Kanemaru
K
K
D000818:Animals; D017304:Annexin A5 / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004996:Ethidium; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011794:Quercetin / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D017304:Annexin A5; D002614:Chelating Agents; D016877:Oxidants; D011794:Quercetin; D006861:Hydrogen Peroxide; D004996:Ethidium; D016650:Fluorescein-5-isothiocyanate; D003676:Deferoxamine; D002118:Calcium