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Sections of Emergency Medicine and Pulmonary and Critical Care, Department of Medicine, The University of Chicago, Chicago, Illinois 60637
Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O2 of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 ± 0.1 to 2.3 ± 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 ± 0.1, as did the site I inhibitors rotenone (1.0 ± 0.1), amytal (1.1 ± 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 ± 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-L-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 ± 0.1) and decreased with exogenous SOD (1.1 ± 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain.
reactive oxygen species; mitochondria
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