The Generation of Hydrogen Peroxide, Superoxide Radical, and Hydroxyl Radical by 6-Hydroxydopamine, Dialuric Acid, and Related Cytotoxic Agents

Hydrogen peroxide, superoxide radical, and hydroxyl radical were all formed during the autoxidation of four cytotoxic agents, namely, 6-hydroxydopamine, 6-aminodopamine, 6,7-dihydroxytryptamine, and dialuric acid. Ascorbic acid and 5-hydroxydopamine, two autoxidizable agents of biological interest but without similar cytotoxic actions, were used as negative controls. The results tend to implicate O2- and ·OH, as well as H2O2, in the molecular mechanisms for cytotoxicity. Production of H2O2 was measured with an oxygen electrode. Accumulation of H2O2 was apparent from the return to solution of approximately one-half of the consumed oxygen after the addition of catalase. Superoxide radicals were detected by reduction of cytochrome c, with inhibition by superoxide dismutase. Although O2- radicals were observed previously by other means during the autoxidation of 6-hydroxydopamine, there was no clear effect in the cytochrome c system. This may have been due, in part, to a previously described scavenging of O2- by 6-hydroxydopamine itself. Hydroxyl radicals were detected by reaction with β-methylthiopropionaldehyde to form ethylene. Catalase or superoxide dismutase inhibited ethylene production. These results point to a reaction between H2O2 and O2- (Haber-Weiss reaction) as a major source of the ·OH radicals. Ethylene production was inhibited by known ·OH trappers, such as benzoate and ethanol, and by a variety of other agents. Catechol and the catecholamines, dopamine and norepinephrine, were very potent inhibitors of ethylene production. The effectiveness of these agents may have been due to a combined action of blocking ·OH formation as well as accelerating its removal. Superoxide dismutase had a biphasic effect in the 6-hydroxydopamine system, inhibitory at early times but causing increased accumulation of ethylene at later times. An explanation for this latter phenomenon may be based on a reaction between O2- and ·OH, as described by other investigators.