OXIDATION OF 2',7'-DICHLOROFLUORESCIN BY PEROXYNITRITE

The simultaneous production of nitric oxide and superoxide anion leads to the formation of peroxynitrite, a potent oxidant which may be an i mportant mediator of cellular injury. Oxidation of dichlorofluorescin to the fluorescent dichlorofluorescein has been used as a marker for c ellular oxidant production. The mechanisms of peroxynitrite-mediated o xidation of dichlorofluorescin to dichlorofluorescein were investigate d. Chemically synthesized peroxy nitrite (50-500 nM) induced the oxida tion of dichlorofluorescin to dichlorofluorescein in a linear fashion. In addition, the simultaneous generation of nitric oxide and superoxi de anion induced the oxidation of dichlorofluorescin to dichlorofluore scein, while nitric oxide (1-10 mu M) alone under aerobic conditions d id not. Peroxynitrite-mediated oxidation of dichlorofluorescin was not inhibited by the hydroxyl radical scavengers mannitol (100 mM) or dim ethylsulfoxide (100 mM). Moreover, peroxynitrite-mediated oxidation of dichlorofluorescin was not dependent upon metal ion-catalyzed reactio ns. Furthermore, dichlorofluorescein formation was diminished at alkal ine pH. These findings suggest that peroxynitrite-mediated dichloroflu orescein formation results directly from the protonation of peroxynitr ite to form the conjugate peroxynitrous acid. L-cysteine was an effici ent inhibitor (K-I approximate to 25 mu M) of dichlorofluorescin oxida tion through competitive oxidation of free sulfhydryls. Urate was a le ss efficient with a maximum inhibition of only 49%. These results demo nstrate that dichlorofluorescin is efficiently oxidized by peroxynitri te. Therefore, under conditions where nitric oxide and superoxide are produced simultaneously, oxidation of dichlorofluorescin may be mediat ed by the formation of peroxynitrite.