Formation of spin trap adducts during the decomposition of peroxynitrite

Peroxynitrite-mediated one-electron oxidations may be an important event in its cytotoxic mechanisms, and yet, free radical formation in the presence of peroxynitrite is difficult to study by EPR-spin trapping because adducts from most spin traps are destroyed by the oxidant. This led to some controversy with regard to the interpretation of experiments in the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), an adequate spin trap to study most of free radicals. In this report we reexamined peroxynitrite-mediate formation of spin-trap adducts. Kinetic studies and EPR experiments with water labeled with 17O are in agreement with the reaction of DMPO with a highly reactive intermediate derived from peroxynitrite to produce the DMPO-hydroxyl radical adduct by a mechanism not involving the oxidation of DMPO to a cation radical followed by water addition. The results cannot discriminate between two mechanisms of DMPO-hydroxyl radical formation, either spontaneous peroxynitrite homolysis to the hydroxyl radical or DMPO-assisted peroxynitrite homolysis. The formation of DMPO adducts during peroxynitrite-mediated oxidation of dimethyl sulfoxide, ethanol, and formate occurs through free radical mechanisms as confirmed by studies of oxygen consumption and product formation. Accordingly, spin-trapping experiments in the presence of 3,5-dibromo-4-nitrosobenzenesulfonic acid, a spin trap that is more resistant to nitrogen dioxide, led to the detection of the methyl and the beta-hydroxyethyl radical during peroxynitrite-mediated oxidation of dimethyl sulfoxide and ethanol, respectively. Oxidation of these hydroxyl radical scavengers to detectable radicals favors the hypothesis that the hydroxyl radical is produced during peroxynitrite homolysis. Bicarbonate was able to modulate peroxynitrite-mediated one-electron oxidations.