PP48 - Prevention of peroxynitrite-induced nitration and oxidation by nitroxides

Abstract

Peroxynitrite, formed in vivo in a reaction between nitric oxide and superoxide radical anion, is a compound of profound pathophysiological importance. The formation of peroxynitrite is intensified in many diseases, including neurodegenerative ones, and its reactions appear to contribute to the development of these diseases. Therefore, prevention of reactions of peroxynitrite may be useful in disease prevention and treatment. The aim of this study was to study the ability of nitroxides of various structures to prevent reactions of nitration and oxidation of model compounds and bovine serum albumin. Eleven nitroxides were used: 2,2,6,6-tetramethylpiperidine (TEMPO), 4-hydroxy-TEMPO, 4-amino-TEMPO, 4-oxo-TEMPO, 4-carboxy-TEMPO, 4-cyano-TEMPO, 4-methoxy-TEMPO, 4-acetamido-TEMPO, 4-nonylamido-TEMPO, 3-carbamoyl-PROXYL and 3-carbamoyl-dehydroPROXYL. Nitroxides were most effective in preventing peroxynitrite-induced decay of fluorescein (2 µM), conditioned by fluorescein nitration (IC50 of 40.0 ± 0.4 nM, 59.8 ± 0.6 nM and 64.4 ± 1,2 nM for the most effective nitroxides, viz. 4-hydroxy-TEMPO, 4-acetamido-TEMPO and 4-amino-TEMPO, respectively, and 3.72 ± 0.05 µM for the least effective 4-nonylamido-TEMPO). Nitroxides had a biphasic effect on the oxidation of dihydrorhodamine 123 (1 µM) by peroxynitrite, both low and high concentrations having a prooxidant effect. A protection window was observed for micromolar concentrations of nitroxides (maximal protective effect at 17.3 ± 2.3, 40 and 50.3 ± 20.7 µM for TEMPO, 4-hydroxy-TEMPO and 4-amino-TEMPO, respectively and > 400 for Pirolin and 4-nonylamido-TEMPO, the extent of maximal protection being 38-90%. Nitroxides (5 nM – 500 µM) were totally ineffective in preventing peroxynitrite-induced thiol oxidation of bovine serum albumin. These results demonstrate that nitroxides are more effective in preventing nitration than oxidation reactions.