Dual activity of nitroxides as pro- and antioxidants: catalysis of copper-mediated DNA breakage and H2O2 dismutation.

Nitroxide antioxidants can be reduced to hydroxylamines or oxidized to oxoammonium cations. Consequently, nitroxides can modify oxidative damage acting as reducing and/or as oxidizing agents, and in many cases the nitroxides are continuously recycled. They provide protection against oxidative stress via various mechanisms including SOD-mimic activity and detoxification of carbon-, oxygen-, and nitrogen-centered radicals, as well as oxidation of reduced transition metals. In contrast to the common concept, according to which the nitroxides' protective effect takes place via inhibition of the Fenton reaction, there are observations suggesting the opposite. In the present investigation, DNA breakage catalyzed by copper served as an experimental model for studying the anti- and pro-oxidative activity of nitroxides. Nitroxides provided protection in the presence of GSH, which is known to facilitate metal-catalyzed DNA damage. In the absence of a reductant, nitroxides enhanced DNA breakage under aerobic conditions with or without added H(2)O(2) and facilitated H(2)O(2) depletion. The rates of nitroxide-catalyzed DNA breakage and H(2)O(2) depletion increased as the concentrations of copper, H(2)O(2), and nitroxide increased. Although the catalytic activity of nitroxides is low, it is sufficient to induce DNA breakage. The efficacy of DNA breakage by the tested piperidine nitroxides correlated with the nitroxide-induced depletion of H(2)O(2) with the exception of the pyrrolidine nitroxide 3-carbamoylproxyl. The results suggest that the nitroxide and the copper are continuously recycled while catalyzing DNA breakage and depletion of H(2)O(2), which serves both as a source of reducing equivalents and as the electron sink.

Kinetics and mechanism of the comproportionation reaction between oxoammonium cation and hydroxylamine derived from cyclic nitroxides.

Cyclic nitroxides demonstrate antioxidative activity in numerous in vitro and in vivo models, which frequently involves the participation of the reduced and oxidized forms of the nitroxide, namely, the hydroxylamine and oxoammonium cation. Generally, cellular reducing equivalents facilitate rapid enzymatic as well as nonenzymatic reduction of nitroxides in the tissue. On the other hand, the reaction of nitroxides with various radicals yields the highly oxidizing oxoammonium cation, which mediates the catalytic effect of nitroxides in selective oxidation of alcohols. Hence, nitroxides might act as both anti- and pro-oxidants. Therefore, the comproportionation reaction between the oxoammonium cation and the hydroxylamine might play a role in lowering the pro-oxidative activity of nitroxides. Although the comproportionation reaction has previously been studied, there is no agreement regarding its kinetic features. We investigated the reaction of the reduced forms of 2,2,6,6-tetramethylpiperidinoxyl (TPO) and 4-OH-2,2,6,6-tetramethylpiperidinoxyl (4-OH-TPO) with the oxoammonium cation derived from TPO at various pHs using rapid-mixing stopped-flow and EPR spectrometry. From the pH dependence of the reaction rate constants we determined the pK(1) of the respective hydroxylamines to be 7.5 and 6.9, respectively. The reduction potentials of the hydroxylamines were determined by cyclic voltammetry, and from their dependence on pH, we obtained the same pK(1) values. The rate constant of the comproportionation reaction does not exceed 20 M(-1) s(-1) in the physiological pH range and, therefore, cannot greatly contribute toward recycling of the nitroxides in the tissue.