Formation of N-nitrosamines and N-nitramines by the reaction of secondary amines with peroxynitrite and other reactive nitrogen species: comparison with nitrotyrosine formation

Abstract

Reactive nitrogen species, including nitrogen oxides (N(2)O(3) and N(2)O(4)), peroxynitrite (ONOO(-)), and nitryl chloride (NO(2)Cl), have been implicated as causes of inflammation and cancer. We studied reactions of secondary amines with peroxynitrite and found that both N-nitrosamines and N-nitramines were formed. Morpholine was more easily nitrosated by peroxynitrite at alkaline pH than at neutral pH, whereas its nitration by peroxynitrite was optimal at pH 8.5. The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at pH <7.5. For the morpholine-peroxynitrite reaction, nitration was enhanced by low concentrations of bicarbonate, but was inhibited by excess bicarbonate. Nitrosation was inhibited by excess bicarbonate. On this basis, we propose a free radical mechanism, involving one-electron oxidation by peroxynitrite of secondary amines to form amino radicals (R(2)N(*)), which react with nitric oxide ((*)NO) or nitrogen dioxide ((*)NO(2)) to yield nitroso and nitro secondary amines, respectively. Reaction of morpholine with NO(*) and superoxide anion (O(2)(*)(-)), which were concomitantly produced from spermine NONOate and by the xanthine oxidase systems, respectively, also yielded nitromorpholine, but its yield was <1% of that of nitrosomorpholine. NO(*) alone increased the extent of nitrosomorpholine formation in a dose-dependent manner, and concomitant production of O(2)(*)(-) inhibited its formation. Reactions of morpholine with nitrite plus HOCl or nitrite plus H(2)O(2), with or without addition of myeloperoxidase or horseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants. Tyrosine was nitrated easily by synthetic peroxynitrite, by NaNO(2) plus H(2)O(2) with myeloperoxidase, and by NaNO(2) plus H(2)O(2) under acidic conditions. Nitrated secondary amines, e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated by reactive nitrogen species.