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

Reactive nitrogen species, including nitrogen oxides (N2O3 and N2O4), perox ynitrite (ONOO-), and nitryl chloride (NO2Cl), have been implicated as caus es of inflammation and cancer. We studied reactions of secondary amines wit h peroxynitrite and found that both N-nitrosamines and N-nitramines were fo rmed. 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 nitromorp holine than nitrosomorpholine was formed at pH <7.5. For the morpholine-per oxynitrite reaction, nitration was enhanced by low concentrations of bicarb onate, but was inhibited by excess bicarbonate. Nitrosation was inhibited b y excess bicarbonate. On this basis, we propose a free radical mechanism, i nvolving one-electron oxidation by peroxynitrite of secondary amines to for m amino radicals (R2N .), which react with nitric oxide (. NO) or nitrogen dioxide (. NO2) 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 s ystems, respectively, also yielded nitromorpholine, but its yield was <1% o f that of nitrosomorpholine. NO . alone increased the extent of nitrosomorp holine 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 H2O2, with or without addition of myeloperoxidase or h orseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants. Tyrosine was nitrated easily by synt hetic peroxynitrite, by NaNO2 plus H2O2 with myeloperoxidase, and by NaNO2 plus H2O2 under acidic conditions. Nitrated secondary amines, e.g., N-nitro proline, could be identified as specific markers for endogenous nitration m ediated by reactive nitrogen species.