Nitrosation and Nitration of 2-Amino-3-methylimidazo[4,5-f ]quinoline by Reactive Nitrogen Oxygen Species

Abstract

Both cooked red meat intake and chronic inflammation/infection are thought to play a role in the etiology of colon cancer. The heterocyclic amine 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) is formed during cooking of red meat and may be involved in initiation of colon cancer. Reactive nitrogen oxygen species (RNOS), components of the inflammatory response, contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible chemical transformation of IQ by RNOS. RNOS were generated by various conditions to react with (14)C-IQ, and samples were evaluated by HPLC. Myeloperoxidase (MPO)-catalyzed reaction was dependent upon both H(2)O(2) and NO(2)(-). This reaction produced an azo-IQ dimer and IQ dimer along with two nitrated IQ products identified by ESI/MS. 2-Nitro-IQ was not detected. Product formation was inhibited by 2 mM cyanide. Reduction in nitrated products observed with 100 mM chloride was not altered with 0.5 mM taurine. Nitrated products were also produced by other conditions, ONOO(-) and NO(2)(-) + HOCl, which generate nitrogen dioxide radical. In contrast, conditions which generate N(2)O(3), such as diethylamine NONOate, produced only small amounts of nitrated products with the major product identified by MS and NMR as N-nitroso-IQ. MPO activation of IQ to bind DNA was dependent upon both H(2)O(2) and NO(2)(-). RNOS generated by ONOO(-) and DEA NONOate also activated IQ DNA binding. The nitrated IQ products were not activated by MPO to bind DNA. In contrast, N-nitroso-IQ was activated to bind DNA by MPO +/- NO(2)(-). HOCl activated N-nitroso-IQ, but not IQ. RAW cells produced N-nitroso-IQ and increased amounts of NO(2)(-)/NO(3)(-), when incubated with 0.1 mM IQ and stimulated with lipopolysaccharide and interferon gamma. Results demonstrate chemical transformation and activation of IQ by RNOS and activation of its N-nitroso product by biological oxidants, events which may contribute to initiation of colon cancer.

Myeloperoxidase Potentiates Nitric Oxide-mediated Nitrosation

“…NO 2 ⅐ -mediated nitrosation was shown to be similar to that observed in cells. We have recently demonstrated RNOS nitrosation of the colon carcinogen 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), forming 2-nitrosoamino-3-methylimidazo [4,5-f]quinoline (N-NO-IQ) (13). The latter binds DNA and is mutagenic.…”

“…1, lower (16). The nitration products of IQ metabolism, 2-amino-5-nitro-3-methylimidazo [4,5-f]quinoline (NO 2 -IQ1) and 2-nitroamino-3-methylimidazo[4,5-f]quinoline (NO 2 -IQ2), were prepared as previously described (13). Ultima-Flo AP was purchased from PerkinElmer Life Sciences.…”

“…The reactions were stopped by adding 10 mM ascorbic acid in dimethylformamide (0.05 ml). Samples were immediately frozen and kept at Ϫ70°C for analysis by HPLC mobile phase 1 (13).…”

“…To assess MPO nonnitrosative oxidation of IQ, [ 14 C]IQ (0.06 mM) was incubated in 100 mM potassium phosphate buffer, pH 5.5, containing 0.1 mM DETAPAC, 1 mM glucose, 15 milliunits/ml glucose oxidase, 0.3 mM NaNO 2 , and 85 nM MPO in a total volume of 0.1 ml for 60 min at 37°C (13). Reactions were started by the addition of [ 14 C]IQ.…”

“…1). Previous studies have demonstrated significant transformation of IQ to a stable N-nitroso product, N-NO-IQ, during autoxidation of NO, using a high flux of NO (ϳ30 M NO/min, 0.1 mM diethylamine NONOate) (13). However, using a flux of NO more likely to occur during inflammation (2.4 M/min, 0.1 mM SpN), N-NO-IQ formation due to autoxidation (0.2 M) was near the limit of detection, 1.6% of the total radioactivity recovered by HPLC (Fig.…”