2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Abstract

Although nitrosation plays an important role in initiation of carcinogenesis, the reactive nitrogen oxygen species (RNOS) mediating this reaction by multiple pathways have not been determined. The heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) was used as a target to investigate RNOS and pathways for potentiation of nitric oxide (NO)-mediated nitrosation. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) oxidizes NO to NO 2 and was used as a tool to investigate NO 2 potentiation of nitrosation. The IQ nitrosation product, 2-nitrosoamino-3-methylimidazo[4,5- f]quinoline ( 14C–N–NO–IQ), was monitored by HPLC. Autoxidation of NO, generated by spermine NONOate (2.4 μM NO/min) for 7.5 min, did not convert 10 μM 14C–IQ to N–NO–IQ. However, the presence of 15 μM CPTIO resulted in 3 μM N–NO–IQ formation. Potentiation by CPTIO occurred at low and high fluxes of NO, 0.075 to 1.2 μM/min, and over a range of IQ to CPTIO ratios of 0.5 to 10. A significant portion of N–NO–IQ formation was insensitive to azide (10 mM) inhibition, suggesting oxidative nitrosylation. NADH (0.02 mM) did not alter nitrosation by autoxidation, but effectively inhibited potentiation by CPTIO. Ascorbic acid (0.2 mM) and 5,5-dimethyl-1-pyrroline N-oxide (30 mM) inhibited nitrosation with or without CPTIO, while superoxide dismutase was not inhibitory. The RNOS produced by CPTIO had a 27-fold greater affinity for IQ than those produced by autoxidation. Results are consistent with NO 2 or a RNOS like NO 2 potentiating IQ oxidative nitrosylation. Nitrosation occurring at both low and high fluxes of NO can contribute to carcinogenesis.