Metabolic nitrite formation from N-nitrosamines: are there other pathways than reductive denitrosation by cytochrome P-450?

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Abstract
To demonstrate whether there are any pathways of nitrite formation from N -nitrosamines other than reductive denitrosaction by cytochrome P-450 we performed the following experiments,(i) An esterified α-hydroxylated nitrosamine was incubated in amicrosomal system to test if nitrite generation is coupled with or linked to the oxidative bioactivation pathway. Simultaneously, inhibitors of microsornal esterases were added to test if the intact molecule or a metabolite from the oxidative metabolism was responsible for nitrite formation,(ii) To check if the superoxide radical anion could be related to the mechanism of nitrite generation, nitrosamines were incubated with a xanthine oxidase/hypoxanthine system, (iii) To test if the OH radical was involved in nitrite formulation, nitrosamines were incubated with an artificial hydroxy-radical generating system (xanthine oxidase/hypoxanthine system supplemented with Fe 2+ /EDTA). Measurable amounts of nitrite were detected after incubation of the esterified-hydroxylated N -nitrosamine when the hydrolysis by microsomal esterases was inhibited by diisopropylfluorophos-phate or paraoxon could and when the N -nitrosamines were incubated with the artificial hydroxy-radical generation system. Nitrite formulation could not be detected in the O 2 - -generating system (xanthine oxidase/hypoxanthine)or when the esterified a-hydroxylated N -nitrosamine was incubated with out inhibition of the microsomal esterase. These results demonstrate that besides reductive denitrosation by cytochrome P-450, nitrite generation from N -nitrosamines can also be caused by hydroxy-redicals. The importance of this possible pathway for the in vivo situation of nitrosamine metabolism is discussed.