Metabolic denitrosation of N-nitrosamines: mechanism and biological consequences.

NADPH-dependent microsomal metabolism of N-nitrosamines results in both oxidative dealkylation and denitrosation of the molecule. For denitrosation, two enzymatic mechanisms have been proposed: (i) cytochrome P450 (P450)-dependent one-electron reduction of the nitrosamine molecule, resulting in the formation of nitric oxide (NO) and secondary and primary amine, and (ii) liberation of NO via an oxidative mechanism mediated by a P450-dependent one-electron abstraction. In order to clarify the mechanism of denitrosation, the metabolism and kinetics of N-nitrosodibenzylamine (NDBzA) and its corresponding secondary amine dibenzylamine were studied. The main metabolites of NDBzA are benzaldehyde, the primary amine benzylamine and nitrite. An important finding is that benzaldehyde is generated more rapidly from dibenzylamine than from the parent NDBzA. During reductive denitrosation of NDBzA, the oxygen atom in benzaldehyde is derived from air, while benzaldehyde generated via the oxidative mechanism of denitrosation receives its oxygen atom from water due to hydrolysis of the intermediary benzylidenebenzylamine. Microsomal incubation of NDBzA in buffer containing 18O-H2O resulted in no incorporation of 18O from water into benzaldehyde, which could be related to the formation of the corresponding benzaldehyde, which could be related to the formation of the corresponding benzylidenebenzylamine. It is concluded that NDBzA is denitrosated by the proposed reductive mechanism. Current belief is that denitrosation leads to detoxification of the NA molecule; however, toxic effects cannot be excluded if the conversion of NO into NO2- and NO3- involves intermediary formation of the NO2 radical.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced demethylation and denitrosation of N-nitrosodimethylamine by human liver microsomes from alcoholics.

The metabolism of N-nitrosodimethylamine (NDMA) was investigated in incubations with human liver microsomes from alcoholics and control patients who suffered from other diseases, but had a histological normal liver. All of the microsomal samples studied were able to metabolize NDMA at various concentrations to both formaldehyde and nitrite. Analysis of the liver microsomes from alcoholics revealed that both enzymatic activities--formaldehyde and nitrite formation--were enhanced several times as compared to the control patients. The results point to the fact that alcoholics metabolize NDMA at a higher rate probably due to the induction of one or more ethanol-inducible human liver cytochromes (cyt.) P450. The question if alcoholics therefore possess a higher risk for carcinogenic events is discussed.

Some aspects of cytochrome P450-dependent denitrosation of N-nitrosamines.

The present paper deals with three aspects of cytochrome P450-dependent denitrosation of N-nitrosamines. (1) Nitrate was found in addition to nitrite as a metabolic product of the denitrosation reaction when N-nitrosamines were incubated with a microsomal system. This could also be shown when nitric oxide was added to the microsomes. (2) In order to determine the amount of denitrosation in vivo, the nitroso group of N-nitroso-N-methylaniline was labelled with the 15N isotope and administered to rats; then, the concentrations of 15N-nitrate and 15-N-nitrite in the urine were quantified by measuring the reaction of nitrate and benzene to nitrobenzene. It is estimated from these data that about 33% of the applied dose of 15N-nitroso-N-methylaniline is denitrosated in vivo. (3) Although N-nitrosodiphenylamine (NDPhA) has been classified as a noncarcinogen, recent long-term and short-term studies have cast some doubt. In order to evaluate the mechanism by which NDPhA exerts its possible genetoxic effects, its metabolism was studied in vitro, and NDPhA and its metabolites were tested for induction of DNA single-strand breaks in rat hepatocytes and in Chinese hamster V79 cells. One metabolite was identified as diphenylamine; others were suspected to be the 4-hydroxylated derivative and its corresponding quinoneimine. NDPhA caused DNA damage in rat hepatocytes but not in V79 cells. Diphenylamine also gave negative results in V79 cells, but its putative metabolite, diphenylhydroxylamine, induced a significant increase in DNA single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

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

To demonstrate whether there are any pathways of nitrite formation from N-nitrosamines other than reductive denitrosation by cytochrome P-450 we performed the following experiments. An esterified alpha-hydroxylated nitrosamine was incubated in a microsomal system to test if nitrite generation is coupled with or linked to the oxidative bioactivation pathway. Simultaneously, inhibitors of microsomal esterases were added to test if the intact molecule or a metabolite from the oxidative metabolism was responsible for nitrite formation. 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. To test if the OH radical was involved in nitrite formation, nitrosamines were incubated with an artificial hydroxy-radical generating system (xanthine oxidase/hypoxanthine system supplemented with Fe2+/EDTA). Measurable amounts of nitrite were detected after incubation of the esterified-hydroxylated N-nitrosamine when the hydrolysis by microsomal esterases was inhibited by diisopropylfluorophosphate or paraoxon and when the N-nitrosamines were incubated with the artificial hydroxy-radical generating system. Nitrite formation could not be detected in the O2(-)-generating system (xanthine oxidase/hypoxanthine) or when the esterified alpha-hydroxylated N-nitrosamine was incubated without inhibition of the microsomal esterases. These results demonstrate that besides reductive denitrosation by cytochrome P-450, nitrite generation from N-nitrosamines can also be caused by hydroxy-radicals. The importance of this possible pathway for the in vivo situation of nitrosamine metabolism is discussed.