Comparative metabolism of N-nitrosopiperidine and N-nitrosopyrrolidine by rat liver and esophageal microsomes and cytochrome P450 2A3.

N-nitrosopiperidine (NPIP) is a potent esophageal carcinogen in rats whereas structurally similar N-nitrosopyrrolidine (NPYR) induces liver, but not esophageal tumors. NPIP is a possible causative agent for human esophageal cancer. Our goal is to explain mechanistically these differing carcinogenic activities in the esophagus. We hypothesize that differences in metabolic activation of these nitrosamines could be one factor accounting for their differing carcinogenicity. alpha-Hydroxylation is the key metabolic activation pathway leading to nitrosamine-induced carcinogenesis. In this study, we examined the alpha-hydroxylation rates of [3,4-(3)H]NPIP and [3,4-(3)H]NPYR by male F344 rat esophageal and liver microsomes. The major alpha-hydroxylation products of NPIP and NPYR, 2-hydroxytetrahydro-2H-pyran (2-OH-THP) and 2-hydroxytetrahydrofuran (2-OH-THF), respectively, were monitored by high performance liquid chromatography with radioflow detection. NPIP or NPYR (4 microM) was incubated with varying concentrations of esophageal microsomes and co-factors. Microsomes converted NPIP to 2-OH-THP with a 40-fold higher velocity than NPYR to 2-OH-THF. Similar results were observed in studies with NPIP and NPYR at substrate concentrations between 4 and 100 micro M. Kinetics of NPIP alpha-hydroxylation were biphasic; K(M) values were 312 +/- 50 and 1600 +/- 312 microM. Expressed cytochrome P450 2A3, found in low levels in rat esophagus, was a good catalyst of NPIP alpha-hydroxylation (K(M) = 61.6 +/- 20.5 microM), but a poor catalyst of NPYR alpha-hydroxylation (K(m) = 1198 +/- 308 micro M). Cytochrome P450 2A3 may play a role in the preferential activation of NPIP observed in rat esophagus. Liver microsomes metabolized NPYR to 2-OH-THF (V(max)/K(M) = 3.23 pmol/min/mg/ microM) as efficiently as NPIP to 2-OH-THP (V(max)/K(M) = 3.80-4.61 pmol/min/mg/ microM). We conclude that rat esophageal microsomes activate NPIP but not NPYR whereas rat liver microsomes activate NPIP and NPYR. These results are consistent with previous findings that tissue-specific activation of nitrosamines contributes to tissue-specific tumor formation.

Effects of ethanol treatment on DNA damage induced in Escherichia coli K-12 in various organs of mice by N-nitro-sonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosopyrrolidine.

DNA damage induced by tobacco-related nitrosamines was quantitatively determined in animal-mediated DNA-repair assays with Escherichia coli K-12 strains. Intraperitoneal administration of N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosopyrrolidine (NPYR) caused dose-dependent genotoxic effects in indicator bacteria recovered from various organs of nitrosamine-pretreated mice. Oral administration of ethanol (6.3 g/kg body wt) 1 h prior to administration of NNN, NNK or NPYR resulted in a substantial reduction of genotoxicity that was more pronounced in the liver as compared to lungs, spleen, kidneys and blood. However, when the same ethanol dose was given 26 h before NNN or NPYR, an increase of DNA damage was found, that was, in most cases, higher in the kidneys than in the liver. Significant enhancement of genotoxic activity was also measured in lungs and spleen, whereas only a marginal increase was detectable in the blood. Repeated administration of smaller ethanol doses (2.0 g/kg body wt at 12 h intervals) for 4 days caused a comparable increase. Similar enhancement of genotoxicity was also measured when acetone (3.5 g/kg) was given orally 15 h before the nitrosamine administration. The stimulating effect of ethanol was dose dependent and was absent when the alcohol was administered 60 h prior to the nitrosamine. Neither ethanol nor acetone had an effect on the genotoxicity of NNK under identical experimental conditions. The same E. coli K-12 strains were used to test NNN, NNK and NPYR in in vitro assays. The ranking order of activation capacity was liver S-9 > kidney S-9 > lung S-9 for all three nitrosamines. Blood S-9 did not markedly activate the nitrosamines. S-9 mixtures prepared from mice that had been treated with ethanol (6.3 g/kg body wt) for 26 h before death activated NNN and NPYR more efficiently than those S-9s from untreated animals. The increase of genotoxic activity was more pronounced with S-9 from kidneys and lungs than from liver. No difference was seen with S-9 from ethanol-treated and untreated mice with NNK.

Direct involvement of human gastric mucosa in the activation of alimentary pro-carcinogens.

Exposure to N-nitroso-compounds and aromatic amines, xenobiotics which require an activation in order to exert their genotoxic potential, is causally associated with gastric cancer. We evaluated the capacity of microsome-containing fractions from human gastric mucosa to activate two model carcinogenic compounds. A 9,000 x g supernatant (S9) was obtained from gastric mucosal specimens and, for comparison, from human liver and aroclor-induced and non induced rat liver. The capacity of the S9 to activate N-nitrosopyrrolidine (NPY) and 2-aminofluorene (2AF) to mutagenic metabolites was tested in the Ames/Salmonella reversion assay, while dimethylnitrosamine (DMN) and aminopyrine (AP) demethylation activities were spectrophotometrically evaluated by using an enzymatic assay of the amount of formaldehyde released following the enzymatic demethylation of the corresponding substrates. Results indicate that human gastric S9 fractions may activate 2AF to a genotoxic derivative and are characterized by DMN and AP demethylase activities higher (p < 0.05) than those of human liver, when expressed in mg/protein (p < 0.05). Although the parameters evaluated can only be considered as a partial measure of the general activating capacity toward dietary and environmental procarcinogens, these results suggest that human gastric mucosa may be directly involved in the metabolic activation of these compounds to mutagenic/carcinogenic species.