Immunoassays for proteins alkylated by nicotine-derived N-nitrosamines.

Polyclonal antibodies recognizing the pyridyloxobutyl (POB) moiety of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were produced in rabbits immunized either with POB-bovine albumin or POB-Sepharose. The POB intermediates necessary to modify the protein were generated by alkaline (pH 9.0) treatment of the synthetic precursor 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone.

Reduction potentials of imine-substituted, biologically active pyridines: possible relation to activity.

Cyclic voltammetry data were obtained for a number of biologically active compounds which incorporate imine substitution on the pyridine nucleus. The reductions in acid (iminium ion formation) were for the most part reversible, and in the range of -0.5 to -0.

Cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks induced by 4-oxo-4-(3-pyridyl)butanal, a metabolite of a tobacco-specific N-nitrosamine.

The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is metabolized by alpha-carbon hydroxylation to reactive diazohydroxides and aldehydes. The aim of this study was to determine the relative ability of one NNK-derived aldehyde, 4-oxo-4-(3-pyridyl)butanal, to induce cytotoxicity, sister-chromatid exchanges (SCEs) and DNA single-strand breaks (SSBs) in V79 cells. Our data demonstrate that this aldehyde is cytotoxic for V79 cells (IC50 = 0.

Tobacco-specific nitrosamines in Canadian cigarettes.

Twenty-five brands of Canadian commercial cigarettes were analyzed for tobacco-specific nitrosamines (TSNA) in tobacco and in mainstream smoke as well as for nitrate in tobacco. Preformed N'-nitrosonor-nicotine (NNN) in the tobacco ranged from 265 ng to 979 ng/cigarette, preformed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) ranged from 465 ng to 878 ng/cigarette. The mainstream smoke concentration for NNN was between 5 ng and 39 ng/cigarette and for NNK between 5 ng and 97 Ng/cigarette.

Metabolism and DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in fetal tissues of the Syrian golden hamster.

The nicotine derived N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent respiratory tract carcinogen in adult Syrian golden hamsters. In this study, the metabolism and genotoxicity of NNK was investigated in fetal hamster trachea and lung tissues. Fetal lung and tracheal explants were cultured in vitro with [5-3H]NNK, and metabolites released into the culture medium were assayed by high-performance liquid chromatography-scintigraphy.

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by hamster respiratory tissues cultured with ellagic acid.

Previous studies have shown that the nicotine-derived N-nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces tracheal papillomas and lung carcinomas in Syrian golden hamsters. In this study, we showed that hamster tracheal and lung explants metabolize NNK by alpha-carbon hydroxylation, pyridine N-oxidation and carbonyl reduction. alpha-Methylene hydroxylation and methyl hydroxylation yield methylating and pyridyloxobutylating intermediates, respectively.

Transplacental genotoxicity of a tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, in Syrian golden hamster.

NNK is abundant in cigarette smoke and is a potent respiratory carcinogen in adult Syrian golden hamsters. Micronucleus (MN) induction in fetal liver and maternal bone marrow polychromatic erythrocytes (PCEs) were assayed after i.p.

In vitro morphological changes induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-(butanone) in fetal hamster respiratory tract tissue.

Previous studies conducted in our laboratories have shown that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized by fetal hamster respiratory tissues to form DNA-alkylating and clastogenic intermediates. This study was conducted to explore if morphological changes compatible with such events could be detected. Explants of fetal hamster tracheas and lungs were exposed in vitro to various concentrations of NNK and investigated by light and electron microscopy.

In vitro and in vivo modulation of the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in hamster lung tissues.

The metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by hamster lung explants was studied. The three major metabolic pathways were alpha-C-hydroxylation (activation), pyridine N-oxidation (deactivation) and carbonyl reduction. alpha-C-Hydroxylation and pyridine N-oxidation were linear with time (0.

In vitro genotoxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone activated by hamster maternal and fetal tissues.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was found to be a potent genotoxic agent, in the V79 cell line, when activated by liver and lung homogenates from polychlorinated biphenyl (Aroclor)-treated adult Syrian golden hamsters. The damage was observed in the metaphase as well as in the anaphase--telophase divisions. Liver and lung homogenates from non-induced adult hamster were less effective in activating NNK.

Assay of 1-nitropropane, 2-nitropropane, 1-azoxypropane and 2-azoxypropane for carcinogenicity by gavage in Sprague-Dawley rats.

1-Nitropropane (1-NP), 2-nitropropane (2-NP), 1-azoxypropane (1-AP) and 2-azoxypropane (2-AP), were assayed for carcinogenicity by gavage in male Sprague-Dawley rats. 2-NP was given at 1 mmol/kg three times per week for 16 weeks. 1-NP (1 mmol/kg), 1-AP (0.

Effects of alpha-deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its analogues substituted with deuterium at the methylene carbon, 4,4-dideutero-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [4,4-D2)NNK], and the methyl carbon, 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone [(CD3)NNK], adjacent to the N-nitroso group were tested for tumorigenicity in F344 rats. Each compound was administered by 60 s.c.

Cellular changes induced by the tobacco-specific carcinogen 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone in the respiratory tract of Syrian golden hamsters.

The N-nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is abundant in cigarette smoke and is a potent pulmonary carcinogen in Syrian golden hamsters. After intratracheal instillation of NNK to Syrian golden hamsters, we observed focal cell death in the trachea and bronchi followed by cellular regeneration. Regenerating cells were not ciliated and led to mucous-cell hyperplasia followed by squamous-cell metaplasia.

Localization of bound metabolites in the respiratory tissues and oesophagus by high-resolution microautoradiography in Fischer 344 rats treated with N'-nitrosonornicotine.

High-resolution microautoradiography for detection of bound radioactivity in Fischer 344 rats given a single subcutaneous dose of 3H-N'-nitrosonornicotine (3H-NNN) showed a labelling of the olfactory region of the nose, which was restricted to the secretory cells of the glands of the lamina propria mucosae (Bowman's glands). In the oesophagus, bound radioactivity was present in the squamous epithelium, most marked in the middle and superficial cell layers. The olfactory nasal mucosa and the oesophagus are targets for the carcinogenicity of NNN in Fischer 344 rats.

Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine.

The tumorigenic activities and DNA methylating abilities in F344 rats of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the structurally related nitrosamine N-nitrosodimethylamine (NDMA) were compared. Groups of 30 male rats were given 60 s.c.

Metabolism and tissue distribution of tobacco-specific N-nitrosamines in the marmoset monkey (Callithrix jacchus).

Three male marmoset monkeys (Callithrix jacchus) were injected i.v. with the tobacco-specific carcinogen [2'-14C]N'-nitrosonornicotine (NNN) (20.

Study of DNA methylation by tobacco-specific N-nitrosamines.

An enzyme-linked immunosorbent assay (BA-ELISA) involving use of biotin-labeled anti-rabbit IgG and avidin-labeled horseradish peroxidase was developed for the measurement of O6-methyl-2'-deoxyguanosine (O6-MedGuo). Up to 5 micrograms of methylated DNA was enzymatically hydrolyzed, and the extent of inhibition of binding of immobilized O6-MedGuo-bovine serum albumin to rabbit anti-O6-MedGuo was measured. Fifty percent inhibition of antigen-antibody binding was achieved with 2.

Measurement of O6-methyldeoxyguanosine in DNA methylated by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone using a biotin-avidin enzyme-linked immunosorbent assay.

Rabbit antibodies raised against O6-methyl-2'-deoxyguanosine (O6-MedGuo) were used to develop a competitive Biotin-Avidin enzyme-linked immunosorbent assay (BA-ELISA) for the quantitation of this adduct in DNA from animals exposed to tobacco-specific N-nitrosamines. The assay was able to detect as little as 30 mumol O6-MedGuo per mol deoxyguanosine. The specificity of the assay was at least 10 000-fold greater for O6-MedGuo than for unmodified nucleosides or 7-methyl-2'-deoxyguanosine.

Microautoradiographic localization of bound metabolites in the nasal cavities of F344 rats treated with the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the tobacco-specific N-nitrosamine, labeled with 14C on the carbonyl group, was given iv or orally to F334 rats. The animals were killed 4 hours later, and the localization of bound radioactivity in the nasal cavities was studied by light microscopic autoradiography. The strongest labeling was observed in the following groups of glands situated in the lamina propria mucosae: 1) Bowman's glands in the olfactory region, 2) the group of serous glands situated in the anterior half of the nasal septum beneath the respiratory epithelium, 3) the group of serous glands in the middle portion of the lateral wall of the nasal cavity, and 4) the lateral nasal gland (Steno's gland) located lateroventrally to the maxillary sinus.

Effects of chronic ethanol consumption on the metabolism and carcinogenicity of N'-nitrosonornicotine in F344 rats.

The effects of chronic ethanol consumption on the carcinogenicity and metabolism of N'-nitrosonornicotine (NNN) in male F344 rats have been investigated. Groups of 26 to 30 rats were maintained on either a control liquid diet (Groups 1, 3, and 5) or an ethanol-containing liquid diet (Groups 2, 4, and 6) for 4 weeks prior to and during treatment with NNN. The carcinogen was injected s.

Perinatal metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in C57BL mice.

Whole-body autoradiography of pregnant C57BL mice given iv injections of carbonyl-14C-labeled 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) revealed that some metabolites were covalently bound to cellular macromolecules of the nasal mucosae, bronchial mucosae, and liver of the mother. Unbound metabolites were present in those tissues and were also localized in the gastrointestinal lumens, kidneys, urinary bladder, eye melanin, and corpora lutea of the ovaries. Autoradiograms taken at the 1-hour survival interval showed labeling of the fetal kidneys and urinary bladder.

Kinetics of DNA methylation by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats.

The carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was injected intravenously (0.41 mmol/kg) into F344 rats. DNA from target organs (lung, liver) and a non-target organ (kidney) was extracted hydrolysed and analysed for methylated guanines by cation-exchange high-performance liquid chromotography-fluorimetry.

Fate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in pregnant and newborn C57BL mice.

Whole-body autoradiography of pregnant C57Bl mice injected intravenously with the tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), indicated that NNK and/or its metabolites can diffuse through the placenta and reach the fetal tissues. During the last days of gestation, nasal, pulmonary and hepatic tissues develop the enzymatic capacity to activate NNK to alkylating species which bind covalently to cellular macromolecules. Within 4 h of the injection, a considerable proportion of NNK metabolites present in the fetal tissues are excreted in the amniotic fluid via the fetal urinary tract.

Carcinogenicity and metabolic activation of tobacco-specific nitrosamines: current status and future prospects.

Over the past decade, research on the carcinogenicity and metabolism of tobacco-specific nitrosamines has provided a basis for understanding their possible roles in human cancer. 4-(N-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone appears to be the most important tobacco-specific nitrosamine, because of its strong carcinogenicity. A large population of smokers and snuff dippers is exposed to significant quantities of this and the other tobacco-specific nitrosamines on a daily basis.