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The glyoxal-deoxyguanosine adduct (gG) is formed from alpha-nitrosamino aldehydes and dG in vitro and in vivo from nitrosamines carrying the 2-hydroxyethyl side-chain as well as from N-nitrosomorpholine. The structures of all of the diastereomeric forms of both the cis and trans isomers of the adduct have been investigated by ab initio calculations and with nuclear magnetic resonance spectroscopy at 500 MHz. The preferred orientation of the OH groups is trans, but at equilibrium a small amount of the cis isomer was observed. The pH-independent equilibrium constant for the hydrolysis of the gG adduct is K = 1.36 x 10(-4) mol/L, and its rate of formation at pH 7.3 is k = 5.3 min-1 mol-1. In acid (pH 2), the hydrolysis of the nucleosidic linkage is nearly twice as rapid as the hydrolysis of gG to glyoxal and dG. We used a gG analogue to explore a number of reductive methods for derivatization of the adduct, but all of the processes either gave low yields or product mixtures which rendered them impractical for derivatizing the adduct in DNA. A 32P-postlabelling method for detection of the pH-sensitive gG adduct has been developed, which permitted detection of the adduct in the liver DNA of male Wistar rats after administration of selected nitrosamines. The levels of adducts found were: N-nitrosodiethanolamine > 2-hydroxyethylmethynitrosamine > N-nitrosomorpholine > 2-hydroxyethyethylnitrosamine. In separate experiments, N-nitrosodiethanolamine gave greater adduct levels than its metabolite 2-hydroxy-N-nitrosomorpholine. Mechanistic pathways for the generation of gG adducts in vitro and in vivo are discussed.

The tobacco-specific nitrosamine, 4-(N-nitrosomethylamino)-1-(3-pyridyl)- 1-butanone (NNK) can be activated to DNA-binding species by two pathways: methylene hydroxylation leads to an equimolar formation of methyldiazohydroxide and 4-oxo-4-(3-pyridyl)-butanal followed by DNA methylation, while methyl hydroxylation leads to DNA pyridyloxobutylation via the 4-(3-pyridyl)-4-oxobutyldiazohydroxide (Hecht et al., 1988; Spratt et al., 1989). We report here that the ketoaldehyde, 4-oxo-4-(3-pyridyl)-butanal, as well as 4-(N-carbethoxy-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNC), a model compound for pyridyloxobutylation, form DNA adducts in vitro, as detected by 32P-postlabelling assays. The two major adduct spots of both compounds were chromatographically indistinguishable, suggesting structurally similar DNA adducts.

N-Nitrosamine formation by bacteria in the achlorhydric stomach has been proposed as an important factor in the development of gastric cancer. Thus, the effect of the presence of bacteria in the stomach on endogenous nitrosation was investigated in rats given omeprazole (an inhibitor of gastric H+, K((+)-ATPase) which reduces gastric secretion sufficiently to allow survival of a bacterial suspension of Escherichia coli or Pseudomonas. When rats were given both thiazolidine 4-carboxylic acid and nitrate, greater endogenous nitrosamine formation was observed in rats receiving omeprazole and an E. coli suspension than in control or omeprazole-treated rats. A similar result was obtained when rats were given morpholine and nitrate. Since the endogenous formation of N-nitrosomorpholine (NMOR) can be evaluated more precisely from the levels of its urinary metabolites, N-nitrosohydroxyethylglycine (NHEG), the metabolism of NMOR was studied in omeprazole-treated rats. In this preliminary study, we showed that 60% of an oral dose of NMOR was excreted as NHEG, while in rats with a higher gastric pH 20% was excreted as NHEG. The amount of endogenously formed NMOR was increased in omeprazole-treated rats given morpholine and nitrite together with bacteria, and greater excretion of unchanged urinary NMOR was observed. Thus, as shown in this in-vivo model, bacteria efficiently reduce nitrate to nitrite and catalyse nitrosation, resulting in increased endogenous formation of N-nitroso compounds in the achlorhydric stomach.

A method was developed to quantify globin adducts of the tobacco-specific nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). Globin adducts of NNK and NNN release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) after mild treatment with a base. HPB was analysed as its pentafluorobenzoate by capillary column gas chromatography with detection by negative-ion chemical ionization-mass spectrometry and selected-ion monitoring. The detection limit for HPB-pentafluorobenzoate was approximately 1 fmol/injection. The method was applied to haemoglobin from snuff dippers, smokers and nonsmokers. Adduct levels were highest in snuff dippers (517 +/- 538 fmol HPB per gram haemoglobin), followed by smokers (79 +/- 189 fmol HPB per gram haemoglobin) and nonsmokers (29.3 +/- 25.9 fmol HPB per gram haemoglobin). The method will be useful in assessing the role of tobacco-specific nitrosamines as causes of cancer in smokers and snuff dippers.

The tissue localization of the DNA adducts O6- and 7-methylguanine induced in the nasal cavity by the nicotine-derived carcinogen 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK, 30 mg/kg intraperitoneally) has been investigated immunocytochemically in male Sprague-Dawley rats. Adduct-specific nuclear staining, indicative of the metabolic activation of NNK to a methylating compound, was observed in both respiratory and olfactory mucosa. In the respiratory epithelium, strong staining was generally observed in areas devoid of goblet cells. Less intense staining was observed both in the serous gland cells and their efferent ducts in the respiratory submucosa, whereas the mucous gland cells were unstained. In the olfactory mucosa, the sustentacular and basal cells of the olfactory epithelium were moderately stained; staining varied substantially from site to site. No DNA adduct was detected in the olfactory cells. Strong nuclear staining, similar to that in the respiratory mucosa, was observed in the cells of the Bowman glands of the olfactory submucosa. A similar distribution of methylated DNA bases in nasal tissues has been observed in rats after exposure to other N-nitrosamines and in Syrian hamsters after exposure to NNK. This finding may indicate that in man the same cell types undergo DNA adduct formation after exposure to NNK and other N-nitrosamines.

We have characterized the metabolism of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in cultured rat hepatocytes and have established the relationship between various metabolic pathways and single-strand breaks (SSB) in DNA. Metabolism of [5-3H]-NNK by carbonyl reduction, alpha-carbon hydroxylation and pyridine N-oxidation was linear from 0.5 to 6 h with 0.25-2 x 10(6) hepatocytes. Using an alkaline elution assay, we observed that NNK induces SSB in DNA in a dose- and time-dependent manner. SSB induced by NNK were rejoined partially within 2 h and totally by 12 h after exposure. NNK-N-oxide produced a smaller number of SSB than NNK, suggesting that pyridine N-oxidation of NNK is a deactivation pathway. Carbonyl reduction of NNK led to 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butan-1-ol (NNAl). Reaction of NNK with methyl magnesium iodide gave 4-(N-nitrosomethylamino)-1-(methyl)-1-(3- pyridyl)butanol-1-ol (1-MeNNAl) 82% yield. NNAl, but not 1-MeNNA1, can be reoxidized to NNK. Doses of 5 mM NNAl and 1-MeNNAl both induced SSB, indicating that NNAl does not require reconversion to NNK to be activated to DNA damaging intermediates. alpha-Methylene hydroxylation resulted in the formation of 4-oxo-4-(3- pyridyl)butanal. At equimolar concentration (5 mM), the aldehyde was more damaging than NNK to hepatocyte DNA. The results of this study demonstrate that NNK is activated by rat hepatocytes and that metabolites formed by alpha-carbon hydroxylation induce SSB.

The carcinogenic activity of endogenously synthesized N-nitroso-bis(2-hydroxy-propyl)amine (NDHPA) was investigated in male Wistar rats administered bis(2-hydroxypropyl)amine (DHPA), mixed into a powdered diet at a concentration of 1%, and NaNO2 dissolved in distilled water at concentrations of 0.15% and 0.3%, for 94 weeks. Urinary excretion of NDHPA clearly demonstrated its endogenous synthesis in rats given 1% DHPA and 0.3% NaNO2, but not in the groups receiving either of these precursors alone. Tumours of the nasal cavity, lung, oesophagus, liver and urinary bladder were found in rats treated with 1% DHPA and 0.15% or 0.3% NaNO2. The incidences of nasal cavity and lung tumours reached 74% and 58% respectively, in rats given 1% DHPA and 0.3% NaNO2. The tumour distribution was almost the same as that seen in rats given NDHPA. These results indicate that endogenously synthesized NDHPA has similar carcinogenic activity to exogenously administered NDHPA in rats.

The inhibitory effect of 145 samples of Chinese tea on the formation of N-nitrosomorpholine was studied in vitro. The rates of inhibition by green tea, crush, tear, curl (CTC) black tea, brick tea, jasmine tea, Oolong tea, sun-dried tea and black tea were positively correlated with their polyphenol contents. An inhibitory effect of green and black tea on endogenous N-nitrosation was also confirmed in humans. Drinking tea after a meal had a greater effect than drinking it before a meal.

Inhibition of enzyme activity by monoclonal antibodies (MAbs) was used to indicate which cytochrome P450 isozymes in Sprague-Dawley rat liver microsomes catalyse hydroxylation of the oesophageal carcinogen N-nitrosomethyl-n-amylamine (NMAA) to give 2- to 5-hydroxy-NMAA (HO-NMAA), formaldehyde and pentaldehyde. Liver microsomes (0.3-0.6 mg protein) were incubated (15 min, 23 degrees C) with 0.4 mg MAb and, after adding NMAA to 6 mM, incubated for 20 min at 37 degrees C. Mixtures were analysed for HO-NMAAs by gas chromatography-thermal energy analysis and for aldehydes by high-performance liquid chromatography of their 2,4-dinitrophenylhydrazones. The percentage inhibition by each MAb indicates the percentage metabolism by the corresponding P450 isozyme(s). These results indicate that the MAb to P450 IIB1 cross-reacts with P450 IIE1 and that the MAb to male-specific constitutive IIC11 cross-reacts with female-specific IIC12. Taking this into account, the main results were as follows. With uninduced male microsomes, 4-hydroxylation was catalysed mainly by IIC11 and demethylation by IIC11 and IIE1. With uninduced female microsomes, P450s reacting with the MAb to IIC11 (probably mainly IIC12) were responsible for most of the 4-hydroxylation and demethylation. With 3-methylcholanthrene-induced male microsomes, most 3-hydroxylation and some depentylation were due to IA1 or IA2. With phenobarbital-induced microsomes, all six reactions, but especially 4-hydroxylation and depentylation, were largely due to IIB1. With Aroclor-induced microsomes, all six reactions were catalysed by IIB1 and IA1 or IA2. The role of P450 IIC11 in 4-(omega-1)-hydroxylation was striking.

Mouse macrophages produced nitrite and N-nitrosomorpholine after incubation with catalase. A macrophage cell line, J774.1 (1 x 10(6) cells/ml), was incubated with catalase (500 U/ml) and morpholine (5 mM); after 48 h incubation at 37 degrees C, macrophages produced nitrite (100 microM) and N-nitrosomorpholine (1 microM). Stimulation of J774.1 cells with catalase enhanced interleukin-1 production and tumour-killing activity against mastocytoma P815 cells. Flow cytometric analysis showed that catalase was bound to the surface of the macrophages.

The enzymatic activation of simple dialkylnitrosamines is widely perceived to involve cytochrome P450-mediated alpha-hydroxylation to generate an unstable alpha-hydroxynitrosamine. This process can also result in the denitrosation of the nitrosamine, presumably through a common intermediate. We present evidence that the critical intermediate is the alkynitrosaminomethyl free radical, which we generated by thermal decomposition of a nitrosamino acid perester. The radical rapidly loses NO to generate an N-alkylmethyleneimine. The radical is also produced during the Ce(IV) oxidation of beta-hydroxynitrosamines after fragmentation, where it not only loses NO but is oxidized further to a cation which reacts with water to form an alpha-hydroxynitrosamine. These results provide models of the activation and detoxication pathways for beta-oxidized nitrosamines.

Aliphatic methylalkylnitrosamines with a chain length of three to six carbon atoms are powerful oesophageal carcinogens in rats and have been shown to methylate target organ DNA preferentially. This class of carcinogens is efficiently metabolized not only in the oesophageal mucosa but also in the mucosa of the nasal and oral cavity, trachea and bronchioli, i.e., tissues derived from the rat ventral entoderm. In order to determine whether more than one cytochrome P450 isozyme is involved in the bioactivation of asymmetrical aliphatic dialkylnitrosamines in these tissues, we have studied the effects of various modulators of nitrosamine metabolism, including dietary zinc deficiency, ethanol and disulfiram, on DNA alkylation by N-nitrosomethyl-n-butylamine (NMBA) and its ethyl analogue N-nitrosoethyl-n-butylamine (NEBA). Formation of O6-methyl- and O6-ethyldeoxyguanosine by a single dose of NMBA and NEBA, respectively, was quantified after a survival time of 6 h by immuno-slot-blot assay. In control rats, methylation of DNA by NMBA was highest in oesophagus, followed by nasal mucosa, liver and lung. Formation of O6-ethyldeoxyguanosine from NEBA, however, was twice as high in liver as in nasal mucosa and lung and four times as high in liver as in oesophagus. In oesophagus, trachea and bronchioli, both nitrosamines were selectively metabolized in mucosal cells. Bioactivation of NMBA and NEBA was almost completely inhibited in nasal mucosa by ethanol. In contrast, a striking interorgan shift in DNA methylation by NMBA from liver (-50%) to lung (+100%), oesophagus (+300%) and nasal mucosa (+400%) was obtained with dietary disulfiram, whereas only 20-50% increases in extrahepatic DNA ethylation were determined for NEBA.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of molecular nitrogen formed as a consequence of nitrosamine alpha-hydroxylation provides a useful means for studying the extent of activation of these compounds in target and nontarget organs and tissues. alpha-Hydroxylation in rat liver and urothelial cells was compared using as substrate doubly 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine (15N-NBHBA), a potent bladder carcinogen in rodents. Both enzyme sources metabolized 15N-NBHBA through the alpha-hydroxylation pathway. 15N2 production was dependent on the amount of substrate incubated. Vmax values for 15N2 production by urothelial cells and by liver postmitochondrial supernatant were 4.47 and 3.21 nmol/mg protein per h, respectively.

Many nitrosamines are metabolized by cytochromes P450, one of which (P450IIE1) has received much attention because of its role in the metabolic activation of N-nitrosodimethylamine. This enzyme exists in man, rat, mouse, hamster and other animal species. It is inducible by fasting, diabetes and exposure to ethanol, acetone, isoniazid, benzene and other chemicals. P450IIE1 is responsible for the low Km form of N-nitrosodimethylamine demethylase and is the major enzyme catalysing the metabolic activation of this carcinogen. In addition, P450IIE1 is the most active P450 species known in the metabolism of N-nitrosoethylmethylamine and N-nitrosopyrrolidine. In the metabolism of N-nitrosobutylmethylamine, P450IIE1 preferentially oxidizes the methyl group over the butyl group, whereas P450IIB1 efficiently oxidizes both the methyl and butyl groups. P450IIB1 also catalyses the alpha-oxygenation of both the pentyl and methyl groups of N-nitrosopentylmethylamine, forming pentaldehyde and formaldehyde at a rate ratio of 2:1, as well as oxygenation at other carbons of the pentyl group. Many nitrosamines are effectively activated in nonhepatic target tissues. The metabolism of 4-(N-nitroso-methylamino)-1-(3-pyridyl)-1-butanone in lung and nasal microsomes is discussed.

N-Nitroso-N-methylaniline (NMA) is a strong oesophageal carcinogen in rats but exhibits few overt genotoxic effects. Previous work from our laboratory established that NMA is readily metabolized by cytochrome P450-catalysed N-demethylation to produce the benzenediazonium ion (BDI), a relatively stable but reactive electrophilic agent. We have also shown that BDI reacts with DNA to form an acid-labile adduct. We have now shown that BDI, generated chemically or by the metabolism of NMA in vitro, reacts with DNA to form a triazene coupling product at the N6-position of adenine residues. This adduct has also been shown to be produced in the liver DNA of rats treated with NMA. Procedures for the isolation of DNA and for analysis of the adduct are presented.

In a series of experiments on formation of N-nitrosamines from precursors in the gastric juice, we investigated individual nitrosation capacity and the influence of inhibitors on this process. In the presence of a molar ratio of 2:1 ascorbic acid:NaNO2, we found increased nitrosamine formation in 34% of gastric juice samples. Ferulic acid and caffeic acid generally suppressed nitrosation at high concentrations and stimulated it at low concentrations. The concentration at which the transition from a stimulating to an inhibitory effect occurs depends on individual differences in the gastric juice.

The mutagenicity of nine carcinogenic N-nitrosopropylamines was studied by the Ames preincubation assay using 9000 g supernatant (S9) fractions or alcohol dehydrogenase. Treatment of animals with polychlorinated biphenyls or phenobarbital resulted in a marked increase in the ability of liver S9 to activate N-nitrosobis(2-hydroxypropyl)amine, N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine, N-nitrosobis(2-oxopropyl)amine, N-nitrosobis(2-acetoxypropyl)amine, N-nitroso-2,6-dimethylmorpholine, N-nitroso(2-hydroxypropyl)methylamine, N-nitroso(2-oxopropyl)methylamine, N-nitroso(2,3-dihydroxypropyl)methylamine and N-nitroso(2,3-dihydroxypropyl)(2-hydroxypropyl)amine to mutagens, whereas 3-methylcholanthrene induction was not effective. All reactions required NADP as a cofactor for mutagenic activation, and nitrogen, carbon monoxide, cytochrome c and metyrapone considerably inhibited their mutagenic activities, whereas 7,8-benzoflavone did not. Five propanol derivatives were not mutagenic in the presence of NAD and alcohol dehydrogenase. We conclude that the phenobarbital-inducible major cytochrome P450 in liver S9 from five animal species tested was selectively involved in mutagenic activation. The same cytochrome in human liver S9 and in lung S9 from three rodent species also activated the mutagenicity of N-nitroso(2-hydroxypropyl)methylamine.

Betel-quid ingredients were found to produce reactive oxygen species, such as superoxide anion and hydrogen peroxide, in vitro. We demonstrated that N'-nitrosonornicotine (NNN) can be converted to its active metabolite, hydrogen peroxide, nonenzymatically in the presence of ferrous ions and ethylenediaminetetracetic acid (EDTA) at pH 7.2. Three ultimate metabolites of NNN--NNN-1-N-oxide, 4-hydroxy-4-(3- pyridyl)butyric acid and 4-oxo-4-(3-pyridyl)butyric acid--and nornicotine were detected by high-performance liquid chromatography. 3H-NNN and 14C-NNN interact with calf thymus DNA in the presence of hydrogen peroxide, ferrous ion and EDTA. The results suggest that formation of reactive oxygen species in the presence of NNN may be a key factor in the initiation of oral tumours in tobacco and betel-quid chewers.

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)

Nitrite was formed on incubation of N-nitrosamines with both microsomal systems and a reconstituted system consisting of cytochrome P-450 and NADPH P-450 reductase from pig liver. Nitrite was not obtained when the nitrosamines were incubated with NADPH P-450 reductase alone or when molecular oxygen or NADPH was omitted. Various inhibitors of the microsomal monooxygenase decreased nitrite generation. Furthermore, nitrite and a substantially higher amount of nitrate could be found in the urine of rats given N-nitrosodiphenylamine. Diphenylamine was also detected. From in-vitro studies, it is concluded that denitrosation of N-nitrosamines is a cytochrome P-450-dependent process, which also occurs in vivo.

The effect of ascorbic acid on the oxidation of N-nitrosopiperidine by a microsomal preparation obtained from guinea-pig liver has been examined. The metabolites from the oxidation of N-nitrosopiperidine were found to be 5-hydroxypentanal (about 1.93%), N-nitroso-3-hydroxypiperidine (about 0.02%), and N-nitroso-4-hydroxypiperidine (about 0.15%). With microsomes separated from the liver of guinea-pigs pretreated with phenobarbital and 3-methylcholranthrene, the yields of N-nitroso-3-hydroxypiperidine and N-nitroso-4-hydroxypiperidine were found to decrease upon increasing the concentration of ascorbic acid in the reaction mixture. In the presence of small amounts of ascorbic acid, the yields of these compounds with untreated liver microsomes apparently increased, while the presence of larger amounts of ascorbic acid caused a decrease in the yields of these metabolites. In contrast with the yields of N-nitroso-3-hydroxypiperidine and N-nitroso-4-hydroxypiperidine, that of 5-hydroxypentanal decreased with increasing amounts of ascorbic acid.

Subsequent to modification of N-nitrosamine metabolism by disulfiram, mucus-producing cells and Clara cells in the respiratory tract are involved increasingly in detoxification as well as in bioactivation of N-nitroso-N-methylbenzylamine and N-nitrosodibutylamine. Overtaxing of these cells or local concentration of antigenic metabolites leads to cytolytic defects in tracheal, bronchial and bronchiolar epithelium, in addition to toxic degenerative lesions. The resulting continuous stimulation of proliferation leads to basal-cell hyperplasia, squamous-cell metaplasia and squamous papillomas. In areas with insufficient differentiation, due to cell proliferation, there is an increased probability that focal mutation, subsequent to alkylation of purine bases, will be passed from one cell generation to the next, with subsequent formation of tumours in the bronchiolo-alveolar region.

Application of the WHO Nitrosation Assay Procedure (NAP test) to a range of potentially nitrosatable drugs has given rise to considerable variations in the formation of volatile N-nitrosamines and N-nitroso compounds as a group. No nitrosation whatsoever was observed with 40 mM nitrite in some instances. In simulating more closely the conditions likely to be encountered in the human stomach, however, the order of susceptibility of the drugs to N-nitrosation has proved to be very different. At a constant nitrite concentration of 25 microM, which is considered to represent the upper limit of those likely to be encountered in the acidic human stomach, the drugs giving rise to the greatest yields of products reacting as N-nitroso compounds from a maximum adult dose were the penicillins, G, V, cloxacillin and ampicillin.

Volatile N-nitrosamines have been detected in the human environment--in work places, such as the rubber, leather and chemical industries, in tobacco smoke and also inside new cars. In order to make risk assessments on the basis of inhalation experiments with animals at dose levels relevant to the human situation, it is important to know the actual absorption rate in the respiratory tract. In this study, 63 female Sprague-Dawley rats were exposed to four nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine and N-nitrosomorpholine) in air. Respiratory parameters were monitored by pneumotachography. After 10 min of inhalation, the exhaled air was collected for 10 min in steps of 2 min intervals and analysed for its content of nitrosamines with a Thermal Energy Analyzer. Inhalation and exhalation were maintained by endotracheal intubation under narcosis with Thalamonal. The influence of this anaesthetic on the urinary excretion of N-nitrosodimethylamine after gavage and inhalation was tested: in comparison with unanaesthetized animals and with a group under ether narcosis, the excretion of the nitrosamine by the Thalamonal-anaesthetized animals was drastically reduced (factor of 20 to 60). Independent of the concentration of inhaled nitrosamine, ranging from 1-450 micrograms/L in air, the relative amounts of exhaled substance 10 min after inhalation were 0.9% N-nitrosodimethylamine, 0.4% N-nitrosodiethylamine, 6% N-nitrosopyrrolidine and 5% N-nitrosomorpholine. Extrapolation of these data back to the first exhalation (i.e., following the last inhalation) revealed that a remarkable amount of substance may be exhaled (up to 30% N-nitrosodimethylamine).

To investigate a possible connection between the pattern of the excreted metabolites of a series of nitrosomethylalkylamines and their tumour spectrum in rats, the urinary metabolites of nitrosomethylalkylamines with alkyl chain lengths from C4 (n-butyl) to C14 (n-tetradecyl) have been studied, using male Fischer rats. All of these compounds induced a high incidence of tumours in the rats, but only those with chain lengths of 8, 10, 12 or 14 carbons induced bladder tumours. The principal metabolite from the nitrosamines with an odd number of carbons in the alkyl chain was nitrosomethyl-2-carboxyethylamine. Nitrososarcosine and nitrosomethyl-3-carboxypropylamine were the principal metabolites from those compounds with even-numbered chains. No trend was discernible between the pattern or yields of these acids and the carcinogenic effectiveness. The principal component in the neutral fractions was nitrosomethyl-2-oxopropylamine (less than or equal to 1% of the dose administered). Very small amounts were obtained from the odd-numbered chain compounds, but serially increasing amounts from the even-numbered chain compounds.

Trimorphamide [N-(1-formamido-2,2,2-trichloroethyl)morpholine], with an acute LD50 of 990 mg/kg, is a systemic fungicide under development. To examine its possible nitrosation in vivo, CFY rats were treated i.g. with 326 mg/kg trimorphamide, suspended in 40 mg/kg sodium nitrite. after 1 hour, another 40 mg/kg sodium nitrite was given i.g. and the animals were killed an hour later. The nitroso derivative was extracted by dichloromethane from the gastric content, after steam distillation. The samples were analysed by gas chromatography (GC) and mass spectrometry (MS). N-Nitrosomorpholine (NMOR) was identified by comparison with authentic samples, both by GC and MS, using retention times and electron impact mass spectra. Quantification was carried out by GC-MS-specific ion monitoring, using the m/e 116 peak. The yield of NMOR ws found to be 0.65%. By the use of the Ames test, NMOR formed in vivo proved to be strongly mutagenic for the strains TA 1535 and TA 100, both in presence and absence of S-9 fraction. Tumour-bearing animals, treated by sodium nitrite plus trimorphamide, are being analysed.

The metabolic fate of the lung carcinogen N-nitrosobis(2-hydroxypropyl)amine (ND2HPA) in male Wistar rats was studied. The blood level after a single intraperitoneal (i.p.) injection of [1-14C]-ND2HPA at a dose of 3 g/kg body weight reached a maximum within 1 h. Most of the administered 14C was eliminated via the urine; 90.8% of the 14C was excreted in urine within 24 h, 5.5% in faeces, and 3.2% in expired air. About 11% of the 14C was detected in bile collected over 24 h. A relatively high concentration of 14C was found in the blood and target organs, such as the lung, liver, thyroid gland and kidney 1 h after treatment. Analysis by high-pressure liquid chromatography showed that the 14C in the blood and urine was mostly accounted for by unchanged ND2HPA, together with smaller amounts of N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (N2HP2OPA). ND2HPA and N2HP2OPA were also detected in the lung and liver of rats 30 min to 12 h after the administration and were present in higher concentrations in the blood and lung than in the liver and pancreas. Besides ND2HPA and N2HP2OPA. N-nitrosomethyl(2-hydroxypropyl)amine (NM2HPA) was also found in urine collected over 6 h. ND2HPA, N2HP2OPA and NM2HPA showed mutagenicity in the Salmonella assay system with metabolic activation by a 9000 X g supernatant of rat liver, and N2HP2OPA was also mutagenic in the presence of a rat lung preparation. These data suggest that N2HP2OPA and NM2HPA might be important intermediates in the metabolic activation of ND2HPA to its ultimate carcinogenic form in rats.

Substitution of N-nitrosodialkylamines with fluorine at specific sites inhibits oxidative metabolism at the respective carbon atoms. The results of in-vitro metabolism studies with N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA) and their fluorinated analogues, N-nitroso-2,2,2-trifluoroethyl-ethylamine (NDEA-F3), N-nitroso-bis(2,2,2-trifluoroethyl)amine (NDEA-F6), N-nitroso-4,4,4-trifluorobutyl-butylamine (NDBA-F3), N-nitroso-bis(4,4,4-trifluo-robutyl)amine (NDBA-F6) and N-nitroso-bis(2,2,3,3,4,4,4-heptafluorobutyl)amine (NDBA-F14), showed effects of fluorination on biotransformation which can explain results of carcinogenicity and mutagenicity experiments; NDEA-F6 and NDBA-F14 were practically not metabolized by microsomal fractions, even though no decrease in binding affinity to cytochrome P450 was observed. Both compounds were not biologically active and were exhaled unchanged in high proportions after oral administration to the rat. Biologically active analogues, NDEA, NDBA, NDBA-F3 and NDBA-F6, were found to be dealkylated at the unfluorinated alkyl chains and, to a lesser extent, at the omega-fluorinated alkyl chains. Detection of corresponding alcohols as hydrolysis products confirmed the generation of electrophilic intermediates by alpha-C-hydroxylation. However, trifluorethanol was detected only in very small proportions from dealkylation of NDEA-F3, although dealkylation occurred almost exclusively at the unfluorinated site.

A sensitive method was developed for quantitation of N-nitrosomorpholine (NMOR) formation in vivo in rats. A major metabolite of NMOR, N-nitroso(2-hydroxyethyl)glycine, was detected by gas chromatography-thermal energy analysis in the urine of rats treated with trace levels of NMOR or treated sequentially with morpholine and sodium nitrite. Since significant levels of 2-hydroxyethylglycine do not seem to occur in rat urine, even after treatment with morpholine, artefact formation was not a problem. Formation of 1 micrograms NMOR in vivo was readily detected. The formation of NMOR from morpholine and sodium nitrite in vivo in rats appeared to depend on the doses of morpholine and [sodium nitrite]2 and occurred in higher yield than reported for formation of N-nitrosoproline, as expected. The formation of NMOR from morpholine and sodium nitrite under the conditions of the carcinogenicity study of Shank and Newberne was quantified. The daily levels of NMOR formation were highly variable, but the mean level was consistent with the tumorigenicity data in that dose-response study.

The technique of Ohshima and Bartsch for measuring the excretion of N-nitrosoproline (NPRO) was applied in a balance trial with cows to estimate the in-vivo formation of N-nitrosamines. In the first trial, a cow with a high milk production received rations of concentrates and hay with low or high contents of nitrate and free proline. Samples were taken from the urine, faeces and milk. In the second trial, two non-lactating cows were put on hay rations with different nitrate and free proline contents, and samples were taken from the rumenal fluid every 15 min to measure the formation of nitrite from the ingested nitrate. Samples of urine and faeces were also taken. It was found that dried roughage may contain considerable amounts of free proline, and the NPRO formed is related to the nitrate content. No NPRO was found in the urine and faeces when the NPRO content of the hay was low, not even when the ingested nitrate was reduced drastically to nitrite or when the concentration of free proline was high. When NPRO was present in the feed, it was recovered from the urine and faeces but was not transmitted to the milk.

Endogenous nitrosation of proline was investigated in smokers and nonsmokers. Volunteers consumed a volume of beetroot juice equivalent to 325 mg nitrate and, 1 h later, 500 mg proline. In separate experiments, volunteers ingested proline alone. Twenty-four-hour urines were collected and analysed for N-nitrosoproline. When proline was ingested alone, there was no significant difference in urinary N-nitrosoproline excretion between smokers and nonsmokers. When beetroot juice and proline were consumed, however, smokers produced approximately 2.5 times as much N-nitrosoproline as nonsmokers. Salivary nitrite levels of smokers and nonsmokers, both before and after consumption of beetroot juice, were not significantly different. Salivary thiocyanate levels were approximately 3.2. times higher in smokers than in nonsmokers. Our results suggest that the higher level of salivary thiocynate in smokers is responsible for the increased rate of endogenous nitrosation of proline in that group when compared with nonsmokers. Oxides of nitrogen in cigarette smoke do not appear to play a significant role.

The biochemical retroaldol-like fragmentation of beta-hydroxynitrosamines has been investigated further. The extent of fragmentation of 2-hydroxy-2-methylpropyl-methylnitrosamine (HMPMN) to N-nitrosodimethylamine and acetone induced by metabolic activation increases as the NADPH level is decreased. 2-Hydroxy-2-phenylethyl-methylnitrosamine (HPhEMN) undergoes competitive oxidation to 2-oxy-2-phenylethyl-methylnitrosamine (OPhEMN) and fragmentation to benzaldehyde and N-nitroso-dimethylamine in the presence of a metabolic activation system from rat liver. The extent of the oxidation was increased by preinduction of the rats with phenobarbital, or separate addition of NADPH and NAD, but was decreased by addition of dimethyl sulfoxide. The fragmentation was observed most readily when oxidation was inhibited or was not induced by cofactors. When HPhEMN was administered to a rat intraperitoneally, benzaldehyde (fragmentation) was found in the urine with OPhEMN and the substrate, but only the last two substances were found in liver and blood. These experiments provide evidence for retroaldol-like fragmentation of beta-hydroxynitrosamines both in vitro and in vivo. In a related investigation, it was found that N-nitroso-N-4-chlorophenyl-2-aminoethanal (NCAE) is extremely reactive and induces spontaneous generation of 4-chlorobenzenediazonium ion in chloroform, as trapped by 2-naphthol. NCAE reacts with dimethylamine in chloroform, benzene or methanol to give N-nitrosodimethylamine and 4-chloroaniline, among other products. This suggests that beta-nitrosaminoaldehydes produced by the biooxidation of their corresponding alcohols could produce cell alteration through alkylation, deamination or transnitrosation.

The principal pathway of nitrosamine metabolism has long been considered to be alpha-hydroxylation. For N-nitrosodialkylamines, this hypothesis requires that a molecule of molecular nitrogen be released for every molecule of nitrosamine that is alpha-hydroxylated. Thus, the quantitative determination of nitrogen formation should provide a measure of the importance of this pathway. This method was applied earlier to the doubly-labelled nitrogen-15 compounds, N-nitrosodimethylamine (NDMA), N-nitrosomethylphenylamine (NMPhA) and N-methyl-N-nitrosourea (MNU), using both a 9 000 X g supernatant fraction of liver and the intact animal as metabolic systems. The in-vitro results were quite different from those obtained in vivo. The majority of the NDMA (67%) and the MNU (88%) were converted to nitrogen in vivo, while NMPhA gave considerably less nitrogen (52%). These results differed by a factor of approximately two from those obtained in vitro (NDMA, 33%; NMPhA, 18.8% and MNU, 96%). Since such differences may be a result of the loss of cellular architecture, we have extended the work to include isolated hepatocytes. It had been shown previously that isolated hepatocytes constitute a practical alternative to in-vivo systems, even though the correlation with in-vivo metabolism appears to depend on the substrate analysed. The values obtained using this system (NDMA, 47%; NMPhA, 23%; and MNU 105%) reconfirm that metabolism may be substrate dependent. As in our previous studies, no mixed nitrogen (15N14N) or labelled nitrogen oxides were found. The data are all consistent with the hypothesis that at least one demethylase for each of the nitrosamine substrates is associated with a cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the urinary metabolites of N-nitrosopiperidine, 3- and 4-hydroxy-N-nitrosopiperidine, N-nitroso-4-piperidone, N-nitrosohexamethyleneimine and N-nitroso-heptamethyleneimine. The N-nitrosopiperidines induce primarily oesophageal tumours in rats; N-nitrosohexamethyleneimine induces oesophageal and liver tumours and N-nitrosoheptamethyleneimine induces oesophageal and lung tumours. The products were identified by gas chromatographic comparison with standards, where possible, and by gas chromatography-mass spectrometry. The percentage of the dose excreted as metabolites in the urine increased with ring size. Hydroxy and ketone derivatives of all three of the N-nitrosoalicyclic amines were found. Pimelic acid was the major metabolite (congruent to 25%) of N-nitrosoheptamethyleneimine; adipic acid was identified as a metabolite of N-nitrosohexamethyleneimine, but not glutaric acid was found from N-nitrosopiperidine.

Chinese gooseberry juice has been found to be an efficient inhibitor of nitrosation both in vitro and in vivo. This activity appears to be due partly to high concentrations of ascorbic acid and partly to an unidentified nitrite scavenger.

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. Incubation of tissue slices with NNK indicated that the nose, the lung and the liver of 13-day-old fetuses could reduce NNK to 4-(methylnitrosamino)-1-(3-pyridyl)butan -1-ol (NNA1), but could not activate NNK by alpha-carbon hydroxylation. However, these activating enzymes were competent in 18-day old fetuses, and the activities increased during the first six days of life. The results provide evidence that NNK could exert genotoxic effects transplacentally and in newborn mice.

Results from animal experiments and studies in human subjects indicated that the amount of nitrosoproline (NPRO) excreted in the 24-h urine, following ingestion of precursors, is an index for the rate of endogenous nitrosation; this method was found to be sensitive, reproducible and could be satisfactorily applied to human subjects in clinical and field studies. N-Nitrosothiazolidine 4-carboxylic acid (NTCA) and its 2-methyl derivative (NMTCA) were also identified in the urine of human subjects. As the respective amino precursors (thiazolidine 4-carboxylic acids) can be formed by reaction of formaldehyde or acetaldehyde with cysteine, measurement of NTCA and NMTCA in urine may provide a further index for endogenous nitrosation in the human body and may also allow monitoring of exposure of human subjects to aldehydes, nitrate and nitrite. The yield of nitroso compounds formed endogenously in the human body was shown to be linked to the intake of precursors, but several inhibitors and catalysts, either as pure substances or occurring in complex mixtures, were shown to modify the nitrosation reaction in vivo. In particular, ingestion of ascorbic acid after nitrate-rich meals was efficient in lowering human exposure to endogenously formed N-nitroso compounds. A dose-response relationship was established for the formation of NPRO in rats in vivo, after concurrent administration of various concentrations of the precursors, L-proline and sodium nitrite. The logarithm of the amount of NPRO formed was found to be proportional to the logarithm of the product of the proline dose and the square of the nitrite dose. On the basis of these results, a kinetic model was formulated allowing the estimation of the daily precursor dose quantity, ([amine][nitrite]2), required to give 50% tumour incidence in rats after two years of feeding. The potential application of this model, for the estimation of carcinogenic risk from endogenously formed N-nitrosamines in humans, is discussed. Our results demonstrate unequivocally the endogenous formation of N-nitroso compounds in the human body, the significance of which in human carcinogenesis remains to be established.

Several beta-oxidized nitrosamines have been shown to induce pancreatic cancer in Syrian hamsters. N-Nitrosobis(2-oxopropyl)amine (ND2OPA) is the most specific, but it also induces either colonic or prostatic tumours in MRC-Wistar rats. In-vivo and in-vitro metabolic studies show that N-nitrosomethyl(2-oxopropyl)amine is a metabolite of ND2OPA in hamsters but not in rats. ND2OPA is also present in higher concentrations in hamster pancreas than in the liver. No similar effect occurs in rats. These factors may explain, in part, the organotropism and species specificity of this nitrosamine.

alpha-Tocopherol has been evaluated as a nitrite scavenger for the prevention of nitrosamine formation in a model system and under practical conditions. alpha-Tocopherol was found to react with nitrosating agents in both lipophilic and aqueous environments. The use of alpha-tocopherol was shown to inhibit aminopyrene-nitrite induced hepatotoxicity in rats and to reduce the amount of NDMA formed in cigarette smoke. Of primary interest is the finding of a significant reduction of NPYR formation in fried bacon. Nitrosamine inhibition was greater with alpha-tocopherol used in combination with sodium ascorbate than with sodium ascorbate alone.

The nitrosation of aminopyrine (4.3 mmol/l) and dimethylamine (4.3 mmol/l) by nitrite (4.3 mmol/l) were studied over the pH range 6-8 in a system containing bile acids and lipid. Both amines were nitrosated to form N-nitrosodimethylamine. The nitrosation of aminopyrine and, to a greater extent, dimethylamine, is enhanced by bile acids. The simultaneous incorporation of ascorbic acid (8.6 mmol/l) and dl-alpha-tocopherol (4.3 mmol/l) was found to be effective in preventing the nitrosation of these two amines. dl-alpha-Tocopheryl acetate was found to be an ineffective blocking agent in this in vitro system.

The carcinogenicity of eleven alpha-oxidized nitrosamines (alpha-acyloxy, alpha-hydroperoxy and alpha-oxo nitrosamines) was tested in F-344 rats. All chemicals were dissolved in olive oil and rats received 10 weekly subcutaneous injections of these chemicals (10 x 5 mg/kg of N-methyl-N-(acetoxymethyl) nitrosamine or equimolar amounts of the other chemicals) at the interscapular region. Subcutaneous tumours were detected in many rats of all groups treated with the chemicals, although no tumour was detected at the injection site in the control group. Lung and thyroid tumours were also observed in many rats of the treated groups, especially those injected with N-alkyl-N-(acetoxymethyl)nitrosamines. Many other tumours developed in the experimental groups, but it is not certain that they were related to the treatment with the chemicals. The results indicate that the chemicals possess local as well as systemic carcinogenicity in F-344 rats. The potent carcinogenic effects at the injection site of the alpha-oxidized nitrosamines, coupled with their direct mutagenic activity, suggest that these derivatives are useful models for the ultimate form of the metabolically activated nitrosamines.

The effect of disulfiram (DSF) on the biological activity of N-nitrosodiethylamine (NDEA) was studied in vivo. We determined its influence on NDEA level, on NDEA-induced DNA damage and on DNA alkylation in the liver and oesophagus. It was found that 500 mg/kg DSF given 2 h before a single dose of 28 mg/kg NDEA inhibited the metabolism and increased the concentrations of NDEA in the organs, especially in the oesophagus. Consequently, DNA damage and the alkylation of DNA are inhibited in both the liver and the oesophagus.

The omega- and omega-1-hydroxylation of N-nitrosodi-n-butylamine (NDBA) has been studied in microsomes from rat liver, lung, intestine and kidney. Both reactions followed at least two enzyme kinetics with low (2-10 microM) and high (1 mM) Km values. Whereas omega-1-hydroxylation was the predominant pathway in liver, omega-hydroxylation was more important in extrahepatic tissues. First-pass metabolism of NDBA in lungs and intestinal mucosa may be of importance in the development of urinary bladder tumours in rats.

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), pyrazole (PY) and ethanol (EtOH) pretreatment on N-nitrosodimethylamine (NDMA), N-nitrosobutylmethylamine (NBMA) and N-nitrosomethylbenzylamine (NMBzA) metabolism were examined in rats. In isolated hepatocytes, PB increased the metabolic decomposition of NBMA and NMBzA, and MC increased that of NBMA; PY and EtOH increased only that of NDMA. In studies of hepatic microsomal dealkylation, PB increased NBMA debutylation and NMBzA debenzylation, and MC increased NBMA debutylation; PY and EtOH increased NDMA demethylation selectively. Several cytochrome P450 (P450) species were active in dealkylating nitrosamines, indicating that the organ-specific carcinogenicity of nitrosamines might be changed by various P450 inducers.

Methods were developed to determine the biological half-life of N'-nitrosonornicotine (NNN) and 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in Syrian golden hamsters and Fischer rats. The formation and elimination of 4-(N-nitrosomethylamino)-1-(-3-pyridyl)-1-butanol (NNA1), the major metabolite of NNK, was determined in the context of this study. The method consisted of extraction of the nitrosamine with ethyl acetate, elution through a Clin-Elut column, and concentration of the sample, followed by gas chromatography-thermal energy analysis. Biological half-lives of NNN, NNK and NNA1 in hamsters were found to be 0.77, 0.25 and 1.78 h, respectively; in rats they were 5.78, 1.78 and 3.56 h. These findings clearly indicate species differences in the pharmacokinetics associated with the distribution and elimination of the tobacco-specific N-nitrosamines.