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The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and N'-nitrosoanatabine (NAT) are found in substantial quantities in unburned tobacco. Although this has been documented in many previous studies, no data are available on the enantiomeric composition of these nitrosamines, which both have a chiral center at their 2'-positions. We used chiral stationary phase gas chromatography with nitrosamine-selective detection to determine the enantiomeric composition of NNN and NAT in moist snuff, chewing tobacco, and cigarette tobacco. (S)-NNN comprised 75.0 +/- 8.83% (SD) (n = 12) of total NNN while (S)-NAT comprised 82.6 +/- 1.44% (n = 12) of total NAT. Levels of the (S)-enantiomers of NNN and NAT were generally similar to those of the corresponding secondary amines, nornicotine and anatabine, suggesting a precursor to product relationship. Nitrosation of (S)-nicotine at pH 7.0 produced >99% (S)-NNN. These results suggest that nornicotine is a significant precursor of NNN in tobacco. The results of this study provide new insights into the structures and precursors of tobacco-specific nitrosamines in tobacco products.

People who use tobacco products are exposed to considerable amounts of N'-nitrosonornicotine (NNN), a well-established esophageal carcinogen in rats. NNN is believed to play a significant role as a cause of esophageal and oral cavity cancer in smokers and snuff dippers. The carcinogenicity of NNN is dependent on its metabolic activation. However, virtually all studies carried out to date on NNN metabolism have used racemic material. In this study, we examined the metabolism of [5-(3)H]-(S)-NNN and [5-(3)H]-(R)-NNN in cultured rat esophagus and in vivo in rats. Cultured rat esophagus metabolized (S)-NNN (1 microM) predominantly to products of 2'-hydroxylation, 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol). In contrast, the major metabolite of (R)-NNN under these conditions was 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), a product of NNN 5'-hydroxylation. The 2'-hydroxylation:5'-hydroxylation metabolite ratio ranged from 6.22 to 8.06 at various time intervals in the incubations with (S)-NNN, while the corresponding ratios were 1.12-1.33 in the experiments with (R)-NNN. These differences were statistically significant (P<0.001). Since 2'-hydroxylation is believed to be the major metabolic activation pathway of NNN in the rat esophagus, the results demonstrate that (S)-NNN is metabolically activated more extensively than (R)-NNN in this tissue, and therefore may be more carcinogenic. Rats were treated with 0.3 mg/kg of [5-(3)H]-(R)-NNN, [5-(3)H]-(S)-NNN, or racemic [5-(3)H]NNN by gavage, and the urinary metabolites were analyzed. The major metabolites were hydroxy acid and keto acid. As in the in vitro studies, products of 2'-hydroxylation predominated in the urine of the rats treated with (S)-NNN while products of 5'-hydroxylation were more prevalent in the rats treated with (R)-NNN. 2'-Hydroxylation:5'-hydroxylation metabolite ratios ranged from 1.66 to 2.04 in the urine at various times after treatment with (S)-NNN, while the ratios were 0.398-0.450 for the rats treated with (R)-NNN (P<0.001). The results of this study provide new insights into NNN metabolism in rats and suggest that the carcinogenicity of (S)-NNN, the predominant enantiomer in tobacco products, may be greater than that of (R)-NNN or racemic NNN.

The metabolism of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was investigated in short-term cultures of monkey lung. Explants from the lungs of two patas monkeys (Erythrocebus patas) and one cynomolgus monkey (Macaca fascicularis) were incubated with 10 microM [5-(3)H]NNK and aliquots were analyzed for NNK metabolites by HPLC at various time points from 1 to 24 h. F344 rat lung tissue metabolism of NNK was assayed under the same conditions. Substantial amounts of metabolites from the alpha-hydroxylation metabolic activation pathway were detected in all cultures. In two of the monkey lung cultures, these metabolite levels were significantly higher than those formed by other pathways. All cultures also metabolized NNK by pyridine-N-oxidation and carbonyl reduction. The metabolism of NNK by cultured monkey lung was generally similar to that observed in rat lung, indicating that there are not major species differences between rodents and nonhuman primates in pulmonary metabolism of NNK.

Tobacco-specific N-nitrosamines are a group of carcinogens derived from the tobacco alkaloids. They are likely causative factors for cancers of the lung, esophagus, pancreas, and oral cavity in people who use tobacco products. The most carcinogenic tobacco-specific nitrosamines in laboratory animals are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N'-nitrosonornicotine (NNN). DNA adduct formation from NNK and NNN has been studied extensively and is reviewed here. NNK is metabolically activated by cytochromes P450 to intermediates which methylate and pyridyloxobutylate DNA. The resulting adducts have been detected in cells and tissues susceptible to NNK carcinogenesis in rodents. The methylation and pyridyloxobutylation pathways are both important in carcinogenesis by NNK. NNK also induces single strand breaks and increases levels of 8-oxodeoxyguanosine in DNA of treated animals. NNAL, which like NNK is a potent pulmonary carcinogen, is also metabolically activated to methylating and pyridyloxobutylating intermediates. NNN pyridyloxobutylates DNA in its rat target tissues, esophagus and nasal mucosa. Methyl and pyridyloxobutyl DNA adducts are detected in human tissues. The methyl adducts most likely result in part from exposure of smokers to NNK, but these adducts are also detected in non-smokers. Some of the methyl adducts detected in non-smokers may be due to environmental tobacco smoke exposure. There are also potential dietary and endogenous sources of these adducts. Pyridyloxobutyl DNA adducts in human tissues result mainly from exposure to tobacco-specific N-nitrosamines. In laboratory animals, DNA adduct formation and carcinogenicity of tobacco-specific N-nitrosamines are closely correlated in many instances, and it is likely that similar relationships will hold in humans., (Copyright 1999 Elsevier Science B.V.)

Tobacco products are the leading cause of avoidable cancer death in the U.S., accounting for approximately 30% of all cancer deaths. While avoidance of tobacco and smoking cessation are clearly the best way to decrease tobacco-related cancer, these approaches have not been uniformly successful. Approximately 25% of the U.S. population over 18 years of age smokes cigarettes, while 6% use smokeless tobacco products; these figures have not changed markedly in recent years. Our approach toward the tobacco and cancer problem is based on an understanding of the carcinogens in tobacco smoke. These carcinogens form the link between nicotine addiction and cancer. In this paper, two strategies for cancer prevention--the development of carcinogen-derived biomarkers and chemopreventive agents--are discussed. Carcinogen-derived biomarkers can provide specific information on individual metabolic activation and detoxification of tobacco carcinogens. This information can be used to assess individual risk for cancer development upon exposure to tobacco products. Chemopreventive agents can be targeted against the important carcinogens in tobacco smoke. Isothiocyanates, strong inhibitors of lung cancer development by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, are discussed as an example of this approach.

Carcinogenic tobacco-specific nitrosamines are present in tobacco products and are believed to play a significant role in human cancers associated with tobacco use. Additional amounts of tobacco-specific nitrosamines could be formed endogenously. We tested this hypothesis by treating rats with nicotine and sodium nitrite and analyzing their urine. Initially, we treated groups of rats with (S)-nicotine (60 micromol/kg) and NaNO2 (180 micromol/kg), (S)-nicotine alone, NaNO2 alone or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 12 nmol/kg) by gavage twice daily for 4 days. We collected urine and analyzed for two metabolites of NNK; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide. We did not detect these metabolites in the urine of rats treated with nicotine alone or nicotine plus NaNO2, indicating that endogenous conversion of nicotine to NNK did not occur. However, the urine did contain N'-nitrosonornicotine (NNN), N'-nitrosoanabasine (NAB) and N'-nitrosoanatabine (NAT). Analysis of the (S)-nicotine used in this experiment demonstrated that it contained trace amounts of nornicotine, anabasine and anatabine. In a second experiment, we used an identical protocol to compare the endogenous nitrosation of this (S)-nicotine with that of synthetic (R,S)-nicotine, which did not contain detectable amounts of nornicotine, anabasine or anatabine. NNN (0.53 x 10(-3)% of nicotine dose), NAB (0.68%) and NAT (2.1%) were detected in the urine of the rats treated with the (S)-nicotine and NaNO2. NNN (0.47 x 10(-3)% of dose), but not NAB or NAT, was present in the urine of the rats treated with synthetic (R,S)-nicotine and NaNO2. NNN probably formed via nitrosation of metabolically formed nornicotine. These results demonstrate for the first time that endogenous formation of tobacco-specific nitrosamines occurs in rats treated with tobacco alkaloids and NaNO2. The potential significance of the results with respect to nitrosamine formation in people who use tobacco products or nicotine replacement therapy is discussed.

Two metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone were quantified in the urine of smokeless tobacco users. The metabolites are 4-(methylnitrosamino) -1-(3-pyridyl)-1-butanol (NNAL) and [4-(methylnitrosamino)-1-(3-pyridyl) but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). The study population consisted of 47 male nonsmokers, of whom 23 were snuff dippers, 13 were tobacco chewers, 3 were users of both products, and 8 were nonusers. The levels of NNAL-Gluc in urine ranged from 0.14-30.3 pmol/mg creatinine with a mean +/- SD of 3.47 +/- 5.86, whereas the levels of NNAL ranged from 0.02-8.73 pmol/mg creatinine with a mean +/- SD of 0.92 +/- 1.59. The mean levels of NNAL-Gluc and NNAL were not significantly different from those measured in a previous study of smokers. The levels of NNAL-Gluc were significantly higher in snuff dippers than in tobacco chewers. The ratio of NNAL-Gluc:NNAL was higher in snuff dippers than in tobacco chewers or smokers. There was no indication of two phenotypes of the NNAL-Gluc:NNAL ratio in smokeless tobacco users, in contrast to previous observations in smokers. Of the 39 smokeless tobacco users in this study, 16 presented with oral leukoplakia. When the total levels of NNAL-Gluc, NNAL, or NNAL-Gluc + NNAL were divided into tertiles, there was a significant association between the presence of leukoplakia and increasing levels of these metabolites; a similar relationship was found between urinary cotinine and leukoplakia. The results of this study demonstrate that there is significant uptake of carcinogenic nitrosamines in smokeless tobacco users, and that such products are not harmless alternatives to cigarettes. Moreover, the urinary biomarkers NNAL-Gluc, NNAL, and cotinine were associated with the presence of leukoplakia, which provides biochemical support for the role of smokeless tobacco products as a cause of oral leukoplakia.

Tobacco-specific N-nitrosamines (TSNA) are the most abundant, strong carcinogens in tobacco smoke. Seven TSNA have been identified in tobacco products: N'-nitrosonornicotine (NNN), N'-nitrosoanabasine (NAB), N'-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL), and 4-(methylnitrosamino)-4-3-pyridyl)butyric acid (iso-NNAC). The syntheses of these compounds are reviewed. The syntheses of 14C- and 3H-labeled NNK as well as metabolites of NNK and NNN are also discussed. Comparative assays for lung tumorigenesis in female A/J mice were carried out for six of the TSNA and for two related compounds, N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR). They yielded the following ranking of potency: NDMA > NNK > NNAL > NPYR > NNN > NAB. Iso-NNAL and iso-NNAC were inactive. These results are also compared with previous assays of TSNA carcinogenicity in rats and hamsters.

The metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was examined in rat pancreatic microsomes. No pyridine N-oxidation or alpha-hydroxylation were observed in these preparations. However, one unidentified metabolite of NNAL (unknown A) and two unknown metabolites of NNK (unknowns B and C) were formed. These metabolites were also detected in rat liver microsomal incubations of NNK and NNAL. Studies using [5-3H]-NNK and [Me-3H]NNK demonstrated that the metabolites contained both the pyridyl and methyl portions of the parent compound. Similar results were obtained with NNAL. Formation of unknown C required active microsomes, NADP+, and an NADPH regenerating system. The regenerating system was not required for the formation of NNAL unknown metabolite A or NNK unknown metabolite B. Chemical characterization of unknowns A and B by NMR, UV, and electrospray ionization MS demonstrated that they are NADP+ analogs in which the nicotinamide portion has been replaced by NNAL or NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol adenosine dinucleotide phosphate [(NNAL)ADP+] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone adenosine dinucleotide phosphate [(NNK)ADP+]. Unknown C was identified as (NNK)ADPH. Both (NNK)ADP+ and (NNK)ADPH were formed from NNK while only (NNAL)ADP+ was produced from NNAL. These NADP+ derivatives were also formed when porcine brain NAD+ glycohydrolase was incubated with NADP+ and NNK or NNAL. These results indicate that NNK and NNAL are substrates for rat liver and pancreatic microsomal NAD+ glycohydrolase-catalyzed transglycosylation reactions.

Tobacco-specific nitrosamines are a group of carcinogens formed from nicotine and related tobacco alkaloids. Two of these compounds, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine, are believed to be involved as causative agents for cancers of the lung, oral cavity, esophagus, and pancreas associated with the use of tobacco products. The goal of the studies described here is to develop biomarkers which will allow us to understand the uptake, metabolic activation, and detoxification of these carcinogens in humans. Two metabolites of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide, have been identified and quantified in human urine. These metabolites allow assessment of NNK uptake in smokers, tobacco chewers, and people exposed to environmental tobacco smoke. NNK and N'-nitrosonornicotine form hemoglobin and DNA adducts upon metabolic activation by alpha-hydroxylation. These adducts release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon hydrolysis. The released 4-hydroxy-1-(3-pyridyl)-1-butanone can be quantified by gas chromatography-mass spectrometry. A subset of smokers and most tobacco chewers have hemoglobin adduct levels which are higher than detected in nonsmokers. 4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts are higher in lung tissue from smokers than from nonsmokers. These data indicate that some smokers and tobacco chewers are capable of metabolically activating NNK or N'-nitrosonornicotine to intermediates which bind to cellular macromolecules and are, therefore, at potentially higher risk for cancer development. The application of these biomarkers to studies on cancer induction by tobacco products is discussed.

This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are believed to be involved in cancers of the lung, esophagus, oral cavity, and pancreas in people who use tobacco products. The adduct dosimetry method employs GC-MS for quantitation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) released by mild base hydrolysis of hemoglobin or acid hydrolysis of DNA as a biochemical marker of the pyridyloxobutylation metabolic activation pathway. Approximately 22% of smokers (n = 101) had elevated levels of HPB released from hemoglobin (range, 200-1600 fmole/g Hb). Adduct levels in snuff dippers ranged from 200-1800 fmole/g Hb. HPB levels in nonsmokers were generally below the detection limit. Acid hydrolysis of lung and tracheal DNA obtained at autopsy and analysis for released HPB revealed levels ranging up to 50 fmole/mg DNA in smokers; the adduct was not detected in nonsmokers. These findings are consistent with data generated in studies of adduct formation by NNK in rats. The biological significance of the HPB-releasing DNA pyridyloxobutylation pathway was compared to that of the DNA methylation pathway in the A/J mouse. These studies demonstrated that the persistence of O6-methylguanine in lung DNA is critical for tumorigenesis by NNK and that pyridyloxobutylation enhances both persistence of O6-methylguanine and tumorigenesis by acetoxymethylmethylnitrosamine. In the rat, the relative roles of methylation and pyridyloxobutylation in lung tumorigenesis by NNK are not as clearly defined.(ABSTRACT TRUNCATED AT 250 WORDS)

The carcinogenic tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) form hemoglobin adducts in laboratory animals and humans. These adducts release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base hydrolysis. HPB released from human hemoglobin can be quantified by gas chromatography-mass spectrometry. It is the only available biochemical marker for determination of exposure to, and metabolic activation of, carcinogens present only in tobacco. Levels of HPB were highest in snuff-dippers, followed by smokers and nonsmokers. Large interindividual variations in adduct levels were observed. The relationship between HPB levels in globin and DNA of rats treated with NNK has been investigated in order to aid in interpretation of the data from humans. These studies have provided the initial database for understanding the metabolic activation of tobacco-specific nitrosamines in humans.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen found in tobacco and tobacco smoke. It is a potent lung tumorigen in rodents and appears to be involved in human cancer induced by tobacco products. NNK induces lung tumors in A/J mice after a single dose; tumor multiplicity is higher when the mice are maintained on an AIN-76A diet than when they are maintained on NIH-07 diet. This paper reviews our recent research using this single-dose model. Bioassays of deuterium substituted analogues of NNK have demonstrated that methylation of DNA by NNK is an important step in lung tumor induction. Arylalkyl isothiocyanates inhibit the metabolic activation of NNK and consequently inhibit its DNA binding and tumorigenesis. Structure activity studies have demonstrated that increasing alkyl chain length leads to increasing efficacy in prevention of NNK tumorigenesis. Thus, 3-phenylpropyl isothiocyanate and 4-phenylbutyl isothiocyanate blocked NNK induced lung tumor formation in A/J mice. Lower doses of longer chain arylalkyl isothiocyanates were even more effective as chemopreventive agents.

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.

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and N'-nitrosonornicotine were quantified in blood samples collected from snuff dippers, smokers, and nonsmokers. Mild base treatment of hemoglobin adducted by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N'-nitrosonornicotine releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB was enriched by solvent partitioning and derivatized to its pentafluorobenzoate. After purification by high performance liquid chromatography, HPB-pentafluorobenzoate was analyzed by capillary column gas chromatography with detection by negative ion chemical ionization mass spectrometry and selected ion monitoring. [4,4-D2]HPB was used as internal standard. The detection limit for HPB-pentafluorobenzoate was approximately 100 amol/injection or 5 fmol/g hemoglobin. Mean adduct levels (fmol HPB/g hemoglobin) were 517 +/- 538 (SD) in snuff dippers, 79.6 +/- 189 in smokers, and 29.3 +/- 25.9 in nonsmokers. Adduct levels in snuff dippers and in a subgroup of smokers were higher than would have been predicted solely based on estimates of exposure to tobacco-specific nitrosamines. The results of this study provide the first measurements of tobacco-specific nitrosamine hemoglobin adducts in humans and suggest new approaches to understanding the metabolic activation of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine in humans.

The yields of volatile N-nitrosamines in cigarette smoke are primarily dependent upon the nitrate content of the tobacco and, to some extent, on the protein content. Cellulose acetate tips, such as those found on most commercial filter cigarettes, selectively remove at least 70% of the volatile N-nitrosamines, independently of the pH of the weakly acidic or weakly alkaline smoke. So far, three tobacco-specific N-nitrosamines have been detected in tobacco and tobacco smoke. During tobacco processing and smoking, N'-nitrosonornicotine is formed by nitrosation of nicotine and, to a minor degree, by nitrosation of nornicotine, whereas 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone originates from oxidative nitrosation of nicotine. N'-Nitrosoanatabine is formed by nitrosation of the second most abundant tobacco alkaloid, anatabine. The tobacco-specific N-nitrosamines in the smoke arise partly from the tobacco by transfer and partly by nitrosation of the alkaloids during smoking (pyrosynthesis). Preliminary results indicate that cellulose acetate filter tips may selectively remove considerable amounts of the nonvolatile nitrosamines from the smoke.

Smokeless tobacco (snuff and chewing tobacco) is reemerging as a popular form of tobacco, particularly among male adolescents. In different regions of the United States, from 8 to 36 percent of male high-school students are regular users. The use of smokeless tobacco has been shown to cause oral-pharyngeal cancer. The strongest link is with cancers of the cheek and gum. White mucosal lesions (leukoplakia) are found in 18 to 64 percent of users, often at the site where the tobacco was held. Other associations have been suggested for cancers of the esophagus, larynx, and pancreas. Nitrosamines, found in high concentrations in smokeless tobacco, most likely have a role in its carcinogenicity. Other health problems include periodontal disease, acute elevations of blood pressure, and dependence. In early 1986, after action at the state level, Congress enacted a federal law requiring health-warning labels on packages of smokeless tobacco and a ban on electronic advertising. Other regulatory measures under consideration include raising state and federal excise taxes, tightening controls on advertising, and prohibiting sales to minors. In view of the recent growth of this problem, policy makers are taking the opportunity to intervene with preventive measures to protect a new generation of tobacco users.

The tobacco-specific carcinogens, N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), were tested for carcinogenicity in F344 rats. Each nitrosamine in trioctanoin was administered by s.c. injection to 12 male and 12 female rats over a period of 20 weeks. The total dose of each nitrosamine was 3.4 mmol. The experiment was terminated after 12 months. NNK induced nasal cavity tumors in 83% of the males and in 83% of the females, liver tumors in 83% of the males and in 100% of the females, and lung tumors in 67% of the males and in 67% of the females. NNN induced nasal cavity tumors in 92% of the males and in 75% of the females. Only one liver tumor and no lung tumors were observed in the NNN-treated rats. These results indicate that, in the F344 rat, NNK is a more powerful carcinogen than is NNN.

The activity and distribution of the metabolic pathways of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and the structurally related nitrosamine, N'-nitrosonornicotine (NNN) were examined in pulmonary cells from F344 rats in order to investigate the mechanisms by which NNK and NNAL, but not NNN, cause lung tumors. The tritium labeled nitrosamines were incubated with Clara cells, alveolar macrophages, alveolar type II cells, or small cells and metabolites were analyzed by HPLC. O6-Methyl-guanine (O6MG) formation was also quantified in the cells incubated with NNK. Clara cells metabolized all compounds more extensively than the other cell types. Total alpha-hydroxylation, carbonyl reduction to NNAL, and pyridine N-oxidation in cells incubated with NNK, as well as concentrations of O6MG in DNA were higher in Clara cells than in other cell types. Carbonyl reduction of NNK predominated over the other metabolic pathways in all cell types. The high activity for alpha-hydroxylation of NNK in Clara cells is consistent with previous studies which proposed that the cell specificity for O6MG formation and the accumulation of this adduct during low-dose exposure to NNK may stem from the presence of a high affinity pathway in Clara cells for NNK activation. Metabolism of NNAL by alpha-hydroxylation, and by reconversion to NNK followed by alpha-hydroxylation were observed. Total alpha-hydroxylation of NNAL was less extensive than alpha-hydroxylation of NNK. NNN was metabolized by both the 2'- and 5'-alpha-hydroxylation pathways. 2'-Hydroxylation of NNN produces the same DNA pyridyloxobutylating agent as does methyl hydroxylation of NNK. However, NNN is not a methylating agent and does not induce lung tumors in rats. Metabolism of NNN by 2'-hydroxylation was, depending on cell type, 41-85% as extensive as total alpha-hydroxylation of NNK, indicating that the rates of formation of the DNA pyridyloxobutylating agent were similar from NNN and NNK. The results of this study demonstrate that Clara cells have a high capacity to metabolically activate NNK, NNAL and NNN and provide further support for the hypothesis that DNA methylation of pulmonary cells is important in NNK carcinogenesis.

A method was developed for the analysis of 1-nitronaphthalene, 1-nitropyrene, and 6-nitrochrysene in mainstream cigarette smoke, using [14C]1-nitronaphthalene, [14C]1-nitropyrene, and [14C]6-nitrochrysene as tracers and internal standards. Cigarette smoke condensate was collected and the appropriate fractions containing the labelled standards were obtained by a series of solvent partitions and column chromatography. Recovery of internal standards ranged from 60 to 70%. The fractions were analyzed by capillary gas chromatography with electron capture detection and by combined capillary gas chromatography-mass spectrometry. 1-Nitronaphthalene (less than 10 ng/cigarette), 1-nitropyrene (less than 10 ng/cigarette), and 6-nitrochrysene (less than 1 ng/cigarette) were not detected in the mainstream smoke of the University of Kentucky 1R1 cigarette, a US commercial 85 mm non-filter cigarette, or a French commercial 70 mm non-filter cigarette.

The metabolism, in the F-344 rat, of the tobacco-specific carcinogens, N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was studied. NNN was hydroxylated at each position of the pyrrolidine ring; 2'-hydroxylation gave 4-hydroxy-1-(3-pyridyl)-1-butanone in vitro and the corresponding acid in vivo, 3'-hydroxylation gave 3'-hydroxyNNN, 4'-hydroxylation gave 4'-hydroxy-NNN and 5'-hydroxylation gave 4-hydroxy-4-(3-pyridyl)butanal (in vitro) and 4-hydroxy-4-(3-pyridyl) butanoic acid (in vivo). The principle ring hydroxylation in the untreated F-344 rat was 5'-hydroxylation. Pyridine N-oxidation was also observed, giving NNN-1-N-oxide as a major metabolite. The principle urinary metabolites of NNN were formed by 5'-hydroxylation and pyridine-N-oxidation. For NNK, a major process was reduction of the carbonyl to give 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanol. alpha-Hydroxylation of both the N-methyl and N-methylene groups was also observed, as was formation of NNK-N-oxide in vitro and in vivo.

The tumorigenic activities in A/J mouse lung of the tobacco-specific nitrosamines, N'-nitrosonornicotine (NNN), 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and several structural analogues were evaluated. The analogues were N-nitrosopyrrolidine (NPYR), 5'-carboxy-N'-nitrosonornicotine (CNNN), N-nitrosoproline (NPRO) and 1-(3-pyridyl)-2-buten-1-one (PBO). The results were as follows (dose in mumol per mouse/lung tumors per mouse): NNN (100/1.8 +/- 1.4); NPYR (100/3.9 +/- 1.5); CNNN (200/0.3 +/- 0.5); CNNN (100/0.5 +/- 0.6); NPRO (100/0.6 +/- 0.7); NNK (20/7.2 +/- 3.4); PBO (20/0.7 +/- 1.0); saline control (0.0.5 +/- 0.7). Several conclusions were drawn from this assay. NNK and NPYR were more tumorigenic than NNN. CNNN was non-tumorigenic and thus appears to have potential as a monitor for endogenous formation of tobacco-specific nitrosamines. The alpha,beta-unsaturated ketone PBO does not appear to be an ultimate tumorigen of NNK or NNN.

N'-Nitrosonornicotine, a potential carcinogen, has been positively identified in unburned tobacco. The amount in commercial U.S. tobacco products is between 1.9 to 88.6 parts per million, one of the highest values of an environmental nitrosamine yet reported. The amount in food and drink rarely exceeds 0.1 part per million. This compound is the first example of a potential organic carcinogen isolated from tobacco.

N'-Nitrosonornicotine (NNN) and N'-nitrosoanabasine (NAB) were synthesized and injected subcutaneously three times weekly in male and female Syrian golden hamsters for 25 weeks (total dose 375 mg). N-Nitrosopiperidine (NPD) served as positive control (total dose 150 mg). Within 83 weeks 12 out of 19 hamsters given NNN had developed tracheal tumors, 1 had a carcinoma of the nasal cavity. During the same time none of the hamsters given NAB developed tumors, whereas all 20 hamsters given NPD developed tracheal tumors and about 50% had additional neoplasms in the nasal cavity. Under the described conditions and in comparison to NPD, NNN appears to be a moderately active tumorigenic agent in the upper respiratory tract of Syrian hamsters, whereas NAB is inactive.

Approaches for assessing molecular dosimetry of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) in humans by measurement of haemoglobin or DNA adducts are discussed. NNK and NNN form haemoglobin adducts in Fischer 344 rats. Acid or base hydrolysis of the globin gives 4-hydroxy-1-(3-pyridyl)-1-butanone, which can be detected in rat blood up to six weeks after injection of NNK; it may be a useful marker for assessing uptake and metabolic activation of NNK and NNN in tobacco consumers. NNK and its major metabolite, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAI), methylated DNA of rat liver, lung and nasal mucosa to similar extents. NNAI is formed in human tissues from NNK, but immunoassays for O6-methyldeoxyguanosine (O6-medGuo) in exfoliated oral cells from snuff-dippers have been negative. NNK is also expected to form pyridyloxobutyl adducts in DNA; 32P-postlabelling assays for these adducts are being developed and appear to hold promise for detecting NNK- or NNN-DNA adducts in vivo.

The effect of chronic ethanol consumption by Syrian golden hamsters (SGH) on the carcinogenicity and target tissue metabolism of N-nitroso-pyrrolidine (NPYR) and N'-nitrosonornicotine (NNN) has been examined. Ethanol-consuming hamsters treated with NPYR developed more nasal cavity and tracheal tumours than controls. Ethanol consumption did not affect the carcino-genicity of NNN. When the metabolism of NPYR and NNN by isolated tracheal rings was examined, it was observed that tracheal rings isolated from ethanol-consuming SGH metabolized NPYR at a higher rate than similar preparations from control animals. In contrast, no differences in the rates of metabolism of NNN were observed.

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. Levels of O6-methylguanine, a promutagenic lesion, and 7-methylguanine were three to eight times higher in the liver than in the lung. Neither base could be detected in the kidneys. The extent of methylation of hepatic DNA by NNK was 35 times lower than observed with an equimolar dose of NDMA by Swann et al. (1983). The levels of the two methylated guanines in liver and lung DNA increased between 4 and 24 h following NNK injection. NNK is metabolized rapidly in F344 rats to 4-(methylnitrosamino)-1(3-pyridyl)-butan-1-ol (NNA1). The relatively slow methylation of hepatic DNA after injection of NNK could be due to a slow release of methylating species from the major circulating metabolite NNA1. This low but sustained level of O6-methylguanine induced by NNK could, in part, explain its carcinogenic potency.

The transplacental tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was assessed in three strains of mice: A/J; C3H/He x C57BL/6 F1 (hereafter called C3B6F1); and Swiss outbred [Cr:NIH(S)]. NNK (100 mg/kg) was administered i.p. on Days 14, 16, and 18 of gestation to A/J and C3H/He mice and on Days 15, 17 and 19 of gestation to the Swiss mice. The effects of postnatal treatment with tumor-promoting agents, including 0.05% sodium barbital in the drinking water until death or a single dose of Aroclor 1254 (a mixture of polychlorinated biphenyls, PCB) given on Postnatal Day 8 or 56, were also examined. Progeny were sacrificed at age 24 wk (A/J) or 72 wk (C3B6F1 and Swiss). Significant incidences of tumors occurred in the lungs of strain A/J progeny and in the livers of male C3B6F1 and Swiss progeny. Lung tumor incidence was 8 of 34 (24%) in the female offspring of the A/J mice treated with NNK, compared with 1 of 39 (3%) in controls (P less than 0.05). A 2-fold difference in lung tumor incidence in male offspring of NNK-treated (4 of 23, 13%) versus control (3 of 48, 6%) A/J mice was not of statistical significance. However, the incidence of lung tumors in NNK-exposed progeny A/J mice in both sexes combined (12 of 66, 18%) was also significantly greater than in controls (4 of 87, 5%). The incidence of liver tumors in the male C3B6F1 mice exposed transplacentally to NNK was 12 of 30 (40%) compared to 8 of 46 (17%) in controls (P less than 0.05). No effects of postnatal sodium barbital or PCB were observed on transplacental NNK tumorigenicity in C3B6F1 mice. The combined incidence of liver carcinoma in male mice in all NNK-treated groups (13 of 141, 9%) was significantly greater (P less than 0.05) than in controls (5 of 144, 3%). In male Swiss mice exposed transplacentally to NNK, the incidence of liver tumors was 3 of 57 (5%) compared to 0 of 35 controls, and postnatal treatment with PCB on Day 56 caused a significant increase (5 of 26, 19%) (P less than 0.05) in the incidence of NNK-induced liver tumors. The combined incidence of liver tumors in the male offspring of the Swiss mice treated with NNK, with or without PCB, was 8 of 83 (10%) which was significantly greater (P less than 0.05) than in controls (0 of 66).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Tobacco and tobacco smoke contain relatively high amounts of four tobacco-specific N-nitrosamines. Of these, N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N'-nitrosoanatabine (NAT) were bioassayed at three dose levels by subcutaneous injections into male and female F344 rats in 60 subdoses amounting in total to 9,3, and 1 mmol/kg. Compared with the solvent control group (trioctanoin), both NNN and NNK induced significant numbers of tumors of the nasal cavity (P less than 0.01) at all three dose levels in both male and female rats. Significant numbers of tumors were also induced by NNK in the lung at all three dose levels and in the liver at the highest dose level (P less than 0.05). In addition to nasal tumors NNN also induced esophageal tumors at a significant rate in male rats at the high and medium dose levels and in female rats at the high level (P less than 0.05); NAT was inactive at the three doses tested. Bioassays at lower dose levels as well as biochemical studies are strongly indicated for NNN and NNK since these nitrosamines occur in relatively high amounts in both chewing tobacco and tobacco smoke.

NNN is the first organic carcinogen isolated from unburned tobacco. It has been found in smoking tobaccos, chewing tobaccos and in snuff in concentrations between 0.3 and 90.0 mug. This appears to be an unusually high concentration for a nitrosamine in an environmental agent. We have presented data which suggest that NNN, and possibly other unknown nitrosamines, are formed during the curing of tobacco and that the nitrate content of tobacco is an important factor in nitrosamine formation. Studies with N'-methylanabasine applied to tobacco plants are currently under way to test the idea that nicotine rather than nornicotine is the major precursor of NNN in processed tobacco. In mice, NNN induces adenomas of the lung. Bioassays with rats have shown that NNN is carcinogenic to the oesophagus and the nasal cavity. These chemical and biological data are consistent with the observation that tobacco chewers face an increased risk of cancer of the oesophagus. This observation does not, of course, rule out the possibility that other tobacco carcinogens are responsible for the increased cancer risk of tobacco chewers.

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. Further research should now focus on the relationship between human exposure to tobacco-specific nitrosamines and the risk of developing tobacco-related cancers. Several important areas can be identified: we need to develop sensitive assays that can be used routinely to quantify the levels of tobacco-specific nitrosamines or their metabolites in human blood, and the levels of their DNA adducts in human tissues; we need to establish, through comparative metabolic and DNA-binding studies, the relationships between the organospecificity of tobacco-specific nitrosamines in experimental animals and that in humans; we may also be enabled to identify naturally-occurring substances that can inhibit carcinogenesis by tobacco-specific nitrosamines. These research approaches will hopefully lead to a reduction in the incidence of tobacco-related cancers.

The formation of tobacco-specific nitrosamines from the major tobacco alkaloid nicotine was examined. Detached leaf tobacco was fed either [2'-14C]nicotine or [2'-14C]nornicotine and air cured. The cured leaf was then analyzed for [2'-14C]N'-nitrosonornicotine ([2'-14C]NNN). The yield of [2'-14C]NNN was 0.007% from nornicotine and 0.009% from nicotine. Because the ratio of nicotine to nornicotine in conventional nicotine-type tobacco is 20-100:1, nicotine is considered to be the major precursor for the carcinogen NNN in tobacco. The formation of other nitrosamines from nicotine in vitro was then studied. Reaction of nicotine with NaNO2 gave rise to NNN, as well as to two other nitrosamines, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(N-methyl-N-nitrosamino)-4-(3-pyridyl)butanal (NNA). Analysis of market products revealed the presence of NNK (0.6-24 microgram/g) in chewing tobacco and snuff. The tumorigenic activity of NNN, NNK, and NNA in strain A mice was studied. NNK induced more lung adenomas per mouse than did NNN, whereas NNA was less active than NNN. In addition, two cases of undifferentiated carcinoma of the salivary glands occurred in the NNN experimental groups.

A chemical analytic method to determine N'-nitrosonornicotine (NNN) and N' -nitrosoanabasine (NAB) in tobacco was developed. NNN was found in the unburned tobacco of commercial products at concentrations betweeen 0.3 and 88.6 mug/g. The highest levels were observed in highly fermented snuff (29.1 mug/g) and fine-cut chewing tobacco (88.6 mug/g). NAB was not detected (smaller than 0.5 ng/g) in any tobacco examined. Two new tobacco components, N' -carbomethoxynornicotine and N' -carbomethoxyanabasine,were found. Possible origins of NNN in tobacco were discussed, especially in relation to concentrations of nitrite, nitate, and alkaloids, and in relation to pH and curing. Biologic implications, including the possible function of NNN (the first organic carcinogen isolated from unburned tobacco) as a causative factor in cancer of the oral cavity in tobacco chewers and betel quid chewers, were also discussed.

[5-3H]4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK), [C3H3]NNK, and [5-3H]N'-nitrosonornicotine ([5-3H]NNN) were administered to F344 rats by i.p. injection. Levels of tritium present per milligram globin, 24 h after treatment were 720 fmol (0.1% of dose) for [5-3H]NNK, 640 fmol for [C3H3]NNK, and 370 fmol for [5-3H]NNN. Tritium was detectable in globin 7-8 weeks after treatment with [5-3H]NNK or [5-3H]NNN. Approximately 10-15% of the bound tritium in the globin of rats treated with [5-3H]NNK was released upon incubation of the globin with dilute NaOH or HCl. The released material was identified as 4-hydroxy-1-(3-pyridyl)-1-butanone; it was detectable in globin for 6 weeks (t1/2 = 9.1 days) after administration of [5-3H]NNK. 4-Hydroxy-1-(3-pyridyl)-1-butanone was also formed upon NaOH treatment of globin isolated from rats injected with [5-3H]NNN or [5-3H]4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone. The formation of 4-hydroxy-1-(3-pyridyl)-1-butanone under these conditions is consistent with a mechanism by which 4-(3-pyridyl)-4-oxobutyldiazohydroxide is produced upon metabolic alpha-hydroxylation of NNK or NNN and binds to globin of hemoglobin, yielding an adduct which is readily hydrolyzed by acid or base. Support for this mechanism was obtained by in vitro experiments. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone released upon base treatment of globin were 50 times greater after incubation of rat hemoglobin with [5-3H]4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone than with either [5-3H]NNK or [5-3H]4-hydroxy-1-(3-pyridyl)-1-butanone. The results of this study suggest methods that might be applicable for assessing the molecular dosimetry of NNK and NNN in individuals exposed to tobacco and tobacco smoke.

Cigarette smoke induces carcinoma of the larynx in Syrian golden hamsters and is active as a tumor promoter in hamsters pretreated with a low dose of a PAH, nitrosamine, or nitrosamide. These tumorigenic effects are only observed with total smoke, but not with the gas phase alone. This demonstrates that the tumorigenic agents reside primarily in the particulate phase. According to fractionation experiments, a number of four- and five-ring aromatic hydrocarbons serve as the major tumor initiators in tobacco smoke. Tumor promoters reside primarily in weakly polaric neutral subfractions and in the weakly acidic portion of the particulate matter and include certain unsaturated hydrocarbons and phenolic compounds. Cocarcinogenic activity is a characteristic feature of tobacco smoke and its particulates. Among the cocarcinogens formed during combustion are catechols and certain nontumorigenic aromatic hydrocarbons and terpenes. Nicotine may also serve as a cocarcinogen as is indicated by preliminary data. The action of tumor promoters and cocarcinogens in tobacco carcinogenesis, the precursors for tobacco smoke promoters and cocarcinogens, and methods for their reduction in smoke are discussed.

A new GC-TEA method for the analysis of tobacco-specific N-nitrosamines (TSNA) has been developed. Four TSNA have thus far been identified; these are N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanabasine (NAB). The method is currently being applied to the development of cigarette filter-tips which will selectively remove these carcinogens from cigarette smoke. Since recent epidemiological studies have established a correlation between snuff dipping and oral cancer, we have analysed leading snuff brands for TSNA. Snuff products from Sweden, Denmark, Bavaria and the USA contained 5-106 mg/kg of the TSNA and the saliva of snuff dippers had TSNA levels of 20-890 micrograms/kg. NNN, NNK and NAB induce benign and malignant tumours of the respiratory tract of mice and rats. We have shown that NNN and NNK induce tumours in the upper respiratory tract of hamsters and that NNK is the most active carcinogen of the TSNA, also inducing adenoma and adenocarcinoma in the hamster lung. The reported chemical analyses and bioassay results support the epidemiological findings on the causal association of tobacco use and cancer in man.

Dry snuff contains high levels of tobacco-specific N-nitrosamines (TSNA); their concentrations exceed by more than 100 times the quantities of nitrosamines found in any other consumer product. The concentrations of TSNA are similar in dry snuff and in the more popular moist snuff. In addition to the four TSNA identified earlier [N'-nitrosonornicotine (NNN), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanabasine (NAB)], two new nitrosamines were detected in snuff, namely 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAl; 0.07-0.15 ppm) and 4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAl; 0.06-1.1 ppm). After oral swabbing with a mixture of NNN and NNK, rats developed tumours of the oral cavity and lung, showing that these TSNA are not only organ-specific carcinogens but can also induce local tumours. After swabbing an extract of snuff containing the same concentrations of NNN and NNK, significantly fewer tumours were induced in the oral cavity and lung, indicating inhibition of the tumorigenic activity of the TSNA by other snuff constituents.

DNA was isolated from tissues of F344 rats 24 h after treatment by s.c. injection with [5-3H]4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK) or [5-3H]N'-nitrosonornicotine ([5-3H]NNN). It was hydrolyzed with acid or at pH 7, 100 degrees C, and the hydrolysates were analyzed by HPLC. The major product in each case was identified as 4-hydroxy-1-(3-pyridyl)-1-butanone, formed by hydrolysis of a DNA adduct. It was detected in DNA from the livers of rats treated with [5-3H]NNK or [5-3H]NNN, and in DNA from lungs of rats treated with [5-3H]NNK. These results demonstrate that 4-(3-pyridyl)-4-oxobutylation of DNA occurs in rats treated with NNK or NNN, and are consistent with the hypothesis that these nitrosamines are metabolically activated by alpha-hydroxylation.