Enantiomeric composition of N'-nitrosonornicotine and N'-nitrosoanatabine in tobacco
Steven G. Carmella,
Edward J. McIntee,
Menglan Chen and
Stephen S. Hecht1
University of Minnesota Cancer Center, Minneapolis, MN 55455, USA
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Abstract
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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.
Abbreviations: GC-MS-SIM, gas chromatographymass spectrometry with selected ion monitoring; GC-TEA, gas chromatography with detection by thermal energy analyzer; 5-MeNNN, 5-methyl-N'-nitrosonornicotine; NAB, N'-nitrosoanabasine; NAT, N'-nitrosoanatabine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N'-nitrosonornicotine.
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Introduction
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Since the first report documenting the presence of p.p.m. quantities of N'-nitrosonornicotine (NNN) in unburned tobacco (1), numerous studies have confirmed this observation and extended it to other tobacco-specific nitrosamines (reviewed in refs 25). Recent studies clearly demonstrate that levels of NNN in tobacco continue to be substantial (46). For example, NNN levels in the five leading brands of moist snuff sold in the USA range from 3.04 to 8.73 µg/g dry weight of tobacco (6). In cigarette tobacco, NNN levels in products from 12 different countries range from 45 to 12 454 ng/cigarette (4). NNN and the related tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are the most abundant strong carcinogens in unburned tobacco (7). Tobacco products also contain considerable amounts of N'-nitrosoanatabine (NAT) and smaller quantities of N'-nitrosoanabasine (NAB) (25). Neither of these nitrosamines displays potent carcinogenicity in laboratory animals (2,3).
There has been a significant and alarming increase in the sales of moist snuff products in the USA (8). Sales of moist snuff increased from 7 700 000 kg in 1972 to 23 900 000 kg in 1994 (8). It has been estimated that each year in the USA ~824 000 young people experiment with smokeless tobacco and 304 000 become regular users (8). Among high school students, 15.8% of males and 1.5% of females were current smokeless tobacco users in 1997 (9). Snuff-dipping, as practiced in North America, is an accepted cause of oral cancer and the tobacco-specific nitrosamines NNN and NNK are believed to play an important role in oral cancer induction by smokeless tobacco products (2,3,7,1013). In rats, a mixture of NNN and NNK applied to the oral cavity induces oral tumors (14).
NNN and NAT have chiral centers at their 2'-positions (Figure 1
), but all studies carried out so far on the analysis of these nitrosamines in tobacco have reported data for racemic material. Enantiomers frequently differ in their biological effects. Therefore, in this study we have determined the enantiomeric composition of NNN and NAT in tobacco.
Nornicotine enantiomers were prepared as described (15). They were nitrosated as reported previously (16). The resulting NNN enantiomers were purified by silica gel (70230 mesh) column chromatography with elution by CHCl3/MeOH 98:2. (S)-NNN (195 mg, 1.1 mmol) and (R)-NNN (182 mg, 1.03 mmol) were isolated as yellowish oils which solidified to off-white solids upon standing at 20°C. The NMR and MS of (S)-NNN and (R)-NNN were identical to those of racemic NNN. Specific rotations (MeOH) were as follows: (S)-nornicotine [
]20ISOdiaD = 33.0 (concentration, in g/100 ml, 0.98), literature value (15), 34.9; (R)-nornicotine [
]20ISOdiaD = +35.2 (1.9), literature value (15), +34.9; (S)-NNN [
]20ISOdiaD = 154 (0.85); (R)-NNN [
]20ISOdiaD = +159 (1.5). NAT enantiomers were assigned by nitrosation of a methanol extract of smokeless tobacco which contains predominantly (S)-anatabine (17) and analysis for (S)-NAT by gas chromatography with detection by a thermal energy analyzer (GC-TEA) as described below.
5-Methyl-N'-nitrosonornicotine (5-MeNNN) was synthesized from methyl 5-methylnicotinate (Lancaster Synthesis Inc., Windham, NH) as described for NNN (16). The overall yield was 24.2%, giving 283 mg (1.48 mmol) of a yellowish solid, mp 5859°C. Its spectral properties were as follows:1H NMR (CDCl3);
8.41 (0.63 H, s, pyr-6H, E isomer), 8.37 (0.63 H, s, pyr-2H, E isomer), 8.32 (0.37 H, s, pyr-6H, Z isomer), 8.20 (0.37 H, s, pyr-2H, Z isomer), 7.32 (0.66 H, s, pyr-4H, E isomer), 7.14 (0.34 H, s, pyr-4H, Z isomer), 5.67 (0.63 H, t, 2'H, E isomer), 5.22 (0.37 H, t, 2'H, Z isomer), 4.714.40 (0.74 H, m, 5'H, Z isomer), 3.943.67 (1.26 H, m, 5'H, E isomer), 2.53 (1.26 H, m, 3'H, E isomer), 2.35 (2 H, s, 5-CH3, E isomer), 2.31 (1H, s, 5-CH3, Z isomer), 2.241.96 (2.75 H, m, 4'H, E + Z isomer; 3'H, Z isomer); ESI MS m/z [M+H]+ 192.
Commercial cigarettes and smokeless tobacco products were purchased on the open market, in April 1999. Research cigarettes and research smokeless tobacco products were chosen for comparison to the enantiomeric composition of nornicotine and anatabine (17). These were obtained from the Tobacco and Health Research Institute, University of Kentucky.
The analytical method is summarized in Figure 2
and is similar to that published previously (18). Extraction buffer, pH 4.5, was prepared from 2 l of 0.1 M citric acid, 1.65 l of 0.2 M Na2HPO4 and ascorbic acid to a final concentration of 20 mM. Tobacco was chopped in a blender into pieces ~1 mm2. A 2.0 g portion of tobacco and 500 ng 5-MeNNN internal standard were added to 40 ml of extraction buffer and the mixture was sonicated for 1.5 h. It was filtered and the tobacco residue was rinsed once with buffer. The filtrates were combined, the pH was adjusted to 5.0 and the solution was extracted three times with 20 ml of ethyl acetate. The extracts were concentrated to dryness, redissolved in 0.5 ml of ethyl acetate and applied to a SEP-PAK Plus silica solid phase extraction cartridge containing 690 mg silica (Waters Corp., Milford, MA) and pre-equilibrated with methylene chloride. The cartridge was eluted with methylene chloride (5 ml) and methylene chloride/ethyl acetate 50:50 (5 ml), then with ethyl acetate (10 ml). The ethyl acetate eluant contained the analytes. An aliquot was set aside for quantitation of total NNN, NAB and NAT as described below. The remainder was brought to dryness, redissolved in 1 ml of H2O and further purified by reverse phase HPLC using a 250x4.6 mm Bondclone C-18 column (Phenomenex, Torrance, CA) eluted at 1 ml/min as follows: 15% MeOH in H2O for 5 min, then a 20 min gradient to 45% MeOH in H2O, then a 5 min gradient back to 15% MeOH, and a hold for 15 min. 3-Acetylpyridine (50 µg) and 3-pyridylcarbinol acetate (50 µg) were used as retention time markers. Fraction 1 containing NNN was collected from 1622 min, e.g. between the apices of the two marker peaks. Fraction 2, which contained NAB, NAT and 5-MeNNN, was collected between 22 and 36 min. These fractions were concentrated to dryness and transferred to a 100 µl vial. The sample was redissolved in CH3CN for analysis by GC-TEA (Thermedics Detection Inc., Chelmsford, MA) using a 30 mx0.25 mm i.d., 0.25 µm film thickness Cyclosil-B column (J & W Scientific, Folsom, CA). This column has a 30% heptakis-(2,3-di-O-methyl-6-O-t-butyldimethylsilyl)-ß-cyclodextrin chiral selector physically dispersed in the DB-1701 stationary phase [14% (cyanopropylphenyl)methylpolysiloxane]. The column was operated with a 2 mx0.53 mm i.d. deactivated fused silica precolumn. The GC was operated in the splitless mode. The initial temperature of the oven was 60°C for 2 min, then it was programmed at 12°C/min to 170°C, then held for 35 min, then increased at 12°C/min to 210°C, and held for 5 min. The carrier gas was He at 1 ml/min. Retention times of the NNN, NAB, NAT and 5-MeNNN enantiomers are summarized in Table I
. For quantitation of total NNN, NAT and NAB we used a 30 mx0.32 mm i.d, 0.25 µm film thickness DB-1301 column [6% (cyanopropylphenyl) methylpolysiloxane; J. & W. Scientific] and a 2 mx0.53 mm i.d. deactivated fused silica precolumn. The flow rate was 2.6 ml/min He and splitless injection at 225°C was used. The oven temperature program was 80°C for 2 min, then 12°C/min to 150°C, then a hold for 15 min, then an increase at 12°C/min to 200°C, then a hold for 10 min. NNN eluted at 19.4 min, NAT at 20.4 min, NAB at 21.8 min and 5-MeNNN at 25.7 min.
Chiral column gas chromatograms of HPLC fraction 1, containing NNN, and fraction 2, containing NAT, NAB and 5-MeNNN, are illustrated in Figure 3A and B
. The identities of the nitrosamines were confirmed by gas chromatographymass spectrometry with selected ion monitoring (GC-MS-SIM) analysis of 1R3 cigarette tobacco as well as 1S2 and 1S3 snuff. A Finnigan TSQ-7000 instrument was operated in the positive ion chemical ionization mode. GC conditions were the same as those used for GC-TEA analysis. Selected ion monitoring of m/z 178 (M+H) and 148 (M+H-NO) was employed for analysis of NNN, m/z 190 and 160 for NAT and m/z 192 and 162 for NAB. In the GC-MS-SIM chromatograms of fraction A, peaks were observed only at the correct retention times for (S)- and (R)-NNN. The ratios of (S)- to (R)-NNN were similar to those observed by GC-TEA. Analysis of fraction B similarly produced peaks for (S)- and (R)-NAT and (S)- and (R)-NAB.

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Fig. 3. Gas chromatography of fractions 1 and 2 containing (A) NNN and (B) NAT and NAB. 5-MeNNN is the internal standard. See Materials and methods for details of the preparation of fractions 1 and 2.
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GC-TEA was used for quantitation of the enantiomers. The GC-TEA chromatograms of fraction 1 were exceptionally clean (Figure 3A
) and the NNN enantiomers were readily quantified. We did not attempt to quantify the enantiomers of NAB (fraction 2, Figure 3B
) because the peaks were small and (R)-NAB co-eluted with (R)-NAT. The results are summarized in Table II
.
Total amounts of the nitrosamines were consistent with published data (16). With respect to chirality, the (S)-enantiomers of both NNN and NAT predominated. (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. The percent (S)-enantiomer was significantly lower for NNN than for NAT (P = 0.0075, t-test). The variation in levels of (S)-NNN, ranging from 63 to 95% of the total, was greater than that of (S)-NAT (8085%). There were generally similar levels of the (S)-enantiomer in the nitrosamines and the corresponding secondary amines, based on seven samples for which data for both were available.
The similarity between the enantiomeric composition of the secondary amines and the corresponding nitrosamines suggests that nornicotine is the precursor to NNN and anatabine is the precursor to NAT. However, our previous studies indicated that nicotine, not nornicotine, is the major precursor to NNN in tobacco (1921). The formation of NNN from nicotine is believed to proceed via iminium ion 3 (Figure 4
; 22). This iminium ion is also hypothesized to be an intermediate in the biosynthesis of nornicotine from nicotine in the tobacco plant (23). If both NNN and nornicotine were formed from nicotine via a common intermediate such as 3, their enantiomeric compositions would be similar. Therefore, although nicotine in tobacco is >99% (S)-nicotine (24), conversion to nornicotine or NNN in tobacco could result in loss of optical purity if iminium ion 3 were in equilibrium with 2, leading to values more similar to those in Table II
[e.g. 6395% (S)-NNN and 70.696.0% (S)-nornicotine]. To test this possibility, we allowed (S)-nicotine (0.1 mmol, >99% (S) based on ref. 24; Aldrich) to react with NaNO2 (0.5 mmol) in 15 ml of 0.1 M KH2PO4 buffer, pH 7.0, at room temperature for 17 h. These conditions have been previously shown to produce NNN, NNK and 4-(methylnitrosamino)-4-(3-pyridyl)butanal in yields of 0.10.2% (22). The enantiomeric composition of the NNN produced in this reaction was >99% (S)-NNN. These results demonstrate that, if 3 is an intermediate in the conversion of nicotine to NNN, racemization of 3 does not occur under these conditions. Thus, the enantiomeric composition of NNN in tobacco is closer to that of nornicotine than nicotine. These results suggest that nornicotine is a more important precursor of NNN in tobacco than nicotine. This is not consistent with our previous data (1921) and at present we have no explanation for the discordant results. It is possible, however, that the conditions of our in vitro experiment are quite different from those prevailing in tobacco.
The results of this study provide new information relevant to the potential role of NNN as a causative agent for cancers associated with the use of moist snuff and other tobacco products. Our results demonstrate that (S)-NNN predominates in moist snuff, chewing tobacco and cigarette tobacco. Virtually all previous metabolic and carcinogenicity studies of NNN have been carried out with racemic material. In our ongoing studies, we hope to define these properties for the individual enantiomers.
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Acknowledgments
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This study was supported by grant CA-81301 from the National Cancer Institute.
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Notes
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1 To whom correspondence should be addressed Email: hecht002{at}tc.umn.edu 
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References
|
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-
Hoffmann,D., Hecht,S.S., Ornaf,R.M. and Wynder,E.L. (1974) N'-nitrosonornicotine in tobacco. Science, 186, 265267.[ISI][Medline]
-
Hoffmann,D. and Hecht,S.S. (1985) Nicotine-derived N-nitrosamines and tobacco related cancer: current status and future directions. Cancer Res., 45, 935944.[ISI][Medline]
-
Hecht,S.S. and Hoffmann,D. (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis, 9, 875884.[Abstract]
-
Spiegelhalder,B. and Bartsch,H. (1996) Tobacco-specific nitrosamines. Eur. J. Cancer Prev., 5, 3338.
-
Hoffmann,D., Brunnemann,K.D., Prokopczyk,B. and Djordjevic,M.V. (1994) Tobacco-specific N-nitrosamines and areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity and relevance to humans. J. Toxicol. Environ. Health, 41, 152.[ISI][Medline]
-
Hoffmann,D., Djordjevic,M.V., Fan,J., Zang,E., Glynn,T. and Connolly,G.N. (1995) Five leading U.S. commercial brands of moist snuff in 1994assessment of carcinogenic N-nitrosamines. J. Natl Cancer Inst., 87, 18621869.[Abstract]
-
International Agency for Research on Cancer. (1985) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 37, Tobacco Habits Other than Smoking; Betel-quid and Areca-nut Chewing; and Some Related Nitrosamines. IARC, Lyon.
-
Tomar,S.L. and Giovino,G.A. (1998) Incidence and predictors of smokeless tobacco use among U. S. youth. Am. J. Public Health, 88, 2026.[Abstract]
-
Anonymous (1998) Tobacco use among high school studentsUnited States, 1997. Morbid. Mortal. Weekly Rep., 47, 229233.[Medline]
-
Magee,P.N. (1989) The experimental basis for the role of nitroso compounds in human cancer. Cancer Surv., 8, 207239.[ISI][Medline]
-
Preston-Martin,S. and Correa,P. (1989) Epidemiological evidence for the role of nitroso compounds in human cancer. Cancer Surv., 8, 459473.[ISI][Medline]
-
Magee,P.N. (1996) Nitrosamines and human cancer: introduction and overview. Eur. J. Cancer Prev., 5, 710.[ISI][Medline]
-
Bartsch,H. and Spiegelhalder,B. (1996) Environmental exposure to N-nitroso compounds (NNOC) and precursors: an overview. Eur. J. Cancer Prev., 5, 1118.[ISI][Medline]
-
Hecht,S.S., Rivenson,A., Braley,J., DiBello,J., Adams,J.D. and Hoffmann,D. (1986) Induction of oral cavity tumors in F344 rats by tobacco-specific nitrosamines and snuff. Cancer Res., 46, 41624166.[Abstract]
-
Seeman,J.I., Chavdarian,C.G. and Secor,H.V. (1985) Synthesis of the enantiomers of nornicotine. J. Org. Chem., 50, 54195421.[ISI]
-
Hu,M.W., Bondinell,W.E. and Hoffmann,D. (1973) Chemical studies on tobacco smoke XXIII. Synthesis of carbon-14 labelled myosmine, nornicotine and N'-nitrosonornicotine. J. Labelled Compounds, 10, 7988.
-
Armstrong,D.W., Wang,X., Lee,J.-T. and Liu,Y.-S. (1999) Enantiomeric composition of nornicotine, anatabine and anabasine in tobacco. Chirality, 11, 8284.[ISI]
-
Hecht,S.S., Adams,J.D. and Hoffmann,D. (1983) Tobacco-specific nitrosamines in tobacco and tobacco smoke. In Preussmann,R., O'Neill,I.K., Eisenbrand,G., Spiegelhalder,B. and Bartsch,H. (eds) Environmental Carcinogens: Selected Methods of Analysis, Vol. 6, N-Nitroso Compounds. IARC, Lyon, pp. 93101.
-
Hecht,S.S., Ornaf,R.M. and Hoffmann,D. (1974) N'-Nitrosonornicotine in tobacco: analysis of possible contributing factors and biologic implications. J. Natl Cancer Inst., 54, 12371244.[ISI]
-
Mirvish,S.S., Sams,J. and Hecht,S.S. (1977) Kinetics of nornicotine and anabasine nitrosation in relation to N'-nitrosonornicotine occurrence in tobacco and to tobacco-induced cancer. J. Natl Cancer Inst., 59, 12111213.[ISI][Medline]
-
Hecht,S.S., Chen,C.B., Hirota,N., Ornaf,R.M., Tso,T.C. and Hoffmann,D. (1978) Tobacco specific nitrosamines: formation from nicotine in vitro and during tobacco curing and carcinogenicity in strain A mice. J. Natl Cancer Inst., 60, 819824.[ISI][Medline]
-
Hecht,S.S., Chen,C.B., Ornaf,R.M., Jacobs,E., Adams,J.D. and Hoffmann,D. (1978) Reaction of nicotine and sodium nitrite: formation of nitrosamines and fragmentation of the pyrrolidine ring. J. Org. Chem., 43, 7276.[ISI][Medline]
-
Leete,E. (1983) Biosynthesis and metabolism of the tobacco alkaloids. In Pelletier,S.W. (ed.) Alkaloids: Chemical and Biological Perspectives. John Wiley & Sons, New York, NY, Vol. 1, pp. 85152.
-
Armstrong,D.W., Wang,X. and Ercal,N. (1998) Enantiomeric composition of nicotine in smokeless tobacco, medicinal products and commercial reagents. Chirality, 10, 587591.[ISI]
Received October 6, 1999;
revised November 18, 1999;
accepted November 29, 1999.