Tobacco-specific nitrosamines

N-Nitrosamines are formed during processing and smoking of tobacco products. Proteins, agricultural chemicals and alkaloids in tobacco products serve as major precursors for volatile, nonvolatile, and tobacco-specific nitrosamines (Figure 1). In this review we will summarize the progress achieved in respect to tobacco nitrosamines since the last ACS symposium in Boston in June of 1978 (J ). Additional papers will review the metabolism of cyclic N-nitrosamines, including that of N -nitrosonornicotine 1) and the correlation between tobacco and alcohol consumption and cancer of the upper alimentary tract (J ). 

Nonvolatile Nitrosamines In Tobacco. A method which we developed several years ago for the analysis of tobacco-specific nitrosamines (TSNA 31) involves extraction of tobacco with buffered ascorbic acid TpH 4.5) followed by partition with ethyl acetate, chromatographic clean-up on silica gel, and analysis by HPLC-TEA (Figure 9). Results obtained with this method for a large spectrum of tobacco products (Table IV), strongly support the concept that the levels of nitrate and alkaloids, and especially the methods for curing and fermentation, determine the yields of TSNA in tobacco products. Recent and as yet preliminary data from snuff analyses indicate that aerobic bacteria play a role in the formation of TSNA during air curing and fermentation. 

Nonvolatile Nitrosamines In Saliva. In vitro experiments had indicated that the tobacco-specific nitrosamines are formed also during snuff dipping (26). Therefore, we analyzed the saliva of snuff dippers and tobacco chewers. A comparison of the results demonstrated the presence of TSNA in saliva at a wide range of concentrations (Table Vl), which could be ascribed to differences in the product, but also to differences in the manner of chewing, and, lastly, to individual factors in each person s saliva. 

Figure 10, Tobacco specific nitrosamines as a function of puff volume (other smoking conditions were kept constant puff duration, 2 s puff frequency, once a minute and butt length, 23 mm).

On the other hand, as discussed at this meeting by Hoffmann et al. (13,14), there are certain tobacco-specific nitrosamines such as nltrosonornlcotlne which form a substantial part of the... 

Chung, F.L. and Xu, Y. (1992). Increased 8-oxodeoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with the tobacco-specific nitrosamine 4-(methyl-nitrosamine)-l-(3-pyridyl)-l-butanone. Carcinogenesis 13, 1269-1272. 

NNAL 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanol NNAL-gluc 4-(methylnitrosammo)-l-(3-pyridyl)-l-butanol glucuronide NNK 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone TSNA tobacco-specific nitrosamines PAH ftolycylcic aromatic hydrocarbons... 

Burton HR, Bush LP, Djordjevic MV (1989a) Influence of temperature and humidity on the accumulation of tobacco-specific nitrosamines in stored burley tobacco, J Agric Food Chem 37 1372-1377... 

Burton HR, Childs GH Jr, Andersen RA, Fleming PD (1989b) Changes in chemical composition of burley tobacco during senescence and curing, 3, Tobacco-specific nitrosamines, J Agric Food Chem 37 426 30... 

Burton HR, Dye NK, Bush LP (1992) Distribution of tobacco constituents in tobacco leaf tissue, 1, Tobacco-specific nitrosamines, nitrate, nitrite and alkaloids, J Agric Food Chem 40 1050-1055 Burton HR, Dye NK, Bush LP (1994) Relationship between tobacco-specific nitrosamines and nitrite from different air cured tobacco varieties, J Agric Food Chem 42 2007-2011 Calafat AM, Polzin GM, Saylor J, Richter P, Ashley DL, Watson CH (2004) Determination of tar, nicotine, and carbon monoxide yields in the mainstream smoke of selected international cigarettes, Tob Control 13 45-51... 

Counts ME, Morton MJ, Lafoon SW, Cox RH, Lipowicz PJ (2005) Smoke composition and potential relationship for international commercial cigarettes smoked with three machine-smoking conditions, Regul Toxicol Pharmacol 41 185-227 DeRoton C, Wiemik A, Wahlberg 1, Vidal B (2005) Factors influencing the formation of tobacco-specific nitrosamines in french air-cured tobaccos in trials and at the farm level, Beitr Tabakforsch 21 305-320... 

Djordjevic MV, Gay SL, Bush LP, Chaplin JF (1989) Tobacco-specific nitrosamine accumulation and distribution in flue-cured tobacco isoUnes, J Agric Food Chem 37 752-756 Djordjevic MV, Sigountos CW, Brunnemann KD Hoffmann D (1990) Tobacco-specific nitrosamine delivery in the mainstream smoke of high- and low-yield cigarettes smoked with varying puff volume. In CORESTA Symposium Information Bulletin, Hellas, October 7-11 1990, p209... 

A tribute to Ernst L. Wynder. Chem Res Toxicol 14 767-790 Idris AM, Nair J, Oshima H, Friesen M, Bronet I, Faustman EM, Bartsch H (1991) Unusually high levels of carcinogenic tobacco-specific nitrosamines in Sudan snuff (toombak). Carcinogenesis 12 1115-1118... 

MacKown CT, Douglas B, Djordjevic MV, Bush LP (1988) Tobacco-specific nitrosamines formation during processing of midvein and lamina fines. J Agric Food Chem 36 1031-1035 McNeill A, Bedi R, Islam S, AUchatib MN, West R (2006) Ixvels of toxins in oral tobacco products in the UK. Tob Control 15 64-67... 

Pancreatic effect. Cigarette smoke, administered to anesthetized rats alone or in combination with iv ethanol infusion, reduced pancreatic blood flow temporarily and increased leukocyte-endothelium interaction (roller p < 0.001, sticker p < 0.01 vs baseline). Cigarette smoke potentiated the impairment of pancreatic capillary perfusion caused by ethanol, and both the number of rolling leukocytes and myeloperoxidase activity levels were increased compared with ethanol or nicotine administration alone h Tobacco-specific nitrosamines, administered to rats, induced pancreatic acinar cell and ductal cell neoplasms. One of the tumors had a mixed ductal-squamous-islet cell components . 

Comparative metabolism of the tobacco-specific nitrosamines 4-(meth-ylnitrosamino)-1 -(3-pyridyl)-1 -butanone and 4- (methylnitrosam ino) -1 - (3 -pyri-dyl)-l -butanol by rat cytochrome P450 2A3 and human cytochrome P450 2A13. Drug Metab Dispos 2003 31(10) 1199-1202. 

Malignant transformation of human bronchial epithelial cells with the tobacco-specific nitrosamine, 4-(methyl-nitrosamino) -1 -(3 -pyridyl) -1 -butanone. Int J Cancer 2003 106(6) 821-826. 

White, J. F. Foley, R. R. Maronpot, and NT108 M. W. Anderson. Role of Clara cells and type II cells in the development of pulmonary tumors in rats and mice following exposure to a tobacco-specific nitrosamine. Exp Lung Res 1991 ... 

Bhide. Effects of tobacco-specific nitrosamines and snuff extract on cell proliferation and activities of ornithine decarboxylase and aryl hydrocarbon hydroxylase in mouse tongue primary epithelial cell cultures. J Cancer Res Clin Oncol 1989 115(6) 558-563. 

Hoffman. A study of tobacco carcinogenesis. XLll. Bioassay in A/] mice of some structural analogues of tobacco-specific nitrosamines. Cancer Lett 1988 42(1-2) 141-145. 

NTl79 Hirota, N. Intranuclear rodlets in undifferentiated carcinomas of salivary glands in strain A mice in a study involving a tobacco specific nitrosamine, N-nitrosonornicotine. Cancer Lett 1979 6(6) 365-369. 

NT181 Hecht, S. S., C. B. Chen, N. Hirota, R. M. Omaf, T. C. Tso, and D. Hoffmann. Tobacco-specific nitrosamines formation from nicotine in vitro and during tobacco curing and carcinogenicity in strain A mice. J Natl Cancer Inst 1978 60(4) 819-824. 

Desai, and S. S. Hecht. Evidence for endogenous formation of tobacco—specific nitrosamines in rats treated with tobacco alkaloids and sodium nitrite. Carcinogenesis 1997 18(3) 587—592. NT257 Mendez-Alvarez, E., R. Soto-Otero, I. Sanchez-Sellero, and M. Lopez-Ri-vadulla Lamas. Inhibition of brain monoamine oxidase by adducts of 1, 2,3,4-tetrahydroisoquinoline with components of cigarette smoke. Life Sci 1997 60(19) 1719-1727. 

The prototype of the tobacco specific nitrosamines, NNN, has been detected in both tobacco smoke and unburned tobacco. Various analytical methods have been used including gas chromatography (GLC) (13,14,15,16) combined GLC-mass spectrometry (17), thin layer chromatography (18) high pressure liquid chromatography (HPLC) (19,20), and combined HPLC-thermal energy analysis (21). 

In tobacco samples examined so far, the levels of NAB were significantly less than those of NNN. In fact, NAB has not yet been detected with certainty in unburned tobacco (15). These findings are in line with the major role of nicotine rather than nornicotine as a precursor to NNN since kinetic studies showed that nornicotine and anabasine were nitrosated at similar rates (25). These rates are relatively high, which suggests that the formation of NNN and NAB could be favored in vivo. When chewing tobacco was incubated with human saliva for 3 hours at 37 and the mixture analyzed for NNN, the concentrations of NNN increased by 44% over that in the chewing tobacco, presumably as a result of further nitrosation (15). Thus, in vivo formation of NNN and NAB could constitute an additional exposure of smokers or chewers to these tobacco specific nitrosamines. 

Since nicotine is the major precursor to NNN in tobacco and tobacco smoke, the reaction of nicotine with sodium nitrite was studied to provide information on formation of other tobacco specific nitrosamines, especially NNK and NNA, which could arise by oxidative cleavage of the l -2 bonds or l -5 bond of nicotine followed by nitrosation (26). The reaction was investigated under a variety of conditions as summarized in Table I. All three nitrosamines were formed when the reaction was done under relatively mild conditions (17 hrs, 20 ). The yields are typical of the formation of nitrosamines from tertiary amines (27). At 90 , with a five fold excess of nitrite, only NNN and NNK were detected. Under these conditions, both NNK and NNA gave secondary products. NNK was nitrosated a to the carbonyl to yield 4-(N-methyl-N-nitrosamino)-2-oximino-l-(3-pyridyl)-1-butanone while NNA underwent cyclization followed by oxidation, decarboxylation and dehydration to give l-methyl-5-(3-pyridyl)pyrazole, as shown in Figure 4. Extensive fragmentation and oxidation of the pyrrolidine ring was also observed under these conditions. The products of the reaction of nicotine and nitrite at 90 are summarized in Table II. 

Figure 5. Formation of tobacco specific nitrosamines from nicotine (22)...

D. and Tso, T.C., "Tobacco Specific Nitrosamines Formation by Nitrosation of Nicotine During Curing of Tobacco and Carcinogenicity in Strain A Mice", J. Natl Cancer Inst., 1978, 60,819-824. 

Two other types of element-specific detector for nitrogen currently in use coupled to SFCs are the nitrogen phosphorus detector (NPD) and the thermal energy analyzer (TEA). The NPD uses a hot, catalytically active solid surface immersed in a layer of dissociated H2 and O2 to form electronegative N and P ions which are detected on a nearby electrode [2]. NPD has been shown to have broad application in SFC, especially in the agrochemical industry [3]. The TEA, as described by Fine et al. [4], uses low-temperature pyrolysis, followed by ozone-induced chemiluminescence, for the detection of compounds containing NO2 groups. The TEA has been used for the determination of tobacco-specific nitrosamines and explosives [5]. Both of these detectors require spedlic standards of the analytes of interest for quantitation... 

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