Tobacco 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 ). 

A6 Coumarin, tobacco nitrosamines, nicotine (to cotinine and 2 -hydroxynicotine) Rifampin, phenobarbital Tranylcypromine, menthofuran, methoxsalen... 

There is insufficient evidence to unequivocally link nitrosamine exposure to elevated risk for human cancer (159). There are, however, a number of specific cases, especially with respect to the tobacco-related nitrosamines, in which exposure to V/-nitroso compounds is of concern. The strongest evidence in this context is probably that relating to oral cancer rates among habitual users of smokeless tobacco (snuff). Oral cancer rates among this group are significantly elevated over those of nonusers, and /V-nitrosonornicotine, and 4-(methylnitrosamino)-l-(3-pyridinyl)-l-butanone [64091 -91 both of... 

Excluding tobacco and tobacco smoke (lf f3), the largest known human exposure to N-nitrosamines is in the industrial sector (4). The area or process air samples containing the largest amount of nitrosamines are in a tire factory, NMOR at the 250 yg/m level (5, a leather tannery, NDMA at the 47 ]xg/wr level (jy, and a rocket fuel factory, NDMA at the 36 yg/m level (7). 

Formation, Occurrence, and Carcinogenicity of iV-Nitrosamines in Tobacco Products... 

In Tobacco. At the time of harvesting, fresh tobacco leaves do not contain measurable amounts of nitrosamines (<5 ppb). However, these compounds are formed during curing, aging and fermentation. Their concentrations depend primarily on the content of proteins, alkaloids, agricultural chemicals and nitrate in the tobacco, as well as on the processing conditions which lead to the reduction of the nitrates. 

The first step in the analysis is extraction of the tobacco with buffer solution (pH 4.5) containing 20 mM ascorbic acid. The nitrosamines are then concentrated by partition with dichloromethane, and a chromatographic clean-up on alumina. In the final step, the concentrate is analyzed by GC-TEA and confirmation of the nitrosamines is obtained by GC-MS (O. If isolated amounts of the nitrosamines are below levels needed for GC-MS confirmation, we employ confirmatory techniques proposed by Krull et a. ( 5). 

In Smoke. We compared the gas chromatograms of nitrosamines in matching aliquots of mainstream smoke derived from Burley type cigarettes that were identical except for the degree of nitrate fertilization during cultivation (Figure 2). This comparison supports the concept that the nitrate concentration in tobacco is a determining factor for the nitrosamine yields in the smoke. The data in Table II confirm this concept. These studies have... 

Figure 2. Effect of nitrate in tobacco on nitrosamine yield in mainstream smoke...

Among the agricultural chemicals used for the cultivation of tobacco crops we find several amines, amides and carbamates. These include dimethyldodecylamine acetate (Penar), maleic hydra-zide-diethanolamine (MH-30), and carbaryl (Sevin) as a representative of the methyl urethanes (Figure 3 , 14), It is known that small quantities of these agents are found as residues in harvested tobacco (15). To date, only diethanolamine (DELA), the water-solubilizer for maleic hydrazide in MH-30, has been studied as a possible precursor for nitrosamines in tobacco and in tobacco smoke. In 1976, more than 1,400 metric tons of maleic hydrazide had been used on U.S. tobacco (16), most of which had been applied as the MH-30 formulation with diethanolamine (14,16). 

In tobacco, we found the highest NDELA values for fresh and aged snuff with 6.8 and 3.2 ppm, respectively. The fermentation process appears to increase NDELA, as was also observed for VNA concentrations. There was clear evidence that tobaccos which had not been treated with MH-30, and cigarette smoke obtained from these tobaccos, were free of NDELA, whereas all MH-30 treated tobaccos and cigarette smoke derived from them showed measurable quantities of this nitrosamine (19). 

In order to ascertain that the NDELA formation does not occur as a result of trapping of the smoke or during the analysis, we added diethanolamine to tobacco prior to extraction with ethyl acetate in the presence of ascorbic acid. The control value for NDELA was 121 ppb and the experiment with 5.5 iqg diethanolamine addition yielded 113 ppb NDELA. For control of the smoke analysis we added 5.5 mg of DELA in the solvent trap and smoked cigarettes known to be free of DELA. Analysis of the trapped material showed no significant quantities of NDELA, so that artifactual formation of this nitrosamine during smoke collection and analysis can be ruled out. 

Currently, we are studying the transfer rate of NDELA from tobacco into smoke as well as the potential of DELA and NDELA to serve as precursors for other nitrosamines in smoke. 

Carcinogenicity of NDELA. Our special interest in NDELA as a constituent of tobacco products and as an environmental agent relates to the observation that this nitrosamine induces carcinoma of the liver as well as of the kidney in rats (21,22) and carcinoma of the nasal cavity and papillomas of the trachea in hamsters (23). Recently, Lijinsky reported that NDELA admini-... 

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. 

Figure 7. Some nitrosamines which can be derived from the tobacco alkaloids.

The alkaloid derived nitrosamines in cigarettes, cigars and pipe tobacco contribute to the quantities of these carcinogens in the smoke because of their partial transfer (32). 

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. 

Tobacco Specific N-Nitrosamines Snuff Dipper Saliva Collected (ng/g)... 

Saliva of 3 non-snufMipping women (controls) was free of nicotine and tobacco-specific N-nitrosamines. 

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).

Nonvolatile Nitrosamines In Tobacco Smoke. Although there are more than 10 million exsmokers in the U.S.A., 53 million adults continue to smoke cigarettes and an additional 10 million still smoke cigars or pipes (39). The cigarette smokers are exposed to about 10 ng of volatile nitrosamines, 20-40 ng of NDELA and, most importantly, to 1-10 pg of tobacco specific N-nitros-amines with each cigarette smoked (Table IV). Similar quantities of the TSNA are found in sidestream smoke. The quantities of TSNA in the smoke are dependent on nitrate, nitrite, tobacco alkaloids and on NNN, NNK and NAT in the tobacco itself (31)>... 

Tobacco Specific N-Nitrosamines Occurrence, Carcinogenicity and Metabolism", ACS Symp. Ser, 1979, 101, 125-152,... 

Hoffmann, D. Adams, J.D. Carcinogenic Tobacco Specific N-Nitrosamines in Snuff and in the Saliva of Snuff Dippers. Submitted. 

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... 

Radioisotope-labeled nitrosamines have proven valuable in development of analytical methods and for demonstrating efficiency of recovery of nitrosamines from tobacco products and smoke (37-39). The very high specific activity required for low part-per-billion determinations has discouraged most analysts from using this approach. Unless a radiochromatographic detector with adequate sensitivity is available, samples must be counted independently of the final chromatographic determination, and one of the advantages of internal standardization, correction for variation in volume injected, is lost. 

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. 

Metabolism of nitrosamines from tobacco generates a series of electrophiles that alkylate DNA, and some of the resulting adducts can also regenerate electrophilic intermediates illustrated in Scheme 9.4. This oxonium ion can be terminally quenched... 

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