Nornicotine nitrosation in saliva and its relation to endogenous synthesis of N'-nitrosonornicotine in humans.

INTRODUCTION: We recently reported that certain amounts of the carcinogen N'-nitrosonornicotine (NNN) can be formed endogenously from nicotine and/or nornicotine in some users of oral nicotine replacement therapy products. Although the acidic environment of the stomach creates the most favorable conditions for nitrosation, this reaction could also occur in the oral cavity in the presence of bacteria that catalyze nitrosation at neutral pH. METHODS: To test the hypothesis that endogenous formation of NNN could occur in the oral cavity, we investigated nitrosation of nicotine and nornicotine in human saliva. To specifically identify NNN as derived from precursors added to saliva, we incubated saliva samples with [pyridine-D(4)]nicotine and [pyridine-D(4)]nornicotine, with and without the addition of nitrite, and subsequently analyzed [pyridine-D(4)]NNN by liquid chromatography-tandem mass spectrometry. RESULTS: Consistent with kinetic studies on nicotine and nornicotine nitrosation, incubation of saliva with [pyridine-D(4)]nornicotine alone produced detectable amounts of [pyridine-D(4)]NNN, whereas only traces of [pyridine-D(4)]NNN were found in samples incubated with [pyridine-D(4)]nicotine and sodium nitrite. Incubation of saliva samples from 10 nonsmoking volunteers with [pyridine-D(4)]nornicotine resulted in the formation of [pyridine-D(4)]NNN in 8 samples, with yields ranging from 0.003% to 0.051% of the added alkaloid. CONCLUSION: Our results demonstrate that NNN can be formed from nornicotine in human saliva without deliberate addition of any other substance. Therefore, nornicotine, as present in tobacco or in nicotine replacement products, is a carcinogen precursor.

Carcinogenic tobacco-specific N-nitrosamines in US cigarettes: three decades of remarkable neglect by the tobacco industry.

BACKGROUND: Modification of tobacco curing methods and other changes in cigarette manufacturing techniques could substantially reduce the levels of tobacco-specific nitrosamines (TSNA), a group of potent carcinogens, in cigarette smoke. In 1999, two major US cigarette manufacturers stated their intent to move towards using tobaccos low in TSNA. There is no information available on current TSNA levels in tobacco of various cigarettes available in the US, particularly in the newer varieties introduced over the past decade. METHODS: Seventeen brands of cigarettes were purchased in April of 2010 from retail stores in Minnesota. TSNA levels were measured in the tobacco filler and smoke of these cigarettes. RESULTS: In all brands, the sum of two potent carcinogenic TSNA--4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine--in cigarette filler averaged 2.54 (± 1.05) µg/g tobacco. This value is virtually identical to the sum of these two carcinogens reported for the tobacco of a US filtered cigarette in 1979. TSNA levels in smoke positively correlated with those in tobacco filler of the same cigarettes. CONCLUSION: We found no indication that any meaningful attempt was made to reduce or at least control TSNA levels in the new varieties of the popular brands Marlboro and Camel introduced over the last decade. In light of the recently granted regulatory authority to the FDA over tobacco products, regulation of TSNA levels in cigarette tobacco should be strongly considered to reduce the levels of these potent carcinogens in cigarette smoke.

Monitoring tobacco-specific N-nitrosamines and nicotine in novel Marlboro and Camel smokeless tobacco products: findings from Round 1 of the New Product Watch.

INTRODUCTION: Information on chemical composition of the new oral "spitless" smokeless tobacco products is scarce, and it is not clear whether there is some variability as a function of purchase place or time due to either unintended or intended manufacturing variations or other conditions. METHODS: We analyzed tobacco-specific N-nitrosamines (TSNA) and nicotine in Marlboro Snus, Camel Snus, and dissolvable Camel products Orbs, Sticks, and Strips that were purchased in various regions of the country during the summer of 2010. RESULTS: A total of 117 samples were received from different states representing six regions of the country. Levels of unprotonated nicotine in Marlboro Snus and Camel Snus varied significantly by regions, with the differences between the highest and the lowest average regional levels being relatively small in Marlboro Snus (∼1.3-fold) and large in Camel Snus (∼3-fold). Some regional variations in TSNA levels were also observed. Overall, Camel Snus had significantly higher TSNA levels than Marlboro Snus, and Camel Strips had the lowest TSNA levels among all novel products analyzed here. The amount of unprotonated nicotine in the dissolvable Camel products was comparable to the levels found in Marlboro Snus. CONCLUSIONS: Our study demonstrates some regional variations in the levels of nicotine and TSNA in Marlboro and Camel novel smokeless tobacco products. Continued monitoring of this category of products is needed as the existing products are being test marketed and modified, and new products are being introduced. This information is particularly important given its relevance to Food and Drug Administration regulation of tobacco products.

Urinary levels of the tobacco-specific carcinogen N'-nitrosonornicotine and its glucuronide are strongly associated with esophageal cancer risk in smokers.

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are tobacco-specific nitrosamines. NNN and NNK can induce cancers of the esophagus and lung, respectively, in laboratory animals, but data on human esophageal cancer are lacking. The association between levels of NNN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), an NNK metabolite, in urine samples collected before diagnosis and risk of esophageal cancer was examined in 77 patients with esophageal cancer and 223 individually matched controls, all current smokers, from a cohort of 18244 Chinese men in Shanghai, China, followed from 1986 to 2008. Urinary total NNN (free NNN plus NNN-N-glucuronide) was significantly higher, whereas the percentage of its detoxification product NNN-N-glucuronide was significantly lower in cases than controls. Odds ratios (95% confidence intervals) of esophageal cancer for the second and third tertiles of total NNN were 3.99 (1.25-12.7) and 17.0 (3.99-72.8), respectively, compared with the first tertile after adjustment for urinary total NNAL and total cotinine and smoking intensity and duration (P(trend) < 0.001). The corresponding figures for the percentage of NNN-N-glucuronides were 0.37 (0.17-0.80) and 0.27 (0.11-0.62) (P(trend) = 0.001). Urinary total NNN and the percentage of NNN-N-glucuronides almost completely accounted for the observed association for urinary total NNAL (free NNAL plus its glucuronides), urinary total cotinine and smoking intensity with esophageal cancer risk. These findings along with results of previous studies in laboratory animals support a significant and unique role of NNN in esophageal carcinogenesis in humans.

Analysis of 23 polycyclic aromatic hydrocarbons in smokeless tobacco by gas chromatography-mass spectrometry.

Smokeless tobacco contains 28 known carcinogens and causes precancerous oral lesions and oral and pancreatic cancer. A recent study conducted by our research team identified eight different polycyclic aromatic hydrocarbons (PAHs) in U.S. moist snuff, encouraging further investigations of this group of toxicants and carcinogens in smokeless tobacco products. In this study, we developed a gas chromatography-mass spectrometry method that allows simultaneous analysis of 23 various PAHs in smokeless tobacco after a simple two-step extraction and purification procedure. The method produced coefficients of variation under 10% for most PAHs. The limits of quantitation for different PAHs varied between 0.3 and 11 ng/g tobacco, starting with a 300 mg sample. The recovery of the stable isotope-labeled internal standards averaged 87%. The method was applied to analysis of 23 moist snuff samples that included various flavors of the most popular U.S. moist snuff brands, as well as 17 samples representing the currently marketed brands of spit-free tobacco pouches, a relatively new type of smokeless tobacco. The sum of all detected PAHs in conventional moist snuff averaged 11.6 (+/-3.7) microg/g dry weight; 20% of this amount was comprised of carcinogenic PAHs. The levels of PAHs in new spit-free tobacco products were much lower than those in moist snuff; the sum of all detected PAHs averaged 1.3 (+/-0.28) microg/g dry weight. Our findings render PAHs one of the most prevalent groups of carcinogens in smokeless tobacco. Urgent measures are required from the U.S. tobacco industry to modify manufacturing processes so that the levels of these toxicants and carcinogens in U.S. moist snuff are greatly reduced.