Levels Of Tobacco-specific N-nitrosamines Research Articles

Variability in addictive and carcinogenic potential of smokeless tobacco products marketed in Mumbai, India: a surveillance study

India has the highest incidence worldwide of smokeless tobacco (SLT)-associated oral cancer, accounting for nearly 70% of all SLT users globally. Nicotine and tobacco-specific N-nitrosamines (TSNA) play critical roles in the addictive and carcinogenic potential, respectively, of SLT products. Our group has previously reported substantial variability in nicotine and TSNA levels across a small SLT product sample in India, calling for systematic surveillance. However, there is no information available on the current levels of these constituents in Indian SLT. We analysed 321 samples representing 57 brands of eight popular types of manufactured SLT products purchased from five local markets in Mumbai, India between August, and September 2019. The sampling locations were Mumbai Central, Kurla, Thane, Vashi, and Airoli. Product pH, moisture content, total and unprotonated (biologically available) nicotine, and TSNA levels were measured at the Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC) in Mumbai. Total nicotine content ranged from 0.45 to 35.1mg/g across products. The unprotonated nicotine fraction contributed 0.1-100% of the total nicotine content. The carcinogenic TSNA levels ranged 0.06-76 ug/g for N'-nitrosonornicotine (NNN), 0.02-19.2 ug/g for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 0.01-6.51 ug/g for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Consistent with our previous study, we observed substantial variations across different brands of the same product type. This is the most extensive and the first within-country study to report brand-specific nicotine and TSNA levels in SLT products marketed in Mumbai, India. Our results show that levels of these constituents remain extremely variable across Indian SLT and are strikingly high in many products. Enhanced public education and continued efforts to reduce SLT use prevalence in India are critical for reducing the global burden of SLT-associated morbidity and mortality. Regulation of nicotine and TSNA levels in SLT products should be considered. This work was supported by the National Institutes of Health (USA) grant R01-TW010651 and, in part, by grants R01-CA180880 and R50-CA211256. The LC-MS/MS analysis was supported in part by XII Plan project funding from the Department of Atomic Energy, Government of India.

Associations between microbial communities and key chemical constituents in U.S. domestic moist snuff.

BackgroundSmokeless tobacco (ST) products are widely used throughout the world and contribute to morbidity and mortality in users through an increased risk of cancers and oral diseases. Bacterial populations in ST contribute to taste, but their presence can also create carcinogenic, Tobacco-Specific N-nitrosamines (TSNAs). Previous studies of microbial communities in tobacco products lacked chemistry data (e.g. nicotine, TSNAs) to characterize the products and identify associations between carcinogen levels and taxonomic groups. This study uses statistical analysis to identify potential associations between microbial and chemical constituents in moist snuff products.MethodsWe quantitatively analyzed 38 smokeless tobacco products for TSNAs using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and nicotine using gas chromatography with mass spectrometry (GC-MS). Moisture content determinations (by weight loss on drying), and pH measurements were also performed. We used 16S rRNA gene sequencing to characterize the microbial composition, and additionally measured total 16S bacterial counts using a quantitative PCR assay.ResultsOur findings link chemical constituents to their associated bacterial populations. We found core taxonomic groups often varied between manufacturers. When manufacturer and flavor were controlled for as confounding variables, the genus Lactobacillus was found to be positively associated with TSNAs. while the genera Enteractinococcus and Brevibacterium were negatively associated. Three genera (Corynebacterium, Brachybacterium, and Xanthomonas) were found to be negatively associated with nicotine concentrations. Associations were also investigated separately for products from each manufacturer. Products from one manufacturer had a positive association between TSNAs and bacteria in the genus Marinilactibacillus. Additionally, we found that TSNA levels in many products were lower compared with previously published chemical surveys. Finally, we observed consistent results when either relative or absolute abundance data were analyzed, while results from analyses of log-ratio-transformed abundances were divergent.

Smokeless Tobacco Products (STPs) Harbour Bacterial Populations with Potential for Oral Carcinogenicity.

Introduction:Smokeless Tobacco Products (STPs) vary significantly in their carcinogenicity, a feature accredited to the variation in the concentrations of carcinogenic chemicals. Tobacco associated bacteria are known to produce Tobacco-specific N-nitrosamines (TSNAs) and hence are determinants of TSNA levels in Tobacco. The primary objective of this study was to conduct a microbiological survey of STPs and to provide a baseline information of the bacterial communities present in the STPs. Materials and Methods:The present study analyzed the constituency of microbial communities in 7 different smokeless Tobacco products including four chewable (T1_CW to T4_CW), two snus (T5_Snus and T6_Snus) and one snuff sample (T7_Snuff) using high-throughput sequencing of the 16S rRNA based next generation sequencing. The Tobacco samples were also analyzed for pH and moisture content. Statistical analysis of the data obtained was done using SPSS software version 20. Pearson’s Correlation was done to analyze the correlation between pH and moisture content of the Tobacco samples. Results:A total of 11 phyla were identified in all smokeless Tobacco products. A total of 36 classes were identified across all smokeless Tobacco products and bacilli was the predominant class in all the products followed by Actinobacteria and Bacteroidia. In species level, a total of 2369 species were identified across all smokeless Tobacco products. In T1 chewable Tobacco products, predominant species was staphylococcus whereas in T2 and T3, Bacillus subtilis and pumilus were predominant. In T4 chewable Tobacco product, Virgibacillus was predominant followed by halodentrificans, staphylococcus epidermidis. In snus 1 and 2, Bacillus pumilus and subtilis were predominant. In snuff, Bacillus cereus was predominant. Snus products had the highest moisture content (15.4% and 14.3%) compared to the chewable Tobacco and snuff products. The snus products analyzed had alkaline values (pH 8.50 and 8.15) and snuff and chewable Tobacco had acidic values ranging from 5.62 to 6.09. Conclusion:The current study demonstrates that ST products differ qualitatively, quantitatively, and in their bacterial composition. There is a possibility that some of these species may contribute to oral carcinogenesis, either by influencing levels of TSNAs or directly inducing chronic inflammation.

Nicotine, Carcinogen, and Toxin Exposure in Long-Term E-Cigarette and Nicotine Replacement Therapy Users: A Cross-sectional Study.

Given the rapid increase in the popularity of e-cigarettes and the paucity of associated longitudinal health-related data, the need to assess the potential risks of long-term use is essential. To compare exposure to nicotine, tobacco-related carcinogens, and toxins among smokers of combustible cigarettes only, former smokers with long-term e-cigarette use only, former smokers with long-term nicotine replacement therapy (NRT) use only, long-term dual users of both combustible cigarettes and e-cigarettes, and long-term users of both combustible cigarettes and NRT. Cross-sectional study. United Kingdom. The following 5 groups were purposively recruited: combustible cigarette-only users, former smokers with long-term (≥6 months) e-cigarette-only or NRT-only use, and long-term dual combustible cigarette-e-cigarette or combustible cigarette-NRT users (n= 36 to 37 per group; total n= 181). Sociodemographic and smoking characteristics were assessed. Participants provided urine and saliva samples and were analyzed for biomarkers of nicotine, tobacco-specific N-nitrosamines (TSNAs), and volatile organic compounds (VOCs). After confounders were controlled for, no clear between-group differences in salivary or urinary biomarkers of nicotine intake were found. The e-cigarette-only and NRT-only users had significantly lower metabolite levels for TSNAs (including the carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL]) and VOCs (including metabolites of the toxins acrolein; acrylamide; acrylonitrile; 1,3-butadiene; and ethylene oxide) than combustible cigarette-only, dual combustible cigarette-e-cigarette, or dual combustible cigarette-NRT users. The e-cigarette-only users had significantly lower NNAL levels than all other groups. Combustible cigarette-only, dual combustible cigarette-NRT, and dual combustible cigarette-e-cigarette users had largely similar levels of TSNA and VOC metabolites. Cross-sectional design with self-selected sample. Former smokers with long-term e-cigarette-only or NRT-only use may obtain roughly similar levels of nicotine compared with smokers of combustible cigarettes only, but results varied. Long-term NRT-only and e-cigarette-only use, but not dual use of NRTs or e-cigarettes with combustible cigarettes, is associated with substantially reduced levels of measured carcinogens and toxins relative to smoking only combustible cigarettes. Cancer Research UK.

Monitoring Tobacco-Specific N-Nitrosamines and Nicotine in Novel Smokeless Tobacco Products: Findings From Round II of the New Product Watch

Analysis of novel smokeless tobacco products purchased in Round I of the New Product Watch (NPW)-a national tobacco monitoring network-demonstrated that some tobacco constituents vary not only across various brands but also regionally and over time within the same product. In this study, we analyzed snus and dissolvable tobacco products that were purchased in Round II of the NPW. We analyzed tobacco-specific N-nitrosamines (TSNA) and nicotine in snus and dissolvable tobacco products that were purchased in various regions of the country during the spring and summer of 2011. The results were compared against the Round I data, across different U.S. regions, and among products. A total of 216 samples were received from different states representing 6 regions of the country. Compared with the previous analyses, TSNA levels increased significantly in Marlboro and Camel Snus and some dissolvable Camel products. The levels of unprotonated nicotine in Marlboro Snus and Camel Snus in this study were not different from Round I but varied significantly by regions; the differences between the highest and the lowest average regional levels were ~3.2-fold in Marlboro Snus ~1.7-fold in Camel Snus. Our results indicate that some novel smokeless tobacco products contain TSNA at the levels found in the conventional moist snuff. Observation of regional variations in unprotonated nicotine content in both Round I and Round II of NPW suggest that manufacturers may tailor the levels of this constituent consistently to different regions.

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

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. 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. 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. 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.

Quantification of four tobacco-specific nitrosamines in cigarette filter tips using liquid chromatography-tandem mass spectrometry

Tobacco-specific N-nitrosamines (TSNA) have been suggested by some scientists to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC-MS/MS method for the determination of TSNA, notably N-nitrosoanabasine (NAB), N-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), extracted from smoked cigarette filter tips. Reporting limits of 0.44, 0.89, 0.91 and 0.91ng mL−1 for NAB, NAT, NNK and NNN respectively were achieved. The newly developed method may find application in the filter analysis methodology for estimating the mouth-level exposure to NAB, NAT, NNK and NNN for cigarette smokers. TSNA levels were determined in mainstream smoke collected on industry standard Cambridge Filter Pads following smoking on a smoking machine. TSNA yields were compared with TSNA levels extracted from cigarette filter tips. However, the observation of the progressive post-smoking accumulation of TSNA during ambient storage of smoked cigarette filter tips potentially compromises use of this technique as an estimate of mouth-level exposure. Storage of smoked cigarette filter tips at sub-ambient temperatures reduced substantially the post-smoking synthesis of TSNA.

Determination of tobacco-specific N-nitrosamines in urine of smokers and non-smokers

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN), N′-nitrosoanabasine (NAB) and N′-nitrosoanatabine (NAT) and are found in tobacco and tobacco smoke. TSNA are of interest for biomonitoring of tobacco-smoke exposure as they are associated with carcinogenesis. Both NNK and NNN are classified by IARC as Group 1 carcinogens. Samples of 24 h urine collections (n = 108) were analysed from smokers and non-smokers, using a newly developed and validated LC-MS/MS method for determining total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major metabolite of NNK), and total NNN, NAB and NAT. TSNA levels in smokers’ urine were significantly higher than in non-smokers. In smokers, urinary excretion of total TSNA correlated significantly (r > 0.5) with markers of smoking dose, such as daily cigarette consumption, salivary cotinine and urinary nicotine equivalents and increased with the ISO tar yield of cigarettes smoked. The correlation between urinary total NNN and the smoking dose was weaker (r = 0.4–0.5). In conclusion, this new method is suitable for assessing tobacco use-related exposure to NNK, NNN, NAB and NAT.

Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). TSNA are suggested to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC–MS/MS method for the determination of total (free and conjugated) TSNA in human urine. The limits of detection (LOD) were 2.0, 0.8, 1.1 and 0.7 pg/ml for NNAL, NNN, NAB and NAT, respectively. Smokers were found to have significantly higher levels of TSNA in their urine than nonsmokers. In conclusion, the newly developed method is suitable for assessing the tobacco use-related exposure to NNK, NNN, NAB and NAT.

Use of Swedish moist snuff, smoking and alcohol consumption in the aetiology of oral and oropharyngeal squamous cell carcinoma. A population-based case-control study in southern Sweden

Conclusions. The results of this study confirm that both smoking of tobacco and alcohol consumption are risk factors for oral and oropharyngeal squamous cell carcinoma (OOSCC). The use of moist snuff had no effect on the risk of OOSCC, probably due to the low levels of tobacco-specific N-nitrosamines in Swedish moist snuff. Objective. The aims of this population-based case-control study in southern Sweden were to establish risk estimates for cigarette and alcohol consumption and to evaluate whether Swedish moist snuff is a risk factor for OOSCC. Material and methods. Between September 2000 and January 2004, 132/165 consecutive cases (80%) diagnosed with OOSCC and 320/396 matched controls (81%) were investigated. All subjects were interviewed and examined according to a standardized protocol. Results. Individuals who drank ≥350 g of alcohol/week showed an increased risk of OOSCC (OR 2.6; 95% CI 1.3–5.4). Total lifetime consumption of tobacco for smoking (>250 kg) had a dose–response effect on the risk of OOSCC (OR 4.7; 95% CI 2.4–9.1). We found no increased risk of OOSCC associated with the use of Swedish moist snuff (OR 1.1; 95% CI 0.5–2.5).

Decreased levels of tobacco-specific N-nitrosamines in moist snuff on the Swedish market.

Moist snuff, or snus, on the Swedish market in 2001 and 2002 was analyzed for tobacco-specific N-nitrosamines (TSNAs) using a recently developed LC-MS/MS method. All samples of moist snuff analyzed were found to contain detectable levels of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In the survey in 2001, all samples except for one were produced by Swedish Match (n = 14), which is the dominating manufacturer on the Swedish snuff market. In the survey in 2002, samples from both Swedish Match (n = 7) and seven smaller manufacturers (n = 20) were analyzed. Total TSNA levels of between 0.15 and 3.0 microg/g wet weight were found. In the survey in 2001 and 2002, the mean level of the total TSNA content in moist snuff was 1.1 microg/g (n = 14) and 1.0 microg/g (n = 27), respectively. The result of the survey shows that the level of TSNAs in moist snuff on the Swedish market has been greatly reduced since the middle of the 1980s. Clearly, efforts have been made by the manufacturers to reduce the level of TSNAs in snuff.

Formation and Analysis of Tobacco-SpecificN-Nitrosamines

Chemical-analytical studies during the past 4 years led to several new observations on the formation of tobacco-specific N-nitrosamines (TSNA) and their occurrence in smokeless tobacco, mainstream smoke (MS), and sidestream smoke (SS) of American and foreign cigarettes. When snuff was extracted by means of supercritical fluid extraction with carbon dioxide containing 10% methanol, analysis of this material confirmed that the extraction with organic solvents had been partially incomplete. Epidemiological studies in the northern Sudan showed a high risk for oral cancer for users of toombak, a home-made oral snuff. Toombak contains 100-fold higher levels of TSNA than commercial snuff in the U.S. and Sweden. The TSNA content in the saliva of toombak dippers is at least ten times higher than that reported in the saliva of dippers of commercial snuff. Biomarker studies have shown corresponding high levels of hemoglobin adducts with metabolites of NNN and NNK as well as for urinary metabolites of NNK. These data supported the epidemiological findings. The analyses of MS of U.S. and foreign cigarettes smoked under FTC conditions revealed comparable data for the smoke of nonfilter cigarettes and filter cigarettes except in the case of low- and ultralow-yield cigarettes, which showed reduced TSNA yields. The MS of cigarettes made from Burley or dark tobacco is exceptionally high in TSNA, primarily because of the high nitrate content of those tobacco types. Taking puffs of larger volume and drawing puffs more frequently, practices observed among most smokers of cigarettes with low nicotine yield, results in high TSNA values in the MS. The formation of the lung carcinogen NNK is favored during the smoldering of cigarettes, between puffs, when SS is generated. Consequently, in most samples from indoor air polluted with environmental tobacco smoke (ETS), the highest concentration of an individual TSNA is that of NNK. When nonsmokers had remained for up to 2 h in a test laboratory with high ETS pollution, they excreted measurable amounts of NNK metabolites in the urine, indicative of the uptake of TSNA.

Five Leading U.S. Commercial Brands of Moist Snuff in 1994: Assessment of Carcinogenic N-Nitrosamines

Moist snuff is the only tobacco product in the United States with increasing sales (an increase of 38.4% between 1981 and 1993) and with increased consumption, primarily by male adolescents aged 12-18 years old and young adults aged 19 years old or older. It is known from previous studies that levels of nicotine and the proportion of unprotonated (free) nicotine, as well as the pH, which affects nicotine delivery, vary considerably among the leading snuff brands. Whether concentrations of major carcinogens, such as the nicotine-derived tobacco-specific N-nitrosamines (TSNAs), like N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), also vary among these brands has not been determined previously. Our purpose was threefold: 1) to determine the concentrations of major carcinogenic nicotine-derived N-nitrosamines in each of the five most popular moist snuff brands; 2) to analyze the quantitative differences in the various snuff components (e.g., NNN) between two major brand categories: a category comprising the brands known to have high levels of unprotonated nicotine (Copenhagen, Skoal fine cut, and Kodiak) versus a category comprising the brands known to have low levels (Hawken and Skoal Bandits); and 3) to compare the differences in the concentrations of nicotine (previously determined), NNN, NNK, and total TSNAs between these two major brand categories. Three boxes of each of the five leading U.S. moist snuff brands were bought in July 1994 from retailers in six areas and transferred immediately to the analytical laboratory. After extraction, N-nitrosamino acids and TSNAs were determined on a gas chromatograph interfaced with a thermal energy analyzer (GC-TEA) and integrator. Each 5-g sample of ground, freeze-dried tobacco was extracted twice, and each extract was analyzed twice by GC-TEA. All P values reported are two sided. Copenhagen, Skoal fine cut, and Kodiak as a group had statistically significant higher levels of nicotine (P = .0017), NNN (P < .0001), NNK (P = .0119), and total TSNAs (P < .0001) than the Hawken and Skoal Bandits group. Concentrations (means +/- SD) of nicotine, NNN, NNK, and total TSNAs comparing the two major brand categories are as follows: nicotine--11.6 +/- 1.01 mg/g versus 6.96 +/- 3.62 mg/g (P = .0017), NNN--7.74 +/- 1.70 micrograms/g versus 4.17 +/- 1.35 micrograms/g (P < .0001), NNK--1.23 +/- 0.68 micrograms/g versus 0.61 +/- 0.41 micrograms/g (P = .0119), and total TSNAs--14.3 +/- 3.82 micrograms/g versus 6.3 +/- 2.56 micrograms/g (P < .0001). The three leading U.S. snuff brands (Copenhagen, Skoal fine cut, and Kodiak; making up 92% of the U.S. market) showed not only high levels of pH, nicotine, and unprotonated (free) nicotine, but also high concentrations of the strongly carcinogenic TSNAs in comparison with the fourth and fifth best selling moist snuff brands, Hawken and Skoal Bandits (3% of the U.S. market).

Effects of storage conditions on levels of tobacco-specific N-nitrosamines and N-nitrosamino acids in U.S. moist snuff

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffects of storage conditions on levels of tobacco-specific N-nitrosamines and N-nitrosamino acids in U.S. moist snuffMirjana V. Djordjevic, Jingrun. Fan, Lowell P. Bush, Klaus D. Brunnemann, and Dietrich. HoffannCite this: J. Agric. Food Chem. 1993, 41, 10, 1790–1794Publication Date (Print):October 1, 1993Publication History Published online1 May 2002Published inissue 1 October 1993https://doi.org/10.1021/jf00034a051RIGHTS & PERMISSIONSArticle Views290Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (598 KB) Get e-Alerts Get e-Alerts

Tobacco-specific N-nitrosamines in the saliva of habitual male snuff dippers.

Saliva was collected every ten minutes from habitual male snuff dippers and analysed for tobacco-specific N-nitrosamines (TSNA). Detectable levels of at least two TSNA were found in all samples collected between 10 and 30 minutes after the snuff had been placed in the mouth. Total concentrations of TSNA up to 241 ng/g were found in the saliva. Trace levels of TSNA were still found in the saliva 20 minutes after the snuff had been removed.