Total Tobacco-specific Nitrosamines Research Articles

Control of tobacco-specific nitrosamines by the Bacillus siamensis: Strain isolation, genome sequencing, mechanism analysis and genetic engineering

Nitrosamines are considered carcinogens that threaten human health and environment. Especially, high contents of Tobacco-specific nitrosamines (TSNAs) are generated during the fermentation process of cigar tobacco. To control the accumulation of TSNAs, one novel strain WD-32 was isolated by comprehensively evaluating the reduction characteristics of nitrate, nitrite, and TSNAs, and this strain was identified as Bacillus siamensis by 16 S rRNA gene analysis and MALDI-TOF MS evaluation. Subsequently, whole genome sequencing of B. siamensis WD-32 was carried out to excavate important genes and enzymes involved, and the possible reduction mechanism of TSNAs was explored. More importantly, the reduction of TSNAs by B. siamensis was significantly promoted by knockout of narG gene. During the practical agricultural fermentation process of the cigar tobacco leaves, the treatment by the WD-32∆narG cells resulted in a 60% reduction of the total TSNAs content compared with the control, and the concentrations of the NNN and NNK were decreased by 69% and 59%, respectively. In summary, this study offers efficient strains for reduction of the TSNAs in cigar tobacco, and provides new insights into the reduction mechanism of TSNAs, which will promote the application of microbial methods in control of TSNAs and nitrite.

Intake of tobacco nitrosamines of smokers in various provinces of China and their cancer risk: A meta-analysis

Nitrosamines are a class of carcinogens which have been detected widely in food, water, some pharmaceuticals as well as tobacco. The objectives of this paper include reviewing the basic information on tobacco consumption and nitrosamine contents, and assessing the health risks of tobacco nitrosamines exposure to Chinese smokers. We searched the publications in English from “Web of Science” and those in Chinese from the “China National Knowledge Infrastructure” in 2022 and collected 151 literatures with valid information. The content of main nitrosamines in tobacco, including 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), total tobacco-specific nitrosamines (TSNA), and N-nitrosodimethylamine (NDMA) were summarized. The information of daily tobacco consumption of smokers in 30 provinces of China was also collected. Then, the intakes of NNN, NNK, NAT, NAB, TSNAs, and NDMA via tobacco smoke were estimated as 1534 ng/day, 591 ng/day, 685 ng/day, 81 ng/day, 2543 ng/day, and 484 ng/day by adult smokers in 30 provinces, respectively. The cancer risk (CR) values for NNN and NNK inhalation intake were further calculated as 1.44 × 10−5 and 1.95 × 10−4. The CR value for NDMA intake via tobacco smoke (inhalation: 1.66 × 10−4) indicates that NDMA is similarly dangerous in tobacco smoke when compared with the TSNAs. In China, the CR values caused by average nitrosamines intake via various exposures and their order can be estimated as the following: smoke (3.75 × 10−4) > food (1.74 × 10−4) > drinking water (1.38 × 10−5). Smokers in China averagely suffer 200% of extra cancer risk caused by nitrosamines in tobacco when compared with non-smokers.

Biodegradation of Nicotine and TSNAs by Bacterium sp. Strain J54.

Background: Microorganisms play an important role in reducing harmful substances in flue-cured tobacco. Numerous studies have been conducted to degrade nicotine by microorganisms.Objectives: The present research deals with the isolation of a potent bacterial strain able to efficiently degrade nicotine and tobacco-specific nitrosamines (TSNAs) in flue-cured tobacco.Material and Methods: Bacterial strain J54, capable of efficiently degrading nicotine and tobacco-specific nitrosamines (TSNAs), was isolated from tobacco leaves and identified. The strain J54 can use nicotine as the sole carbon and nitrogen source and could effectively degrade nicotine while growing in a nicotine isolation medium (NIM) medium.Results: Compared with the control (CK), the total TSNAs content in the tobacco flue-cured eaves after being sprayed with a solution of the J54 strain was found to decrease by 26.22%. Therein, the degradation rates of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N’-nitrosonornicotine (NNN), N’-nitrosoanatabine (NAT), and N′-nitrosoanabasine (NAB) were 24.01%, 26.27%, 28.6%, and 1.83%, respectively.Conclusions: Bacterial strain J54, was isolated from tobacco leaves and identified as a bacterium, which is similar to Bacillus altitudinis based on its morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. To our knowledge, this is the first report of the isolation and characterization of a Bacillus sp. strain that can efficiently degrade nicotine and TSNAs. The findings pave the way for the application of new biotechnologies for the degradation of nicotine and TSNAs by microorganisms.

Effect of Potassium Source and Rate on Yield, Quality, and Tobacco-Specific Nitrosamines in Dark and Burley Tobacco

Field trials were conducted in Princeton, Murray, and Lexington, KY in 2016–2018 to determine response of dark and burley tobacco to potassium source (potassium sulfate or potassium chloride) and potassium rate (0, 93, 186, or 279 kg K ha−1). Field sites that showed higher potential for potassium yield response were selected based on low soil test potassium levels from soil samples collected in early spring each year. All potassium applications were made between 1 and 10 days before transplanting. Significant yield responses to potassium were seen in 5 of 12 trials at sites that had initial soil test potassium levels of ≤150 kg K ha−1. Although cured leaf chloride levels were >1% on average where potassium chloride was used, negative effects on cured leaf moisture were only seen in 1 of 12 trials, and negative effects on quality grade index were not seen in any trial. The most consistent effect of potassium chloride application seen in this research was a 28% reduction in average total tobacco-specific nitrosamines (TSNA) compared to potassium sulfate application. These results showing lower TSNA from potassium chloride applications, along with minimal effects on moisture and quality grade index, may cause the tobacco industry to reconsider the long-standing preference for potassium sulfate as the potassium source for tobacco production.

Tobacco-Specific Nitrosamines (NNAL, NNN, NAT, and NAB) Exposures in the US Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).

The tobacco-specific nitrosamines (TSNAs) are an important group of carcinogens found in tobacco and tobacco smoke. To describe and characterize the levels of TSNAs in the Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014), we present four biomarkers of TSNA exposure: N'-nitrosonornicotine, N'-nitrosoanabasine, N'-nitrosoanatabine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which is the primary urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. We measured total TSNAs in 11 522 adults who provided urine using automated solid-phase extraction coupled to isotope dilution liquid chromatography-tandem mass spectrometry. After exclusions in this current analysis, we selected 11 004 NNAL results, 10 753 N'-nitrosonornicotine results, 10 919 N'-nitrosoanatabine results, and 10 996 N'-nitrosoanabasine results for data analysis. Geometric means and correlations were calculated using SAS and SUDAAN. TSNA concentrations were associated with choice of tobacco product and frequency of use. Among established, every day, exclusive tobacco product users, the geometric mean urinary NNAL concentration was highest for smokeless tobacco users (993.3; 95% confidence interval [CI: 839.2, 1147.3] ng/g creatinine), followed by all types of combustible tobacco product users (285.4; 95% CI: [267.9, 303.0] ng/g creatinine), poly tobacco users (278.6; 95% CI: [254.9, 302.2] ng/g creatinine), and e-cigarette product users (6.3; 95% CI: [4.7, 7.9] ng/g creatinine). TSNA concentrations were higher in every day users than in intermittent users for all the tobacco product groups. Among single product users, exposure to TSNAs differed by sex, age, race/ethnicity, and education. Urinary TSNAs and nicotine metabolite biomarkers were also highly correlated. We have provided PATH Study estimates of TSNA exposure among US adult users of a variety of tobacco products. These data can inform future tobacco product and human exposure evaluations and related regulatory activities.

Biomarkers of Exposure among Adult Smokeless Tobacco Users in the Population Assessment of Tobacco and Health Study (Wave 1, 2013-2014).

Monitoring population-level toxicant exposures from smokeless tobacco (SLT) use is important for assessing population health risks due to product use. In this study, we assessed tobacco biomarkers of exposure (BOE) among SLT users from the Wave 1 (2013-2014) of the Population Assessment of Tobacco and Health (PATH) Study. Urinary biospecimens were collected from adults ages 18 and older. Biomarkers of nicotine, tobacco-specific nitrosamines (TSNA), polycyclic aromatic hydrocarbons (PAH), volatile organic compounds (VOC), metals, and inorganic arsenic were analyzed and reported among exclusive current established SLT users in comparison with exclusive current established cigarette smokers, dual SLT and cigarette users, and never tobacco users. In general, SLT users (n = 448) have significantly higher concentrations of BOE to nicotine, TSNAs, and PAHs compared with never tobacco users; significant dose-response relationships between frequency of SLT use and biomarker concentrations were also reported among exclusive SLT daily users. Exclusive SLT daily users have higher geometric mean concentrations of total nicotine equivalent-2 (TNE2) and TSNAs than exclusive cigarette daily smokers. In contrast, geometric mean concentrations of PAHs and VOCs were substantially lower among exclusive SLT daily users than exclusive cigarette daily smokers. Our study produced a comprehensive assessment of SLT product use and 52 biomarkers of tobacco exposure. Compared with cigarette smokers, SLT users experience greater concentrations of some tobacco toxicants, including nicotine and TSNAs. Our data add information on the risk assessment of exposure to SLT-related toxicants. High levels of harmful constituents in SLT remain a health concern.

Effect of Ordering Method on Tobacco-Specific Nitrosamines (TSNAs) Content in Dark Air-Cured and Burley Tobacco

Tobacco-specific nitrosamines (TSNAs) are known carcinogens in cured tobacco. They are produced primarily during the curing process, but agronomic practices occurring in the field as well as handling practices after curing may also influence TSNA levels, particularly if cured leaf is stored at high moisture. After curing and during market preparation, the cured leaf must be supple to avoid breakage. Ideally, this is after a period of wet weather during which the leaf absorbs moisture and comes into order or case. Often the weather remains dry for long periods after curing, and growers resort to artificial ordering to take down a sufficient amount of their crop to work on for several days, during which time the tobacco is bulked. The effect of this artificial ordering on TSNAs during short-term storage is not known. Field experiments were conducted in each of 3 years at two locations in Kentucky to evaluate TSNA accumulation following several ordering methods in dark air-cured and burley tobacco types. Treatments included natural ordering and variants of steaming and misting, which are both commonly used artificial ordering methods. At the Princeton location, samples were taken within 24 hr after the ordering treatments were done. In Lexington, samples were taken sequentially at takedown, after ordering, and after 14 d in the bulk. There were limited and inconsistent differences in total TSNAs between methods of ordering, and the TSNA levels were not affected by the moisture content of the leaf during bulking. There was a significant increase in TSNAs in the 24-hr period between takedown and bulking, which cannot be explained. We conclude that, in Kentucky, growers should use ordering methods that are best suited for their production system, but this may not be the case in warmer climates.

Characterization of mainstream cigarette smoke aerosol by size-fractionated chemical analysis of nicotine, solanesol, and tobacco-specific nitrosamines

The distribution of key chemicals in mainstream cigarette smoke (MCS) particles of different sizes have implications for respiratory dose. In this study, a differential mobility spectrometer (DMS) and an electrical low-pressure impactor (ELPI) were used to characterize MCS particles generated from three cigarettes (3R4F, Dunhill, and Hongtashan). In addition, the ELPI was used to collect smoke particles of three different aerodynamic diameters (144, 431, and 722 nm) from 3R4F MCS, which were analyzed for nicotine, solanesol and tobacco-specific nitrosamines (TSNAs). The number concentration, particle size distribution, and count median diameter (CMD) of the MCS particles characterized by DMS and ELPI were compared quantitatively. The observed difference in the physical characterization results of MCS particles may be due to a myriad of discrepancies between the two techniques. Chemical analyses of the three ELPI selected particle sizes showed that, after normalization to total particulate matter (TPM), nicotine and TSNAs were distributed evenly, whereas solanesol showed a tendency to increase with increasing particle diameter. Notably, the distribution of nicotine and four TSNAs was independent of the three particle sizes; however, the amount of solanesol showed a slight dependency on particle size, varying by less than 23.4% over the size range considered.

Acetylsalicylic acid application decreased tobacco-specific nitrosamines and its precursors but maintained quality of air-cured burley tobacco (Nicotiana tabacum L.)

Burley tobacco (Nicotiana tabacum L.) is an important raw material in the production of Chinese-style blended cigarettes. However, the high levels of tobacco-specific nitrosamines (TSNA) have devalued the industrial use of burley tobacco and limited its sustainable use. The objective of this paper was to investigate the effects of acetylsalicylic acid (ASA) application on accumulation of TSNA, its precursors and quality of air-cured burley tobacco (Nicotiana tabacum L. cv. DaBai 1). Field experiments were conducted to determine the contents of TSNA and the precursors and the sensory quality of burley tobacco under 0, 0.1, 0.3mM of ASA in 2014 and 2015 at Dazhou, Sichuan Province, China. The results showed that TSNA, nitrate, nitrite and alkaloids levels were significantly reduced by the application of ASA, of which the total TSNA were significantly lower by 10.40–24.75%, and two of the strong animal carcinogens in tobacco products, i.e. N-nitrosonornicotine (NNN) and 4-(methylnitrosamino) -1- (3- pyridyl) -1- butanone (NNK) were reduced by 9.01–24.23% and 11.43–26.86%, respectively. Year had significant effects on TSNA and the TSNA contents in 2014 were higher than in 2015. Interestingly, the sensory quality of burley tobacco remained or was even better after application of ASA. In conclusion, this study revealed that ASA application was able to reduce the levels of TSNA and its precursors in sustaining burley tobacco industry potentially.

Tobacco-Specific Nitrosamines in the Tobacco and Mainstream Smoke of U.S. Commercial Cigarettes.

Tobacco-specific nitrosamines (TSNAs) are N-nitroso-derivatives of pyridine-alkaloids (e.g., nicotine) present in tobacco and cigarette smoke. Two TSNAs, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are included on the Food and Drug Administration's list of harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco. The amounts of four TSNAs (NNK, NNN, N-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT)) in the tobacco and mainstream smoke from 50 U.S. commercial cigarette brands were measured from November 15, 2011 to January 4, 2012 using a validated HPLC/MS/MS method. Smoke samples were generated using the International Organization of Standardization (ISO) and Canadian Intense (CI) machine-smoking regimens. NNN and NAT were the most abundant TSNAs in tobacco filler and smoke across all cigarette brands, whereas NNK and NAB were present in lesser amounts. The average ratios for each TSNA in mainstream smoke to filler content is 29% by the CI smoking regimen and 13% for the ISO machine-smoking regimen. The reliability of individual TSNAs to predict total TSNA amounts in the filler and smoke was examined. NNN, NAT, and NAB have a moderate to high correlation (R2 = 0.61-0.98, p < 0.0001), and all three TSNAs individually predict total TSNAs with minimal difference between measured and predicted total TSNA amounts (error < 7.4%). NNK has weaker correlation (R2 = 0.56-0.82; p < 0.0001) and is a less reliable predictor of total TSNA quantities. Tobacco weight and levels of TSNAs in filler influence TSNA levels in smoke from the CI machine-smoking regimen. In contrast, filter ventilation is a major determinant of levels of TSNAs in smoke by the ISO machine-smoking regimen. Comparative analysis demonstrates substantial variability in TSNA amounts in tobacco filler and mainstream smoke yields under ISO and CI machine-smoking regimens among U.S. commercial cigarette brands.

Analysis of Tobacco-Specific Nitrosamines in the Common Smokeless Tobacco Afzal in Oman.

There is a lack of awareness regarding the carcinogenicity of Afzal, an illegal smokeless tobacco product (STP) widely used among the Omani youth. Previous research has shown that certain types of tobacco-specific nitrosamines (TSNAs) are associated with oral and lung cancers. This study therefore aimed to assess levels of four common TSNAs in a randomly selected sample of Afzal. This study was carried out at Sultan Qaboos University in Muscat, Oman, between April and September 2013. A random sample of Afzal was analysed for four types of TSNAs using high-performance liquid chromatography-tandem mass spectrometry. The four types of TSNAs analysed were 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB). As a reference product, a sample of laboratory-manufactured American moist snuff (Centers for Disease Control and Prevention, Atlanta, Georgia, USA) was also used to confirm the accuracy and precision of the analysis. The analysis revealed total TSNA levels of 3.573 μg/g in the Afzal sample. Mean levels of NNN, NNK, NAT and NAB were 1.205, 1.015, 0.809 and 0.545 μg/g, respectively. Levels of the two most abundant TSNAs (NNN and NNK) found in the Afzal sample exceeded international regulatory limits. Afzal users therefore need to be educated regarding the potential health risks associated with their STP use. Stricter implementation of current legislation is recommended to reduce the availability and usage of Afzal in Oman.

Measurement of Nicotine, Tobacco-Specific Nitrosamines, and Additives in the Filler and Gas from Japanese Brand Snuff

The World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC) reported that understanding the use and impact of smokeless tobacco (SLT) products is complicated by product diversity. Many different SLT products with different characteristics are used worldwide. ZERO STYLE STIX(TM) (sold by Japan Tobacco Inc.) is a brand of snuff, a type of smokeless tobacco. Our objective was to determine the constituents of the gas from SLT and analyze the ingredients in tobacco fillers. ZERO STYLE STIX smokeless tobacco was released in the Japanese market in 2010. Nicotine, menthol, and tobacco-specific nitrosamines in the smokeless tobacco fillers were determined by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). The gaseous compounds were collected by a smoking machine using two smoking protocols, i.e., the ISO and Health Canada Intense methods. Nicotine and menthol in the gas were determined by GC/MS. Nicotine, menthol, and the total tobacco-specific nitrosamines were detected in the tobacco fillers. The level of menthol in the snuff was more than ten times that of nicotine. The determined levels of the two components of the gas from the snuff were higher when using the Health Canada Intense protocol than when using the ISO protocol. In addition, flavors other than menthol were emitted from the smokeless tobacco. The new type of snuff introduced in the Japanese market in 2010 contained added flavors, and was attractive smokeless tobacco. Flavors in tobacco products need to regulate on the basis of FCTC 9 and 10 in JAPAN.

Nicotine levels and presence of selected tobacco-derived toxins in tobacco flavoured electronic cigarette refill liquids.

Background. Some electronic cigarette (EC) liquids of tobacco flavour contain extracts of cured tobacco leaves produced by a process of solvent extraction and steeping. These are commonly called Natural Extract of Tobacco (NET) liquids. The purpose of the study was to evaluate nicotine levels and the presence of tobacco-derived toxins in tobacco-flavoured conventional and NET liquids. Methods. Twenty-one samples (10 conventional and 11 NET liquids) were obtained from the US and Greek market. Nicotine levels were measured and compared with labelled values. The levels of tobacco-derived chemicals were compared with literature data on tobacco products. Results. Twelve samples had nicotine levels within 10% of the labelled value. Inconsistency ranged from −21% to 22.1%, with no difference observed between conventional and NET liquids. Tobacco-specific nitrosamines (TSNAs) were present in all samples at ng/mL levels. Nitrates were present almost exclusively in NET liquids. Acetaldehyde was present predominantly in conventional liquids while formaldehyde was detected in almost all EC liquids at trace levels. Phenols were present in trace amounts, mostly in NET liquids. Total TSNAs and nitrate, which are derived from the tobacco plant, were present at levels 200–300 times lower in 1 mL of NET liquids compared to 1 gram of tobacco products. Conclusions. NET liquids contained higher levels of phenols and nitrates, but lower levels of acetaldehyde compared to conventional EC liquids. The lower levels of tobacco-derived toxins found in NET liquids compared to tobacco products indicate that the extraction process used to make these products did not transfer a significant amount of toxins to the NET. Overall, all EC liquids contained far lower (by 2–3 orders of magnitude) levels of the tobacco-derived toxins compared to tobacco products.

Decreased tobacco-specific nitrosamines by microbial treatment with Bacillus amyloliquefaciens DA9 during the air-curing process of burley tobacco.

Tobacco specific nitrosamines (TSNA) mainly consisting of N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are a group of toxic components threatening human health. To inhibit TSNA formation in tobacco leaves, a high nitrite reductive strain with low nitrate reduction ability was isolated and applied to tobacco leaves in an attempt to lower the nitrite precursor of TSNA. By morphology, physiology, biochemistry, and 16S rDNA sequence analysis, the strain DA9 was identified as Bacillus amyloliquefaciens. Under the optimized fermentation parameters (glucose 40 g/L, NH4Cl 4 g/L, corn steep liquor 8 g/L, MnSO4 0.01 g/L, KH2PO4 1.0 g/L, MgSO4 0.3 g/L, initial pH 7.0, inoculum age 6 h, inoculum size 3%, temperature 37 °C), the maximum cell dentisity of 1.2 × 10(9) CFU/mL was obtained at 36 h. The DA9 cell suspensions were applied in the air-curing process of the Burley tobacco (Eyan 6) leaves. The treatment by DA9 cells lowered 32% of the nitrite content and 47% of total TSNA content in the tobacco leaves, and the concentrations of the NNN, NNK, and NAT were decreased by 48%, 12%, and 35%, respectively. Collectively, this study provides a promising strain and a novel strategy for decreasing TSNA during the air-curing process.

Measurement of Hazardous Chemical Constituents and Mutagenic Activity in Fillers and Mainstream Smoke from Neo Cedar

To determine constituents of fillers and mainstream smoke from Neo Cedar. Neo Cedar is a second-class over-the-counter (OTC) drug and similar to cigarettes in a number of ways. In particular, the design and usage are very similar to those of cigarettes. For the fillers of the drug, the levels of nicotine, tobacco-specific nitrosamines (TSNA), and heavy metals, and mutagenicity were determined using the methods for cigarette products. For the mainstream smoke, the levels of tar, nicotine, carbon monoxide (CO), TSNA, polycyclic aromatic hydrocarbons (PAH), and carbonyl compounds were also determined using the methods for cigarettes. The mainstream smoke from the drug were collected with a smoking machine using two smoking protocols (ISO and Health Canada Intense methods). The nicotine and total TSNA levels in the fillers of the drug averaged 2.86 mg/g and 185 ng/g, respectively. The nine species of heavy metals were also detected in the fillers of the drug. The levels of nicotine, tar, CO, TSNA, PAH, and carbonyl compounds of mainstream smoke from the drug were higher when determined using the HCI regime than when using the ISO regime. The mutagenicity of the mainstream smoke determined using the HCI regime was also higher than that determined using the ISO regime. In this study, all constituents of Neo Cedar were determined by methods for cigarette products. The drug had a ventilation hole on its filter. Thus, its constituents are different from those determined by the smoking protocols. Neo Cedar users should be careful of higher exposure to the hazardous gases owing to smoking patterns.

Multi-analyte approach for determining the extraction of tobacco constituents from pouched snus by consumers during use

BackgroundSnus is a smokeless oral tobacco product with a significant history of use in Sweden, where it is regulated under food legislation. Users place a small porous sachet or a pinch of loose snus between the upper jaw and cheek for approximately one hour, leading to partial intake of tobacco constituents. To understand user exposure to tobacco, a multi-analyte approach based on the extraction of pouches by methanol, ethanol and water was validated and applied to the measurement of various constituents, including nicotine, four tobacco-specific nitrosamines (TSNAs), propylene glycol, water, ammonium, nitrate, sodium, chloride, linalool, citronellol, linalyl acetate and geraniol, extracted from snus pouches during use by human consumers.ResultsAfter validation against established single-analyte methods, the multi-analyte approach was used to determine constituent levels in snus pouches before and after one hour of use. Although the concentrations in the snus pouches varied from nanogram (e.g. TSNAs) to milligram (e.g. nicotine, sodium and propylene glycol) quantities (25.1 ng to 35.3 mg per 1 g pouch), the mean percentage extracted varied only from 19.2% for linalyl acetate to 37.8% for the TSNA 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) among all constituents analyzed. The TSNAs, some of which are known carcinogens, showed the highest percentage extraction (range 34.6%–37.8%). Measurement variability was low for all analytes, ranging from 2.4% (total TSNAs, NAT) to 9.5% (geraniol). By contrast, inter-subject variability ranged from 6.7% (NAB) to 52.2% (linalyl acetate), and was greater than 20% for eight of the constituents analyzed. Intra-subject variability ranged from 3.4% (citronellol) to 29.7% (geraniol).ConclusionsGenerally, less than a third of each constituent tested was extracted during one hour of snus use, independent of constituent concentration. The variable nature of in-use extraction was shown to be driven by inter-subject variability. The results provide insight into possible mechanisms controlling constituent extraction in the mouth during snus use, and provide reference data for the development of in-vitro laboratory systems for estimating extraction of tobacco constituents from snus.

Assessment of Tobacco Specific Nitrosamines in Tobacco Genotypes

The tobacco-specific nitrosamines (TSNAs) are one of the most important groups of carcinoigens in tobacco leaf, knowing the difference of TSNAs contents in tobacco genotypes should be beneficial for breeding low TSNAs cultivars which could decrease the harmfulness to human health in tobacco products. For this purpose, 8 genotypes (include burley, sun-cured and flue-cured tobacco) were selected as materials, the TSNAs contents of uncured and cured leaves were assessed with LC-MS/MS. The results showed that TSNAs contents were significantly increased after curing in all genotypes. The mean TSNAs contents of leaves before and after curing in burley were the highest among three types of tobacco, and the contents in sun-cured tobacco were the lowest. Coefficient of variation (CV) of TSNAs among genotypes in burley was higher than that in flue-cured and sun-cured tobacco, and the CV in sun-cured tobacco was the lowest. The positive correlation was observed in NNN, NAT and total TSNAs contents between uncured and cured leaves in three types of tobacco. Therefore, the TSNAs contents in uncured leaves could be considered as evaluation index to screen tobacco cultivars with the low TSNAs contents.

Catalytic degradation of tobacco-specific nitrosamines by ferric zeolite

New strategy of using ferric zeolite ZSM-5 and Y to adsorb and catalytically degrade tobacco-specific nitrosamines (TSNA) is reported in this article. These zeolites were modified by both aqueous ion-exchange with FeSO4 solution and wet-impregnation of Fe(NO3)3, and characterized by XRD, UV–vis DRS, H2-TPR and temperature-programmed surface reaction (TPSR). Apart from laboratory tests, these samples were put into tobacco rods of cigarette to assess their real performance of reducing TSNA content in smoke, and the determination of TSNA was carried out by LC–MS/MS technique with the assistance of internal-standards. The ion-exchanged zeolites are more active than their impregnated analogs, and the ferric Y zeolite could selectively reduce 26% of total TSNA in mainstream smoke of Burley type tobacco. Catalytic breaking of the chemical bond in NNO group of nitrosamines by zeolite is the key step of decomposition of these carcinogens.

RNA interference (RNAi)‐induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves

Technologies for reducing the levels of tobacco product constituents that may contribute to unwanted health effects are desired. Target compounds include tobacco-specific nitrosamines (TSNAs), a class of compounds generated through the nitrosation of pyridine alkaloids during the curing and processing of tobacco. Studies have reported the TSNA N'-nitrosonornicotine (NNN) to be carcinogenic in laboratory animals. NNN is formed via the nitrosation of nornicotine, a secondary alkaloid produced through enzymatic N-demethylation of nicotine. Strategies to lower nornicotine levels in tobacco (Nicotiana tabacum L.) could lead to a corresponding decrease in NNN accumulation in cured leaves. The major nicotine demethylase gene of tobacco has recently been isolated. In this study, a large-scale field trial was conducted to evaluate transgenic lines of burley tobacco carrying an RNA interference (RNAi) construct designed to inhibit the expression of this gene. Selected transgenic lines exhibited a six-fold decrease in nornicotine content relative to untransformed controls. Analysis of cured leaves revealed a commensurate decrease in NNN and total TSNAs. The inhibition of nicotine demethylase activity is an effective means of decreasing significantly the level of a key defined animal carcinogen present in tobacco products.

Comparative Analysis of Tobacco-Specific Nitrosamines and Total N-Nitroso Compounds in Moldovan Cigarette Tobacco

While previous studies have evaluated levels of tobacco-specific nitrosamines (TSNA) and total N-nitroso compounds (NOC) in tobacco, there are no reports in the literature on TSNA and total NOC in the same tobacco products. We compared levels of TSNA, total NOC, and NOC precursors (NOCP) in tobacco of cigarettes purchased in Moldova and in some tobacco types commonly used for the manufacturing of Moldovan cigarettes. Cigarette tobaccos included those from non-Moldovan, traditional Moldovan, and blended Moldovan cigarettes. The results demonstrate that tobacco of non-Moldovan cigarettes contains higher TSNA and NOC levels (mean, 16 and 63 nmol/g tobacco, n = 6) than that of Moldovan cigarettes (mean, 5 and 23 nmol/g tobacco, n = 25). TSNA and NOC levels were also generally higher in tobacco of blended than in traditional Moldovan cigarettes. NOCP levels in Moldovan and non-Moldovan cigarette tobacco were similar as follows: 29000 +/- 30000 and 33000 +/- 28000 nmol/g tobacco (mean +/- SD). Total NOC were strongly correlated with total TSNA levels (r = 0.66; P < 0.0001). These findings demonstrate that current technologies involved in the manufacture of some blended cigarettes create conditions that favor N-nitrosation of alkaloids and other tobacco constituents.