Tobacco-specific Nitrosamines Concentrations 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.

Direct analysis of tobacco specific nitrosamines in tobacco products using a molecularly imprinted polymer-packed column.

Tobacco specific nitrosamines (TSNAs) are highly carcinogenic by-products in tobacco samples, and their presence is regulated by the Food and Drug Administration. Molecularly imprinted polymers (MIPs) are synthetic polymers that have been "imprinted" with a template analyte in a co-polymer system, and can selectively extract analytes from complex matrices. MIPs can be incorporated into online systems, replacing traditional high performance liquid chromatography (HPLC) columns. MIP material specific for TSNAs was packed into an empty HPLC column using a slurry packing technique. The developed method with the MIP-packed HPLC column was validated on a LC-MS/MS system for the quantitation of N-nitrosonornicotine (NNN) and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in commercial tobacco products. The method was linear over .1-10 ng/ml (.4-10 μg/g) for NNN and NNK. The limit of detection (LOD) was .03 ng/ml (12 μg/g) and the limit of quantitation (LOQ), .1 ng/ml (.4 μg/g). All column uniformity parameters with the exception of theoretical plate number were within the accepted criteria (% RSD values <15%). Theoretical plate number was <250, owing to the large (50 μm) sized MIP particles. Twenty-six tobacco products contained TSNA concentrations that were consistent with reported literature values. The TSNA-MIP based HPLC column effectively replaced a traditional reverse phase HPLC column, and was used for the direct analysis of nicotine and tobacco products without extensive sample preparation prior to instrumental analysis.

Thirdhand Exposures to Tobacco-Specific Nitrosamines through Inhalation, Dust Ingestion, Dermal Uptake, and Epidermal Chemistry.

Tobacco-specific nitrosamines (TSNAs) are emitted during smoking and form indoors by nitrosation of nicotine. Two of them, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are human carcinogens with No Significant Risk Levels (NSRLs) of 500 and 14 ng day-1, respectively. Another TSNA, 4-(methylnitrosamino)-4-(3-pyridyl) butanal (NNA), shows genotoxic and mutagenic activity in vitro. Here, we present additional evidence of genotoxicity of NNA, an assessment of TSNA dermal uptake, and predicted exposure risks through different pathways. Dermal uptake was investigated by evaluating the penetration of NNK and nicotine through mice skin. Comparable mouse urine metabolite profiles suggested that both compounds were absorbed and metabolized via similar mechanisms. We then investigated the effects of skin constituents on the reaction of adsorbed nicotine with nitrous acid (epidermal chemistry). Higher TSNA concentrations were formed on cellulose and cotton substrates that were precoated with human skin oils and sweat compared to clean substrates. These results were combined with reported air, dust, and surface concentrations to assess NNK intake. Five different exposure pathways exceeded the NSRL under realistic scenarios, including inhalation, dust ingestion, direct dermal contact, gas-to-skin deposition, and epidermal nitrosation of nicotine. These results illustrate potential long-term health risks for nonsmokers in homes contaminated with thirdhand tobacco smoke.

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.

Complex estimation system for smokeless nicotine containing products

Smokeless nicotine containing products are very diverse and offered in flavored tobacco/non tobacco forms, packed in portioned sachets of different mass, dimensions, and colors, with different nicotine content. In RF of chewing tobacco is regulated by Federal law № 268, sucking tobacco (snus) is prohibited for wholesale and retail trade according to FL № 456. Federal law № 303 equates consuming of non tobacco nicotine containing products for chewing and sucking to tobacco consumption. Absence of regulations for nicotine containing products with similar package, but different in composition and physiological effect requires elaboration methods for complex estimation of smokeless products with methods verification in order to their control. Monitoring of studies conducted in various scientific centers on the issue of toxicological assessment of smokeless tobacco / non-tobacco nicotine-containing products has been carried Analysis of consumer’s indicators of smokeless products includes organoleptic testing and measuring nicotine content as toxicity indicator. It is discovered that nicotine content in studied samples varies from 0.3 to 7.6 %. Quantity of absorbed nicotine during cigarette smoking is criterion of sufficient nicotine concentration in smokeless products, extracted through oral mucosa. Base (carrier) for non tobacco products is usually powdered microcrystal cellulose or grinded plant matter (peppermint) of different flavor and color. Structure of smokeless products is defined and their identification features are offered, including extra feature – tobacco presence in product, defined by presence of tobacco specific nitrosamines. As the result of the researches adoption of nicotine content limit of 20 – 30 mg per portion is offered. Complex estimation system for smokeless products is elaborated. It includes: organoleptic testing of consumer’s properties, spectrophotometric method for nicotine content measuring, thermogravimetric determination of the mass fraction of moisture, optical microscopy and luminiscence analysis for defining product structure in order to its identification, gas chromatography for defining humectants and LC-MS for measuring content of tobacco specific nitrosaminescontent.

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.

Chemical analysis of snus products from the United States and northern Europe.

IntroductionSnus is an oral tobacco product that originated in Sweden. Snus products are available as fine-cut loose tobacco or in pre-portioned porous “pouches.” Some snus products undergo tobacco pasteurization during manufacturing, a process that removes or reduces nitrite-forming microbes, resulting in less tobacco-specific nitrosamine content in the product. Some tobacco companies and researchers have suggested that snus is potentially less harmful than traditional tobacco and thus a potential smoking cessation aid or an alternative to continued cigarette consumption. Although snus is available in various countries, limited information exists on snus variants from different manufacturers.MethodsMoisture, pH, nicotine, and tobacco-specific N’-nitrosamines (TSNAs) were quantified in 64 snus products made by 10 manufacturers in the United States and Northern Europe (NE). Reported means, standard errors, and differences are least-square (LS) estimates from bootstrapped mixed effects models, which accounted for correlation among repeated measurements. Minor alkaloids and select flavors were also measured.ResultsAmong all product types, moisture (27.4%–59.5%), pH (pH 5.87–9.10), total nicotine (6.81–20.6 mg/g, wet), unprotonated nicotine (0.083–15.7 mg/g), and total TSNAs (390–4,910 ng/g) varied widely. The LS-mean unprotonated nicotine concentration of NE portion (7.72 mg/g, SE = 0.963) and NE loose (5.06 mg/g, SE = 1.26) snus were each significantly higher than US portion snus (1.00 mg/g, SE = 1.56). Concentrations of minor alkaloids varied most among products with the highest total nicotine levels. The LS-mean NNN+NNK were higher in snus sold in the US (1360 ng/g, SE = 207) than in NE (836 ng/g, SE = 132) countries. The most abundant flavor compounds detected were pulegone, eucalyptol, and menthol.ConclusionPhysical and chemical characteristics of US and NE products labeled as snus can vary considerably and should not be considered “equivalent”. Our findings could inform public health and policy decisions pertaining to snus exposure and potential adverse health effects associated with snus.

Comparison of TSNAs concentration in saliva according to type of tobacco smoked

ObjectiveTo compare tobacco-specific nitrosamines (TSNAs) measured in saliva according to different types of tobacco smoked in a sample of smokers of the city of Barcelona (Spain). MethodsWe used data from a cross-sectional study of a sample of the adult smoking population of Barcelona, Spain in 2013–2014 (n = 165). We classified smokers in five groups according to the type of tobacco smoked: a) manufactured cigarettes only, b) roll-your-own (RYO) cigarettes only, c) dual smokers (both manufactured and RYO cigarettes), d) manufactured plus other types of tobacco products different from RYO and e) other types of tobacco products different from manufactured and RYO cigarettes. We calculated the geometric mean (GM) and geometric standard deviation (GSD) of TSNAs concentration in saliva (pg/mL), including N’-nitroaonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) according to the five tobacco groups. We also described all TSNAs concentration in each tobacco group stratified by the number of cigarettes smoked per day. ResultsSmokers from the RYO cigarette group had higher TSNAs concentration than smokers from the manufactured cigarette group: 13 pg/mL vs 4.9 pg/mL of NNN, 1.9 pg/mL vs 1.7 pg/mL in NNK and 1.1 pg/mL vs 0.9 pg/mL of NNAL. There were significant differences in NNN concentrations between smokers of RYO vs manufactured cigarettes. The higher the number of cigarettes smoked, the higher the TSNAs concentrations. After adjusted by number of cigarettes smoked, there were not statistically significant differences in TSNAs between RYO and manufactured cigarettes. ConclusionsOur data shows that RYO cigarette is at least as hazardous as manufactured cigarettes. Regulating RYO tobacco prices could be an effective strategy to control tobacco use.

Temporal Variations in Cigarette Tobacco Bacterial Community Composition and Tobacco-Specific Nitrosamine Content Are Influenced by Brand and Storage Conditions.

Tobacco products, specifically cigarettes, are home to microbial ecosystems that may play an important role in the generation of carcinogenic tobacco-specific nitrosamines (TSNAs), as well as the onset of multiple adverse human health effects associated with the use of these products. Therefore, we conducted time-series experiments with five commercially available brands of cigarettes that were either commercially mentholated, custom-mentholated, user-mentholated, or non-mentholated. To mimic user storage conditions, the cigarettes were incubated for 14 days under three different temperatures and relative humidities (i.e., pocket, refrigerator, and room). Overall, 360 samples were collected over the course of 2 weeks and total DNA was extracted, PCR amplified for the V3V4 hypervariable region of the 16S rRNA gene and sequenced using Illumina MiSeq. A subset of samples (n = 32) was also analyzed via liquid chromatography with tandem mass spectrometry for two TSNAs: N’-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Comparative analyses of the five tobacco brands revealed bacterial communities dominated by Pseudomonas, Pantoea, and Bacillus, with Pseudomonas relatively stable in abundance regardless of storage condition. In addition, core bacterial operational taxonomic units (OTUs) were identified in all samples and included Bacillus pumilus, Rhizobium sp., Sphingomonas sp., unknown Enterobacteriaceae, Pantoea sp., Pseudomonas sp., Pseudomonas oryzihabitans, and P. putida. Additional OTUs were identified that significantly changed in relative abundance between day 0 and day 14, influenced by brand and storage condition. In addition, small but statistically significant increases in NNN levels were observed in user- and commercially mentholated brands between day 0 and day 14 at pocket conditions. These data suggest that manufacturing and user manipulations, such as mentholation and storage conditions, may directly impact the microbiome of cigarette tobacco as well as the levels of carcinogens.

Analysis of Variability in Curing Conditions and Tobacco-Specific Nitrosamines Within Barns of Dark Air-Cured Tobacco

Significant variability in cured-leaf tobacco-specific nitrosamine (TSNA) content is commonly observed when sampling within dark air-curing barns. This variability may be due to inconsistency in the curing environment within different areas of the barn. A study was initiated in 2012, through support from a CORESTA Study Grant, to evaluate if cured-leaf TSNA content is related to microenvironmental conditions in the barn. Low-converter (TRsc) and high-converter (TRHC) selections of TR Madole dark tobacco were air cured in barns near Princeton and Lexington, KY. Temperature and relative humidity were measured with data loggers placed at 27 different locations within each barn for the duration of curing. There were no significant effects of individual data logger placement in either variety selection on hours above 24°C temperature, hours above 80% relative humidity, or TSNA; therefore, we investigated these data within the 3-dimensional aspects of tier, room, and bent within each barn. There were various effects of tier, room, and bent on temperature, relative humidity, and TSNA. Temperature data followed an understandable pattern across tiers in the barn within each year and location; however, relative humidity and TSNA were more difficult to characterize adequately. There was a significant relationship between hours above 24°C and TSNA, but not hours above 80% relative humidity. This study has shown that the effect of within-barn position on TSNA cannot be easily predicted.

Tobacco, Microbes, and Carcinogens: Correlation Between Tobacco Cure Conditions, Tobacco-Specific Nitrosamine Content, and Cured Leaf Microbial Community.

Tobacco-specific nitrosamines are carcinogenic N-nitrosamine compounds present at very low levels in freshly harvested tobacco leaves that accumulate during leaf curing. Formation of N-nitrosamine compounds is associated with high nitrate levels in the leaf at harvest, and nitrate is presumed to be the source from which the N-nitrosation species originates. More specifically, nitrite is considered to be a direct precursor, and nitrite is linked with N-nitrosation in many environmental matrices where it occurs via microbial nitrate reduction. Here, we initiate work exploring the role of leaf microbial communities in formation of tobacco-specific nitrosamines. Leaves from burley tobacco line TN90H were air cured under various temperature and relative humidity levels, and 22 cured tobacco samples were analyzed for their microbial communities and leaf chemistry. Analysis of nitrate, nitrite, and total tobacco-specific nitrosamine levels revealed a strong positive correlation between the three variables, as well as a strong positive correlation with increasing relative humidity during cure conditions. 16S rRNA gene amplicon sequencing was used to assess microbial communities in each of the samples. In most samples, Proteobacteria predominated at the phylum level, accounting for >90% of the OTUs. However, a distinct shift was noted among members of the high tobacco-specific nitrosamine group, with increases in Firmicutes and Actinobacteria. Several OTUs were identified that correlate strongly (positive and negative) with tobacco-specific nitrosamine content. Copy number of bacterial nitrate reductase genes, obtained using quantitative PCR, did not correlate strongly with tobacco-specific nitrosamine content. Incomplete denitrification is potentially implicated in tobacco-specific nitrosamine levels.

Evidence supporting product standards for carcinogens in smokeless tobacco products.

Smokeless tobacco products sold in the United States vary significantly in yields of nicotine and tobacco-specific nitrosamines (TSNA). With the passage of the Family Smoking Prevention and Tobacco Control Act, the Food and Drug Administration now has the authority to establish product standards. However, limited data exist determining the relative roles of pattern of smokeless tobacco use versus constituent levels in the smokeless tobacco product in exposure of users to carcinogens. In this study, smokeless tobacco users of brands varying in nicotine and TSNA content were recruited from three different regions in the U.S. Participants underwent two assessment sessions. During these sessions, demographic and smokeless tobacco use history information along with urine samples to assess biomarkers of exposure and effect were collected. During the time between data collection, smokeless tobacco users recorded the amount and duration of smokeless tobacco use on a daily basis using their diary cards. Results showed that independent of pattern of smokeless tobacco use and nicotine yields, levels of TSNA in smokeless tobacco products played a significant role in carcinogen exposure levels. Product standards for reducing levels of TSNA in smokeless tobacco products are necessary to decrease exposure to these toxicants and potentially to reduce risk for cancer.

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.

Trapping tobacco specific N-nitrosamines in Chinese-Virginia type tobacco extracting solution by porous material

Liquid adsorption of tobacco specific nitrosamines (TSNA) in the tobacco extracting solution by porous materials is reported, with the monitoring of standard LC–MS/MS technique, in order to reduce the environmental cancerogens in smoke at the source. Three types of adsorbent, zeolite, mesoporous silica and activated carbon are utilized to trap nitrosamines in the extracting solution of Chinese-Virginia type tobacco with low TSNA content, and their performance was measured by spectrophotometric and LC–MS/MS methods. Both measurements gave the same variation tendency on the nitrosamines analysis, enabling the photometric method to be the simple and feasible tool in preliminary tests. The influence of pore size and cations of zeolite along with the organic modification of mesoporous silica with chloroisopropyl triethoxysilane were carefully investigated, and the optimal liquid conditions were determined, in which CaA zeolite and modified mesoporous silica CAM48-10 sample can capture 34 and 46 % of TSNA in the tobacco extracting solution, offering the efficient candidate for controlling the carcinogen like nitrosamines in environment.

A comprehensive evaluation of the toxicology of experimental, non-filtered cigarettes manufactured with different circumferences

Context: Historical work indicates that cigarette circumference may affect the toxicological profile of experimental cigarettes.Objective: Studies were conducted to examine the effect of different cigarette circumferences on (1) selected mainstream smoke constituents including concentrations of tobacco specific nitrosamines (TSNA) in smoke and (2) mutagenicity and cytotoxicity of cigarette smoke condensate.Materials and methods: Analytical chemistry, Salmonella mutagenicity and cytotoxicity assays were used to evaluate the composition and biological activity of mainstream smoke from experimental, non-filtered cigarettes manufactured with four different circumferences (17.0–27.1 mm).Results: Most smoke constituents, including TSNA, decreased with decreasing cigarette circumference; however, amounts of hydrogen cyanide increased in a non-circumference dependent manner. Mutagenicity and cytotoxicity also decreased slightly with decreasing cigarette circumference.Conclusion: Cigarette circumference may have a minor role in the toxicological profile of experimental cigarettes, with a so-far-unidentified mechanism.

Changes in TSNA Contents during Tobacco Storage and the Effect of Temperature and Nitrate Level on TSNA Formation

Samples of burley, sun-cured, and flue-cured tobacco from the main producing areas of relevant tobacco types in China were collected to study the changes in tobacco-specific nitrosamine (TSNA) contents during storage and to investigate the effect of storage temperature and tobacco nitrate level on TSNA formation of cured tobacco. Contents of TSNAs in burley and sun-cured tobacco increased substantially during 1 year under natural storage environment, with total TSNA content increasing about 215% for both tobacco types. The most rapid increase occurred during the high temperature season. Temperature had a significant promoting effect on TSNA formation during storage. Storage temperature as high as 27 °C for 12 days was enough to induce the increase of TSNA formation, while the most significant effect was shown when the temperature was above 30 °C. The increased rate of accumulation became greater as the temperature increased. Total TSNA content in air-cured burley tobacco after the treatment of 60 °C for 24 days was 772% higher than that in the low temperature control. Different types of tobacco showed different results in terms of the response of TSNA formation to high temperature. TSNA formation in flue-cured tobacco did not increase after high-temperature treatment for 36 days, while burley and sun-cured tobacco saw a dramatic increase of TSNA content. This difference could be explained by the fact that burley tobacco and sun-cured tobacco usually had more than 10 times the nitrate content than flue-cured tobacco. As the nitrate nitrogen increased in cured burley tobacco, TSNA formation during leaf storage at high temperature significantly increased. Addition of nitrate onto flue-cured tobacco to the level equivalent to burley tobacco followed by high-temperature treatment increased the TSNA concentration comparable to burley tobacco. The interaction between high temperature and abundant nitrate content in cured tobacco could be responsible for TSNA formation during storage.

Application of exogenous substances reduces tobacco-specific nitrosamines content by regulating biosynthesis of nicotine and nitrite in burley tobacco

Tobacco-specific nitrosamines (TSNAs) are carcinogenic chemicals found in tobacco plants. The increasing health consciousness of individuals had led to an increased interest in research on reducing TSNAs content. The aim of this study was to use a pot experiment in which exogenous substances were applied to burley tobacco to dissect the mechanism of TSNAs production. The results indicated that spraying the exogenous substances IAA, NAA, SA and combination thereof on burley tobacco after topping decreased TSNAs content by 2.69–29.4 % in upper leaves and 0.23–39.3 % in middle leaves without affecting total sugar, total nitrogen, potassium and chlorine contents. The application of exogenous substances could down-regulate expression of the NR gene and the activity of the NR enzyme, resulting in less accumulation of the TSNAs precursor nitrite. The exogenous substances significantly reduced nicotine accumulation, which was consistent with low enzyme activities and the down-regulated expressions of genes involved in nicotine biosynthesis, especially significant in the case of quinolinate phosphoribosyltransferase. These results suggested that the application of exogenous substances on burley tobacco after topping could reduce TSNAs content which may be attributed to the regulation of exogenous substances on nitrite and nicotine. This also implies one potential improvement to agronomic practices aimed at controlling the accumulation of TSNAs in burley tobacco.

Determination of tobacco-specific nitrosamines in replacement liquids of electronic cigarettes by liquid chromatography–tandem mass spectrometry

A liquid chromatography–tandem mass spectrometric method was described to detect tobacco-specific nitrosamines (TSNAs) in replacement liquids of electronic cigarettes. Solid-phase extraction (SPE) and liquid–liquid extraction (LLE) were compared to each other to select the optimum clean-up method. Under the established condition, the limits of quantification of N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanabasine (NAB), and N′-nitrosoanatabine (NAT) were 0.06, 0.07, 0.06, and 0.04μg/L respectively, by using 0.5mL of replacement liquids, respectively, and the relative standard deviation was less than 10% at concentrations of 5.0 and 25.0μg/L. The concentrations of TSNAs were measured in concentration ranges of 0.34–60.08μg/L (64.8% detection frequency) for NNN, 0.22–9.84μg/L (88.6% detection frequency) for NNK, 0.11–11.11μg/L (54.3% detection frequency) for NNB, and 0.09–62.19μg/L (75.2% detection frequency) for NAT in 105 replacement liquid brands from 11 electronic cigarette companies purchased in the Korean market.

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.