Trends Over Time in Tobacco-Specific Nitrosamines (TSNAs) in Whole Tobacco and Smoke Emissions From Cigarettes Sold in Canada.
ABSTRACT: Introduction:Tobacco-specific nitrosamines (TSNAs) are potent carcinogens. Levels of TSNAs can be modified through manufacturing practices. In the 2000s, TSNA levels in cigarettes sold in Canada were reduced by changes in tobacco curing processes. The current study examined TSNA levels over the following decade to examine trends over time. Methods:Data submitted to Health Canada under the Tobacco Reporting Regulations were used to examine whole tobacco constituents for 1809 brands and mainstream smoke emissions for 191 brands manufactured by Canada's three leading cigarette companies from 2005 through 2011/12 using one-way analysis of variances (ANOVAs) and linear regression models. Results:Levels of N-nitrosoanatabine (NAT) (p < .001) and 4-(methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) (p < .001) in whole tobacco showed significant differences over time, decreasing between 2005 and 2007, and generally increasing from 2007 through 2012. Levels of all TSNAs in mainstream smoke emissions reflected a similar pattern: N-nitrosoanabasine (NAB) (p < .001), NAT (p < .001), NNK (p < .001), and N'-nitrosonornicotine (NNN) (p = .021). Linear regression analyses showed that TSNA levels varied by manufacturer over time in whole tobacco for NAT, NNK, and NNN (p < .001 for all), and in smoke emissions for NAB, NAT, NNK, and NNN (p < .001 for all). Conclusions:The findings indicate that levels of TSNAs in whole tobacco and smoke emissions of cigarettes sold in Canada increased from 2007 through 2011/12, following initial reductions over the previous 2 years. Differences in TSNA levels between companies raise questions about manufacturing practices that may be responsible for these changes. Although increased levels of carcinogenic TSNAs may be alarming, it remains unclear whether these differences translate into differences in health risk. Implications:The wide variation of TSNAs within the Canadian market across time and across cigarette companies demonstrates the feasibility of reducing the levels of these potent carcinogens. Although it is unclear whether changes made to levels of TSNAs will result in less tobacco-related disease, the tobacco industry bears a responsibility to minimize the harm from smoking to the fullest extent possible.
Project description: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.
Project description:Burley tobaccos (Nicotiana tabacum) display a nitrogen-use-deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco-specific nitrosamines (TSNAs). Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen-assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field-grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well-documented animal carcinogens found in tobacco products.
Project description:Our recent studies on tobacco smoke carcinogen and toxicant biomarkers and cancer risk among male smokers in the Shanghai Cohort Study showed that exposure to tobacco-specific nitrosamines (TSNA) and polycyclic aromatic hydrocarbons (PAH) is prospectively associated with the risk of cancer. These findings support the hypothesis that the smokers' cancer risk is a function of the dose of select tobacco carcinogens and highlight the importance of understanding the factors that affect the intake of these carcinogens by smokers. Given that tobacco constituent exposures are driven, at least in part, by the levels of these constituents in cigarette smoke, we measured mainstream smoke TSNA and PAH levels in 43 Chinese cigarette brands that participants of the Shanghai Cohort Study reported to smoke. In all brands analyzed here, mainstream smoke levels of NNN and NNK, the two carcinogenic TSNA, were generally relatively low, averaging (±SD) 16.8(±25.1) and 14.2(±9.5) ng/cigarette, respectively. The levels of PAH were comparable to those found in U.S. cigarettes, averaging 15(±9) ng/cigarette for benzo[a]pyrene, 119(±66) ng/cigarette for phenanthrene and 37(±19) ng/cigarette for pyrene. Our findings indicate that the generally low levels of NNN and NNK are most likely responsible for the relatively low levels of the corresponding biomarkers in the urine of the Shanghai Cohort Study participants as compared to those found in the U.S. smokers, supporting the role of the levels of these constituents in cigarette smoke in smokers' exposures. Our findings also suggest that, in addition to smoking, other sources contribute to Chinese smokers' exposure to PAH.
Project description:Bangladesh exhibits the second highest rate of smokeless tobacco (SLT) product usage in the world, and this has been associated with the high upper aerodigestive tract cancer incidence in this country. The goal of the present study was to examine the levels of the highly carcinogenic tobacco-specific nitrosamines (TSNAs) in Bangladeshi SLT products and compare these levels to that observed in SLT brands from southeast Asia and the USA. The levels of TSNAs and nicotine were determined by LC-MS/MS in twenty-eight SLT brands and several tobacco additives from Bangladesh, as well as several SLT brands from India, Pakistan and the USA. The levels of N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB) in Bangladeshi SLT brands ranged from 1.1-59, 0.15-34, 0.79-45, and 0.037-13 ?g/g SLT powder, respectively. The mean levels of the highly carcinogenic TSNAs (NNN+NNK) were 7.4-, 2.4-, and 63-fold higher in Bangladeshi SLT products as compared to SLT brands from the USA, India and Pakistan, respectively; these trends were also observed for NAT and NAB. Similar mean levels of nicotine were observed in the Bangladeshi brands (31 mg/g powder) versus brands from the USA (25 mg/g powder) and India (20 mg/g powder); they were 3-fold higher than brands from Pakistan (10 mg/g powder). Gul SLT brands exhibited the highest pH and the highest levels of unprotonated nicotine. The high levels of TSNAs in Bangladeshi SLT brands may be an important factor contributing to the high rates of upper aerodigestive tract cancer in Bangladesh.
Project description:Tobacco-specific nitrosamines (TSNAs) have been of concern to the public health community for decades and their reduction through agricultural practices, plant breeding, and tobacco processing has also been a decades-long industry effort. Despite those efforts, TSNAs, though lower, continue to be constituents of concern in tobacco products. This paper examines the TSNA levels of dark air-cured, dark fire-cured, and burley tobaccos purchased in the United States by U.S. Smokeless Tobacco Company LLC (USSTC) and of nine finished USSTC moist smokeless tobacco products. TSNA values of the incoming purchased tobaccos and the finished products showed considerable variability. For the incoming tobaccos, the coefficient of variation was generally more than 100 % for each tobacco type and for each of the measured TSNAs. The relative TSNA variability of the finished tobacco products was also considerable, averaging approximately 25 %. It was also found that the measured values for the finished products averaged well above the proposed FDA NNN proposed product standard of 1.0 ?g/g dry weight. Because of the large variability in NNN values, products would have to average well below FDA's proposed product standard to be consistently compliant.
Project description:BACKGROUND:Nicotine is a stimulant and potent parasympathomimetic alkaloid that accounts for 96-98% of alkaloid content. A reduction in the amount of nicotine in cigarettes to achieve a non-addictive level is necessary. We investigated whether replacing tobacco root with eggplant by grafting can restrict nicotine biosynthesis and produce tobacco leaves with ultra-low nicotine content, and analyzed the gene expression differences induced by eggplant grafting. RESULTS:The nicotine levels of grafted tobacco leaves decreased dramatically. The contents of nornicotine, anabasine, NNN, NNK, NAT, total TSNAs and the nicotine of mainstream cigarette smoke decreased, and the contents of amino acids and the precursors of alkaloids increased in grafted tobacco. Eggplant grafting resulted in the differential expression of 440 genes. LOC107774053 had higher degrees in two PPI networks, which were regulated by LOC107802531 and LOC107828746 in the TF-target network. CONCLUSIONS:Replacing tobacco root with eggplant by grafting can restrict nicotine biosynthesis and produce tobacco leaves with ultra-low or zero nicotine content. The differential expression of LOC107774053 may be associated with eggplant grafting.
Project description:The association of tobacco smoke with decreased cell motility and wound healing is well documented; however, the cellular mechanisms and specific toxic tobacco constituents responsible for this effect are not well understood. Tobacco-specific N-nitrosamines (TSNAs) are among the most important classes of carcinogens found in tobacco products. The TSNA N'-nitrosonornicotine (NNN) is present at relatively high levels in tobacco and its smoke, as well as second- and third-hand smoke. To investigate the cellular pathways that are perturbed upon NNN exposure, we employed a quantitative proteomic approach, utilizing stable isotope labeling by amino acids in cell culture and mass spectrometry, to assess the NNN-induced alteration of protein expression in GM00637 human skin fibroblast cells. With this approach, we were able to quantify 2599 proteins, 191 of which displayed significantly changed expression following NNN exposure. One of the main findings from our proteomic analysis was the down-regulation of six different subunits of myosin, particularly nonmuscle myosin II heavy chain, isoforms A, B, and C. In addition, we found the altered expression of several extracellular matrix proteins and proteins involved in cellular adhesion. Together, our quantitative proteomic results suggested that NNN exposure may interfere with fibroblast motility. An in vitro scratch wound assay result supported that NNN exposure reduced the ability of dermal fibroblast to migrate into the scratched area. The results from the present study offer novel insights into the cellular mechanisms of NNN toxicity and identify NNN as a specific tobacco constituent that contributes to decreased fibroblast migration.
Project description:The content of tobacco-specific nitrosamines (TSNAs) possessing carcinogenic properties has been an important area of research since replacement liquids were introduced for e-cigarettes. A method for determining N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT), and N'-nitrosoanabasine (NAB) in replacement liquids for electronic cigarettes was developed using liquid chromatography-tandem mass spectrometry with electrospray ionisation (HPLC-ESI-MS/MS) in the multiple reaction monitoring mode. The sample preparation of replacement liquids was accomplished via the ultrasound-assisted solvent extraction of a porous membrane packed sample. The sample preparation proved to be successful in extracting the analytes, with recoveries from 87% to 105%, with coefficients of variation < 4.9%. Moreover, the linearity and limits of detection and quantitation (LOD, LOQ), together with repeatability and accuracy, were determined for the developed method. The proposed sample preparation and developed chromatographic method were successfully applied to the determination of TSNAs in 9 replacement liquid samples. The NNK and NNN were found to be most frequently detected (89 and 67%, respectively), with concentration ranges from 1.2-54.3 ng/mL and 4.1-30.2 ng/mL, respectively, while NAT was detected with frequency of 22% with range 1.7-2.5 ng/mL and NAB were found to be below the LOD in all samples.
Project description:This study shows that residual nicotine from tobacco smoke sorbed to indoor surfaces reacts with ambient nitrous acid (HONO) to form carcinogenic tobacco-specific nitrosamines (TSNAs). Substantial levels of TSNAs were measured on surfaces inside a smoker's vehicle. Laboratory experiments using cellulose as a model indoor material yielded a > 10-fold increase of surface-bound TSNAs when sorbed secondhand smoke was exposed to 60 ppbv HONO for 3 hours. In both cases we identified 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal, a TSNA absent in freshly emitted tobacco smoke, as the major product. The potent carcinogens 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone and N-nitroso nornicotine were also detected. Time-course measurements revealed fast TSNA formation, with up to 0.4% conversion of nicotine. Given the rapid sorption and persistence of high levels of nicotine on indoor surfaces-including clothing and human skin-this recently identified process represents an unappreciated health hazard through dermal exposure, dust inhalation, and ingestion. These findings raise concerns about exposures to the tobacco smoke residue that has been recently dubbed "thirdhand smoke." Our work highlights the importance of reactions at indoor interfaces, particularly those involving amines and NO(x)/HONO cycling, with potential health impacts.
Project description:4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic of the four tobacco-specific nitrosamines (TSNAs) and it needs to be metabolically activated to exert its carcinogenic effect on humans. For the simultaneous intake of NNK and other compounds with similar molecular structures in the context of tobacco smoke, whether (R,S)-N-nitrosoanatabine (NAT), (R,S)-N-nitrosoanabasine (NAB) and nicotine contribute to the inhibitory potency of the cytochrome P450 (CYP) enzyme-catalyzed NNK metabolism or not needs to be investigated. In the in vitro study, 4-oxo-4-(3-pyridyl) butanal (OPB), 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-oxo-4-(3-pyridyl) butanoic acid (OPBA) were established as the products of the CYP2A13-catalyzed NNK metabolism and the kinetic parameters were calculated from the Michaelis-Menten equation. Addition of NAT, NAB or nicotine resulted in a competitive inhibition for the NNK metabolism catalyzed by CYP2A13. The inhibition constant Ki values were calculated to be 0.21 ?M (NAT), 0.23 ?M (NAB) and 8.51 ?M (nicotine) for OPB formation; 0.71 ?M (NAT), 0.87 ?M (NAB) and 25.01 ?M (nicotine) for HPB formation and 0.36 ?M (NAT), 0.50 ?M (NAB) and 6.57 ?M (nicotine) for OPBA formation, respectively. In addition, the study of the transformation of the three metabolites revealed OPB was not only an end product but also an intermediate product of the CYP2A13-catalyzed NNK metabolism. These results suggest that structurally similar tobacco constituents with weak or no carcinogenicity influence the metabolic activation of NNK, which interferes with its carcinogenicity to some extent.