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Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific N -nitrosamines (TSNAs), such as N '-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.

Smoking constitutes a major global health problem. As it triggers various health hazards including cancers, cardiac and pulmonary illness, it is imperative to understand the mechanism of action of various smoke constituents on our cellular processes. Various in vitro studies have compiled the affinity of cigarette smoke constituents on various nicotinic acetylcholine receptors (nAChRs). But the nature of the intermolecular interactions contributing to this affinity and the key amino acids in the receptor active sites involved in this are not investigated so far. Here, we are examining the interaction of α7nAChR and α4β2nAChR on nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosornicotine (NNN), the physiologically significant constituents in smoke, through molecular docking and dynamics simulations study. The docking of α4β2nAChR structure with the ligands nicotine, NNK and NNN yielded docking scores of -41.45 kcal/mol, -59.28 kcal/mol and -54.60 kcal/mol, respectively, and that of α7nAChR receptor molecule with the ligands yielded docking scores of -59.54 kcal/mol, -71.06 kcal/mol and -70.86 kcal/mol, respectively. The study showed that NNK exhibited the highest affinity with the ligands which was confirmed by dynamics simulation. But higher stability of interactions as surmised from Molecular dynamics simulations was found for nicotine with α4β2nAChR and NNN with α7nAChR. The findings validate the in vitro studies comparing the affinities of these compounds. The study will be useful in formulating effective nAChR agonists to treat neurological disorders and antagonists for smoke deaddiction and improve health standards.Communicated by Ramaswamy H. Sarma.

Smokeless tobacco products expose adult and youth tobacco users to various addictive and carcinogenic constituents that can cause long-term nicotine dependence and oral cancers. In this study, nicotine, benzo[a]pyrene (B[a]P), N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acetaldehyde, crotonaldehyde, formaldehyde, moisture, and pH levels in 16 smokeless tobacco products were measured on a wet-weight basis (wwb). In addition, change in analytical variability with increasing replicate measurements was assessed. Total nicotine in the products varied from 6.2 to 35.5 mg/g. The percentage of total nicotine in the unprotonated form ranged from 0.1 to 62%; whereas, product moisture varied from 7.4 to 57%. The quantities of harmful and potentially harmful constituents (HPHCs) range from 0.46 to 179.9 ng/g for B [a]P, 270-12206 and 81-20716 ng/g for NNN and NNK, respectively, and 0.33-6.85 and 0.13-5.67 μg/g for acetaldehyde and formaldehyde, respectively. This study shows wide variation in smokeless tobacco product HPHC quantities. The results also show that analytical variability stabilizes after seven replicate measurements., (Published by Elsevier Inc.)

Smoking has been recognized by the World Health Organization (WHO) as the fifth highest threat to humanity. Smoking, a leading disease promoter, is a major risk factor for non-communicable diseases (NCDs) such as cancer, cardiovascular disease, diabetes, and chronic respiratory diseases. NCDs account for 63% of all deaths worldwide. Passive smoking is also a health risk. Globally, more than a third of all people are regularly exposed to harmful smoke. Air pollution is a common global problem in which pollutants emitted into the atmosphere undergo a series of physical or chemical reactions to produce various oxidation products, which are often referred to as secondary pollutants. Secondary pollutants include ozone (O 3 ), sulfur trioxide (SO 3 ), nitrogen dioxide (NO 2 ), and respirable particulate matter (PM). It is worth mentioning that third-hand smoke (THS), formed by the reaction of nicotine with second-hand smoke (SHS) caused by indoor O 3 or nitrous acid (HONO), is a major indoor secondary pollutant that cannot be ignored. As a form of indoor air pollution that is relatively difficult to avoid, THS exists in any corner of the environment where smokers live. In this paper, we summarize the important research progress on the main components, detection, and toxicity of THS and look forward to future research directions. Scientific understanding of THS and its hazards will facilitate smoking bans in indoor and public places and raise public concern for how to prevent and remove THS.

The tobacco-specific N -nitrosamines 4-( N -nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N '-nitrosonornicotine (NNN) always occur together and exclusively in tobacco products or in environments contaminated by tobacco smoke. They have been classified as "carcinogenic to humans" by the International Agency for Research on Cancer. In 1998, we published a review of the biochemistry, biology and carcinogenicity of tobacco-specific nitrosamines. Over the past 20 years, considerable progress has been made in our understanding of the mechanisms of metabolism and DNA adduct formation by these two important carcinogens, along with progress on their carcinogenicity and mutagenicity. In this review, we aim to provide an update on the carcinogenicity and mechanisms of the metabolism and DNA interactions of NNK and NNN.

Nitrate accumulation in tobacco ( Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies., Competing Interests: LB, PC, AH, SK, HL, JS, and SG were employed by PMI R&D, Philip Morris Products S.A., Neuchatel, Switzerland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bovet, Campanoni, Lu, Hilfiker, Kleinhans, Laparra, Schwaar, Lewis, Matsuba, Ma, Dewey and Goepfert.)

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 μg/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., Competing Interests: All authors were employees of Altria Client Services LLC or U.S. Smokeless Tobacco Company at the time the research was done, and those companies funded the work., (© 2020 The Authors. Published by Elsevier B.V.)

Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN.

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R 2  = 0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R 2  = 0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To 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)., Methods: We 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., Results: Smokers 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., Conclusions: Our 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: In addition to direct slide microscopy, traditional culture method (TCM) has long been considered as a gold standard method for the diagnosis of cutaneous leishmaniasis (CL). However, TCM is relatively expensive and time-consuming compared to the newly introduced microculture method (MCM), which has shown to be sensitive and rapid diagnostic method elsewhere for different Leishmania parasite species other than Leishmania (L.) aethiopica . The objective of this study was to evaluate the diagnostic performance of MCM for the diagnosis of CL caused by L. aethiopica., Methods: One hundred forty-three lesion aspirates were collected from 124 suspected CL patients prospectively based on their consecutive series. Portion of the aspirates were cultured in duplicate in TCM with modified Novy-MacNeal-Nicolle (NNN) in tissue culture flask and microcapillary tubes containing RPMI 1640 with 10% fetal bovine serum (FBS) for MCM. Smears on glass slides from the remaining portion of the aspirate were used for direct microscopy to detect the parasite after stained with Giemsa staining solution. Up on a consensus, positive result in any two of the three tests was used as a reference standard to analyze sensitivity., Results: As per consensus standard criteria, 52 of the lesions were qualified to evaluate MCM versus TCM. Forty-eight lesion samples were positive by MCM, 36 by TCM, and 37 by smear microscopy. The representative DNA from parasite culture isolates revealed the causative Leishmania parasite was L. aethiopica by ITS1 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Culturing L. aethiopica in vitro by MCM is more sensitive (92.3%) than by TCM (69.2%), P  = 0.003. The median time for L. aethiopica promastigotes emergence in the culture was 3 days for MCM and 6 days for TCM, P  < 0.001., Conclusions: Our finding indicated that MCM is a sensitive and a rapid culturing method for the isolation of L. aethiopica than TCM and smear microscopy., Competing Interests: This study was ethically approved by Scientific and ethical review Office of Ethiopian Public Health Institute (EI/SERO/SERC/45/2002) and AHRI/ALERT Ethics (PO07/12) committee. Written consent was obtained from all study participants.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

According to the World Health Organization, the consumption of tobacco products is the single largest cause of preventable deaths in the world, exceeding the total aggregated number of deaths caused by diseases such as AIDS, tuberculosis, and malaria. An important element in the evaluation of the health risks associated with the consumption of tobacco products is the assessment of the internal exposure to the tobacco constituents responsible for their addictive (e.g. nicotine) and carcinogenic (e.g. N-nitrosamines such as NNN and NNK) properties. However, the assessment of the serum levels of these compounds is often challenging from an analytical standpoint, in particular when limited sample volumes are available and low detection limits are required. Currently available analytical methods often rely on complex multi-step sample preparation procedures, which are prone to low analyte recoveries and ex-vivo contamination due to the ubiquitous nature of these compounds as background contaminants. In order to circumvent these problems, we report a facile and highly sensitive method for the simultaneous quantification of nicotine, cotinine, NNN, and NNK in serum samples. The method relies on a simple "one pot" liquid-liquid extraction procedure and isotope dilution ultra-high pressure (UPLC) hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry. The method requires only 10μL of serum and presents a limit of quantification of 0.02nmol (3000pg/mL) nicotine, 0.6pmol (100pg/mL) cotinine, 0.05pmol NNK (10pg/mL), and 0.06pmol NNN (10pg/mL), making it appropriate for pharmacokinetic evaluations., (Published by Elsevier B.V.)

Research conducted during past decades to reduce the level of the tobacco specific nitrosamine N-nitrosonornicotine (NNN) and its precursor nornicotine in tobacco yielded identification of three tobacco genes encoding for cytochrome P450 nicotine demethylases converting nicotine to nornicotine. We carried out trials to investigate the effect of using tobaccos containing three non-functional nicotine demethylase genes on the selective reduction of NNN in cigarette tobacco filler and mainstream smoke. Our results indicate that the presence of non-functional alleles of the three genes reduces the level of nornicotine and NNN in Burley tobacco by 70% compared to the level observed in currently available low converter (LC) Burley tobacco varieties. The new technology, named ZYVERT™, does not require a regular screening process, while a yearly selection process is needed to produce LC Burley tobacco seeds for NNN reduction. The reduction of NNN observed in smoke of blended prototype cigarettes is proportional to the inclusion level of tobacco having ZYVERT™ technology. Inclusion of Burley tobacco possessing the new trait into a typical American blend resulted in a selective reduction of NNN in cigarette smoke, while the levels of other Harmful and Potentially Harmful Constituents (HPHC) currently in the abbreviated list provided by the US Food and Drug Administration are statistically equivalent in comparison with the levels obtained in reference prototype cigarettes containing LC Burley., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The aim of this study was to investigate the effectiveness of an educational intervention on home nursing care plans based on NANDA, Nursing Interventions Classification, and Nursing Outcomes Classification for registered nurses working at primary healthcare settings in Greece., Methods: This is a quasi-experimental study without a control group. The sample consisted of 19 registered nurses. The study tool was a questionnaire administered pre- and post-educational intervention., Findings: The intervention improved their skills on nursing diagnoses' nomination, proper formulation, and individualization of defining characteristics, but it did not improve them in desired outcomes formulation., Conclusions: A significant effect of an educational intervention on nursing care plans was demonstrated., Implications for Nursing Practice: Nurses' knowledge and attitudes are important for understanding and integrating documentation within the nursing process., (© 2015 NANDA International, Inc.)

N'-Nitrosonornicotine (NNN) is considered to be one of the most carcinogenic compounds of the four conventionally measured tobacco-specific N-nitrosamines (TSNAs). In order to evaluate the significance of metabolic activation for the carcinogenic potential of NNN, its catalysis by different phase I enzymes and its interaction with nicotine and nicotine-derived TSNAs need to be investigated. Using an in vitro model system, NNN was found to interact with various cytochrome P450 enzymes, predominantly CYP2A13. Mass-spectrometric analysis confirmed the presence of various predicted NNN metabolites, including 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid) but little amount of 4-oxo-4-(3-pyridyl) butanal (OPB), which was somewhat different from in vitro NNK metabolism. Addition of nicotine, N'-Nitrosoanatabine (NAT), N'-Nitrosoanabasine (NAB) resulted in a competitive inhibition for NNN metabolism. The inhibition constant Ki value was calculated as 0.98 μM (nicotine), 1.37 μM (NAT), 0.71 μM (NAB) for HPB formation, 1.35 μM (nicotine), 1.35 μM (NAT), 1.01 μM (NAB) for hydroxy acid formation and 8.40 μM (nicotine), 3.40 μM (NAT), 3.04 μM (NAB) for OPB formation, respectively. These results implied that CYP2A13 is the most efficient enzyme to metabolize NNN in vitro and structurally similar tobacco constitutes including nicotine, NAT and NAB influence the metabolic activation of NNN, which may further interfere in its carcinogenicity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Smoke-free laws are expected to reduce smoking habits and exposure to secondhand smoke. The objective of this study was the measurement of tobacco specific carcinogens (TSNAs) in oral fluid to assess the most suitable biomarker of cancer risk associated with tobacco smoke., Methods: TSNAs, N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), as well as nicotine and cotinine were measured in oral fluid samples from 166 smokers and 532 non-smokers of the adult population of Barcelona, Spain. A simple method with an alkaline single liquid-liquid extraction with dichloromethane/isopropanol was used and lower limits of quantification for cotinine, NNN, NNK and NNAL were set at 0.10ng/mL, 1.0, 2.0 and 0.50pg/mL respectively. The NNN/cotinine ratio was also calculated., Results: NNN was the most abundant TSNA present in oral fluid with a significant difference between smokers and non-smokers (mean concentrations of 118 and 5.3pg/mL, respectively, p<0.001). NNK and NNAL were detectable in fewer samples. NNN and cotinine concentrations had a moderate correlation within both groups (Spearman's rank correlation coefficient of 0.312, p<0.001 in smokers and 0.279, p=0.022 in non-smokers). NNN/cotinine ratio was significantly higher (p<0.001) in non-smokers than in smokers, in line with equivalent findings for the NNAL/cotinine ratio in urine., Conclusions: TSNAs are detectable in oral fluid of smokers and non-smokers. NNN is the most abundant, in line with its association with esophageal and oral cavity cancers. The NNN/cotinine ratio confirms the relative NNN increase in second hand smoke. Findings provide a new oral fluid biomarker of cancer risk associated with exposure to tobacco smoke., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The evolution of the levels of tobacco-specific N-nitrosamines (TSNA), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mainstream (MS) cigarette smoke is investigated based on smoke and tobacco chemistry data of cigarette brands sold by Philip Morris International (PMI) between 2000 and 2014. A total of 315 cigarette samples representing a wide range of product and design characteristics manufactured by PMI between 2008 and 2014 were analyzed and compared to a previously published dataset of PMI brands manufactured in 2000. The data indicate that there is a substantial reduction of NNN and NNK levels in tobacco fillers and MS cigarette smoke per mg of tar and per mg of nicotine using Health Canada Intense (HCI) machine-smoking regime. This observed reduction in NNN and NNK levels in MS cigarette smoke is also supported by the downward trend observed on NNN and NNK levels in USA flue-cured Virginia and Burley tobacco lots from 2000 to 2014 crops, reflecting effectiveness of measures taken on curing and agricultural practices designed to minimize TSNA formation in tobacco., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

This study reports on variation in the yields of N-nitrosonornicotine (NNN) and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) from the mainstream smoke of cigarette brands and describes factors affecting this variation. Yields of NNN and NNK from smoke and tobacco filler, which is blended and cut tobacco filled in a cigarette tube, together with cigarette design parameters, were measured for 11 commercial cigarette brands, which were available in Japan from 2009 to 2013. The ranges of the relative standard deviations (RSDs) for NNN from the smoke of each brand were 5.0%-29.9% under International Organization for Standardization (ISO) and 6.3%-26.3% under Health Canada Intense (HCI) smoking regime. When these RSDs were compared with those of tar, nicotine, and carbon monoxide yields in each brand, they were found to be much higher for all of the brands under the HCI, and higher for most of the brands under the ISO smoking regime. In addition, the RSDs of NNN and NNK in smoke were mostly higher than those of KY3R4F which was manufactured in a single batch. It was identified that variation in NNN yields from tobacco filler mainly contribute to variation in NNN yields from smoke., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Regulation of the "neuronal" nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

The objective of this work was to characterize trends over time in urinary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) among cigarette smokers in the US. We identified 35 studies presenting data that either reported, or could be converted to, common units of total urinary NNAL excretion as pmol/mg creatinine. The studies spanned 18years, reported urinary NNAL excretion estimates for 61 defined populations, and included a combined total of 3941 study participants. Analyses show that urinary NNAL excretion trends downward with study publication year, and the trend is statistically significant. The trend does not appear to be accounted for by a reduction in cigarettes smoked per day by study participants over the same time period. This trend is consistent with reductions in tobacco specific nitrosamine (TSNA) levels in both cigarette tobacco filler and mainstream cigarette smoke observed over the past decade and with efforts by the tobacco industry and the agricultural community to reduce levels of TSNAs in tobacco and cigarette smoke., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

A nicotine part-filter method can be applied to estimate smokers' mouth level exposure (MLE) to smoke constituents. The objectives of this study were (1) to generate calibration curves for 47 smoke constituents, (2) to estimate MLE to selected smoke constituents using Japanese smokers of commercially available cigarettes covering a wide range of International Organization for Standardization tar yields (1-21mg/cigarette), and (3) to investigate relationships between MLE estimates and various machine-smoking yields. Five cigarette brands were machine-smoked under 7 different smoking regimes and smoke constituents and nicotine content in part-filters were measured. Calibration curves were then generated. Spent cigarette filters were collected from a target of 50 smokers for each of the 15 brands and a total of 780 filters were obtained. Nicotine content in part-filters was then measured and MLE to each smoke constituent was estimated. Strong correlations were identified between nicotine content in part-filters and 41 out of the 47 smoke constituent yields. Estimates of MLE to acetaldehyde, acrolein, 1,3-butadiene, benzene, benzo[a]pyrene, carbon monoxide, and tar showed significant negative correlations with corresponding constituent yields per mg nicotine under the Health Canada Intense smoking regime, whereas significant positive correlations were observed for N-nitrosonornicotine and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Alkaloids represent an extensive group of nitrogen-containing secondary metabolites that are widely distributed throughout the plant kingdom. The pyridine alkaloids of tobacco (Nicotiana tabacum L.) have been the subject of particularly intensive investigation, driven largely due to the widespread use of tobacco products by society and the role that nicotine (16) (see Fig. 1) plays as the primary compound responsible for making the consumption of these products both pleasurable and addictive. In a typical commercial tobacco plant, nicotine (16) comprises about 90% of the total alkaloid pool, with the alkaloids nornicotine (17) (a demethylated derivative of nicotine), anatabine (15) and anabasine (5) making up most of the remainder. Advances in molecular biology have led to the characterization of the majority of the genes encoding the enzymes directly responsible the biosynthesis of nicotine (16) and nornicotine (17), while notable gaps remain within the anatabine (15) and anabasine (5) biosynthetic pathways. Several of the genes involved in the transcriptional regulation and transport of nicotine (16) have also been elucidated. Investigations of the molecular genetics of tobacco alkaloids have not only provided plant biologists with insights into the mechanisms underlying the synthesis and accumulation of this important class of plant alkaloids, they have also yielded tools and strategies for modifying the tobacco alkaloid composition in a manner that can result in changing the levels of nicotine (16) within the leaf, or reducing the levels of a potent carcinogenic tobacco-specific nitrosamine (TSNA). This review summarizes recent advances in our understanding of the molecular genetics of alkaloid biosynthesis in tobacco, and discusses the potential for applying information accrued from these studies toward efforts designed to help mitigate some of the negative health consequences associated with the use of tobacco products., (Copyright © 2013 Elsevier Ltd. All rights reserved.)