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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.)

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.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To meet the requirement of environment protection, the adsorption of volatile nitrosamines, which are carcinogenic compounds in tobacco smoke, by zeolite is investigated in this article, along with the temperature programmed surface reaction of nitrosamines on zeolite to assess the catalytic capability of the porous material on the decomposition of the carcinogens. The actual function of zeolite to reduce the level of nitrosamines in cigarette smoke is also studied. The influences of zeolite pore's structure and the surface acid-basic properties of the adsorbent on the adsorption are explored in terms of geometric matching content and adsorbent-adsorbate interaction. Moreover, the role played by the cation of zeolite in the adsorption of volatile nitrosamines is discussed and the possible attracting range of the sodium cation in basic zeolite to pull the nitrosamine molecule is tentatively calculated. Owing to the specific ability of capturing nitrosamines, zeolite is proven to be the most effective additive encased within a cigarette as the functional additive for lowering the nitrosamines content of smoke to protect the environment and public health. Other porous oxides such as amorphous silica and alumina are also utilized in these experiments, and their actual functions to trap nitrosamines as well as their potential application as the candidates for cigarette additives are discussed and prospected, too. [ABSTRACT FROM AUTHOR]