Tobacco-specific Nitrosamines Research Articles

Passive exposure to electronic cigarette aerosol in pregnancy: A case study of a family

BackgroundPassive exposure to the aerosols of electronic cigarettes (e-cigarettes) has been little studied. We assessed this exposure in late pregnancy in a woman and her 3-year-old child, exposed through e-cigarette use by another household member. MethodsThis prospective longitudinal case study involved a family unit consisting of an e-cigarette user, a pregnant woman who delivered an infant during the study, and the couple's older 3-year-old son. At 31, 36, and 40 weeks of the pregnancy, we measured biomarkers (nicotine metabolites, tobacco-specific nitrosamines, propanediols, glycerol, and metals) in the urine and hair of all three participants and in the saliva of the adults, in cord blood at delivery, and in the breast milk at the postpartum period. ResultsSamples from the e-cigarette user showed quantifiable concentrations of all analytes assessed (maximum urinary cotinine concentration, 4.9 ng/mL). Among samples taken from the mother, nicotine and its metabolites were found mainly in urine and also in saliva and hair, but not in cord blood. During the postpartum period, we found cotinine concentrations of 2.2 ng/mL in the mother's urine and 0.22 ng/mL in breast milk; 1,2-propanediol was generally detected in urine and saliva, but not in cord blood or breast milk. The maximum urinary cotinine concentration in the 3-year-old child was 2.6 ng/mL and propanediols also were detected in his urine. Nitrosamines were not detected in samples taken from the mother or the 3-year-old. Metals found in the refill liquid were detected at low levels in both the mother and the 3-year-old. ConclusionsWe detected low but not negligible concentrations of e-cigarette–related analytes (including cord blood and breast milk) in an exposed pregnant non-user and in a 3-year-old child also living in the home. Passive exposure to e-cigarette aerosols cannot be disregarded and should be assessed in larger observational studies.

Accuracy improvement of determination of seven minor tobacco alkaloids in mainstream cigarette smoke using analyte protectants by gas chromatography-mass spectrometry

Tobacco alkaloids are important precursors of carcinogenic tobacco-specific nitrosamines. Therefore, accurate quantification of tobacco alkaloids is highly important. This study investigates the compensation effects of novel analyte protectants (APs) for matrix effects (MEs) to determine seven minor tobacco alkaloids (nornicotine, myosmine, anabasine, anatabine, nicotyrine, 2,3′-bipyridine, and cotinine) in mainstream cigarette smoke with high accuracy and robustness. By comparing the heights and shapes of the chromatographic peaks before and after the addition of APs to standard solutions prepared in dichloromethane and cigarette smoke solutions, the compensation effects of 12 APs and their combinations on the MEs of seven minor tobacco alkaloids were evaluated, and the best combination of 2-pyridylethylamine (2 mg/mL)+1,2-decanediol (1 mg/mL) was identified. This AP combination could effectively improve the shapes and increase the heights (by 7–371%) of chromatographic peaks for standard solutions prepared in dichloromethane and cigarette smoke solutions. Before the addition of this AP combination, the slope ratios of the calibration curves for two types of standard solutions of the seven target chemicals were 71.4–159.8%, while they were 87.4–105.6% after the addition, indicating that this AP combination reduced the matrix difference between pure solvent and cigarette smoke solution. After adding the AP combination, the standard curves of solutions prepared in dichloromethane showed good linearity (r2 ≥ 0.999), the spiked recoveries were between 80.9% and 119.6%, and the inter- and intraday precisions were between 1.5–9.5% and 3.1–8.5%, respectively. Three commercial cigarette samples and one mixed standard solution were also tested under four different instrument working conditions to verify the long-term accuracy and ruggedness of the approach in routine real-world analysis of the method. The results showed that the RSD values were higher (3.5–25.4%) without the AP combination than that (<6.7%) with the AP combination. Because of its high accuracy, precision, and robustness, this method has good practical prospects.

UHPLC-MS/MS method for the simultaneous determination of nicotine and tobacco-specific nitrosamines NNN and NNK for use in preclinical studies

A new method was developed and validated for the simultaneous determination of nicotine and tobacco-specific nitrosamines (TSNAs) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in two different tests matrices: porcine buccal epithelium tissue and phosphate buffered saline (PBS) extracts of smokeless tobacco products. The novelty of this work is in the development of a liquid chromatography tandem mass spectrometry method that can provide simultaneous quantification of trace levels of TSNAs and high concentrations of nicotine in biological media. Precision, accuracy, and stability were evaluated during method validation to ensure the method was fit for purpose. Several sample preparation and extraction methods were evaluated to minimize matrix effects and maximize analyte recoveries. The method was accurate in the range of 81.1% – 117%; repeatability was estimated in the range of 1.5% – 13.6% across multiple concentrations. The linear regression correlation coefficient (R2) was greater than 0.9959 for all analytes, and the limit of detection (LOD) was determined for nicotine, NNK, and NNN at 1 ng/mL 0.005 ng/mL, and 0.006 ng/ mL, respectively. Our method was found to be appropriate for the analysis of nicotine, NNN, and NNK in the porcine buccal epithelium and PBS extracts of smokeless tobacco products.Graphical abstract

Exposure to secondhand aerosol from electronic cigarettes at homes: A real-life study in four European countries

Electronic cigarette (e-cigarette) use emits potentially hazardous compounds and deteriorates indoor air quality. Home is a place where e-cigarettes may frequently be used amid its increasing prohibition in public places. This study assessed the real-life scenario of bystanders' exposure to secondhand e-cigarette aerosol (SHA) at home. A one-week observational study was conducted within the TackSHS project in four countries (Greece, Italy, Spain, and the United Kingdom) in 2019 including: 1) homes of e-cigarette users living together with a non-user/non-smoker; and 2) control homes with no smokers nor e-cigarette users. Indoor airborne nicotine, PM2.5, and PM1.0 concentrations were measured as environmental markers of SHA. Biomarkers, including nicotine and its metabolites, tobacco-specific nitrosamines, propanediol, glycerol, and metals were measured in participants' saliva and urine samples. E-cigarette use characteristics, such as e-cigarette refill liquid's nicotine concentration, e-cigarette type, place of e-cigarette use at home, and frequency of ventilation, were also collected. A total of 29 e-cigarette users' homes and 21 control homes were included. The results showed that the seven-day concentrations of airborne nicotine were quantifiable in 21 (72.4 %) out of 29 e-cigarette users' homes; overall, they were quite low (geometric mean: 0.01 μg/m3; 95 % CI: 0.01–0.02 μg/m3) and were all below the limit of quantification in control homes. Seven-day concentrations of PM2.5 and PM1.0 in e-cigarette and control homes were similar. Airborne nicotine and PM concentrations did not differ according to different e-cigarette use characteristics. Non-users residing with e-cigarette users had low but significantly higher levels of cotinine, 3′-OH-cotinine and 1,2-propanediol in saliva, and cobalt in urine than non-users living in control homes. In conclusion, e-cigarette use at home created bystanders' exposure to SHA regardless of the e-cigarette use characteristics. Further studies are warranted to assess the implications of SHA exposure for smoke-free policy.

Associations between urinary biomarkers of oxidative stress and biomarkers of tobacco smoke exposure in smokers

Oxidative stress can contribute to the development of diseases, and may originate from exposures to toxicants commonly found in air pollution and cigarette smoke such as polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs). Yet, associations between these exposures and oxidative stress biomarkers are poorly characterized. We report here novel associations between 14 exposure biomarkers of PAHs and VOCs, and two oxidative stress biomarkers; 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-isoprostaglandin F2α (8-isoprostane) in urine obtained from smokers participating in an ongoing clinical study (ESTxENDS, NCT03589989). We also assessed associations between six biomarkers of tobacco smoke exposure (metabolites of nicotine and tobacco-specific nitrosamines (TSNAs)) and both oxidative stress biomarkers. We then quantified the relative importance of each family of the 20 exposure biomarkers on oxidative stress. Participating smokers (153 men and 117 women, median age 44 years) had on average smoked 25 [2–62] years and smoked about 17 [5–40] cigarettes per day at the time of the study. Multiple linear regression results showed an association between 8-oxodG concentrations and the following metabolites in decreasing relative importance: PAHs (beta coefficient β = 0.105, p-value <0.001, partial R2 = 0.15) > VOCs (β = 0.028, p < 0.001, partial R2 = 0.09) > nicotine (β = 0.226, p < 0.001, partial R2 = 0.08); and between 8-isoprostane concentrations and metabolites of PAHs (β = 0.117, p < 0.001, partial R2 = 0.14) > VOCs (β = 0.040, p < 0.001, partial R2 = 0.14) > TSNAs (β = 0.202, p = 0.003, partial R2 = 0.09) > nicotine (β = 0.266, p < 0.001, partial R2 = 0.08). Behavioral factors known to contribute to oxidative stress, including sleep quality, physical activity, and alcohol consumption, did not play a significant role. Exposures to PAHs and VOCs among smokers were significantly associated with oxidative stress.

Anabasine and Anatabine Exposure Attributable to Cigarette Smoking: National Health and Nutrition Examination Survey (NHANES) 2013-2014.

Anabasine and anatabine are minor alkaloids in tobacco products and are precursors for tobacco-specific nitrosamines (TSNAs). The levels of these two compounds have been used to differentiate tobacco product sources, monitor compliance with smoking cessation programs, and for biomonitoring in TSNA-related studies. The concentrations of urinary anabasine and anatabine were measured in a representative sample of U.S. adults who smoked cigarettes (N = 770) during the 2013–2014 National Health and Nutrition Examination Survey (NHANES) study cycle, which was the first cycle where urinary anabasine and anatabine data became available. Weighted geometric means (GM) and geometric least squares means (LSM) with 95% confidence intervals were calculated for urinary anabasine and anatabine categorized by tobacco-use status [cigarettes per day (CPD) and smoking frequency] and demographic characteristics. Smoking ≥20 CPD was associated with 3.6× higher anabasine GM and 4.8× higher anatabine GM compared with smoking <10 CPD. Compared with non-daily smoking, daily smoking was associated with higher GMs for urinary anabasine (1.41 ng/mL vs. 6.28 ng/mL) and anatabine (1.62 ng/mL vs. 9.24 ng/mL). Urinary anabasine and anatabine concentrations exceeded the 2 ng/mL cut point in 86% and 91% of urine samples from people who smoke (PWS) daily, respectively; in comparison, 100% of them had serum cotinine concentrations greater than the established 10 ng/mL cut point. We compared these minor tobacco alkaloid levels to those of serum cotinine to assess their suitability as indicators of recent tobacco use at established cut points and found that their optimal cut point values would be lower than the established values. This is the first time that anabasine and anatabine are reported for urine collected from a U.S. population-representative sample of NHANES study participants, providing a snapshot of exposure levels for adults who smoked during 2013–2014. The results of this study serve as an initial reference point for future analysis of NHANES cycles, where changes in the national level of urinary anabasine and anatabine can be monitored among people who smoke to show the effect of changes in tobacco policy.

Levels of nicotine and tobacco-specific nitrosamines in oral nicotine pouches

BackgroundNicotine pouches without tobacco are new products that deliver nicotine into the body via the oral mucosa. There is a lack of independent research on the chemical composition and product...

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.

Formulation of smokeless tobacco products with a wide range of pH to study nicotine pharmacokinetics and pharmacodynamics

The rate of nicotine absorption from tobacco products is a determinant of addiction potential and other detrimental health effects. Oral nicotine bioavailability from moist snuff smokeless tobacco (ST) is influenced by nicotine content, pH, flavors, and tobacco cut. For use in a clinical study testing the effect of pH on nicotine pharmacokinetics, four investigational ST products that differed only in pH were produced. A commercial ST product (Copenhagen Long Cut Original, pH 7.7) was modified with citric acid monohydrate (23 mg/g tobacco) or sodium carbonate (4.6 and 11 mg/g) to create products with pH 5.0, 8.2, and 8.6, respectively. All products – including the original product with pH 7.7 – were individually packaged (approximately 2 g) in aluminum foil pouches and stored frozen (−20 °C); pH, nicotine, tobacco-specific nitrosamines, moisture content, and mold and yeast counts were tested for up to 19 months to verify stability. Remarkable stability was demonstrated in this packaging/storage combination. For example, pH from all products were within 0.1 pH units and never exceeded 0.2 units. Nicotine concentration averaged 9.07 mg/g at baseline, maximal deviations from baseline in the four products averaged 0.30 mg/g. Similarly, TSNA, moisture content, yeast, and mold did not materially change. This study illustrates a method of investigational tobacco products formulation by manipulating a single design feature (or component) with the purpose of independently and systematically assessing its influence on nicotine bioavailability in a clinical study.

A chemometric experimental design with three-step stacking capillary electrophoresis for analysis of five tobacco-specific nitrosamines in cigarette products

Tobacco-specific nitrosamines (TSNAs) as carcinogens endanger our health and life from cigarette products. However, the safe range of TSNAs levels in commercial cigarette products has not yet been established. For the purpose of safety and supervision, a three-step stacking approach including field amplified sample injection (FASI), sweeping, and analyte focusing by micelle collapse (AFMC), was developed for the simultaneous determination of five TSNAs levels in cigarette products. This approach also involved aspects of chemometric experimental design, including fractional factorial design and central composite design. After the multilevel optimization of the experimental design, the five TSNAs were well separated. The LOD (S/N = 3) values of the N´-nitrosonornicotine (NNN), N´-nitrosoanatabine (NAT), N´-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the FASI-sweeping-AFMC CE approach were 1.000 ng/mL, 0.500 ng/mL, 0.125 ng/mL, 1.000 ng/mL, and 0.500 ng/mL respectively. The results of relative standard deviation (RSD) and relative error (RE) were all less than 3.35%, demonstrating good precision and accuracy. Finally, this novel approach was further applied to monitor three commercial cigarette products, and a range of 250.1–336.6 ng/g for NNN, 481.6–526.7 ng/g for NAT, 82.2–247.6 ng/g for NAB, 167.7–473.7 ng/g for NNAL, and 39.4–246.7 ng/g for NNK could be observed among these. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of five TSNAs levels in cigarette products and could serve as a tool for assays of quality control of nitrosamines.

Trends in Analytical Methods for Analysis of Tobacco Products: An Overview

Abstract. Currently, due to the strong trends in policies to reduce smoking, several concerns related to quality control and regulation of tobacco products have been raised, as a great variety of products are available to consumers in the market. Considering that development of robust and selective analytical methods for tobacco and derivative products has been an important and necessary task over the years, however laborious, due to the complexity of the matrices. This work presents an overview of advances, innovation, and trends in analytical methods, focused on the chromatographic analysis of nicotine, tobacco-specific nitrosamines (TSNAs), humectants, pesticides, polycyclic aromatic hydrocarbons (PAHs) and sugars in different tobacco products. Gas chromatography (GC) coupled with different detectors is widely used to analyze nicotine, humectants and TSNAs in tobacco leaves and derivative products, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been frequently used to analyze TSNAs in mainstream cigarette smoke and smokeless tobacco products. Pesticides were mainly analyzed in tobacco using GC or LC coupled with mass detector (MS), while PAHs and sugars were generally analyzed in tobacco and smoke using LC-MS and GC-MS techniques, respectively. In addition to already established methods and despite the lack of a worldwide standardization of methods, significant efforts have been made to develop analytical procedures for a wide variety of tobacco products, with a broad range of innovative chromatographic methods available. In this sense, a potential trend is the possibility of simultaneous determination of multiple components aiming to reduce the analysis time. The present study examined the main works that developed or improved analytical methods for identifying and quantifying priority compounds in different tobacco matrices and aims to contribute to future research with this objective, in addition, to promoting standardization of technical terms used in this analytical area. It is noteworthy that some of the methods mentioned here have not been validated and further studies are needed in order to obtain reproducible analytical methods for regulatory purposes.&#x0D; Keywords: chromatography analyses; tobacco leaves; cigarette; cigarette smoke; smokeless tobacco products.&#x0D;

Structure-based study to identify alkaloids as promising cytochrome P450 (CYP1A1) inhibitors: An in silico approach using virtual screening, molecular dynamic simulations, and binding free energy calculation.

Carcinogens present in smokeless tobacco (SLT) like tobacco-specific nitrosamines can be metabolized by the cytochrome P450 (CYP450) enzyme. Functionally, the CYP450 enzyme resides in a heme pigment to perform the catalytic activity. The CYP1A1 is one of the main extrahepatic CYP450 enzymes known to detoxify toxic substances and activate carcinogens. The CYP1A1 inhibition by potential inhibitors reduce the chance of oral cancer. The current study aimed to explore more about the inhibitor binding site and identification of lead alkaloids, that could work as putative inhibitors against target CYP1A1. In respect, we have performed docking studies, virtual screening of alkaloids, and natural product libraries against CYP1A1 followed by molecular dynamicsimulations and binding free energy calculations. Docking studies of tobacco-specific nitrosamine (TSNA) products and their similar carcinogen analogs revealed that the heme group is bound to the floor of the bowl-shaped cavity whereas carcinogens are bound to the roof of the rounded shape cavity. Furthermore, virtual screening and binding free energy calculations revealed Tomatidine as a putative inhibitor against CYP1A1. On the basis of altogether outcomes of the current study, we have concluded that the addition of lead-hit alkaloid Tomatidine and others in SLT products may be working as a supplement that could be able to reduce the expression of human CYP1A1 and suppresses carcinogenic by-products formations.

Composition and Ecological Functionality of Fungal Communities Associated with Smokeless Tobacco Products Mainly Consumed in India.

ABSTRACTThe microbial communities present in smokeless tobacco products (STPs) perform critical steps in the synthesis of carcinogens, mainly tobacco-specific nitrosamines (TSNAs). Most studies emphasize the bacterial component, and the mycobiome of STPs has not been well characterized. In this study, we investigated the fungal communities in the different categories of STPs by sequencing the internal transcribed spacer (ITS) rRNA region of the fungal genome. The ecological character of the fungal community associated with STPs was determined by using FUNGuild. Our results indicated that Ascomycota and Basidiomycota were the most abundant fungal phyla across all STPs. The predominant fungal genera in STPs were Pichia, Sterigmatomyces, and Mortierella. The α-diversity varied significantly across the STPs based on observed, Fisher, and Shannon indices. Using SparCC cooccurrence network analysis, significant positive correlations of 58.5% and negative connections of 41.5% were obtained among fungal genera identified in STPs. Furthermore, the functional predictions by FUNGuild determined that STPs possessed high abundances of saprotroph and pathotroph-saprotroph-symbiotroph fungal trophic groups. At the functional guild level, the qiwam samples contained high abundances of soil saprotrophs, while plant pathogens were prevalent in pan-masala samples. These results suggest that various fungal populations reside in STPs and interrelate with each other and can contribute to the synthesis of TSNAs. This study has established the basis for future large-scale investigations of STP-associated mycobiota and the impact of such mycobiota in oral carcinogenesis in STP users via inflammation and carcinogens (TSNAs and mycotoxins).IMPORTANCE Smokeless tobacco products (STPs) contain complex microbial communities that influence the synthesis of carcinogens, such as tobacco-specific nitrosamines (TSNAs). Research on STP-associated bacterial populations revealed connections between bacterial metabolism and TSNA synthesis. The abundance of the fungal population may also have an impact on the production of TSNAs. This study examined STPs popularly used in India, and diverse fungal communities were identified in these STPs. Pichia, Sterigmatomyces, and Mortierella were the predominant fungal genera in the STPs. High abundances of saprotroph and pathotroph-saprotroph-symbiotroph trophic groups in STPs could affect the degradation of tobacco products and the synthesis of TSNAs.

The Relationship between Social Support and Vaping Behavior of Senior High Age Adolescents in Surabaya

Background: Smoking has a negative impact on health in people of all ages, especially adolescents. With technological advancements, cigarettes have taken on a new form: e-cigarettes. The US Food and Drug Administration (FDA) has begun to prohibit the use of e-cigarettes because they contain hazardous and carcinogenic tobacco specific nitrosamines and diethylene glycol (DEG). However, e-cigarettes are becoming increasingly popular in Indonesia, with an increasing number of users. Objectives: To investigate the relationship between social support on smoking/vaping behaviors of senior high age adolescents in Surabaya. Methods: This is a quantitative study with a cross-sectional and observational analytic design. The total number of respondents in this study was 145, as determined by the snowball sampling method. The independent variable is social support. Social support consists of instrumental support, informational support, emotional support, and appraisal support. The behavior of smoking/vaping is the dependent variable. Results: The relationship among variables was measured by using Pearson Chi-Square Test. The result shows that there is a relationship of instrumental support (p=0.000), informational support (0.019), emotional support (p=0.000), and appraisal support (p=0.000) on smoking / vaping behavior in senior high age adolescents. Conclusions: There is a relationship between social support and smoking / vaping behavior of high school age adolescents in Surabaya. Social support is important since it can influence the adolescents smoking behavior. It is hoped the family such as parents can provide a better understanding of the effect of smoking on health and for adolescents should be more selective with whom they hang out with and have the courage to refuse a friend's invitation to smoke.

Minor Tobacco Alkaloids as Biomarkers to Distinguish Combusted Tobacco Use From Electronic Nicotine Delivery Systems Use. Two New Analytical Methods.

Biomarkers for the use of electronic nicotine delivery systems (ENDS) are desirable for studies of the health effects of electronic cigarettes and related devices. However, the aerosols inhaled from these devices do not contain substances that are unique to this class of products, i.e., substances that are not present in cigarette smoke or those that do not have common environmental or dietary sources. Consequently, identifying selective biomarkers for ENDS use remains a challenge. If co-use of conventional tobacco products can be definitively ruled out, then nicotine and its metabolites are suitable for assessing exposure. Self-reports from questionnaires are often used to obtain information on product use. But self-reports may not always be accurate, and are not amenable to obtaining quantitative information on exposure. An alternative approach is to use selective biomarkers for conventional tobacco products to definitively rule out their use. In this article, we describe two new LC-MS/MS methods for the minor tobacco alkaloids anabasine, anatabine, nicotelline, anatalline, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite, all biomarkers that are selective for the use of conventional tobacco products. Applications of these biomarkers in studies of ENDS use and dual use of ENDS and conventional tobacco products are also discussed.

Delineating the Bacteriome of Packaged and Loose Smokeless Tobacco Products Available in North India.

Smokeless tobacco product (STP) consumption is a significant public health threat across the globe. STPs are not only a storehouse of carcinogens and toxicants but also harbor microbes that aid in the conversion of tobacco alkaloids to carcinogenic tobacco-specific nitrosamines (TSNAs), thereby posing a further threat to the health of its consumers. The present study analyzed the bacterial diversity of popular dry and loose STPs by 16S rRNA gene sequencing. This NGS-based investigation revealed four dominant phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria and identified 549 genera, Prevotella, Bacteroides, and Lactobacillus constituting the core bacteriome of these STPs. The most significantly diverse bacteriome profile was displayed by the loose STP Mainpuri kapoori. The study further predicted the functional attributes of the prevalent genera by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) algorithm. Genes encoding for nitrate and nitrite reduction and transport enzymes, antibiotic resistance, multi-drug transporters and efflux pumps, secretion of endo- and exotoxin, and other pro-inflammatory molecules were identified. The loose STPs showed the highest level of nitrogen metabolism genes which can contribute to the synthesis of TSNAs. This study reveals the bacteriome of Indian domestic loose STPs that stagger behind in manufacturing and storage stringencies. Our results raise an alarm that the consumption of STPs harboring pathogenic genera can potentially lead to the onset of several oral and systemic diseases. Nevertheless, an in-depth correlation analysis of the microbial diversity of STPs and their elicit impact on consumer health is warranted. KEY POINTS: • Smokeless tobacco harbors bacteria that aid in synthesis of carcinogenic nitrosamines. • Most diverse bacteriome profile was displayed by loose smokeless tobacco products. • Pathogenic genera in these products can harm the oral and systemic health of users.

Comparative and analytical characterization of the oral bacteriome of smokeless tobacco users with oral squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) is the most common type of head and neck cancer worldwide. Smokeless tobacco (SLT) has been well proven for its role in oral carcinogenesis due to the abundance of several carcinogens. However, the role of inhabitant microorganisms in the oral cavity of smokeless tobacco users has not yet been well explored in the context of OSCC. Therefore, the present investigation was conceived to analyze the oral bacteriome of smokeless tobacco users having OSCC (CP group). With the assistance of illumina-based sequencing of bacterial-specific V3 hypervariable region of 16S rDNA gene, 71,969 OTUs (operational taxonomic units) were categorized into 18 phyla and 166 genera. The overall analysis revealed that the oral bacteriome of the patients with OSCC, who were smokeless tobacco users, was significantly different compared to the healthy smokeless tobacco users (HTC group) and non-users (HI users). The appearance of 14 significantly abundant genera [FDR (false discovery rate) adjusted probability value of significance (p value) < 0.05] among the CP group showed the prevalence of tobacco-specific nitrosamines forming bacteria (Staphylococcus, Fusobacterium, and Campylobacter). The functional attributes of the oral bacteriome of the CP group can also be correlated with the genes involved in oncogenesis. This study is the first report on the oral bacteriome of Indian patients with OSCC who were chronic tobacco chewers. The results of the present study will pave the way to understand the influence of smokeless tobacco on the oral bacteriome of OSCC patients. KEY POINTS: • Oral bacteriome of OSCC patients differ from healthy smokeless tobacco (SLT) users and SLT non-users. • Smokeless tobacco influences the oral bacteriome of OSCC group. • Oral bacteriome specific diagnostics may be developed for pre-diagnosis of oral cancer.

Tobacco-specific nitrosamine 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA) causes DNA damage and impaired replication/transcription in human lung cells.

Thirdhand smoke (THS) is a newly described health hazard composed of toxicants, mutagens and carcinogens, including nicotine-derived tobacco specific nitrosamines (TSNAs), one of which is 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA). Although TSNAs are generally potent carcinogens, the risk of NNA, which is specific to THS, is poorly understood. We recently reported that THS exposure-induced adverse impact on DNA replication and transcription with implications in the development of cancer and other diseases. Here, we investigated the role of NNA in THS exposure-induced harmful effects on fundamental cellular processes. We exposed cultured human lung epithelial BEAS-2B cells to NNA. The formation of DNA base damages was assessed by Long Amplicon QPCR (LA-QPCR); DNA double-strand breaks (DSBs) and NNA effects on replication and transcription by immunofluorescence (IF); and genomic instability by micronuclei (MN) formation. We found increased accumulation of oxidative DNA damage and DSBs as well as activation of DNA damage response pathway, after exposure of cells to NNA. Impaired S phase progression was also evident. Consistent with these results, we found increased MN formation, a marker of genomic instability, in NNA-exposed cells. Furthermore, ongoing RNA synthesis was significantly reduced by NNA exposure, however, RNA synthesis resumed fully after a 24h recovery period only in wild-type cells but not in those deficient in transcription-coupled nucleotide excision repair (TC-NER). Importantly, these cellular effects are common with the THS-exposure induced effects. Our findings suggest that NNA in THS could be a contributing factor for THS exposure-induced adverse health effect.

Genetic variants in CYP2B6 and HSD17B12 associated with risk of squamous cell carcinoma of the head and neck.

Polycyclic aromatic hydrocarbons (PAH) and tobacco-specific nitrosamines (TSNA) metabolism-related genes play an important role in the development of cancers. We assessed the associations of genetic variants in genes involved in the metabolism of PAHs and TSNA with riskof squamous cell carcinoma of the head and neck (SCCHN) in European populations using two published genome-wide association study datasets. In the single-locus analysis, we identified two SNPs (rs145533669 and rs35246205) in CYP2B6 to be associated with risk of SCCHN (P=1.57 × 10-4 and .004, respectively), two SNPs (EPHX1 rs117522494 and CYP2B6 rs145533669) to be associated with risk of oropharyngeal cancer (P=.001 and .004, respectively), and one SNP (rs4359199 in HSD17B12) to be associated with risk of oral cancer (P=.006). A significant interaction effect was found between rs4359199 and drinking status on risks of SCCHN and oropharyngeal cancer (P < .05). eQTL and sQTL analyzes revealed that two SNPs (CYP2B6 rs35246205 and HSD17B12 rs4359199) were correlated with alternative splicing or mRNA expression levels of the corresponding genes in liver cells (P < .05 for both). In silico functional annotation suggested that these two SNPs may regulate mRNA expression by affecting the binding of transcription factors. Results from phenome-wide association studies presented significant associations between these genes and risks of other cancers, smoking behavior and alcohol dependence (P < .05). Thus, our study provided some insight into the underlying genetic mechanism of head and neck cancer, which warrants future functional validation.

Metabolism and DNA Adduct Formation of Tobacco-Specific N-Nitrosamines.

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.