Tobacco-specific Nitrosamines Research Articles

Smokeless tobacco and cigarette smoking: chemical mechanisms and cancer prevention.

Tobacco products present a deadly combination of nicotine addiction and carcinogen exposure resulting in millions of cancer deaths per year worldwide. A plethora of smokeless tobacco products lead to unacceptable exposure to multiple carcinogens, including the tobacco-specific nitrosamine N'-nitrosonornicotine, a likely cause of the commonly occurring oral cavity cancers observed particularly in South-East Asian countries. Cigarettes continue to deliver a large number of carcinogens, including tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons and volatile organic compounds. The multiple carcinogens in cigarette smoke are responsible for the complex mutations observed in critical cancer genes. The exposure of smokeless tobacco users and smokers to carcinogens and toxicants can now be monitored by urinary and DNA adduct biomarkers that may be able to identify those individuals at highest risk of cancer so that effective cancer prevention interventions can be initiated. Regulation of the levels of carcinogens, toxicants and nicotine in tobacco products and evidence-based tobacco control efforts are now recognized as established pathways to preventing tobacco related cancer.

A Chemometric Experimental Design with Three-Step Stacking Capillary Electrophoresis for Analysis of Five Tobacco-Specific Nitrosamines in Cigarette Products

A Chemometric Experimental Design with Three-Step Stacking Capillary Electrophoresis for Analysis of Five Tobacco-Specific Nitrosamines in Cigarette Products

High Levels of the Carcinogenic Tobacco-Specific Nitrosamine NNAL and Associated Findings in Children of Smokers: A Case Series.

High levels of NNAL, the tobacco smoke exposure (TSE) biomarker of the carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), indicate future cancer risk. A prior study of smokers’ children revealed NNAL levels as high as active smokers. Therefore, we conducted a case series to examine the sociodemographics, TSE and clinical patterns, and other TSE biomarker levels in 9 children with extreme NNAL levels of >200 pg/ml to generate hypotheses and explore potential causes and implications. We identified 0 to 4-year-olds who presented to an emergency setting and lived with ⩾1 smoker who were part of a parental tobacco cessation trial (n = 461). Of these children, 52 had urinary NNAL, cotinine, and N-oxides results (n = 52). Nine children (17.3%) had NNAL levels >200 pg/ml, ranging from 206.4 to 1399.0 pg/ml (Median (Mdn) = 489.2 pg/ml; Interquartile Range (IQR) = 222.7-1289.3 pg/ml). The cotinine Mdn (IQR) was 38.5 (10.3-102.2) ng/ml and the N-oxides Mdn (IQR) = 93.8 (24.7-109.6) pg/ml. While all biomarker levels were alarmingly high, these young children would not have been flagged for very high cancer risk based on urinary cotinine levels alone. This underscores the critical role of comprehensive TSE biomarker measurement in capturing different TSE exposure patterns and assessing children’s future risk for cancer and other TSE-related morbidities.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9–10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

Bacteriome of Moist Smokeless Tobacco Products Consumed in India With Emphasis on the Predictive Functional Potential.

Smokeless tobacco products (STPs) carry assorted microbial population that contributes to carcinogens synthesis like tobacco-specific nitrosamines (TSNAs). Extensive exploration of microbiota-harboring STPs is required to understand their full carcinogenic potential. Here, we applied 16S rRNA gene sequencing to investigate bacteriome present in moist STPs immensely consumed in India (Khaini, Moist-snuff, Qiwam, and Snus). Further, the functional metagenome was speculated by PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to assign the abundance of genes related to nitrogen metabolism, bacterial toxins, antibiotic drug resistance and other pro-inflammatory molecules. Highly diverse bacterial communities were observed in all moist STPs. Taxonomic analysis revealed a total of 549 genera belonging to four major phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Overall, the core bacterial genera Acinetobacter, Bacillus, Prevotella, Acetobacter, Lactobacillus, Paracoccus, Flavobacterium, and Bacteroides were significantly abundant in moist STPs. Elevated moisture-holding products like Moist-snuff and Qiwam harbor rich bacterial species diversity and showed similar bacteriome composition. Furthermore, Qiwam products showed the highest level of genes associated with nitrogen metabolism, antibiotic resistance, toxins, and pro-inflammation (predicted by PICRUSt) which can contribute to the synthesis of TSNAs and induction of oral cancer. The present broad investigation of moist STPs-associated bacteriome prevalence and their detailed metabolic potential will provide novel insight into the oral carcinogenesis induced by STPs.

Shotgun metagenome sequencing of a Sudanese toombak snuff tobacco: genetic attributes of a high tobacco-specific nitrosamine containing smokeless tobacco product.

The most alarming aspect of the Sudanese toombak smokeless tobacco is that it contains high levels of highly toxic tobacco-specific nitrosamines (TSNAs). Understanding the microbiology of toombak is of relevance because TSNAs are an indirect result of microbial-mediated nitrate reductions. We conducted shotgun metagenomic sequencing on a toombak product for which relevant features are presented here. The microbiota was composed of over 99% Bacteria. The most abundant taxa included Actinobacteria, specifically the genera Enteractinococcus and Corynebacterium, while Firmicutes were represented by the family Bacillaceae and the genus Staphylococcus. Selected gene targets were nitrate reduction and transport, antimicrobial resistance, and other genetic transference mechanisms. Canonical nitrate reduction and transport genes (i.e. nar) were found for Enteractinococcus and Corynebacterium while various species of Staphylococcus exhibited a notable number of antimicrobial resistance and genetic transference genes. The nitrate reduction activity of the microbiota in toombak is suspected to be a contributing factor to its high levels of TSNAs. Additionally, the presence of antimicrobial resistance and transference genes could contribute to deleterious effects on oral and gastrointestinal health of the end user. Overall, the high toxicity and increased incidences of cancer and oral disease of toombak users warrants further investigation into the microbiology of toombak.

Comparison of the effects of three types of heating tobacco system and conventional cigarettes on indoor air quality

Environmental tobacco smoke (ETS) from conventional cigarettes is reported to affect indoor air quality (IAQ) in various real indoor environments. Recently, Japan Tobacco Inc. introduced three types of tobacco product that are heated rather than combusted. These comprise one direct heating tobacco system and two in-direct heating tobacco systems. In this study, the impact of using these products on IAQ was evaluated in an environmentally controlled chamber. Two environmental conditions, simulating restaurant and residential spaces, were examined. Under the same conditions, cigarette smoking and the presence of people only were used as positive and negative controls, respectively. The indoor air concentrations of 48 constituents (tobacco-specific nitrosamines, carbonyls, volatile organic compounds, total volatile organic compounds, polycyclic aromatic hydrocarbon, polycyclic aromatic amines, mercury, metals, ETS markers, propylene glycol, glycerol, carbon monoxide, carbon dioxide, suspended particle matter, ammonia, and nitrogen oxides) were measured. Compared with the presence of people, the concentrations of some constituents were actually increased when using heating tobacco products under both environmental conditions, simulating restaurant and residential spaces. However, the constituent concentrations were lower than those obtained by cigarette smoking, except for propylene glycol and glycerol, and below the exposure limits for constituents in air, as defined by air quality guidelines or regulations. Based on these data, the use of heating tobacco systems in appropriate indoor environments has less impacts compared to conventional cigarettes.Article HighlightsWe measured the indoor air concentrations of chemical constituents generated when using three heating tobacco systems with different heating mechanisms in two environment conditions simulating restaurant and residential spaces (positive control: when smoking cigarettes, negative control: the presence of people only).In the measurement and analysis method used this study, it was possible to find not only that the air concentration generated when using the heating tobacco systems in this study were considerably lower than that when smoking cigarettes, but also the differences of the concentrations between heating tobacco systems with different heating mechanisms.We showed some constituents which actually increased the air concentrations when using heating tobacco systems compared with the presence of people only.

Patterns of Carbon-Bound Exogenous Compounds in Patients with Lung Cancer and Association with Disease Pathophysiology.

Asymptomatic anthracosis is the accumulation of black carbon particles in adult human lungs. It is a common occurrence, but the pathophysiologic significance of anthracosis is debatable. Using in situ high mass resolution matrix-assisted laser desorption/ionization (MALDI) fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry imaging analysis, we discovered noxious carbon-bound exogenous compounds, such as polycyclic aromatic hydrocarbons (PAH), tobacco-specific nitrosamines, or aromatic amines, in a series of 330 patients with lung cancer in highly variable and unique patterns. The characteristic nature of carbon-bound exogenous compounds had a strong association with patient outcome, tumor progression, the tumor immune microenvironment, programmed death-ligand 1 (PD-L1) expression, and DNA damage. Spatial correlation network analyses revealed substantial differences in the metabolome of tumor cells compared with tumor stroma depending on carbon-bound exogenous compounds. Overall, the bioactive pool of exogenous compounds is associated with several changes in lung cancer pathophysiology and correlates with patient outcome. Given the high prevalence of anthracosis in the lungs of adult humans, future work should investigate the role of carbon-bound exogenous compounds in lung carcinogenesis and lung cancer therapy. SIGNIFICANCE: This study identifies a bioactive pool of carbon-bound exogenous compounds in patient tissues associated with several tumor biological features, contributing to an improved understanding of drivers of lung cancer pathophysiology.

Secondhand smoke exposure in school children in Malta assessed through urinary biomarkers

School children may be exposed to secondhand smoke (SHS) either at home, in transit or in social gatherings permitting smoking in their presence. Questionnaires about SHS often underestimate prevalence and extent of exposure. A more accurate tool is the use of biomarkers such as cotinine (COT) and trans-3′-hydrocycotinine (3HC) as biomarkers of SHS exposure, alongside 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a reduction product in the body of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), both potent carcinogens. We measured urinary COT, 3HC and total NNAL using sensitive and specific high-performance LC-MS/MS methods. The limit of quantification (LOQ) for each assay were 0.05 ng/mL, 0.1 ng/mL and 0.25 pg/mL respectively. The aim of this study was to evaluate the exposure to SHS of school children (9–11 years), from five public schools in the island of Malta, from questionnaire information about smoking at home and verify it by urinary biomarker data of COT, 3HC and NNAL. These biomarkers were measurable in 99.4%, 95.4% and 98.3% of the participating children respectively. From the children reporting smoking at home, 11% had a history of asthma and had COT, 3HC and NNAL geometric mean concentrations double compared to the non-asthmatic group. In has been confirmed that non-smokers exposed to SHS and THS have a higher NNAL/COT ratio than the group identified as smokers according to specific and defined COT threshold levels (despite the fact that a priori, the entire study group was composed of non-smokers). The implication of high measured levels of urinary NNAL in children should be of concern given its potency. A main effects multifactor ANOVA model was developed and the children's house and school locations and the smoking frequency were statistically significant to predict the levels of the three metabolites. For 3HC only, the status of the employment of the mother was also an important predictor.

Determination of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) arising from tobacco smoke in airborne particulate matter

The most important tobacco-specific nitrosamine found in cigarette smoke and formed in ageing smoke after cigarettes are extinguished is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). It is formed from nitrosation of nicotine, under particular conditions both in indoor and outdoor environments. NNK has been classified as a potent lung carcinogen which is expected to be found primarily in the particle-phase and to be stable in particulate matter. In this study tests have been carried out to show that a bisulfate-treated filter is more efficient than an untreated filter to collect both nicotine and NNK, and that the latter is stable in outdoor particulate matter. To characterize NNK in the outdoor environment, airborne samples were collected from 11 cities in USA, UK, Hong Kong and Malta with characteristics varying from low to high population densities and from urban to suburban to rural, and with desert characteristics and distinct climates. It has been shown that airborne particle + gas phase nicotine and particle-phase NNK behave in a linearly correlated manner. A seasonal analysis was carried out on a subset of data available from five sites in California, where the load of NNK in PM10 is driven by long range transport of the air masses passing over densely populated cities. In the winter season, the load of NNK in PM is higher than in summer in a statistically significant manner. The contamination of PM with NNK shows variability, but is observed at all sites. This paper highlights the potential risk of chronic exposure to NNK in particulate matter by the inhalation pathway.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Histone Acetylation via α7nAChR-Mediated PI3K/Akt Activation and Its Impact on γ-H2AX Generation.

A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in early development: A wide-scope metabolomics assay in zebrafish embryos

The tobacco-specific nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic and ubiquitous environmental pollutant for which toxic activity has been thoroughly investigated in murine models and human tissues. However, its potential deleterious effects on vertebrate early development are yet poorly understood. In this work, we characterized the impact of NNK exposure during early developmental stages of zebrafish embryos, a known alternative model for mammalian toxicity studies. Embryos exposed to different NNK concentrations were monitored for lethality and for the appearance of malformations during the first five days after fertilization. LC-MS based untargeted metabolomics was subsequently performed for a wide-scope assay of NNK-related metabolic alterations. Our results revealed the presence of not only the parental compound, but also of two known NNK metabolites, 4-Hydroxy-4-(3-pyridyl)-butyric acid (HPBA) and 4-(Methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanol (NNAL-N-oxide) in exposed embryos likely resulting from active CYP450-mediated α-hydroxylation and NNK detoxification pathways, respectively. This was paralleled by a disruption in purine and pyrimidine metabolisms and the activation of the base excision repair pathway. Our results confirm NNK as a harmful embryonic agent and demonstrate zebrafish embryos to be a suitable early development model to monitor NNK toxicity.

Sustainable sorbent derived from discarded cigarette butts for elimination of tobacco specific nitrosamines carcinogen

In order to control the increasingly serious pollutions of tobacco specific nitrosamines (TSNA) and discarded cigarette butts (DCB) that caused by smoking, new sustainable carbonaceous sorbent was prepared to follow the new idea of “cleaning environment with recycle of waste”. For this propose, DCB was used as the carbon source and aluminum nitrate as the pore-forming agent to form the sorbent with a developed porosity and the well-exposed metal oxides. One-pot synthesis was adopted without template and washing, in order to recycle waste with a sustainable technology. New sorbent could selectively capture 25% of TSNA among hundreds of components in tobacco extract solution or 35 mg g − 1 of 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) in aqueous solution, quite better than activated carbon and zeolite NaZSM-5. In addition, the sorbent was able to catalyze the degradation of the adsorbed NNK in 472–584 K to eliminate the environment carcinogen at relative low temperature. Moreover, cell test demonstrates that the sorbent derived from DCB was cell-safe and therefore widely available. The application of this new sorbent will control both pollutions of TSNA and DCB at the same time, which will protect environment and public health effectively. • Recycle of waste to clean environment was demonstrated in this paper. • Using discarded cigarette butts as the raw material to prepare new sorbent. • One-pot synthesis of the alumina doped carbon without template and washing. • Sustainable sorbent effectively capture tobacco specific nitrosamines carcinogen. • New sorbent is cell-safe and therefore widely available.

Lung Tumor Growth Promotion by Tobacco-Specific Nitrosamines Involves the β2-Adrenergic Receptors-Dependent Stimulation of Mitochondrial REDOX Signaling.

Aims: Lung cancer is the leading cause of cancer death worldwide, and tobacco smoking is a recognized major risk factor for lung tumor development. We analyzed the effect of tobacco-specific nitrosamines (TSNAs) on human lung adenocarcinoma metabolic reprogramming, an emergent hallmark of carcinogenesis. Results: A series of in vitro and in vivo bioenergetic, proteomic, metabolomic, and tumor biology studies were performed to analyze changes in lung cancer cell metabolism and the consequences for hallmarks of cancer, including tumor growth, cancer cell invasion, and redox signaling. The findings revealed that nicotine-derived nitrosamine ketone (NNK) stimulates mitochondrial function and promotes lung tumor growth in vivo. These malignant properties were acquired from the induction of mitochondrial biogenesis induced by the upregulation and activation of the beta-2 adrenergic receptors (β2-AR)-cholinergic receptor nicotinic alpha 7 subunit (CHRNAα7)-dependent nitrosamine canonical signaling pathway. The observed NNK metabolic effects were mediated by TFAM overexpression and revealed a key role for mitochondrial reactive oxygen species and Annexin A1 in tumor growth promotion. Conversely, ectopic expression of the mitochondrial antioxidant enzyme manganese superoxide dismutase rescued the reprogramming and malignant metabolic effects of exposure to NNK and overexpression of TFAM, underlining the link between NNK and mitochondrial redox signaling in lung cancer. Innovation: Our findings describe the metabolic changes caused by NNK in a mechanistic framework for understanding how cigarette smoking causes lung cancer. Conclusion: Mitochondria play a role in the promotion of lung cancer induced by tobacco-specific nitrosamines. Antioxid. Redox Signal. 36, 525-549.

A Low-Cost, High-Throughput Digital Image Analysis of Stain Patterns on Smoked Cigarette Filter Butts to Estimate Mainstream Smoke Exposure.

Standard machine smoking protocols provide useful information for examining the impact of design parameters, such as filter ventilation, on mainstream smoke delivery. Unfortunately, their results do not accurately reflect human smoke exposure. Clinical research and topography devices in human studies yield insights into how products are used, but a clinical setting or smoking a cigarette attached to such a device may alter smoking behavior. To better understand smokers’ use of filtered cigarette products in a more natural environment, we developed a low-cost, high-throughput approach to estimate mainstream cigarette smoke exposure on a per-cigarette basis. This approach uses an inexpensive flatbed scanner to scan smoked cigarette filter butts and custom software to analyze tar-staining patterns. Total luminosity, or optical staining density, of the scanned images provides quantitative information proportional to mainstream smoke-constituent deliveries on a cigarette-by-cigarette basis. Duplicate sample analysis using this new approach and our laboratory’s gold-standard liquid chromatography/tandem mass spectrometry (LC/MS/MS) solanesol method yielded comparable results (+7% bias) from the analysis of 20 commercial cigarettes brands (menthol and nonmentholated). The brands varied in design parameters such as length, filter ventilation, and diameter. Plots correlating the luminosity to mainstream smoked-nicotine deliveries on a per-cigarette basis for these cigarette brands were linear (average R2 > 0.91 for nicotine and R2 > 0.83 for the tobacco-specific nitrosamine NNK), on a per-brand basis, with linearity ranging from 0.15 to 3.00 mg nicotine/cigarette. Analysis of spent cigarette filters allows exposures to be characterized on a per-cigarette basis or a “daily dose” via summing across results from all filter butts collected over a 24 h period. This scanner method has a 100-fold lower initial capital cost for equipment than the LC/MS/MS solanesol method and provides high-throughput results (~200 samples per day). Thus, this new method is useful for characterizing exposure related to filtered tobacco-product use.

Molecular docking and oxidation kinetics of 3-phenyl coumarin derivatives by human CYP2A13

CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs. To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site. CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The Km-values of the other coumarins varied 0.85–97 µM, Vmax-values of the oxidation reaction varied 0.25–60 min−1, and intrinsic clearance varied 26–6190 kL/min*mol CYP2A13). Km of 6-chloro-3-(3-hydroxyphenyl)-coumarin was 0.85 (0.55–1.15 95% confidence limit) µM and Vmax 0.25 (0.23–0.26) min−1, whereas Km of 6-hydroxy-3-(3-hydroxyphenyl)-coumarin was 10.9 (9.9–11.8) µM and Vmax 60 (58–63) min−1. Docking analyses demonstrated that 6-chloro or 6-methoxy and 3-(3-hydroxyphenyl) or 3-(4-trifluoromethylphenyl) substituents of coumarin increased affinity to CYP2A13, whereas 3-triazole or 3-(3-acetate phenyl) or 3-(4-acetate phenyl) substituents decreased it. The active site of CYP2A13 accepts more diversified types of coumarin substrates than the hepatic CYP2A6 enzyme. New sensitive and convenient profluorescent CYP2A13 substrates were identified, such as 6-chloro-3-(3-hydroxyphenyl)-coumarin having high affinity and 6-hydroxy-3-(3-hydroxyphenyl)-coumarin with high intrinsic clearance.

Microorganisms: crucial players of smokeless tobacco for several health attributes.

Global consumption of smokeless tobacco (SLT) reached 300 million users worldwide majorly from middle-income countries. More than 4000 chemical compounds represent it as one of the noxious consumable products by humans. Besides toxicants/carcinogens, the heavy microbial load on smokeless tobacco further keeps human health at higher risk. Several of these inhabitant microbes participate in biofilm formation and secrete endotoxin/mycotoxins and proinflammatory-like molecules, leading to several oral diseases. Tobacco-associated bacteria exhibit their role in tobacco-specific nitrosamines (TSNAs) formation and acetaldehyde production; both are well-documented carcinogens. Moreover, tobacco exhibits the potential to alter the oral microbiome and induce dysbiotic conditions that lead to the onset of several oral and systemic diseases. Traditional cultivation approaches of microbiology provide partial information of microbial communities of a habitat; therefore, microbiomics has now been employed to study the metagenomes of entire microbial communities. In the past 5 years, few NGS-based investigations have revealed that SLT harbors four dominant phyla (Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes) dominating Bacillus spp. and/or Pseudomonas spp. However, functional characterization of their genetic elements will be a more informative attribute to understand the correlation between inhabitant microbial diversity and their relatedness concerning abundance and diseases. This review provides an update on the microbial diversity of SLT and its associated attributes in human health. KEY POINTS: • Heavy microbial load on smokeless tobacco alarms for poor oral hygiene. • Inhabitant microorganisms of SLT participate in TSNA and biofilm formation. • SLTs alter the oral microbiome and causes oral dysbiosis.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Home Smoking Bans and Urinary NNAL Levels to Measure Tobacco Smoke Exposure in Chinese American Household Pairs.

Home smoking bans can reduce tobacco smoke exposure, but little is known about the impact for Chinese American household pairs. In this study of 202 household pairs with low acculturation, 53.9% reported a home smoking ban, 31.7% had inconsistent reports, and 14.4% reported no ban. With decreasing home smoking ban enforcement, more nonsmokers had tobacco smoke exposure (66.1%–86.2%) as measured by the tobacco-specific nitrosamine biomarker urine NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol). Despite reported bans, about one-quarter of nonsmokers still reported tobacco smoke exposure at home (23.6%–30%) within the past 2 months and three-quarters reported outdoor exposure. In adjusted regression analyses of geometric mean NNAL ratios, nonsmokers in households with no ban had over two times higher levels than nonsmokers in households with a ban: adjusted log NNAL ratio = 2.70 (95% CI 1.21, 6.03). Higher smoker NNAL level and nonsmoker English fluency were also significantly associated with nonsmoker NNAL levels. Nonsmoker levels in households with an inconsistent ban were not significantly different compared to those with a ban. Although home smoking bans were generally associated with lower NNAL levels, tobacco smoke exposure in this immigrant population with low English proficiency was higher than that of the general population. From a health equity standpoint, there is a need for broader implementation and enforcement of comprehensive smoke-free policies.

Sexual identity differences in biomarkers of tobacco exposure among women in a national sample

BackgroundSexual minority women are consistently at increased risk for tobacco use compared to heterosexual women. Neither biomarkers of nicotine exposure nor biomarkers of tobacco toxicant exposure have been examined by sexual identity. MethodsThis study used interview and biomarker data from women in the biomarker core sample of Wave 1 of the Population Assessment of Tobacco and Health (PATH) study (2013–2014; n = 4930). We examined associations of sexual identity with nicotine exposure (measured with urinary cotinine and TNE-2) and with tobacco-specific nitrosamines (measured with urinary NNAL). Multivariable regression modeling was used to examine these associations among the full biomarker core sample, among past 30-day tobacco users, and among exclusive established cigarette users before and after controlling for tobacco use quantity and intensity. ResultsIn the full biomarker sample of women, prior to adjusting for tobacco use quantity and intensity, bisexual women had significantly higher cotinine, TNE-2, and NNAL levels compared to heterosexual women. Among exclusive established cigarette users, gay/lesbian women had significantly higher NNAL compared to heterosexual women prior to adjusting for tobacco quantity and intensity. No differences by sexual identity were found after adjusting for tobacco use quantity and intensity. ConclusionsThis is the first study to demonstrate differences in biological markers of tobacco exposure by sexual identity among women in the U.S. This has important public health implications as greater exposure to both nicotine and to tobacco-specific nitrosamines are strongly linked to cancer risk.