Tobacco-specific N-nitrosamines Research Articles

Heterogeneity of Harmful Constituent Profiles in Smokeless Tobacco Products from Five African Countries.

Addictive, toxic, and carcinogenic constituents present in smokeless tobacco (SLT) products are responsible for the harmful effects associated with SLT use. There are limited data on levels of such constituents in SLT products used in Africa, a region with high prevalence of SLT use and the associated morbidity and mortality. Manufactured and custom-made SLT products were purchased from five African countries (South Africa, Uganda, Mauritania, Nigeria, and Zambia) using a standard approach for sample collection, labeling, and storage. Moisture content, pH, total and unprotonated (biologically available) nicotine, five tobacco-specific N-nitrosamines (TSNA), 10 polycyclic aromatic hydrocarbons (PAH), five metals and metalloids (As, Cd, Cr, Ni, and Pb), nitrate, and nitrite were analyzed. A total of 54 samples representing 15 varieties of manufactured SLT products and 13 varieties of custom-made SLT products were purchased and analyzed. In all samples, the total nicotine ranged from 1.6 to 20.5 mg/g product and unprotonated nicotine accounted for 5.3-99.6% of the total nicotine content. The sum of all five TSNA ranged from 1.6 to 100 μg/g product, with significant within-country variations observed across both the manufactured and custom-made varieties. Significant variations were also found for PAH, metals and metalloids, nitrates, and nitrites. This is the most comprehensive report on the chemical profiling of products from African countries. This is also the first study illustrating the variability of harmful constituents within the same types and brands of African SLT. Our findings emphasize the need for consumer education and interventions to reduce SLT use in Africa. The data reported here can be useful to regulators in considering measures to prevent the occurrence of high levels of known toxicants and carcinogens in manufactured products.

Brain distribution and metabolic profiling of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats investigated by UHPLC-HRMS/MS following peripheral administration

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known to be a tobacco-specific N-nitrosamine and has peripheral carcinogenic properties. It can also induce oxidative stress, glial cell activation, and neuronal damage in the brain. However, the distribution and metabolic characteristics of NNK in the central nervous system are still unclear. Here, a sensitive and effective UHPLC-HRMS/MS method was established to identify and investigate the metabolites of NNK and their distribution in the rat brain. In addition, the pharmacokinetic profiles were simultaneously investigated via blood-brain synchronous microdialysis. NNK and its seven metabolites were well quantified in the hippocampus, cortex, striatum, olfactory bulb, brain stem, cerebellum, and other regions of rat brain after peripheral exposure (5mg/kg, i.p.). The average content of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in all brain regions was at least threefold higher than that of NNK, indicating a rapid carbonyl reduction of NNK in the brain. Lower concentrations of pyridine N-oxidation products in the cortex, olfactory bulb, hippocampus, and striatum might be related to the poor detoxification ability in these regions. Compared to α-methyl hydroxylation, NNK and NNAL were more inclined to the α-methylene hydroxylation pathway. Synchronous pharmacokinetic results indicated that the metabolic activity of NNK in the brain was different from that in the blood. The mean α-hydroxylation ratio in the brain and blood was 0.037 and 0.161, respectively, which indicated poor metabolic activity of NNK in the central nervous system.

Smokeless tobacco: a ticking time bomb for Pakistan

Oral cancers are one of the most prevalent cancers worldwide with almost 300,000 cases reported annually1. In Pakistan, it is the leading type of cancer in men and women after breast cancer. Each year around 16,000 new cases of oral cancers are documented with 5500 cases resulting in death2. There are multiple etiological factors for oral cancers among which the use of smokeless tobacco (SLT) is at the top of the list. SLT is available in different consumable forms such as chewable tobacco, betel leaf, snuff (naswar), Betelnut, etc., which contain a number of carcinogenic substances3. Studies have revealed that naswar is the most common etiological factor of oral cancers among the Pakistani population as compared to the other forms of SLT3. The basic constituents of naswar are slaked lime, powdered tobacco, and indigo which contain more potent carcinogens such as unionized nicotine and tobacco-specific N-nitrosamines, that are specifically harmful to the oral cavity4. It can be used through different routes either inhaled nasally or by placing inside the mouth under the tongue. The nicotine is absorbed into the oral mucosa which is further aided by the increased pH of the mouth by slaked lime5. It has been observed historically that the use of SLT products is very commonly acceptable in Pakistani culture3. The sale and production of naswar are not well regulated and it is being sold by different vendors at cheaper prices and in different package sizes with no such instructions and health warnings written on it1,2. Moreover, some scientific data implicates it as replacement therapy for tobacco smoke cessation as it is a cheaper nicotine substitute1. In 2005, Pakistan formalized the WHO “Framework Convention on Tobacco Control (FCTC),” which addresses the details of the regulation and control of tobacco and SLT products by regulating its price, taxation, packaging, labeling, advertisement, promotion, and sponsorship5. In the last few years, Pakistan has made significant progress in smoking cessation policies but regulation of STL production and taxing is still lagging5,6. A case-control study, conducted in Khyber Pakhtunkhwa, Pakistan, revealed that 68% (men) and 38% (women) of oral cancer patients in Pakistan are due to naswar use. It was also shown that those who used naswar ever in their lifetime have 20 times more risk to develop oral cancers compared with the nonusers of naswar1. Thus, the increasing incidence of oral cancers with the relative use of naswar sets a ground basis for the implementation of strict policies regarding the manufacture and sales of SLT. Studies have provided shreds of evidence that there is a marked decrease in tobacco product use among young people by applying a ban on their promotion and advertisement. Health warnings and relevant constituents written on packages can also be an important tool for minimizing SLT use6. Sticking firmly to the FCTC protocols of smokeless tobacco use is the dire need of time. Furthermore, awareness regarding the harmful effects of STL use should prevail on urban and rural levels as there is a great lack of knowledge among the local population. Ethical approval None. Sources of funding None. Author contribution S.F., O.A.S., G.S., and A.K.: wrote the manuscript. O.A.S. and S.A.: review editing, formatting, and referencing. Conflict of interest disclosure The authors declare that they have no financial conflict of interest with regard to the content of this report. Research registration unique identifying number (UIN) N/A. Guarantor Omer Ahmed Shaikh. Financial Disclosure The author(s) received no financial support for the research, authorship, and/or publication of this article.

Does the Red Shift in UV-Vis Spectra Really Provide a Sensing Option for Detection of N-Nitrosamines Using Metalloporphyrins?

N-nitrosamines are widespread cancerogenic compounds in human environment, including water, tobacco products, food, and medicinal products. Their presence in pharmaceuticals has recently led to several recalls of important medicines from the market, and strict controls and tight limits of N-nitrosamines are now required. Analytical determination of N-nitrosamines is expensive, laborious, and time-inefficient making development of simpler and faster techniques for their detection crucial. Several reports published in the previous decade have demonstrated that cobalt porphyrin-based chemosensors selectively bind N-nitrosamines, which produces a red shift of characteristic Soret band in UV-Vis spectra. In this study, a thorough re-evaluation of metalloporphyrin/N-nitrosamine adducts was performed using various characterization methods. Herein, we demonstrate that while N-nitrosamines can interact directly with cobalt-based porphyrin complexes, the red shift in UV-Vis spectra is not selectively assured and might also result from the interaction between impurities in N-nitrosamines and porphyrin skeleton or interaction of other functional groups within the N-nitrosamine structure and the metal ion within the porphyrin. We show that pyridine nitrogen is the interacting atom in tobacco-specific N-nitrosamines (TSNAs), as pyridine itself is an active ligand and not the N-nitrosamine moiety. When using Co(II) porphyrins as chemosensors, acidic and basic impurities in dialkyl N-nitrosamines (e.g., formic acid, dimethylamine) are also UV-Vis spectra red shift-producing species. Treatment of these N-nitrosamines with K2CO3 prevents the observed UV-Vis phenomena. These results imply that cobalt-based metalloporphyrins cannot be considered as selective chemosensors for UV-Vis detection of N-nitrosamine moiety-containing species. Therefore, special caution in interpretation of UV-Vis red shift for chemical sensors is suggested.

Investigation of exposure biomarkers in human plasma following differing levels of tobacco-specific N-nitrosamines and nicotine in cigarette smoke

Tobacco-specific N-nitrosamines (TSNAs) are strong carcinogens widely found in tobacco products, environmental tobacco smoke, lake, and wastewater. The main objective of this study was to investigate the effects of cigarette smoke with different yields of TSNAs (NNK, NNN, NAT, NAB) and nicotine on the levels of biomarkers of exposure in smokers’ plasma. Three hundred healthy volunteers were recruited comprising 60 smokers of each of 3 mg, 8 mg and 10 mg ISO tar yield cigarettes and 60 smokers who smoked 10 mg, 8 mg, and 3 mg for 14 days sequentially and 60 non-smokers. All study participants were male, aged from 21 to 45 years old, and were recruited from a same unit in Hebei, China. We measured the levels of NNAL, NAT, NNN, NAB and cotinine in plasma from 240 smokers and 60 non-smokers using a novel method established by online two-dimensional solid phase extraction-liquid chromatography-tandem mass spectrometry. The results showed that NNAL, NAT, NNN, NAB and cotinine in the plasma of smokers smoking cigarette with low TSNAs and nicotine were lower than that with high TSNAs and nicotine. When smokers switched from higher to lower TSNA yields of cigarettes, their plasma NNAL, NAT, NNN, NAB levels significantly decreased. The plasma concentrations of NNAL were significantly correlated with those of cotinine, NNN, NAT and NAB for smokers (p < 0.001). Similarly, the plasma concentrations of cotinine were significantly correlated with those of NNN, NAT and NAB for smokers (p < 0.001). The plasma NNAL, NAT, NNN, NAB and cotinine levels for smokers were significantly higher than those for non-smokers. These findings suggested that the total NNAL, NNN, NAT, NAB and cotinine in plasma were valid and reliable biomarkers for human exposure to TSNAs and nicotine.

Associations between microbial communities and key chemical constituents in U.S. domestic moist snuff

BackgroundSmokeless tobacco (ST) products are widely used throughout the world and contribute to morbidity and mortality in users through an increased risk of cancers and oral diseases. Bacterial populations in ST contribute to taste, but their presence can also create carcinogenic, Tobacco-Specific N-nitrosamines (TSNAs). Previous studies of microbial communities in tobacco products lacked chemistry data (e.g. nicotine, TSNAs) to characterize the products and identify associations between carcinogen levels and taxonomic groups. This study uses statistical analysis to identify potential associations between microbial and chemical constituents in moist snuff products.MethodsWe quantitatively analyzed 38 smokeless tobacco products for TSNAs using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and nicotine using gas chromatography with mass spectrometry (GC-MS). Moisture content determinations (by weight loss on drying), and pH measurements were also performed. We used 16S rRNA gene sequencing to characterize the microbial composition, and additionally measured total 16S bacterial counts using a quantitative PCR assay.ResultsOur findings link chemical constituents to their associated bacterial populations. We found core taxonomic groups often varied between manufacturers. When manufacturer and flavor were controlled for as confounding variables, the genus Lactobacillus was found to be positively associated with TSNAs. while the genera Enteractinococcus and Brevibacterium were negatively associated. Three genera (Corynebacterium, Brachybacterium, and Xanthomonas) were found to be negatively associated with nicotine concentrations. Associations were also investigated separately for products from each manufacturer. Products from one manufacturer had a positive association between TSNAs and bacteria in the genus Marinilactibacillus. Additionally, we found that TSNA levels in many products were lower compared with previously published chemical surveys. Finally, we observed consistent results when either relative or absolute abundance data were analyzed, while results from analyses of log-ratio-transformed abundances were divergent.

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.

Improving on estimates of the potential relative harm to health from using modern ENDS (vaping) compared to tobacco smoking

BackgroundAlthough the harm to health from electronic nicotine delivery systems (ENDS) compared to smoked tobacco remains highly uncertain, society and governments still need to know the likely range of the relative harm to inform regulatory policies for ENDS and smoking.MethodsWe identified biomarkers with specificity of association with different disease groupings e.g., volatile organic compound (VOCs) for chronic obstructive pulmonary disease; and tobacco-specific N´-nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) for all cancers. We conducted a review of recent studies (post January 2017) that compared these biomarkers between people exclusively using ENDS and those exclusively smoking tobacco. The percentage differences in these biomarkers, weighted by study size and adjusted for acrolein from other sources, were used as a proxy for the assumed percentage difference in disease harm between ENDS and smoking. These relative differences were applied to previously modelled estimates of smoking-related health loss (in health-adjusted life-years; HALYs).ResultsThe respective relative biomarker levels (ENDS vs smoking) were: 28% for respiratory diseases (five results, three studies); 42% for cancers (five results, four studies); and 35% for cardiovascular (seven results, four studies). When integrated with the HALY impacts by disease, the overall harm to health from ENDS was estimated to be 33% that of smoking.ConclusionsThis analysis, suggests that the use of modern ENDS devices (vaping) could be a third as harmful to health as smoking in a high-income country setting. But this estimate is based on a limited number of biomarker studies and is best be considered a likely upper level of ENDS risk given potential biases in our method (i.e., the biomarkers used being correlated with more unaccounted for toxicants in smoking compared to with using ENDS).

An in Vitro Study of Constituents Released from Smokeless Tobacco Products into Human Saliva.

Smokeless tobacco (ST) products are used worldwide, and consumption is increasing in the USA. Although ST products are considered to occupy a different position on the tobacco product continuum of risk compared to combusted tobacco products, they can still lead to health problems, including cancer, dental problems and changes in heart rate and blood pressure. Therefore, the determination of harmful and potentially harmful constituents released from ST products into human saliva is important. Four certified reference ST products were tested in this study: loose leaf chewing tobacco (3S1), Swedish-style snus (1S4), snus (1S5) and moist snuff (3S3). These certified reference ST products are manufactured for research purposes, not for human consumption. The reference ST products were used in this study because they have been well characterized and are intended and designed to represent commercial ST products. The reference ST products were incubated in human saliva at 37°C with a range of incubation times for the evaluation of constituents released from these products into human saliva. In this study, alkaloids (nicotine and cotinine), tobacco-specific N-nitrosamines (TSNAs) (N'-nitrosornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) and benzo[α]pyrene (B[α]P) in the reference ST products and saliva samples were determined by gas chromatography--mass spectrometry (GC--MS), gas chromatography--flame ionization detection (GC--FID), or ultra-performance liquid chromatography--tandem mass spectrometry (UPLC--MS-MS). Our results indicate that the amounts of each constituent released from the reference ST products were altered by the tobacco cut size and product format (pouched or unpouched). The constituents (TSNAs and alkaloids) in moist snuff and loose leaf chewing tobacco were released faster compared to those in Swedish-style snus and snus. B[α]P was only detected in reference moist snuff samples, and only 3.4% of the total B[α]P was released into human saliva after incubation for 60 min, whereas higher percentages of total TSNAs and alkaloids were released at different rates from the four reference ST products.

Correlation between smokeless tobacco (Gutkha) and biomarkers of oxidative stress in plasma with cardiovascular effects

Tobacco products are widely consumed around the world in smoking and smokeless tobacco (SLT) forms. Analysis of smokeless tobacco consumption suggested that the effects of nicotine and tobacco-specific N-nitrosamines, the main ingredients of smokeless tobacco are attractive to study because its consumption often results in biochemical changes of plasma parameters and markers of oxidative stress development. Smokeless tobacco users generally consume the most commonly available SLT products like khaleja brand of gutkha and mahak chaini brand of khaini 3–5 times per day. We found a significant increase in plasma glucose levels, total cholesterol, triglycerides, and a significant decrease in high-density lipoprotein (HDL) cholesterol indicative of atherosclerosis risk. We also found that the plasma peroxynitrites (ONOO−), nitric oxide (NO), lipid peroxidation (LPO), and protein carbonyls (PCO) levels were significantly elevated. Plasma nicotine and cotinine levels were significantly elevated in study subjects, suggesting that nicotine could be responsible for the oxidative and nitrosative stress indirectly inducing cardiovascular risk. There was a strong correlation of nicotine with reactive oxygen species (ROS), reactive nitrogen species (RNS), cholesterol, and creatinine in exposed smokeless tobacco (gutkha) consumers. These data demonstrate SLT users are at high cardiovascular risk due to nicotine-induced free radicals and oxidative damage.

Mitochondrial Transcription Factor A Binds to and Promotes Mutagenic Transcriptional Bypass of O4-Alkylthymidine Lesions.

O2- and O4-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a quantitative proteomic method to discover regioisomeric O2- and O4-n-butylthymidine (O2- and O4-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O2-nBudT- and O4-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O2-nBudT-containing DNA, whereas three high-mobility group (HMG) proteins (i.e., HMGB1, HMGB2, and mitochondrial transcription factor A (TFAM)) exhibit preferential binding to O4-nBudT-bearing DNA. We further demonstrated that TFAM binds directly and selectively with O4-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O4-nBudT and O4-pyridyloxobutylthymidine, which is a DNA adduct induced by tobacco-specific N-nitrosamines, in vitro and in human cells. Together, we explored, for the first time, the interaction proteomes of O-alkyldT lesions, and our study expanded the functions of TFAM by revealing its capability in the recognition of O4-alkyldT-bearing DNA and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.

Smokeless Tobacco Products (STPs) Harbour Bacterial Populations with Potential for Oral Carcinogenicity.

Introduction:Smokeless Tobacco Products (STPs) vary significantly in their carcinogenicity, a feature accredited to the variation in the concentrations of carcinogenic chemicals. Tobacco associated bacteria are known to produce Tobacco-specific N-nitrosamines (TSNAs) and hence are determinants of TSNA levels in Tobacco. The primary objective of this study was to conduct a microbiological survey of STPs and to provide a baseline information of the bacterial communities present in the STPs. Materials and Methods:The present study analyzed the constituency of microbial communities in 7 different smokeless Tobacco products including four chewable (T1_CW to T4_CW), two snus (T5_Snus and T6_Snus) and one snuff sample (T7_Snuff) using high-throughput sequencing of the 16S rRNA based next generation sequencing. The Tobacco samples were also analyzed for pH and moisture content. Statistical analysis of the data obtained was done using SPSS software version 20. Pearson’s Correlation was done to analyze the correlation between pH and moisture content of the Tobacco samples. Results:A total of 11 phyla were identified in all smokeless Tobacco products. A total of 36 classes were identified across all smokeless Tobacco products and bacilli was the predominant class in all the products followed by Actinobacteria and Bacteroidia. In species level, a total of 2369 species were identified across all smokeless Tobacco products. In T1 chewable Tobacco products, predominant species was staphylococcus whereas in T2 and T3, Bacillus subtilis and pumilus were predominant. In T4 chewable Tobacco product, Virgibacillus was predominant followed by halodentrificans, staphylococcus epidermidis. In snus 1 and 2, Bacillus pumilus and subtilis were predominant. In snuff, Bacillus cereus was predominant. Snus products had the highest moisture content (15.4% and 14.3%) compared to the chewable Tobacco and snuff products. The snus products analyzed had alkaline values (pH 8.50 and 8.15) and snuff and chewable Tobacco had acidic values ranging from 5.62 to 6.09. Conclusion:The current study demonstrates that ST products differ qualitatively, quantitatively, and in their bacterial composition. There is a possibility that some of these species may contribute to oral carcinogenesis, either by influencing levels of TSNAs or directly inducing chronic inflammation.

Chemical analysis of snus products from the United States and northern Europe.

IntroductionSnus is an oral tobacco product that originated in Sweden. Snus products are available as fine-cut loose tobacco or in pre-portioned porous “pouches.” Some snus products undergo tobacco pasteurization during manufacturing, a process that removes or reduces nitrite-forming microbes, resulting in less tobacco-specific nitrosamine content in the product. Some tobacco companies and researchers have suggested that snus is potentially less harmful than traditional tobacco and thus a potential smoking cessation aid or an alternative to continued cigarette consumption. Although snus is available in various countries, limited information exists on snus variants from different manufacturers.MethodsMoisture, pH, nicotine, and tobacco-specific N’-nitrosamines (TSNAs) were quantified in 64 snus products made by 10 manufacturers in the United States and Northern Europe (NE). Reported means, standard errors, and differences are least-square (LS) estimates from bootstrapped mixed effects models, which accounted for correlation among repeated measurements. Minor alkaloids and select flavors were also measured.ResultsAmong all product types, moisture (27.4%–59.5%), pH (pH 5.87–9.10), total nicotine (6.81–20.6 mg/g, wet), unprotonated nicotine (0.083–15.7 mg/g), and total TSNAs (390–4,910 ng/g) varied widely. The LS-mean unprotonated nicotine concentration of NE portion (7.72 mg/g, SE = 0.963) and NE loose (5.06 mg/g, SE = 1.26) snus were each significantly higher than US portion snus (1.00 mg/g, SE = 1.56). Concentrations of minor alkaloids varied most among products with the highest total nicotine levels. The LS-mean NNN+NNK were higher in snus sold in the US (1360 ng/g, SE = 207) than in NE (836 ng/g, SE = 132) countries. The most abundant flavor compounds detected were pulegone, eucalyptol, and menthol.ConclusionPhysical and chemical characteristics of US and NE products labeled as snus can vary considerably and should not be considered “equivalent”. Our findings could inform public health and policy decisions pertaining to snus exposure and potential adverse health effects associated with snus.

Smokeless tobacco induced biophysical and biochemical alterations in the plasma, erythrocytes, and platelets of panmasala users: Subsequent biological effects

Aim & backgroundSmokeless tobacco (SLT) products are extensively consumed throughout the world including India. These products act as the primary addictive agents, due to the presence of nicotine among other tobacco products to humans and animals and its quitting is difficult. Higher the exposure of SLT products more is the toxic effects and alterations in erythrocytes and platelets. ObjectivesThe products of smokeless tobacco could cause increase in the concentrations of oxidants (free radicals), decrease the activities antioxidant enzymes, activate the process of programmed cell death through enhanced expression of inducible nitric oxide synthase. Smokeless tobacco products represent a major modifiable risk factor for the development of redox imbalance through the enhanced production of reactive oxygen species and diminished activities of antioxidant enzymes in plasma, bio-membranes of erythrocytes, and platelets and induction of apoptosis in the blood. Materials and methodsThe protein expression of inducible nitric oxide synthase (iNOS) was studied by western blot and gene expression of apoptotic proteins, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) was evaluated by RT-PCR technique. Membrane fluidity of erythrocytes and platelets was studied by the fluorescence method. ResultsThe results of the present study revealed that significantly elevated levels of iNOS enzyme in plasma, erythrocyte, and platelet membranes of panmasala users. We found that gene expression levels of Bcl2, Bax, IL-6, caspase proteins (Caspase 8, Caspase 10, and Caspase 12) are greater and decreased levels of TNF-α with no significant change in blood of smokeless tobacco users in comparison with normal controls. In addition, there were substantial significantly higher in concentrations of nicotine, cotinine, and epinephrine in the plasma of panmasala users than non-tobacco users. Panmasala can be caused a significant increase in nitroxidative stress marker (LPO, NO, and ONOO−) values and significant decrease in the levels of antioxidant enzymes in erythrocytes and platelets. ConclusionOn the basis of the present study results, it may be concluded that the chronic use of panmasala than any smokeless tobacco products may be a contributory risk factor or may give conclusive idea and has been associated with the expansion of the development of structural and functional alterations in the erythrocyte and platelet membranes induced oxidative damage and apoptosis, possibly further enhanced by nicotine and tobacco-specific N-nitrosamines. SLT exposure had implicated a threat and enormous implications on public health and is required to prove that may not be viewed as a safe alternative to any tobacco products.

Quantification of Nicotine and Cotinine in Plasma, Saliva, and Urine by HPLC Method in Chewing Tobacco Users.

Background:Nicotine acts as major alkaloid of all tobacco products including smokeless tobacco (SLT) forms. The mode of SLT consumption is in the form of chewing under the cheek or lip and induced biochemical alterations in the plasma, saliva, and urine. Materials and Methods:The smokeless tobacco products like Raja or blue bull tobacco brands are widely consumed by human male volunteers under the age of 18-30 years for the period of 3 years consisting of 30g per day. The concentrations of nicotine and cotinine in samples of plasma, saliva, and urine are quantified by the method of HPLC. The remaining variables of plasma are evaluated by auto analyzer and spectrophotometric methods. Results:The analysis of results presented that significant increase in the levels of nicotine and cotinine in plasma, saliva, and urine of chewing tobacco users. The lipid profile (Cholesterol, triglycerides, HDL-C, and LDL-C), liver marker enzymes (SGOT, SGPT, and ALP), kidney markers (Creatinine, urea, and uric acid), glucose, and the remaining variables are present within normal range observed in SLT users. The lipid peroxidation (LPO), nitric oxide (NO) (NO2 and NO3), protein carbonyls (PCO), and peroxynitrites (ONOO-) are reported to be higher levels in the plasma of experimental subjects in comparison with normal controls. The various brands of tobacco varieties (Raja, madhu chhap, hans chhap, miraj, badshah, blue bull, and swagat gold tobacco) are presented. Conclusion:The chewing tobacco users exhibited greater amounts of nicotine and cotinine are at risk of cardiovascular due to nicotine has cardiovascular effects, and oral cancer disease complications in the future for chronic consumption of smokeless tobacco products due to the presence of carcinogens of tobacco-specific N-nitrosamines.

The Bacterial Communities of Little Cigars and Cigarillos Are Dynamic Over Time and Varying Storage Conditions.

Despite their potential importance with regard to tobacco-related health outcomes, as well as their hypothesized role in the production of tobacco-specific N-nitrosamines, bacterial constituents of tobacco products lack characterization. Specifically, to our knowledge, there has been no comprehensive characterization of the effects of storage conditions on the bacterial communities associated with little cigars and cigarillos. To address this knowledge gap, we characterized the bacterial community composition of the tobacco and wrapper components of the following four products: Swisher Sweets Original; Swisher Sweets, Sweet Cherry; Cheyenne Cigars Full Flavor 100’s; and Cheyenne Menthol Box. Each product was stored under three different conditions of temperature and relative humidity to mimic different user storage conditions: room (20°C 50% RH), refrigerator (5°C 18% RH) and pocket (25°C 30% RH). On days 0, 5, 9 and 14, subsamples were collected, the wrapper and tobacco were separated, and their total DNA was extracted separately and purified. Resulting DNA was then used in PCR assays targeting the V3 V4 region of the bacterial 16S rRNA gene, followed by sequencing using Illumina HiSeq 300bp PE. Resulting sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package, followed by analyses in R using the Phyloseq and Vegan packages. A single bacterial phylum, Firmicutes, dominated in the wrapper subsamples whereas the tobacco subsamples were dominated by Proteobacteria. Cheyenne Menthol Box (CMB) samples were characterized by significant differential abundances for 23 bacterial operational taxonomic units (OTUs) in tobacco subsamples and 27 OTUs in the wrapper subsamples between day 0 and day 14 under all conditions. OTUs from the genera Acinetobacter and Bacillus significantly increased in the CMB tobacco subsamples, and OTUs from Bacillus, Streptococcus, Lactobacillus, and Enterococcus significantly increased in the CMB wrapper subsamples over time. These initial results suggest that the bacterial communities of little cigars and cigarillos are dynamic over time and varying storage conditions.

Magnetic solid-phase extraction of tobacco-specific N-nitrosamines using magnetic graphene composite as sorbent.

Tobacco-specific N-nitrosamines are carcinogenic components in mainstream cigarette smoke. To explore tobacco-specific N-nitrosamine release levels in cigarettes, a magnetic solid-phase extraction procedure using magnetic graphene composite as sorbent for fast enrichment of tobacco-specific N-nitrosamine was developed. Under optimal conditions, a tobacco-specific N-nitrosamine determination method was successfully proposed by combining magnetic solid-phase extraction procedure and high-performance liquid chromatography coupled with tandem mass spectrometry. The method's limit of detection for tobacco-specific N-nitrosamines in mainstream cigarette smoke ranged from 0.018 to 0.057ng/cigarette. Good linearities were obtained with correlation coefficients above 0.9992. The accuracies of tobacco-specific N-nitrosamines in a spiked mainstream cigarette smoke sample were from 89.3 to 109.4%, with a relative standard deviation of less than 11.2%. The proposed method has the merits of rapidity and high sensitivity. Finally, the method was successfully applied to tobacco-specific N-nitrosamine analysis in real samples.

Highly Efficient Degradation of Tobacco Specific N‐Nitrosamines by TiO2 Mesocrystals with Robust and Tailored Microporous Structure

AbstractIt is essential to control tobacco specific N‐nitrosamines (TSNAs) pollution for the protection of public health, which were identified as the most abundant carcinogens in tobacco and tobacco smoke. Herein, we provide microporous TiO2 mesocrystals as a new material for the remove of TSNAs with high degradation efficiency. The anatase TiO2 mesocrystals with tunable microporous structure were synthesized by a facile method under the acid solution. The TiO2 mesocrystals were firstly used to the remove of TSNAs through photocatalytic degradation. It's exciting that TSNAs of tobacco extraction solution can be almost entirely reduced (up to 97%) by anatase TiO2 mesocrystals after 30 mins through photocatalytic degradation. Moreover, it's found that the microporous size of mesocrystals play a key role on the degradation capability. The TiO2 mesocrystals with microporous size comparable to the size of TSNAs molecule exhibit the best degradation performance. This finding offers a robust microporous material and efficient method for removing the carcinogen like TSNAs as well as other pollutants in environment.

Identification of 5-hydroxymethylfurfural in cigarette smoke extract as a new substrate metabolically activated by human cytochrome P450 2A13

Cytochrome P450 2A13 (CYP2A13) is an extrahepatic enzyme mainly expressed in the human respiratory system and is reported to mediate tobacco-specific N-nitrosamines (TSNA) metabolism in cigarette smoke. This study aimed to identify other new substrates of CYP2A13 in cigarette smoke and their corresponding respiratory toxicity. Following separation by HPLC, GC–MS/MS, NMR and cytotoxicity assays in BEAS-2B cells stably expressing CYP2A13 (B-2A13), 5-Hydroxymethylfurfural (5-HMF) was screened and identified in the 4–5 min section of cigarette smoke extract (CSE). In vitro metabolism results showed that CYP2A13 mediated the fast clearance of 5-HMF and formed the metabolite 5-HMF acid (5-HMFA). CSE 5-HMF (CSE-5-HMF) showed cytotoxicity similar to that of standard 5-HMF in B-2A13 and B-2A5 cells, which was inhibited by 8-methoxypsoralen (8-MOP), a CYP enzyme inhibitor. Mouse CYP2A5, a homologous CYP enzyme to CYP2A13, shares many substrates with CYP2A13 in cigarette smoke. Thus, CYP2A5−/− mice were generated to explore the role of CYP2A5 in 5-HMF bioactivation. Compared with CYP2A5−/− mice, WT mice showed serious histological lung and nasal olfactory mucosa damage, as well as increased inflammatory cells and elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid. Besides, nasal microsomes undertook fast 5-HMFA formation in WT mice than that in CYP2A5−/− mice, which could be inhibited by 8-MOP. This study is the first to identify 5-HMF as a new toxic substrate of human CYP2A13 in cigarette smoke, it may play a potential role in cigarette smoke-induced respiratory injuries.

Solid‐phase extraction of tobacco‐specific N‐nitrosamines with a mixed‐mode hydrophobic/cation‐exchange sorbent

AbstractA mixed‐mode hydrophobic/cation‐exchange sorbent was used for the solid‐phase extraction of tobacco‐specific N‐nitrosamines. Several parameters including sample solution pH, washing solution, type and volume of desorption solution, and amount of sorbent were investigated. By combining solid‐phase extraction and high‐performance liquid chromatography and tandem mass spectrometry, we successfully developed a method for tobacco‐specific N‐nitrosamines determination. The limit of detection for target analytes in our method ranged from 0.012 to 0.037 ng/mL, and the correlation coefficients were larger than 0.9994 for all target analytes indicating good linearities. Intra‐day (n = 5) and inter‐day (n = 3) precision showed good reproducibility, with relative standard deviations less than 11.7 and 10.9%, respectively. The results show that our method is capable of measuring tobacco‐specific N‐nitrosamines in tobacco, smokeless tobacco products and mainstream cigarette smoke. No significant deviation was found between our method and a similar analysis using standard methods.