From cultivation to cancer: formation of N-nitrosamines and other carcinogens in smokeless tobacco and their mutagenic implications.

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

What Can We Learn about the Bias of Microbiome Studies from Analyzing Data from Mock Communities?

It is known that data from both 16S and shotgun metagenomics studies are subject to biases that cause the observed relative abundances of taxa to differ from their true values. Model community analyses, in which the relative abundances of all taxa in the sample are known by construction, seem to offer the hope that these biases can be measured. However, it is unclear whether the bias we measure in a mock community analysis is the same as we measure in a sample in which taxa are spiked in at known relative abundance, or if the biases we measure in spike-in samples is the same as the bias we would measure in a real (e.g., biological) sample. Here, we consider these questions in the context of 16S rRNA measurements on three sets of samples: the commercially available Zymo cells model community; the Zymo model community mixed with Swedish Snus, a smokeless tobacco product that is virtually bacteria-free; and a set of commercially available smokeless tobacco products. Each set of samples was subject to four different extraction protocols. The goal of our analysis is to determine whether the patterns of bias observed in each set of samples are the same, i.e., can we learn about the bias in the commercially available smokeless tobacco products by studying the Zymo cells model community?

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

BACKGROUND: Smokeless 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. METHODS: We 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. RESULTS: Our 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.

Characterization of Total and Unprotonated (Free) Nicotine Content of Nicotine Pouch Products.

INTRODUCTION: Nicotine pouch products, oral smokeless products that contain nicotine but no tobacco leaf material, have recently entered the US marketplace. Available data indicate sales of these products in the United States have increased since 2018; however, the extent of use among US youth and adults is uncertain. METHODS: To assay the chemistry of these emerging tobacco products, we analyzed 37 nicotine pouch brands from six total manufacturers. Almost all of the products had flavor descriptors (36 of 37), such as mint, licorice, coffee, cinnamon, and fruit. The amount of free nicotine, the form most easily absorbed, was calculated for each product using total nicotine, product pH, the appropriate pKa, and the Henderson-Hasselbalch equation. RESULTS: Nicotine pouch products varied in pouch content mass, moisture content (1.12%‒47.2%), alkalinity (pH 6.86‒10.1), and % free nicotine (7.7%‒99.2%). Total nicotine content ranged from 1.29 to 6.11 mg/pouch, whereas free nicotine ranged from 0.166 to 6.07 mg/pouch. These findings indicate that nicotine and pH levels found in some of these nicotine pouches are similar to conventional tobacco products, such as moist snuff and snus, and that most of these pouch products are flavored. CONCLUSIONS: Although these products likely lack many tobacco-related chemicals, each product analyzed contained nicotine, which is both addictive and can harm human health. Given that nicotine pouches may appeal to a spectrum of users, from novice to experienced users, it is important to include these emerging tobacco products in tobacco control research, policy, and practice. IMPLICATIONS: These "tobacco-free" nicotine pouches have similar pH and nicotine content to conventional tobacco products, such as moist snuff and snus. Although they lack many tobacco-related chemicals, most are highly flavored which could increase experimentation from new users. Given that nicotine pouches may appeal to a spectrum of users, from novice to experienced users, in terms of their flavors and nicotine content, it is important to examine and include these emerging tobacco products as they relate to tobacco control research, policy, and practice.

Smokeless tobacco products harbor diverse bacterial microbiota that differ across products and brands.

Smokeless tobacco products contain numerous chemical compounds, including known human carcinogens. Other smokeless tobacco constituents, including bacteria, may also contribute to adverse health effects among smokeless tobacco users. However, there is a lack of data regarding the microbial constituents of smokeless tobacco. Our goal was to characterize the bacterial microbiota of different smokeless tobacco products and evaluate differences across product types and brands. DNA was extracted from 15 brands of smokeless tobacco products (including dry snuff, moist snuff, snus, and Swedish snus) and 6 handmade products (e.g., toombak) using an enzymatic and mechanical lysis approach. Bacterial community profiling was performed using PCR amplification of the V1-V2 hypervariable region of the 16S rRNA gene, followed by 454 pyrosequencing of the resulting amplicons and sequence analysis using the QIIME package. Total viable counts were also determined to estimate the number of viable bacteria present in each product. Average total viable counts ranged from 0 to 9.35 × 107 CFU g-1. Analysis of the 16S rRNA gene sequences revealed high bacterial diversity across the majority of products tested: dry snuff products where characterized by the highest diversity indices compared to other products. The most dominant bacterial phyla across all products were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Significant differences in both bacterial community composition and in silico predicted gene content were observed between smokeless tobacco product types and between brands of specific smokeless tobacco products. These data are useful in order to comprehensively address potential health risks associated with the use of smokeless tobacco products.

Restoring the Duality between Principal Components of a Distance Matrix and Linear Combinations of Predictors, with Application to Studies of the Microbiome.

Appreciation of the importance of the microbiome is increasing, as sequencing technology has made it possible to ascertain the microbial content of a variety of samples. Studies that sequence the 16S rRNA gene, ubiquitous in and nearly exclusive to bacteria, have proliferated in the medical literature. After sequences are binned into operational taxonomic units (OTUs) or species, data from these studies are summarized in a data matrix with the observed counts from each OTU for each sample. Analysis often reduces these data further to a matrix of pairwise distances or dissimilarities; plotting the first two or three principal components (PCs) of this distance matrix often reveals meaningful groupings in the data. However, once the distance matrix is calculated, it is no longer clear which OTUs or species are important to the observed clustering; further, the PCs are hard to interpret and cannot be calculated for subsequent observations. We show how to construct approximate decompositions of the data matrix that pair PCs with linear combinations of OTU or species frequencies, and show how these decompositions can be used to construct biplots, select important OTUs and partition the variability in the data matrix into contributions corresponding to PCs of an arbitrary distance or dissimilarity matrix. To illustrate our approach, we conduct an analysis of the bacteria found in 45 smokeless tobacco samples.

Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis.

The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.

Characterization of SPECTRUM Variable Nicotine Research Cigarettes.

OBJECTIVE: To provide researchers an extensive characterization of the SPECTRUM variable nicotine research cigarettes. METHODS: Data on cigarette physical properties, nicotine content, harmful and potentially harmful constituents in the tobacco filler was compiled. RESULTS: Data on physical properties, concentrations of menthol, nicotine and minor alkaloids, tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, ammonia, and toxic metals in the filler tobacco for all available varieties of Spectrum research cigarettes are provided. The similarity in the chemistry and physical properties of SPECTRUM cigarettes to commercial cigarettes renders them acceptable for use in behavioral studies. Baseline information on harmful and potentially harmful constituents in research tobacco products, particularly constituent levels such as minor alkaloids that fall outside typical ranges reported for commercial, provide researchers with the opportunity to monitor smoking behavior and to identify biomarkers that will inform efforts to understand the role of nicotine in creating and sustaining addiction. CONCLUSIONS: Well characterized research cigarettes suitable for human consumption are an important tool in clinical studies for investigating the physiological impacts of cigarettes delivering various levels of nicotine, the impact of reduced nicotine cigarettes on nicotine addiction, and the relationship between nicotine dose and smoking behavior.

Comprehensive chemical characterization of Rapé tobacco products: Nicotine, un-ionized nicotine, tobacco-specific N'-nitrosamines, polycyclic aromatic hydrocarbons, and flavor constituents.

Rapé, a diverse group of smokeless tobacco products indigenous to South America, is generally used as a nasal snuff and contains substantial amount of plant material with or without tobacco. Previously uncharacterized, rapé contains addictive and harmful chemicals that may have public health implications for users. Here we report % moisture, pH, and the levels of total nicotine, un-ionized nicotine, flavor-related compounds, tobacco-specific N-nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) for manufactured and hand-made rapé. Most rapé products were mildly acidic (pH 5.17-6.23) with total nicotine ranging from 6.32 to 47.6 milligram per gram of sample (mg/g). Calculated un-ionized nicotine ranged from 0.03 to 18.5 mg/g with the highest values associated with hand-made rapés (pH 9.75-10.2), which contain alkaline ashes. In tobacco-containing rapés, minor alkaloid levels and Fourier transform infrared spectra were used to confirm the presence of Nicotiana rustica, a high nicotine tobacco species. There was a wide concentration range of TSNAs and PAHs among the rapés analyzed. Several TSNAs and PAHs identified in the products are known or probable carcinogens according to the International Agency for Research on Cancer. Milligram quantities of some non-tobacco constituents, such as camphor, coumarin, and eugenol, warrant additional evaluation.

Role of dihydrolipoamide dehydrogenase in regulation of raffinose transport in Streptococcus pneumoniae.

Streptococcus pneumoniae strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the raf operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50- to 80-fold reduction in expression of the raf operon genes aga (alpha-galactosidase) and rafEFG (raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of raf operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of aga and rafEFG, but DLDH did not regulate rafK or the putative regulatory genes rafR and rafS. DLDH also bound directly to RafK both in vitro and in vivo, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDH-negative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport in vivo.