Six-Week Actual Use Study to Evaluate the Impact of Oral Tobacco-Derived Nicotine Pouches on Cigarette Smoking and Smokeless Tobacco Product Use Behaviors

Background Oral tobacco-derived nicotine (OTDN) products are a rapidly emerging, innovative category of noncombustible tobacco products. These products are tobacco-leaf free and do not contain or generate many of the harmful and potentially harmful constituents (HPHCs) found in combustible cigarettes or smokeless tobacco (ST) products. OTDN products may therefore offer adult tobacco consumers a potentially reduced harm alternative. However, little is known about OTDN product use patterns or their ability to substitute for more harmful tobacco products.Methods We conducted an open-label, 2-phase, actual use study designed to characterize OTDN pouch product (on!® nicotine pouches - NP) use patterns, including impact on other tobacco use behaviors among adult tobacco consumers interested in using NP. Adult current cigarette smokers (AS) and/or adult ST users (ASTU) (n = 1,147 complete) who were not planning to quit were offered free choice of a portfolio of NP (seven flavors at five nicotine levels) to use at-home, ad-libitum, for 6-weeks following an initial 5-day trial. Participants responded to daily electronic surveys that captured the amount, frequency, and topography (e.g., time in mouth, placement in mouth) of NP and other tobacco product use. Results The majority (99 to 100%) of study participants used NP throughout the 6-week period. Participants used ~5-6 pouches/day of a variety of flavors and nicotine levels. At the end of 6 weeks, a modest proportion of AS (27%) and a substantial proportion of ASTU (71%) reported no use of cigarettes or ST respectively, while reporting continued use of NPs. Additionally, 39% of AS and 14% of ASTU reduced daily consumption of cigarettes or ST products, respectively by 50-99%. Conclusions Participants found NPs acceptable with a sizeable proportion completely switching away from or substantially reducing (50-99%) their cigarette or ST consumption by week 6. Availability of a variety of flavors and nicotine levels appeared to facilitate the conversion to NP. These data suggest that, under the conditions of the study, NP can be a potential substitute for cigarettes or ST products and may offer harm reduction potential for adult smokers and smokeless tobacco users not interested in quitting.Trial Registration Note: This study is an observational study with tobacco products currently available in the marketplace for adult tobacco consumers. Since there is no intervention per se and we did not collect any biosamples this is not a “clinical study” and there is no appropriate registry for such behavioral observational studies, we do not believe that this requirement is applicable.

Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

E-cigarette, combustible, and smokeless tobacco product use combinations among youth in the United States, 2014-2019

ABSTRACTIntroductionYouth e-cigarette use has been rising, however U.S. prevalence data are generally reported without disaggregating by individuals’ use of other tobacco products. It is not clear how the proportion of youth e-cigarette users naïve to all combustible tobacco is changing.MethodsAnnual prevalence estimates of ever and current (defined as past 30-day use) tobacco use prevalence by school type are reported using the 2014-2019 National Youth Tobacco Surveys (NYTS) with mutually exclusive categories of e-cigarette, smokeless tobacco, and/or combustible tobacco product use. T-tests were used to compare annual estimates with the preceding year. The annual percent change (APC) for each category from 2014-2018 were analyzed using JoinPoint regression. Data for 2019 were reported separately due to the change in survey format from paper to electronic.ResultsCurrent use of only e-cigarettes among HS students who never used combustible tobacco increased significantly from 2014-2018 (APC = +42.4%, 95% CI: 0.7, 101.3); by 2019, prevalence peaked at 9.2% (95% CI: 8.2, 10.2) among never combustible users and 8.3% (95% CI: 7.3, 9.3) among former combustible users. This coincided with significant declines in use of only combustible tobacco (APC=-14.5%, 95% CI: −18.3, −10.5).ConclusionsUse of only e-cigarettes among US youth with no history of combustible tobacco use has increased substantially over time, even as combustible tobacco use continues to plummet. Of the 17.5% (95% CI: 15.7, 19.0) of HS students who currently used only e-cigarettes (but not other tobacco) in 2019, more than half have no history of combustible tobacco use.

Effect of Smokeless Tobacco Product, Afzal, on the Reproductive Hormones and Gonadal Pathology of Wistar Rats

Afzal is a common smokeless tobacco product (STP) in Oman, and it is believed to contain toxins that may affect the reproductive hormones and hence reproductive function. This study assessed the effect of Afzal on the gonads of Wistar rats. In order to assess gonad toxicity induced by this STP, an aqueous extract of Afzal was added to drinking water to be administrated orally to Wistar albino rats (n = 72) classified as young (4 weeks old) and adult (20 weeks old) of both genders weighing between 60-80 g and 150-240 g respectively for 8 weeks. The rats were divided into 3 groups; control (received distilled water instead of Afzal extract), low-dose (received 3 mg nicotine/kg body weight/day) and high-dose (received 6 mg nicotine/kg body weight/day). At the termination of the study, the rats were euthanized and their blood samples and ovaries were collected for biochemical and histopathological investigations. Testosterone and estradiol hormones showed a significant decrease (P<0.05( in Afzal-treated groups (low and high doses) compared with the control. Histopathological findings revealed the damaging effects manifested as a reduction in the number of the germ cells with deformed organization and in fatty and fibrous degenerations in testes and ovaries. Afzal was found to have adverse effect on the reproductive hormones and gonadal pathology in Wistar rats of both genders, and hence users of Afzal need to consider the risk associated with its frequent use.

An exploration of smokeless tobacco product nucleic acids: a combined metagenome and metatranscriptome analysis

Smokeless tobacco (ST) products are used worldwide and are a major public health concern. In addition to harmful chemicals found in these products, microbes found in ST products are believed to be responsible for generating harmful tobacco-specific nitrosamines (TSNAs), the most abundant carcinogens in ST. These microbes also contribute endotoxins and other pro-inflammatory components. A greater understanding of the microbial constituents in these products is sought in order to potentially link select design aspects or manufacturing processes to avoidable increases in harmful constituents. Previous studies looked primarily at bacterial constituents and had not differentiated between viable vs nonviable organisms, so in this study, we sought to use a dual metatranscriptomic and metagenomic analysis to see if differences exist. Using high-throughput sequencing, we observed that there were differences in taxonomic abundances between the metagenome and metatranscriptome, and in the metatranscriptome, we also observed an abundance of plant virus RNA not previously reported in DNA-only studies. We also found in the product tested, that there were no viable bacteria capable of metabolizing nitrate to nitrite. Therefore, the product tested would not be likely to increase TSNAs during shelf storage. We tested only a single product to date using the strategy presented here, but succeeded in demonstrating the value of using of these methods in tobacco products. These results present novel findings from the first combined metagenome and metatranscriptome of a commercial tobacco product.