Epigenomic Dysregulation in Youth Vapers: Implications for Disease Risk Assessment.

Despite the ongoing epidemic of youth vaping, the long-term health consequences of electronic cigarette use are largely unknown. We report the effects of vaping versus smoking on the oral cell methylome of healthy young vapers and smokers relative to non-users. Whereas vapers and smokers differ in number of differentially methylated regions (DMRs) (831 vs 2,863), they share striking similarities in the distribution and patterns of DNA methylation, chromatin states, transcription factor binding motifs, and pathways. There is substantial overlap in DMR-associated genes between vapers and smokers, with the shared subset of genes enriched for transcriptional regulation, signaling, tobacco use disorders, and cancer-related pathways. Of significance is the identification of a common hypermethylated DMR at the promoter of "Hypermethylated In Cancer 1" (HIC1), a tumor suppressor gene frequently silenced in smoking-related cancers. Our data support a potential link between epigenomic dysregulation in youth vapers and disease risk. These novel findings have significant implications for public health and tobacco product regulation.

Exploring the Utility of Long Non-Coding RNAs for Assessing the Health Consequences of Vaping.

Electronic cigarette (e-cig) use, otherwise known as "vaping", is widespread among adolescent never-smokers and adult smokers seeking a less-harmful alternative to combustible tobacco products. To date, however, the long-term health consequences of vaping are largely unknown. Many toxicants and carcinogens present in e-cig vapor and tobacco smoke exert their biological effects through epigenetic changes that can cause dysregulation of disease-related genes. Long non-coding RNAs (lncRNAs) have emerged as prime regulators of gene expression in health and disease states. A large body of research has shown that lncRNAs regulate genes involved in the pathogenesis of smoking-associated diseases; however, the utility of lncRNAs for assessing the disease-causing potential of vaping remains to be fully determined. A limited but growing number of studies has shown that lncRNAs mediate dysregulation of disease-related genes in cells and tissues of vapers as well as cells treated in vitro with e-cig aerosol extract. This review article provides an overview of the evolution of e-cig technology, trends in use, and controversies on the safety, efficacy, and health risks or potential benefits of vaping relative to smoking. While highlighting the importance of lncRNAs in cell biology and disease, it summarizes the current and ongoing research on the modulatory effects of lncRNAs on gene regulation and disease pathogenesis in e-cig users and in vitro experimental settings. The gaps in knowledge are identified, priorities for future research are highlighted, and the importance of empirical data for tobacco products regulation and public health is underscored.

A Multi-Year Characterization of Confiscated Vaping Products from Virginia School Youth.

The United States (US) Food and Drug Administration's (FDA) regulatory oversight over electronic cigarettes (e-cigs) includes access restriction for persons <21 years of age and flavor restrictions for "cartridge-based" products. Despite the restrictions, consumption by US youth perseveres. Studies on youth e-cig use are limited by the reliability and accuracy of self-reports. As an alternative to self-reports, the current study examined nicotine, cannabinoid, and unlabeled e-cigs and other vaping products confiscated from Virginia public schools to characterize trends among students. Findings highlight a shift from JUUL and pod-based products to single use disposable e-cigs following the FDA flavor restrictions on cartridge-based e-cigs. Chemical analysis of e-liquids by gas chromatography-mass spectrometry identified a wide variety of flavorants and an increase in the prevalence of synthetic coolants. Most confiscated products were nicotine salt formulations, but the prevalence of cannabinoid-based vaping products increased. The popularity of flavored disposable e-cigs highlights the need for further restrictions to reduce youth consumption. The increasing use of synthetic coolants instead of menthol may suggest that manufacturers are employing tactics to bypass regulations. Continued youth access to e-cigs and the abundance of cannabinoid-based products is problematic from health and safety perspectives. Continued research incorporating confiscated product analysis can be used to understand youth access to vaping products and evolutions in manufacturing practices.

Comparative study of the effects of cigarette smoke versus next-generation tobacco and nicotine product extracts on inflammatory biomarkers of human monocytes.

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.

An assessment of vaping-induced inflammation and toxicity: A feasibility study using a 2-stage zebrafish and mouse platform.

It is currently understood that tobacco smoking is a major cause of pulmonary disease due to pulmonary/lung inflammation. However, due to a highly dynamic market place and an abundance of diverse products, less is known about the effects of e-cigarette (E-cig) use on the lung. In addition, varieties of E-cig liquids (e-liquids), which deliver nicotine and numerous flavor chemicals into the lungs, now number in the 1000s. Thus, a critical need exists for safety evaluations of these E-cig products. Herein, we employed a "2-stage in vivo screening platform" (zebrafish to mouse) to assess the safety profiles of e-liquids. Using the zebrafish, we collected embryo survival data after e-liquid exposure as well as neutrophil migration data, a key hallmark for a pro-inflammatory response. Our data indicate that certain e-liquids induce an inflammatory response in our zebrafish model and that e-liquid exposure alone results in pro-inflammatory lung responses in our C57BL/6J model, data collected from lung staining and ELISA analysis, respectively, in the mouse. Thus, our platform can be used as an initial assessment to ascertain the safety profiles of e-liquid using acute inflammatory responses (zebrafish, Stage 1) as our initial metric followed by chronic studies (C57BL/6J, Stage 2).

Aerosol, vapor, or chemicals? College student perceptions of harm from electronic cigarettes and support for a tobacco-free campus policy.

Objective: This study is the first to examine the influence of e-cigarette emission phrasing on perceived harm of secondhand exposure, and whether harm perception was associated with support for a tobacco-free campus policy. Participants: In the fall 2018 and spring 2019 semesters, 52 sections of a college English course (N = 791 students) were cluster randomized to one of three conditions ("vapor," "aerosol," or "chemicals") assessing harm of secondhand exposure to e-cigarette emissions. Methods: Regression models adjusted for demographic characteristics, tobacco use, and other potential confounders. Results: Compared to the "vapor" condition, "chemicals" and "aerosol" conditions were associated with increased odds of perceiving secondhand exposure to e-cigarettes to be harmful/very harmful (AOR = 2.0, p < 0.01). Greater perceived harm of secondhand e-cigarette exposure was associated with increased odds of supporting a tobacco-free campus policy (AOR = 2.22, p < 0.001). Conclusions: Health campaigns should use accurate terminology to describe e-cigarette emissions, rather than jargon that conveys lower risk.

Comparative study of the effects of cigarette smoke versus next generation tobacco and nicotine product extracts on endothelial function.

Tobacco smoking and hemodynamic forces are key stimuli for the development of endothelial dysfunction. As an alternative to smoking, next generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their potential pro-inflammatory and atherogenic effects on the endothelium. In this study, we analyzed key parameters of endothelial function after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine. All experiments were performed under atheroprotective high laminar or atherogenic low flow with primary human endothelial cells. Treatment with 3R4F, but not alternative smoking products, reduced endothelial cell viability and wound healing capability via the PI3K/AKT/eNOS(NOS3) pathway. Laminar flow delayed detrimental effects on cell viability by 3R4F treatment. 3R4F stimulation led to activation of NRF2 antioxidant defense system at nicotine concentrations ≥0.56 µg/ml and increased expression of its target genes HMOX1 and NQO1. Treatment with HTP revealed an induction of HMOX1 and NQO1 at dosages with ≥1.68 µg/ml nicotine, whereas e-cig and nicotine exposure had no impact. Analyses of pro-inflammatory genes revealed an increased ICAM1 expression under 3R4F treatment. 3R4F reduced VCAM1 expression in a dose-dependent manner; HTP treatment had similar but milder effects; e-cig and nicotine treatment had no impact. SELE expression was induced by 3R4F under static conditions. High laminar flow prevented this upregulation. Stimulation with laminar flow led to downregulation of CCL2 (MCP-1). From this downregulated level, only 3R4F increased CCL2 expression at higher concentrations. Finally, under static conditions, all components increased adhesion of monocytes to endothelial cells. Interestingly, only stimulation with 3R4F revealed increased monocyte adhesion under atherosclerosis-prone low flow. In conclusion, all product categories activated anti-oxidative or pro-inflammatory patterns. NGP responses were typically lower than in 3R4F exposed cells. Also, 3R4F stimulation led to an impaired endothelial wound healing and induced a pro-inflammatory phenotype compared to NGP treatment.

Development of a standardized new cigarette smoke generating (SNCSG) system for the assessment of chemicals in the smoke of new cigarette types (heat-not-burn (HNB) tobacco and electronic cigarettes (E-Cigs)).

To systematically regulate new types of cigarettes for which their safety has yet to be verified, such as heat-not-burn (HNB) products and electronic cigarettes (E-Cigs), the identification of chemicals in the new cigarette smoke is necessary. However, this is challenging due to the large number of new cigarette types and their different vaporization approaches. To address this issue, we herein report the development of a standardized new cigarette smoke generating (SNCSG) system based on heating-temperature control, which is able to generate smoke for all types of new cigarettes. Validation of the developed system was also carried out through analysis of the carbonyl compounds (e.g., formaldehyde and acetaldehyde) in the new cigarette smoke of HNB products and E-Cigs generated by the SNCSG system under different heating temperatures. The analytical results were used to validate the SNCSG system by comparison with those of previous studies. In all new cigarette smoke samples, the formaldehyde and acetaldehyde concentrations increased dramatically upon increasing the heating temperatures, especially over the reference heating range of each HNB device (mean concentration (µg/cigarette, n = 5 (HNB and E-Cig samples)): formaldehyde = 0.373-5.841 (250-320 °C), and acetaldehyde = 0.088-27.60 (250-320 °C). In the case of the HNB samples, the concentration differences determined by the heating temperatures of the tobacco stick were statistically significant, with p-values (ANOVA) of 1.85E-10 (formaldehyde) and 1.73E-08 (acetaldehyde). In the majority of smoke samples, acrolein and propionaldehyde were detected under relatively high heating temperature conditions (>250 °C) at 0.50 ± 1.76 µg/(cigarette or 10 µL), while acetone was detected under low heating temperature conditions (<250 °C) at 0.09 ± 0.17 µg/(cigarette or 10 µL). These results indicate that the developed SNCSG system could be suitable for application in the regulation of new types of cigarettes, regardless of the cigarette type and heating approach.

Piloting a clinical laboratory method to evaluate the influence of potential modified risk tobacco products on smokers' quit-related motivation, choice, and behavior.

Research methods are needed that can predict whether the availability of potential modified risk tobacco products (MRTPs) may influence smokers' quit-related motivation, choice, and behavior. This pilot study assessed the primary outcomes of feasibility and adherence to address this need using an electronic cigarette (ECIG) as a model MRTP. Cigarette smokers were randomly assigned to use only their own brand of cigarettes (OB-only) or a second-generation ECIG (18 ng/ml nicotine) plus their OB cigarettes (ECIG+OB) ad libitum for four weeks. Participants logged products using a mobile device, collected used cigarette filters, and provided saliva samples every day for analysis of cotinine. They returned to the lab once per week to provide a breath sample and accept or decline a choice to quit all tobacco products (i.e., cigarettes and/or ECIGs). They also returned for a one-month follow-up visit. Of those participants randomized (n = 60), 56.7% completed the 4-week intervention and 40.0% completed the follow-up visit. The primary reason for withdrawal was poor adherence with mobile device use. Comparable numbers of participants in each group chose to make a quit attempt, although more OB-only participants chose to quit during the first two weeks and more ECIG+OB participants during the last two weeks. With protocol modifications to reduce participation burden, the current method might ultimately be used by regulators to predict how smokers' quit-related motivation, choice, and behavior are influenced by current and future MRTPs.