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Our purpose was to test the hypothesis that ultrasonic cigarettes (u-cigarettes), which operate at relatively low temperatures, produce aerosols that are less harmful than heated-coil pod-style electronic cigarettes (e-cigarettes). The major chemicals in SURGE u-cigarette fluids and aerosols were quantified, their cytotoxicity and cellular effects were assessed, and a Margin of Exposure risk assessment was performed on chemicals in SURGE fluids. Four SURGE u-cigarette flavor variants (Blueberry Ice, Watermelon Ice, Green Mint, and Polar Mint) were evaluated. Flavor chemicals were quantified in fluids and aerosols using gas chromatography/mass spectrometry. Cytotoxicity and cell dynamics were assessed using the MTT assay, live-cell imaging, and fluorescence microscopy. WS-23 (a coolant) and total flavor chemical concentrations in SURGE were similar to e-cigarettes, while SURGE nicotine concentrations (13-19 mg/mL) were lower than many fourth generation e-cigarettes. Transfer efficiencies of dominant chemicals to aerosols in SURGE ranged from 44-100%. SURGE fluids and aerosols had four dominant flavor chemicals (>1 mg/mL). Toxic aldehydes were usually higher in SURGE aerosols than in SURGE fluids. SURGE fluids and aerosols had aldehyde concentrations significantly higher than pod-style e-cigarettes. Chemical constituents, solvent ratios, and aldehydes varied among SURGE flavor variants. SURGE fluids and aerosols inhibited cell growth and mitochondrial reductases, produced attenuated and round cells, and depolymerized actin filaments, effects that depended on pod flavor, chemical constituents, and concentration. The MOEs for nicotine, WS-23, and propylene glycol were

BackgroundOur previous infodemiological study was performed by manually mining health-effect data associated with electronic cigarettes (ECs) from online forums. Manual mining is time consuming and limits the number of posts that can be retrieved. ObjectiveOur goal in this study was to automatically extract and analyze a large number (>41,000) of online forum posts related to the health effects associated with EC use between 2008 and 2015. MethodsData were annotated with medical concepts from the Unified Medical Language System using a modified version of the MetaMap tool. Of over 1.4 million posts, 41,216 were used to analyze symptoms (undiagnosed conditions) and disorders (physician-diagnosed terminology) associated with EC use. For each post, sentiment (positive, negative, and neutral) was also assigned. ResultsSymptom and disorder data were categorized into 12 organ systems or anatomical regions. Most posts on symptoms and disorders contained negative sentiment, and affected systems were similar across all years. Health effects were reported most often in the neurological, mouth and throat, and respiratory systems. The most frequently reported symptoms and disorders were headache (n=939), coughing (n=852), malaise (n=468), asthma (n=916), dehydration (n=803), and pharyngitis (n=565). In addition, users often reported linked symptoms (eg, coughing and headache). ConclusionsOnline forums are a valuable repository of data that can be used to identify positive and negative health effects associated with EC use. By automating extraction of online information, we obtained more data than in our prior study, identified new symptoms and disorders associated with EC use, determined which systems are most frequently adversely affected, identified specific symptoms and disorders most commonly reported, and tracked health effects over 7 years.

Starting in the 1970s, individuals, businesses and the public have increasingly benefited from policies prohibiting smoking indoors, saving thousands of lives and billions of dollars in healthcare expenditures. Smokefree policies to protect against secondhand smoke exposure, however, do not fully protect the public from the persistent and toxic chemical residues from tobacco smoke (also known as thirdhand smoke) that linger in indoor environments for years after smoking stops. Nor do these policies address the economic costs that individuals, businesses and the public bear in their attempts to remediate this toxic residue. We discuss policy-relevant differences between secondhand smoke and thirdhand smoke exposure: persistent pollutant reservoirs, pollutant transport, routes of exposure, the time gap between initial cause and effect, and remediation and disposal. We examine four policy considerations to better protect the public from involuntary exposure to tobacco smoke pollutants from all sources. We call for (a) redefining smokefree as free of tobacco smoke pollutants from secondhand and thirdhand smoke; (b) eliminating exemptions to comprehensive smoking bans; (c) identifying indoor environments with significant thirdhand smoke reservoirs; and (d) remediating thirdhand smoke. We use the case of California as an example of how secondhand smoke-protective laws may be strengthened to encompass thirdhand smoke protections. The health risks and economic costs of thirdhand smoke require that smokefree policies, environmental protections, real estate and rental disclosure policies, tenant protections, and consumer protection laws be strengthened to ensure that the public is fully protected from and informed about the risks of thirdhand smoke exposure.

Little is known about the chemical exposures that electronic cigarette (EC) users receive and emit during JUUL vaping and if exposures produce symptoms dose dependently. This study examined chemical exposure (dose), retention, symptoms during vaping, and the environmental accumulation of exhaled propylene glycol (PG), glycerol (G), nicotine, and menthol in a cohort of human participants who vaped JUUL “Menthol” ECs. We refer to this environmental accumulation as “EC exhaled aerosol residue” (ECEAR). Chemicals were quantified using gas chromatography/mass spectrometry in JUUL pods before and after use, lab-generated aerosols, human exhaled aerosols, and in ECEAR. Unvaped JUUL “Menthol” pods contained ∼621.3 mg/mL of G, ∼264.9 mg/mL of PG, ∼59.3 mg/mL of nicotine, ∼13.3 mg/mL of menthol, and ∼0.1 mg/mL of the coolant WS-23. Eleven experienced male EC users (aged 21–26) provided exhaled aerosol and residue samples before and after vaping JUUL pods. Participants vaped ad libitum for 20 min, while their average puff count (22 ± 6.4) and puff duration (4.4 ± 2.0) were recorded. The transfer efficiency of nicotine, menthol, and WS-23 from the pod fluid into the aerosol varied with each chemical and was generally similar across flow rates (9–47 mL/s). At 21 mL/s, the average mass of each chemical retained by the participants who vaped 20 min was 53.2 ± 40.3 mg for G, 18.9 ± 14.3 mg for PG, 3.3 ± 2.7 mg for nicotine, and 0.5 ± 0.4 mg for menthol, with retention deduced to be ∼90–100% for each chemical. There was a significant positive relationship between the number of symptoms during vaping and total chemical mass retained. ECEAR accumulated on enclosed surfaces where it could contribute to passive exposure. These data will be valuable to researchers studying human exposure to EC aerosols and agencies that regulate EC products.

IntroductionBoth spill over and spill back of SARS-CoV-2 virus have been reported on mink farms in Europe and the United States. Zoonosis is a public health concern as dangerous mutated forms of the virus could be introduced into the human population through spillback.MethodsThe purpose of our study was to determine the SARS-CoV-2 entry mechanism using the mink lung epithelial cell line (Mv1Lu) and to block entry with drug inhibitors.ResultsMv1Lu cells were susceptible to SARS-CoV-2 viral pseudoparticle infection, validating them as a suitable disease model for COVID-19. Inhibitors of TMPRSS2 and of endocytosis, two pathways of viral entry, were tested to identify those that blocked infection. TMPRSS2 inhibitors had minimal impact, which can be explained by the apparent lack of activity of this enzyme in the mink and its localization within the cell, not on the cell surface.DiscussionDyngo4a, a small molecule endocytosis inhibitor, significantly reduced infection, supporting the conclusion that the entry of the SARS-CoV-2 virus into Mv1Lu cells occurs primarily through endocytosis. The small molecule inhibitors that were effective in this study could potentially be used therapeutically to prevent SARS-CoV-2 infection in mink populations. This study will facilitate the development of therapeutics to prevent zoonotic transmission of SARS-CoV-2 variants to other animals, including humans.

The relationship between the use of tobacco products and SARS-CoV-2 infection is poorly understood and controversial. Few studies have examined the effect of electronic cigarettes (ECs) on SARS-CoV-2 infection. We tested the hypothesis that EC fluids and aerosols with nicotine promote SARS-COV-2 infection by increasing viral entry into human respiratory epithelial cells. Responses of BEAS-2B cells to JUUL aerosols or their individual constituents were compared using three exposure platforms: submerged culture, air-liquid-interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex system, which produces authentic heated EC aerosols. In general, nicotine and nicotine + propylene glycol/vegetable glycerin aerosols increased ACE2 (angiotensin converting enzyme 2) levels, the SARS-CoV-2 receptor; and increased the activity of TMPRSS2 (transmembrane serine protease 2), an enzyme essential for viral entry. Lentivirus pseudoparticles with spike protein were used to test viral penetration. Exposure to nicotine, EC fluids, or aerosols altered the infection machinery and increased viral entry into cells. While most data were in good agreement across the three exposure platforms, cells were more responsive to treatments when exposed at the ALI in the Cultex system, even though the exposures were brief and intermittent. While both nicotine and JUUL aerosols increased SARS-CoV-2 infection, JUUL significantly decreased the effect of nicotine alone. These data support the idea that vaping can increase the likelihood of contracting COVID-19 and that e-liquid composition may modulate this effect.

The design of popular disposable electronic cigarettes (ECs) was analyzed, and the concentrations of WS-23, a synthetic coolant, in EC fluids were determined for 22 devices from 4 different brands. All products contained WS-23 in concentrations that ranged from 1.0 to 40.1 mg/mL (mean = 21.4 ± 9.2 mg/mL). To determine the effects of WS-23 on human bronchial epithelium in isolation of other chemicals, we exposed EpiAirway 3-D microtissues to WS-23 at the air liquid interface (ALI) using a cloud chamber that generated aerosols without heating. Proteomics analysis of exposed tissues revealed that the cytoskeleton was a major target of WS-23. BEAS-2B cells were exposed to WS-23 in submerged culture to validate the main results from proteomics. F-actin, which was visualized with phalloidin, decreased concentration dependently in WS-23 treated BEAS-2B cells, and cells became immotile in concentrations above 1.5 mg/mL. Gap closure, which depends on both cell proliferation and migration, was inhibited by 0.45 mg/mL of WS-23. These data show that WS-23 is being added to popular EC fluids at concentrations that can impair processes dependent on the actin cytoskeleton and disturb homeostasis of the bronchial epithelium. The unregulated use of WS-23 in EC products may harm human health.

BackgroundThe increased popularity of electronic cigarettes (e-cigarettes) has been linked to the abundance of flavoured products that are attractive to adolescents and young adults. In the last decade, e-cigarette designs have evolved through four generations that include modifications in battery power, e-cigarette liquid (e-liquid) reservoirs and atomiser units. E-liquids have likewise evolved in terms of solvent use/ratios, concentration and number of flavour chemicals, use of nicotine salts and acids, the recent increased use of synthetic cooling agents and the introduction of synthetic nicotine. Our current objective was to evaluate and compare the evolving composition of tobacco-flavoured e-liquids over the last 10 years.MethodsOur extensive database of flavour chemicals in e-liquids was used to identify trends and changes in flavour chemical composition and concentrations.ResultsTobacco-flavoured products purchased in 2010 and 2011 generally had very few flavour chemicals, and their concentrations were generally very low. In tobacco-flavoured refill fluids purchased in 2019 and Puff Bar Tobacco e-cigarettes, the total number and concentration of flavour chemicals were higher than expected. Products with total flavour chemicals >10 mg/mL contained one to five dominant flavour chemicals (>1 mg/mL). The most frequently used flavour chemicals in tobacco e-liquids were fruity and caramellic.ConclusionsThere is a need for continuous surveillance of e-liquids, which are evolving in often subtle and harmful ways. Chemical constituents of tobacco flavours should be monitored as they clearly can be doctored by manufacturers to have a taste that would appeal to young users.

BackgroundThirdhand smoke (THS) exposure correlated with significant metabolism of carcinogenic chemicals and the potential to cause detrimental health effects. Human harm research of THS exposure is limited to one other study and overall, there is a general lack of knowledge of the human health responses to THS exposure.MethodsThis was a clinical investigation to evaluate the health effects of 3-h dermal THS exposure from urine and plasma. 10 healthy, non-smoking subjects were recruited for dermal exposure for 3 h exposed to clothing impregnated with filtered clean air or THS. Exposures to clean air or THS occurred 20-30 days apart.FindingsIn THS-exposed group, there was a significant elevation of urinary 8-OHdG, 8-isoprostane, protein carbonyls. The THS 3-h exposure identified proteomics pathways of inflammatory response (p=2.18 × 10-8), adhesion of blood cells (p=2.23 × 10-8), atherosclerosis (p=2.78 × 10-9), and lichen planus (p=1.77 × 10-8). Nine canonical pathways were significantly activated including leukocyte extravasation signaling (z-score=3.0), and production of nitric oxide and reactive oxygen in macrophages (z-score=2.1). The THS 22-h proteomics pathways revealed inflammation of organ (p=3.09 × 10-8), keratinization of the epidermis (p=4.0 × 10-7), plaque psoriasis (p=5.31 × 10-7), and dermatitis (p=6.0 × 10-7). Two activated canonical pathways were production of nitric oxide and reactive oxygen in macrophages (z-score=2.646), and IL-8 signaling (z-score=2.0).InterpretationThis is a clinical study demonstrating that acute dermal exposure to THS mimics the harmful effects of cigarette smoking, alters the human plasma proteome, initiates mechanisms of skin inflammatory disease, and elevates urinary biomarkers of oxidative harm.FundingFunding was provided by the Tobacco Related Disease Research Program (TRDRP) 24RT-0037 TRDRP, 24RT-0039 TRDRP, and 28PT-0081 TRDRP.

The popularity of disposable fourth-generation electronic cigarettes (ECs) among young adults and adolescents has been increasing since the ban on flavored cartridge EC products such as JUUL. Although the constituents and toxicity of some cartridge-based fourth-generation ECs, such as JUUL, have been studied, limited data exist for other disposable ECs such as Puff. The purpose of this study was to determine flavor chemicals, synthetic coolants, and nicotine concentrations in 16 disposable Puff devices, evaluate the cytotoxicity of the different flavors from the Puff brand using in vitro assays, and investigate the health risks of synthetic coolants in EC products. Gas chromatography/mass spectrometry was used to identify and quantify chemicals in Puff EC fluids. One hundred and twenty-six flavor chemicals were identified in Puff fluids, and 16 were >1 mg/mL. WS-23 (2-isopropyl-N,2,3-trimethylbutyramide) was present in all products, and concentrations ranged from 0.8 to 45.1 mg/mL. WS-3 (N-ethyl-p-menthane-3-carboxamide) concentrations ranged from 1.5 to 16.4 mg/mL in 6/16 products. Nicotine concentrations ranged from 40.6 to 52.4 (average 44.8 mg/mL). All unvaped fluids were cytotoxic at dilutions between 0.1 and 10% in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. The cytotoxicity of Puff fluids was highly correlated with total chemical concentrations, nicotine, WS-23, both synthetic coolants, and synthetic coolants plus ethyl maltol. Lower concentrations of WS-23 than those in the fluids adversely affected cell growth and morphology. Concentrations of synthetic coolants exceeded levels used in consumer products. The margin of exposure data showed that WS-3 and WS-23 concentrations were high enough in Puff products to present a health hazard. Our study demonstrates that disposable Puff ECs have high levels of cytotoxic chemicals. The data support the regulation of flavor chemicals and synthetic coolants in ECs to limit potentially harmful health effects.

BackgroundThe Food and Drug Administration (FDA) has recently banned flavours from pod-style electronic cigarettes (e-cigarettes), except for menthol and tobacco. JUUL customers have quickly discovered that flavoured disposable e-cigarettes from other manufacturers, such as Puff, are readily available. Our goal was to compare flavour chemicals, synthetic coolants and pulegone in mint-flavoured/menthol-flavoured e-cigarettes from JUUL and Puff, evaluate the cytotoxicity of the coolants and perform a cancer risk assessment for pulegone, which is present in both JUUL pods and disposable Puff products.MethodsIdentification and quantification of chemicals were performed using gas chromatography/mass spectrometry. Cytotoxicity of the coolants was evaluated with BEAS-2B cells using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cancer risk of pulegone was calculated using the margin of exposure (MOE).ResultsMenthol was the dominant flavour chemical (>1 mg/mL) in all products from both manufacturers. Minor flavour chemicals (

BackgroundThe health effects caused by electronic cigarette (e-cigarette) use are not well understood. ObjectiveOur purpose was to document the positive and negative short-term health effects produced by e-cigarette use through an analysis of original posts from three online e-cigarettes forums. MethodsData were collected into Microsoft Access databases and analyzed using Cytoscape association graphics, frequency distributions, and interactomes to determine the number and type of health effects reported, the organ systems affected the frequency of specific effects, and systems interactions. ResultsA total of 405 different symptoms due to e-cigarette use were reported from three forums. Of these, 78 were positive, 326 were negative, and one was neutral. While the reported health effects were similar in all three forums, the forum with the most posts was analyzed in detail. Effects, which were reported for 12 organ systems/anatomical regions, occurred most often in the mouth and throat and in the respiratory, neurological, sensory, and digestive systems. Users with negative symptoms often reported more than one symptom, and in these cases interactions were often seen between systems, such as the circulatory and neurological systems. Positive effects usually occurred singly and most frequently affected the respiratory system. ConclusionsThis is the first compilation and analysis of the health effects reported by e-cigarette users in online forums. These data show that e-cigarette use can have wide ranging positive and negative effects and that online forums provide a useful resource for examining how e-cigarette use affects health.

The concentrations of elements/metals, nicotine, flavor chemicals and acids were compared in the e-liquids of unused and used first-generation electronic cigarettes (ECs) that were stored for 5-10 years. Metal analysis was performed using inductively coupled plasma optical emission spectroscopy; nicotine and flavor chemical analyses were performed using gas chromatography/mass spectroscopy. Of the 22 elements analyzed, 10 (aluminum, chromium, copper, iron, lead, nickel, selenium, silicon, tin, zinc) were often found in the e-liquids. Five elements had the highest average concentrations: copper (1161.6 mg/L), zinc (295.8 mg/L), tin (287.6 mg/L), nickel (71.1 mg/L), and lead (50.3 mg/L). Nicotine concentrations were always lower than label concentrations indicated. Of the 181 flavor chemicals analyzed, 11 were detected in at least one sample, with hydroxyacetone being present in all samples. In used products, some flavor chemicals appeared to be by-products of heating. E-liquids with the highest concentrations of acids and the lowest pH levels also had the highest concentrations of elements/metals. Metal concentrations in e-liquids increased after use in some products, and some metal concentrations, such as nickel, were high enough to be a health concern. Leachates from discarded ECs could contribute toxic metals/chemicals to the environment, supporting the need for better regulation of atomizer design, composition, and disposal.

Dermal exposure to nicotine is common due to the widespread use of tobacco products. Here, we assessed the effects of nicotine at concentrations found in thirdhand smoke (THS) contaminated environments and electronic cigarette (EC) spills or leaks on a 3D human skin model (EpiDermTM) and on submerged keratinocyte cultures. Air liquid interface treatment of EpiDermTM with 10 or 400 μg/mL of nicotine for 24 h followed by proteomics analysis showed altered pathways related to inflammation, protein synthesis, cell–cell adhesion, apoptosis, and mitochondrial function. Submerged cultured keratinocytes were used to validate the proteomics data and further characterize the response of skin cells to nicotine. Mitochondrial phenotype changed from networked to punctate in keratinocytes treated with 10 or 400 μg/mL of nicotine for 48 h and 24 h, respectively. After 72 h, all concentrations of nicotine caused a significant decrease in the networked phenotype. In Western blots, keratinocytes exposed to 400 μg/mL of nicotine had a significant decrease in mitofusin 2, while mitofusin 1 decreased after 72 h. The shift from networked to punctate mitochondria correlated with a decrease in mitofusin 1/2, a protein needed to establish and maintain the networked phenotype. Mitochondrial changes were reversible after a 24 h recovery period. Peroxisomes exposed to 400 μg/mL of nicotine for 24 h became enlarged and were fewer in number. Nicotine concentrations in THS and EC spills altered the proteome profile in EpiDermTM and damaged organelles including mitochondria and peroxisomes, which are involved in ROS homeostasis. These changes may exacerbate skin infections, inhibit wound healing, and cause oxidative damage to cells in the skin.

Lung cancer is the leading cause of cancer-related deaths worldwide, with increased risk being associated with unresolved or chronic inflammation. Agricultural and livestock workers endure significant exposure to agricultural dusts on a routine basis; however, the chronic inflammatory and carcinogenic effects of these dust exposure is unclear. We have developed a chronic dust exposure model of lung carcinogenesis in which mice were intranasally challenged three times a week for 24 weeks, using an aqueous dust extract (HDE) made from dust collected in swine confinement facilities. We also treated mice with the omega-3-fatty acid lipid mediator, aspirin-triggered resolvin D1 (AT-RvD1) to provide a novel therapeutic strategy for mitigating the inflammatory and carcinogenic effects of HDE. Exposure to HDE resulted in significant immune cell influx into the lungs, enhanced lung tumorigenesis, severe tissue pathogenesis, and a pro-inflammatory and carcinogenic gene signature, relative to saline-exposed mice. AT-RvD1 treatment mitigated the dust-induced inflammatory response but did not protect against HDE + NNK-enhanced tumorigenesis. Our data suggest that chronic HDE exposure induces a significant inflammatory and pro-carcinogenic response, whereas treatment with AT-RvD1 dampens the inflammatory responses, providing a strong argument for the therapeutic use of AT-RvD1 to mitigate chronic inflammation.

The health benefits of switching from tobacco to electronic cigarettes (ECs) are neither confirmed nor well characterized. To address this problem, we used RNA-seq analysis to compare the nasal epithelium transcriptome from the following groups (n = 3 for each group): (1) former smokers who completely switched to second generation ECs for at least 6 months, (2) current tobacco cigarette smokers (CS), and (3) non-smokers (NS). Group three included one former cigarette smoker. The nasal epithelial biopsies from the EC users vs. NS had a higher number of differentially expressed genes (DEGs) than biopsies from the CS vs. NS and CS vs. EC sets (1817 DEGs total for the EC vs. NS, 407 DEGs for the CS vs. NS, and 116 DEGs for the CS vs. EC comparison). In the EC vs. NS comparison, enriched gene ontology terms for the downregulated DEGs included cilium assembly and organization, whereas gene ontologies for upregulated DEGs included immune response, keratinization, and NADPH oxidase. Similarly, ontologies for cilium movement were enriched in the downregulated DEGs for the CS vs. NS group. Reactome pathway analysis gave similar results and also identified keratinization and cornified envelope in the upregulated DEGs in the EC vs. NS comparison. In the CS vs. NS comparison, the enriched Reactome pathways for upregulated DEGs included biological oxidations and several metabolic processes. Regulator effects identified for the EC vs. NS comparison were inflammatory response, cell movement of phagocytes and degranulation of phagocytes. Disease Ontology Sematic Enrichment analysis identified lung disease, mouth disease, periodontal disease and pulmonary fibrosis in the EC vs. NS comparison. Squamous metaplasia associated markers, keratin 10, keratin 13 and involucrin, were increased in the EC vs. NS comparison. Our transcriptomic analysis showed that gene expression profiles associated with EC use are not equivalent to those from non-smokers. EC use may interfere with airway epithelium recovery by promoting increased oxidative stress, inhibition of ciliogenesis, and maintaining an inflammatory response. These transcriptomic alterations may contribute to the progression of diseases with chronic EC use.

BackgroundGiven the high concentrations of nicotine and flavor chemicals in EC (electronic cigarette) fluids, it is important to determine how efficiently they transfer to aerosols, how well they are retained by users (exposure), and if they are exhaled into the environment where they settle of surfaces forming ECEAR (EC exhaled aerosol residue).ObjectivesTo quantify the flavor chemicals and nicotine in refill fluids, inhaled aerosols, and exhaled aerosols. Then deduce their retention and contribution to ECEAR.MethodsFlavor chemicals and nicotine were identified and quantified by GC-MS in two refill fluids, smoking machine-generated aerosols, and aerosols exhaled by 10 human participants (average age 21; 7 males). Machine generated aerosols were made with varying puff durations and two wattages (40 and 80). Participants generated exhale ad libitum; their exhale was quantified, and chemical retention and contribution to ECEAR was modeled.Results"Dewberry Cream" had five dominant (≥1 mg/mL) flavor chemicals (maltol, ethyl maltol, vanillin, ethyl vanillin, furaneol), while "Cinnamon Roll" had one (cinnamaldehyde). Nicotine transferred well to aerosols irrespective of topography; however, transfer efficiencies of flavor chemicals depended on the chemical, puff volume, puff duration, pump head, and EC power. Participants could be classified as "mouth inhalers" or "lung inhalers" based on their exhale of flavor chemicals and nicotine and retention. Lung inhalers had high retention and exhaled low concentrations of EC chemicals. Only mouth inhalers exhaled sufficient concentrations of flavor chemicals/nicotine to contribute to chemical deposition on environmental surfaces (ECEAR).ConclusionThese data help distinguish two types of EC users, add to our knowledge of chemical exposure during vaping, and provide information useful in regulating EC use.

Our goal was to evaluate the effects of EC refill fluids and EC exhaled aerosol residue (ECEAR) on cultured human keratinocytes and MatTek EpiDerm™, a 3D air liquid interface human skin model. Quantification of flavor chemicals and nicotine in Dewberry Cream and Churrios refill fluids was done using GC-MS. The dominant flavor chemicals were maltol, ethyl maltol, vanillin, ethyl vanillin, benzyl alcohol, and furaneol. Cytotoxicity was determined with the MTT and LDH assays, and inflammatory markers were quantified with ELISAs. Churrios was cytotoxic to keratinocytes in the MTT assay, and both fluids induced ROS production in the medium (ROS-Glo™) and in cells (CellROX). Exposure of EpiDerm™ to relevant concentrations of Dewberry Cream and Churrios for 4 or 24 h caused secretion of inflammatory markers (IL-1α, IL-6, and MMP-9), without altering EpiDerm™ histology. Lab made fluids with propylene glycol (PG) or PG plus a flavor chemical did not produce cytotoxic effects, but increased secretion of IL-1α and MMP-9, which was attributed to PG. ECEAR derived from Dewberry Cream and Churrios did not produce cytotoxicity with Epiderm™, but Churrios ECEAR induced IL-1α secretion. These data support the conclusion that EC chemicals can cause oxidative damage and inflammation to human skin.

StemCellQC is a video bioinformatics software tool for the quantitative analysis of human pluripotent stem cell (hPSC) colonies. Our objective was to use StemCellQC to evaluate and compare various experimental culture conditions, cell lines, and treatments and to demonstrate its applicability to PSC problems. Seven key features were identified that provided useful information on PSC morphology, dynamic behavior, and viability. Colony attachment was better on laminin-521 than on Matrigel and Geltrex. Growth rates were similar on each matrix when data were normalized. The brightness/area ratio feature showed greater cell death in colonies grown on Matrigel and Geltrex than on laminin-521 further contributing to an overall greater yield of cells on laminin-521. Four different PSC culture media performed similarly; however, one medium produced batch-to-batch variation in colony morphology and dynamic features. Two embryonic and one induced pluripotent stem cell line showed significant differences in morphology, growth rates, motility, and death rates. Cells from the same vial that became phenotypically different in culture showed measurable differences in morphology, brightness, and motility. Likewise, differentiating and undifferentiated colonies varied in growth rate, intensity, and motility. Three pluripotent cell lines treated with a low concentration of cinnamaldehyde, a chemical used in consumer products, showed adverse effects and differed in their sensitivity to treatment. Our data demonstrate various applications of StemCellQC which could be used in basic and translational research, toxicological and drug testing, and clinical facilities engaged in stem cell therapy.

BackgroundThe Food and Drug Administration (FDA) has recently banned flavours from pod-style electronic cigarettes (e-cigarettes), except for menthol and tobacco. JUUL customers have quickly discovered that flavoured disposable e-cigarettes from other manufacturers, such as Puff, are readily available. Our goal was to compare flavour chemicals, synthetic coolants and pulegone in mint-flavoured/menthol-flavoured e-cigarettes from JUUL and Puff, evaluate the cytotoxicity of the coolants and perform a cancer risk assessment for pulegone, which is present in both JUUL pods and disposable Puff products.MethodsIdentification and quantification of chemicals were performed using gas chromatography/mass spectrometry. Cytotoxicity of the coolants was evaluated with BEAS-2B cells using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cancer risk of pulegone was calculated using the margin of exposure (MOE).ResultsMenthol was the dominant flavour chemical (>1 mg/mL) in all products from both manufacturers. Minor flavour chemicals (

SignificanceAutomated understanding of human embryonic stem cell (hESC) videos is essential for the quantified analysis and classification of various states of hESCs and their health for diverse applications in regenerative medicine.AimThis paper aims to develop an ensemble method and bagging of deep learning classifiers as a model for hESC classification on a video dataset collected using a phase contrast microscope.ApproachThe paper describes a deep learning-based random network (RandNet) with an autoencoded feature extractor for the classification of hESCs into six different classes, namely, (1) cell clusters, (2) debris, (3) unattached cells, (4) attached cells, (5) dynamically blebbing cells, and (6) apoptotically blebbing cells. The approach uses unlabeled data to pre-train the autoencoder network and fine-tunes it using the available annotated data.ResultsThe proposed approach achieves a classification accuracy of 97.23  ±  0.94  %   and outperforms the state-of-the-art methods. Additionally, the approach has a very low training cost compared with the other deep-learning-based approaches, and it can be used as a tool for annotating new videos, saving enormous hours of manual labor.ConclusionsRandNet is an efficient and effective method that uses a combination of subnetworks trained using both labeled and unlabeled data to classify hESC images.

Thirdhand smoke (THS) is an environmental contaminant that may cause adverse health effects in smokers and nonsmokers. Currently, time-consuming analytical methods are necessary to assess chemicals in THS repositories, like upholstered furniture and clothing. Our goal was to develop a rapid, accessible method that can be used to measure THS contamination in common household fabrics and to evaluate remediation. Cotton, terry cloth, polyester, and wool were exposed to THS for various times in a controlled laboratory environment and then extracted in various media at room temperature or 60 °C to develop an autofluorescent method to quantify THS. Concentrations of nicotine and related alkaloids in the extracts were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). The autofluorescence of extracts was proportional to the time and amount of THS exposure received by cotton and terry cloth. Extracts of polyester and wool did not show autofluorescence unless heat was applied during extraction. Nicotine, nicotine alkaloids, and TSNA concentrations were higher in THS extracts from cotton and terry cloth than extracts of polyester and wool carpet, in agreement with the autofluorescence data. For fabrics spiked with 10 mg of nicotine, extraction efficiency was much higher from terry cloth (7 mg) than polyester (0.11 mg). In high relative humidity, nicotine recovery from both cotton and polyester was 80% (~8 mg). Our results provide a simple, rapid method to assess THS contaminants in household fabrics and further show that THS extraction is influenced by fabric type, heat, and humidity. Thus, remediation of THS environments may need to vary depending on the fabric reservoirs being treated. Understanding the dynamics of THS in fabrics can help set up appropriate remediation policies to protect humans from exposure.

BackgroundThe atomizers of electronic cigarettes (ECs) contain metals that transfer to the aerosol upon heating and may present health hazards. This study analyzed 4th-generation EC pod atomizer design features and characterized their elemental/metal composition.MethodsEleven EC pods from six brands/manufacturers were purchased at local shops and online. Pods were dissected and imaged using a Canon EOS Rebel SL2 camera. Elemental analysis and mapping of atomizer components was done using a scanning electron microscope coupled with an energy dispersive x-ray spectrometer.ResultsEC pods varied in size and design. The internal atomizer components were similar across brands except for variations occurring mainly in the wicks and filaments of some products. The filaments were either Elinvar (nickel, iron, and chromium) (36.4%), nichrome (36.4%), iron-chromium (18.2%), or nickel (9%). Thick wires present in 55% of the atomizers were mainly nickel and were joined to filaments by brazing. Wire-connector joints were Elinvar. Metal air tubes were made of Elinvar (50%), nickel, zinc, copper, and tin (37.5%), and nickel and copper (12.5%). Most of the wick components were silica, except for two pods (PHIX and Mico), which were mainly ceramic. Connectors contained gold-plated nickel, iron-chromium multiple alloys of nickel, zinc, gold, iron, and copper. Wick chambers were made of Elinvar. Outer casings were either nickel, copper-tin, or nickel-copper alloys. Magnets were nickel with minor iron, copper, and sulfur. Some frequently occurring elements were high in relative abundance in atomizer components.ConclusionsThe atomizers of pods are similar to previous generations, with the introduction of ceramic wicks and magnets in the newer generations. The elements in EC atomizers may transfer into aerosols and adversely affect health and accumulate in the environment.

Flavor chemicals in electronic cigarette (EC) fluids, which may negatively impact human health, have been studied in a limited number of countries/locations. To gain an understanding of how the composition and concentrations of flavor chemicals in ECs are influenced by product sale location, we evaluated refill fluids manufactured by one company (Ritchy LTD) and purchased worldwide. Flavor chemicals were identified and quantified using gas chromatography/mass spectrometry (GC/MS). We then screened the fluids for their effects on cytotoxicity (MTT assay) and proliferation (live-cell imaging) and tested authentic standards of specific flavor chemicals to identify those that were cytotoxic at concentrations found in refill fluids. A total of 126 flavor chemicals were detected in 103 bottles of refill fluid, and their number per/bottle ranged from 1-50 based on our target list. Two products had none of the flavor chemicals on our target list, nor did they have any nontargeted flavor chemicals. A total of 28 flavor chemicals were present at concentrations ≥1 mg/mL in at least one product, and 6 of these were present at concentrations ≥10 mg/mL. The total flavor chemical concentration was ≥1 mg/mL in 70% of the refill fluids and ≥10 mg/mL in 26%. For sub-brand duplicate bottles purchased in different countries, flavor chemical concentrations were similar and induced similar responses in the in vitro assays (cytotoxicity and cell growth inhibition). The levels of furaneol, benzyl alcohol, ethyl maltol, ethyl vanillin, corylone, and vanillin were significantly correlated with cytotoxicity. The margin of exposure calculations showed that pulegone and estragole levels were high enough in some products to present a nontrivial calculated risk for cancer. Flavor chemical concentrations in refill fluids often exceeded concentrations permitted in other consumer products. These data support the regulation of flavor chemicals in EC products to reduce their potential for producing both cancer and noncancer toxicological effects.

IntroductionChemical elements and their toxicity were evaluated in electronic cigarette (EC) solvents, fluids, and aerosols.Aims and methodsElement identification and quantification in propylene glycol (PG), glycerin (G), refill fluids before and after use, and aerosols was done using inductively coupled plasma optical emission spectrometry. Cytotoxicity and oxidative stress were evaluated using in vitro assays.ResultsSeven elements were present in PG, G, and popular refill fluids, and they transferred to aerosols made with ECs. Selenium was in all products (0.125-0.292 mg/L), while arsenic, aluminum, and tin were frequently in solvent and refill fluid samples at lower concentrations. Iron, chromium, copper, nickel, zinc, and lead were only detected in fluid after EC use, indicating they came from heated atomizers. Elements transferred most efficiently to aerosols made with second-/third-generation ECs. Of the elements in fluid, selenium and arsenic were the most cytotoxic to human bronchial epithelial cells (BEAS-2B) and pulmonary fibroblasts in the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Selenium increased superoxide production in mitochondria and nucleoli and elevated selenoprotein H in nucleoli of BEAS-2B cells at concentrations found in EC aerosols (10 nM or 0.002 mg/L).ConclusionsElements in EC aerosols came from both e-fluids and atomizing units. Within second-/third-generation products, transfer became more efficient as power increased. In vitro responses occurred at concentrations of selenium found in some EC aerosols. Human exposure to chemical elements in ECs could be reduced by regulating (decreasing) allowable EC power and by improving the purity of PG and G.ImplicationsPG, G, refill fluids, and e-fluids contained potentially toxic chemical elements that transferred to aerosols. Transfer was more efficient in second- and third-generation EC products and increased as power increased. Selenium and arsenic were the most cytotoxic of the elements tested in the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Selenium tetrachloride-induced oxidative stress in BEAS-2B cells, but not in human pulmonary fibroblasts. All fluids contained selenium above the concentration that induced oxidative stress in human bronchial epithelial cells. Selenium increased superoxide in mitochondria and nucleoli and increased selenoprotein H, a redox responsive DNA-binding protein that is upregulated by superoxide and an indicator of nucleolar stress. EC users are exposed to elements in aerosols, which may with chronic exposure contribute to diseases associated with oxidative stress.

Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL® cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL® cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease.

ObjectivesTo determine if urinary biomarkers of effect and potential harm are elevated in electronic cigarette users compared with non-smokers and if elevation correlates with increased concentrations of metals in urine.Study design and settingThis was a cross-sectional study of biomarkers of exposure, effect and potential harm in urine from non-smokers (n=20), electronic cigarette users (n=20) and cigarette smokers (n=13). Participant's screening and urine collection were performed at the Roswell Park Comprehensive Cancer Center, and biomarker analysis and metal analysis were performed at the University of California, Riverside.ResultsMetallothionein was significantly elevated in the electronic cigarette group (3761±3932 pg/mg) compared with the non-smokers (1129±1294 pg/mg, p=0.05). 8-OHdG (8-hydroxy-2'-deoxyguanosine) was significantly elevated in electronic cigarette users (442.8±300.7 ng/mg) versus non-smokers (221.6±157.8 ng/mg, p=0.01). 8-Isoprostane showed a significant increase in electronic cigarette users (750.8±433 pg/mg) versus non-smokers (411.2±287.4 pg/mg, p=0.03). Linear regression analysis in the electronic cigarette group showed a significant correlation between cotinine and total metal concentration; total metal concentration and metallothionein; cotinine and oxidative DNA damage; and total metal concentration and oxidative DNA damage. Zinc was significantly elevated in the electronic cigarette users (584.5±826.6 µg/g) compared with non-smokers (413.6±233.7 µg/g, p=0.03). Linear regression analysis showed a significant correlation between urinary zinc concentration and 8-OHdG in the electronic cigarette users.ConclusionsThis study is the first to investigate biomarkers of potential harm and effect in electronic cigarette users and to show a linkage to metal exposure. The biomarker levels in electronic cigarette users were similar to (and not lower than) cigarette smokers. In electronic cigarette users, there was a link to elevated total metal exposure and oxidative DNA damage. Specifically, our results demonstrate that zinc concentration was correlated to oxidative DNA damage.

This study compared the cytotoxicity of IQOS aerosols to smoke from Marlboro Red (MR) and 3R4F reference cigarettes. Aerosol/smoke solutions were tested as the gas vapor phase (GVP), particulate phase (total particulate matter or TPM), or whole aerosol/smoke (WA), the latter being what smokers actually inhale. Cytotoxicities were evaluated using the LDH, MTT and neutral red uptake (NRU) assays in conjunction with eight different cell types, mainly from the respiratory system. Most test solutions did not compromise the plasma membranes of cells (LDH). However, mitochondrial activity (MTT) and dye uptake/lysosomal activity (NRU) were equally depressed by IOQS aerosols and cigarette smoke solutions at the high concentrations. Our NRU data with mouse NIH/3T3 transformed fibroblasts were similar to those previously reported by the IQOS manufacturer and showed little cytotoxicity in the NRU assay. In both studies with NIH/3T3 cells, the results were significantly different from 3RF4 cigarette smoke, suggesting reduced toxicity with IQOS. However, by expanding evaluations to a broader spectrum of cells that included respiratory system cells and by including higher concentrations of GVP, as well as WA, cytotoxicity equivalent to that of Marlboro Red and 3R4F cigarettes was frequently observed with IQOS aerosols in the MTT and NRU assays.

BackgroundAcute liver failure (ALF) is a complicated clinical syndrome associated with high mortality, with liver transplantation as the only treatment option. Treatment of mesenchymal stem cells has shown a potential therapeutic option for acute liver failure. However, the lack of random clinical trials and large non-human primate studies makes it necessary to assess the efficacy and safety in the clinic.MethodsWe treated the monkeys with peripheral delivery of human umbilical MSCs (hUC-MSCs) and investigated the role of hUC-MSCs in modulating the progress of acute liver failure.ResultsThe use of early peripheral infusion of human umbilical cord MSC infusion did not improve liver regeneration or modulate adaptive immunity. However, it significantly suppressed the hepatic aggregation and maturation of circulating monocytes and their IL-6 secretion, greatly improving liver histology, systemic homeostasis, and survival.ConclusionsOur study reveals the critical role of monocyte-derived IL-6 in initiating and accelerating acute liver failure and hUC-MSC treatment can disrupt the development of the inflammatory cascade by inhibiting monocyte activation. Early hUC-MSC treatment disrupts the development of the inflammatory cascade, indicating a potential clinical solution for acute liver failure.

Our purpose was to examine the effect of model, puffing topography (voltage, air-flow, puff interval), and method of collection on 19 elements/metals in aerosols from six tank-style electronic cigarettes (EC). Aerosols were collected from six brands using a cold trap or impinger and various puffing topographies. 19 elements were quantified using inductively coupled plasma optical emission spectroscopy. 16 elements/metals were present and quantified in the aerosols. The total concentrations of elements/metals ranged from 43 to 3,138 µg/L with the impinger method of collection and 226 to 6,767 µg/L with the cold trap method. The concentrations of individual elements were often similar across brands and across topographies. Some elements (e.g., zinc) were present in most aerosols, while others (e.g., cadmium, titanium, vanadium) were rarely found. Concentrations of some elements (e.g., lead) increased in aerosols as voltage/power increased. The model with fewest metal parts in the atomizer had the fewest metals in its aerosols. Most elements/metals in the aerosols have been found previously in the atomizers of EC. All tank-style aerosols had elements/metals that appeared to originate in the atomizers, and concentrations increased with increasing power. Concentrations of some elements were high enough to be a health concern.

BackgroundElectronic cigarettes (ECs) are nicotine delivery devices that produce aerosol without combustion of tobacco; therefore, they do not produce sidestream smoke. Nevertheless, many users exhale large clouds of aerosol that can result in passive exposure of non-users. Analogous to thirdhand cigarette smoke, the exhaled aerosol also settles on indoor surfaces where it can produce a residue. We refer to this residue as EC exhaled aerosol residue (ECEAR). Our objective was to determine if exhaled EC aerosol transferred from a vape shop in a multiple-tenant retail building, where it was produced, to a nearby business (field site) where it could deposit as ECEAR.MethodsWe examined the build-up of ECEAR in commonly used materials (cotton towel and paper towels) placed inside the field site across from the vape shop. Materials were subjected to short-term (days) and long-term (months) exposures. Nicotine, other alkaloids and tobacco-specific nitrosamines (TSNAs) were identified and quantified in controls and field site samples using analytical chemical techniques.ResultsNicotine and other alkaloids were detected after 1 day of exposure in the field site, and these chemicals generally increased as exposure times increased. TSNAs, which have been linked to carcinogenesis, were also detected in short-term and long-term exposed samples from the field site.ConclusionsIn a multiple-tenant retail building, chemicals in EC aerosol travelled from a vape shop into an adjacent business where they deposited forming ECEAR. Regulatory agencies and tenants occupying such buildings should be aware of this potential environmental hazard.

The design of electronic cigarette (EC) atomizing units has evolved since their introduction over 10 years ago. The purpose of this study was to evaluate atomizer design in ECs sold between 2011-2017. Atomizers from 34 brands representing three generations of ECs were dissected and photographed using a stereoscopic microscope. Five distinct atomizer design categories were identified in first generation products (cig-a-like/cartomizer) and three categories were found in the third generation. Atomizers in most cig-a-like ECs contained a filament, thick wire, wire joints, air-tube, wick, sheath, and fibers, while some later models lacked some of these components. Over time design changes included an increase in atomizer size; removal of solder joints between wires; removal of Polyfil fibers; and removal of the microprocessor from Vuse. In second and third generation ECs, the reservoirs and batteries were larger, and the atomizing units generally lacked a thick wire, fibers, and sheath. These data contribute to an understanding of atomizer design and show that there is no single design for ECs, which are continually evolving. The design of the atomizer is particularly important as it affects the performance of ECs and what transfers into the aerosol.

BackgroundSince their release in 2004, electronic cigarettes (ECs) and their atomizers have undergone significant evolution.ObjectiveThe purpose of this study was to evaluate and compare the elemental/metal composition of atomizers in cartomizer and tank style ECs produced over a 5-year period.MethodsPopular cartomizer and tank models of ECs were dissected and photographed using a stereoscopic microscope, and elemental analysis of EC atomizers was done using scanning electron microscopy coupled with energy dispersive x-ray spectroscopy.ResultsEight elements/metals were found in most products across and within brands purchased at different times. These included chromium, nickel, copper, silver, tin, silicon, aluminum, and zinc. Iron and lead were found in some but not all products, while manganese, cobalt, molybdenum, titanium, and tungsten were only found in a few of the products. The metals used in various components were often similar in cartomizer and tank models. Filaments were usually chromium and nickel (nichrome), although in some newer products, the filament also contained iron, copper, and manganese. The thick wire in earlier products was usually copper coated with silver, while in some newer products, the thick wire was predominantly nickel. In all products, the wick was silica, and sheaths, when present, were fiberglass (silicon, oxygen, calcium, aluminum, magnesium). Wire-to-wire joints were either brazed or clamped with brass (copper and zinc), and air-tube-to-thick wire joints, when present, were usually soldered with tin. Tank style products generally lacked a thick wire and sheaths.ConclusionIn general, atomizer components in ECs were remarkably similar over time and between brands. Certain elements/metals were consistently found in most models from all generations, and these should be studied carefully to determine if their transfer to aerosols affects user's health and if their accumulation in trash affects the environment.

Whereas JUUL electronic cigarettes (ECs) have captured the majority of the EC market, with a large fraction of their sales going to adolescents, little is known about their cytotoxicity and potential effects on health. The purpose of this study was to determine flavor chemical and nicotine concentrations in the eight currently marketed prefilled JUUL EC cartridges ("pods") and to evaluate the cytotoxicity of the different variants (e.g., "Cool Mint" and "Crème Brulee") using in vitro assays. Nicotine and flavor chemicals were analyzed using gas chromatography-mass spectrometry in pod fluid before and after vaping and in the corresponding aerosols. 59 flavor chemicals were identified in JUUL pod fluids, and 3 were >1 mg/mL. Duplicate pods were similar in flavor chemical composition and concentration. Nicotine concentrations (average 60.9 mg/mL) were significantly higher than those of any EC products we have previously analyzed. The transfer efficiency of individual flavor chemicals that were >1 mg/mL and nicotine from the pod fluid into aerosols was generally 35-80%. All pod fluids were cytotoxic at a 1:10 dilution (10%) in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. Most aerosols were cytotoxic in these assays at concentrations between 0.2 and 1.8%. The cytotoxicity of collected aerosol materials was highly correlated with nicotine and ethyl maltol concentrations and moderately to weakly correlated with total flavor chemical concentration and menthol concentration. Our study demonstrates that (1) some JUUL flavor pods have sufficiently high concentrations of flavor chemicals that may make them attractive to youth and (2) the concentrations of nicotine and some flavor chemicals (e.g., ethyl maltol) are high enough to be cytotoxic in acute in vitro assays, emphasizing the need to determine if JUUL products will lead to adverse health effects with chronic use.

ImportanceNo previous studies have shown that acute inhalation of thirdhand smoke (THS) activates stress and survival pathways in the human nasal epithelium.ObjectiveTo evaluate gene expression in the nasal epithelium of nonsmoking women following acute inhalation of clean air and THS.Design, setting, and participantsNasal epithelium samples were obtained from participants in a randomized clinical trial (2011-2015) on the health effects of inhaled THS. In a crossover design, participants were exposed, head only, to THS and to conditioned, filtered air in a laboratory setting. The order of exposures was randomized and exposures were separated by at least 21 days. Ribonucleic acid was obtained from a subset of 4 healthy, nonsmoking women.ExposuresBy chance, women in the subset were randomized to receive clean air exposure first and THS exposure second. Exposures lasted 3 hours.Main outcomes and measuresDifferentially expressed genes were identified using RNA sequencing with a false-discovery rate less than 0.1.ResultsParticipants were 4 healthy, nonsmoking women aged 27 to 49 years (mean [SD] age, 42 [10.2] years) with no chronic diseases. A total of 389 differentially expressed genes were identified in nasal epithelium exposed to THS, while only 2 genes, which were not studied further, were affected by clean air. Enriched gene ontology terms associated with stress-induced mitochondrial hyperfusion were identified, such as respiratory electron transport chain (q = 2.84 × 10-3) and mitochondrial inner membrane (q = 7.21 × 10-6). Reactome pathway analysis identified terms associated with upregulation of DNA repair mechanisms, such as nucleotide excision repair (q = 1.05 × 10-2). Enrichment analyses using ingenuity pathway analysis identified canonical pathways related to stress-induced mitochondrial hyperfusion (eg, increased oxidative phosphorylation) (P = .001), oxidative stress (eg, glutathione depletion phase II reactions) (P = .04), and cell survival (z score = 5.026).Conclusions and relevanceThis study found that acute inhalation of THS caused cell stress that led to the activation of survival pathways. Some responses were consistent with stress-induced mitochondrial hyperfusion and similar to those demonstrated previously in vitro. These data may be valuable to physicians treating patients exposed to THS and may aid in formulating regulations for the remediation of THS-contaminated environments.

Stem cells provide a sensitive model to study exposure to toxicants, such as cigarette smoke. Electronic cigarettes (ECs) are popular nicotine delivery devices, often targeted to youth and pregnant mothers. However, little is known about how chemicals in ECs might affect neural stem cells, and in particular their mitochondria, organelles that maintain cell functionality and health. Here we show that the mechanism underlying EC-induced stem cell toxicity is stress-induced mitochondrial hyperfusion (SIMH), a transient survival response accompanied by increased mitochondrial oxidative stress. We identify SIMH as a survival response to nicotine, now widely available in EC refill fluids and in pure form for do-it-yourself EC products. These observed mitochondrial alterations combined with autophagy dysfunction to clear damaged mitochondria could lead to faulty stem cell populations, accelerate cellular aging, and lead to acquired mitochondriopathies. Any nicotine-containing product may likewise stress stem cells with long-term repercussions for users and passively exposed individuals. VIDEO ABSTRACT.

BackgroundElectronic cigarette (EC) users may exhale large clouds of aerosol that can settle on indoor surfaces forming ECEAR (EC exhaled aerosol residue). Little is known about the chemical composition or buildup of this residue.ObjectiveOur objective was to identify and quantify ECEAR chemicals in two field sites: an EC user's living room and a multi-user EC vape shop.MethodsWe examined the buildup of ECEAR in commonly used materials (cotton, polyester, or terrycloth towel) placed inside the field sites. Materials were subjected to different lengths of exposure. Nicotine, nicotine alkaloids, and tobacco-specific nitrosamines (TSNAs) were identified and quantified in unexposed controls and field site samples using analytical chemical techniques.ResultsNicotine and nicotine alkaloids were detected in materials inside the EC user's living room. Concentrations of ECEAR chemicals remained relatively constant over the first 5 months, suggesting some removal of the chemicals by air flow in the room approximating a steady state. ECEAR chemicals were detected in materials inside the vape shop after 6 h of exposure and levels continually increased over a month. By 1 month, the nicotine in the vape shop was 60 times higher than in the EC user's living room. ECEAR chemical concentrations varied in different locations in the vape shop. Control fabrics had either no detectable or very low concentrations of chemicals.ConclusionsIn both field sites, chemicals from exhaled EC aerosols were deposited on indoor surfaces and accumulated over time forming ECEAR. Non-smokers, EC users, and employees of vape shops should be aware of this potential environmental hazard.

We identified the most popular electronic cigarette (EC) refill fluids using an Internet survey and local and online sales information, quantified their flavor chemicals, and evaluated cytotoxicities of the fluids and flavor chemicals. "Berries/Fruits/Citrus" was the most popular EC refill fluid flavor category. Twenty popular EC refill fluids were purchased from local shops, and the ingredient flavor chemicals were identified and quantified by gas chromatography-mass spectrometry. Total flavor chemical concentrations ranged from 0.6 to 27.9 mg/ml, and in 95% of the fluids, total flavor concentration was greater than nicotine concentration. The 20 most popular refill fluids contained 99 quantifiable flavor chemicals; each refill fluid contained 22 to 47 flavor chemicals, most being esters. Some chemicals were found frequently, and several were present in most products. At a 1% concentration, 80% of the refill fluids were cytotoxic in the MTT assay. Six pure standards of the flavor chemicals found at the highest concentrations in the two most cytotoxic refill fluids were effective in the MTT assay, and ethyl maltol, which was in over 50% of the products, was the most cytotoxic. These data show that the cytotoxicity of some popular refill fluids can be attributed to their high concentrations of flavor chemicals.

We characterized the flavor chemicals in a broad sample of commercially available electronic cigarette (EC) refill fluids that were purchased in four different countries. Flavor chemicals in 277 refill fluids were identified and quantified by gas chromatography-mass spectrometry, and two commonly used flavor chemicals were tested for cytotoxicity with the MTT assay using human lung fibroblasts and epithelial cells. About 85% of the refill fluids had total flavor concentrations >1 mg/ml, and 37% were >10 mg/ml (1% by weight). Of the 155 flavor chemicals identified in the 277 refill fluids, 50 were present at ≥1 mg/ml in at least one sample and 11 were ≥10 mg/ml in 54 of the refill fluids. Sixty-one% (170 out of 277) of the samples contained nicotine, and of these, 56% had a total flavor chemical/nicotine ratio >2. Four chemicals were present in 50% (menthol, triacetin, and cinnamaldehyde) to 80% (ethyl maltol) of the samples. Some products had concentrations of menthol ("Menthol Arctic") and ethyl maltol ("No. 64") that were 30 times (menthol) and 100 times (ethyl maltol) their cytotoxic concentration. One refill fluid contained cinnamaldehyde at ~34% (343 mg/ml), more than 100,000 times its cytotoxic level. High concentrations of some flavor chemicals in EC refill fluids are potentially harmful to users, and continued absence of any regulations regarding flavor chemicals in EC fluids will likely be detrimental to human health.

OBJECTIVE:To evaluate performance of the I quit original smoking (iQOS) heat-not-burn system as a function of cleaning and puffing topography, investigate the validity of manufacturer's claims that this device does not burn tobacco and determine if the polymer-film filter is potentially harmful. METHODS:iQOS performance was evaluated using five running conditions incorporating two different cleaning protocols. Heatsticks were visually and stereomicroscopically inspected preuse and postuse to determine the extent of tobacco plug charring (from pyrolysis) and polymer-film filter melting, and to elucidate the effects of cleaning on charring. Gas chromatography-mass spectrometry headspace analysis was conducted on unused polymer-film filters to determine if potentially toxic chemicals are emitted from the filter during heating. RESULTS:For all testing protocols, pressure drop decreased as puff number increased. Changes in testing protocols did not affect aerosol density. Charring due to pyrolysis (a form of organic matter thermochemical decomposition) was observed in the tobacco plug after use. When the manufacturer's cleaning instructions were followed, both charring of the tobacco plug and melting of the polymer-film filter increased. Headspace analysis of the polymer-film filter revealed the release of formaldehyde cyanohydrin at 90°C, which is well below the maximum temperature reached during normal usage. DISCUSSION:Device usage limitations may contribute to decreases in interpuff intervals, potentially increasing user's intake of nicotine and other harmful chemicals. This study found that the tobacco plug does char and that charring increases when the device is not cleaned between heatsticks. Release of formaldehyde cyanohydrin is a concern as it is highly toxic at very low concentrations.

This study characterizes dynamic and apoptotic blebbing in human embryonic stem cells (hESC), identifies dynamic blebbing as a bottleneck to successful cell attachment during passaging, and demonstrates that dynamic blebbing can be rapidly stopped by plating cells on recombinant human laminin. In freshly plated hESC, dynamic and apoptotic blebbing differed in time of occurrence, bleb retraction rate, mitochondrial membrane potential, and caspase 3&7 activation. While dynamic blebbing can be controlled with drugs that inhibit myosin II, these methods have off-target effects and are not suitable for clinical applications. Recombinant human laminin-521 or addition of laminin-111 to Matrigel provided a safe method to drastically decrease dynamic blebbing and improve cell attachment with proteins normally found in the inner cell mass. Inhibition of focal adhesion kinase, which is activated by binding of integrins to laminin, prolonged dynamic blebbing and inhibited attachment. These data show that hESC bind rapidly to laminins through an integrin, which activates focal adhesion kinase that in turn downregulates dynamic blebbing. Laminins enabled hESC to rapidly attach during passaging, improved plating efficiency, enabled passaging of single pluripotent stem cells, and avoided use of inhibitors that have non-specific off-target effects. These data provide a strategy for improving hESC culture using biologically safe recombinant human proteins.

There is a critical need for better analytical methods to study mitochondria in normal and diseased states. Mitochondrial image analysis is typically done on still images using slow manual methods or automated methods of limited types of features. MitoMo integrated software overcomes these bottlenecks by automating rapid unbiased quantitative analysis of mitochondrial morphology, texture, motion, and morphogenesis and advances machine-learning classification to predict cell health by combining features. Our pixel-based approach for motion analysis evaluates the magnitude and direction of motion of: (1) molecules within mitochondria, (2) individual mitochondria, and (3) distinct morphological classes of mitochondria. MitoMo allows analysis of mitochondrial morphogenesis in time-lapse videos to study early progression of cellular stress. Biological applications are presented including: (1) establishing normal phenotypes of mitochondria in different cell types; (2) quantifying stress-induced mitochondrial hyperfusion in cells treated with an environmental toxicant, (3) tracking morphogenesis in mitochondria undergoing swelling, and (4) evaluating early changes in cell health when morphological abnormalities are not apparent. MitoMo unlocks new information on mitochondrial phenotypes and dynamics by enabling deep analysis of mitochondrial features in any cell type and can be applied to a broad spectrum of research problems in cell biology, drug testing, toxicology, and medicine.

Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.

Acute liver failure (ALF) is associated with high mortality, and a poor understanding of the underlying pathophysiology has resulted in a lack of effective treatments so far. Here, using an amatoxin-induced rhesus monkey model of ALF, we panoramically revealed the cellular and molecular events that lead to the development of ALF. The challenged monkeys with toxins underwent a typical course of ALF including severe hepatic injury, systemic inflammation and eventual death. Adaptive immune was not noticeably disturbed throughout the progress of ALF. A systematic examination of serum factors and cytokines revealed that IL-6 increase was the most rapid and drastic. Interestingly, we found that IL-6 was mainly produced by circulating monocytes. Furthermore, ablation of monocyte-derived IL-6 in mice decreased liver injury and systemic inflammation following chemical injection. Our findings reveal a critical role of circulating monocytes in initiating and accelerating ALF, indicating a potential therapeutic target in clinical treatment for ALF.

ObjectivesEpithelial-to-mesenchymal transition (EMT) is the initial step enabling the metastasis of cancer cells, which often leads to death. Although smoking is a major risk factor for lung cancer, there is still widespread use of conventional cigarettes. Recently, the tobacco industry has been transformed by the introduction of electronic cigarettes (ECs), which have lower levels of carcinogens and may provide a safer alternative. Here, we investigate the ability of EC liquids and aerosols to induce an EMT in A549 lung cancer cells.Materials and methodsHuman adenocarcinoma alveolar basal epithelial cells (A549) were exposed to EC liquids and aerosols from a popular product for 3-8 days. Live cell imaging, EMT biomarker analysis, and machine learning/image processing algorithms were used to characterize changes associated with EMT.ResultsLong-term exposure of A549 cells to menthol or tobacco-flavored EC liquids or aerosols induced an EMT that was characterized by acquisition of a fibroblast-like morphology, loss of cell-to-cell junctions, internalization of E-cadherin, increased motility, and upregulation of other EMT markers. The EMT was concurrent with plasma membrane to nuclear translocation of active β-catenin.ConclusionThis is the first known study to show an EMT of lung cancer cells during exposure to EC products. Because an EMT is an initial step leading to metastasis, an intractable problem that often leads to patient death, this critical finding has significant implications for former or heavy cigarette smokers who are using EC and may be at risk for lung cancer or who may already have a lung tumor.

Thousands of electronic cigarette refill fluids are commercially available. The concentrations of nicotine and the solvents, but not the flavor chemicals, are often disclosed on product labels. The purpose of this study was to identify and quantify flavor chemicals in 39 commercial refill fluids that were previously evaluated for toxicity. Twelve flavor chemicals were identified with concentrations ≥1 mg/ml: cinnamaldehyde, menthol, benzyl alcohol, vanillin, eugenol, p-anisaldehyde, ethyl cinnamate, maltol, ethyl maltol, triacetin, benzaldehyde, and menthone. Transfer of these flavor chemicals into aerosols made at 3V and 5V was efficient (mean transfer = 98%). We produced lab-made refill fluids containing authentic standards of each flavor chemical and analyzed the toxicity of their aerosols produced at 3V and 5V using a tank Box Mod device. Over 50% of the refill fluids in our sample contained high concentrations of flavor chemicals that transferred efficiently to aerosols at concentrations that produce cytotoxicity. When tested with two types of human lung cells, the aerosols made at 5V were generally more toxic than those made at 3V. These data will be valuable for consumers, physicians, public health officials, and regulatory agencies when discussing potential health concerns relating to flavor chemicals in electronic cigarette products.

BackgroundAs thousands of electronic cigarette (e-cigarette) refill fluids continue to be formulated and distributed, there is a growing need to understand the cytotoxicity of the flavouring chemicals and solvents used in these products to ensure they are safe. The purpose of this study was to compare the cytotoxicity of e-cigarette refill fluids/solvents and their corresponding aerosols using in vitro cultured cells.MethodsE-cigarette refill fluids and do-it-yourself products were screened in liquid and aerosol form for cytotoxicity using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The sensitivity of human pulmonary fibroblasts, lung epithelial cells (A549) and human embryonic stem cells to liquids and aerosols was compared. Aerosols were produced using Johnson Creek's Vea cartomizer style e-cigarette.ResultsA hierarchy of potency was established for the aerosolised products. Our data show that (1) e-cigarette aerosols can produce cytotoxic effects in cultured cells, (2) four patterns of cytotoxicity were found when comparing refill fluids and their corresponding aerosols, (3) fluids accurately predicted aerosol cytotoxicity 74% of the time, (4) stem cells were often more sensitive to aerosols than differentiated cells and (5) 91% of the aerosols made from refill fluids containing only glycerin were cytotoxic, even when produced at a low voltage.ConclusionsOur data show that various flavours/brands of e-cigarette refill fluids and their aerosols are cytotoxic and demonstrate the need for further evaluation of e-cigarette products to better understand their potential health effects.

ObjectivesElectronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown.Materials and methodsWe have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene.Results and conclusionWe observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro.

New methods are needed to reduce dynamic blebbing which inhibits cell attachment and survival during passaging of pluripotent stem cells. We tested the hypothesis that activation of the P2X7 receptor by extracellular ATP during passaging initiates dynamic blebbing. The P2X7 receptor was present in human embryonic stem cells (hESC), but not in differentiating cells. Extracellular ATP concentrations were 14× higher in medium during passaging. Addition of ATP to culture medium prolonged dynamic blebbing and inhibited attachment. Inhibition of P2X7 by specific drugs or by siRNA significantly reduced dynamic blebbing and improved cell attachment. When cells were incubated in calcium chelators (EGTA or BAPTA), blebbing decreased and attachment improved. Calcium influx was observed using Fura-4 when ATP was added to culture medium and inhibited in the presence of the P2X7 inhibitor. Over-expressing activated Rac in hESC reduced blebbing and promoted cell attachment, while a Rac inhibitor prolonged blebbing and reduced attachment. These data identify a pathway involving P2X7 that initiates and prolongs dynamic blebbing during hESC passaging. This pathway provides new insight into factors that increase dynamic blebbing and identifies new targets, such as P2X7, that can be used to improve the culture of cells with therapeutic potential.