Classic Tobacco Research Articles

Tobacco Smoking or Nicotine Phenotype and Severity of Clinical Presentation at the Emergency Department (SMOPHED): Protocol for a Noninterventional Observational Study.

In the last few years, several nicotine products have become available as alternatives to smoking tobacco. While laboratory and limited clinical studies suggest that these devices are less toxic compared to classic tobacco cigarettes, very little is known about their epidemiological impact. Visiting the emergency department (ED) often represents the first or even the only contact of patients with the health care system. Therefore, a study conducted at the ED to assess the impact of these products on health can be reliable and reflect a real-life setting. The aim of this noninterventional observational study (SMOPHED study) is to analyze the association between the severity of clinical presentation observed during ED visits among patients using various nicotine products and the subsequent outcomes, specifically hospitalization and mortality. Outcomes (hospitalization and mortality in the ED) will be examined in relation to various patterns of nicotine products use. We plan to enroll approximately 2000 participants during triage at the ED. These individuals will be characterized based on their patterns of tobacco and nicotine consumption, identified through a specific questionnaire. This categorization will allow for a detailed analysis of how different usage patterns of nicotine products correlate with the clinical diagnosis made during the ED visits and the consequent outcomes. Enrollment into the study started in March 2024. We enrolled a total of 901 participants in 1 month (approximately 300 potential participants did not provide the informed consent to participate). The data will be analyzed by a statistician as soon as the database is completed. Full data will be published by December 2024. There is substantial debate about the harm reduction potential of alternative nicotine products in terms of their smoking-cessation and risk-reduction potential. This study represents an opportunity to document epidemiological data on the link between the use of different types of nicotine products and disease diagnosis and severity during an ED visit, and thus evaluate the harm reduction potential claims for these products. PRR1-10.2196/54041.

E-Cigarette "Tobacco Flavor," How Do I Name Thee? Let Me Count the Ways….

In recent years, hundreds of jurisdictions around the world have enacted policies that prohibit sales of flavored e-cigarettes. In most cases, these jurisdictions permit the sale of tobacco flavored e-cigarettes. We sought to understand how tobacco flavor in e-cigarette advertisements were presented. Our sample included 2966 U.S. English-language e-cigarette ads compiled by a market research firm, Numerator, that ran from January 2018 to December 2020. We describe the prevalence of different tobacco flavor names in our sample and classified each name into one of the following thematic categories: Traditional Tobacco, Pipe/Cigar Tobacco, Sensory Expectancies, Color, Physical Attribute, Place, and non-characterizing Concept flavor. In our sample, 28% (n = 832/2966) of ads promoted at least one tobacco flavored e-cigarette product (e-cigarette device or liquid). Across the 832 ads, we counted 1019 tobacco flavored products and identified 51 unique tobacco flavor names. The most common tobacco flavor names were Traditional Tobacco names like "Tobacco" (n = 393), "Classic tobacco" (n = 107) and 'Original' (n = 59). Some names were associated with Color (eg, 'Golden tobacco'; n = 153), Sensory Expectancy (eg, 'Rich tobacco'; n = 148), Place where tobacco cultivation takes place (eg, 'Carolina tobacco'; n = 83), Physical Attributes of tobacco (eg, 'Cut tobacco'; n = 17) and non-characterizing Concept flavor (eg, 'Freedom juice'; n = 14). Few tobacco flavors suggested a type of Pipe/Cigar Tobacco (eg, "Cavendish"; n = 4). We identified 51 different tobacco flavor names, highlighting the practice describing the flavor beyond "tobacco." Future research can investigate whether these flavor names and descriptors influence consumers' perceptions, including perceived risks, of e-cigarette products. Flavors are used to market e-cigarettes. Globally, many jurisdictions restrict flavored e-cigarette sales, but few restrict tobacco flavor. This study identified 51 unique ways tobacco flavored e-cigarettes have been named in a sample of U.S. English-language ads, suggesting diversified ways to market "tobacco." We identified several sub-categories of tobacco flavor names that rely on Color, Sensory Expectancies, and non-characterizing Concept flavor attributes. As e-cigarette flavor restrictions that exempt tobacco flavor are still prevalent, this study highlights the need for continued monitoring of naming conventions of tobacco flavored products and examination of how nuanced flavor names influence perceptions and expectations.

USERS OF TOBACCO AND NICOTINE PRODUCTS: KEY BEHAVIORAL TYPES

Aim. Establishing the type of tobacco behavior and the main motives and reasons for smoking among consumers of tobacco products.
 Material and Methods. A survey involved 817 individuals aged 18-44 in the Lviv region. Various types of tobacco products were identified among the interviewees: 96 respondents were smokers of electronic nicotine delivery systems (ENDS), 93 respondents were smokers of heated tobacco products (HTPs), and 228 respondents were smokers of classic cigarettes. A sociological survey using Horn's questionnaire was conducted to determine the types of behavior of smokers.
 Results and Discussion. The dominant type of smoking behavior, both among smokers of classic cigarettes or tobacco products for heating (HTP), and among users of nicotine through electronic cigarettes (ENDS) was the "Support" type, the second-ranked - "Playing with a cigarette", the third-ranked - "Thirst". The largest number of smokers by the behavioral type "Playing with a cigarette" was found among HTP smokers - 55.91±5.15 cases per 100 respondents. The "Thirst" type is most typical in smokers of classic cigarettes - 39.91±3.24 cases per 100 respondents. The "Support" type was the leading one for male smokers in the groups of classic cigarette smokers (47.09±3.81 cases per 100 men) and ENDS smokers (47.62±7.71 cases per 100 men), while for HTP smokers - "Playing with a cigarette" (54.55±7.51 cases per 100 men). In the group of female smokers in all studied groups, the "Support" type was the dominant type of behavior. At the same time, among female ENDS smokers, two types of behavior were dominant, namely - "Support" and "Playing with a cigarette" with an identical rate of 44.44±6.76 cases per 100 women.
 Conclusions. Determining the types of behavior of a smoker and the main reasons and motives for smoking will allow choosing a method of smoking cessation and increasing the effectiveness of quitting tobacco and nicotine use.

Examining Flavor Appeal (Liking), Emotions and Openness to Use in Adults Who Do Not Use E-cigarettes

Objective: In this study, we examined emotional profiles, the pattern of intensities of evoked emotions, and the associations between emotions and openness in the use flavored e-cigarettes among adults who do not currently use e-cigarettes – younger/never smoked and older/currently smoked. Methods: We conducted an Internet experiment in women (N = 141) who do not currently use e-cigarettes. Experiment stimuli appeared as flavor names (classic tobacco, cool mint, fresh strawberry, top-shelf bourbon) and were randomly presented to participants who rated their evoked emotions using the EsSense™ Profile. Overall liking and openness to use were assessed per flavor. Seemingly unrelated regression models were constructed to determine factors associated with openness. Results: All flavors evoked greater intensities of negative emotions (disgusted, worried, and guilty) in younger/never smoked adults. The majority of the ratings on positive emotions (enthusiastic, interested, and satisfied) were positively and significantly correlated with flavor liking, whereas disgusted, was negatively correlated with flavor liking. Perceived harm was high whereas overall liking and openness were low. Flavor liking was significantly greater for “classic tobacco” in older/currently smoked (p < .001). Conclusions: Low intensities of positive emotions to flavor names suggest disinterest in e-cigarettes as a class product irrespective of flavor.

Effect of menthol/mint-flavored pods on young JUUL E-cigarette users’ subjective experience, puffing behavior, and nicotine exposure: A pilot study

BackgroundRecent regulations have banned all flavors except menthol/mint and classic tobacco from pod-based e-cigarette devices such as JUUL. However, menthol/mint flavor can present a potential risk given its increasing popularity among young people in the US and its puffing and nicotine-enhancing properties. This study examines the impact of menthol/mint flavor manipulation on users’ puffing behavior, subjective experience, and nicotine exposure among young people. MethodsJUUL users (n = 33, 18–24 years) attended two 60-min ad libitum e-cigarette use sessions (menthol/mint flavor vs. classic tobacco flavor) in a cross-over design. Puff topography and plasma nicotine concentration were measured, and participants completed subjective experience questionnaires. ResultsFollowing the use of the menthol/mint-flavored pod, increases were observed in measures of satisfaction, pleasurable/interest to use, willingness to use again, enjoyment, urge to vape, product appeal, taste, and concentration (p < .05 for all). For example, compared to the classic tobacco flavor, participants experienced significantly more satisfaction of the product (4.24 vs. 3.09; p = .001) and sensation enjoyment of the product (3.55 vs. 2.48; p = .002) when using the menthol/mint flavor. While means of the plasma nicotine boost and puff parameters were lower in the classic tobacco condition compared to the menthol/mint flavor condition, no statistical significance was observed between the two conditions (p > .05 for all). ConclusionsResults of this pilot study suggest that menthol/mint-flavor increases e-cigarette usersʼ subjective experience significantly. Regulating menthol/mint flavor is a potentially promising strategy to curb e-cigarette use among young people.

Flavored and Nicotine-Containing E-Cigarettes Induce Impaired Angiogenesis and Diabetic Wound Healing via Increased Endothelial Oxidative Stress and Reduced NO Bioavailability.

The prevalent use of electronic cigarettes (e-cigarettes) has increased exponentially in recent years, especially in youth who are attracted to flavored e-cigarettes. Indeed, e-cigarette or vaping product use-associated lung injury (EVALI) cases started to emerge in the United States in August 2019, resulting in 2807 hospitalized cases and 68 deaths as of 18 February 2020. In the present study, we investigated, for the first time, whether flavored and nicotine containing e-cigarettes induce endothelial dysfunction to result in impaired angiogenesis and wound healing particularly under diabetic condition. Nicotine containing e-cigarettes with various contents of nicotine (0, 1.2%, 2.4%), and flavored e-cigarettes of classic tobacco, mint, menthol, and vanilla or fruit from BLU (nicotine 2.4%) or JUUL (nicotine 3%), were used to treat endothelial cells in vitro and streptozotocin-induced diabetic mice in vivo. Endothelial cell superoxide production, determined by dihydroethidium (DHE) fluorescent imaging and electron spin resonance (ESR), was markedly increased by exposure to e-cigarette extract (e-CSE) in a nicotine-content dependent manner, while nitric oxide (NO) bioavailability detected by DAF-FM fluorescent imaging was substantially decreased. All of the different flavored e-cigarettes examined also showed significant effects in increasing superoxide production while diminishing NO bioavailability. Endothelial cell apoptosis evaluated by caspase 3 activity was markedly increased by exposure to e-CSE prepared from flavored and nicotine containing e-cigarettes. Endothelial monolayer wound assays revealed that nicotine-containing and flavored e-cigarettes induced impaired angiogenic wound repair of endothelial cell monolayers. Furthermore, vascular endothelial growth factor (VEGF) stimulated wound healing in diabetic mice was impaired by exposure to e-CSEs prepared from nicotine-containing and flavored e-cigarettes. Taken together, our data demonstrate for the first time that flavored and nicotine-containing e-cigarettes induce endothelial dysfunction through excessive ROS production, resulting in decreased NO bioavailability, increased endothelial cell apoptosis, and impairment in angiogenesis and wound healing, especially under diabetic condition. These responses of endothelial dysfunction likely underlie harmful effects of e-cigarettes in endothelial-rich organs, such as heart and lungs. These data also indicate that rigorous regulation on e-cigarette use should be enforced in diabetic and/or surgical patients to avoid severe consequences from impaired angiogenesis/wound healing.

Vaping /E‐liquid Exposure Causes Dysregulation of Neutrophil Extracellular Trap formation

IntroductionElectronic cigarettes (e‐cigarettes) vaporize e‐liquids containing propylene glycol and vegetable glycerine (PG/VG), nicotine, and flavors. The inhaled aerosol from e‐liquids may be potentially toxic or harmful. Although the short and long‐term health consequences of e‐cigarettes use are poorly understood, their use has risen steeply among adolescents and young adults. Neutrophils play an essential role in innate immune and are the first responders during infections. Thus, this study aims to test whether vaping can affect neutrophil function. We hypothesized that vaping e‐cigarettes and exposure to e‐liquids activate neutrophils, cause degranulation, and induce extracellular traps (NETs) through altered calcium signaling.Method and MaterialsNeutrophils were isolated from human blood samples of non‐vapor control (n=9) and e‐cigarette users (n=5) and studied within 2 hours. To measure changes in cytosolic calcium, neutrophils were loaded with Fluo‐4. Intracellular calcium responses were monitored on a Tecan multi‐plate reader following stimulation by 1 µM thapsigargin and 1 µM fMLP/100ng per ml LPS. In addition, since elevations in intracellular calcium trigger neutrophil activation and degranulation, elastase activity in the media samples was measured using a specific fluorogenic substrate (MAA‐3133, © vivitide, LLC.) at excitation/emission wavelengths 380/460 nm. To study NET formation, neutrophils were labeled with SYTOX Green dye and then exposed to different concentrations of Juul e‐liquids (menthol, classic tobacco flavors) and Bidi Stick flavors (Arctic, Gold, and Solar). Data acquisition was performed with Cytation‐5 Cell Imaging Plate Reader, then analysis and representative images were captured by confocal microscopy.ResultsNeutrophils from e‐cigarette users (n=5 vapers) showed significantly (P &lt; 0.001) increased intracellular calcium responses to thapsigargin and fMLP/LPS treatments compared to the non‐user controls (n=9). Additionally, vapers' neutrophils also secreted more elastase (P &lt; 0.001) compared to the control non‐users, indicating that e‐cigarette users may be susceptible to increased systemic protease activity. Our data also indicated that Juul and Bidi Stick e‐liquids stimulated neutrophils to induce NETs in a dose‐dependent manner. Furthermore, the study revealed that Juul e‐liquid menthol flavor with 5% nicotine could significantly (P &lt; 0.001) release NETs than equivalent nicotine ± benzoic acid alone.SummaryCirculating neutrophils are normally quiescent. However, our data indicated that circulating neutrophils from vapers were abnormally active and degranulated by released elastase. Additionally, e‐cigarette liquids dysregulated neutrophils by increasing NETs production. Overall, we speculate that vaping imbalanced innate immune system and may lead to abnormal responses to the infection, inappropriate inflammatory responses, and adverse health effects.

Effect of flavour manipulation on ENDS (JUUL) users’ experiences, puffing behaviour and nicotine exposure among US college students

SignificanceElectronic nicotine delivery system (ENDS) use has continued to increase exponentially among young people in the USA, with unique flavours being one of the most cited reasons for use. Yet,...

Acute Exposure to E‐Cigarette Vapor Promotes Neutrophil‐Platelet Aggregation in Murine Pulmonary Microvasculature

More than 1 in 4 high school students use electronic nicotine delivery systems (ENDS) in the United States, with usage rising over 130% since 2017. It is well known that smoking can cause cardiovascular disease and thrombosis; however, few studies have investigated the adverse effects of ENDS use on the cardiovascular system, especially in the lungs. Neutrophils and platelets are known to undergo activation and proinflammatory phenotypic changes when exposed to ENDS vapor, and their aggregation could promote vascular occlusion and further inflammation. We hypothesize that platelet and neutrophil recruitment will be increased in mice exposed to ENDS vapor, and that higher rates of aggregate formation will be observed in the pulmonary vasculature of these subjects. To investigate lung vascular pathology following ENDS use, we have applied an innovative spinning disk confocal microscopy approach to visualize real-time interactions of blood cells in the lungs of live mice. Identifying the presence of platelet-neutrophil aggregates in the lung due to ENDS vapor exposure would suggest that ENDS users may be at higher risk of atherosclerotic and thrombotic complications that with chronic use could eventually result in respiratory disease. Mice were subjected to 3 consecutive days of ENDS vapor inhalation using a Juul device with Virginia Classic Tobacco e-liquid (5% nicotine) for 3 hours each day. Mice subjected to room air sham inhalation constituted the control group. 24 hours after completion of the inhalation course, mice were anesthetized to facilitate microsurgery enabling fluorescence microscopy of the living lung. Neutrophils and platelets were labeled with fluorescently tagged antibodies to compare overall presence and aggregation within the lung microvasculature (Fig. 1). Preliminary data suggest significant (p<0.01) increases (Fig. 2) in platelet (2.59-fold), neutrophil (2.06-fold), and platelet-neutrophil aggregate (2.02-fold) presence in mice exposed to ENDS vapor compared to untreated mice. Collectively, this illustrates that ENDS vapor induces a pro-inflammatory state in the pulmonary vasculature in as little as one day after limited exposure. These findings will prove useful in future studies seeking to model the consequences of long-term ENDS use, which will ultimately enhance both clinician monitoring of patients at risk of such complications and public awareness of the potential consequences of ENDS use.

Flavor and product messaging are the two most important drivers of electronic cigarette selection in a choice-based task

Electronic cigarette use—vaping—is increasingly popular. Various product factors may influence an individual’s choice of e-cigarette. To provide an evidence base for e-cigarette regulation, a better understanding of the role different product attributes play in product preferences is needed. Here, we used conjoint analysis to quantify different factors that influence e-cigarettes choices, including flavors, nicotine level, customizability, or use of e-cigarettes to manage appetite/food craving. Young adults completed a set of choice-based conjoint tasks online. Choice Based Conjoint analysis (CBC) was used to determine utility scores for each attribute. Young adults (n = 587) who vaped at least once per week were included in analyses; gender differences were explored. Flavor was the most important attribute (48.1%), followed by product messaging (21.0%) and nicotine level (15.3%). Within flavor, confectionery and fruit flavors had the highest utility scores, while classic menthol and tobacco flavors had the lowest. Men and women differed in flavors, nicotine levels, and product messaging that appealed most. Among young adults who vape weekly, flavor is the most important factor in e-cigarette preferences. Gender also factors into e-cigarette preferences, especially for preferred nicotine level. Understanding why individuals choose particular e-cigarette products will help inform public health efforts and policy making.

Comparison of the biological impact of aerosol of e-vapor device with MESH® technology and cigarette smoke on human bronchial and alveolar cultures

Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than cigarette smoke (CS) exposure. Although many studies on e-vapor products have tested the effects of liquid formulations on cell cultures, few have evaluated the effects of aerosolized formulations. We examined the effects of acute exposure to the aerosol of an e-vapor device that uses the MESH® technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette on human organotypic bronchial epithelial culture and alveolar triculture models. In contrast to 3R4F CS exposure, exposure to the IQOS MESH aerosol (Classic Tobacco flavor) did not cause cytotoxicity in bronchial epithelial cultures or alveolar tricultures despite its greater concentrations of deposited nicotine (3- and 4-fold, respectively). CS exposure caused a marked decrease in the frequency and active area of ciliary beating in bronchial cultures, whereas IQOS MESH aerosol exposure did not. Global mRNA expression and secreted protein profiles revealed a significantly lower impact of IQOS MESH aerosol exposure than 3R4F CS exposure. Overall, our whole aerosol exposure study shows a clearly reduced impact of IQOS MESH aerosol relative to CS in bronchial and alveolar cultures, even at greater nicotine doses.

Abstract 15203: Acute Electronic Cigarette Exposure Decreases Heart Rate Variability in C57BL/6 Mice

Background: Despite a lack of empirical data, an unprecedented rise in electronic cigarettes (ECs) has been widely perceived as safe. Multiple studies support a causal link between tobacco cigarette (TC) use and a decline in cardiovascular (CV) health; however, there is a paucity of data with EC use. A study showed EC use decreased HRV in humans via increased sympathetic activation- a known mechanism predictive of CV disease from TC use. We aimed to develop a mouse model to determine if acute EC exposure modifies HRV in mice. Methods: Telemetry devices were implanted in the abdomen of 6 eight-week-old C57BL/6 mice to monitor ECG activity continuously. Mice underwent a 1-hr acclimation phase in exposure chambers followed by a 1-hour exposure to air (control). EC exposures consisted of two 15-min sessions (4-sec puff/26-sec air) to BluPlus® 2.4% nicotine (Classic Tobacco) with a 15-min period of aerosolized PBS added as a secondary control. For each exposure episode, a 45-min post-exposure event followed. Ponemah v6.20 software was used for HRV analysis of the time domain. A linear mixed-effects model with a robust estimator was used to determine the relationship between air vs. combined EC events and PBS (n = 1,109). R (Version R3.6.3) was utilized to test for statistical significance at α = 0.05. Results: EC exposures significantly decreased the standard deviation of NN intervals (SDNN) and root mean square of successive differences (RMSSD) by 5.00 and 9.34 units as compared to air exposures, suggesting reductions in total and short-term autonomic variability (Table 1). Both parameters increased in the post-EC periods as compared with the EC exposures. Conversely, PBS vs. air exposure showed a marked increase in SDNN and RMSSD. Conclusion: Short-term exposures to ECs decreased HRV in mice, consistent with similar results in human studies, suggesting that ECs could increase cardiovascular risk in an acute manner.

HPV Detection in Head and Neck Squamous Cell Carcinomas: What Is the Issue?

Besides classic tobacco and alcohol risk factors, human papillomavirus (HPV) plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), and notably oropharynx squamous cell carcinomas (OPSCCs). HPV-induced OPSCCs have a different biological behavior and a better prognosis compared to non-HPV-induced OPSCCs and the eighth-edition TNM classification now separates these two entities. Therefore, determining the HPV status of patients with OPSCC is now essential for treatment, prognosis, and development of clinical trials. In this review, after reminding essential steps of HPV implication in the cell cycle, we describe the existing tools that are currently feasible in routine practice according to facilities available in health structures, with their benefits and drawbacks: HPV PCR, E6/E7 mRNA RT-PCR, E6/E7 mRNA in situ hybridization, HPV DNA in situ hybridization, and P16 immunochemistry. Besides these traditional HPV detection tools, novel diagnostic approaches are being evaluated for HPV-induced OPSCC “ultrastaging.” E6 humoral response and ddPCR-detecting HPVct DNA are two techniques performed on blood and are therefore non-invasive. Baseline E6 humoral levels could have a prognostic value, and HPVct DNA could be helpful for HPV OPSCC recurrence monitoring. At last, next-generation sequencing (NGS)-based “capture HPV” is a technique feasible on biopsies and circulating DNA material. It helps characterize HPV integration status and sites, and it could define prognostic subgroups in HPV-induced OPSCC. These novel precision detection tools could be further integrated in the care of patients with HPV-induced OPSCC.

Application of High-Resolution Mass Spectrometry and a Theoretical Model to the Quantification of Multifunctional Carbonyls and Organic Acids in e-Cigarette Aerosol.

Electronic (e-) cigarette aerosol (particle and gas) is a complex mixture of chemicals, of which the profile is highly dependent on device operating parameters and e-liquid flavor formulation. The thermal degradation of the e-liquid solvents propylene glycol and glycerol often generates multifunctional carbonyls that are challenging to quantify because of unavailability of standards. We developed a theoretical method to calculate the relative electrospray ionization sensitivities of hydrazones of organic acids and carbonyls with 2,4-dinitrophenylhydrazine based on their gas-phase basicities (ΔGdeprotonation). This method enabled quantification by high-performance liquid chromatography-high-resolution mass spectrometry HPLC-HRMS in the absence of chemical standards. Accurate mass and tandem multistage MS (MSn) were used for structure identification of vaping products. We quantified five simple carbonyls, six hydroxycarbonyls, four dicarbonyls, three acids, and one phenolic carbonyl in the e-cigarette aerosol with Classic Tobacco flavor. Our results suggest that hydroxycarbonyls, such as hydroxyacetone, lactaldehyde, and dihydroxyacetone can be significant components in e-cigarette aerosols but have received less attention in the literature and have poorly understood health effects. The data support the radical-mediated e-liquid thermal degradation scheme that has been previously proposed and emphasize the need for more research on the chemistry and toxicology of the complex product formation in e-cigarette aerosols.

Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of Classic Tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESH\u2122 technology

We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air–liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.

Microglia Activation and Gene Expression Alteration of Neurotrophins in the Hippocampus Following Early-Life Exposure to E-Cigarette Aerosols in a Murine Model.

Recent epidemiological data indicate that the popularity of electronic cigarettes (e-cigarettes), and consequently nicotine use, is rising in both adolescent and adult populations. As nicotine is a known developmental neurotoxin, these products present a potential threat for those exposed during early life stages. Despite this, few studies have evaluated the toxicity of e-cigarettes on the developing central nervous system. The goal of this study was to assess neurotoxicity resulting from early-life exposure to electronic cigarette aerosols in an in vivo model. Specifically, studies here focused on neuro-parameters related to neuroinflammation and neurotrophins. To accomplish this, pregnant and neonatal C57BL/6 mice were exposed to aerosols produced from classic tobacco flavor e-cigarette cartridges (with [13 mg/ml] and without nicotine) during gestation (∼3 weeks) and lactation (∼3 weeks) via whole-body inhalation. Exposure to e-cigarette aerosols with and without nicotine caused significant reductions in hippocampal gene expression of Ngfr and Bdnf, as well as in serum levels of cytokines IL-1β, IL-2, and IL-6. Exposure to e-cigarette aerosols without nicotine enhanced expression of Iba-1, a specific marker of microglia, in the cornus ammonis 1 region of the hippocampus. Overall, our novel results indicate that exposure to e-cigarette aerosols, with and without nicotine, poses a considerable risk to the developing central nervous system. Consequently, e-cigarettes should be considered a potential public health threat, especially early in life, requiring further research and policy considerations.

HPV infection and p53 and p16 expression in esophageal cancer: are they prognostic factors?

BackgroundEsophageal squamous cell carcinoma (ESCC) is a highly lethal malignant tumor. Currently, Human papillomavirus (HPV) is suggested as a potential risk factor for esophageal cancer (EC) in addition to the classic risk factors, alcohol and tobacco, but this hypothesis still remains contradictory. We sought to investigate wether HPV and well-known biomarkers (p16 and p53) and patient-related factors that may have impact on survival of ESCC.MethodsWe conducted a prospective cohort study. By using multiplex PCR, we determined the prevalence of high risk HPV in ESCC, and evaluated the immunohistochemical expression of p16 and p53, molecular markers related to esophageal carcinogenesis in order to verify the potential influence of these variables in patients’s survival. Survival rates were estimated using Kaplan-Meier methods. A multivariate confirmatory model was performed using Cox proportional hazards regression.ResultsTwelve (13.8%) of 87 patients were HPV-DNA positive. Positive reactions of p16 and p53 were 10.7% and 68.6%, respectively. Kaplan-Meier analysis indicated that men (p = 0.025) had poor specific-cancer survival and a shorter progression-free survival (p = 0.050) as compared to women; III or IV clinical stage (p < 0.019) had poor specific-cancer survival and a shorter progression-free survival (p < 0.001) compared to I and II clinical stage; not submitted to surgery (<0.001) and not submitted to chemoradiotherapy (p = 0.039) had a poor specific-cancer survival, as well. The multivariate analysis showed that HPV, p16 and p53 status are not predictive parameters of progression-free and specific-cancer survival.ConclusionHPV infection and p53 and p16 expression are not prognostic factors in ESCC.

Role of sweet and other flavours in liking and disliking of electronic cigarettes

ObjectiveTo examine the extent to which the perception of sweet and other flavours is associated with liking and disliking of flavoured electronic cigarettes (e-cigarettes).Methods31 participants (13 females/18 males; 12 sole/19...

A C. elegans model of electronic cigarette use: Physiological effects of e-liquids in nematodes.

BackgroundElectronic cigarettes (e-cigs) have recently become very popular particularly among the younger generation. These nicotine delivery devices are viewed as a preferable alternative to more conventional forms of tobacco use and are thought to reduce the risk of chronic obstructive pulmonary disease, the third leading cause of death worldwide. However, there is very little data available on the consequences of e-cig use, though recently nicotine-independent inflammatory responses have been reported. The genetic model organism Caenorhabditis elegans is a soil nematode whose cell biology is remarkably well conserved with mammals. Here, we used C. elegans to test the physiologic effects of e-liquids used to refill e-cigs.MethodsLarval worms were exposed from hatching onwards to low concentrations (0.2 %) of e-liquids, distilled e-liquid vapor, propylene glycol (PG), or M9 buffer as a negative control. E-liquids tested included grape, menthol, and V2 Red “classic tobacco” flavors. Nicotine (48 ppm) was tested as a second level variable. Stereotypical physiological outputs were then measured, including developmental rate, fecundity, locomotion, lifespan, and the induction of canonical stress signaling pathways.ResultsA small but significant impairment of developmental rate and brood size was observed for PG and V2 Red treated worms compared to the negative control. Worms treated with e-liquids containing nicotine fared significantly worse than those that did not, but vaporization did not increase toxicity. Finally, both PG and V2 Red e-liquid induced an oxidative stress response in the absence of nicotine.ConclusionsPG exposure is sufficient to induce an oxidative stress response in nematodes, while nicotine is not. Both PG and nicotine independently influence physiologic measures of health and viability. The e-liquid flavorings did not significantly impact outcomes and there was no evidence for vaporization altering toxicity. These data suggest that the major physiologically significant component of e-liquids besides nicotine is likely the common solvent PG. We conclude that C. elegans are an appropriate model to rapidly assess parameters that may contribute to the basic cell biological effects of e-cigs.