The E-liquid flavoring vanillin alters energy and autophagic pathways in human proximal tubule (HK-2) epithelial cells.

The use of flavored e-liquids in electronic nicotine delivery systems (ENDS) has become very popular in recent years, but effects of these products have not been well characterized outside the lung. In this study, acute exposure to the popular flavoring vanillin (VAN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0-1000 µM VAN for 24 or 48 h and cellular stress responses were determined. Mitochondrial viability using MTT assay showed a significant decrease between the control and 1000 µM group by 48 h. Seahorse XFp analysis showed significantly increased basal respiration, ATP production, and proton leak after 24 h exposure. By 48 h exposure, these parameters remained significantly increased in addition to non-mitochondrial respiration and maximal respiration. Glycolytic activity after 24 h exposure showed significant decreases in glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. The autophagy markers microtubule-associated protein 1A/1B light chain 3 (LC3B-I and LC3B-II) were probed via western blotting. The ratio of LC3B-II/LC3B-I was significantly increased after 24 h exposure to VAN, but by 48 h this ratio significantly decreased. The mitophagy marker PINK1 showed an increasing trend at 24 h, and its downstream target Parkin was significantly increased between the control and 750 µM group only. Finally, the oxidative stress marker 4-HNE was significantly decreased after 48 h exposure to VAN. These results indicate that acute exposure to VAN in the kidney HK-2 model can induce energy and autophagic changes within the cell.

The e-liquid flavoring cinnamaldehyde induces cellular stress responses in human proximal tubule (HK-2) kidney cells.

Flavored e-liquid use has become popular among e-cigarette users recently, but the effects of such products outside the lung are not well characterized. In this work, acute exposure to the popular flavoring cinnamaldehyde (CIN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0-100 µM CIN for 24-48 h and cellular stress responses were assessed. Mitochondrial viability via MTT assay was significantly decreased at 20 µM for 24 and 48 h exposure. Seahorse XFp analysis showed significantly decreased mitochondrial energy output at 20 µM by 24 h exposure, in addition to significantly reduced ATP Synthase expression. Seahorse analysis also revealed significantly decreased glycolytic function at 20 µM by 24 h exposure, suggesting inability of glycolytic processes to compensate for reduced mitochondrial energy output. Cleaved caspase-3 expression, a mediator of apoptosis, was significantly increased at the 24 h mark. C/EBP homologous protein (CHOP) expression, a mediator of ER-induced apoptosis, was induced by 48 h and subsequently lost at the highest concentration of 100 µM. This decrease was accompanied by a simultaneous decrease in its downstream target cleaved caspase-3 at the 48 h mark. The autophagy marker microtubule-associated protein 1 A/1B light chain 3 (LC3B-I and LC3B-II) expression was significantly increased at 100 µM by 24 h. Autophagy-related 7 (ATG7) protein and mitophagy-related proteins PTEN-induced putative kinase 1 (PINK1) and PARKIN expression were significantly reduced at 24 and 48 h exposure. These results indicate acute exposure to CIN in the kidney HK-2 model induces mitochondrial dysfunction and cellular stress responses.

Does removing menthol cigarettes in convenience stores reduce susceptibility to cigarette smoking? An experimental investigation in young people.

BACKGROUND: Evidence for the effectiveness of menthol cigarette bans comes mostly from studies of adults that smoke. This experiment evaluated whether the absence of menthol products from a convenience store influenced young people's susceptibility to cigarette smoking after they shopped in the store. METHODS: This experiment took place in the RAND StoreLab (RSL), a life-sized research convenience store. A three-group, between-subjects design was used. Study conditions differed in the mix of flavored tobacco products the RSL displayed: 1) All tobacco-, sweet-, and menthol-flavors displayed; 2) only tobacco- and menthol-flavors displayed; and 3) only tobacco-flavors displayed. Participants were randomly assigned to shop in the RSL under one of these conditions and after shopping, completed measures of their susceptibility to cigarette smoking, one measure for menthol cigarettes and one for unflavored cigarettes (scores on each susceptibility measure was dichotomized: 0 = not susceptible; 1 = susceptible). RESULTS: Multivariable logistic regression assessed the main effects of condition on susceptibility to smoking menthol and unflavored cigarettes. There was no condition effect on susceptibility to smoking unflavored cigarettes. However, removing menthol-flavored products significantly increased participants' susceptibility to smoking menthol cigarettes compared to when all flavored products were available (OR = 3.66, 95% CI [1.33, 10.03]). This significant effect was only found among young people with some pre-existing risk of cigarette smoking (OR = 5.92, 95% CI [1.81, 19.39]). CONCLUSION: Results suggest the need to consider that menthol bans could unintentionally increase the appeal of menthol cigarettes among youth already at risk of smoking.

Acute effects of flavored Black and mild cigars among young adult cigarette smokers.

The U.S. Food and Drug Administration proposed new product standards that would ban characterizing flavors (other than tobacco) in cigars. To inform this regulatory action, we compared physiological effects, use behavior, and subjective effects of four popular cigar flavors in cigar-naïve young adult cigarette smokers. Across five laboratory visits, participants (n = 25) used and evaluated own brand (OB) cigarettes or Black & Mild cigars (original, wine, apple, and cream flavors). Linear mixed models tested differences in saliva nicotine, exhaled carbon monoxide (CO), heart rate (HR), blood pressure (BP), puff topography, and subjective effects (p < .05). Compared to all cigars, OB resulted in higher nicotine boost (953 vs. < 300 ng/ml) and lower CO boost (4 vs. 8-9 ppm). Nicotine boost for original cigars (283 ng/ml) was significantly higher than wine (190 ng/ml). All products significantly increased HR/BP relative to baseline, but across time wine and apple cigars were associated with significantly lower HR than OB and BP effects varied. Relative to OB, participants took approximately 0.5 s longer puffs for all cigars and took significantly larger puffs (+ 21%-24%) of original, wine, and apple cigars. OB was rated more positively than all cigars, which had similar subjective effects. Wine cigars were disliked most and were less effective in reducing tobacco abstinence symptoms than OB; cream cigars were harsher and had stronger flavor intensity than original. The consistency in toxicant exposure, use behavior, and subjective effects across cigar flavors, including original, highlights the need for product standards to interpret characterizing flavors subject to prohibition broadly. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

A discrete choice experiment on price and flavour effects on the appeal of nicotine products: a pilot study among young adults in Switzerland.

AIMS: To explore the effects of prices and flavour availability on the appeal of different tobacco and nicotine products, including conventional cigarettes, Electronic Nicotine Delivery Systems (ENDS) and Heated Tobacco Systems (HTS) among an adult population in Switzerland. METHODS: We performed a Discrete Choice Experiment among a group of Swiss aged ≥18 years via the online recruiting platform Prolific in a convenience sample. Our sample included both non-smokers and smokers. We used a within-subject, alternative-specific block design in a series of choice sets including different smoking products. We fixed the attributes of nicotine content (high or medium) and harmfulness (in years of life lost) for each product. Attributes of interest included price (ranging from CHF 5 to 25 in increments of 5) and flavour (fruity/menthol vs none/tobacco flavour). We performed a conditional logistic regression on the attributes' influence on the appeal of cigarettes, ENDS and HTS. RESULTS: A total of 108 out of 153 participants (n = 25 smokers and n = 83 non-smokers, completion rate = 71%) successfully completed our pilot survey experiment. We found that, in general, increasing the price of combustible cigarettes, ENDS and HTS by one standard deviation (around CHF 7) reduced their appeal by approximately 66% (relative risk [RR]: 0.34; 95% CI: 0.28-0.42). Unflavoured alternative nicotine products were found to be less appealing than flavoured products, especially for non-smokers, with a 86% decrease in appeal (RR: 0.14; 95% CI: 0.13-0.16). For non-smokers, an increase in price by one standard deviation was associated with a decrease in the appeal of any product by approximately 19% (RR: 0.81; 95% CI: 0.72-0.92). For smokers, the effect sizes were smaller, but overall, the appeal of all products decreased with increasing prices and reduced flavours. CONCLUSIONS: Our Discrete Choice Experiment suggests that, for the Swiss context, limiting the availability of flavours for alternative smoking products has the potential to reduce their appeal to non-smokers by 86% and that a small but significant increase in prices to CHF 15 for cigarettes, ENDS and HTS could lead to a major (around 66%) decrease in their appeal.

Impact of tobacco flavoring on oral nicotine consumption in C57BL/6J mice.

BACKGROUND: The continued use of flavors in tobacco products has been a prominent factor in their popularity, yet little is known regarding their role in nicotine dependence. This study aimed to investigate the impact of tobacco flavoring on oral nicotine consumption in mice using the two-bottle choice (2BC) test and assessed the potential impact of age and sex in their interactions. METHODS: Adolescent and adult male and female C57BL/6J mice were used. First, voluntary consumption of tobacco flavor concentrate from a commercial electronic cigarette liquid vendor (Avail Vapor LLC) was measured; then, the effects of tobacco flavoring in combination with nicotine were examined. In one approach, tobacco flavor concentration was kept constant while nicotine concentration varied, and in the second, nicotine was kept constant while the tobacco flavor concentration varied. RESULTS: Overall, tobacco flavoring decreased oral nicotine consumption in mice, and its effects were sex- and age-dependent. Although females consumed the tobacco-flavored solution at a slightly higher rate than males, male mice were more sensitive to the effects of the combination (nicotine + tobacco). Furthermore, adolescent mice showed a starker reduction in nicotine consumption in the presence of tobacco flavoring compared to adult mice. This attenuation was most likely due to a basal aversion to the tobacco flavoring itself, thus, creating a negative synergistic effect with nicotine. CONCLUSIONS: Tobacco flavoring increases aversion to nicotine in the 2BC test in C57BL6J mice, suggesting that some flavors may diminish rather than enhance oral nicotine consumption in rodents.

Effect of D-Limonene Nanoemulsion Edible Film on Banana (Musa sapientum Linn.) Post-Harvest Preservation.

D-limonene (4-isopropenyl-1-methylcyclohexene) is an important compound in several citrus essential oils (such as orange, lemon, tangerine, lime, and grapefruit). It has been used as a flavoring agent and as a food preservative agent, with generally recognized as safe (GRAS) status. D-limonene has been well-studied for its anti-inflammatory, antioxidant, anti-cancer, and antibacterial properties. The antibacterial activity of D-limonene against food-borne pathogens was investigated in this study by preparing a D-limonene nanoemulsion. The D-limonene solution and nanoemulsion have been prepared in six concentrations, 0.04%, 0.08%, 0.1%, 0.2%, 0.4%, and 0.8% (v/v), respectively, and the antibacterial activity was tested against four food-borne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, and Escherichia coli). The results showed that the D-limonene nanoemulsion had good nanoscale and overall particle size uniformity, and its particle size was about 3~5 nm. It has been found that the D-limonene solution and nanoemulsion have a minimal inhibitory concentration of 0.336 mg/mL, and that they could inhibit the growth of microorganisms efficiently. The data indicate that the D-limonene nanoemulsion has more antibacterial ability against microorganisms than the D-limonene essential oil. After bananas are treated with 1.0% and 1.5% D-limonene nanoemulsion coatings, the water loss of the bananas during storage and the percentage of weight loss are reduced, which can inhibit the activity of pectinase. The application of a biocoating provides a good degree of antibacterial activity and air and moisture barrier properties, which help with extending the shelf life of bananas.

Toxicity, genotoxicity, and carcinogenicity of 2-methylfuran in a 90-day comprehensive toxicity study in gpt delta rats.

2-Methylfuran (2-MF) exists naturally in foods and is used as a flavoring agent. Furan, the core structure of 2-MF, possesses hepatocarcinogenicity in rodents. Accumulation of toxicological information on furan derivatives is needed to elucidate their carcinogenic mode of action. In the current study, we examined the comprehensive toxicological studies of 2-MF using gpt delta rats. 2-MF was intragastrically administered to groups of 10 male and 10 female Sprague-Dawley gpt delta rats at a dose of 0, 1.2, 6, or 30 mg/kg/day for 13 weeks. Effects of 2-MF on the hepatobiliary system including an increase in serum alkaline phosphatase were observed in the 6 and 30 mg/kg groups, and cholangiofibrosis was found in the 30 mg/kg group. The no observed adverse effect level was set at 1.2 mg/kg/day for both sexes and 1.14 mg/kg/day was determined as the benchmark dose low. The acceptable daily intake was calculated to be 11.4 µg/kg/day. Increases in the number and areas of glutathione S-transferase placental form-positive foci in the 30 mg/kg group were apparent, suggesting the hepatocarcinogenicity of 2-MF in rats. By contrast, the lack of increase in in vivo mutagenicity in the liver implied that 2-MF hepatocarcinogenesis may not involve genotoxic mechanisms.

E-liquids and vanillin flavoring disrupts retinoic acid signaling and causes craniofacial defects in Xenopus embryos.

Environmental teratogens such as smoking are known risk factors for developmental disorders such as cleft palate. While smoking rates have declined, a new type of smoking, called vaping is on the rise. Vaping is the use of e-cigarettes to vaporize and inhale an e-liquid containing nicotine and food-like flavors. There is the potential that, like smoking, vaping could also pose a danger to the developing human. Rather than waiting for epidemiological and mammalian studies, we have turned to an aquatic developmental model, Xenopus laevis, to more quickly assess whether e-liquids contain teratogens that could lead to craniofacial malformations. Xenopus, like zebrafish, has the benefit of being a well-established developmental model and has also been effective in predicting whether a chemical could be a teratogen. We have determined that embryonic exposure to dessert flavored e-liquids can cause craniofacial abnormalities, including an orofacial cleft in Xenopus. To better understand the underlying mechanisms contributing to these defects, transcriptomic analysis of the facial tissues of embryos exposed to a representative dessert flavored e-liquid vapor extract was performed. Analysis of differentially expressed genes in these embryos revealed several genes associated with retinoic acid metabolism or the signaling pathway. Consistently, retinoic acid receptor inhibition phenocopied the craniofacial defects as those embryos exposed to the vapor extract of the e-liquid. Such malformations also correlated with a group of common differentially expressed genes, two of which are associated with midface birth defects in humans. Further, e-liquid exposure sensitized embryos to forming craniofacial malformations when they already had depressed retinoic acid signaling. Moreover, 13-cis-retinoic acid treatment could significantly reduce the e-liquid induced malformation in the midface. Such results suggest the possibility of an interaction between retinoic acid signaling and e-liquid exposure. One of the most popular and concentrated flavoring chemicals in dessert flavored e-liquids is vanillin. Xenopus embryos exposed to this chemical closely resembled embryos exposed to dessert-like e-liquids and a retinoic acid receptor antagonist. In summary, we determined that e-liquid chemicals, in particular vanillin, can cause craniofacial defects potentially by dysregulating retinoic acid signaling. This work warrants the evaluation of vanillin and other such flavoring additives in e-liquids on mammalian development.

Phenolic acid metabolites of polyphenols act as inductors for hormesis in C. elegans.

INTRODUCTION: Aging represents a major risk factors for metabolic diseases, such as diabetes, obesity, or neurodegeneration. Polyphenols and their metabolites, especially simple phenolic acids, gained growing attention as a preventive strategy against age-related, non-communicable diseases, due to their hormetic potential. Using Caenorhabditis elegans (C. elegans) we investigate the effect of protocatechuic, gallic, and vanillic acid on mitochondrial function, health parameters, and the induction of potential hormetic pathways. METHODS: Lifespan, heat-stress resistance and chemotaxis of C. elegans strain P X 627, a specific model for aging, were assessed in 2-day and 10-day old nematodes. Mitochondrial membrane potential (ΔΨm) and ATP generation were measured. mRNA expression levels of longevity and energy metabolism-related genes were determined using qRT-PCR. RESULTS: All phenolic acids were able to significantly increase the nematodes lifespan, heat-stress resistance and chemotaxis at micromolar concentrations. While ΔΨm was only affected by age, vanillic acid (VA) significantly decreased ATP concentrations in aged nematodes. Longevity pathways, were activated by all phenolic acids, while VA also induced glycolytic activity and response to cold. CONCLUSION: While life- and health span parameters are positively affected by the investigated phenolic acids, the concentrations applied were unable to affect mitochondrial performance. Therefore we suggest a hormetic mode of action, especially by activation of the sirtuin-pathway.

Nicotine-free vapor inhalation produces behavioral disruptions and anxiety-like behaviors in mice: Effects of puff duration, session length, sex, and flavor.

Electronic-cigarette's (ECIGs) popularity has grown over the last decade and changed the way individuals administer nicotine. Preclinical research is imperative for understanding the addictive properties and health-risks associated with ECIG use; however, there is not a standard dosing regimen used across research laboratories. The main objective was to determine how vapor puff durations, administration session length, and flavored e-liquid alter general and mood-disorder related behaviors while providing a foundation of vapor administration parameters. Adult male and female C57BL/6 mice were exposed to several nicotine-free unflavored vapor puff durations (1, 3, 6, or 10 s) and vapor administration session lengths (10 and 30 min) then measured on the following assays: locomotor activity (LMA), tail suspension test (TST), and light-dark test. The effects of mecamylamine and the time-course of vapor-induced depression of LMA also were assessed. Additionally, mice were exposed to flavored (strawberry and adventurers tobacco blend) vapor inhalation and measured on locomotor activity, tail suspension test, and light-dark test. Following both 10 and 30 min vapor administration session, there was a puff duration-dependent decrease in distance traveled, time in center, and rearing. The vapor-induced depression of LMA was not mediated by nicotine or nicotinic acetylcholine receptor (nAChR) activation and lasted 60-90 min. The 10 s puff duration produced an anxiogenic-like effect in the light-dark test by decreasing the time spent in the light side. Vapor inhalation did not significantly alter TST behavior. No significant effects of sex or flavor were found. The anxiogenic-like effects of nicotine-free vapor inhalation are concerning as many adolescents vape nicotine-free flavored e-liquid, and there is an association between ECIGs and mood disorders. Additionally, these studies demonstrate that vapor puff duration, but not vapor administration session length, is an important variable to consider during research design as it can become a confounding variable and alter baseline behaviors.

Self-assembled 5-fluorouracil-cinnamaldehyde nanodrugs for greatly improved chemotherapy in vivo.

The preferred cancer treatment is to achieve a high therapeutic effect as well as reduce side effects. In this study, we developed carrier-free nano drugs based on 5-fluorouracil (5FU) and cinnamaldehyde (CA) to meet the above goals. Two model drugs were spliced by acetal linkage and ester bond, which could self-assemble into nano drug particles (5FU-CA NPs) with a size of ∼170 nm. In vitro cell experiments showed 5FU-CA NPs were efficiently internalized by HepG2 cells. They then quickly exerted dual drug activities by the cleavage of acetal and ester bond, resulting in enhanced cell-killing efficacy and apoptosis. Synergistic mechanisms were achieved via the anti-metabolic effects mediated by 5FU-COOH and the oxidative damage induced by CA. In vivo anti-tumor evaluation further indicated that 5FU-CA NPs had higher tumor growth inhibition than 5FU-COOH/CA mixture (5FU-COOH + CA) and exhibited lower systemic toxicity under the same reducing dose of each drug. Overall, this is a successful synergistic anti-tumor attempt through rational self-assembly of drugs with different mechanisms and it can be extrapolated to other agents.

The effects of inhaled flavors on intravenous nicotine.

Menthol is the only available flavor in combusted tobacco cigarettes; however, e-cigarettes are available in thousands of flavors. Research on flavors and rewarding properties of nicotine is limited. The present study sought to examine the acute rewarding effects of flavors inhaled from an e-cigarette, in combination with intravenous (IV) nicotine among cigarette smokers. In the present study, 24 menthol-preferring young adult (aged 18 to 30) cigarette smokers were tested under 3 different e-cigarette flavor conditions (menthol, green apple, or menthol + green apple) in a within-subject cross-over design. During each test session, each participant received 3 IV infusions (saline, 0.25 mg/70 kg nicotine, 0.5 mg/70 kg nicotine) administered 1 hr apart. The main outcome measures assessed cardiovascular, subjective, and cognitive domains. Compared with green apple or green apple + menthol, menthol produced higher ratings of "cooling" (ps < 0.01). Craving was rated higher following administration of green apple and the combined menthol + apple flavor compared to menthol alone (ps < 0.05). As expected, IV-nicotine dose-dependently increased the ratings of subjective liking/disliking and peak heart rate, improved cognitive performance, and reduced smoking urges (all ps < 0.05). These subjective, cognitive, and physiological effects of nicotine were not affected by any flavor condition. The present findings did not support an interaction between IV-nicotine dose and inhaled flavor for acute effects of nicotine. Green apple flavor, alone or in combination with menthol, could result in higher craving or insufficiently alleviate craving, relative to menthol flavor alone. Additional research is warranted to examine extended exposure to inhaled flavors on the rewarding and addictive effects of nicotine. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Investigation on the Antibacterial Activity of Electronic Cigarette Liquids (ECLs): A Proof of Concept Study.

BACKGROUND: The key ingredients of e-cigarettes liquid are commonly propane-1,2-diol (also called propylene glycol) and propane-1,2,3-triol (vegetal glycerol) and their antimicrobial effects are already established. The nicotine and flavors which are often present in e-liquids can interfere with the growth of some microorganisms. OBJECTIVE: The effect of combining these elements in e-liquids is unknown. The aim of the study was to investigate the possible effects of these liquids on bacterial growth in the presence or absence of nicotine and flavors. METHODS: Susceptibilities of pathogenic strains (Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Enterococcus faecalis and Sarcina lutea) were studied by means of a multidisciplinary approach. Cell viability and antioxidant assays were also evaluated. RESULTS: All e-liquids investigated showed antibacterial activity against at least one pathogenic strain. Higher activity was correlated to the presence of flavors and nicotine. DISCUSSION: In most cases, the value of minimal bactericidal concentration is equal to the value of minimal inhibitory concentration showing that these substances have a bactericidal effect. This effect was observed in concentrations up to 6.25% v/v. Antioxidant activity was also correlated to the presence of flavors. Over time, the viability assay in human epithelial lung A549 cells showed a dose-dependent inhibition of cell growth. CONCLUSION: Our results have shown that flavors considerably enhance the antibacterial activity of propane-1,2-diol and propane-1,2,3-triol. This study provides important evidence that should be taken into consideration in further investigative approaches, to clarify the different sensitivity of the various bacterial species to e-liquids, including the respiratory microbiota, to highlight the possible role of flavors and nicotine.

Electronic Cigarette Liquid Constituents Induce Nasal and Tracheal Sensory Irritation in Mice in Regionally Dependent Fashion.

INTRODUCTION: Electronic cigarettes (e-cigs) are currently used by millions of adults and adolescents worldwide. Major respiratory symptoms, such as coughing reported by e-cig users, including patients with e-cig, or vaping, product use-associated lung injury (EVALI), indicate e-cig constituent-induced sensory irritation. However, e-cig constituent-induced nociceptive activity in nasal and tracheal respiratory epithelia (RE) and neuronal activation in the trigeminal ganglia and brainstem nuclei, which receive airway chemosensory inputs have not been examined and compared. Comparisons of physiological responses between freebase nicotine and nicotine salts are also missing. AIMS AND METHODS: Event-related potential (ERP) was recorded electrophysiologically to assess mouse nasal and tracheal RE chemosensory responses to various flavorings, nicotine, including freebase and nicotine salts, e-liquid mixtures, and tussigenic stimuli. Also, mice were subjected to inhalation exposure to aerosol of a vanilla-flavored e-liquid or air (control), and the activated-trigeminal nociceptive neurons and brainstem neurons were examined using immunohistochemistry. RESULTS: Individual constituents and mixtures of e-liquids, capsaicin, and citric and acetic acids evoked significantly larger ERP in the nose than in the trachea with the exception of menthol. ERP responses to freebase nicotine were significantly larger than protonated nicotine. Four nicotine salts (benzoate, lactate, levulinate, and salicylate) induced similar responses. Compared with air-exposed mice, e-liquid aerosol-exposed mice showed a significant increase in numbers of activated trigeminal nociceptive neurons and brainstem neurons in the spinal trigeminal nucleus, paratrigeminal nucleus, and nucleus tractus solitarius. CONCLUSIONS: E-liquid constituents region-dependently stimulate airway nociceptive chemosensory systems, and freebase nicotine is more potent than protonated nicotine. IMPLICATIONS: Neural abnormalities have been implicated in the development of nasal and respiratory illnesses. The higher sensitivity of the nasal nociceptive chemosensory system to nicotine and flavorings may indicate a health risk for e-liquid aerosol-induced upper airway illnesses via neurogenic alteration and warrants further investigation.

Electronic Cigarette Refill Fluids Sold Worldwide: Flavor Chemical Composition, Toxicity, and Hazard Analysis.

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.

Cigarillos Compromise the Mucosal Barrier and Protein Expression in Airway Epithelia.

Smoking remains a leading cause of preventable morbidity and mortality worldwide. Despite a downward trend in cigarette use, less-regulated tobacco products, such as cigarillos, which are often flavored to appeal to specific demographics, such as younger people, are becoming increasingly popular. Cigar/cigarillo smoking has been considered a safer alternative to cigarettes; however, the health risks associated with cigar in comparison with cigarette smoking are not well understood. To address this knowledge gap, we characterized the effects of multiple brands of cigarillos on the airway epithelium using ex vivo and in vivo models. To analyze these effects, we assessed the cellular viability and integrity of smoke-exposed primary airway cell cultures. We also investigated the protein compositions of apical secretions from cigarillo-exposed airway epithelial cultures and BAL fluid of cigarillo-exposed mice through label-free quantitative proteomics and determined the chemical composition of smoke collected from the investigated cigarillo products. We found that cigarillo smoke exerts similar or greater effects than cigarette smoke in terms of reduced cell viability; altered protein levels, including those of innate immune proteins; induced oxidative-stress markers; and greater nicotine delivery to cells. The analysis of the chemical composition of the investigated cigarillo products revealed differences that might be linked to the differential effects of these products on cell viability and protein abundance profiles, which have been associated with a range of health risks in the context of airway biology. These findings contradict the assumption that cigarillos might be safer and less harmful than cigarettes. Instead, our results indicate that cigarillo smoke is associated with equal or greater health risks and the same or increased airway toxicity compared with cigarette smoke.

Monosodium glutamate alters the function and morphology of the parotid gland in Sprague Dawley rats / Glutamato monosódico altera la función y morfología de la glándula parótida en ratas Sprague Dawley

Monosodium glutamate (MSG) is a flavor enhancer widely used in the food industry, with obesogenic properties, in addition to causing alterations in the oral cavity. The aim of the study was to observe the morphofunctional changes in the parotid gland after the administration of MSG in rats. 18 newborn male Sprague Dawley rats were used, divided into three groups (Control group; MSG1 group: 4 mg/g weight of monosodium glutamate, 5 doses, kept for 8 weeks, and MSG2 group: 4 mg/g weight of MSG, 5 doses, kept for 16 weeks). The body mass index (BMI) was calculated, and the salivary flow, pH, a-amylase activity, Na, Cl, K and Ca were analyzed by quantitative analysis. After euthanasia by ketamine/xylazine overdose, parotid volume was analyzed and stereology was performed. MSG administration caused an increase in BMI and a decrease in parotid volume as well as a reduction in salivary flow and pH and an increase in a-amylase activity, also increasing the salivary sodium and chlorine levels. Alterations in the normal stereological parameters of the gland were observed. Exposure to MSG caused morphofunctional alterations at parotid gland.

El glutamato monosódico (MSG), es un potenciador del sabor ampliamente utilizado en la industria alimentaria. Diversos estudios han propuesto la relación entre éste y el desarrollo de obesidad, además de provocar alteraciones en la cavidad oral. El objetivo del estudio fue observar los cambios morfofuncionales a nivel de la glándula parótida, posterior a la administración de MSG en ratas. Se utilizaron 18 ratas neonatas Sprague Dawley machos, divididas en tres grupos según su tiempo de exposición y dosis a MSG (Grupo Control, Grupo MSG1: 4 mg/g peso de glutamato monosódico, 5 dosis, mantenidas 8 semanas, Grupo MSG2: 4 mg/g peso de MSG, 5 dosis, mantenidas 16 semanas. Fue calculado el índice de masa corporal (BMI), además de ser analizado el flujo salival, pH, actividad de α-amilasa, y Na, Cl, K y Ca mediante análisis semicuantitativo. Luego de la eutanasia por sobredosis de ketamina/xilasina, las glándulas parótidas fueron extraídas y analizado su volumen y fueron procesadas para histología, y estudio estereológico. La administración de MSG causó aumento en BMI y disminución del volumen parotídeo, además de disminución del flujo y pH salival, así como aumento en actividad de la a-amilasa, aumentando además los niveles de sodio y cloro salival. Fueron observadas alteraciones a nivel de los parámetros estereológicos normales de la glándula. La exposición a MSG causó alteraciones morfofuncionales a nivel parotídeo, observándose una disminución del volumen de la glándula, acompañado de alteraciones en el adenómero y conductos estriados de la glándula, implicados en la producción, secreción y modificación de la saliva, la cual se vio alterada, en el flujo, pH, y en sus componentes.

Cinnamaldehyde changes the dynamic balance of glucose metabolism by targeting ENO1.

AIMS: Hepatic glucose metabolism involves a variety of catabolic and anabolic pathways, and the dynamic balance of glucose metabolism is regulated in response to environmental and nutritional changes. The molecular mechanism of glucose metabolism in liver is complex and has not been fully elucidated so far. In this study, we hope to elucidate the target and mechanism of cinnamaldehyde (CA) in regulating glucose metabolism. MATERIALS AND METHODS: Molecular image tracing and magnetic capture in combination with an alkynyl-CA probe (Al-CA) was used to show CA covalently binds to α-enolase (ENO1) in both mouse liver and HepG2 cells. Accurate metabolic flow assays subsequently demonstrated that the utilization of glycogenic amino acids and the biosynthesis of tricarboxylic acid (TCA) cycle intermediates were strengthened, which was detected using nontargeted and targeted metabolomics analyses. KEY FINDINGS: Our study shows that CA covalently bonds with ENO1, which affects the stability and activity of ENO1 and changes the dynamic balance of glucose metabolism. The interruption of gluconeogenic reflux by ENO1 enhanced TCA cycle, and eventually led to a decrease in blood glucose and the improvement of mitochondrial efficiency. SIGNIFICANCE: These results provide a detailed description of how CA maintains the dynamic balance of glucose utilization and improves energy metabolism.

Impact of E-Cigarette Liquid Flavoring Agents on Activity of Microsomal Recombinant CYP2A6, the Primary Nicotine-Metabolizing Enzyme.

Nicotine is the primary psychoactive chemical in both traditional and electronic cigarettes (e-cigarettes). Nicotine levels in both traditional cigarettes and e-cigarettes are an important concern for public health. Nicotine exposure due to e-cigarette use is of importance primarily due to the addictive potential of nicotine, but there is also concern for nicotine poisoning in e-cigarette users. Nicotine concentrations in e-liquids vary widely. Additionally, there is significant genetic variability in the rate of metabolism of nicotine due to polymorphisms of CYP2A6, the enzyme responsible for the metabolism of approximately 80% of nicotine. Recent studies have shown CYP2A6 activity is also reduced by aromatic aldehydes such as those added to e-liquids as flavoring agents, which may increase nicotine serum concentrations. However, the impacts of flavored e-liquids on CYP2A6 activity are unknown. In this study, we investigated the impact of three flavored e-liquids on microsomal recombinant CYP2A6. Microsomal recombinant CYP2A6 was challenged at e-liquid concentrations ranging up to 0.125% (v/v) and monitored for metabolic activity using a probe molecule approach. Two e-liquids exhibited dose-dependent inhibition of CYP2A6 activity. Mass spectrometry was conducted to identify flavoring agents in flavored e-liquids that inhibited CYP2A6. Microsomal recombinant CYP2A6 was subsequently exposed to flavoring agents at concentrations ranging from 0.03 µM to 500 µM. Cinnamaldehyde and benzaldehyde were found to be the most potent inhibitors of microsomal CYP2A6 of the flavoring agents tested, with identified IC50 values of 1.1 µM and 3.0 µM, respectively. These data indicate certain aromatic aldehyde flavoring agents are potent inhibitors of CYP2A6, which may reduce nicotine metabolism in vivo. These findings indicate an urgent need to evaluate the effects of flavoring agents in e-cigarette liquids on the pharmacokinetics of nicotine in vivo.