Pharmacologic Inhibition of Transient Receptor Potential Ion Channel Ankyrin 1 Counteracts 2-Chlorobenzalmalononitrile Tear Gas Agent-Induced Cutaneous Injuries.

Deployment of the tear gas agent 2-chlorobenzalmalononitrile (CS) for riot control has significantly increased in recent years. The effects of CS have been believed to be transient and benign. However, CS induces severe pain, blepharospasm, lachrymation, airway obstruction, and skin blisters. Frequent injuries and hospitalizations have been reported after exposure. We have identified the sensory neuronal ion channel, transient receptor potential ankyrin 1 (TRPA1), as a key CS target resulting in acute irritation and pain and also as a mediator of neurogenic inflammation. Here, we examined the effects of pharmacologic TRPA1 inhibition on CS-induced cutaneous injury. We modeled CS-induced cutaneous injury by applying 10 µl CS agent [200 mM in dimethyl sulfoxide (DMSO)] to each side of the right ears of 8- to 9-week-old C57BL/6 male mice, whereas left ears were applied with solvent only (DMSO). The TRPA1 inhibitor HC-030031 or A-967079 was administered after CS exposure. CS exposure induced strong tissue swelling, plasma extravasation, and a dramatic increase in inflammatory cytokine levels in the mouse ear skin. We also showed that the effects of CS were not transient but caused persistent skin injuries. These injury parameters were reduced with TRPA1 inhibitor treatment. Further, we tested the pharmacologic activity of advanced TRPA1 antagonists in vitro. Our findings showed that TRPA1 is a crucial mediator of CS-induced nociception and tissue injury and that TRPA1 inhibitors are effective countermeasures that reduce key injury parameters when administered after exposure. Additional therapeutic efficacy studies with advanced TRPA1 antagonists and decontamination strategies are warranted. SIGNIFICANCE STATEMENT: 2-Chlorobenzalmalononitrile (CS) tear gas agent has been deployed as a crowd dispersion chemical agent in recent times. Exposure to CS tear gas agents has been believed to cause transient acute toxic effects that are minimal at most. Here we found that CS tear gas exposure causes both acute and persistent skin injuries and that treatment with transient receptor potential ion channel ankyrin 1 (TRPA1) antagonists ameliorated skin injuries.

Synthetic nicotine has arrived.

The introduction of a new product line of the popular disposable electronic cigarette brand Puffbar, advertised as containing synthetic nicotine, has drawn attention to the increasing use of synthetic nicotine in marketed products and its uncertain regulatory status. A search of the Truth Tobacco Industry Documents revealed that the industry considered using synthetic nicotine already in the 1960s, efforts that were abandoned due to high costs and insufficient purity. Recent patents revealed renewed efforts to develop more efficient strategies for the synthesis of nicotine. Nicotine exists as two stereoisomers, S-nicotine and R-nicotine. While S-nicotine is the prevalent (>99%) form of nicotine in tobacco, a market-leading form of synthetic nicotine contains both stereoisomers at equal amounts, raising concerns about inaccurate labelling and the poorly understood health effects of R-nicotine. Other manufacturers, including a leading vendor of pharmaceutical grade nicotine, developed stereospecific strategies to synthesise pure S-nicotine, now added to electronic cigarette products marketed in the USA and UK. While S-nicotine and R-nicotine can be differentiated by enantioselective High Performance Liquid Chromatography (HPLC), differentiation of synthetic (fossil-derived) from tobacco-derived S-nicotine will require development of methods to measure carbon isotope (14C or 13C) content. Vendors claim that the FDA has no authority to regulate synthetic nicotine as a tobacco product, allowing them to circumvent the premarket tobacco product application process. However, legal analysis suggests that FDA may have the authority to regulate synthetic nicotine as a drug. Alternatively, Congress needs to include nicotine from any source within the legal definition of tobacco products.

Ice flavours and non-menthol synthetic cooling agents in e-cigarette products: a review.

E-cigarettes with cooling flavours have diversified in ways that complicate tobacco control with the emergence of: (1) Ice-hybrid flavours (eg, 'Raspberry Ice') that combine cooling and fruity/sweet properties; and (2) Products containing non-menthol synthetic cooling agents (eg, Wilkinson Sword (WS), WS-3, WS-23 (termed 'koolada')). This paper reviews the background, chemistry, toxicology, marketing, user perceptions, use prevalence and policy implications of e-cigarette products with ice-hybrid flavours or non-menthol coolants. Scientific literature search supplemented with industry-generated and user-generated information found: (a) The tobacco industry has developed products containing synthetic coolants since 1974, (b) WS-3 and WS-23 are detected in mass-manufactured e-cigarettes (eg, PuffBar); (c) While safe for limited oral ingestion, inhalational toxicology and health effects from daily synthetic coolant exposure are unknown and merit scientific inquiry and attention from regulatory agencies; (d) Ice-hybrid flavours are marketed with themes incorporating fruitiness and/or coolness (eg, snow-covered raspberries); (e) WS-23/WS-3 concentrates also are sold as do-it-yourself additives, (f) Pharmacology research and user-generated and industry-generated information provide a premise to hypothesise that e-cigarette products with ice flavours or non-menthol cooling agents generate pleasant cooling sensations that mask nicotine's harshness while lacking certain aversive features of menthol-only products, (g) Adolescent and young adult use of e-cigarettes with ice-hybrid or other cooling flavours may be common and cross-sectionally associated with more frequent vaping and nicotine dependence in convenience samples. Evidence gaps in the epidemiology, toxicology, health effects and smoking cessation-promoting potential of using these products exist. E-cigarettes with ice flavours or synthetic coolants merit scientific and regulatory attention.

Synthetic cooling agent in oral nicotine pouch products marketed as 'Flavour-Ban Approved'.

BACKGROUND: US sales of oral nicotine pouches (ONPs) have rapidly increased, with cool/mint-flavoured ONPs the most popular flavour category. Restrictions on sales of flavoured tobacco products have either been implemented or proposed by several US states and localities. Zyn, the most popular ONP brand, is marketing Zyn Chill and Zyn Smooth as 'Flavour-Ban Approved' or 'unflavoured', probably to evade flavour bans and increase product appeal. At present, it is unclear whether these ONPs are indeed free of flavour additives that can impart pleasant sensations such as cooling. METHODS: Sensory cooling and irritant activities of 'Flavour-Ban Approved' Zyn ONPs, Chill and Smooth, along with minty varieties (Cool Mint, Peppermint, Spearmint, Menthol), were analysed by Ca2+ microfluorimetry in HEK293 cells expressing the cold/menthol (TRPM8) or menthol/irritant receptor (TRPA1). Flavour chemical content of these ONPs was analysed by gas chromatography/mass spectrometry. RESULTS: Zyn Chill ONP extracts robustly activated TRPM8, with much higher efficacy (39%-53%) than the mint-flavoured ONPs. In contrast, mint-flavoured ONP extracts elicited stronger TRPA1 irritant receptor responses than Chill extracts. Chemical analysis demonstrated that Chill exclusively contained WS-3, an odourless synthetic cooling agent, while mint-flavoured ONPs contained WS-3 together with mint flavourants. CONCLUSIONS: ONP products marketed as 'Flavour-Ban Approved' or 'unflavoured' contain flavouring agents, proving that the manufacturer's advertising is misleading. Synthetic coolants such as WS-3 can provide a robust cooling sensation with reduced sensory irritancy, thereby increasing product appeal and use. Regulators need to develop effective strategies for the control of odourless sensory additives used by the industry to bypass flavour bans.

Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment.

INTRODUCTION: Menthol, through its cooling and pleasant sensory effects, facilitates smoking and tobacco product initiation, resulting in the high popularity of mint/menthol-flavored E-cigarettes. More recently, E-cigarette vendors started marketing synthetic cooling agents as additives that impart a cooling effect but lack a characteristic minty odor. Knowledge about content of synthetic coolants in US-marketed E-cigarette products and associated health risks is limited. AIMS AND METHODS: E-liquid vendor sites were searched with the terms "koolada", "kool/cool", "ice", or WS-3/WS-23, denoting individual cooling agents, and relevant refill E-liquids were purchased. "Ice" flavor varieties of Puffbar, the most popular disposable E-cigarette brand, were compared with non-"Ice" varieties. E-liquids were characterized, and synthetic coolants quantified using GC/MS. Margin of exposure (MOE), a risk assessment parameter, was calculated to assess the risk associated with synthetic coolant exposure from E-cigarette use. RESULTS: WS-3 was detected in 24/25 refill E-liquids analyzed. All Puffbar flavor varieties contained either WS-23 (13/14) or WS-3 (5/14), in both "Ice"- and non-"Ice" flavors. Modeling consumption of WS-3 from vaped E-liquids, resulted in MOEs below the safe margin of 100 for most daily use scenarios. MOEs for WS-23 were <100 for 10/13 Puffbar flavors in all use scenarios. Puffbar power specifications are identical to Juul devices. CONCLUSIONS: Synthetic cooling agents (WS-3/WS-23) were present in US-marketed E-cigarettes, at levels that may result in consumer exposures exceeding safety thresholds set by regulatory agencies. Synthetic coolants are not only found in mint- or menthol-flavored products but also in fruit- and candy-flavored products, including popular disposable E-cigarette products such as Puffbar. IMPLICATIONS: Synthetic cooling agents are widely used in "kool/cool"- and "ice"-flavored E-liquids and in E-liquids without these labels, both as a potential replacement for menthol or to add cooling "notes" to nonmenthol flavors. These agents may be used to bypass current and future regulatory limits on menthol content in tobacco products, and not just E-cigarettes. Because synthetic cooling agents are odorless, they may not fall under the category of "characterizing flavor", potentially circumventing regulatory measures based on this concept. Regulators need to consider the additional health risks associated with exposure to synthetic cooling agents.

Toxic effects of chlorine gas and potential treatments: a literature review.

Chlorine gas is one of the highly produced chemicals in the USA and around the world. Chlorine gas has several uses in water purification, sanitation, and industrial applications; however, it is a toxic inhalation hazard agent. Inhalation of chlorine gas, based on the concentration and duration of the exposure, causes a spectrum of symptoms, including but not limited to lacrimation, rhinorrhea, bronchospasm, cough, dyspnea, acute lung injury, death, and survivors develop signs of pulmonary fibrosis and reactive airway disease. Despite the use of chlorine gas as a chemical warfare agent since World War I and its known potential as an industrial hazard, there is no specific antidote. The resurgence of the use of chlorine gas as a chemical warfare agent in recent years has brought speculation of its use as weapons of mass destruction. Therefore, developing antidotes for chlorine gas-induced lung injuries remains the need of the hour. While some of the pre-clinical studies have made substantial progress in the understanding of chlorine gas-induced pulmonary pathophysiology and identifying potential medical countermeasure(s), yet none of the drug candidates are approved by the U.S. Food and Drug Administration (FDA). In this review, we summarized pathophysiology of chlorine gas-induced pulmonary injuries, pre-clinical animal models, development of a pipeline of potential medical countermeasures under FDA animal rule, and future directions for the development of antidotes for chlorine gas-induced lung injuries.

Urgent reconsideration of lung edema as a preventable outcome in COVID-19: inhibition of TRPV4 represents a promising and feasible approach.

Lethality of coronavirus disease (COVID-19) during the 2020 pandemic, currently still in the exponentially accelerating phase in most countries, is critically driven by disruption of the alveolo-capillary barrier of the lung, leading to lung edema as a direct consequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We argue for inhibition of the transient receptor potential vanilloid 4 (TRPV4) calcium-permeable ion channel as a strategy to address this issue, based on the rationale that TRPV4 inhibition is protective in various preclinical models of lung edema and that TRPV4 hyperactivation potently damages the alveolo-capillary barrier, with lethal outcome. We believe that TRPV4 inhibition has a powerful prospect at protecting this vital barrier in COVID-19 patients, even to rescue a damaged barrier. A clinical trial using a selective TRPV4 inhibitor demonstrated a benign safety profile in healthy volunteers and in patients suffering from cardiogenic lung edema. We argue for expeditious clinical testing of this inhibitor in COVID-19 patients with respiratory malfunction and at risk for lung edema. Perplexingly, among the currently pursued therapeutic strategies against COVID-19, none is designed to directly protect the alveolo-capillary barrier. Successful protection of the alveolo-capillary barrier will not only reduce COVID-19 lethality but will also preempt a distressing healthcare scenario with insufficient capacity to provide ventilator-assisted respiration.

Activation of a nerve injury transcriptional signature in airway-innervating sensory neurons after lipopolysaccharide-induced lung inflammation.

The lungs and the immune and nervous systems functionally interact to respond to respiratory environmental exposures and infections. The lungs are innervated by vagal sensory neurons of the jugular and nodose ganglia, fused together in smaller mammals as the jugular-nodose complex (JNC). Whereas the JNC shares properties with the other sensory ganglia, the trigeminal (TG) and dorsal root ganglia (DRG), these sensory structures express differential sets of genes that reflect their unique functionalities. Here, we used RNA sequencing (RNA-seq) in mice to identify the differential transcriptomes of the three sensory ganglia types. Using a fluorescent retrograde tracer and fluorescence-activated cell sorting, we isolated a defined population of airway-innervating JNC neurons and determined their differential transcriptional map after pulmonary exposure to lipopolysaccharide (LPS), a major mediator of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) after infection with gram-negative bacteria or inhalation of organic dust. JNC neurons activated an injury response program, leading to increased expression of gene products such as the G protein-coupled receptor Cckbr, inducing functional changes in neuronal sensitivity to peptides, and Gpr151, also rapidly induced upon neuropathic nerve injury in pain models. Unique JNC-specific transcripts, present at only minimal levels in TG, DRG, and other organs, were identified. These included TMC3, encoding for a putative mechanosensor, and urotensin 2B, a hypertensive peptide. These findings highlight the unique properties of the JNC and reveal that ALI/ARDS rapidly induces a nerve injury-related state, changing vagal excitability.

Chemical Adducts of Reactive Flavor Aldehydes Formed in E-Cigarette Liquids Are Cytotoxic and Inhibit Mitochondrial Function in Respiratory Epithelial Cells.

INTRODUCTION: Flavor aldehydes in e-cigarettes, including vanillin, ethyl vanillin (vanilla), and benzaldehyde (berry/fruit), rapidly undergo chemical reactions with the e-liquid solvents, propylene glycol, and vegetable glycerol (PG/VG), to form chemical adducts named flavor aldehyde PG/VG acetals that can efficiently transfer to e-cigarette aerosol. The objective of this study was to compare the cytotoxic and metabolic toxic effects of acetals and their parent aldehydes in respiratory epithelial cells. AIMS AND METHODS: Cell metabolic assays were carried out in bronchial (BEAS-2B) and alveolar (A549) epithelial cells assessing the effects of benzaldehyde, vanillin, ethyl vanillin, and their corresponding PG acetals on key bioenergetic parameters of mitochondrial function. The potential cytotoxic effects of benzaldehyde and vanillin and their corresponding PG acetals were analyzed using the LIVE/DEAD cell assay in BEAS-2B cells and primary human nasal epithelial cells (HNEpC). Cytostatic effects of vanillin and vanillin PG acetal were compared using Click-iT EDU cell proliferation assay in BEAS-2B cells. RESULTS: Compared with their parent aldehydes, PG acetals diminished key parameters of cellular energy metabolic functions, including basal respiration, adenosine triphosphate production, and spare respiratory capacity. Benzaldehyde PG acetal (1-10 mM) increased cell mortality in BEAS-2B and HNEpC, compared with benzaldehyde. Vanillin PG acetal was more cytotoxic than vanillin at the highest concentration tested while both diminished cellular proliferation in a concentration-dependent manner. CONCLUSIONS: Reaction products formed in e-liquids between flavor aldehydes and solvent chemicals have differential toxicological properties from their parent flavor aldehydes and may contribute to the health effects of e-cigarette aerosol in the respiratory system of e-cigarette users. IMPLICATIONS: With no inhalation toxicity studies available for acetals, data from this study will provide a basis for further toxicological studies using in vitro and in vivo models. This study suggests that manufacturers' disclosure of e-liquid ingredients at time of production may be insufficient to inform a comprehensive risk assessment of e-liquids and electronic nicotine delivery systems use, due to the chemical instability of e-liquids over time and the formation of new compounds.

Differences in flavourant levels and synthetic coolant use between USA, EU and Canadian Juul products.

BACKGROUND: 'Juul' is the dominant US e-cigarette brand and was recently introduced to Canada, UK, France, Germany and Italy, with several flavours available across countries. US/Canadian products are sold with 5%, 3% and 1.5% (Canada only) nicotine content, whereas European Union (EU) regulation limits nicotine content to 1.7%. The differential nicotine content raises the question if flavour profiles and Juul device power output differ between countries. METHODS: 'Mint', 'Vanilla' and 'Mango' e-liquids from all six countries were purchased in 2019 and analysed by GC/MS for their principal flavourant and nicotine content. In addition, device power specifications were compared for devices purchased from the respective countries. RESULTS: Compositions of Juul e-liquids from the USA and Canada were identical and differed from the EU-marketed liquids, in which principal flavourant concentrations were significantly lower. EU Juul 'Mint' e-liquids contained a synthetic coolant, N-ethyl-p-menthane-3-carboxamide (WS-3), absent in US/Canadian products. US/Canadian 'Mango' e-liquid contained triethyl-citrate, an emulsifier. Nicotine contents matched label information, and devices had identical power specifications. CONCLUSIONS: Tested US/Canadian Juul e-liquids contained higher flavour concentrations than EU products, likely reflecting adaptation to user preferences. In EU, 'Mint' e-liquid, menthol is partially substituted with the synthetic coolant WS-3 that elicits a cooling effect like menthol but lacks its distinct 'minty' odour. The inhalational safety of WS-3 is unknown. The use of an emulsifier in US/Canadian 'Mango' Juul e-liquid may be necessary to keep the product homogeneous. Similar power specifications of devices between countries suggest that nicotine aerosol delivery is likely proportional to the e-liquid nicotine content.

ETAR and protein kinase A pathway mediate ET-1 sensitization of TRPA1 channel: A molecular mechanism of ET-1-induced mechanical hyperalgesia.

Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor that has been widely known as a pain mediator involved in various pain states. Evidence indicates that ET-1 sensitizes transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in vivo. But the molecular mechanisms still remain unknown. We aim to explore whether ET-1 sensitizes TRPA1 in primary sensory neurons and the molecular mechanisms. Ca2+ imaging, immunostaining, electrophysiology, animal behavioral assay combined with pharmacological experiments were performed. ET-1 sensitized TRPA1-mediated Ca2+ responses in human embryonic kidney (HEK)293 cells as well as in cultured native mouse dorsal root ganglion (DRG) neurons. ET-1 also sensitized TRPA1 channel currents. ET-1 sensitized TRPA1 activated by endogenous agonist H2O2. ETA receptor (ETAR) colocalized with TRPA1 in DRG neurons. ET-1-induced TRPA1 sensitization in vivo was mediated via ETAR and protein kinase A (PKA) pathway in HEK293 cells and DRG neurons. Pharmacological blocking of ETAR, PKA, and TRPA1 significantly attenuated ET-1-induced mechanical hyperalgesia in mice. Our results suggest that TRPA1 acts as a molecular target for ET-1, and sensitization of TRPA1 through ETAR-PKA pathway contributes to ET-1-induced mechanical hyperalgesia. Pharmacological targeting of TRPA1 and ETAR-PKA pathway may provide effective strategies to alleviate pain conditions associated with ET-1.

IL-33/ST2 signaling excites sensory neurons and mediates itch response in a mouse model of poison ivy contact allergy.

Poison ivy-induced allergic contact dermatitis (ACD) is the most common environmental allergic condition in the United States. Case numbers of poison ivy ACD are increasing due to growing biomass and geographical expansion of poison ivy and increasing content of the allergen, urushiol, likely attributable to rising atmospheric CO2 Severe and treatment-resistant itch is the major complaint of affected patients. However, because of limited clinical data and poorly characterized models, the pruritic mechanisms in poison ivy ACD remain unknown. Here, we aim to identify the mechanisms of itch in a mouse model of poison ivy ACD by transcriptomics, neuronal imaging, and behavioral analysis. Using transcriptome microarray analysis, we identified IL-33 as a key cytokine up-regulated in the inflamed skin of urushiol-challenged mice. We further found that the IL-33 receptor, ST2, is expressed in small to medium-sized dorsal root ganglion (DRG) neurons, including neurons that innervate the skin. IL-33 induces Ca2+ influx into a subset of DRG neurons through neuronal ST2. Neutralizing antibodies against IL-33 or ST2 reduced scratching behavior and skin inflammation in urushiol-challenged mice. Injection of IL-33 into urushiol-challenged skin rapidly exacerbated itch-related scratching via ST2, in a histamine-independent manner. Targeted silencing of neuronal ST2 expression by intrathecal ST2 siRNA delivery significantly attenuated pruritic responses caused by urushiol-induced ACD. These results indicate that IL-33/ST2 signaling is functionally present in primary sensory neurons and contributes to pruritus in poison ivy ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy ACD.

High-Intensity Sweeteners in Alternative Tobacco Products.

INTRODUCTION: Sweeteners in tobacco products may influence use initiation and reinforcement, with special appeal to adolescents. Recent analytical studies of smokeless tobacco products (snuff, snus, dissolvables) detected flavorants identical to those added to confectionary products such as hard candy and chewing gum. However, these studies did not determine the levels of sweeteners. The objective of the present study was to quantify added sweeteners in smokeless tobacco products, a dissolvable product, electronic cigarette liquids and to compare with sweetener levels in confectionary products. METHODS: Sweetener content of US-sourced smokeless tobacco, electronic cigarette liquid, and confectionary product samples was analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). RESULTS: All smokeless products contained synthetic high intensity sweeteners, with snus and dissolvables exceeding levels in confectionary products (as much as 25-fold). All snus samples contained sucralose and most also aspartame, but no saccharin. In contrast, all moist snuff samples contained saccharin. The dissolvable sample contained sucralose and sorbitol. Ethyl maltol was the most common sweet-associated component in electronic cigarette liquids. DISCUSSION: Sweetener content was dependent on product category, with saccharin in moist snuff, an older category, sucralose added at high levels to more recently introduced products (snus, dissolvable) and ethyl maltol in electronic cigarette liquid. The very high sweetener concentrations may be necessary for the consumer to tolerate the otherwise aversive flavors of tobacco ingredients. Regulation of sweetener levels in smokeless tobacco products may be an effective measure to modify product attractiveness, initiation and use patterns. IMPLICATIONS: Dissolvables, snus and electronic cigarettes have been promoted as risk-mitigation products due to their relatively low content of nitrosamines and other tobacco toxicants. This study is the first to quantify high intensity sweeteners in snus and dissolvable products. Snus and dissolvables contain the high intensity sweetener, sucralose, at levels higher than in confectionary products. The high sweetness of alternative tobacco products makes these products attractive to adolescents. Regulation of sweetener content in non-cigarette products is suggested as an efficient means to control product palatability and to reduce initiation in adolescents.

Menthol decreases oral nicotine aversion in C57BL/6 mice through a TRPM8-dependent mechanism.

BACKGROUND: Nicotine is a major oral irritant in smokeless tobacco products and has an aversive taste. Mentholated smokeless tobacco products are highly popular, suggesting that menthol increases their palatability and may facilitate initiation of product use. While menthol is known to reduce respiratory irritation by tobacco smoke irritants, it is not known whether this activity extends to oral nicotine and its aversive effects. STUDY DESIGN: The two-bottle choice drinking assay was used to characterise aversion and preference in C57BL/6 mice to a range of menthol concentrations (10-200 µg/mL). Then, effects of menthol on oral nicotine aversion were determined. Responses were compared with those in mice deficient in the cold/menthol receptor, TRPM8, expressed in trigeminal sensory neurons innervating the oral cavity. RESULTS: Mice showed aversion to menthol concentrations of 100 µg/mL and above. When presented with a highly aversive concentration of nicotine (200 µg/mL), mice preferred solutions with 50 or 100 µg/mL menthol added over nicotine alone. In contrast to wild-type mice, Trpm8-/- showed a strong aversion to mentholated (100 µg/mL) nicotine (200 µg/mL) and preferred nicotine alone. Trpm8-/- mice show aversion to lower concentrations of menthol than wild-type mice. CONCLUSIONS: Oral menthol can reduce the aversive effects of oral nicotine and, at higher concentrations, acts as an irritant by itself. Menthol's effects in relation to nicotine require TRPM8, the cool temperature sensing ion channel that activates analgesic and counterirritant mechanisms. These mechanisms may underlie preference for menthol-containing smokeless tobacco products and may facilitate initiation of product use.

TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury.

The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.