Research paper Tob Control: first published as 10.1136/tobaccocontrol-2016-053209 on 3 October 2016. Downloaded from Menthol decreases oral nicotine aversion in C57BL/6 mice through a TRPM8-dependent mechanism Lu Fan,1 Shrilatha Balakrishna,1 Sairam V Jabba,2 Pamela E Bonner,2 Seth R Taylor,1 Marina R Picciotto,1 Sven-Eric Jordt1,2 ▸ Additional material is ABSTRACT acrolein and to cigarette smoke itself.67These published online only. To view Background Nicotine is a major oral irritant in mice had never been exposed to cigarette smoke please visit the journal online (http://dx.doi.org/10.1136/ smokeless tobacco products and has an aversive taste. before, mimicking initiating users. Higher levels tobaccocontrol-2016-053209). Mentholated smokeless tobacco products are highly of cotinine were detected in the mice inhaling popular, suggesting that menthol increases their mentholated cigarette smoke than in the mice 1Department of Psychiatry, Yale Tobacco Center of Regulatory palatability and may facilitate initiation of product use. inhaling non-mentholated smoke, implying that men- 7 Science (TCORS), Yale School While menthol is known to reduce respiratory irritation thol increases nicotine uptake. These effects were of Medicine, New Haven, by tobacco smoke irritants, it is not known whether this observed at menthol levels present in smoke of Connecticut, USA activity extends to oral nicotine and its aversive effects. commercial menthol cigarettes.7 Together these 2Department of Study design The two-bottle choice drinking assay data suggested that menthol may facilitate smoking Anesthesiology, Duke 6–8 University School of Medicine, was used to characterise aversion and preference in initiation by reducing aversion to noxious smoke. Durham, North Carolina, USA C57BL/6 mice to a range of menthol concentrations It is currently controversial whether menthol’s (10–200 mg/mL). Then, effects of menthol on oral counterirritant activity extends to oral nicotine and Correspondence to nicotine aversion were determined. Responses were its aversive effects. Most studies examining the Dr Sven-Eric Jordt, Department fi of Anesthesiology, Duke compared with those in mice de cient in the cold/ effects of menthol in the context of tobacco use University School of Medicine, menthol receptor, TRPM8, expressed in trigeminal were performed in chronic smokers and provide no Box 3094 MS27, Durham, sensory neurons innervating the oral cavity. information about menthol’s behavioural effects NC 27710-3094, USA; Results Mice showed aversion to menthol during initiation of tobacco use. Menthol, at high [email protected] concentrations of 100 mg/mL and above. When levels, can also be perceived as irritating.45A Received 27 May 2016 presented with a highly aversive concentration of human psychophysical study demonstrated that Accepted 13 September 2016 nicotine (200 mg/mL), mice preferred solutions with menthol applied to the tongue at irritating concen- copyright. Published Online First 50 or 100 mg/mL menthol added over nicotine alone. In trations caused desensitisation to nicotine later 3 October 2016 contrast to wild-type mice, Trpm8−/− showed a strong applied to the same location.4 Another human aversion to mentholated (100 mg/mL) nicotine (200 mg/ study using mentholated nicotine gums observed mL) and preferred nicotine alone. Trpm8−/− mice show that menthol reduced pain and irritation elicited by aversion to lower concentrations of menthol than wild- nicotine, but only transiently, and some test partici- type mice. pants perceived the high menthol concentrations in Conclusions Oral menthol can reduce the aversive this study as irritating.5 effects of oral nicotine and, at higher concentrations, Pain and irritation are initiated when noxious http://tobaccocontrol.bmj.com/ acts as an irritant by itself. Menthol’s effects in relation chemicals activate peripheral sensory nerves.9 to nicotine require TRPM8, the cool temperature sensing Smoke irritants such as acrolein activate the sensory ion channel that activates analgesic and counterirritant irritant receptor, TRPA1, in nerve endings in the mechanisms. These mechanisms may underlie preference mucosa of the throat and nasal passages.10 Oral for menthol-containing smokeless tobacco products and nicotine activates nicotinic acetylcholine receptors may facilitate initiation of product use. (nAChRs) in sensory nerves innervating the tongue and oral cavity.11 12 Nicotine also activates TRPA1, albeit higher concentrations are required than for INTRODUCTION nAChRs.13 The bitter taste associated with nicotine Smokeless tobacco products (snuff, chewing is likely mediated by bitter taste receptors in oral tobacco, snus and dissolvables) containing menthol taste buds that signal to gustatory nerves.11 14 on September 28, 2021 by guest. Protected are highly popular.12Menthol adds a fresh, Menthol also has complex interactions with cooling and minty taste, sensations that are reminis- sensory nerves and their receptors. The cooling cent of other tobacco products such as menthol effect of menthol is mediated by the cold/menthol cigarettes and of confectionary products such as receptor, TRPM8, expressed in a subset of neurons chewing gum and mint lozenges. Menthol may sensitive to cool temperature.15 16 Activation of increase the palatability of smokeless products that these neurons by menthol is thought to underlie contain tobacco ingredients with aversive flavours, menthol’s analgesic and counterirritant effects. thereby facilitating initiation of product use.3 Indeed, menthol failed to inhibit smoke-induced Nicotine levels in smokeless tobacco products are respiratory irritation when mice were treated with a sufficient to be perceived as irritating by humans, TRPM8 inhibitor before exposure. The irritant eliciting burning and tingling sensations in the effects of menthol at higher concentration are 45 17 18 To cite: Fan L, mouth. Nicotine also has an aversively bitter likely mediated by TRPA1. Balakrishna S, Jabba SV, taste.5 In recent studies in mice, we demonstrated Mice are obligate nasal breathers, and access for et al. Tob Control 2016;25: that menthol inhibits the respiratory irritation inhaled air to the oral cavity is normally blocked. – ii50 ii54. response to cigarette smoke irritants such as Thus, oral and respiratory sensing are separated, ii50 Fan L, et al. Tob Control 2016;25:ii50–ii54. doi:10.1136/tobaccocontrol-2016-053209 Research paper Tob Control: first published as 10.1136/tobaccocontrol-2016-053209 on 3 October 2016. Downloaded from allowing selective study of oral effects. Here we used the two- filled with plain water and drank ad libitum. Control groups bottle choice paradigm in mice to investigate the effects of were provided with water only throughout the experiment. menthol on oral aversion to nicotine. The role of TRPM8 in Liquid consumption from each tube was measured by comparing these effects was investigated by comparing responses of wild- weights of the drinking tubes before and after the choice drinking type mice with responses of TRPM8-deficient mice. periods. Investigators tasked with filling and swapping tubes were different from investigators reading fluid consumption. The latter METHODS were blinded to the tube contents. Animals Male C57/BL6 mice (adults aged 11 weeks) purchased from Menthol-flavoured solutions Charles River Laboratories were maintained in a L(−) menthol was used in all studies, the minty and cooling 19 AAALAC-certified facility in a temperature and humidity con- menthol isomer added to tobacco products. Menthol solutions trolled room at a 12-hour light–dark cycle with unlimited access (10–200 mg/mL) were prepared by dissolving L(−)-menthol to food and water. Trpm8–/– mice were a gift from David Julius (Acros Organics) in distilled deionised H2O (ddH2O). Nicotine (University of California, San Francisco) and backcrossed into solutions (200 mg/mL) were prepared fresh daily by dissolving – m C57BL/6 background using marker-assisted backcrossing nicotine base (Sigma) in dd H2Oor10200 g/mL menthol (Charles River Laboratories, Wilmington, Massachusetts, USA). solutions. Drinking tubes with nicotine solutions were protected All experimental protocols were approved by the Institutional from light by covering with aluminium foil. Animal Care and Use Committees of Duke University and Yale University. No adverse events were noted during the course of Statistical analysis the described experiments. For flavour preference study, data were averaged across the 4 days. One-way ANOVA was used to compare daily averaged Two-bottle choice assay liquid consumption among groups, and Tukey post hoc was Mice were housed individually in Med Associates behavioural used to compare between individual groups. In addition, a chambers with water supply from drinking tubes manufactured two-way ANOVA was performed to determine if daily liquid from 25 mL serological pipette tubes with 0.2 mL graduations consumption differed from day to day within individual groups and stainless steel sipper tubes (Ancare, Part Number, OT99.5, due to changes in tolerance over the period of experiment. with steel collars at a 90° angle). Tubes were placed in each cage Paired t-test was used to compare consumed liquid volume at an equal distance from food. Locations of the tubes were between the two choices within each group (*p<0.05; switched every 24 hours to control for side preferences. Each **p<0.01; ***p<0.0005; ****p<0.0001). tube contained about 20 mL of liquid. The tubes were calibrated copyright. for fluid