Effects of Menthol on Nicotine Pharmacokinetic, Pharmacology and Dependence in Mice

Abstract

Although menthol, a common flavoring additive to cigarettes, has been found to impact the addictive properties of nicotine cigarettes in smokers little is known about its pharmacological and molecular actions in the brain. Studies were undertaken to examine whether the systemic administration of menthol would modulate nicotine pharmacokinetics, acute pharmacological effects (antinociception and hypothermia) and withdrawal in male ICR mice. In addition, we examined changes in the brain levels of nicotinic receptors of rodents exposed to nicotine and menthol. Administration of i.p. menthol significantly decreased nicotine's clearance (2-fold decrease) and increased its AUC compared to i.p. vehicle treatment. In addition, menthol pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg, s.c.) for periods up to 180 min post-nicotine administration. Repeated administration of menthol with nicotine increased the intensity of mecamylamine-precipitated withdrawal signs in mice exposed chronically to nicotine. The potentiation of withdrawal intensity by menthol was accompanied by a significant increase in nicotine plasma levels in these mice. Western blot analyses of α4 and β2 nAChR subunit expression suggests that chronic menthol impacts the levels and distribution of these nicotinic subunits in various brain regions. In particular, co-administration of menthol and nicotine appears to promote significant increase in β2 and α4 nAChR subunit expression in the hippocampus, prefrontal cortex and striatum of mice. Surprisingly, chronic injections of menthol alone to mice caused an upregulation of β2 and α4 nAChR subunit levels in these brain regions. Because the addition of menthol to tobacco products has been suggested to augment their addictive potential, the current findings reveal several new pharmacological molecular adaptations that may contribute to its unique addictive profile.

Fig 1. Time course of nicotine plasma concentrations in mice pretreated with menthol.

Nicotine was administered (2.5 mg/kg s.c.), 30 min after pretreatment with vehicle or menthol (100 mg/kg i.p.). Each time point represents the mean ± SEM of 7 to 10 animals. For the vehicle pretreatment, values for nicotine plasma levels at 2 h were below the limits of detection. Each point represents the mean ± SE of 8–12 mice. Nic = nicotine.

Fig 2. Effects of menthol on the time course of nicotine’s effects in (A) the tail-flick test, (B) the hot-plate assay, and (C) body temperature in mice.

Animals were pretreated with either menthol (100 mg/kg i.p.) or vehicle and 30 min later received nicotine (2.5 mg/kg, s.c.). A control group (vehicle/vehicle) is also represented in all three tests. Mice were tested at different time points after injection. Each point represents the mean ± SE of 8–12 mice. *p < 0.05 compared with vehicle/nicotine group.

Fig 3. Effect of (A) pretreatment time and (B) dose of menthol’s effect in the tail-flick.

(A) Mice pretreated with vehicle or menthol (100 mg/kg, i.p.) and various time points (5, 30, 60 and 120 min) later, they were given nicotine (2.5 mg/kg, s.c.). Mice were then evaluated for antinociception 45 min after nicotine administration. (B) Mice pretreated with vehicle or different doses of menthol (10, 50, 100 or 200 mg/kg, i.p.) and 30 min later, they were given nicotine (2.5 mg/kg, s.c.). Mice were then evaluated for antinociception 45 min after nicotine administration. Each point represents the mean ± SE of 8–12 mice. *p < 0.05 compared with control.

Fig 4. Effects of menthol pretreatment on nicotine-induced antinociception and hypothermia dose-response curves in mice.

Vehicle or menthol (100 mg/kg, ip) was administered 30 min before various doses of nicotine (0.5, 1.5, 2, and 2.5 mg/kg s.c.) and mice were tested in (A) the tail-flick test, (B) the hot-plate test, and (C) hypothermia. Each point represents the mean ± SE of 8–12 mice.

Fig 5. Effects of menthol on nicotine withdrawal signs. (A) Elevated plus-maze test, (B) Somatic signs and in (C) Hyperalgesia.

Withdrawal from nicotine induced a: A) no significant change in anxiety- related behavior, B) significant increase in somatic signs, and C) a significant decrease in hot plate latency. Compared to vehicle, pretreatment with menthol (100 mg/kg, i.p. for 7 days) A) significantly increased expression of anxiety-related behavior; B) a further increase in somatic signs, and C) a decrease in hot plate latency in mice. Each point represents the mean ± S.E.M. of 6–8 mice per group. *P < 0.05 compared with control MP/Saline/Vehicle group. #P < 0.05 compared with MP/Nicotine/ Vehicle group. MP = minipump; Nic = nicotine; Sal = saline; Veh = vehicle.

Fig 6. Menthol mediated changes in nAChR expression in the brain.

Western Blot detection of α4 and β2 nAChR subunits in membrane fractions of treated mice. Representative immunoblots showing the expression of α4 and β2 nAChR subunit bands in (A) Hippocampus, (B) Striatum, (C) Prefrontal Cortex, and (D) all three regions when expressed relative to nicotine treatment alone. GAPDH was used as a loading control and a normalizing factor for protein loading across lanes. Histograms show average values of optical density (OD) measurements of the immunoreactive bands relative to the vehicle lane (n = 6 mice per group; and all samples were run in triplicates). Student’s test *p < 0.05, **p < 0.01.