Fig 1.
Effect of L-menthol on the murine respiratory irritation response to acrolein.
(A) Change in duration of braking (DB) during exposure to 3 ppm acrolein, 40 ppm L-menthol vapor or the combination. Data are presented as the 1 minute average DB level during the baseline (-5 to 0 minute) or the exposure (0 to 15 minute) period, expressed as mean ± SEM (n = 4–7 mice per group). Repeated measures ANOVA followed by Newman-Keuls test indicated the response to the combination was significantly lower than that to acrolein alone (p<0.001). At all exposure times, the response to acrolein+L-menthol was virtually identical to that of L-menthol alone.(B) Effect of 8 or 54 ppm L-menthol on responses to 3, 7 or 11 ppm acrolein. Data are presented as the average DB during the 15 minute exposure (corrected for baseline), expressed as mean ± SEM (n = 5–7 per group). The responses in both the 8 and 54 ppm L-menthol groups were significantly lower than for acrolein alone, and differed significantly from each other (p<0.0001, two-factor ANOVA).
Fig 2.
Dose-response relationships of effects of TRPM8 agonists (L-menthol, eucalyptol) on respiratory irritation by acrolein or cyclohexanone in mice.
(A) Logarithmic concentration response curve for the effect of L-menthol (9, 14, 18 or 39 ppm) on acrolein (4 ppm)-induced irritation. Data are presented as the average DB level during the 15 minute exposure (corrected for baseline), expressed as mean ± SEM (n = 5–7 mice per group). Shown is the log linear plot of the response (percent of control) in the acrolein-L-menthol groups. The response to acrolein was attenuated at all L-menthol exposure concentrations (p<0.0001 ANOVA followed by Newman-Keuls test). A significant correlation (r2 = 0.99, p<0.01) was observed, with an apparent IC50 of 4 ppm (95% confidence limits, 3.3–4.9 ppm). (B). Logarithmic concentration response curve for the effect of L-menthol (8, 18. 24 pr 40 ppm) on cyclohexanone (1500 ppm)-induced irritation. Data are presented as the average DB level during the 15 minute exposure (corrected for baseline), expressed as mean ± SEM (n = 5–8 mice per group). Shown is the log linear plot of the response (percent of control) in the cyclohexanone-L-menthol groups. The response to cyclohexanone was significantly attenuated by L-menthol in the 24 and 40 ppm L-menthol groups (p<0.05, ANOVA followed by Newman-Keuls test). A significant correlation (r2 = 0.99, p<0.01) was observed, with an apparent IC50 of 19 ppm (95% confidence limits, 16–24 ppm). This IC50 is higher than that observed for attenuation of the irritant response to acrolein (Fig. 1C). (C) Effect of eucalyptol on the irritation response to acrolein. Data are presented as the average DB level during the 15 minute exposure (corrected for baseline) expressed as mean ± SEM (n = 5–8 mice per group). The response to 3 ppm acrolein was attenuated by eucalyptol (p<0.0001, ANOVA) with the responses in the 60, 90, 140 and 300 ppm groups all being significantly lower than the response to acrolein alone (* p<0.05 compared to cyclohexanone alone, Newman-Keuls test). Log linear regression analysis of the response (percent of control) in the acrolein-eucalyptol groups revealed a significant correlation (r2 = 0.99, p<0.01) with an apparent IC50 of 73 ppm (95% confidence limits, 69–80 ppm). (D) Effect of eucalyptol on the irritation response to cyclohexanone. Data are presented as the average DB level during the 15 minute exposure (corrected for baseline) expressed as mean ± SEM (n = 5–8 mice per group). The response to 1500 ppm cyclohexanone was attenuated by eucalyptol (p<0.0001, ANOVA) with the response in the 390 and 730 ppm being significantly lower than cyclohexanone alone (*, p<0.05 compared to cyclohexanone alone, Newman-Keuls test). Log-linear regression analysis of the response (percent of control) in the cyclohexanone-eucalyptol groups revealed a significant correlation (r2 = 0.96, p<0.01), with an apparent IC50 of 305 ppm (95% confidence limits, 220–470 ppm). This IC50 value is higher than the IC50 observed for eucalyptol attenuation of acrolein-induced irritation (Fig. 1C).
Fig 3.
Effect of TRPM8 inhibitor, AMG2850 (15 mg/kg), on inhibition of 3 ppm acrolein response by 7 ppm L-menthol or 800 ppm eucalyptol.
Data are presented as the average DB level during the 15 minute exposure (corrected for baseline), expressed as mean ± SEM (n = 3–5 mice per group). Data were analyzed by ANOVA (p<0.001) followed by Newman-Keuls test; bars with differing superscripts differ from each other at the p<0.05 level. The counterirritant effects of L-menthol were absent in AMG2850-pretreated mice.
Fig 4.
Effect of L-menthol on the murine respiratory irritation response to cigarette smoke.
Time course of response to smoke alone and smoke + L-menthol and smoke + L-menthol in AMG2850-pretreated mice (30 mg/kg). Data are presented as the 1 minute average DB level during the baseline (-5 to 0 minute) or the exposure (0 to 8 minute) period. AMG2850 did not modulate the response to menthol alone (data not shown.). (A) At a smoke concentration of 9 mg/m3 (80 ppm carbon monoxide) and L-menthol concentration of 19 ppm, the response to smoke was significantly attenuated by L-menthol, and the attenuation was blocked by AMG2850 (repeated measures ANOVA, p<0.001, Newman-Keuls; p values indicated in the figure, n = 4–12 mice per group). (B) At a smoke concentration of 30 mg/m3 (195 ppm carbon monoxide) and L-menthol concentration of 50 ppm, the response was significantly attenuated by L-menthol (repeated measures ANOVA; p value indicated in the figure, n = 4–6 mice per group).
Fig 5.
Effect of L-menthol on murine ventilation response to high concentration of cigarette smoke and on serum cotinine levels.
(A) Breathing patterns of a mouse at baseline (top), during exposure to 300 mg/m3 (>400 ppm carbon monoxide) side stream cigarette smoke (middle), and side stream smoke combined with 60 ppm L-menthol vapor (bottom). Shown are representative recordings of respiratory flow rate (inspiration downward, expiration upward). For the sake of clarity, the recordings are representative of minutes 6–9 of exposure when the effects of smoke and menthol were maximal and at equilibrium, see Fig. 5B. The prolonged braking at the onset of expiration during smoke exposure is readily apparent and is indicated by the heavy bars. This effect is measured as the duration of braking (DB) during each expiration. (B) Time course of the response to 300 mg/m3 smoke (>400 ppm carbon monoxide). Data are presented as the 1 minute average DB level during the baseline (-5 to 0 minute) or the exposure (0 to 20 minute) period, expressed a mean ± SEM (n = 7–8 mice per group). The response was significantly attenuated by 60 ppm L-menthol (repeated measures ANOVA, p value provided in the figure), particularly at the start of exposure. (C) Cotinine levels in blood drawn immediately after 20 minute exposure to 300 mg/m3 smoke or 300 mg/m3 smoke +60 ppm L-menthol. Data are expressed as mean ± SEM (n = 7–8 mice per group). These were the same mice whose breathing response is shown in (B) above. The two groups differed from each other (*) at p = 0.015 level (t-test). The airborne nicotine levels averaged 30 mg/m3 and were identical in the smoke-alone and smoke + L-menthol groups. Cotinine levels in control (non-exposed) mice averaged less than 1 ng/ml.
Table 1.
Breathing patterns in mice exposed to smoke, menthol or the combination 1