Inhibition of Human Liver Microsomal (S)-Nicotine Oxidation by

The results of our in vitro studies herein show that (−)-menthol and synthetic .... I. Opole , G.-L. Whembolua , M. S. Mayo , J. S. Ahluwalia , N. L...
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Chem. Res. Toxicol. 2003, 16, 988-993

Inhibition of Human Liver Microsomal (S)-Nicotine Oxidation by (-)-Menthol and Analogues James M. MacDougall,† Keith Fandrick,† Xiaodong Zhang,† Scott V. Serafin,‡ and John R. Cashman*,† Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, California 92126, and Department of Chemistry, University of California, Riverside, California 92507 Received March 21, 2003

(-)-Menthol is a widely used flavoring ingredient present in mouthwash, foods, toothpaste, and cigarettes; yet, the pharmacological effects of menthol have not been widely studied. Mentholated cigarette smoking may increase the risk for lung cancer. Many African American smokers smoke mentholated cigarettes, and African Americans have a significantly higher incidence of lung cancer as compared with whites. There may be a relationship between the incidence of lung cancer and the type of cigarette smoked because the use of mentholated cigarettes by white smokers is significantly less and the incidence of lung cancer is less. The mechanism whereby (-)-menthol could increase the health risk of smoking is not known. The results of our in vitro studies herein show that (-)-menthol and synthetic congeners inhibit the microsomal oxidation of nicotine to cotinine and the P450 2A6-mediated 7-hydroxylation of coumarin. Replacement of the alcohol oxygen atom of menthol with other heteroatoms increased the potency of P450 2A6 inhibition. Thus, the Ki value of (-)-menthol for inhibition of microsomal nicotine oxidation was 69.7 µM but neomenthyl thiol possesses a Ki value of 13.8 µM. Menthylamine inhibited nicotine oxidation with a Ki value of 49.8 µM, but its hydroxylamine derivative gave an IC50 value of 2.2 µM. A series of 16 menthol derivatives and putative metabolites were procured or chemically synthesized and tested as inhibitors of P450 2A6. While highly potent inhibition of P450 2A6 was not observed for the menthol analogues examined, it is nevertheless possible that smoking mentholated cigarettes leads to inhibition of nicotine metabolism and allows the smoker to achieve a certain elevated dose of nicotine each day. This may be another example of self-medication to obtain the desired effect of nicotine.

Introduction (-)-Menthol[(-)-p-menthan-3-ol;(l)-menthol;(1R,2S,5R)(-)-menthol; hereafter referred to as menthol] is a monoterpenoid obtained from peppermint or mint oils and prepared commercially by the hydrogenation of the phenolic compound thymol. Menthol is used as a flavoring ingredient in a large number of consumer products, including foods, toothpaste, mouthwash, and cigarettes. Despite its widespread use, there are surprisingly few studies of the physiological or pharmacological effects of menthol. In animal models, menthol has shown some toxicities (1) including depressant effects on the central nervous system (2), but in humans, menthol is relatively safe although menthol has a small cardio-accelerating effect (3). In rodents, high doses of menthol may exert a depressant effect on the central nervous system (2, 4), but in humans, it is not clear whether menthol has any psychoactivity at doses achieved with food or beverages. There may be a relationship between the incidence of lung cancer and the type of cigarette smoked because African American smokers have a significantly higher lung cancer rate as compared with white smokers (5) and African American smokers also prefer to smoke mentholated cigarettes. As many as 91% of young black females and 87% of young black male smokers use mentholated * To whom correspondence should be addressed. Tel: (858)458-9305. Fax: (858)458-9311. E-mail: [email protected]. † Human BioMolecular Research Institute. ‡ University of California.

cigarettes as compared with 34 and 24% of white female and male smokers, respectively (6). It is possible that mentholated cigarette smoking increases the risk for lung cancer (7). Paradoxically, black smokers report smoking 35% fewer cigarettes per day than white smokers (8). Blacks also report smoking more of each cigarette than whites. Despite smoking fewer cigarettes, blacks have significantly higher rates of smoking-related diseases for any smoking level (9). Black smokers also have higher serum cotinine levels (mean, 485.1 ng/mL) as compared to whites (mean, 354.3 ng/mL), higher concentrations of expired air carbon monoxide, and higher urine levels of the tobacco specific lung carcinogen 4-methylamino-1-(3pyridyl)-1-butanone (10). In humans (3) and in the rabbit (12), menthol is efficiently metabolized to menthol glucuronide as well as hydroxylated metabolites. However, menthol metabolism is highly species-dependent because dogs only eliminate 5% of a dose as menthol glucuronide (12). Oxidations of the methyl and isopropyl groups of menthol have been reported to provide major metabolites in rat after administration of menthol for up to 20 days (13). The major biliary metabolite of menthol in rat is menthol glucuronide; a series of p-menthane diols, triols, and carboxylic acids (some excreted as their glucuronic acid conjugates) are excreted in the urine (14). Although the acute toxicity of menthol is low (2) and negative in a range of genotoxicity tests (15, 16), it is remarkable that the complete details of the metabolism of menthol in humans are lacking.

10.1021/tx0340551 CCC: $25.00 © 2003 American Chemical Society Published on Web 07/17/2003

Inhibition of Nicotine Oxidation by Menthol

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Table 1. IC50 Values for Inhibition of Coumarin and Nicotine Oxidation by CYP2A6

b

entry

inhibitor

IC50 (µM) coumarin 7-hydroxylation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

(-)-menthol (+)-menthol (+)-neomenthol thymol (-)-menthoxyacetic acid (-)-menthone (R)-(+)-pulegone (S)-(-)-pulegone neomenthyl thioacetate neomenthyl thiol neomenthylmethyl sulfide (-)-menthone ethylenethioketal (-)-menthyl chloride menthylamine (-)-menthone oxime menthyl hydroxylamine

70.49 37.77 52.77 67.49 >400 67.54 129.5 139.4 5.15 2.19a 103.1b 35.11c 132.2 16.11 24.61d 2.24e

SD 10.70 5.087 6.162 4.842 9.752 24.58 15.25 0.916 0.3291 8.168 8.505 27.16 2.57 1.614 0.26

highest concentration of inhibitor (µM) 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 100

a Microsomal IC values for inhibition of nicotine oxidation to cotinine as described in the Materials and Methods was 0.73 ( 0.16 µM. 50 IC50 value is 29.4 ( 6.15 µM. c IC50 value is 10.9 ( 2.4 µM. d IC50 value is 14.2 ( 2.9 µM. e IC50 value is 2.0 ( 0.5 µM.

Figure 1. Chemical structures of 1, 9, 10, and 14.

The molecular mechanism whereby menthol could increase health risk is unknown but could include increased duration or deepness of inhalation and smoking particulate material due to the “wetness” or cooling effect of menthol (10). Another mechanism could involve an increased enhancement or adsorption of smoking products (17, 18) including tobacco smoke-derived carcinogens (19). Yet another mechanism could involve inhibition of metabolism of nicotine by menthol or a metabolite of menthol thereby enhancing the toxicity of mentholated cigarettes. Thus, menthol could serve as an inhibitor of nicotine and/or cotinine metabolism and increase the serum levels of nicotine and/or cotinine while at the same time decreasing the need for smoking. This work provides a summary of in vitro studies investigating the latter mechanism.

Materials and Methods Chemicals. Menthol (compound 1, Figure 1), trans-p-menth6-en-2,8-diol and related compounds (Table 1), and (S)-nicotine and (S)-cotinine were obtained from Aldrich Chemical Co. (Milwaukee, WI). The amino analogue of menthol (menthylamine 14) was purchased from Maybridge Chemical Co. (Isle of Palms, SC). [N-Methyl-3H]nicotine (69.5 Ci/mmol) was purchased from NEN Life Sciences (Boston, MA) and combined with nonradiolabeled (S)-nicotine to afford working stocks. Glucose6-phosphate dehydrogenase, glucose-6-phosphate, NADP+, and diethyleneaminetetracetic acid (DETAPAC) were obtained from Sigma (Milwaukee, WI). Pooled human liver, male rat liver, and male cynomolgus monkey liver microsomes and P450 2A6 cytochrome P450 reductase supersomes were obtained from BD Gentest Corp. (Woburn, MA). The human liver microsomes had the following functional activities (nmol/min/mg of protein): phenacetin O-deethylase (0.17), coumarin 7-hydroxylase (2), (S)mephenytoin N-demethylase (0.05), diclofenac 4′-hydroxylase (1.7), (S)-mephenytoin 4′-hydroxylase (0.04), bufuralol 1′-hydroxylase (0.11), chlorzoxazone 6-hydroxylase (1.8), testosterone 6β-hydroxylase (6.1), and methyl p-tolyl sulfide oxidase (4.6). The functional activity of the pooled male rat liver microsomes

(nmol/min/mg of protein) was as follows: testosterone 6βhydroxylase (5.8) and nicotine oxidase (2.2); total P450 content was 1.8 nmol/mg of protein. The male monkey liver microsomes had the following functional activities (nmol/min/mg of protein): testosterone 6β-hydroxylase (9.7), p-nitrophenol hydroxylase (1.4), lauric acid hydroxylase (2.2), and total P450 content (1.2 nmol/mg of protein). The P450 2A6 cytochrome P450 reductase supersomes had a specific activity of 2.6 nmol/min/ mg of protein and a total P450 content of 0.13 nmol/mg of protein. A source of crude aldehyde oxidase was obtained from liver microsomal supernatant from male Sprague-Dawley rats isolated as described previously (20). Chemical Syntheses. 1. General. Synthetic reactions were conducted in flame or oven-dried glassware under an atmosphere of dry nitrogen and mixed by magnetic stirring. N,NDimethylformamide (DMF) and Me2SO were dried by passage through a column of neutral alumina and stored over 4 Å molecular sieves. Dichloromethane (CH2Cl2) was distilled from calcium hydride immediately prior to use. TLC was done on Merck silica gel 60 plates (250 µM, particle size 5-20 µM, pore size 60 Å, with fluorescent indicator). Flash column chromatography was done with silica gel 60 (230-400 mesh). Rf data refer to the mixture of solvents in which the column chromatography was carried out unless otherwise indicated. Iodine was used for TLC spot visualization. 1H NMR spectra were recorded at 500 MHz using a Varian NMR (NuMega Resonance, San Diego, CA). Chemical shifts were reported in ppm (δ) relative to CDCl3 at 7.26 ppm. High-resolution mass spectrometry was done at the University of California Riverside, using a HewlettPackard 5989A GCMS. The GC column was an HP-5ms (30 m × 0.25 mm i.d. × 0.25 mm film thickness). The analytical conditions were as follows: helium flow rate, 1.2 mL/min; 2 mL injection, 16:1 split; detector temp, 230 °C (source), 100 °C (quad); oven ramp, 50 °C (10 min), 20 °C/min to 300 °C (15 min). The purity of synthetic compounds was determined by GC analysis and was g97.0% in all cases. 2. Neomenthyl Thioacetate (9). A mixture of potassium thioacetate (0.78 g, 6.87 mmol) and (-)-menthyl chloride (1.0 g, 5.72 mmol) in Me2SO (4 mL) was heated at 110 °C for 20 h. The resulting black mixture was cooled to room temperature and diluted with ethyl acetate (EtOAc) (120 mL), water (10 mL), and brine (10 mL). The separated organic layer was washed with brine (3 × 10 mL), dried over Na2SO4, filtered, and concentrated to a dark brown oil. Flash chromatography on silica gel with hexanes as an eluant provided the title compound (307 mg, 25%) as a yellow oil: Rf ) 0.4 (hexanes/EtOAc, 25:1, v/v). 1H NMR (CDCl3): δ 4.09-4.07 (m, 1H), 2.32 (s, 3H), 1.84-1.79 (m, 2H), 1.73-1.70 (m, 2H), 1.65-1.55 (m, 1H), 1.43-1.39 (m, 1H), 1.351.29 (m, 1H), 1.15-1.10 (m, 1H), 0.93-0.82 (10H). EI GC/MS: m/z calcd for C12H22OS (M+), 214.1391; found, 214.1393 at 13.9 min (g99%).

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3. Neomenthyl Thiol (10). A solution of 9 in CHCl3/MeOH (1:1, v:v) (4 mL) at 0 °C was degassed for 2 min by nitrogen bubbling, and sodium thiomethoxide (NaSMe) (33 mg, 0.47 mmol) was added. An additional 110 mg (1.57 mmol) of NaSMe was added during the next hour. The mixture was poured into a solution of 10% HCl (2 mL) in water (10 mL) and extracted with CH2Cl2 (2 × 30 mL). The combined organic extracts were washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated to provide a brown oil. Purification by flash chromatography on silica gel using hexanes as an eluant provided the title compound as a colorless oil (44 mg, 54%): Rf ) 0.58. 1H NMR (CDCl3): δ 3.51-3.49 (m, 1H), 1.87-1.84 (m, 1H), 1.72-1.66 (m, 3H), 1.49-1.47 (m, 2H), 1.41-1.36 (m, 1H), 1.31-1.20 (m, 2H), 1.04-1.02 (m, 1H), 0.91-0.83 (m, 9H). EI GC/MS: m/z calcd for C10H20S (M+), 172.1286; found, 172.1288 at 10.9 min (97.0%). 4. Neomenthylmethyl Sulfide (11). (-)-Menthyl chloride (0.21 mL, 1.14 mmol) was dissolved in DMF (2 mL), NaSMe (144 mg, 2.05 mmol) was added, and the resulting mixture was heated at 100 °C for 15 h under an air-cooled condenser. After it was cooled to room temperature, the dark brown mixture was diluted with EtOAc (30 mL) and washed with water (5 × 2 mL) and brine (5 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to a brown oil. The oil was purified by flash chromatography on silica gel using hexanes as an eluant to provide the title compound as a colorless oil (72 mg, 34%): Rf ) 0.24. 1H NMR: δ 3.01 (m, 1H), 2.06 (s, 3H), 1.981.89 (m, 2H), 1.74-1.61 (m, 3H), 1.19-1.05 (m, 4H), 0.95-0.87 (three overlapping doublets, 9H). EI GC/MS: m/z calcd for C11H22S (M+), 186.1442; found, 186.1436 at 12.2 min (g99%). 5. Ethylene Thioketal of (-)-Menthone (12). 1,2-Ethanedithiol (1.2 mL, 12.9 mmol), followed by BF3-OEt2 (1.8 mL, 14.2 mmol), was added by syringe to a 0 °C solution of (-)-menthone (2.0 g, 12.9 mmol) in CH2Cl2 (50 mL). The mixture was stirred at 0 °C for 45 min at which time an additional 0.3 mL of 1,2ethanedithiol and 0.45 mL of BF3-OEt2 were added by syringe. After the mixture was stirred for an additional 38 min, the reaction was judged complete by TLC. The mixture was diluted with CH2Cl2 (50 mL) and washed with water (10 mL) followed by brine (10 mL). The organic layer was separated and dried over Na2SO4, filtered, and concentrated to a colorless oil. Flash chromatography on silica gel using hexanes as an eluant provided the title compound as a colorless oil (2.10 g, 71%); Rf ) 0.24. 1H NMR: δ 3.32-3.18 (m, 4H), 2.39-2.36 (m, 1H), 2.172.14 (m, 1H), 1.76-1.66 (m, 3H), 1.62-1.57 (m, 1H), 1.51-1.43 (m, 2H), 1.27-1.19 (m, 1H), 0.97-0.86 (9H). EI GC/MS: m/z calcd for C12H22S2 (M+), 230.1163; found, 230.1170 at 17.0 min (98.4%). 6. Oxime of (-)-Menthone (15). NaOH pellets (1.3 g, 32.50 mmol) were added to a mixture of (-)-menthone (1.0 g, 6.48 mmol) and NH2OH-HCl (0.72 g, 10.43 mmol) in EtOH/water (5:1, v/v) (3.0 mL) over 5 min. The mixture was lowered into a 100 °C oil bath and heated at reflux for an additional 10 min, cooled to room temperature, and poured into a solution of concentrated HCl (3.5 mL) in water (23.5 mL). The mixture was cooled to 0 °C with stirring, and the resulting white precipitate was collected by filtration on a Bu¨chner funnel. The collected precipitate was washed with 5 °C water (10 mL) and dried under high vacuum to provide 471 mg (43%) of the title compound as a white solid: Rf ) 0.19 (using hexane/EtOAc, 9:1, v/v). 1H NMR (CD3OD): δ 2.72 (dd, J ) 13.4, 4.7 Hz, 1H), 2.12-2.08 (m, 1H), 2.00 (dd, J ) 13.4, 7.9 Hz, 1H), 1.88-1.79 (m, 4H), 1.51-1.45 (m, 1H), 1.23-1.18 (m, 1H), 0.96 (d, J ) 6.7 Hz, 3H), 0.92 (d, J ) 6.7 Hz, 3H), 0.88 (d, J ) 6.7 Hz, 3H). EI GC/MS: m/z calcd for C10H19NO (M+), 169.1467; found, 169.1462 at 12.3 min (g99%). 7. Menthyl Hydroxylamine (16). Menthone oxime 15 (250 mg, 1.48 mmol) and NaBH3CN (139 mg, 2.22 mmol) were dissolved in MeOH (1.5 mL). Three crystals of bromocresol green were added. A blue solution was obtained. Several drops of 10% concentrated HCl were added so that the solution just turned yellow. The solution was vigorously stirred at room temperature,

MacDougall et al. and additional drops of 10% HCl were added periodically over 10 min to maintain the yellow color. After 2.75 h, the MeOH was removed by concentration under reduced pressure. Brine (2 mL) was added to the residue, and the pH was raised to ca. pH 12 by the addition of 5% aqueous NaOH. The solution was extracted with CH2Cl2 (20 mL × 2), and the organic extracts were dried over Na2SO4, filtered, and concentrated to a viscous white oil (173 mg, 68%) that consisted of a 3.5 to 2.2 mixture of diastereomers as determined by GC analysis: Rf (using hexanes/ EtOAc, 9:1,v/v) ) 0.18 (major diastereomer), 0.10 (minor diastereomer). 1H NMR (CDCl3): δ 3.31 (m, 1H), 2.66 (dt, J ) 10.6, 7.2 Hz, 1H), 2.22-2.18 (m, 1H), 2.13-2.11 (m, 1H), 2.00-1.99 (m, 1H), 1.75-0.80 (33H). EI GC/MS: m/z calcd for C10H21NO (M+), 171.1623; found, 171.1623 at 12.2 min (96.6%). Metabolic Incubations. 1. Nicotine C-Oxidase. Microsomal incubations (0.25 mL) with [3H]-(S)-nicotine were done with a radiometric assay. Stocks of diluted human liver microsomes (0.7 mg protein), an NADPH-generating system, DETAPAC (0.5 mM), potassium phosphate buffer (pH 7.4), and rat liver microsome supernatant (350 µg) as a source of aldehyde oxidase were combined and maintained on ice. For an assay, either vehicle or inhibitor was added and reactions were initiated by adding [3H]nicotine. Each incubation was thoroughly mixed and placed in a 37 °C water bath with constant shaking. Incubations were stopped at the appropriate time by addition of 0.25 mL of cold acetonitrile and placed on ice. The incubation mixture was thoroughly mixed, and a 25 µL aliquot of the supernatant was applied to the loading zone of a Whatman LK5DF TLC plate containing 1 mg each of nicotine and cotinine as standards for UV-visualization. After it was air-dried, the plate was developed in CH2Cl2/methanol/20% trichloroacetic acid (9:10:1, v/v). The following bands (Rf values) were scraped into scintillation vials for counting: nicotine (0.35) and cotinine (0.55). Control experiments established that using this procedure, losses of radioactivity due to evaporation was practically nil. 2. Coumarin 7-Hydroxylase. Coumarin 7-hydroxylase activity was determined following the general method of Greenlee and Poland (21). Briefly, buffer (0.1 M Tris, pH 7.5), cofactors (0.5 mM NADP+, 2.0 mM glucose-6-phosphate, 1 IU glucose-6phosphate dehydrogenase, 0.6 mM DETAPAC, and 3 mM MgCl2), P450 2A6 cytochrome P450 reductase supersomes (1 pmol), and inhibitors (0.4-400 µM) were combined at 4 °C. The incubations were initiated by the addition of 3 µM coumarin. After they were incubated for 15 min at 37 °C, the reactions were stopped by the addition of 0.75 mL of CH3CN/CH3COOH (80:20, v/v). After a brief centrifugation, fluorescence was measured using a Wallac Victor2 1420 Multilabel Counter (PerkinElmer, Shelton, CT). The coumarin metabolite, 7-hydroxycoumarin, was measured using an excitation wavelength of 355 nm and emission wavelength of 460 nm, and the concentration was determined by comparison to an external standard curve. Data were reported as 3-6 determinations ( SD. 3. Testosterone 6β-Hydroxylase. Testosterone 6β-hydroxylase activity was determined in the presence of adult human liver microsomes by the method of Buters et al. (22). As described above, the microsomes used formed 6.1 nmol 6β-hydroxy testosterone/min/mg of protein. Incubations were stopped by addition of cold acetonitrile, evaporated to dryness, and reconstituted in 100 µL of methanol. Analysis was done with HPLC using an Altex Ultrasphere ODS column with UV-vis detection at 254 nm employing an eluant of water/acetonitrile/methanol (30:10: 60, v/v). Identification of Metabolites of Menthol. We examined the metabolism of (-)- and (+)-menthol in the presence of rat, monkey, and human liver microsomes. As described above, microsomes used possessed significant P450 functional activity. Incubations were done as described above except 30 samples were run in a preparative fashion with 1.6 µg of menthol per reaction in a sealed tube for 1 h. Incubations were terminated by addition of 0.25 mL of cold CH2Cl2/2-propanol (95:5, v/v) and mixed thoroughly, and the organic layer was separated from

Inhibition of Nicotine Oxidation by Menthol the aqueous portion by centrifugation. The organic material was pooled and dried with sodium sulfate and either analyzed directly or concentrated by evaporating with a stream of argon to a minimum volume. Samples were analyzed using a 5890 Series II GC (Hewlett-Packard, Palo Alto, CA) coupled to a 5989A MS Engine (Hewlett-Packard) MS. An Agilent J&W DB5ht column (30 m × 50 µm) separated the samples before passing them into the EI or CI source, 70 eV, of the MS. Evaporated samples were reconstituted in dry ether, CH2Cl2, or pentane for direct injection into the GC/MS. After each injection at 200 °C, each sample was held at 50 °C for 1 min and then the temperature was increased by 10 °C every minute until elution to the detector (300 °C). In the EI mode, analysis of menthol, trans-p-menth-6-ene-2,8-diol, p-menthan-2,8-diol, or diol metabolites of menthol all showed negligible molecular ions. In the CI mode, strong molecular ions corresponding to the anticipated products were observed.

Results The effects of menthol, congeners, and putative metabolites were studied in vitro for their effect on the 7-hydroxylation of coumarin. As compared with compounds 1 and 3, data of Table 1 indicated that highly purified P450 2A6-mediated coumarin 7-hydroxylation was stereoselectively inhibited by (+)-menthol 2. An aliphatic six-membered ring appeared to be a necessary substructure for P450 2A6 inhibitory potency because the phenol 4 was greater than 2-fold less active than menthol. The nucleophilic menthyl oxygen atom was required for P450 2A6 inhibition because there was no measurable inhibition with the carboxylic acid 5 and a ketone analogue of menthol ((-)-menthone 6) possessed significantly decreased activity. The putative R,β-unsaturated ketone metabolite of menthol (i.e., R- or S-pulegone, 7 and 8, respectively) was examined as an inhibitor of P450 2A6. Both pulegone isomers were similarly poor inhibitors of P450 2A6. Next, we investigated the influence of nucleophilic heteroatom substitution of the menthol oxygen atom on P450 2A6 inhibitory potency. The thiol analogue 10 was a potent inhibitor of coumarin 7-hydroxylase possessing an IC50 value of 2.2 µM. The thioacetate analogue 9 was less potent of a P450 2A6 inhibitor than the thiol 10, and it was possible that enzymatic and/or nonenzymatic hydrolysis to thiol 10 contributed to inhibitor potency. That a free thiol was required to provide maximum inhibitor potency was observed from the fact that blocking the thiol as a methyl thioether 11 or as a thioketal 12 significantly reduced inhibitor potency. The alkylating agent menthyl chloride 13 possessed moderate potency. While not as nucleophilic as compared with thiol 10, the nitrogen-containing analogues were less potent inhibitors but showed interesting structure-activity relationship features. Menthylamine 14 and its oxime analogue 15 were moderate inhibitors of P450 2A6, but the highly nucleophilic hydroxylamine 16 was a potent P450 2A6 inhibitor. Several potent inhibitors of coumarin 7-hydroxylase were also tested for their effect on nicotine oxidation to cotinine. Previously, the human liver microsomes were judged to possess good P450 activity including P450 2A6, the isoform responsible for approximately 90% of nicotine oxidation (20, 23). In the presence of human liver microsomes supplemented with an NADPH-generating system and rat liver microsomal supernatant (as a source of aldehyde oxidase), the major initial metabolite of nicotine was cotinine. Preliminary studies showed that the rate of cotinine formation was linearly dependent on

Chem. Res. Toxicol., Vol. 16, No. 8, 2003 991 Table 2. Effect of (-)-Menthol and Congeners on Microsomal Oxidation of (S)-Nicotinea inhibitor

Kmapp (µM)b

Ki (µM)

none 1 14 9 10

23.0 ( 2.4 102.5 ( 14.7 90.0 ( 11.9 292.2 ( 34.5 766.6 ( 58.1

69.7 ( 5.6 49.8 ( 4.2 64.5 ( 7.1 13.8 ( 1.9

a Results are the mean ( SEM for 3-5 determinations with human liver microsomes as described in the Materials and Methods. b Inhibitor was present at 100 µM.

protein (0-1 mg) and time (0-15 min). The relative IC50 values and rank order for inhibition of human microsomemediated nicotine C-oxidation was in reasonable agreement with the IC50 values determined for the inhibition of 7-hydroxycoumarin formation (Table 1). Thus, the inhibitory potency of microsomal C-oxidation of nicotine (IC50 values in micromolar) followed the rank order: 10 (0.7) > 16 (2.0) > 12 (10.9) > 15 (14.2) > 11 (29.4). The inhibition of the microsomal C-oxidation of nicotine was examined in greater detail (Table 2). The kinetic constants for the oxidation of nicotine catalyzed by human liver microsomes were calculated from the rate of cotinine formation at variable substrate concentrations. The KM and Vmax values obtained from double reciprocal plots of velocity vs substrate concentration in the absence of inhibitor were 23.0 µM and 0.4 nmol/min/ mg of protein, respectively. As shown by the kinetic constants listed in Table 2, menthol was a competitive inhibitor of human liver microsome-mediated cotinine formation. In the presence of excess menthol, the concentration required for half-maximal activity was increased to 102.5 µM. The Ki value from Dixon analysis for inhibition of cotinine formation by menthol was 69.7 µM (Table 2). In parallel to the recombinant P450 2A6 studies that examined whether chemical modification of menthol could increase the potency of inhibition, the amino, thioacetate, and thiol derivatives were tested (Table 2). As shown in Table 2, the potency of inhibition followed the order: thiol 10 > amine 14 > thioacetate 9. In the presence of human liver microsomes, we hypothesized that the thioacetate was hydrolyzed to the thiol and this was presumably the compound showing the most potent inhibitory activity. To examine the selectivity of the inhibition of P450 2A6 by menthol and analogues, we also evaluated the effect of several select agents on testosterone 6β-hydroxylase activity as a functional marker for inhibition of human microsomal P450 3A4. The IC50 values for inhibition of testosterone hydroxylase by menthol and its enantiomer (+)-menthol were 666 ( 62.8 and 300 ( 33.4 µM, respectively. The selectivity ratio of P450 3A4/P450 2A6 inhibition for (-)- and (+)-menthol was 21.5 and 2.3, respectively. For menthylamine 14, no inhibition of testosterone 6β-hydroxylase activity was observed up to 400 µM inhibitor concentration. To determine whether the relatively moderate potency of inhibition of P450 2A6 by menthol was due to loss of inhibitor via substrate oxidation of menthol, preparative scale reactions were run with human liver microsomes to investigate whether menthol was metabolized in the presence of human liver microsomes. As a control, we also studied the metabolic stability of (-)- and (+)-menthol in the presence of adult rat and monkey liver microsomes.

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Rat liver microsomes converted both (-)- and (+)menthol enantiomers to diols. However, in the EI GC/ MS studies, the diol rapidly dehydrated to produce M•+-O or M•+-OH2 species. Hence, the menthol-derived diols lost oxygen in the EI experiments so that it appeared that only the monohydroxy compound (parent menthol) was present. The GC retention times of the diols were very different from menthol but experiments with CI GC/MS provided evidence for the correct molecular ion. The CI GC/MS traces suggested that a diol was a prominent metabolite of menthol in the presence of rat liver microsomes, based on the retention time (i.e., as compared with an authentic sample of p-menthan-2,8diol and molecular ion). A sample of p-menthan-2,8-diol, prepared by catalytic hydrogenation of trans-p-menth6-ene-2,8-diol and analyzed by GC/MS, eluted in the same GC region as the diol metabolite observed. Analysis of CI and EI GC/MS studies suggested that the metabolite fragmentation pattern was similar to that of the commercially available p-menthan-2,8-diol when adjustment was made for the difference in molecular weight. A GC/ MS analysis with ammonia CI of the extract from the rat liver microsome incubation confirmed the results for the formation of menthol derived diol. Adult monkey liver microsomes catalyzed the formation of a diol from menthol, and significant stereoselectivity was observed. GC/MS analysis showed that menthol was oxidized, but (+)-menthol was not. A diol product, comparable to those described above, was detected for one menthol isomer but not for the other. The extract from incubation of menthol in the presence of monkey liver microsomes showed two GC/MS peaks that corresponded to formation of two diols that represented about 1-2% relative to unmetabolized menthol. Somewhat surprising, competent human liver microsomes failed to form detectable amounts of menthol metabolites. This result was verified twice. Finally, we examined the metabolism of menthol in the presence of P450 2A6 cytochrome P450 reductase supersomes and NADPH. We also observed no detectable amount of diol metabolites formed. It was possible that menthol was metabolized to a small degree but below the limits of detection. It was also possible that a minor metabolite, if formed, potently inactivated P450 2A6 present and prevented further menthol oxidation. However, under the conditions of the assay, menthol appeared to be sufficiently stable to rule out the involvement of a major metabolite responsible for P450 2A6 inhibition.

Discussion The difference in the way various ethnic groups smoke cigarettes and/or metabolize nicotine is undoubtedly related to inherited genetic variation and may contribute to health risks of smoking cigarettes. Metabolism of nicotine via the P450 2A6-dependent pathway to initially form cotinine in African Americans is very efficient (24) as compared with that in Latinos and whites, and in Chinese Americans, it is the least efficient (25). The incidence of lung cancer can be used as a population marker of cigarette smoking-related disease. African Americans have the highest rates of lung cancer, and Asians and Latinos have the lowest rates of lung cancer (26). African Americans may have a similar prevalence of cigarette smoking as whites but may be exposed to

MacDougall et al.

more toxins in each cigarette (10, 24, 27). The use of mentholated cigarettes may be associated with increased health risks of smoking (7) because African Americans predominantly smoke mentholated cigarettes and have the highest rates of lung cancer (26). The underlying molecular mechanism as to how mentholated cigarettes increase the health risk of smoking is unknown, but among the many possibilities, mentholated cigarettes may allow a longer lung smoke retention time (28) or increase the permeability and diffusibility of smoke constituents (29). Another hypothetical mechanism is that mentholated cigarettes may be smoked to afford inhibition of nicotine metabolism to achieve a certain dose of nicotine each day to obtain the desired pharmacological effect of nicotine. If menthol or a metabolite of menthol potently inhibits P450 2A6 and decreases nicotine metabolism, this could explain the use of mentholated cigarettes in efficient metabolizers of nicotine. An understanding of the molecular basis for mentholated cigarette use may aid in an understanding of the demographics of their use and also in the design and development of smoking cessation medications (30). To examine the latter mechanism, the effects of menthol, menthol derivatives, and some possible metabolites of menthol were examined as inhibitors of P450 2A6. Kinetic analysis showed that menthol and heteroatom congeners inhibited human liver microsomal nicotine oxidation and P450 2A6-mediated coumarin 7-hydroxylase. Tables 1 and 2 list the IC50 and Ki values, respectively, for the effect of menthol, menthol congeners, and various menthol metabolites on coumarin 7-hydroxylase and/or nicotine oxidase, two functional substrates for P450 2A6. Inspection of the molecular structure of menthol reveals that the compound is lipophilic and possesses a nucleophilic hydroxyl group that could interact with the heme iron of P450 2A6. The IC50 values for menthol and closely related congeners indicated that these compounds act as moderate inhibitors of P450 2A6 (Table 1). If the hydroxyl group is replaced by a more nucleophilic amino or thiol functional group that has higher affinity for hemoprotein iron, the efficiency of inhibition is increased. This is consistent with a mechanism of selective binding to the prosthetic group of P450 2A6. Another mechanistic possibility, that a metabolite of menthol is responsible for potent inhibition of P450 2A6 in vivo, is still a possibility, but none of the putative metabolites of menthol tested herein (Table 1) likely possess sufficient potency to inhibit P450 in vivo and cause a detectable perturbation in nicotine metabolism. We also found no evidence that a prominent metabolite of menthol was formed in the presence of human liver microsomes although we could not rule out the formation of a minor, highly potent inhibitory metabolite. In mentholated cigarettes, approximately 0.3-0.7% of tobacco by weight is menthol (31). In highly mentholated cigarettes, up to 1% of the tobacco product by weight is menthol and this translates to 64 µmol menthol/g of tobacco. For a clinically relevant inhibitory effect, the concentration of menthol would need to exceed the Ki value of 69.7 µM. However, the smoking route of administration, and the fact that menthol does not distribute between a free base and a protonated form as does nicotine, and the volatility of menthol may afford more efficient dosing and this could increase the bioavailability of menthol and the pharmacological effect. P450 2A6 also further metabolizes cotinine and activates tobacco-related

Inhibition of Nicotine Oxidation by Menthol

nitrosamines (32), and it is possible that individuals that smoke mentholated cigarettes have elevated cotinine levels and decreased nitrosamine activation due to inhibition of P450 2A6. One possible conclusion is that the health risk for mentholated smokers may come from a contribution of additional factors besides the bioactivation of nitrosamines, but this requires further study. In summary, menthol is an inhibitor of P450 2A6mediated coumarin 7-hydroxylase and the human liver microsomal oxidation of nicotine. Replacement of the oxygen atom of menthol with a nitrogen or sulfur atom also results in menthol congeners that potently inhibit nicotine oxidation. The thioacetate analogue of menthol probably acts as a prodrug and is hydrolyzed to the thiol by microsomal esterases, and it is likely that the thiol formed possesses the most potent inhibitory activity. That the more nucleophilic heteroatom-containing menthol analogues have the greatest inhibitory potency is consistent with the P450 2A6 prosthetic group as the site of inhibition. Individuals that smoke mentholated cigarettes may do so to elevate the plasma levels of nicotine by inhibiting nicotine metabolism and clearance. For smokers that smoke mentholated cigarettes, the increased plasma area under the curve for nicotine and tobacco-related chemicals (3, 10, 28) may contribute to the increased health risks associated with smoking mentholated cigarettes.

Acknowledgment. We thank Theresa Operana for help with the assays. Financial support for this study was provided by the University of California Tobacco Related Disease Research Program (Grant 9RT-0196 to J.R.C.) and a Cornelius Hopper Diversity Award to Theresa Operana.

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(12) (13) (14) (15)

(16) (17)

(18)

(19) (20)

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(23) (24)

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