Introduction

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Pennyroyal oil is a volatile oil obtained from the leaves of the plants Mentha pulegium and Hedeoma pulegoides (Guenther, 1949), and it is thought to induce abortion (Gleason et al., 1969). However, it does so at lethal or near-lethal doses, so that its action is unpredictable and dangerous (Anderson et al., 1996; Bakerink et al., 1996). (R)-(+)-Pulegone is the major constituent of pennyroyal oil, and it is responsible for pennyroyal hepatotoxicity (Gordon et al., 1982). Pulegone is metabolized to several metabolites, of which menthofuran appears to be the major proximate toxin, based on toxicokinetic studies (Thomassen et al., 1988) (fig. 1). We now report that menthofuran is a potent, mechanism-based inactivator of human liver CYP2A6.¹

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Chemical structure of (R)-(+)-menthofuran.

	Materials and Methods

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Materials. N-Acetylcysteine, N-acetyllysine, caffeine, catalase, chlorzoxazone, deferoxamine mesylate, dextromethorphan, 7-hydroxycoumarin, glutathione, NADP⁺, and superoxide dismutase were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium cyanide and methoxylamine hydrochloride were purchased from Aldrich Chemical Co. (Milwaukee, WI). Semicarbazide hydrochloride was purchased from Eastman Kodak Co. (Rochester, NY). Glucose-6-phosphate and glucose-6-phosphate dehydrogenase (yeast, grade II) were purchased from Boehringer-Mannheim (Indianapolis, IN). The peroxidase rabbit IgG staining kit (Immunopure ABC kit) and diaminobenzidine/metal enhancer kit were purchased from Pierce Chemical Co. (Rockford, IL). Coumarin was from Merck (Rahway, NJ), and (R)-(+)-menthofuran was purchased from Fluka (Buchs, Switzerland). Human liver microsomes were prepared as described previously (Rettie et al., 1989). Purified expressed human CYP2A6, rat NADPH-P450 reductase, and human cytochrome b₅ were obtained as described previously (Koenigs et al., 1997). Human CYP2E1 was expressed and purified as reported (Chen et al., 1996). The cDNA for human CYP2A6 was kindly provided by Dr. Frank J. Gonzalez, National Institutes of Health (Bethesda, MD).

CYP2A6 Inactivation Assays. Menthofuran (0-10 µM) was incubated with human liver microsomes (HL109) or purified CYP2A6 and the NADPH-generating system to determine its ability to inhibit CYP2A6 in a mechanism-based manner. After exposure of the enzyme to menthofuran and NADPH, aliquots of the mixture were transferred to a vial containing coumarin and the NADPH-generating system, to determine the amount of enzyme activity remaining. Coumarin 7-hydroxylase activity was used as a marker for CYP2A6 activity (Miles et al., 1990).

Dialysis Experiments. Incubations were conducted as described above for CYP2A6 inactivation assays, for 20 min, in the absence or presence of menthofuran (50 µM). The reaction mixtures were dialyzed against 25 mM potassium phosphate buffer, pH 7.4 (3 × 1 liter), for 4 hr at 4°C, using Slide-A-lyzer membranes.

NADPH Dependence and Effects of Trapping Agents. The effects of trapping agents were determined by coincubating menthofuran (5 µM) in the presence of NADPH and trapping agents for 20 min in the preactivation step.

Covalent Binding to CYP2A6. Reconstituted CYP2A6 (100 pmol) was incubated with menthofuran (50 µM) at 30°C for 60 min, in the absence or presence of a NADPH-generating system. Trapping experiments were performed using reconstituted CYP2A6, menthofuran, and NADPH in the presence of glutathione (10 mM), N-acetyllysine (10 mM), methoxylamine (10 mM), or semicarbazide (10 mM). The adducted proteins were detected by Western blotting, using an antibody directed against metallothionein conjugated with a -ketoenal metabolite of menthofuran (Thomassen et al., 1992; Skiles et al., 1994; Anderson et al., 1996).

Preparation of Immunogens. To 5 mg of horse kidney metallothionein (0.735 µmol) in 20 ml of acidic aqueous solution (adjusted to pH 5 with 1 N hydrochloric acid) was added 31.2 mg (147 µmol, 200-fold molar excess) of ,'-dimethoxydihydromenthofuran [a protected form of the -ketoenal 2(Z)-(2'-keto-4'-methylcyclohexyldene)propanal] in 0.5 ml of ethanol. A 0.2-ml aliquot of the reaction mixture was removed at time 0, 0.5, 3, and 18 hr. To each aliquot, 0.8 ml of 0.1 M phosphate buffer, pH 7.5, was added, and then the total amount of thiols was determined spectrometrically (412 nm) with Ellman's reagent. After 18 hr of stirring at 4°C, the reaction mixture was transferred to cellulose dialysis tubing and dialyzed against 10 mM ammonium bicarbonate, pH 7.8 (2 × 3 liters), at 4°C for 48 hr. The dialysate was transferred to plastic vials, lyophilized, and stored at 80°C until immunization. Immunization was performed by injecting modified metallothionein sc into rabbits. The rabbits received two booster shots. The antisera were found to recognize protein adducts formed by the microsomal oxidation of menthofuran. Characterization of the antibody will be reported separately.

Western Blotting. After 10% resolving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the separated proteins were blotted to a polyvinyldifluoride membrane, as described previously (Sambrook et al., 1989). The membrane was blocked with 3% nonfat dry milk in TBS overnight at 4°C with shaking, followed by three 10-min periods of washing with TBS. The membrane was incubated with the primary antibody (diluted 1:500 in TBS) for 3 hr, followed by three 10-min periods of washing with TBS. The secondary antibody used was included in the ABC kit from Pierce. The membrane was incubated with the biotinylated goat anti-rabbit antibody for 30 min, followed by three 10-min periods of washing with TBS. The membrane was then incubated with avidin-biotin complex solution for 30 min, followed by washing (3 × 10 min) with TBS. The membrane was developed with diaminobenzidine/metal-enhanced developer.

Partition Ratio Experiments. The partition ratio was determined by quantifying the metabolites of menthofuran (mintlactone and isomintlactone) by GC/MS after a 60-min incubation. A reconstituted system of purified expressed CYP2A6 (100 pmol), rat NADPH-P450 reductase, and human cytochrome b₅ was incubated at 30°C in 250 µl (final volume) of an incubation mixture containing 100 mM potassium phosphate buffer, pH 7.4, 0.5 mM NADP⁺, 10 mM glucose-6-phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase, and menthofuran. The internal standard was acetophenone (10 µM). The GC/MS analysis has been described previously (Anderson et al., 1996).

CYP1A2, CYP2D6, CYP2E1, and CYP3A4 Inactivation Assays. Selected assays were used as probes for the activities of CYP1A2, CYP2D6, CYP2E1, and CYP3A4. Caffeine N³-demethylation was used as a probe for CYP1A2 activity (Tassaneeyakul et al., 1992), dextromethorphan O-demethylation for CYP2D6 activity (Dayer et al., 1989), chlorzoxazone 6-hydroxylation for CYP2E1 activity (Peter et al., 1990), and dextromethorphan N-demethylation for CYP3A4/5 activity (Jacqz-Aigrain et al., 1993). Each assay was carried out with incubations that were conducted as described above for CYP2A6 inactivation by menthofuran.

	Results

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Time-Dependent Inhibition of Human CYP2A6 by Menthofuran. Inhibition of CYP2A6 activity by menthofuran was found to be time- and concentration-dependent in both human microsomal and reconstituted CYP2A6 incubations (figs. 2a and 3a). The K_i values for menthofuran were determined from nonlinear regression analysis of the data to be 2.5 and 0.84 µM for CYP2A6 in human liver microsomes and for the reconstituted purified enzyme, respectively (figs. 2b and 3b). The k_inact values were determined to be 0.22 and 0.25 min¹ for the microsomal and purified enzymes, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Menthofuran-mediated inactivation of CYP2A6 activity in human liver microsomes (HL109) in the presence of a NADPH-generating system (a) and double-reciprocal plot of the relationship between the inactivation rate and the menthofuran concentration (b). In a, results are averages of duplicate values. The concentrations of menthofuran in the inactivation assay were 0 µM (), 0.5 µM (), 1 µM (), 2.5 µM (), 5 µM (), and 10 µM ().

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Menthofuran-mediated inactivation of purified expressed CYP2A6 in the presence of a NADPH-generating system (a) and double-reciprocal plot of the relationship between the inactivation rate and the menthofuran concentration (b). In a, results are averages of duplicate values. The concentrations of menthofuran in the inactivation assay were 0 µM (), 0.1 µM (), 0.5 µM (), 1 µM (), and 5 µM ().

NADPH Dependence and Effects of Trapping Agents. In our studies, the concentration of menthofuran (5 µM) and the inactivation time (20 min) were chosen so that approximately 10% of the enzyme activity remained (table 1). Inactivation of CYP2A6 by menthofuran was demonstrated to be NADPH dependent. The protective effects of various trapping agents on the inactivation were also determined. The nucleophilic trapping agents gluthathione and N-acetylcysteine failed to protect CYP2A6 from inactivation. Sodium cyanide protected 78% of the CYP2A6 activity. The free iron scavenger deferoxamine failed to protect CYP2A6 from inactivation. Furthermore, superoxide dismutase and catalase, scavengers of reactive oxygen species, did not protect the enzyme.

CYP1A2, CYP2D6, CYP2E1, and CYP3A4 Inactivation. Little inactivation of CYP1A2, CYP2D6, CYP2E1, or CYP3A4 by menthofuran was observed at concentrations greater than those required to completely inactivate CYP2A6 (table 2). Complete inactivation of CYP2A6 occurred under those conditions at 10 µM (fig. 2a).

Covalent Binding of Menthofuran Metabolites to CYP2A6. Immunochemical methods were used to detect CYP2A6 adducts with reactive metabolites of menthofuran. Western blots of proteins incubated with menthofuran in the presence of NADPH showed a band corresponding in molecular mass to CYP2A6 (~52 kDa) and a band corresponding in molecular mass to NADPH-P450 reductase (~76 kDa) that were absent when NADPH was not present in the incubation mixture (fig. 4, lanes 1 and 2). Various trapping agents did not protect CYP2A6 from adduction. However, glutathione, methoxylamine, and semicarbazide prevented adduction to NADPH-P450 reductase, as shown by the intensities of bands at ~76 kDa (fig. 4). N-Acetyllysine, on the other hand, did not change the intensities of the bands observed, compared with control.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Western blot of CYP2A6 adducted with reactive menthofuran metabolite in the presence of NADPH (lane 1), in the absence of NADPH (lane 2), or in the presence of NADPH and the trapping agents glutathione (10 mM) (lane 3), N-acetyllysine (10 mM) (lane 4), methoxylamine (10 mM) (lane 5), and semicarbazide (10 mM) (lane 6). The bands at ~52 and ~76 kDa correspond to proteins with the molecular masses of CYP2A6 and NADPH-P450 reductase, respectively.

Partition Ratio of Menthofuran with CYP2A6. The menthofuran metabolite formation/CYP2A6 inactivation partition ratio was determined using an incubation time of 60 min, to ensure complete inactivation of CYP2A6. The ratio was 3.5 ± 0.6 nmol/nmol of P450.

	Discussion

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Menthofuran was previously shown to be metabolically activated to chemically reactive intermediates that are capable of covalent binding to cellular proteins (Thomassen et al., 1992). Investigations with inhibitors and inducers of hepatic P450 demonstrated an association between hepatocellular damage caused by menthofuran and its metabolic activation and covalent binding to target organ proteins.

In this study we observed inactivation of CYP2A6 by menthofuran. The inactivation of CYP2A6 by menthofuran was determined to be mechanism-based, by considering the following observations. 1) Menthofuran inactivated CYP2A6 in a time- and concentration-dependent manner (figs. 2 and 3). 2) The loss of activity was NADPH dependent (table 1). 3) The loss of activity was irreversible and was not recovered by extensive dialysis. 4) Inactivation was not protected against by exogenous nucleophiles. However, sodium cyanide was able to protect CYP2A6 from inactivation, at least in part because of inhibition of CYP2A6 activity (table 1). 5) Inactivation was not a consequence of the generation of reactive oxygen species, because catalase and superoxide dismutase did not protect CYP2A6 from inactivation (table 1).

Menthofuran was found to be a relatively efficient inactivator of CYP2A6, with a K_i of ~1 µM, a k_inact of ~0.2 min¹ (figs. 2 and 3), and a partition ratio of ~3.5 nmol/nmol. It was highly selective for CYP2A6, because several other P450 isoforms were not inactivated at concentrations of menthofuran significantly greater than those required for inactivation of CYP2A6 (table 2).

Interestingly, oxidative metabolites of menthofuran formed adducts with CYP2A6 and with NADPH-P450 reductase (fig. 4). The intensities of the CYP2A6 bands were unchanged when various trapping agents were used in the incubations. The intensities of the bands corresponding to NADPH-P450 reductase were affected by various trapping agents. The decreases in the intensities of the bands demonstrate that the reactive electrophilic metabolite that leaks out of the active site of CYP2A6 is a soft electrophile (trapped with glutathione and methoxylamine) and contains an aldehyde group (trapped with semicarbazide). It is unclear whether these adducts are those that lead to inactivation of CYP2A6; adduction of the reductase does not apparently affect its ability to reduce other P450 isoforms, inasmuch as CYP2E1 activity in human liver microsomes is unaffected under conditions in which inactivation of CYP2A6 occurs (table 2).