(r)-(1)-menthofuran Is a Potent, Mechanism-based Inactivator of Human Liver Cytochrome P450 2a6

(R)-(1)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 (CYP or P450) 2A6. Menthofuran caused a timeand concentration-dependent loss of CYP2A6 activity. The inactivation of CYP2A6 was characterized by a Ki of 2.5 mM and a kinact of 0.22 min 21 for human liver microsomes and a Ki of 0.84 mM and a kinact of 0.25 min 21 for purified expressed CYP2A6. Addition of various nucleophiles, a chelator of iron, or scavengers of reactive oxygen species or extensive dialysis failed to protect CYP2A6 from inactivation. An antibody to metallothionein conjugates of a suspected reactive metabolite of menthofuran was used to detect reactive menthofuran metabolite adducts with CYP2A6. These adducts were formed only in the presence of NADPH-P450 reductase and NADPH. Glutathione, methoxylamine, and semicarbazide did not prevent adduction of reactive menthofuran metabolites to CYP2A6, however. The menthofuran metabolite formation/CYP2A6 inactivation partition ratio was determined to be 3.5 6 0.6 nmol/nmol of P450. Menthofuran was unable to inactivate CYP1A2, CYP2D6, CYP2E1, or CYP3A4 in a timeand concentration-dependent manner. 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)-(1)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. Materials and Methods 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)-(1)-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 mM) was incubated with human liver microsomes (HL109) or purified CYP2A6 and the NADPHgenerating system to determine its ability to inhibit CYP2A6 in a mechanismbased 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 mM). The reaction mixtures were dialyzed against 25 mM potassium phosphate buffer, pH 7.4 (3 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 mM) 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 mM) 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 presThis research was supported by National Institutes of Health Grants GM25418 and GM32165 (to S.D.N.). 1 Abbreviations used are: CYP or P450, cytochrome P450; TBS, Tris-buffered