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COMPARATIVE DISPOSITION OF (R)-(+)-PULEGONE IN B6C3F1 MICE AND F344 RATS

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

(Received October 10, 2002; accepted April 2, 2003)

	Abstract

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Pulegone is a monoterpene ketone that is usually associated with the herb pennyroyal but is also found in the essential oils from many other mint species. It is the major constituent of pennyroyal oil. Pennyroyal is used as a flavoring and fragrance and as an herbal medicine to induce menstruation and abortion. A disposition study of ¹⁴C-pulegone in B6C3F1 mice and F344 rats has been conducted at doses from 0.8 to 80 mg/kg. Mice excrete 85 to100% of the dose in 24 h. Rats excrete only 59 to 81% of the administered radioactivity in the same time, primarily in urine and feces, with a trace in respired air. Consequently, tissue concentrations are lower in mice than in rats. Male rats tend to have higher tissue concentrations, especially in kidney, than female rats have, but this sex difference is not seen in mice. The residual radioactivity at 24 h demonstrates potential for accumulation of pulegone-derived material in several tissues following multiple doses. The metabolic profile is complex in both species, with at least three pathways involving hydroxylation, reduction, or conjugation with glutathione as first steps. Mercapturic acid pathway metabolites were detected in bile in mice and both bile and urine in rats.

Toxicity studies in laboratory animals have focused primarily on the acute hepatotoxicity of pulegone. However, pulmonary toxicity and cyst-like lesions in the brain have also been reported in mice and rats, respectively (Thorup et al., 1983). Mechanistic studies have demonstrated that hepatotoxicity, at least in part, involves metabolism of pulegone to menthofuran and further metabolism to a reactive -ketoenal. Covalent binding of ¹⁴C-pulegone-derived radioactivity, evidence for the formation of reactive intermediates, was observed in the liver, kidney, and lung of mice receiving 280 mg/kg i.p. (McClanahan et al., 1989). A single i.p. dose of 150 mg/kg pulegone has been shown to deplete glutathione in plasma and liver of rats. Hepatotoxicity was increased if glutathione synthesis was inhibited with buthione-[S,R]-sulfoximine (Thomassen et al., 1990). Mice receiving hepatotoxic doses of pulegone had decreased glutathione levels within 3 h, followed by a rapid rise in plasma glutamic-pyruvic transferase (Gordon et al., 1982).

Pulegone metabolism has received considerable attention primarily due to interest in its metabolic activation. In vivo and in vitro studies have shown that pulegone is metabolized by the cytochrome P450 enzyme system (McClanahan et al., 1989; Madyastha and Raj, 1990). Khojasteh-Bakht et al. (1999) determined human CYP2E1, CYP1A2, and CYP2C19 to be active in metabolism of pulegone to menthofuran. The cytochromes P450 responsible for pulegone metabolism in rodents have not been reported. Recently, 14 urinary metabolites of pulegone in rats were identified, 13 of which are phase II metabolites and several are mercapturic acids (Chen et al., 2001). Glutathione conjugates excreted in bile of rats have been partially characterized by liquid chromatography/MS¹ (Thomassen et al., 1991).

There is little information about the in vivo disposition of pulegone at doses and routes of administration likely to be used in subchronic and chronic toxicology studies. Therefore, the present work investigates the disposition of ¹⁴C-pulegone after a single oral dose of 0.8, 8, or 80 mg/kg in male and female F344 rats and B6C3F1 mice. The rationale for 80 mg/kg as the high dose in this distribution study is based on a 28-day gavage study in rats by Thorup et al. (1983), which found some toxicity to the liver and brain and a substantial decrease in weight gain at 80 mg/kg/day. Multiple-dose studies in female rats and mice were also conducted to investigate accumulation in tissues and the effect of repeat dosing on pulegone metabolism.

	Materials and Methods

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Chemicals. ¹⁴C-labeled pulegone (Fig. 1), specific activity 60.0 mCi/mmol, radiochemical purity 97.44%, was obtained from Wizard Laboratories, Inc. (West Sacramento, CA). Unlabeled pulegone, purity 98%, phenylmethylsulfonyl fluoride, and 4-dimethylaminoantipyrine (aminopyrine) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Emulphor was obtained from BASF Corp. (Mt. Olive, NJ). Nembutal was obtained from Abbott Laboratories (Abbott Park, IL).

View larger version (8K): [in this window] [in a new window]   FIG. 1. Structure of pulegone. *, location of 14C label.

Animals and Treatments. The Institutional Animal Care and Use Committee approved all animal procedures. Male and female B6C3F1 mice and F344 rats were obtained from Taconic Farms, Inc. (Germantown, NY). Female mice were 6 to 7 weeks old and 15 to 21 g. Male mice were 6 to 7 weeks old and 17 to 25 g. Female rats were 2 to 13 weeks old and 160 to 195 g. Male rats were 11 weeks old and 225 to 333 g. Animals were housed individually in glass or plastic metabolism cages and provided with food (NIH #31) and water ad libitum.

For oral single dose studies, rats or mice were dosed by gavage with 0.8, 8.0, or 80 mg of pulegone/kg in corn oil, 4 ml/kg for rats and 10 ml/kg for mice at 40 µCi/kg. Animals were sacrificed at 2, 8, 24, or 72 h after dosing. For i.v. studies, animals were dosed with 0.8 mg of pulegone/kg, 40 µCi/kg, 1 ml/kg in 8:1:1 water/Emulphor/ethanol. Four animals were dosed for each of these experiments. In multiple dose disposition studies, animals were dosed once daily by gavage at 80 mg of pulegone/kg in corn oil on 4 consecutive days. Animals were sacrificed 24 h after the last dose. Groups were dosed with either four ¹⁴C-labeled (40 µCi/kg) doses (tissue accumulation study) or three non-radioactive doses and the fourth radiolabeled (pretreatment study). Four animals were dosed for each of these study groups.

Female mice and rats were dosed with vehicle or 80 mg/kg unlabeled pulegone once daily for 4 consecutive days, and liver microsomes were prepared 24 h after the last dose. Two or three animals were dosed for each of the study groups.

For bile studies, two male mice were anesthetized with Nembutal; the bile duct was cannulated as described by Griffin et al. (1997). The mice were dosed i.v. by the jugular vein with 0.8 mg of pulegone/kg, 40 µCi/kg, 10 ml/kg in 8:1:1 0.9% saline/Emulphor/ethanol. Bile was collected for 2 to 3 h at room temperature.

Sample Collection and Analysis. The rate and routes of excretion of pulegone-derived radioactivity were measured in 24- and 72-h single-dose experiments, and multiple-dose experiments, with urine and feces collections at 4, 8, 24, 48, and 72 h. The amount of pulegone-derived radioactivity eliminated in expired air was determined by pulling air through the cage by means of a vacuum system. The flow rate was 0.4 to 0.8 l/min. Air exiting the cage passed through a trap containing 200 to 400 ml of ethanol for collection of volatile ¹⁴C, then through a trap containing 200 to 400 ml of a mixture of ethylene glycol monomethyl ether and ethanolamine (7:3, v/v) for collection of expired ¹⁴CO₂. Traps were changed at 4, 8, 12, and 24 h after ¹⁴C-pulegone administration. The trapping efficiency of the solutions was maintained by passing the intake air through calcium sulfate and soda lime to reduce moisture and CO₂ content. Total exhaled radioactivity was determined by counting triplicate 1-ml aliquots of each trapping solution in EcoLume (ICN, Research Products Division, Costa Mesa, CA) in a Beckman Coulter LS 6500 liquid scintillation counter (LSC) (Beckman Coulter, Inc., Fullerton, CA).

Urine aliquots (in triplicate) were added to EcoLume and counted for ¹⁴C in the LSC, and the remainder was stored at –20°C. Feces were air-dried, weighed, and ground to a powder using a ceramic mortar and pestle. The cages were rinsed with distilled water at the end of each collection period, and the radioactivity in the rinse was included in the urine total.

Animals were euthanized with CO₂, blood was drawn by cardiac puncture, and tissues (blood, liver, kidney, lung, muscle, skin, fat, brain, testes or uterus, forestomach, glandular stomach, small intestine, and large intestine) were collected. Tissue and feces aliquots (triplicate 50- to 100-mg samples from rats, up to 100 mg from mice) were oxidized in a PerkinElmer 306 Biological Sample Oxidizer (PerkinElmer Life Sciences, Boston, MA) and counted in the LSC for determination of total ¹⁴C content.

HPLC Analysis of Urine and Bile. Urine samples were centrifuged at low g before analysis. Bile samples were directly analyzed without further purification. HPLC analyses were carried out using a Beckman Coulter System Gold model 126 solvent module, a model 168 photodiode array detector, and an IN/US (Tampa, FL) ß-Ram flow detector equipped with a liquid cell (500 µl) for radiochemical detection. A linear gradient system from 100% A (0.1% trifluoroacetic acid in H₂O) to 50% A and 50% B (CH₃CN) over 28 min on a Metachem (Torrance, CA) Inertsil C18 5-µm column (4.6 x 250 mm) at a flow rate of 1.5 ml/min was used for all studies.

Determination of Liver Microsomal Aminopyrine Demethylase. Livers removed from euthanized animals were homogenized individually in 1:1 (w/v) 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% KCl, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride, and then fractionated by centrifugation at 10,000g for 30 min and 105,000g for 70 min. Microsomal pellets were suspended in a wash buffer of 100 mM sodium pyrophosphate containing 0.1 mM EDTA and recentrifuged at 105,000g for 70 min. Microsomal protein was stored at –80°C following resuspension in 10 mM potassium phosphate buffer (pH 7.4) containing 0.25 M sucrose. The total protein concentrations in the microsomal fractions were determined using the Bio-Rad (Hercules, CA) protein assay based on the Bradford dye-binding procedure. N-Demethylation of aminopyrine was measured by the method of Gibson and Skett (1986).

Statistics. Statistical analysis used JMP Software (SAS Institute, Inc., Cary, NC) and consisted of an analysis of variance followed by pairwise comparison using a Tukey-Kramer test. Values were considered statistically significant at p < 0.05.

	Results

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Excretion. The majority of pulegone-derived radioactivity excreted in the 24 h postdosing is via urine (Table 1). Mice excreted more pulegone-derived radioactivity in 24 h than did rats, primarily due to a consistently higher percentage of dose excreted in urine. In the first 24 h, urine and feces together account for 86 to 101% of the orally administered dose in mice and 59 to 81% in rats. There appears to be nonlinearity in excretion with dose in that a larger fraction of an oral dose is excreted in urine by rats at the lowest dose compared with the highest. The opposite trend is seen in mice, although the differences reach statistical significance only for female mice. The excretion of radioactivity following i.v, administration is similar to that after an oral dose in both species, implying nearly complete absorption.

The data in Table 1 account for nearly all the radioactivity administered to mice, but only 70 to 90% of that given to rats. At least part of the deficit is in the intestinal contents. Data (not shown) from selected experiments showed about 10% of pulegone-derived radioactivity in the gut contents of rats at 24 h, whereas mice had less than 0.2% at that time point. An additional small amount of radioactivity is excreted via the lungs. In 24-h studies, respired ¹⁴CO₂ and volatiles combined account for about 4% of the administered dose (80 mg/kg, oral) in mice and 2% in rats. Total urinary excretion accounted for 65 and 57% and fecal excretion 26 and 35% of the administered dose in female and male rats, respectively, 72 h after oral administration of 8 mg/kg pulegone (data not shown).

Tissue Distribution. The total radioactivity remaining in tissues at 24 h is shown in Table 1. Although there is generally a higher percentage of radioactivity remaining in tissue as the dose increases, the differences rarely reach statistical significance.

The concentration of ¹⁴C-pulegone-derived radioactivity in blood, liver, kidney, and lungs 24 h after administration is presented in Table 2. These four tissues generally had the highest concentration of radioactivity of all tissues examined, excluding the digestive tract tissues. Although not presented in Table 2, radioactivity content was determined for all tissues listed under Materials and Methods in each study; typical data for these tissues are included in Table 3. Several trends are noted in the concentration of pulegone-derived radioactivity in the tissues in Table 2. 1) As expected from the excretion results, tissue concentrations are lower in mice compared with rats. 2) Where the differences are significant, males of both species generally have higher concentrations of radioactivity present in tissues compared with females. 3) The concentration of radioactivity is much higher in male rat kidney compared with the concentration in female rat and mouse kidney. It should be noted that the concentration of pulegone-derived radioactivity in brain, a reported target tissue, is the lowest of the tissues analyzed (Table 3). The change in tissue concentration with time for an 8 mg/kg dose is shown in Fig. 2. Tissue concentrations were highest in mice at 2 h, in male rats at 8 h, and in female rats at 2 to 8 h, indicating the rapid absorption of an oral dose of pulegone, more rapid in mice than in rats.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Time course for tissue distribution of 14C-pulegone-derived radioactivity following a single oral 8 mg/kg dose.

Dose-dependent effects are most evident when the data are calculated as percentage of dose remaining in the tissue (Table 2). There is a clear nonlinear relationship for the percentage of dose remaining versus dose for the male rat. Interestingly, the downward trend in kidney is opposite the trend in the other three tissues. In contrast, the percentage of dose remaining in tissues from female rats is either almost constant, e.g., liver and lung, or increasing, e.g., blood, across the range of doses. A nonlinear relationship between percentage of dose remaining in tissue and dose is apparent for female but not male mice.

Multiple-Dose Experiments. Two types of multiple-dose studies were performed. The pretreatment study was designed to determine whether there was induction or inhibition of metabolism, and the bioaccumulation study was designed to quantitate buildup of pulegone-derived material during a repeat-dose toxicity study. Because there was relatively little pulegone-derived radioactivity in mice 24 h after administration, a bioaccumulation study was not performed.

Tissue distribution and excretion data from the multiple dosing studies are shown in Tables 3 and 4. The pretreatment study determined the disposition of a single radiolabeled dose of pulegone after three daily doses of unlabeled pulegone. Changes in tissue distribution following pretreatment were minor, with the exception of the glandular stomach and forestomach of female rats. Tissue/blood ratios were little changed by pretreatment. Pretreatment does seem to have an effect on urinary excretion; 55% of a single dose was excreted in urine in the first 24 h (Table 1), whereas 71% was excreted in the first 24 h in pretreated animals. Aminopyrine N-demethylase, a nonspecific measure of cytochrome P450 metabolism, was not altered by pretreatment (4.2 ± 1.2 versus 5.7 ± 2.2 nmol/mim/mg protein, control versus treated). The results from a similar study in mice also indicate minor changes in tissue distribution.

Results of the bioaccumulation study are also shown in Table 3. In those tissues where significant increases occurred, there was approximately a 3-fold increase in pulegone-derived radioactivity compared with a single dose. Tissue/blood ratios were not changed by multiple doses except in the gastrointestinal tract. The concentration was decreased in forestomach and glandular stomach. Decreased radioactivity in the stomach resulting from both multiple dose studies may be due to increased cell turnover in these tissues because of repeated irritation from the pulegone.

Characterization of Urinary and Biliary Metabolites of Pulegone in Mice. The urinary metabolites in rats have been separated by HPLC to reveal 13 major radiolabeled peaks A through M (Fig. 3), in which 14 major metabolites and some minor metabolites were characterized (Chen et al., 2001). Peak A was present in urine of male rats but not distinct in urine of females. Urine samples (24-h) from female mice dosed with pulegone (80 mg/kg) were analyzed by the same HPLC method to reveal several major radiolabeled peaks corresponding to peaks A through I in rats (Fig. 3). One radiolabeled peak A' was more abundant in urine of mice compared with rats. Fractions containing these peaks were collected and analyzed by MS and HPLC as described by Chen et al. (2001). The molecular weights, HPLC retention time, and UV spectra of the urinary metabolites from mice were compared with those of the characterized metabolites from rats. The identified urinary metabolites in mice and rats are shown in Fig. 4. Three hydroxypulegone glucuronides (C1, D1, and D2) were identified in mouse urine. Two other glucuronic acid metabolites derived from the hydroxypulegones, C2 and E3, were also produced by mice. Four hydroxymenthone/isomenthone glucuronides (E2, F1, F2, and G1) have been identified in urine of rats. They also appeared in urine of mice. Peak A' was isolated and characterized by negative ion electrospray ionization-MS/MS. Two metabolites with m/z 361 and one metabolite with m/z 245 were detected. Their tandem MS and tentative assignments were as follows: m/z 361 (M – H⁺), 343 (M – H₃O⁺), 303 (M – H⁺, acetone), 193 (glucuronide ion), 157 (glucuronide ion – 2H₂O), formed as the result of 1,4-addition of H₂O to hydroxypulegone glucuronides; m/z 361 (M – H⁺), 343 (M – H₃O⁺), 325 (M – H⁺ – 2H₂O), 193 (glucuronide ion), 175 (glucuronide ion – H₂O), consistent with formation of a hydroxylated product of hydroxymenthone/isomenthone glucuronides; m/z 245 (M – H⁺), 165 (M – H⁺ – SO₃), consistent with formation of a hydrated product of metabolite M. There were no major radiolabeled peaks eluting after 22 min in the urinary metabolite profile of mice (Fig. 3); therefore, it appeared that mercapturic acids K and L and the aromatic metabolites J and M were not present in urine of mice. MS analysis did not detect other mercapturic acid metabolites in urine of mice. The parent compound, pulegone, was not detected in the urine of mice or rats after single or multiple doses.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Representative HPLC radiochromatograms of pulegone-derived radioactivity excreted in cumulative 24-h urine of a female rat (top) and a female mouse (bottom) receiving a single oral dose (80 mg/kg, 40 µCi/kg) in corn oil. Major metabolites are marked; peak A (see text) from rats is predominantly found in males.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Metabolic pathways proposed for the biotransformation of pulegone in mice and rats. a, hydroxylation followed by glucuronidation (C1, D1, D2, and E1) or further metabolism (C2, E3, J, and M); b, reduction to give menthone/isomenthone, followed by hydroxylation/glucuronidation (E2, F1, F2, and G1); c, formation of glutathione conjugates (I and II) followed by conversion to mercapturic acids [K (III) and L (IV)] and further hydroxylation of L to give B1. Metabolites B1, E1, J, M, K, and L were not observed in mouse urine. Metabolites I, II, K (III), and L (IV) were observed in mouse bile.

Bile samples (0–50 min, 50 min to 1 h 45 min, and 1 h 45 min to 2 h 45 min) from male mice dosed with pulegone (0.8 mg/kg, i.v.) were analyzed by the same HPLC method as above to reveal several major radiolabeled peaks (Fig. 5). Peaks I and II, which were distinct only at the first time point (Fig. 5A), comigrated with 8-(glutathion-S-yl)menthone/isomenthone prepared as described before (Chen et al., 2001). Peaks III and IV comigrated with 8-(N-acetylcystein-S-yl)menthone/isomenthone isolated from urine of rats dosed with pulegone (Chen et al., 2001). Peaks III and IV consisted of 2.4% and 4.6% of total radioactivity in bile of a male mouse at 0 to 50 min. Fractions containing these four peaks were isolated by HPLC and analyzed by negative ion electrospray ionization-MS. Except for peak IV, containing a small peak with m/z 314, the other fractions did not give peaks matching the proposed metabolites. The concentration of the metabolites might be below the detection limit of MS.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Representative HPLC radiochromatograms of pulegone-derived radioactivity excreted in bile of a male mouse receiving a single intravenous dose (0.8 mg/kg, 40 µCi/kg, 10 ml/kg) in 8:1:1 0.9% saline/Emulphor/ethanol at various time points (A, 0–50 min; B, 50 min to 1 h 45 min; and C, 1 h 45 min to 2 h 45 min). Peaks I and II coeluted with 8-(glutathion-S-yl)menthone/isomenthone, and peaks III and IV comigrated with 8-(N-acetylcystein-S-yl)menthone/isomenthone.

	Discussion

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The most striking observation in the tissue distribution data is the concentration of pulegone-derived radioactivity in male rat kidney. The concentration is 10-fold greater than that in females at the low dose. As the dose is increased, the difference becomes less and is about 3-fold greater at the high dose. Although male mouse kidney concentrations are also higher than the corresponding female kidney concentration, the fold differences are not as great (Fig. 2). One explanation for the difference between male and female rats is the presence of _2u-globulin in the males (Melnick and Kohn, 1999; Swenberg and Lehman-McKeeman, 1999). _2u-Globulin, a protein produced in the liver of male rats, binds to small organic molecules. The protein is normally catabolized in the kidney, but when the binding site is occupied, the catabolism is inhibited, resulting in buildup of the complex in the kidney (Melnick and Kohn, 1999; Swenberg and Lehman-McKeeman, 1999). Pulegone, or pulegone metabolites, may bind to this protein, resulting in higher concentrations of radioactivity in kidney. Protein binding is also consistent with the percentage of dose data in Table 2. In the other rat tissues, from both males and females, the percentage of dose remaining after 24 h increases with dose or does not change. The percentage of dose remaining in male rat kidney decreases with dose but remains high relative to that in females. This may result from saturation of available binding sites of the limited amount of _2u-globulin present.

Liver is the target organ most closely associated with pulegone toxicity (Gordon et al., 1982). As would be expected, the concentration of pulegone-derived radioactivity is high in this tissue. Of the data shown in Table 2, only the male rat kidney has a higher concentration than liver, and that is true only for the two lower doses. The residual radioactivity in liver amounts to as much as 3.3% of the administered dose, indicating potential accumulation of pulegone-derived material in this organ following repeated daily exposure, as in a chronic toxicity study. As expected, accumulation of radioactivity in liver was demonstrated in the present multiple-dose experiment (Table 3). Liver is the only tissue with a tissue/blood ratio greater than 1 in the bioaccumulation study. Although the concentration of pulegone-derived radioactivity is greater in most tissues after four labeled doses compared with one, this seems to be more related to rate of total clearance than to bioaccumulation. Bioaccumulation generally implies that material is absorbed from the blood and sequestered in tissue, resulting in tissue/blood ratios greater than 1.

Other reported target tissues of pulegone toxicity, lung and brain, have relatively low tissue concentrations of pulegone-derived radioactivity. Lung contains concentrations of radioactivity similar to those in blood in both the single- and multiple-dose studies. The brain has the lowest concentration of radioactivity of the tissues measured; the values for brain in Table 3 are typical of all the distribution studies.

Because the toxicity of pulegone depends in part on metabolic activation by cytochromes P450 (Mizutani et al., 1987) and presents the possibility of induction or inhibition of its own metabolism, we were interested in the effect of multiple dosing on the disposition of the chemical. Hepatotoxic doses of pulegone, five daily doses of 400 mg/kg, decreased the amount of hepatic cytochromes P450 in rats based on total cytochrome P450 content and N-demethylase activity with a general substrate, aminopyrine. These parameters were also decreased following single doses of 200 but not 100 mg/kg (Moorthy et al., 1989). In the pretreatment study, distribution of a radiolabeled pulegone dose of 80 mg/kg following three daily 80 mg/kg doses of unlabeled pulegone showed some subtle differences from a single dose in naive animals. In general, tissue concentrations were lower and excretion via urine was increased in pretreated rats. This result seems more consistent with induction than reduction of enzyme activity; however, hepatic aminopyrine demethylase activity was not changed by the pretreatment. It appears that changes in oxidase activity induced by relatively nontoxic doses of pulegone are minor, and those that have been observed may be more a result of hepatotoxicity. For example, Moorthy et al. (1989) report an LD₅₀ of 245 mg/kg; they report decreases in enzymatic activity beginning at 200 mg/kg.

The biotransformation of pulegone in rodents results in numerous metabolites (Fig. 4). The identity of 14 rat urinary metabolites was reported (Chen et al., 2001). From the urinary metabolites identified, it was concluded that pulegone was metabolized by three major pathways in rats: 1) hydroxylation to give monohydroxylated pulegones followed by glucuronidation or further metabolism; 2) reduction of the carbon-carbon double bond to give diastereomeric menthone/isomenthone, followed by hydroxylation and glucuronidation: and 3) Michael addition of glutathione to pulegone followed by further metabolism via the mercapturic acid pathway to give diastereomeric 8-(N-acetylcystein-S-yl)menthone/isomenthone. A portion of these two mercapturic acids undergoes further hydroxylation (Fig. 4). In comparison, although most of the metabolites from the first two pathways were also identified, there were no detectable mercapturic acids appearing in mouse urine. Two aromatic metabolites, J and M, identified in rat urine were also not detectable in mouse urine. However, one of the components in peak A' may be a hydration product of metabolite M.

Glucuronides of hydroxylated pulegone and hydroxylated, reduced pulegone, as well as glutathione conjugates leading to K and L, were previously partially characterized in the bile of rats treated with pulegone (Thomassen et al., 1991). In addition, other glutathione conjugate metabolites with molecular mass 2 or 4 Da less and mercapturic acids with molecular mass 2 Da less than K and L were also detected in rat bile. Analysis of bile of mice dosed intravenously with pulegone (0.8 mg/kg) demonstrated that metabolites K and L were major biliary metabolites. Glutathione conjugates leading to K and L may also appear in mouse bile (Fig. 5).

Glutathione plays an important role in the toxicity of pulegone in both rats and mice. Pulegone has been shown to deplete glutathione in both species (Gordon et al., 1982; Thomassen et al., 1990). These studies also showed that depletion of glutathione by pretreatment with either buthione-[S,R]-sulfoximine or diethylmaleate resulted in increased hepatotoxicity of pulegone in rats and mice, respectively. Our metabolism studies suggest that direct reaction of pulegone and metabolites of pulegone with glutathione could result in depletion of glutathione in both species. It is not clear why the mercapturic acids are excreted only in bile in mice and both bile and urine in rats (Thomassen et al., 1991; Chen et al., 2001). Mercapturic acid synthesis is usually considered to be an interorgan process involving liver and kidney. A pathway that involves biliary-hepatic cycling has also been demonstrated (Hinchman and Ballatori, 1994). By either pathway, the final step in mercapturic acid synthesis, N-acetylation, is generally considered to take place in the liver. Whether the mercapturic acid is carried back to the kidney and excreted in urine or directly excreted in bile may be due to differences in substrate specificity of transporters, dose, route of exposure, or other factors (Hinchman and Ballatori, 1994).

In summary, the disposition data indicate that in a chronic study, male rats may not tolerate as high a dose of pulegone as female rats or mice. Pulegone is well absorbed in both species, but cleared more slowly by male rats than female rats or mice. There is evidence that pulegone or pulegone metabolites may bind to _2u-globulin in male rats. The metabolite profile is complex in both species, with at least three pathways involving hydroxylation, reduction, or conjugation with glutathione as first steps.

	Footnotes

 
¹ Abbreviations used are: MS, mass spectrometry; LSC, liquid scintillationcounter; HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. L. T. Burka, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709. E-mail: burka{at}niehs.nih.gov

	References

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