Metabolism of R-(+)-menthofuran in rats : Its relevance to R-(+)-pulegone mediated toxicity

Abstract

Metabolism of R-(+)-Menthofuran In Vivo and In Vitro: Its Relevance to R-(+)-Pulegone Mediated Toxicity

Pennyroyal oil from Mentha pulegium is widely used as a flavoring agent and also as a herbal medicine to terminate pregnancy. The major constituent of this oil is R-(+)-pulegone (1), a monoterpene ketone. R-(+)-Menthofuran (2), a furanoterpene, is one of the minor constituents of pennyroyal oil. It was established earlier by various investigators that R-(+)-pulegone is a potent hepatotoxin and pneumotoxin in animals. We have also observed earlier that massive hepatotoxicity is accompanied by an increase in serum glutamate pyruvate transaminase (SGPT) and a decrease in glucose-6-phosphatase upon treatment of rats with R-(+)-pulegone. Pretreatment of rats with phenobarbital (PB) or diethylmaleate (DEM) potentiated the hepatotoxicity caused by R-(+)-pulegone, whereas pretreatment with 3-methylcholanthrene (3-MC) or piperonyl butoxide protected from it. On the basis of these experiments, it was suggested that PB-induced cytochrome P-450 catalyzed reactive metabolite(s) may be responsible for the hepatotoxicity mediated by R-(+)-pulegone. Our earlier findings from biochemical, light, and electron microscopic studies suggested that pulegone is a potent hepatotoxin. However, the mechanism by which pulegone caused hepatotoxicity had not been established.

Experiments carried out in our laboratory as well as in Prof. S.D. Nelson’s laboratory in the USA have clearly demonstrated that R-(+)-menthofuran is a proximate toxin responsible for the hepatotoxicity mediated by R-(+)-pulegone. We have also demonstrated that incubation of R-(+)-pulegone with rat liver microsomes in the presence of NADPH resulted in covalent binding of radioactive material to macromolecules. All these results suggest the involvement of liver microsomal cytochrome P-450 in the biotransformation of R-(+)-pulegone to reactive metabolite(s) responsible for toxicity. NADPH-dependent covalent binding was inhibited in the presence of cysteine and semicarbazide, suggesting that the reactive metabolite formed could be an aldehyde. In fact, we proposed earlier the formation of an ?,?-unsaturated-?-ketoaldehyde from R-(+)-menthofuran, which could be responsible for hepatotoxicity mediated by R-(+)-pulegone. However, experimental proof to substantiate this hypothesis had not been established.

The present investigation was initiated to probe further the mechanism and the chemical basis of R-(+)-pulegone mediated toxicity. Metabolic studies with R-(+)-pulegone and R-(+)-menthofuran were undertaken both in vivo and in vitro. Very little is known about the metabolic fate of menthofuran in mammals. We have also carried out studies using PB-induced rat liver microsomes as well as a reconstituted cytochrome P-450 system to support the in vivo findings. Besides, earlier studies on the metabolism of R-(+)-pulegone in vivo were re-investigated to characterize some metabolites of R-(+)-pulegone that were missed in earlier investigations.

Since R-(+)-menthofuran has been shown to be a proximate toxin responsible for the hepatotoxicity mediated by R-(+)-pulegone, its metabolic fate in rats was investigated. The metabolites isolated and characterized from urine of rats dosed with R-(+)-menthofuran were: p-cresol (3), 5-methyl-2-cyclohexenone (4), 3-methylcyclohexanone (5), 3-methylcyclohexanol (6), 4-hydroxy-4-methylcyclohexenone (7), geranic acid (8), nerolic acid (9), benzoic acid (10), and 2-[2’-keto-4’-methylcyclohexyl]-propionic acid (11). Besides, GC-MS analysis of the urine extract of rats dosed with menthofuran showed the presence of propanaldehyde (12), which supports the mechanism proposed for the cleavage of the exocyclic double bond in R-(+)-pulegone. The study demonstrated the ability of the rat system to metabolize R-(+)-menthofuran by different pathways (Fig. 1). Some of the pathways proposed are further supported by in vitro studies carried out using PB-induced rat liver microsomes as well as the reconstituted PB-induced cytochrome P-450 system.

The most significant pathway appears to be the one involved in the formation of p-cresol (3) from R-(+)-menthofuran. In fact, p-cresol is one of the major metabolites formed from R-(+)-menthofuran. It is believed that both p-cresol and an ?,?-unsaturated-?-ketoaldehyde are responsible for most of the toxicity mediated by R-(+)-pulegone/R-(+)-menthofuran. The mode of formation of these two metabolites from R-(+)-menthofuran has also been established by in vivo studies. PB-induced rat liver microsomes in the presence of NADPH and O? readily convert menthofuran to an ?,?-unsaturated-?-ketoaldehyde (13). This reactive metabolite possibly could have been formed through epoxidation of the 2,3-double bond of the furan ring. The structure assigned to ?,?-unsaturated-?-ketoaldehyde was further supported by trapping this metabolite as a cinnoline derivative.

One can envisage the formation of 4-hydroxy-4-methylcyclohexenone and p-cresol from ?,?-unsaturated-?-ketoaldehyde (Fig. 1). This assumption was firmly established by various experiments carried out in vitro. Rat liver PB-induced microsomes convert 4-methyl-2-cyclohexenone (14) to 4-hydroxy-4-methylcyclohexenone and p-cresol in the presence of NADPH and O?. The above transformations were also demonstrated using a reconstituted cytochrome P-450 system. It was shown that the hydroxylation of 4-methyl-2-cyclohexenone is stereospecific. This conclusion was drawn on the basis of hydroxylation studies carried out using (±)-4-methyl-2-cyclohexenone as the substrate and subjecting the unreacted substrate to stereochemical analysis (circular dichroism studies and optical rotation measurement). These observations clearly suggest why S-(?)-pulegone is less toxic than R-(+)-pulegone.

The stereospecific hydroxylation of 4-methyl-2-cyclohexenone prevents the formation of p-cresol from S-(?)-pulegone. On the basis of both in vivo and in vitro studies, a possible mechanism for the formation of p-cresol and ?,?-unsaturated-?-ketoaldehyde has been proposed (Fig. 2).

The metabolism of R-(+)-menthofuran carried out in vivo and in vitro clearly suggested that p-cresol is one of the major metabolites formed and stereoselective hydroxylation at C-5 in pulegone is one of the important reactions taking place during metabolism. A detailed study on the metabolism of R-(+)-menthofuran prompted us to re-investigate the metabolic fate of R-(+)-pulegone in rats. Eight new metabolites have been isolated from rat urine in addition to the six metabolites isolated by earlier investigators. The new metabolites isolated and characterized were 5-hydroxypulegone (15), 7-hydroxypulegone (16), piperitone (17), piperitenone (18), 8-hydroxypulegone (19), p-cresol (3), geranic acid (8), and nerolic acid (9). Based on these results, metabolic pathways for the biotransformation of R-(+)-pulegone in rats have been proposed (Fig. 3).

Although R-(+)-menthofuran has been identified as one of the metabolites of R-(+)-pulegone both in vivo and in vitro, the enzyme system involved in this conversion had not been characterized. We have now shown that incubation of R-(+)-pulegone with PB-induced rat liver microsomes in the presence of NADPH and O? resulted in the formation of menthofuran and 2-[2’-keto-4’-methylcyclohexylidene]-propanol (20, 9-hydroxypulegone) as the major and minor metabolite, respectively. When isopulegone (21) was used as the substrate, the major metabolite formed was shown to be 2-[2’-keto-4’-methylcyclohexyl]-prop-2-en-1-ol (22) and the minor metabolite was shown to be menthofuran (Fig. 4). These transformations were also demonstrated using a reconstituted PB-induced cytochrome P-450 system. Thus, we have established the selective allylic methyl oxidation of R-(+)-pulegone, which is the first step involved in the formation of R-(+)-menthofuran from R-(+)-pulegone, and this step is mediated by the PB-induced cytochrome P-450 system.

Although R-(+)-menthofuran has been identified as a proximate toxin responsible for the hepatotoxicity mediated by R-(+)-pulegone, its effects on hepatic drug-metabolizing enzymes as well as hepatic injury have not been evaluated after the exposure of rats to this monoterpene furan. Oral administration of R-(+)-menthofuran (300 mg/kg) once daily for three days caused a decrease in the levels of liver microsomal cytochrome P-450 (55%) with a concomitant increase in the levels of cytochrome P-420, a non-physiological form of cytochrome P-450. However, the levels of cytochrome b? were increased by 30% as compared to control. Massive hepatotoxicity accompanied by an increase in serum glutamate pyruvate transaminase (SGPT), decrease in glucose-6-phosphatase and aminopyrine N-demethylase were also observed upon treatment with R-(+)-menthofuran. The hepatotoxic effects of R-(+)-menthofuran were both dose- and time-dependent. It was also demonstrated that pretreatment of rats with phenobarbital potentiated the hepatotoxicity, whereas pretreatment with 3-methylcholanthrene protected from it. This suggested that a PB-induced cytochrome P-450 catalyzed reactive metabolite(s) may be responsible for the hepatotoxicity caused by R-(+)-menthofuran.

In conclusion, we have demonstrated for the first time that during the metabolism of R-(+)-pulegone/R-(+)-menthofuran, one of the major metabolites formed is p-cresol. We have also shown by both in vivo and in vitro studies the formation of an ?,?-unsaturated-?-ketoaldehyde from R-(+)-menthofuran, the proximate toxin of R-(+)-pulegone. Thus, R-(+)-pulegone/R-(+)-menthofuran-mediated toxicity could be due to the formation of both ?,?-unsaturated-?-ketoaldehyde and p-cresol. It was also demonstrated that PB-induced rat liver microsomal cytochrome P-450 system plays an important role in the formation of these two metabolites. In fact, it has been established that stereospecific hydroxylation of 4-methyl-2-cyclohexenone resulted in the formation of p-cresol, and this observation provides a meaningful explanation why S-(?)-pulegone is less toxic than R-(+)-pulegone.