Studies on the metabolism of structural analogues of a potent hepatotoxin, R-(+)- pulegone, a monoterpene ketone, in both mammalian and microbial systems

Abstract

Pennyroyal oil from Mentha pulegium has been widely used as a fragrance component, a flavoring agent, and also as an herbal medicine to terminate pregnancy. R-(+)-pulegone (I), a monoterpene ketone, is the major constituent of this oil and is a potent hepatotoxin and pneumotoxin. The bioactivation of this compound to reactive metabolites is mostly responsible for the observed toxicity. Pretreatment studies suggest that PB-induced cytochrome P-450 catalyzed reactive metabolite(s) may be responsible for the hepatotoxicity caused by R-(+)-pulegone. Our earlier biochemical, light, and electron microscopic studies showed that R-(+)-pulegone caused necrosis of the liver. It has been demonstrated that R-(+)-pulegone (I) gets extensively metabolized following two major pathways. One of the major pathways is initiated through the regiospecific hydroxylation of R-(+)-pulegone to 9-hydroxypulegone, which upon intramolecular cyclization followed by dehydration yields menthofuran. The second major pathway is involved in the stereoselective hydroxylation of R-(+)-pulegone at the C-5 position to form 5-hydroxypulegone, which gets transformed to piperitenone. In fact, most of the metabolites of R-(+)-pulegone are derived from menthofuran and piperitenone. Menthofuran, the proximate toxin, accounts for nearly half of the toxicity mediated by R-(+)-pulegone, suggesting that the remaining 50% of the toxicity is caused by metabolites derived independently of menthofuran. The available evidence suggests that both the major pathways (5-hydroxylation and 9-hydroxylation pathways) involved in the metabolism of R-(+)-pulegone yield reactive metabolites which can generate toxic effects. However, the contribution of the individual pathways towards R-(+)-pulegone mediated toxicity has not been assessed.

The characteristic structural features of R-(+)-pulegone are the presence of an ?-isopropylidene ketone unit and a chiral centre at C-5. Both these structural features appear to be necessary for the compound to elicit maximum toxicity. It is known that inversion of configuration of the C-5 methyl group in R-(+)-pulegone markedly affects the hepatotoxic potential. Thus, S-(-)-pulegone is approximately one-third as hepatotoxic as its enantiomer, R-(+)-pulegone. It would be interesting to assess the hepatotoxic potential of a compound which is structurally similar to R-(+)-pulegone but without a chiral centre at the C-5 position. One can envisage such a situation if the C-5 hydrogen in R-(+)-pulegone is replaced by another methyl group, as in 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (II), a compound structurally similar to R-(+)-pulegone. In this compound (II), the metabolic pathway initiated by C-5 hydroxylation is blocked due to an additional methyl substitution at the C-5 position. It would be interesting to find out whether compound II gets metabolized following a pathway initiated through regiospecific hydroxylation of the methyl group which is syn to the carbonyl group (C-10 methyl), or if compound II is not accepted by the hydroxylase system as a substrate. To understand the role of the chiral centre at C-5 in R-(+)-pulegone mediated toxicity, metabolic studies with 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (compound II) were undertaken both in vivo and in vitro.

Studies carried out so far clearly indicate that some of the structural features of R-(+)-pulegone are the important determinants for its hepatotoxic response. However, efforts have not been made to find out whether reduction of ring size in R-(+)-pulegone would affect its hepatotoxic potential. To explore the role of ring size in R-(+)-pulegone mediated toxicity, metabolic studies with R-(+)-4-methyl-2-(1-methylethylidene)-cyclopentanone (III) and 5-methyl-2-(1-methylethylidene)-cyclopentanone (IV) were undertaken in both in vivo and in vitro. Compounds III and IV are structurally very similar and have the same functional groups as in R-(+)-pulegone (I), except there is a contraction in the ring size.

The present study describes the isolation and characterization of several novel metabolites from the urine of rats dosed with compounds II, III, and IV. Some of the metabolites isolated and characterized are hitherto not known. The effects of these compounds (II, III, and IV) on hepatic microsomal enzymes in vivo are also reported. A number of liver microsomal enzymes and SGPT levels were studied 24 hours after the intraperitoneal administration of compounds II, III, and IV to rats separately. The effects of these compounds II, III, and IV on the hepatic microsomal enzymes were compared to the effects observed after a single dose of intraperitoneal administration of known hepatotoxins viz. R-(+)-pulegone (I) and CCl? to rats. It was observed that intraperitoneal administration of a single dose of R-(+)-pulegone or CCl? to rats caused marked decrease in microsomal cytochrome P-450, aminopyrine N-demethylase, and glucose-6-phosphatase activities. During this period, a significant increase in SGPT levels was also observed. The effects of intraperitoneal administration of a single dose of compound II resulted in 26%, 23%, and 41% decrease in cytochrome P-450, glucose-6-phosphatase, and aminopyrine N-demethylase activities respectively, after 24 hours of administration. An 11-fold increase in SGPT level was also observed. However, the decrease in the level of cytochrome P-450 and glucose-6-phosphatase and increase in SGPT value after the administration of test compounds were considerably more in the case of R-(+)-pulegone and CCl? than with compound II, indicating that II is considerably less toxic than R-(+)-pulegone. It was noticed that hepatotoxic effects of II were dose-dependent. Pretreatment of rats with PB, prior to administration of II, resulted in potentiation of hepatotoxicity as evidenced by a significant increase in SGPT levels, whereas pretreatment with 3-MC protected from it. It appears that PB-induced cytochrome P-450 catalyzed reactive metabolite(s) may be responsible for the toxicity caused by compound II.

The intraperitoneal administration of a single dose of compounds III and IV and estimation of the microsomal enzymes clearly indicated that the levels of cytochrome P-450, aminopyrine N-demethylase, and glucose-6-phosphatase activities were not significantly affected. Even the increase in SGPT levels was only marginal. Pretreatment of rats with PB for four days prior to the administration of compounds III and IV did not alter significantly the levels of hepatic microsomal enzymes and SGPT.

The present study represents the characterization of various metabolites isolated from the urine of the rats dosed with II, III, and IV. Compound II was administered orally (250 mg/kg body weight/day) to rats for 5 days. Various urinary metabolites were isolated and characterized (Fig. 1A). Incubation of compound (II) with PB-induced rat liver microsomes in the presence of NADPH resulted in the formation of a furanoterpene (lib). The formation of lib was inhibited to a significant extent by carbon monoxide, metyrapone, SKF 525-A, and cytochrome c, suggesting the participation of PB-induced microsomal cytochrome P-450 system in the conversion of II to lib.

Metabolic disposition of R-(+)-4-methyl-2-(1-methylethylidene)-cyclopentanone (III) was examined in rats. Compound (III) was administered orally (250 mg/kg body weight/day). The urinary metabolites isolated and characterized are presented in Fig. 1B. Incubation of compound (III) with PB-induced rat liver microsomes in the presence of NADPH resulted in the formation of metabolites, Illa, Illb, IIIc, IIId, and IIIe.

Metabolic disposition of 5-methyl-2-(1-methylethylidene)-cyclopentanone (IV, DL-camphorone) was examined in rats. Compound (IV) was administered orally (250 mg/kg body weight/day). Seven metabolites were isolated and characterized (Fig. 1C). Incubation of compound (IV) with PB-induced rat liver microsomes in the presence of NADPH resulted in the formation of metabolites, IVa, IVb, and IVc.

Compounds II, III, and IV elicited type I binding spectrum with PB-induced microsomes. The value of spectral dissociation constant (K?) calculated from the double reciprocal plot for these compounds II, III, and IV with PB-induced microsomes were 38.5 ?M, 35.7 ?M, and 29 ?M, respectively.

The biotransformation of 5-methyl-2-(1-ethyl-1-propylidene)-cyclohexanone, 2,2-dimethyl-5-(1-methylethylidene)-cyclopentanone, and 5-methyl-2-(1-ethyl-1-propylidene)-cyclopentanone were also studied using PB-induced microsomes.

It is of interest to know whether microbes bring about transformation in R-(+)-pulegone and closely related compounds in a manner similar to what has been observed in the mammalian system, and if so, then it would be easier to prepare some of the reactive metabolites in large quantities which can be used for their detailed study in the mammalian system. Earlier Mucor piriformis was shown to carry out novel and preparatively useful transformations of steroids and alkaloids. This versatile fungal strain was used in the present study to carry out transformations of R-(+)-pulegone and related terpenoids.

Biotransformation of a monoterpene ketone, R-(+)-pulegone (I), a potent hepatotoxin was studied using a fungal strain, Mucor piriformis. Eight metabolites, namely 5-hydroxypulegone, piperitenone, 6-hydroxypulegone, 3-hydroxypulegone, 5-methyl-2-(1-hydroxy-1-methylethyl)-2-cyclohexene-1-one, 3-hydroxy isopulegone, 7-hydroxypiperitenone, and 7-hydroxypulegone were isolated from the fermentation medium and identified. The organism initiates transformation either by hydroxylation at the C-5 position or hydroxylation of the ring methylenes, the former being the major activity.

Based on the identification of the metabolites, pathways for the biotransformation of R-(+)-pulegone have been proposed. The mode of transformation of S-(-)-pulegone by this organism was shown to be similar to that of its R-(+)-enantiomer. When isopulegone (X) was used as the substrate, the organism isomerized it to pulegone (I), which was then transformed to various metabolites.

Similarly, we have studied the biotransformation of pulegol, 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone, 5-methyl-2-(1-ethyl-1-propylidene)-cyclohexanone, menthone, 1-menthol, 1,8-cineol, D- and L-carvones, 4-methyl-2-(1-methylethylidene)-cyclopentanone, DL-camphorone, 2,2-dimethyl-5-(1-methylethylidene)-cyclopentanone, and 5-methyl-2-(1-ethyl-1-propylidene)-cyclopentanone in M. piriformis. We have also demonstrated the quantitative conversion of L- and D-carvone to (+)-dihydrocarvone and (-)-isodihydrocarvone, respectively. In this chemo-enzymatic method, M. piriformis and pyridinium chlorochromate (PCC) were used as reagents.

We have demonstrated that 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (II) is significantly less toxic than R-(+)-pulegone. Compound II is predominantly metabolized following a pathway initiated through regiospecific hydroxylation of a methyl syn to the carbonyl. The PB pretreatment studies indicated that the hepatotoxicity mediated by 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (II) could be due to the metabolic activation of this compound in the presence of a specific cytochrome P-450 system.

Intraperitoneal administration of compounds III and IV did not alter the rat liver drug metabolizing enzyme activities. This suggests that reduction of the ring size in R-(+)-pulegone completely abolishes its hepatotoxic potential. This is exemplified by our studies using compounds III and IV.

M. piriformis appears to be a very versatile fungal strain with the ability to carry out oxidation of various monoterpenes, regio- and stereospecifically and in a highly efficient manner. A comparison of the transformations of various monoterpenes carried out by M. piriformis with that of PB-induced rat liver microsomes reveals a striking similarity in the mode of transformation (metabolism) of these terpenoids. The ability of M. piriformis to mimic mammalian metabolism and to perform novel biotransformations clearly demonstrates that microbial systems represent an attractive alternative to the use of actual mammalian systems or chemical synthesis of metabolites.