Biotransformations of R-(+)-pulegone and menthofuran invitro: Chemical basis for toxicity

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Summary

Incubation of R-(+)-pulegone(I) with PB-induced rat liver microsomes in the presence of NADPH resulted in the formation of menthofuran(II) and 2′-Z-[2′-keto-4′-methylcyclohexylidene] propanol (III, 9-hydroxy pulegone) as the major and minor metabolites, respectively. When isopulegone(IV) was used as the substrate, the major metabolite formed was shown to have identical GC-MS fragmentation pattern to that of synthetic 2-[2′-keto-4′-methylcyclohexyl]prop-2-en-1-ol (V) and the minor metabolite was shown to be menthofuran (II). Transformation of menthofuran (II) by microsomes in the presence of NADPH yielded a metabolite identified as 2-Z-(2′-keto-4′-methyl cyclohexylidene) propanal (VI, pulegone-8-aldehyde). Formation of this α , β - unsaturated aldehyde was further confirmed by trapping it as cinnoline derivative by adding semicarbazide to the assay medium. The toxicity mediated by pulegone is discussed in the light of these observations.

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    When S-(−)-pulegone was administered, higher amounts of pulegone, piperitone and benzoic acid and lower amounts of menthofuran were present in the urine, compared to the urine of R-(+)-pulegone treated rats (Madyastha and Gaikwad, 1998). The formation of a γ-ketoenal intermediate from the oxidiation of menthofuran that binds to cellular proteins has been proposed to be related to the observed heptatotoxicity of pulegone (Madyastha and Raj, 1990; McClanahan et al., 1989; Nelson et al., 1992; Thomassen et al., 1992). The higher levels of menthofuran observed in the administration of R-versus S- pulegone was proposed to be correlated with the higher hepatotoxicity of the R-entantiomer observed in mice (Gordon et al., 1982).

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    In the second major pathway, (4R)-(+)-pulegone is subjected to stereoselective hydroxylation at the C-5 position to form 5-hydroxypulegone, which in turn dehydrates to form p-mentha-1,4(8)-diene-3-one-piperitenone [28]. ( R)(+)-Menthofuran is converted to an α,β-unsaturated-γ-ketoaldehyde, by studies carried out in vivo and in vitro [26,29,30]. Although our results in flies clearly showed the conversion of (4R)(+)-pulegone and menthone into menthofuran, no transformation of this last terpene into further metabolites could be demonstrated, at least in our range of sensitivity (1 μg of terpene/fly).

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    The stereochemical analysis of the unreacted substrate indicated that the reaction is stereoselective and one of the enantiomers is present in excess.The oxygen uptake studies carried out using reconstituted cytochrome P-450 system with R-(+)-pulegone and ±4-methyl-2-cyclohexenone revealed that they were oxygenated at the fastest rates of 11.8 and 1.33 nmol/min/mg protein (νmax) and km were found to be 1.38 and 2.0 μM, respectively. The regio- and stereoselectivity of liver microsomal cytochrome P-450 system play an important role in the metabolism of R-(+)-pulegone [4,6]. Although both the isopropylidene methyl groups in pulegone are allylic to the double bond, it is the methyl group syn to the carbonyl that gets oxidized by the cytochrome P-450 system, which results in the formation of menthofuran, the proximate toxin.

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