benzofurans and pulegone

(R)-(+)-Menthofuran is the proximate toxic metabolite of pulegone, the major constituent of the pennyroyal oil, that contributes significantly to the hepatotoxicity resulting from ingestion of this folklore abortifacient pennyroyal oil. Recently, menthofuran was shown to be metabolized by cytochrome P450 to form (R)-2-hydroxymenthofuran. In this paper it is demonstrated that glutathione S-transferase (GST) catalyzes the tautomerization of 2-hydroxymenthofuran to mintlactone and isomintlactone, apparently without the formation of stable glutathione (GSH) conjugates. The reaction strictly required GSH; S-methyl GSH, which binds to the active site and leaves the active site Tyr-9 partly ionized, did not support GST-catalyzed isomerization. It was also determined that the tautomerization reaction requires the active site tyrosine, Tyr-9. The rat GSTA1-1 mutant (Y9F), with the active site tyrosine replaced with phenylalanine, demonstrated no catalytic activity. Rat cytosolic GST A1-1, in the presence of GSH, tautomerized 2-hydroxymenthofuran with apparent K(M) and V(max) values of 110 microM and 190 nmol/min/nmol GST, respectively. However, the site-directed mutant (F220Y), in which Tyr-9 and GSH in the binary complex [GST. GSH] have lower pK(a)s, exhibited K(M) and V(max) values of 97 microM and 280 nmol/min/nmol GST, respectively. Similarly, human liver cytosol catalyzed the tautomerization of 2-hydroxymenthofuran in a GST-dependent reaction. The mechanism most consistent with the data is a general-base catalyzed isomerization with GS(-) serving to deprotonate the substrate to initiate the reaction.

Topics: Amino Acid Substitution; Animals; Benzofurans; Catalysis; Cyclohexane Monoterpenes; Cytosol; Glutathione Transferase; Humans; Isoenzymes; Isomerism; Kinetics; Lactones; Liver; Menthol; Molecular Structure; Monoterpenes; Mutagenesis, Site-Directed; Rats; Recombinant Proteins

1. R-(+)-Pulegone was administered orally to rats and the urinary metabolites were investigated. Six metabolites were isolated and purified using column and thin layer chromatographic techniques. Metabolites were identified by i.r., n.m.r. and mass spectral analyses. 2. The neutral metabolites isolated from urine of rats treated with pulegone (I) were: pulegone (II), 2-hydroxy-2(1'-hydroxy-1'-methylethyl)-5-methylcyclohexanone (III), 3,6-dimethyl-7a-hydroxy-5,6,7,7a-tetrahydro-2(4H)-benzofuranone (V) and menthofuran (VII). Metabolites II and III were also excreted in conjugated form. 3. Acidic metabolites isolated from urine of rats treated with pulegone (I) were: 5-methyl-2(1'-methyl-1'-carboxyethylidene)cyclohexanone (IV) and 5-methyl-5-hydroxy-2(1'hydroxy-1'-carboxyethyl)cyclohexanone (VI).

Topics: Administration, Oral; Animals; Benzofurans; Chemical and Drug Induced Liver Injury; Chromatography, Thin Layer; Cyclohexane Monoterpenes; Cyclohexanones; Magnetic Resonance Spectroscopy; Male; Mass Spectrometry; Menthol; Monoterpenes; Rats