Detoxification of α- and β-Thujones (the Active Ingredients of Absinthe): Site Specificity and Species Differences in Cytochrome P450 Oxidation in Vitro and in Vivo

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Abstract
α- and β-Thujones are active ingredients in the liqueur absinthe and in herbal medicines and seasonings for food and drinks. Our earlier study established that they are convulsants and have insecticidal activity, acting as noncompetitive blockers of the γ-aminobutyric acid (GABA)-gated chloride channel, and identified 7-hydroxy-α-thujone as the major metabolite and 4-hydroxy-α- and -β-thujones and 7,8-dehydro-α-thujone as minor metabolites in the mouse liver microsome-NADPH system. We report here unexpected site specificity and species differences in the metabolism of the thujone diastereomers in mouse, rat, and human liver microsomes and human recombinant P450 (P450 3A4), in orally treated mice and rats, and in Drosophila melanogaster. Major differences are apparent on comparing in vitro microsome-NADPH systems and in vivo urinary metabolites. Hydroxylation at the 2-position is observed only in mice where conjugated 2R-hydroxy-α-thujone is the major urinary metabolite of α-thujone. Hydroxylation at the 4-position gives one or both of 4-hydroxy-α- and -β-thujones depending on the diastereomer and species studied with conjugated 4-hydroxy-α-thujone as the major urinary metabolite of α- and β-thujones in rats. Hydroxylation at the 7-position of α- and β-thujones is always a major pathway, but the conjugated urinary metabolite is minor except with β-thujone in the mouse. Site specificity in glucuronidation favors excretion of 2R-hydroxy- and 4-hydroxy-α-thujone glucuronides rather than those of three other hydroxythujones. Two dehydro metabolites are observed from both α- and β-thujones, the 7,8 in the P450 systems and the 4,10 in urine. Two types of evidence establish that P450-dependent oxidations of α- and β-thujones are detoxification reactions: three P450 inhibitors block the metabolism of α- and β-thujones and strongly synergize their toxicity in Drosophila; six metabolites assayed are less potent than α- and β-thujones as inhibitors of [3H]ethynylbicycloorthobenzoate binding to the GABAA receptor in mouse brain membranes and as toxicants to Drosophila.