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

Höld, Karin M.; Sirisoma, Nilantha; Casida, John E.

doi:10.1021/tx000242c

Cited by 70 publications

(42 citation statements)

References 18 publications

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“…The new products are 2-hydroxy-α-thujone, 2-hydroxy-β-thujone, 7,8-dehydro-α-thujone, 7,8-dehydro-β-thujone, and carvacrol (Scheme 2). The 2-hydroxythujone and the 7,8-dehydrothujone isomers were detected in earlier studies of the metabolism of α-and β-thujone by mammalian liver (26,30,31), but were not specifically identified in the previous work with purified bacterial enzymes (14). As indicated by the "Other" column in Table 1, a few small unidentified peaks remain in the gas chromatogram, but the percent of the metabolites represented by these products has greatly decreased compared to the earlier study: from 6 to 1.5% for P450 BM3 and α-thujone, 25 to 14.6% for P450 BM-3 and β-thujone, 19 to 3.5% for P450 cam and α-thujone, and 3 to 1.2% for P450 cam and β-thujone.…”

Section: Discussionmentioning

confidence: 93%

Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes

Jiang

Montellano

2005

Biochemistry

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Cytochromes P450 cam and P450 BM3 oxidize α-and β-thujone into multiple products, including 7-hydroxy-α-(or β-)thujone, 7,8-dehydro-α-(or β-)thujone, 4-hydroxy-α-(or β-)thujone, 2-hydroxy α-(or β-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 ± 0.3 × 10 10 s −1 to 12.5 ± 3 × 10 10 s −1 for trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[ 2 H]-α-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450 cam active site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of α-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450 cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate-and enzyme-dependent.The regio-and stereoselective hydroxylation of hydrocarbons by cytochrome P450 enzymes is a chemically demanding but physiologically important reaction. Hydroxylation is required, for example, for the conversion of cholesterol into a panoply of mammalian sterol hormones and of arachidonic acid into vasodilator and vasoconstrictor signaling molecules (1,2). The mechanism of carbon hydroxylation has been thought for three decades to involve hydrogen abstraction by the catalytic ferryl species followed by recombination of the resulting substrate carbon radical with the equivalent of an iron-bound hydroxyl radical (3,4). The ferryl species involved in this reaction is generated by reductive activation of molecular oxygen at the iron † This work was supported by grant GM25515 from the National Institutes of Health.

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Section: Discussionmentioning

confidence: 93%

Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes

Jiang

Montellano

2005

Biochemistry

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“…A growing number of naturally occurring phytochemicals in foods and herbal supplements have been shown to be substrates or inactivators of P450 enzymes. Some examples include bergamottin, a component of grapefruit juice (He et al, 1998); capsaicin from chili peppers (Surh and Lee, 1995); diallylsulfide found in garlic oil (Brady et al, 1991); numerous isothiocyanates found in all cruciferous vegetables (Conaway et al, 1996;Goosen et al, 2000); I3,II8-bia-piogenin, hypericin, hyperforin, and other components of St. John's Wort (Orbach, 2000); roquefortine, a secondary cyclopeptide metabolite generated by Penicillium reoquefortine and other Penicillium species (Aniant et al, 2001); reservatol, a component of red wine (Chan and Delucchi, 2000); and thujones found in the liqueur absinthe (Höld et al, 2001).…”

Section: Liver Microsomal P450mentioning

confidence: 99%

The Licorice Root Derived Isoflavan Glabridin Inhibits the Activities of Human Cytochrome P450S 3A4, 2B6, and 2C9

Kent¹,

Aviram²,

Rosenblat³

et al. 2002

Drug Metabolism and Disposition

131

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ABSTRACT:The potent antioxidants licorice root extract and glabridin, an isoflavan purified from licorice root extract, were tested for their ability to modulate the activities of several cytochrome P450 (P450) enzymes. P450 3A4, the major human drug metabolizing P450 enzyme, was inactivated by licorice root extract and by glabridin in a time-and concentration-dependent manner. The inactivation was NADPH-dependent and was not reversible by extensive dialysis. Further analysis showed that the loss in enzymatic activity correlated with a loss in the P450-reduced CO spectrum and with a loss of the intact heme moiety. In contrast, incubations of P450 3A4 with similar concentrations of 2,4-dimethylglabridin and NADPH did not lead to inactivation of P450 3A4. P450 2B6 was also inactivated by glabridin in a time-and concentration-dependent manner. The majority of the glabridin-inactivated P450 2B6 was able to form a reduced CO spectrum suggesting that the heme was not modified with this isoform. High-performance liquid chromatography analysis of the P450 heme confirmed that incubations with glabridin and NADPH did not result in the destruction of the heme moiety. The activity of P450 2C9 was competitively inhibited by glabridin, whereas P450 2D6 and P450 2E1 were virtually unaffected. The data show that glabridin can serve as a substrate for at least three human P450 enzymes and that depending on the isoform, metabolism of glabridin can lead to mechanism-based inactivation or inhibition of the P450. Heme and reduced CO spectral analysis also indicated that glabridin inactivated P450s 2B6 and 3A4 by different mechanisms. Liver microsomal P4501 enzymes are involved in the metabolism of endogenous substrates such as fatty acids, cholesterol, and steroids. These enzymes also carry out an important function in the catalysis and ultimate clearance of many structurally distinct xenobiotics such as drugs, pesticides, carcinogens, and environmental pollutants (Porter and Coon, 1991; Rendic and DiCarlo, 1997). P450 3A4, the major hepatic and intestinal P450 enzyme in humans, metabolizes more than 50% of clinically used drugs such as cyclosporine A, dihydropyridines, ethynylestradiol, midazolam, terfenadine, and triazolam. On average, P450 2B6 comprises approximately 0.2% of human liver P450 and is responsible for the metabolism of roughly 3% of all drugs such as ketamine (Yanagihara et al., 2001), orphenadrine, secobarbital, phenobarbital, dexamethasone, and rifampin (Chang et al., 1997). The P450 2C family comprises approximately 18% of the P450 enzymes found in human liver and are responsible for the metabolism of at least 25% of all drugs including tolbutamide, diclofenac, (S)-warfarin, phenytoin, and hexobarbital.

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“…Thujone ( -thujone in particular) is an epileptiform convulsant and is widely regarded as the "active" ingredient in absinthe (5,6). It is certainly at the root of absinthe's reputation as being more drug than drink.…”

Section: Introductionmentioning

confidence: 99%

Chemical Composition of Vintage Preban Absinthe with Special Reference to Thujone, Fenchone, Pinocamphone, Methanol, Copper, and Antimony Concentrations

Lachenmeier

Nathan-Maister

Breaux

et al. 2008

J. Agric. Food Chem.

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Thirteen samples of authentic absinthe dating from the preban era (i.e., prior to 1915) were analyzed for parameters that were hypothesized as contributing to the toxicity of the spirit, including naturally occurring herbal essences (thujone, pinocamphone, fenchone), methanol, higher alcohols, copper, and antimony. The total thujone content of preban absinthe was found to range between 0.5 and 48.3 mg/L, with an average concentration of 25.4 ( 20.3 mg/L and a median concentration of 33.3 mg/L. The authors conclude that the thujone concentration of preban absinthe was generally overestimated in the past. The analysis of postban and modern commercial absinthes (2003)(2004)(2005)(2006) showed that the encompassed thujone ranges of all absinthes are quite similar, disproving the supposition that a fundamental difference exists between preban and modern absinthes manufactured according to historical recipes. Analyses of pinocamphone, fenchone, base spirits, copper, and antimony were inconspicuous. All things considered, nothing besides ethanol was found in the absinthes that was able to explain the syndrome "absinthism".

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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

Cited by 70 publications

References 18 publications

Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes

Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes

The Licorice Root Derived Isoflavan Glabridin Inhibits the Activities of Human Cytochrome P450S 3A4, 2B6, and 2C9

Chemical Composition of Vintage Preban Absinthe with Special Reference to Thujone, Fenchone, Pinocamphone, Methanol, Copper, and Antimony Concentrations

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