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Detoxification of r- and β-Thujones (the Active Ingredients of Absinthe): Site Specificity and Species Differences in Cytochrome P450 Oxidation in Vitro and in Vivo Karin M. Ho¨ld, Nilantha S. Sirisoma, and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 114 Wellman Hall, University of California, Berkeley, California 94720-3112 Received November 24, 2000
R- 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-R-thujone as the major metabolite and 4-hydroxy-R- and -β-thujones and 7,8-dehydro-R-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 microsomeNADPH systems and in vivo urinary metabolites. Hydroxylation at the 2-position is observed only in mice where conjugated 2R-hydroxy-R-thujone is the major urinary metabolite of R-thujone. Hydroxylation at the 4-position gives one or both of 4-hydroxy-R- and -β-thujones depending on the diastereomer and species studied with conjugated 4-hydroxy-R-thujone as the major urinary metabolite of R- and β-thujones in rats. Hydroxylation at the 7-position of R- 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 2Rhydroxy- and 4-hydroxy-R-thujone glucuronides rather than those of three other hydroxythujones. Two dehydro metabolites are observed from both R- 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 R- and β-thujones are detoxification reactions: three P450 inhibitors block the metabolism of R- and β-thujones and strongly synergize their toxicity in Drosophila; six metabolites assayed are less potent than R- and β-thujones as inhibitors of [3H]ethynylbicycloorthobenzoate binding to the GABAA receptor in mouse brain membranes and as toxicants to Drosophila.
Introduction The thujone diastereomers have a long history of use in alcoholic beverages and herbal medicines and as food additives. Absinthe with R-thujone (RT)1 as its principal active ingredient was a major European liqueur during the 1800s, with notable aficionados being Vincent van Gogh, Henri de Toulouse-Lautrec, and Charles Baudelaire (1-5). This liqueur, made from wormwood oil, is now banned in most of the world because of epidemic health problems (4). Although the direct use of RT in food is not allowed in the United States, human exposure to the diastereomers continues because of their presence in 20 approved flavorings and food additives, and also in * To whom correspondence should be addressed. Phone: (510) 6425424. Fax: (510) 642-6497. E-mail:
[email protected]. 1 Abbreviations: GABA, γ-aminobutyric acid; is, internal standard; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; MOX, methyloxime; TMS, trimethylsilyl; UDPGA, 5′-diphosphoglucuronic acid; RT, R-thujone; βT, β-thujone; 2ROHRT, 2R-hydroxy-R-thujone; 2ROHβT, 2R-hydroxy-β-thujone; 4,10DHT, 4,10-dehydrothujone; 4OHRT, 4-hydroxy-R-thujone; 4OHβT, 4-hydroxy-β-thujone; 7OHRT, 7-hydroxy-Rthujone; 7OHβT, 7-hydroxy-β-thujone; 7,8DHRT, 7,8-dehydro-Rthujone; 7,8DHβT, 7,8-dehydro-β-thujone.
fragrances, perfumes and Vicks VapoRub (6). Sources of RT- and β-thujone (βT) mixtures for these uses are medicinal plants and essential oils such as wormwood and cedarleaf oils. On this basis, the United States National Toxicology Program has recommended thujone for possible genotoxicity, neurotoxicity, reproductive toxicity, chronic toxicity and carcinogenicity testing in rats and mice (6). The toxicity of RT and βT is primarily attributable to their action as noncompetitive blockers of the γ-aminobutyric acid (GABA)-gated chloride channel (7). This explains their convulsant action in mammals and probably their insecticidal activity (7-9). RT is more toxic than βT to mice (10) and more potent at the GABA-gated chloride channel (7). RT also has porphyrogenic properties and is potentially hazardous to patients with acute hepatic porphyrias (11). Metabolic fate studies are an essential step in understanding thujone toxicology. The metabolism of RT is partially understood in mice in vitro and in vivo (7). The major metabolite is 7-hydroxy-R-thujone (7OHRT) in the mouse liver microsome-NADPH system and in the brain
10.1021/tx000242c CCC: $20.00 © 2001 American Chemical Society Published on Web 04/13/2001
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Ho¨ ld et al. was from Pharmacia Biotech (Piscataway, NY). Conjugates were cleaved by β-glucuronidase/arylsulfatase (Helix pomatia EC 3.2.1.31/EC 3.1.6.1) from Roche Diagnostics (Indianapolis, IN). Glucuronidation reactions used digitonin and uridine 5′-diphosphoglucuronic acid (UDPGA) from Sigma (St. Louis, MO). P450 inhibitors and insecticide synergists used were piperonyl butoxide from Endura (Bologna, Italy), diethyl phenyl phosphorothionate (dietholate) from Zeneca (Richmond, CA), and O-propyl O-(2-propynyl) phenylphosphonate from FMC (Middleport, NY). [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) (38 Ci/ mmol) was from NEN Life Science Products (Boston, MA).
Figure 1. Metabolic pathways for R-thujone (RT) and β-thujone (βT). The metabolites are designated by the position of hydroxylation (2ROHRT, 2ROHβT, 4OHRT, 4OHβT, 7OHRT, 7OHβT) and dehydrogenation (7,8DHRT, 7,8DHβT and 4,10DHT).
of intraperitoneally treated mice (7). Some hydroxylation also occurs at the 4-position to give 4-hydroxy-R- and 4-hydroxy-β-thujones (4OHRT and 4OHβT) and the 7,8dehydro derivative (7,8DHRT) is formed (7) (Figure 1). These studies involve only the indicated metabolites of RT in mice and therefore their relevance to other species and to βT is not known. The present investigation examines the metabolic fate of the thujone diastereomers in different species and both in vitro and in vivo. It considers the metabolites of RT and βT in mouse, rat, and human liver microsomes and a human recombinant P450 (P450 3A4), in the urine of orally treated mice and rats (free and after deconjugation) and in adult fruit flies (Drosophila melanogaster). There are three goals: define the site specificity and species differences in P450 oxidation of RT and βT in vitro and in vivo; examine the contribution of site specificity in glucuronidation to the patterns of urinary metabolites; determine the biological activity of the metabolites in relation to metabolic activation or detoxification.
Materials and Methods Caution: The thujone diastereomers are convulsants and GABA-gated chloride channel blockers. The derivatization agents are harmful if swallowed, inhaled, or absorbed through the skin and should be used only under a fume hood while wearing suitable protection. Chemicals. The sources of RT and βT and preparation of their hydroxy and dehydro derivatives of >99% purity as synthetic standards (compounds in Figure 1 without brackets) are given by Ho¨ld et al. (12) and Sirisoma et al. (13). Reagents from Pierce (Rockford, IL) for metabolite derivatization were N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) to prepare trimethylsilyl (TMS) derivatives of alcohols and a 2% solution of methoxyamine hydrochloride in pyridine to obtain methyloxime (MOX) derivatives of ketones. Sephadex LH-20
GC/MS Analysis of r- and β-Thujones and Their Metabolites. Analyses were carried out on a HP5890 gas chromatograph coupled to a HP5971 Mass Selective Detector (HewlettPackard, Palo Alto, CA) in the positive chemical ionization mode. The GC was equipped with a DB-5 fused silica gel capillary column (30 m, 0.25 mm i.d., 0.25 µm film thickness) (J&W Scientific, Folsom, CA). The temperature program was first 80200 °C at 5 °C/min and then 200-300 °C at 20 °C/min, holding this final temperature for 2 min. Helium was used as the carrier gas and methane as the reagent gas. Temperatures were 250 °C for the injection port and 280 °C for the detector. A sample aliquot of 1 µL was injected splitless onto the column. The GC/ MS was operated both in the full scan and in the selected ion monitoring modes, measuring m/z 135 for RT and βT and m/z 151 for the hydroxythujones (2ROHRT, 2ROHβT, 4OHRT, 4OHβT, 7OHRT, and 7OHβT), the dehydrothujones (7,8DHRT, 7,8DHβT, and 4,10DHT) and (S)-(-)-carvone [used as the internal standard (is)]. GC/MS Analysis of TMS and MOX Derivatives. TMS ethers, formed by adding MSTFA (50 µL) to the extract residue and heating for 30 min at 80 °C, were analyzed with a 1.0 µL aliquot by GC/MS. MOX derivatives were produced by adding the MOX solution (100 µL) to the extract residue and heating for 60 min at 80 °C, then cleanup on a column (20 × 5 mm) of Sephadex LH-20 with chloroform/hexane (1:1). The MOX derivatives appeared in the first 1 mL of eluant. The solvent was removed under a gentle stream of nitrogen and the residue was dissolved in ethyl acetate (50 µL) for GC/MS analysis. Metabolism in P450 Systems. Liver microsomes from male albino Swiss-Webster mice (average weight 30 g) and male albino rats (average weight 150 g) (Harlan, Indianapolis, IN) were prepared by centrifugation of a 20% (w/v) homogenate in phosphate buffer (100 mM, pH 7.4) at 10000g for 20 min, recovery of the supernatant fraction, and centrifugation at 100000g for 1 h. The microsomal pellet was washed once with phosphate buffer by centrifugation at 100000g for 1 h, and suspended in 0.25 M sucrose, 20 mM Tris base, and 5.4 mM EDTA (pH 7.4). Other P450 sources examined were pooled human microsomes, human P450 3A4 Supersomes [baculovirusinfected insect cells (BTI-TN-5B1-4) containing human P450 3A4 cDNA] and insect cell control Supersomes obtained from Gentest (Woburn, MA). Protein concentrations determined with the bicinchoninic acid protein assay (Pierce) were as follows (mg/ mL): mouse microsomes 16, rat microsomes 10, pooled human microsomes 20, P450 3A4 5.1, and insect cell control 5.0. The total P450 content (nmol/mg protein) was 1.1 for mouse, 0.59 for rat, 0.49 for human, and 0.20 for P450 3A4. The microsomes were stored frozen at -80 °C. Liver microsomes (1 mg of protein) or P450 3A4 (0.25 mg of protein) or insect control microsomes (1 mg of protein) and NADPH (1 mM final concentration) were incubated with RT or βT [30 µg added in ethanol (0.2 µM final concentration)] in 100 mM phosphate pH 7.4 buffer (1 mL) for 1 h at 37 °C. Each reaction mixture was cooled on ice and the is (0.5 µg) was added. The mixture was saturated with sodium chloride and extracted with ethyl acetate (3 mL) for 30 min by gentle rocking. After centrifugation at 900g for 10 min, the organic extract was removed and almost completely evaporated under a stream of nitrogen at room temperature and reconstituted in ethyl acetate (50 µL) for GC/MS analysis of a 1.0 µL aliquot. Controls involved
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enzyme incubations without substrate to recognize endogenous peaks. Metabolism in Mice and Rats. Mice and rats as above were treated orally with RT or βT (40 mg/kg) in propylene glycol (2.0 µL/g mouse or 1.3 µL/g rat). Urine was collected for 18 h in metabolic cages providing free access to food and water. The urine (500 µL for mouse and 2 mL for rat) was mixed with 200 mM phosphate buffer pH 7.0 (1 mL), followed by saturation with sodium chloride and extraction with ethyl acetate (4 mL). Analysis of the unconjugated metabolites in the organic phase then proceeded as with the microsomal sample. For analysis of the conjugate fraction, the residual ethyl acetate (∼1%) was evaporated under a stream of nitrogen and the pH of the aqueous phase was adjusted to 5.5 with 1 N HCl and 200 mM acetate buffer (pH 5.5). The is (0.5 µg) was added, followed by β-glucuronidase/arylsulfatase (50 µL; 0.23 and 0.7 units, respectively). The mixture was incubated for 16 h at 37 °C, then the pH was adjusted back to 7.0 and the mixture was extracted and analyzed as described for the unconjugated metabolites. Urine from mice and rats treated with carrier solvent only served as controls. Feces was not analyzed because of the greater complexity of the matrix resulting in higher background noise. Glucuronidation. Microsomal glucuronidation of hydroxythujones (synthetic standards) was studied by incubating 2ROHRT, 2ROHβT, 4OHRT, 4OHβT, or 7OHRT [30 µg added in propylene glycol (0.2 µM final concentration)] with mouse microsomal protein (1 mg) and digitonin (1 mg) in 10 mM MgCl2, 100 mM sodium phosphate buffer, pH 7.4 (final volume 1 mL). The reaction was started by addition of UDPGA (2 mM final concentration) and allowed to proceed for 60 min at 37 °C. This procedure was based on a method developed for carvone metabolites (14). The unconjugated metabolites of RT and βT and the glucuronide conjugates were processed and analyzed as above. Control experiments leaving out either microsomes or UDPGA gave no conjugate formation. Studies with Drosophila. Adult insects (Canton-S, wildtype strain) were used to study the metabolism of RT and βT and their toxicity alone and with three P450-inhibiting synergists. The test chamber was a glass tube (12 × 75 mm; 6 mL volume). Ethyl acetate (100 µL) containing the synergist (0 or 1 µL) was added to each tube and evaporated under nitrogen to leave a synergist film. Twenty flies were placed in each tube, which was then closed with a single layer of Parafilm. After 1 h the flies were cooled on ice and transferred to a new test chamber containing a filter paper strip (Whatman No. 1, 8 × 65 mm). RT or βT (45 µg) in propylene glycol (5 µL) was injected with a 10 µL syringe through the Parafilm onto the filter paper following which the tube was resealed with a second piece of Parafilm. Survival was recorded after 2 h at 25 °C as flies that could still move. After 18 h, the tubes were placed on ice and metabolites were recovered by homogenizing the Drosophila in ethyl acetate (2 mL) followed by centrifugation. The ethyl acetate was concentrated under a stream of nitrogen at room temperature and reconstituted in ethyl acetate (50 µL) for GC/ MS analysis. The same type of assay was used for toxicity determinations in which five flies were placed in each tube and the test compound (50 µg) was introduced in propylene glycol (5 µL) with survival recorded after 1.5 h. Inhibition of [3H]EBOB Binding. GABA-depleted mouse brain membranes were prepared as described previously (15). RT, βT, and the synthetic standards (added in Me2SO, final concentration 1%) were incubated with the membranes (200 µg of protein) and [3H]EBOB (0.7 nM) in 1.0 mL of 10 mM sodium phosphate (pH 7.5) buffer containing 200 mM sodium chloride at 37 °C (16). After 70 min the mixtures were filtered through GF/C glass fiber filters using a cell harvester, followed by rinsing twice with 5 mL of ice-cold 0.9% sodium chloride. Nonspecific binding was determined in the presence of 5 µM R-endosulfan. Specific binding was defined as the difference between total and nonspecific binding.
Figure 2. Representative GC/MS extracted ion chromatograms (m/z 135 and 151) of R- and β-thujone metabolites in mouse microsome-NADPH system and urine of mice and rats after deconjugation. Compound designations and tR values (min) are as follows: 1, RT 6.3; 2, 2ROHRT 8.1; 3, 4OHRT 8.0; 4, 4OHβT 8.2; 5, 7OHRT 9.9; 6, 7,8DHRT 6.6; 7, 4,10DHT 7.3; 8, βT 6.4; 9, 2ROHβT 9.8; 10, 7OHβT 10.3; 11, 7,8DHβT 7.0. Additional designations are u, for unidentified hydroxythujones; e, for endogenous and is for internal standard.
Results Recognition and Analysis of Metabolites. GC/MS is a convenient technique to recognize and analyze the metabolites of RT and βT. Mouse microsome-NADPH systems and deconjugated urine of mice and rats give representative GC profiles of RT and βT metabolites shown in Figure 2. The metabolites are quantitated as relative total peak area from extracted ion chromatograms. The numbers of metabolites both identified and unidentified evident by GC/MS, each at higher tR than the parent compound, are six from RT and five from βT in the microsome-NADPH system, five from RT and three from βT in mouse urine, and four from RT and three from βT in rat urine. No metabolites were detected in any case without microsomes or in the absence of NADPH (data not shown). Identification of Metabolites. GC/MS chromatograms of RT and βT and their metabolites (Figure 3) revealed two dehydro compounds (dehydrothujones) eluting shortly after the parent compound and several monohydroxy metabolites (hydroxythujones) of longer tR. Although not detailed here, a very small amount (∼2%) of an aldehyde metabolite (m/z 167) was detected only in rat urine after RT administration. The method of analysis used would not detect any acidic metabolites. Isomer designations for resolved metabolites were then made by comparing the tR values and fragmentation patterns with synthetic standards. [Data for synthetic standards are given by Sirisoma et al. (13) and for the metabolites in the Supporting Information]. Metabolite resolution was sometimes enhanced by forming the TMS and MOX derivatives (Figure 3). All derivatizations went
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Figure 3. Representative GC/MS selected ion chromatograms for R-thujone and metabolites in mouse liver microsome-NADPH system analyzed directly and after TMS and MOX derivatization. Upper figure: underivatized metabolite mixture for ions at m/z 135 and 151 with compound designations and tR values as in Figure 2. Middle figure: TMS derivatives of metabolite mixture for ions at m/z 135 and 151. Designations of TMS derivatives and tR values (min) are as follows: 2, 2ROHRT-TMS 11.3; 3, 4OHRT-TMS 12.1; 4, 4OHβT-TMS 11.0; 5, 7OHRT-TMS 12.9. Lower figure: MOX derivatives of metabolite mixture for ions at m/z 180 and 182. The most abundant MOX derivative of the two diastereomers is labeled. Designations of MOX derivatives and tR values (min) are as follows: 1, RT-MOX 8.3; 2, 2ROHRT-MOX 10.9; 3, 4OHRT-MOX 10.8; 4, 4OHβT-MOX 11.0; 5, 7OHRT-MOX 12.0; 6, 7,8DHRT-MOX 8.8.
to completion, except 7OHRT-TMS and 7OHβT-TMS where substantial amounts of nonderivatized compound remained. MOX derivatization resulted in the formation of geometrical isomers (syn and anti) with one more prominent than the other. Identification of metabolites involved comparison with synthesized standards relative to tR values and fragmentation patterns for the parent compound and its TMS and MOX derivatives. Site Specificity and Species Differences in Cytochrome P450 Oxidation in Vitro and in Vivo (Table 1). (1) 2-Hydroxythujones. 2ROHRT and 2ROHβT are prominent metabolites of RT and βT in the mouse microsome-NADPH system with 9 and 15%, respectively, of the total metabolite peak area but are not detected (
