α-Thujone: γ-Aminobutyric acid type A receptor modulation - </strong><strong>Materials and Methods</strong><strong>

Materials and Methods

Sources were: α-thujone (~99% purity) from Fluka; wormwood oil (3.2% α-and 35% β-thujone) from Lhasa Karnak (Berkeley, CA) and absinthe with 0.4 ppm α-thujone, 5 ppm β-thujone, and 50% (vol/vol) ethanol labeled Herring Absenta (Zaragoza, Spain) with concentrations based on analyses in this laboratory; picrotoxinin, diazepam, and sodium phenobarbital from Sigma; dieldrin and α-endosulfan from Chem Service (West Chester, PA); [3H]ethynylbicycloorthobenzoate ([3H]E-BOB) (38 Ci/mmol) from NEN. Although not detailed here, 7-hydroxy-α-thujone, 4-hydroxy-α-thujone, 4-hydroxy-β-thujone, 7,8-dehydro-α-thujone, and a thujol/neothujol mixture were synthesized as standards for comparison with metabolites.


Toxicity to Mice. Male albino Swiss-Webster mice (22-28 g) were treated i.p. with the test compound by using propylene glycol (2 μl/g body weight) as the carrier vehicle. Prophylactic i.p. treatments also were examined for their effect on α-thujone toxicity (100 mg/kg) individually with ethanol (0.5 or 1.0 g/kg as 20% and 40% solutions in saline, 20 min pretreatment), diazepam (1 mg/kg, 15 min pretreatment), or phenobarbital (15 mg/kg, 15 min pretreatment).

Toxicity to Drosophila. Fruit flies (Drosophila melanogaster) were used in two types of assays: comparing two strains known to be different in sensitivity to insecticidal chloride channel blockers and comparing α-thujone and its metabolites for toxicity to the susceptible strain. The median lethal concentration (LC50) was determined for α-thujone and dieldrin with two strains of Drosophila: a dieldrin-resistant RdlMD-RR strain (22, 23) (obtained from the Bloomington Drosophila Stock Center at Indiana University, Bloomington) and the Canton-S, wild-type sensitive (S) strain. The test chamber was a glass tube (12 x 75 mm) containing a filter paper strip (Whatman no. 1, 8 x 65 mm). Five adult flies were placed in the tube, which then was closed with a single layer of parafilm. A solution of α-thujone or dieldrin in propylene glycol (5 μl) was injected with a 10-μl syringe through the parafilm onto the filter paper after which the tube was covered with a second piece of parafilm. Mortality was recorded after 8 h at 25°C as flies that could not move. The experiment was repeated four times to prepare dosage mortality curves for calculation of resistance ratios (LC50Rdl/LC50S).

Effect on [3H]EBOB Binding in Mouse Brain Membranes. Mouse brain membranes were prepared and depleted of GABA as described (24). For inhibitor potency assays, the membranes (200 μg protein) were incubated with the test compound (added in DMSO, final concentration 1%) 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 for 70 min (25). Scatchard analyses were performed with no inhibitor and with 5 and 25 μM α-thujone by using [3H]EBOB at 0.08-26 nM. The inhibitory potency also was compared for ethanol and absinthe (based on ethanol content) with that for ethanol containing 5 μM α-thujone. The incubated mixtures were filtered through GF/C glass fiber filters, then rinsed twice with 5 ml of ice-cold 0.9% sodium chloride, by using a cell harvester. Specific binding was considered to be the difference between total binding and nonspecific binding determined in the presence of 5 μM α-endosulfan {a potent GABA type A (GABAA) receptor antagonist and specific inhibitor of [3H]EBOB binding}.

Effect on GABA-Induced Whole-Cell Currents. Rat dorsal root ganglion neurons were prepared and cultured as described (26). Currents were induced by 10-msec pulses of 300 μM GABA and recorded by using the whole-cell patch clamp technique. The GABA-induced inward current of this preparation was carried by chloride ions through open chloride channels (27). Each cell was tested for the degree of suppression caused by bath application of α-thujone to determine the concentration for 50% inhibition (IC50).

GC-MS Identification and Analysis of α-Thujone and Metabolites. Standard analytical methods of GC-MS and derivatization of alcohol and ketone functionalities were applied to α-thujone and its metabolites. Analyses used the 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 initial column temperature of 80°C was programmed to 200°C at the rate of 5°C/min, followed by an increase at 20°C/min to 300°C where it was maintained for 2 min. The carrier gas and reagent gas were helium and methane, respectively. Temperatures of the injection port and detector were 250°C and 280°C, respectively. The mass spectrometer was operated in the positive chemical ionization mode. One microliter was injected splitless onto the column. For quantitation, the GC-MS was operated in the selected ion monitoring (SIM) mode, measuring m/z 135 for α-thujone and m/z 151 for the hydroxythujones, dehydrothujone, and (S)-(—)-carvone (internal standard). The concentration of each analyte was determined from least-squares equations generated from peak-area ratios of α-thujone, 7-hydroxy-α-thujone, and the internal standard. Identification of α-thujone and metabolites involved comparison with standards by cochromatography and MS fragmentation patterns as parent compounds and two derivatives. Trimethylsilyl ethers were formed on reaction of alcohols with N-methyl-N-trimethylsilyltrifluoroacetamide and methyloximes on coupling ketones with methoxyamine. These derivatization procedures and MS fragmentation patterns also allowed assignment of some metabolites as hydroxythujones without specifying the position of hydroxylation.

Fig. 2. Drosophila of the dieldrin-resistant (Rdl) strain are also resistant to α-thujone. The susceptible (S) strain is Canton S. Concentration is shown on a logarithmic scale and mortality on a probit scale.
Fig. 3. α-Thujone and 7-hydroxy-α-thujone inhibit [3H]EBOB binding to mouse brain membranes. (A)IC50determination for α-thujone and 7-hydroxy-α-thujone (mean α SEM, n α 4). (B) Scatchard plots as average of duplicate measurements for [3H]EBOB alone (Kd 2.8 nM and Bmax 1,700 fmolμMg protein) and with α-thujone at 5 μM(Kd4.1 and Bmax1,700) and 25 μM(Kd7.2 and Bmax 1,700).

Enzymatic Metabolism. Rabbit or mouse liver cytosol (1 mg protein) or washed mouse liver microsomes (1 mg protein) and NADPH (or other cofactor, 1 mM final concentration) were incubated with α-thujone (30 μg, 0.2 μM final concentration) in 100 mM phosphate, pH 7.4 buffer (1 ml) for1hat 37°C. For analysis the internal standard S-carvone (0.05 μg) was added in ethanol (10 μl), and the mixture was saturated with sodium chloride and extracted with ethyl acetate (3 ml) for 30 min by gentle rocking. The organic extract, recovered by centrifugation at 900 g, was almost completely evaporated (but never to dryness) under a stream of nitrogen at room temperature and reconstituted in ethyl acetate (50 μl) for GC-MS analysis. Recovery values by this procedure for α-thujone and the major metabolite were >60% with no degradation during GC.

Analysis of Brain. Mice were treated i.p. with a-thujone. At appropriate times thereafter the animals were killed and whole brains were removed for analysis. They were rinsed and homogenize in 10 ml of 100 mM phosphate, pH 7.4 buffer. The internal standard was added as above. The mixtures were centrifuged at 1,500 x g for 10 min. The pellet was resuspended in 2ml of phosphate buffer, sonicated for 1 min, and centrifuged, and the supernatant fractions were combined. The samples were extracted with ethyl acetate (6 ml) and analyzed as described in Enzymatic Metabolism.



α-Thujone Is a Convulsant. The i.p. LD50 of α-thujone in mice is about 45 mg/kg, generally with 0% and 100% mortality at 30 and 60 mg/kg, respectively. Mice at the higher dose undergo a tonic convulsion leading to death within 1 min whereas at 30-45 mg/kg they exhibit tail-raising within the first 2 min, followed by flexion of the trunk and clonic activity of the forelimbs, progressing to generalized and protracted tonic/clonic convulsions that ultimately result in death or recovery. Intraperitoneal administration of diazepam or phenobarbital 15 min before α-thujone at 100 mg/kg results in almost all of the mice surviving this otherwise lethal dose. Ethanol i.p. pretreatment at 1 gαkg (but not at 0.5 gαkg) also protects against the lethal effects of α-thujone at 100 mg/kg.

α-Thujone Cross-Resistance in Drosophila Strain Resistant to Dieldrin. Flies of the Rdl strain (>55-fold resistant to dieldrin; LC50 >275 μg/tube for Rdl versus 5 μg/tube for S) are 5-fold resistant to α-thujone (LC50 65 μgα/tube for Rdl versus 12 μg/tube for S) (Fig. 2). This finding establishes moderately high insecticidal activity for α-thujone and cross-resistance in the dieldrin-resistant strain.

α-Thujone Inhibition of [3H]EBOB Binding. The IC50of α-thujone for [3H]EBOB binding in mouse brain membranes is 13 ± 4 μM (Fig. 3A). The binding of α-thujone is competitive with that of [3H]EBOB based on Scatchard analysis (Fig. 3B). For comparison, other IC50 values are 29 ± 8 μM for α-thujone, 37 ± 8 μM for wormwood oil (calculated as molecular weight of thujone), and 0.6 ± 0.1 μM for picrotoxinin (inhibition curves not shown).


α-Thujone Modulation of the GABAA Receptor-Chloride Channel. The currents induced by 300 μM GABA are suppressed with 30 μM bath-applied α-thujone and there is full reversal on washing with α-thujone-free solution (Fig. 4 A and B). The IC50 for α-thujone is 21 μM in suppressing the GABA-induced currents (Fig. 4C).


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