Scatchard analyses were performed with no inhibitor and with 5 and 25 μM α-thujone by using EBOB at 0.08–26 nM. For inhibitor potency assays, the membranes (200 μg protein) were incubated with the test compound (added in DMSO, final concentration 1%) and EBOB (0.7 nM) in 1.0 ml of 10 mM sodium phosphate, pH 7.5 buffer containing 200 mM sodium chloride at 37☌ for 70 min ( 25). Mouse brain membranes were prepared and depleted of GABA as described ( 24). Effect on EBOB Binding in Mouse Brain Membranes. The experiment was repeated four times to prepare dosage mortality curves for calculation of resistance ratios (LC 50 Rdl/LC 50 S). Mortality was recorded after 8 h at 25☌ as flies that could not move. 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.
Five adult flies were placed in the tube, which then was closed with a single layer of parafilm. The test chamber was a glass tube (12 × 75 mm) containing a filter paper strip (Whatman no. The median lethal concentration (LC 50) was determined for α-thujone and dieldrin with two strains of Drosophila: a dieldrin-resistant Rdl MD-RR strain ( 22, 23) (obtained from the Bloomington Drosophila Stock Center at Indiana University, Bloomington) and the Canton-S, wild-type sensitive ( S) strain.
Public mistrust of synthetic pharmaceuticals and pesticides has led to the increasing popularity of herbal medicines and botanical insecticides even though they have not been subjected to the same rigorous tests of safety and evaluation of toxicological mechanisms ( 13– 15).įruit 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. Artemesia absinthium and wormwood oil have insecticidal properties ( 11), and α-thujone was one of the two most toxic monoterpenoids tested against western corn rootworm larvae ( 12). Extracts of wormwood were used to control gastrointestinal worms with records back to ancient Egyptian times ( 4). This monoterpenoid occurs in many plants, including Artemesia species, sage, and the Thuja tree ( 4). α-Thujone also is reported to have antinociceptive activity in mice ( 10). The content of β-thujone often exceeds that of α-thujone depending on the plant source, but the β-diastereomer (Fig. 1) generally is considered to be the principal active ingredient of wormwood oil and toxic principle in absinthe ( 2). Thus, α-thujone in absinthe and herbal medicines is a rapid-acting and readily detoxified modulator of the GABA-gated chloride channel. The other metabolites assayed are also detoxification products. The major 7-hydroxy metabolite attains much higher brain levels than α-thujone but is less toxic to mice and Drosophila and less potent in the binding assay.
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α-Thujone is quickly metabolized in vitro by mouse liver microsomes with NADPH (cytochrome P450) forming 7-hydroxy-α-thujone as the major product plus five minor ones (4-hydroxy-α-thujone, 4-hydroxy-β-thujone, two other hydroxythujones, and 7,8-dehydro-α-thujone), several of which also are detected in the brain of mice treated i.p. Most definitively, GABA-induced peak currents in rat dorsal root ganglion neurons are suppressed by α-thujone with complete reversal after washout. Third, α-thujone is a competitive inhibitor of ethynylbicycloorthobenzoate binding to mouse brain membranes. Second, a strain of Drosophila specifically resistant to chloride channel blockers is also tolerant to α-thujone.
First, the poisoning signs (and their alleviation by diazepam and phenobarbital) in mice are similar to those of the classical antagonist picrotoxinin. Four observations establish that α-thujone is a modulator of the γ-aminobutyric acid (GABA) type A receptor. This study elucidates the mechanism of α-thujone neurotoxicity and identifies its major metabolites and their role in the poisoning process. It is also the active ingredient of wormwood oil and some other herbal medicines and is reported to have antinociceptive, insecticidal, and anthelmintic activity. Α-Thujone is the toxic agent in absinthe, a liqueur popular in the 19th and early 20th centuries that has adverse health effects.
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