α-Thujone: γ-Aminobutyric acid type A receptor modulation - Discussion
This study establishes that α-thujone modulates the GABAA receptor based on four observations. Comparison with picrotoxinin, the classical GABAA receptor antagonist, revealed similar poisoning signs and in both cases alleviation of the toxicity by diazepam, phenobarbital, and ethanol (28, 29). Drosophila with a single point mutation in the Rdl GABA receptor subunit of Ala302 to Ser conferring resistance to dieldrin (22, 23) is also resistant to α-thujone, albeit to a lesser degree. α-Thujone is a competitive inhibitor of [3H]EBOB binding, i.e., of the noncompetitive blocker site of the GABA-gated chloride channel (25). Most importantly, electrophysiological studies establish that in dorsal root ganglion neurons α-thujone is a reversible modulator of the GABAA receptor.
Absinthe and wormwood oil contain not only α-thujone as their purported active ingredient but also many other candidate toxicants, including α-thujone and ethanol in the case of absinthe. α-Thujone is less toxic than α-thujone to mice (10) and Drosophila and in addition is 2.3-fold less potent in the [3H]E-BOB assay (this investigation). Ethanol also enhances neuronal GABAAreceptor function (30) and therefore might suppress the blocking action of α-thujone in absinthe. However, ethanol does not alter the inhibitory action of α-thujone on [3H]EBOB binding. The α-and α-thujone content of the absinthe sample examined here (0.4 and 5 ppm or 2.6 and 33 μM, respectively) may be a contributing factor in the somewhat greater potency of absinthe (based on ethanol content) than of ethanol per se in the [3H]EBOB assay. However, the 10 ppm (66 μM) upper limit of the European Commission (6) and particularly the 260 ppm
(1710 μM) thujone content of old absinthe (6) would give a detectable to major inhibitory effect beyond that of the ethanol content. Current low levels of α-and α-thujone in absinthe are of much less toxicological concern than the ethanol content (6).
α-Thujone as other monoterpenes is easily metabolized. The single report on metabolism identifies thujol and neothujol probably as conjugates in the urine of thujone-treated rabbits (21). We find enzymatic reduction (possibly by a cytosolic ketone reductase) (31) of α-thujone to thujol and neothujol in low yield by rabbit but not mouse liver cytosol with NADPH. The mouse liver microsomal P450 system rapidly converts α-thujone to 7-hydroxy-α-thujone (major), the diastereomers of 4-hy-droxythujone (minor), and other hydroxythujones (minor). Interestingly, the major sites of P450 hydroxylation at the 4-and 7-positions are those involving intermediate tertiary radicals that are more stable than secondary and primary radicals. Dehydro-α-thujone also is observed and may arise from dehydration of the 7-hydroxy compound as a biological reaction because this possible conversion is not an artifact during the extraction and analysis procedure. The various hydroxythujones probably are not the terminal metabolites because they are expected to undergo conjugation and excretion. However, the presence of hydroxythujones in the brain suggests their potential importance in the neurotoxicity.
Metabolic detoxification is a dominant feature of a-thujone neurotoxicity in mice. There are two principal candidate toxicants, α-thujone and its 7-hydroxy metabolite. The 7-hydroxy compound is present in brain at much higher levels than the parent α-thujone suggesting possible conversion in situ, but this oxidation was not observed on incubation of a-thujone with brain microsomes and NADPH. a-Thujone compared with 7-hydroxy-α-thujone is 56 fold more potent in the [3H]EBOB binding assay and much more toxic to mice and houseflies. It appears that all of the metabolites studied here are detoxification products, i.e., less toxic than α-thujone. However, the level in brain of 7-hydroxy-α-thujone is several-fold greater than that of α-thujone (e.g., 29 and 11 ppm, respectively, at the time of severe poisoning signs), suggesting that either one or both may contribute to the toxic manifestations.
This study establishes that α-thujone acts at the noncompetitive blocker site of the GABAA receptor and is rapidly detoxified, thereby providing a reasonable explanation for some of the actions of absinthe other than those caused by ethanol, and allowing more meaningful evaluation of risks involved in the continued use of herbal medicines containing α-thujone.
Helpful comments were provided by Wilfred Arnold (University of Kansas Medical Center, Kansas City) and Jeffrey Bloomquist (Virginia Polytechnic Institute, Blacksburg). We thank our former coworker Neil Jacobson for initiating our interest in α-thujone action and our laboratory colleagues Gary Quistad and Susan Sparks for help and advice. This project was supported by National Institute of Environmental Health Sciences Grant R01 ES08419 to J.E.C. and National Institute of Neurological Disorders and Stroke Grant R01 NS14143 to T.N.