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Metabolism of α-Thujone by Liver Enzymes.
Incubation of α-thujone with rabbit (but not mouse) liver cytosol gives thujol and neothujol, identified by GC-MS comparison with authentic standards per se and by forming trimethylsilyl (but not methyl-oxime) derivatives. This enzymatic reduction depends on NADPH but occurs in small yield. Metabolism in mouse liver microsomes is a much more facile reaction and gives no thujol or neothujol but instead different products. α-Thujone is stable on incubation with mouse liver microsomes alone but is almost completely metabolized when NADPH (but not NADP, NADH, or NAD) also is added. Six NADPH-dependent microsomal metabolites are evident by GC-MS, each at higher retention time than the parent α-thujone (Fig. 6). The first-eluting metabolite is identical in GC and MS features to synthetic 7,8-dehydro-α-thujone. The next five metabolites each are converted to trimethylsilyl and methyloxime derivatives, indicating the presence of both an alcohol substituent and a ketone functionality. Synthesis of various hydroxythujones and their comparison with the metabolites (directly, and as trimethylsilyl ethers and methyloximes) identifies the major product as 7-hydroxy-α-thujone and two minor metabolites as the diastereomers of 4-hydroxythujone.
|Fig. 6. Representative GC-MS-selected ion monitoring chromatograms for α-thujone and metabolites extracted from the mouse liver microsome-NADPH (P450) system and the brain of α-thujone-treated mice (50 mg/kg, i.p., 10 min after treatment). The major metabolite is 7-hydroxy-α-thujone. Four minor hydroxythujone metabolites are as follows: 1) 4-hydroxy-α; 3) 4-hydroxy-α;2 and 4) others. Dehydro refers to 7,8-dehydro-α-thujone. Shaded peaks not derived from α-thujone are an endogenous substance (end) and the internal standard (IS). All thujone-derived metabolites fall within the chromatographic region shown.|
Metabolites in the Brain of α-Thujone-Treated Mice. The brain contains α-thujone, dehydro-α-thujone, and four hydroxythujones (7-hydroxy-α major plus 4-hydroxy-α, 4-hydroxy-α, and one other) also observed in the liver P450 system (Fig. 6). Identifications are based on retention times and MS fragmentation patterns both direct and as trimethylsilyl and methyloxime derivatives. The brain levels of α-thujone and 7-hydroxy-α-thujone are dose-and time-dependent after i.p. injection of α-thujone (Fig. 7). Importantly, α-thujone appears at much lower levels and is less persistent than 7-hydroxy-α-thujone. At severely toxic α-thujone doses (40-60 mg/kg) the levels in brain at 30 min are 0.3-1.0 ppm for α-thujone and 1.5-8.4 ppm for 7-hydroxy-α-thujone (Fig. 7A) with much higher levels (11 and 29 ppm for α-thujone and 7-hydroxy-α-thujone, respectively) at 2.5 min (Fig. 7B) when the poisoning signs are most intense. The minor hydroxythujone metabolites are detectable only up to 20 min after the 50 mg/kg α-thujone dose.
Biological Activities of Metabolites. Synthetic standards of the metabolites shown in Fig. 1 except the 4-hydroxy-α-thujone diastereomers were compared with α-thujone for potency as toxicants to mice and Drosophila and inhibitors of [3H]EBOB binding. The discriminating levels used were 50 mg/kg i.p. for mice and 50 αgαtube for the S strain of Drosophila. With mice, α-thujone is lethal, whereas 7-hydroxy-α-thujone, dehydro-α-thujone, and thujolαneothujol are not lethal. With Drosophila, α-thujone gives complete mortality, dehydro-α-thujone gives 70% mortality, and 7-hydroxy-α-thujone and thujolαneothujol give about 30% mortality. In the [3H]EBOB binding assay, 7-hydroxy-α-thujone gives an IC50 value of 730 ± 265 μM versus 13 ± 4 μM for α-thujone (Fig. 3A), whereas the value for dehydro-α-thujone is 149 ± 10 μM (inhibition curve not shown).
|Fig. 7. Brain levels of a-thujone and 7-hydroxy-a-thujone as a function of dose and time in mice treated i.p. with a-thujone. Average of determinations on two mice except for a single determination at 5 min. (A) Dose studies at 30 min after treatment. (B) Time studies at 50 mg/kg.|
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