Identification and structure characterization of S-containing metabolites of 3-tert-butyl-4-hydroxyanisole in rat urine and liver microsomes.
After ip administration of 3-tert-butyl-4-hydroxyanisole (3-BHA) to rats, two previously undocumented metabolites 2-tert-butyl-5-methylthiohydroquinone (TBHQ-5-SMe) and 2-tert-butyl-6-methylthiohydroquinone (TBHQ-6-SMe) were identified in the urine by comparison with the authentic samples by GC/MS. In addition to these metabolites, 3-tert-butyl-4,5-dihydroxyanisole was also detected in the urine hydrolyzed by beta-glucuronidase/sulfatase. Administration of tert-butylhydroquinone (TBHQ), an O-demethylated metabolite of 3-BHA, also resulted in the formation of the S-containing metabolites, TBHQ-5-SMe and TBHQ-6-SMe. After incubation of TBHQ with rat liver microsomes in the presence of glutathione (GSH), two metabolites were isolated and purified by HPLC. The metabolites were identified as 2-tert-butyl-5-(glutathion-S-yl)hydroquinone and 2-tert-butyl-6-(glutathion-S-yl)hydroquinone by 1H- and 13C-NMR spectrometry and by fast atom bombardment-mass spectrometry. The formation of TBHQ-GSH conjugates required NADPH, molecular oxygen, and GSH. Cytochrome P-450 inhibitors such as SKF 525-A and metyrapone markedly inhibited the formation of TBHQ-GSH conjugates in vitro. These results suggest that TBHQ is converted by cytochrome P-450-mediated monooxygenases to a reactive metabolite, 2-tert-butyl-p-benzoquinone (TBQ), which then conjugates with GSH to form TBHQ-GSH conjugates. GSH S-transferase activities do not seem to play a role in GSH conjugation reaction to TBQ because cytosol fraction from rat liver homogenates did not enhance the microsome-mediated production of TBHQ-GSH conjugates.
Department of Chemistry, School of Pharmacy, Hokuriku University, Kanazawa, Japan.
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Tajima
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Hashizaki
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Yamamoto
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Mizutani
D000818:Animals; D002083:Butylated Hydroxyanisole / Q000378:metabolism*; D065607:Cytochrome P-450 Enzyme Inhibitors; D008401:Gas Chromatography-Mass Spectrometry; D005978:Glutathione / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D065607:Cytochrome P-450 Enzyme Inhibitors; D006873:Hydroquinones; D002083:Butylated Hydroxyanisole; C051362:3-tert-butyl-4-hydroxyanisole; C018855:2-tert-butylhydroquinone; D005978:Glutathione