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Targeting hepatic thioredoxin reductase by the acetaminophen metabolite N‐acetyl‐p‐benzoquinone (844.15)
Author(s) -
Jan YiHua,
Dragomir AnaChristina,
Gardner Carol,
Heck Diane,
Laskin Debra,
Laskin Jeffrey
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.844.15
Subject(s) - thioredoxin reductase , selenocysteine , chemistry , biochemistry , cysteine , glutathione , enzyme , thioredoxin , metabolite
Acetaminophen is biotransformed by cytochrome P450's in hepatocytes to an electrophilic metabolite, N‐acetyl‐p‐benzoquinone imine (NAPQI). This reactive species is known to bind to glutathione and a variety of hepatic proteins, an important downstream event mediating acetaminophen hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a critical cellular antioxidant which contains a selenocysteine in its C‐terminal redox center, an accessible target for electrophilic modification. In the present studies, we characterized the reaction of NAPQI with TrxR. Acetaminophen treatment of mice (300 mg/kg, i.p.) caused a marked time‐dependent inhibition of the activities of both cytosolic TrxR1 and mitochondrial TrxR2. Using purified rat liver TrxR1, we found that inactivation of the enzyme was correlated with APAP metabolism by P450s. This suggested that enzyme inhibition was a result of the formation of reactive metabolites of acetaminophen. To examine the mechanism underlying the inhibitory actions of acetaminophen, the effects of NAPQI on purified TrxR were characterized. We found that NAPQI‐mediated enzyme inhibition required reduced TrxR. A recombinant mutant enzyme, in which selenocysteine was replaced with cysteine was only weakly inhibited by NAPQI indicating that selenocysteine residues in the redox motifs of the enzyme are critical for enzyme inactivation. This was supported by our findings that alkylation of TrxR with biotin‐conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. Moreover, LC‐MS/MS analysis confirmed that NAPQI selectively modified cysteine 59, cysteine 497 and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. Collectively, these data demonstrate that reactive intermediates of acetaminophen can target TrxR demonstrating a novel mechanism by which this analgesic induces oxidative stress and hepatotoxicity. Grant Funding Source : Supported by AR055073, ES004738, CA132624, and ES005022.