Acetaminophen reactive intermediates target hepatic thioredoxin reductase

Area of Science:

• Biochemistry
• Toxicology
• Cellular Biology

Background:

• Acetaminophen (APAP) metabolism generates N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite implicated in liver injury.
• Mammalian thioredoxin reductase (TrxR) is a crucial cellular antioxidant enzyme containing selenocysteine (Sec) in its active site, making it susceptible to electrophilic attack.

Purpose of the Study:

• To investigate whether NAPQI directly targets and inhibits TrxR.
• To elucidate the mechanism by which APAP-induced hepatotoxicity occurs, focusing on TrxR as a potential target.

Main Methods:

• In vivo studies using mice treated with APAP to assess TrxR activity.
• In vitro enzyme assays using purified rat liver TrxR1 and NAPQI.
• Site-directed mutagenesis of TrxR to investigate the role of cysteine and selenocysteine residues.
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify NAPQI modification sites on TrxR.

Main Results:

• APAP treatment in mice led to significant inhibition of both cytosolic TrxR1 and mitochondrial TrxR2.
• NAPQI directly inhibited purified TrxR1, with NADPH-reduced TrxR being more sensitive than the oxidized form.
• NAPQI was confirmed to modify cysteine and selenocysteine residues within the redox centers of TrxR, leading to enzyme inactivation.
• TrxR's ability to mediate menadione redox cycling was less sensitive to NAPQI than disulfide reduction.

Conclusions:

• APAP-reactive metabolites, specifically NAPQI, directly target and inhibit TrxR.
• The inhibition of TrxR by NAPQI is mediated through modification of critical cysteine and selenocysteine residues.
• This interaction represents a novel mechanism contributing to APAP-induced hepatotoxicity and oxidative stress.