The Covalent Binding of [¹⁴C]Acetaminophen to Mouse Hepatic Microsomal Proteins:  The Specific Binding to Calreticulin and the Two Forms of the Thiol:Protein Disulfide Oxidoreductases

Abstract

Numerous in vitro studies have indicated that acetaminophen is activated by mouse hepatic microsomal cytochrome P450 to form N-acetylbenzoquinone imine. This in turn covalently binds through a Michael addition to protein sulfhydryl and amino groups. Although acetaminophen adducts of several cytosolic proteins have been purified after its administration in vivo, no adducts of specific microsomal proteins have been reported. We find that, after the in vitro incubation of mouse hepatic microsomes with [ring-14C] acetaminophen in the presence of an NADPH generating system, 95% of the bound radioactivity was associated with adducts to three intraluminal microsomal proteins: calreticulin and the two forms of thiol:protein disulfide oxidoreductase, Q2 and Q5. The acetaminophen bound to 0.35, 1.32, and 0.25 mol/mol of the three proteins, respectively. Sequencing of the 14C-labeled tryptic peptides indicated that the acetaminophen bound to lysine 103 of Q2, lysines 202, 209 or 210 and 354 of Q5 and lysines 233 or 239 of calreticulin. No adducts of cysteine residues were observed. Our data might suggest that acetaminophen hepatotoxicity results from the formation of the reactive metabolite within the endoplasmic reticulum. This then binds to these essential proteins and blocks the posttranslational modification of secretory and membrane proteins. This inhibition could then lead to cellular injury and death.

Metabolism of [14C]GSK977779 in Rats and Its Implication with the Observed Covalent Binding

“…The observation of high plasma nonextractable radioactivity led to further investigation of the reversibility of the observed binding. SEC and SDS-PAGE have been used in the past to investigate the covalent versus noncovalent binding to plasma proteins (Eling et al, 1977;Zhou et al, 1996;Walsh et al, 2002). SEC and SDS-PAGE of selected plasma samples from rats dosed with […”

Liquid Chromatography/Tandem Mass Spectrometry Detection of Covalent Binding of Acetaminophen to Human Serum Albumin

“…Previous methods that have been used to investigate covalent binding of APAP to proteins in vivo include several immunoassays (Roberts et al, 1987;Bartolone et al, 1988;Matthews et al, 1996;James et al, 2001) and the use of radiolabeled APAP (Axworthy et al, 1988;Zhou et al, 1996;Qiu et al, 1998). Still, these methods often lack the necessary sensitivity when concerning human serum samples (Zhou, 2003) quantify covalent protein binding of APAP.…”

“…At therapeutic doses, APAP is primarily metabolized by phase II enzymes to stable glucuronic acid and sulfate conjugates. A small proportion of the drug is bioactivated by cytochromes P450 to a reactive N-acetyl-p-benzoquinoneimine (NAPQI) intermediate that under normal conditions is detoxified by conjugation to glutathione (GSH) (Gibson et al, 1996;Zhou et al, 1996;Qiu et al, 1998). When taken in overdoses, the high levels of NAPQI produced will deplete the GSH stores, resulting in strongly increased covalent binding to liver proteins, oxidative stress, and ultimately to severe hepatotoxicity (Bessems and Vermeulen, 2001).…”

Role of endoplasmic reticulum stress in drug‐induced toxicity

“…It will be interesting to determine whether diclofenac‐derived p ‐benzoquinone imines can bind covalently to the microsomal PDI and calreticulin, as does APAP‐derived NAPQI (Zhou et al. 1996). Finally, it is noteworthy that indomethacin was also found to induce ER stress in hepatic cells (Franceschelli et al.…”