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Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches

Mast, Carole, Lyan, Bernard, Joly, Charlotte, Centeno, Delphine, Giacomoni, Franck, Martin, Jean-François, Mosoni, Laurent, Dardevet, Dominique, Pujos-Guillot, Estelle, Papet, Isabelle
Journal of proteomics 2015 v.120 pp. 194-203
acetaminophen, cysteine, detection limit, diet, glutathione, hepatotoxicity, hydrolysates, hydrolysis, liver, liver protein, mass spectrometry, metabolomics, oxidation, proline, protein hydrolysates, protein synthesis, proteolysis, rats, thiols, tryptophan, tyrosine
Liver protein can be altered under paracetamol (APAP) treatment. APAP-protein adducts and other protein modifications (oxidation/nitration, expression) play a role in hepatotoxicity induced by acute overdoses, but it is unknown whether liver protein modifications occur during long-term treatment with non-toxic doses of APAP. We quantified APAP-protein adducts and assessed other protein modifications in the liver from rats under chronic (17 days) treatment with two APAP doses (0.5% or 1% of APAP in the diet w/w). A targeted metabolomic method was validated and used to quantify APAP-protein adducts as APAP-cysteine adducts following proteolytic hydrolysis. The limit of detection was found to be 7ng APAP–cysteine/mL hydrolysate i.e. an APAP–Cys to tyrosine ratio of 0.016‰. Other protein modifications were assessed on the same protein hydrolysate by untargeted metabolomics including a new strategy to process the data and identify discriminant molecules. These two complementary mass spectrometry (MS)-based metabolic approaches enabled the assessment of a wide range of protein modifications induced by chronic treatment with APAP.APAP-protein adducts were detected even in the absence of glutathione depletion and hepatotoxicity, i.e. in the 0.5% APAP group, and increased by 218% in the 1% APAP group compared to the 0.5% APAP group. At the same time, the untargeted metabolomic method revealed a decrease in the binding of cysteine, cysteinyl–glycine and GSH to thiol groups of protein cysteine residues, an increase in the oxidation of tryptophan and proline residues and a modification in protein expression. This wide range of modifications in liver proteins occurred in rats under chronic treatment with APAP that did not induce hepatotoxicity.