title: Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, BP 10448, F-63000 Clermont-Ferrand, France; INRA, UMR 1019, UNH, CRNH Auvergne, F-63000 Clermont-Ferrand, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Mast
authors.affiliation: Plateforme d'Exploration du Métabolisme, INRA, Centre Clermont-Ferrand-Theix, UMR 1019 , Nutrition Humaine, Saint-Genès-Champanelle, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Lyan
authors.affiliation: Plateforme d'Exploration du Métabolisme, INRA, Centre Clermont-Ferrand-Theix, UMR 1019 , Nutrition Humaine, Saint-Genès-Champanelle, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Joly
authors.affiliation: Plateforme d'Exploration du Métabolisme, INRA, Centre Clermont-Ferrand-Theix, UMR 1019 , Nutrition Humaine, Saint-Genès-Champanelle, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Centeno
authors.affiliation: Plateforme d'Exploration du Métabolisme, INRA, Centre Clermont-Ferrand-Theix, UMR 1019 , Nutrition Humaine, Saint-Genès-Champanelle, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Giacomoni
authors.affiliation: Plateforme d'Exploration du Métabolisme, INRA, Centre Clermont-Ferrand-Theix, UMR 1019 , Nutrition Humaine, Saint-Genès-Champanelle, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Martin
authors.affiliation: Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, BP 10448, F-63000 Clermont-Ferrand, France; INRA, UMR 1019, UNH, CRNH Auvergne, F-63000 Clermont-Ferrand, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Mosoni
authors.affiliation: Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, BP 10448, F-63000 Clermont-Ferrand, France; INRA, UMR 1019, UNH, CRNH Auvergne, F-63000 Clermont-Ferrand, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Dardevet
authors.affiliation: Plateforme d'Exploration du Métabolisme, INRA, Centre Clermont-Ferrand-Theix, UMR 1019 , Nutrition Humaine, Saint-Genès-Champanelle, France.
authors.forename: Estelle
authors.initials: E
authors.lastname: Pujos-Guillot
authors.affiliation: Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, BP 10448, F-63000 Clermont-Ferrand, France; INRA, UMR 1019, UNH, CRNH Auvergne, F-63000 Clermont-Ferrand, France. Electronic address: Isabelle.Papet@clermont.inra.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Papet
mesh_terms: D000082:Acetaminophen / Q000008:administration & dosage*; D018712:Analgesics, Non-Narcotic / Q000008:administration & dosage; D000818:Animals; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling / Q000379:methods; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D055442:Metabolome / Q000187:drug effects / Q000502:physiology*; D020543:Proteome / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D018712:Analgesics, Non-Narcotic; D020543:Proteome; D000082:Acetaminophen
keywords: Chronic treatment; Mass spectrometry; Metabolomics; Oxidized residue; Paracetamol; Post-translational modification