title: Purified acetaminophen-glutathione conjugate is able to induce oxidative stress in rat liver mitochondria.
abstract: Acetaminophen overdose is the most often cause of acute liver injury. The toxic mechanism is linked to formation of an active metabolite that reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). This compound has been recognized to be non-toxic generally. Our preliminary results showed, however, that APAP-SG could possess a toxic effect too. Therefore, the aim of our study was to prepare, purify and to test possible toxicity of APAP-SG. We prepared APAP-SG using organic synthesis. The conjugate was purified by preparative HPLC and its structure was confirmed using mass spectrometry. Final purity of APAP-SG was >98 %. We estimated a toxic effect of APAP-SG in isolated rat liver mitochondria using a fluorescent ROS probe. We assessed ROS production in presence of complex I or complex II substrates. The increase of ROS-dependent fluorescence in presence of glutamate/malate was 104 ± 13 % and 130 ± 10 % in 1 mM and 5 mM APAP-SG, respectively, in comparison with controls. ROS production related to presence of complex II substrate was enhanced 4-times in APAP-SG (5 mM) treated mitochondria (compared to controls). We conclude, we proved our hypothesis that APAP-SG conjugate is able to induce a mitochondrial impairment leading to enhanced ROS production.
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic. Tomas.Rousar@upce.cz
authors.forename: T
authors.initials: T
authors.lastname: Roušar
authors.forename: E
authors.initials: E
authors.lastname: Nýdlová
authors.forename: P
authors.initials: P
authors.lastname: Česla
authors.forename: P
authors.initials: P
authors.lastname: Staňková
authors.forename: O
authors.initials: O
authors.lastname: Kučera
authors.forename: P
authors.initials: P
authors.lastname: Pařík
authors.forename: Z
authors.initials: Z
authors.lastname: Červinková
mesh_terms: D000082:Acetaminophen / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000633:toxicity; D000818:Animals; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D008293:Malates; D017382:Reactive Oxygen Species; D000082:Acetaminophen; D018698:Glutamic Acid; C030298:malic acid; C034780:3-(glutathion-S-yl)acetaminophen