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MECHANISMS OF PROTECTION AGAINST ACETAMINOPHEN HEPATOTOXICITY BY GLUTATHIONE AND N-ACETYLCYSTEINE
C Saito2, C Zwingmann1, H Jaeschke2
1Centre de recherche, Centre hospitalier de l’Universite de Montreal, Hopital Saint-Luc, Montreal, QC; 2Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA
The glutathione (GSH) precursor N-acetylcysteine (NAC) is used to treat patients with acetaminophen (APAP) overdose in the clinic for up to 48h. However, the mechanism of this protection remains incompletely understood. To address this issue, fasted C3HeB/FeJ mice were treated with 300 mg/kg AAP and then received intravenously either 0.65 mmol/kg GSH, NAC or a combination of the individual amino acids of GSH (glycine, glutamic acid and cysteine) at 1.5 h after APAP. The animals were sacrificed at 6 h and liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining) and hepatic GSH levels were determined. APAP alone caused severe liver injury with peroxynitrite formation and DNA fragmentation, all of which was attenuated by all treatments. However, GSH (–82%) and the amino acids (–79%) were more effective than NAC (–46%) in preventing liver injury. 13C-labeled compounds were administered concomitantly with GSH and amino acids at different time points to measure synthesis rate of GSH and precursors, substrate flux and pathways through the mitochondrial Krebs cycle and tissue ATP levels by NMR (nuclear magnetic resonance) spectroscopy. It was observed that the reduced liver injury with GSH or individual amino acid treatment correlated with an enhanced GSH recovery rate and an increased substrate supply for the mitochondrial Krebs cycle compared to APAP alone. Consequently, hepatic ATP levels were fully restored after treatment with GSH or amino acids compared to a 35% and 56% reduction, respectively, at 1.5 and 6 h after APAP treatment alone. In contrast, NAC had only a partial effect on these parameters, which correlated with the limited protection. Thus, delayed treatment with GSH and NAC protect against AAP overdose by a dual mechanisms, i.e. by enhancing hepatic GSH levels (scavenging of NAPQI and reactive oxygen) and by supporting mitochondrial energy metabolism.