title: Mechanisms of acetaminophen-induced cell death in primary human hepatocytes.
abstract: Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6h after APAP and a partial protection when given at 15 h.
  
  
  CONCLUSIONS
  These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Yuchao
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Mitchell R
authors.initials: MR
authors.lastname: McGill
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Dorko
authors.affiliation: Department of Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Kumer
authors.affiliation: Department of Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Schmitt
authors.affiliation: Department of Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Jameson
authors.initials: J
authors.lastname: Forster
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA. Electronic address: hjaeschke@kumc.edu.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Jaeschke
mesh_terms: D000082:Acetaminophen / Q000037:antagonists & inhibitors / Q000633:toxicity*; D000111:Acetylcysteine / Q000494:pharmacology; D000328:Adult; D000368:Aged; D018712:Analgesics, Non-Narcotic / Q000633:toxicity*; D000931:Antidotes / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008930:Mitochondria, Liver / Q000187:drug effects; D028361:Mitochondrial Diseases / Q000139:chemically induced / Q000378:metabolism; D009336:Necrosis / Q000473:pathology; D061251:Primary Cell Culture; D011506:Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D055815:Young Adult
chemical_list: D018712:Analgesics, Non-Narcotic; D000931:Antidotes; D011506:Proteins; D000082:Acetaminophen; D048031:JNK Mitogen-Activated Protein Kinases; D005978:Glutathione; D000111:Acetylcysteine
keywords: Acetaminophen protein adducts; Drug-induced liver injury; Mitochondrial dysfunction; Oncotic necrosis; c-Jun-N-terminal kinase