Acetaminophen hepatotoxicity and mechanisms of its protection by N-acetylcysteine: a study of Hep3B cells.
Acetaminophen (AAP) hepatotoxicity, resulting in centrilobular necrosis, is frequently encountered following suicidal attempts, especially by adolescents, but also after its excessive use in infants. The subcellular and molecular sequences leading to hepatocellular cell death are not yet clear. We therefore investigated AAP hepatotoxicity by using cultured hepatoma-derived cells (Hep3B) exposed to AAP and N-acetylcysteine (NAC), used as a protective agent. Specifically, we studied the role of apoptosis and oxidative damage as putative mechanisms of AAP-associated cytotoxicity. Hep3B cells were exposed to AAP (5-25 mM) and NAC (5 mM) for different time periods. Cell viability was assessed by the Alamar Blue Reduction Test and LDH. Oxidative damage was evaluated by measuring reactive oxygen species (ROS) and glutathione. AAP-induced apoptosis was investigated by flow cytometry and transmission electron microscopy. We found that: 1. In Hep3B cells, AAP causes a time- and concentration-dependent cytotoxic effect, leading to oxidative stress, mitochondrial dysfunction, alterations of membrane permeability and apoptosis; 2. In the course of AAP cytotoxicity, the generation of ROS appears as an early event which precedes decrease of viability, LDH leakage, glutathione depletion and apoptosis; 3. NAC protects Hep3B cells from AAP-induced oxidative injury, but does not prevent apoptosis.
Manov
Irena
I
Pediatric Research and Electron Microscopy Unit, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Hirsh
Mark
M
Iancu
Theodore C
TC
D000082:Acetaminophen / Q000037:antagonists & inhibitors / Q000633:toxicity*; D000111:Acetylcysteine / Q000494:pharmacology*; D018712:Analgesics, Non-Narcotic / Q000037:antagonists & inhibitors / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000187:drug effects / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D005434:Flow Cytometry; D016166:Free Radical Scavengers / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
D018712:Analgesics, Non-Narcotic; D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D000082:Acetaminophen; D005978:Glutathione; D000111:Acetylcysteine