N-acetylcysteine does not protect HepG2 cells against acetaminophen-induced apoptosis.
Acetaminophen in large doses is well-known as hepatotoxic, and early therapy with N-acetylcysteine is frequently life-saving. However, in later stages of acetaminophen poisoning, treatment with N-acetylcysteine is not always effective. Although some of the pathways of acetaminophen toxicity and the effect of N-acetylcysteine have been elucidated, in depth information on this process is still lacking. Hepatoma-derived HepG2 cultured cells were exposed to acetaminophen (5 and 10 mM), with or without N-acetylcysteine (5 mM), for 24 and 48 hr. For the assessment of oxidative damage, apoptosis and necrosis, we followed redox status, glutathione content, nuclear fragmentation, phosphatidylserine externalization and ultrastructural changes. Variations in Ca2+ level and number of mitochondrial dense granules were also studied. Acetaminophen treatment of HepG2 cells caused oxidative damage and apoptosis. Significant decrease of cellular redox potential and glutathione content were time- and concentration-dependent. The protective effect of N-acetylcysteine was expressed by an increase of intracellular glutathione and of the level of metabolic reduction of the redox indicator Alamar Blue. The apoptogenic effect of acetaminophen was assessed by flow cytometry of annexin V binding, nuclear hypodiploidity, intracellular Ca2+, as well as by ultrastructural examination. Beyond 24 hr of acetaminophen exposure, necrosis was also noticed. We conclude that acetaminophen-induced oxidative damage in HepG2 cultured cells can be prevented by exposure to N-acetylcysteine. However, apoptosis, either early or late, here demonstrated, is not avoided by exposure to N-acetylcysteine. N-Acetylcysteine did not prevent acetaminophen-induced plasma membrane asymmetry, nuclear damage, alterations of Ca2+ homeostasis and ultrastructural changes.
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*; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000032:analysis; D002413:Cations, Divalent; D045744:Cell Line, Tumor / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D005434:Flow Cytometry; D005978:Glutathione / Q000032:analysis; D018197:Hepatoblastoma; D006801:Humans; D008113:Liver Neoplasms; D008854:Microscopy, Electron; D009336:Necrosis; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D010718:Phosphatidylserines / Q000032:analysis
D000975:Antioxidants; D002413:Cations, Divalent; D010718:Phosphatidylserines; D000082:Acetaminophen; D005978:Glutathione; D002118:Calcium; D000111:Acetylcysteine