Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases.
OBJECTIVE
In liver diseases, reactive oxygen species (ROS) are involved in cell death and liver injury, but the mechanisms are not completely elucidated. To elucidate the mechanisms of hepatocyte cell death induced by the ROS superoxide anions and hydrogen peroxide, primary cultures of hepatocytes were exposed to the superoxide anion donor menadione (10-50 micromol/L) or H2O2 (1-5 mmol/L). Hepatocytes were also treated with caspases and MAPKs inhibitors, superoxide dismutase (PEG-SOD) and SNAP, a nitric oxide donor. Apoptosis was determined by measuring caspase-9, -6, -3 activation and cleaved PARP, and necrotic cell death by Sytox Green staining.


RESULTS
(1) Menadione (50 micromol/L) induces JNK phosphorylation, caspase-9, -6, -3 activation, PARP cleavage and apoptosis. Superoxide anions-induced apoptosis is dependent on JNK activity. Menadione (50 micromol/L) induces the phosphorylation of ERK1/2 and this attenuates cell death. (2) H2O2 increases necrotic cell death at high concentration or when H2O2 detoxification is impaired. H2O2 does not activate MAPKs signalling. (3) PEG-SOD prevents ERK1/2-, JNK- phosphorylation, caspase activation and apoptosis induced by menadione. Glutathione depletion increases menadione-induced apoptosis. (4) SNAP abolishes menadione-induced apoptosis but increases necrotic cell death.


CONCLUSIONS
In normal hepatocytes, superoxide anions-induced caspase activation and apoptosis is dependent on JNK activity and totally abolished by superoxide scavengers.
Conde de la Rosa
Laura
L
Center for Liver, Digestive and Metabolic Diseases, University Medical Center Groningen, Groningen, P.O. 30.001, 9700 RB, The Netherlands.
Schoemaker
Marieke H
MH
Vrenken
Titia E
TE
Buist-Homan
Manon
M
Havinga
Rick
R
Jansen
Peter L M
PL
Moshage
Han
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D009336:Necrosis; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013047:Specific Pathogen-Free Organisms; D013481:Superoxides / Q000378:metabolism*; D024483:Vitamin K 3 / Q000494:pharmacology; D014815:Vitamins / Q000494:pharmacology
D016166:Free Radical Scavengers; D016328:NF-kappa B; D016877:Oxidants; D017382:Reactive Oxygen Species; D014815:Vitamins; D013481:Superoxides; D024483:Vitamin K 3; D006861:Hydrogen Peroxide; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020169:Caspases; D005978:Glutathione