tert-Butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals.
tert-Butyl hydroperoxide (TBHP) mobilizes arachidonic acid (AA) from membrane phospholipids in rat hepatocytes under cytotoxic conditions, thus leading to an increase in intracellular AA, which precedes cell death. In the present work, the involvement of lipid peroxidation, thiol status, and reactive oxygen species (ROS) in the intracellular AA accumulation induced by 0.5 mM TBHP was studied in rat hepatocytes. Cells treated with TBHP maintained viability and energy status at 10 min. However, TBHP depleted GSH, as well as inducing lipid peroxidation and ROS formation, detected by dichlorofluorescein (DCF) fluorescence. TBHP also significantly increased (32.5%) the intracellular [14C]-AA from [14C]-AA-labelled hepatocytes. The phospholipase A(2) (PLA(2)) inhibitor, mepacrine, completely inhibited the [14C]-AA response. The addition of antioxidants to the cell suspensions affected the TBHP-induced lipid response differently. The [14C]-AA accumulation correlated directly with ROS and negatively with endogenous GSH. No correlation between [14C]-AA and lipid peroxidation was found. Promethazine prevented lipid peroxidation and did not affect the [14C]-AA increase. We conclude that TBHP stimulates the release of [14C]-AA from membrane phospholipids through a PLA(2)-mediated mechanism. Endogenous GSH and ROS play a major role in this effect, while lipid peroxidation-related events are unlikely to be involved. Results suggest that specific ROS generated in iron-dependent reactions, different from lipid peroxyl radicals, are involved in PLA(2) activation, this process being important in TBHP-induced hepatocyte injury.
Martín
C
C
Department of Physiology, University of the Basque Country, E-48080, Bilbao, Spain.
Martínez
R
R
Navarro
R
R
Ruiz-Sanz
J I
JI
Lacort
M
M
Ruiz-Larrea
M B
MB
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D005609:Free Radicals / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D015227:Lipid Peroxidation / Q000187:drug effects; D008055:Lipids / Q000502:physiology; D008297:Male; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D013438:Sulfhydryl Compounds / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
D002250:Carbon Radioisotopes; D005609:Free Radicals; D008055:Lipids; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D016718:Arachidonic Acid; D020122:tert-Butylhydroperoxide; D005978:Glutathione