Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum.
The purpose of the present work was to study the mechanisms involved in apoptosis induced by oxidative stress in rat hepatocytes. We focused on the apoptotic signaling molecules cytochrome c, Bcl-2 and Bax. Rat hepatocytes were exposed for 1 h to increasing concentrations of tert-butylhydroperoxide (t-BHP). Using lactate dehydrogenase (LDH) leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, we observed that a concentration of t-BHP of 0.4-0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored. Malondialdehyde and 8-oxo-guanine formation indicates that t-BHP induces oxidative stress and damage. However, at 0.4 mM t-BHP, these oxidative molecular changes as well as LDH leakage no longer progress after the first hour of t-BHP exposure, suggesting the activation of some defense mechanisms. Western blot analysis of cytochrome c shows that its level increases in the cytosol while that of Bax decreases in this fraction as a result of t-BHP treatment. Moreover, there is a loss of Bcl-2 from mitochondria while, in contrast, Bax accumulates in this organelle following t-BHP treatment. However, cytochrome c appears to be relocalized to the endoplasmic reticulum as its presence in microsomes is greatly enhanced. We suggest that t-BHP triggers apoptosis through a step that involves cytochrome c release from mitochondria. This event is stimulated by Bcl-2 disappearance from mitochondria and Bax recruitment. Neutralization of excess cytosolic cytochrome c is achieved by its relocalization to the endoplasmic reticulum, hence triggering the down-regulation of apoptotic signals.
Haidara
Khadidja
K
Université du Québec à Montréal, Département de Chimie, Montreal, QC, Canada. baba@total.net
Morel
Isabelle
I
Abalea
Valerie
V
Gascon Barre
Marielle
M
Denizeau
Francine
F
D000818:Animals; D017209:Apoptosis; D006690:Bisbenzimidazole; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000032:analysis; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D009336:Necrosis; D018384:Oxidative Stress; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051381:Rats; D051028:bcl-2-Associated X Protein; D020122:tert-Butylhydroperoxide
C494828:Bax protein, rat; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D020122:tert-Butylhydroperoxide; D007770:L-Lactate Dehydrogenase; D006690:Bisbenzimidazole