Early steps in bilirubin-mediated apoptosis in murine hepatoma (Hepa 1c1c7) cells are characterized by aryl hydrocarbon receptor-independent oxidative stress and activation of the mitochondrial pathway.
Unconjugated bilirubin (UCB), the end product of heme catabolism, causes apoptosis in cells of the central nervous system, endothelial cells, and hepatotoma cells. However, the molecular mechanisms that contribute to UCB cytotoxicity remain unclear. The purpose of this study was to characterize the sequence of early events leading to UCB-mediated cytotoxicity in murine hepatoma Hepa 1c1c7 cells. In the present study, UCB (5-50 microM) was found to markedly increase the intracellular generation of reactive oxygen species (ROS) in a concentration-dependent manner, which is significantly elevated by 30 min post-treatment. This generation of ROS by UCB is not dependent on aryl hydrocarbon receptor (Ahr) signaling, as cells deficient in the Ahr (C12 cells) or the Ahr nuclear translocator protein (Arnt; C4 cells) were as efficient at generating ROS as wild type (WT) Hepa 1c1c7 cells. Mitochondrial membrane depolarization, evaluated with the lipophilic cationic dye, JC-1, occurred at least by 2 h after treatment with 50 muM UCB. Analysis of the caspase cascade demonstrated that activation of caspase-9 preceded activation of caspase-3. No conversion of procaspase-2 to active caspase-2 was detected in this study. These results demonstrate that UCB-mediated apoptosis in Hepa 1c1c7 cells is associated with increased oxidative stress and that caspase-9, and definitely not caspase-2, is the initiator caspase for apoptosis in UCB-treated Hepa 1c1c7 cells.
Oakes
Garth H
GH
Department of Physiology & Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Bend
John R
JR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001663:Bilirubin / Q000378:metabolism / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D017382:Reactive Oxygen Species; D018336:Receptors, Aryl Hydrocarbon; D020169:Caspases; D001663:Bilirubin