Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes.
We have previously shown that, in trout hepatocytes, exposure to a high dose of copper (Cu) leads to disruption of Ca(2+) homeostasis and elevated formation of reactive oxygen species (ROS), with the latter ultimately causing cell death. In the present study, we aimed at identifying, using a lower Cu concentration, the role of mitochondria in this scenario, the potential involvement of the mitochondrial permeability transition (MPT), and the mode of cell death induced by the metal. Incubation with 10 muM Cu resulted in a strong stimulation of ROS formation, and after 2 h of exposure a significant increase of both apoptotic and necrotic cells was seen. Co-incubation of Cu-treated hepatocytes with the iron-chelator deferoxamine significantly inhibited ROS production and completely prevented cell death. The origin of the radicals generated was at least partly mitochondrial, as visualized by confocal laser scanning microscopy. Furthermore, ROS production was diminished by inhibition of mitochondrial respiration, but since this also aggravated the elevation of intracellular Ca(2+) induced by Cu, it did not preserve cell viability. In a sub-population of cells, Cu induced a decrease of mitochondrial membrane potential and occurrence of the MPT. Cyclosporin A, which did not inhibit ROS formation, prevented the onset of the MPT and inhibited apoptotic, but not necrotic, cell death. Cu-induced apoptosis therefore appears to be dependent on induction of the MPT, but the prominent contribution of mitochondria to ROS generation also suggests an important role of mitochondria in necrotic cell death.
Krumschnabel
Gerhard
G
Institut für Zoologie und Limnologie and Center for Molecular Biosciences Innsbruck (CMBI), Abteilung für Okophysiologie, Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria. Gerhard.Krumschnabel@uibk.ac.at
Manzl
Claudia
C
Berger
Christian
C
Hofer
Bettina
B
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D003300:Copper / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects*; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects*; D009336:Necrosis; D017686:Oncorhynchus mykiss / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects*; D010539:Permeability / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism
D017382:Reactive Oxygen Species; D003300:Copper; D002118:Calcium