Cadmium-induced apoptosis through the mitochondrial pathway in rainbow trout hepatocytes: involvement of oxidative stress.
Cadmium (Cd) induces oxidative stress and apoptosis in trout hepatocytes. We therefore investigated the involvement of the mitochondrial pathway in the initiation of apoptosis and the possible role of oxidative stress in that process. This study demonstrates that hepatocyte exposure to Cd (2, 5 and 10 microM) triggers significant caspase-3, but also caspase-8 and -9 activation in a dose-dependent manner. Western-blot analysis of hepatocyte mitochondrial and cytosolic fractions revealed that cytochrome c (Cyt c) was released in the cytosol in a dose-dependent manner, whereas the pro-apoptotic protein Bax was redistributed to mitochondria after 24 and 48 h exposure. We also found that the expression of anti-apoptotic protein Bcl-xL, known to be regulated under mild oxidative stress to protect cells from apoptosis, did not change after 3 and 6 h exposure to Cd, then increased after 24 and 48 h exposure to 10 microM Cd. In the second part of this work, two antioxidant agents, 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) (100 microM) and N-acetylcysteine (NAC, 100 microM) were used to determine the involvement of reactive oxygen species (ROS) in Cd-induced apoptosis. Simultaneously exposing trout hepatocytes to Cd and TEMPO or NAC significantly reduced caspase-3 activation after 48 h and had a suppressive effect on caspase-8 and -9 also, mostly after 24 h. Lastly, the presence of either one of these antioxidants in the treatment medium also attenuated Cd-induced Cyt c release in cytosol and the level of Bax in the mitochondria after 24 and 48 h, while high Bcl-xL expression was observed. Taken together, these data clearly evidenced the key role of mitochondria in the cascade of events leading to trout hepatocyte apoptosis in response to Cd and the relationship that exists between oxidative stress and cell death.
Laboratoire de Toxicologie Cellulaire, Moléculaire et Génomique, INRA--Centre de Recherches de Sophia-Antipolis, UMR INRA-UNSA 1112, 400 Route des Chappes, BP 167, 06903 Sophia-Antipolis, France. risso@antibes.inra.fr
C
C
Risso-de Faverney
N
N
Orsini
G
G
de Sousa
R
R
Rahmani
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002104:Cadmium / Q000633:toxicity*; D020169:Caspases / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species / Q000378:metabolism; D018709:Statistics, Nonparametric; D013997:Time Factors; D051028:bcl-2-Associated X Protein
D000975:Antioxidants; D003497:Cyclic N-Oxides; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D002104:Cadmium; D045304:Cytochromes c; D020169:Caspases; C003959:TEMPO; D000111:Acetylcysteine