Analysis of oxidative processes and of myelin figures formation before and after the loss of mitochondrial transmembrane potential during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis: comparison with various pro-apoptotic chemicals

• Published in: Biochemical pharmacology 2002-08, Vol.64 (3), p.527-541
• Main Authors: Miguet-Alfonsi, Carole, Prunet, Céline, Monier, Serge, Bessède, Ginette, Lemaire-Ewing, Stéphanie, Berthier, Arnaud, Ménétrier, Franck, Néel, Dominique, Gambert, Philippe, Lizard, Gérard
• Format: Article
• Language: English
• Subjects: Analysis of Variance; Apoptosis; Caspase 3; Caspases - metabolism; Cell Nucleus - drug effects; Cell Nucleus - ultrastructure; DNA - drug effects; DNA - metabolism; Enzyme Activation; Enzyme Inhibitors - pharmacology; Humans; Hydroxycholesterols - pharmacology; Ketocholesterols - pharmacology; Membrane Potentials - drug effects; Microscopy, Electron; Mitochondria - drug effects; Mitochondria - physiology; Mitochondria - ultrastructure; Myelin Sheath - chemistry; Oxidative Stress - drug effects; Oxidative Stress - physiology; Poly(ADP-ribose) Polymerases - metabolism; U937 Cells
• ISSN: 0006-2952

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol) 7beta-hydroxycholesterol and 7-ketocholesterol are potent inducers of cell death and probably play central roles in atherosclerosis. As suggested by our previous investigations, 7-ketocholesterol might be a causative agent of vascular damage by inducing apoptosis and enhancing superoxide anion (O2*-) production. To determine the precise relationships between cytotoxicity and oxidative stress, the ability of oxysterols oxidized at C7 to induce apoptosis, to stimulate O2*- production and to promote lipid peroxidation was compared with different pro-apoptotic chemicals: antitumoral drugs (VB, Ara-C, CHX, and VP-16) and STS. All compounds, except 7alpha-hydroxycholesterol, induced apoptosis characterized by the occurrence of cells with fragmented and/or condensed nuclei, loss of mitochondrial potential, caspase-3 activation, PARP degradation, and internucleosomal DNA fragmentation. The highest proportion of apoptotic cells was found with antitumoral drugs and STS, whereas the highest overproduction of O2*- detected before and after the loss of mitochondrial potential was obtained with 7beta-hydroxycholesterol and 7-ketocholesterol. Overproduction of O2*- was always correlated with enhanced lipid peroxidation. Vit E was only capable to significantly counteract apoptosis and oxidative stress induced by 7beta-hydroxycholesterol, 7-ketocholesterol, VB and STS. By electron and fluorescence microscopy, myelin figures evocating autophagic vacuoles were barely observed under treatment with 7beta-hydroxycholesterol and 7-ketocholesterol, and their formation occurring before the loss of mitochondrial potential was reduced by Vit E. In the presence of 7alpha-hydroxycholesterol, no enhancement of O2*- production, no lipid peroxidation, and no formation of myelin figures were observed. Collectively, our data demonstrate, that there can be a more or less important stimulation of oxidative stress during apoptosis. They also suggest that enhancement of O2*- production associated with lipid peroxidation during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis could contribute to in vivo vascular injury, and that myelin figures could constitute suitable markers of oxysterol-induced cell death.