title: 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.
abstract: 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.
authors.affiliation: CHU/Hôpital du Bocage, Laboratoire de Biochimie Médicale, Inserm U498, BP 1542, 21034 Cedex, Dijon, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Miguet-Alfonsi
authors.forename: Céline
authors.initials: C
authors.lastname: Prunet
authors.forename: Serge
authors.initials: S
authors.lastname: Monier
authors.forename: Ginette
authors.initials: G
authors.lastname: Bessède
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Lemaire-Ewing
authors.forename: Arnaud
authors.initials: A
authors.lastname: Berthier
authors.forename: Franck
authors.initials: F
authors.lastname: Ménétrier
authors.forename: Dominique
authors.initials: D
authors.lastname: Néel
authors.forename: Philippe
authors.initials: P
authors.lastname: Gambert
authors.forename: Gérard
authors.initials: G
authors.lastname: Lizard
mesh_terms: D000704:Analysis of Variance; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007653:Ketocholesterols / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D009186:Myelin Sheath / Q000737:chemistry; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D020298:U937 Cells
chemical_list: D004791:Enzyme Inhibitors; D006888:Hydroxycholesterols; D007653:Ketocholesterols; C011724:cholest-5-en-3 beta,7 alpha-diol; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C003001:7-ketocholesterol