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Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells.

Authors
Jiang-JF; Borisenko-GG; Osipov-A; Martin-I; Chen-RW; Shvedova-AA; Sorokin-A; Tyurina-Y; Potapovich-A; Tyurin-VA; Graham-SH; Kagan-VE
Source
J Neurochem 2004 Sep; 90(5):1036-1049
NIOSHTIC No.
20031536
Abstract
Cyclo-oxygenase-2 (COX-2) is believed to induce neuronal oxidative stress via production of radicals. While oxygen radicals are not directly involved in COX-2-catalytic cycle, superoxide anion radicals have been repeatedly reported to play a critical role in COX-2-associated oxidative stress. To resolve the controversy, we characterized production of free radicals in PC12 cells in which COX-2 expression was manipulated either genetically or by direct protein transfection and compared them with those generated by a recombinant COX-2 in a cell-free system. Using spin-traps alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide and 4-((9-acridinecarbonyl) amino)-2,2,6,6- tetramethylpiperidine-1-oxyl (Ac-Tempo), we observed arachidonic acid (AA)-dependent production of carbon-centered radicals by heme-reconstituted recombinant COX-2. No oxygen radicals or thiyl radicals have been detected. COX-2 also catalyzed AA-dependent one-electron co-oxidation of ascorbate to ascorbate radicals. Next, we used two different approaches of COX-2 expression in cells, PCXII cells which express isopropyl-1-thio-beta-D-galactopyranoside inducible COX-2, and PC12 cells transfected with COX-2 using a protein delivery reagent, Chariot. In both models, COX-2-dependent AA-induced generation of carbon-centered radicals was documented using spin-traps and Ac-Tempo. No oxygen radical formation was detected in COX-2-transfected cells by either spin-traps or fluorogenic probe, dihydroethidium. In the presence of ascorbate, AA-induced COX-2-dependent ascorbate radicals were detected. AA caused a significant and selective oxidation of one of the major phospholipids, phosphatidylserine (PS). PS was not a direct substrate for COX-2 but was co-oxidized in the presence of AA. The radical generation and PS oxidation were inhibited by COX-2 inhibitors, niflumic acid, nimesulide, or NS-398. Thus, COX-2 generated carbon-centered radicals but not oxygen radicals or thiyl radicals are responsible for oxidative stress in AA-challenged PC12 cells overexpressing COX-2.
Keywords
Exposure-levels; Exposure-assessment; Exposure-methods; Neurological-reactions; Neurological-system; Neuropharmacology; Neurophysiological-effects; Neurophysiology; Neurotoxic-effects; Neurotoxicology; Neurotoxicity; Neurotransmitters; Cell-biology; Cell-differentiation; Cell-function; Cell-metabolism; Oxygen-toxicity; Chemical-analysis; Chemical-hypersensitivity; Chemical-properties; Chemical-structure; Chemical-synthesis
Contact
Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
CODEN
JONRA9
Publication Date
20040901
Document Type
Journal Article
Fiscal Year
2004
NTIS Accession No.
NTIS Price
Issue of Publication
5
ISSN
0022-3042
NIOSH Division
HELD
Source Name
Journal of Neurochemistry
State
WA; MA
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