title: Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity.
abstract: Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO(*)) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.
authors.affiliation: Free Radical Biochemistry Laboratory, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
authors.forename: Marion
authors.initials: M
authors.lastname: Kappler
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Gerry
authors.forename: Erin
authors.initials: E
authors.lastname: Brown
authors.forename: Linzi
authors.initials: L
authors.lastname: Reid
authors.forename: David S
authors.initials: DS
authors.lastname: Leake
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gieseg
mesh_terms: D000578:Amidines / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism / Q000494:pharmacology; D009336:Necrosis / Q000201:enzymology / Q000473:pathology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010545:Peroxides / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D000578:Amidines; D017304:Annexin A5; D016877:Oxidants; D010545:Peroxides; C049375:perhydroxyl radical; D017665:Hydroxyl Radical; C046728:2,2'-azobis(2-amidinopropane); D053148:Caspase 3; D005978:Glutathione