title: Superoxide dismutase and hydrogen peroxide cause rapid nitric oxide breakdown, peroxynitrite production and subsequent cell death.
abstract: Isolated copper/zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) together with hydrogen peroxide (H(2)O(2)) caused rapid breakdown of nitric oxide (NO) and production of peroxynitrite (ONOO(-)) indicated by the oxidation of dihydrorhodamine-1,2,3 (DHR) to rhodamine-1,2,3. The breakdown of NO by this reaction was inhibited by cyanide (CN(-)) or by diethyldithiocarbamate (DETC), both Cu/Zn-SOD inhibitors, and the conversion of DHR to rhodamine-1,2,3 was inhibited by incubating Cu/Zn-SOD with either CN(-) or with high levels of H(2)O(2) or by including urate, a potent scavenger of ONOO(-). In the presence of phenol, the reaction of SOD, H(2)O(2) and NO caused nitration of phenol, which is known to be a footprint of ONOO(-) formation. H(2)O(2) addition to macrophages (cell line J774) expressing the inducible form of NO synthase (i-NOS) caused rapid breakdown of the NO they produced and this was also inhibited by CN(-) and by DETC. Subsequent ONOO(-) production by the macrophages, via this reaction, was inhibited by CN(-), high levels of H(2)O(2) or by urate. H(2)O(2) addition to i-NOS macrophages also caused cell death which was, in part, prevented by DETC or urate. We also found inhibition of mitochondrial respiration with malate and pyruvate as substrates, when isolated liver mitochondria were incubated with Cu/Zn-SOD, H(2)O(2) and NO. Inhibition of mitochondrial respiration was partly prevented by urate. The production of ONOO(-) by SOD may be of significant importance pathologically under conditions of elevated H(2)O(2) and NO levels, and might contribute to cell death in inflammatory and neurodegenerative diseases, as well as in macrophage-mediated host defence.
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. agmb@mole.bio.cam.ac.uk
authors.forename: A G
authors.initials: AG
authors.lastname: McBride
authors.forename: V
authors.initials: V
authors.lastname: Borutaité
authors.forename: G C
authors.initials: GC
authors.lastname: Brown
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D004563:Electrochemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008264:Macrophages; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects / Q000502:physiology; D009566:Nitrates / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry; D052247:Nitric Oxide Synthase Type II; D020112:Rhodamine 123 / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000494:pharmacology*
chemical_list: D009566:Nitrates; D012235:Rhodamines; C058319:dihydrorhodamine 123; D020112:Rhodamine 123; C039642:peroxynitric acid; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D013482:Superoxide Dismutase