Superoxide dismutase and hydrogen peroxide cause rapid nitric oxide breakdown, peroxynitrite production and subsequent cell death

Isolated copper/zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxi de dismutase (Mn-SOD) together with hydrogen peroxide (H2O2) caused rapid b reakdown of nitric oxide (NO) and production of peroxynitrite (ONOO-) indic ated by the oxidation of dihydrorhodamine-1,2,3 (DHR) to rhodamine-1,2,3. T he breakdown of NO by this reaction was inhibited by cyanide (CN-) or by di ethyldithiocarbamate (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 H2O2 or by including urate, a potent scavenger of ONOO-. In the presence of phenol, the reaction of SOD, H2O2 and NO cause d nitration of phenol, which is known to be a footprint of ONOO- formation. H2O2 addition to macrophages (cell line J774) expressing the inducible for m 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 2O2 or by urate. H2O2 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 iso lated liver mitochondria were incubated with Cu/Zn-SOD, H2O2 and NO. Inhibi tion of mitochondrial respiration was partly prevented by urate. The produc tion of ONOO- by SOD may be of significant importance pathologically under conditions of elevated H2O2 and NO levels, and might contribute to cell dea th in inflammatory and neurodegenerative diseases, as well as in macrophage -mediated host defence. (C) 1999 Elsevier Science B.V. All rights reserved.