ESR studies on the production of reactive oxygen intermediates by rat liver microsomes in the presence of NADPH or NADH

Abstract

Experiments were carried out using spin-trapping ESR spectroscopy to evaluate in a quantitative and kinetic manner the production of reactive oxygen intermediates by rat liver microsomes. Comparisons between the effectiveness of NADH and that of NADPH in catalyzing microsomal generation of reactive oxygen intermediates were made. Superoxide production was determined by assaying the generation of superoxide dismutase-sensitive stable nitroxyl radicals formed from 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine. Identical spectra were produced when microsomes were incubated with NADH or NADPH; reaction rates were linear for at least 10 min of reaction and were about three- to fourfold greater with NADPH. In the presence of iron, microsomes catalyze the production of hydroxyl radical during electron transfer. Initial experiments utilizing 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) as the spin-trapping agent proved unsatisfactory for the microsomal system as adequate kinetics could not be determined in view of the rapid decay of the DMPO-OH adduct, as well as secondary adducts such as DMPO-CH3 or DMPO-hydroxyethyl (HER). The spin-trapping agent alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone (POBN) was evaluated. POBN-OH adducts were somewhat more stable but also rapidly decayed after 2 to 3 min. However, production of POBN-CH3 and POBN-HER adducts was proportional to that of microsomal protein, increased with time over a 5- to 15-min period, and then was relatively stable. The redox cycling agent paraquat increased POBN-HER adduct formation twofold. Formation of the adduct with either NADH or NADPH required an iron catalyst and did not occur in the presence of the iron chelator desferrioxamine. Ferric EDTA was most reactive in catalyzing production of the adduct, ferric DTPA was 60 to 70% as effective, and ferric ATP was 20 to 30% as effective as ferric EDTA, with both reductants. Irrespective of the iron complex, rates of POBN-HER formation with NADH were about 70% those of NADPH. Formation of the adduct was inhibited by catalase, mannitol, and GSH, but not superoxide dismutase. These experiments support the usefulness of POBN plus ethanol for kinetic studies on the production of .OH by microsomes and validate that microsomes in the presence of an iron catalyst generate .OH not only with NADPH as cofactor, but also with NADH.