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NAD(P)H:Quinone Oxidoreductase 1: Role as a Superoxide Scavenger

2004; American Society for Pharmacology and Experimental Therapeutics; Volume: 65; Issue: 5 Linguagem: Inglês

10.1124/mol.65.5.1238

1521-0111

David Siegel, Daniel L. Gustafson, Donna L. Dehn, Jin Yi Han, Preecha Boonchoong, Lawrence J. Berliner, David Ross,

Vitamin C and Antioxidants Research

Experiments using purified recombinant human NAD(P)H:quinone oxidoreductase 1 (NQO1) revealed that the auto-oxidation of fully reduced protein resulted in a 1:1 stoichiometry of oxygen consumption to NADH oxidation with the production of hydrogen peroxide. The rate of auto-oxidation of fully reduced NQO1 was markedly accelerated in the presence of superoxide (O(2)(*)(-)), whereas the addition of superoxide dismutase greatly inhibited the rate of auto-oxidation. The ability of reduced NQO1 to react with O(2)(*)(-) suggested a role for NQO1 in scavenging O(2)(*)(-), and this hypothesis was tested using established methods for O(2)(*)(-) production and detection. The addition of NQO1 in combination with NAD(P)H resulted in inhibition of dihydroethidium oxidation, pyrogallol auto-oxidation, and elimination of a potassium superoxide-generated ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide:O(2)(*)(-) adduct signal (electron spin resonance). Kinetic parameters for the reduction of O(2)(*)(-) by NQO1 were estimated using xanthine/xanthine oxidase as the source of O(2)(*)(-) and after NQO1-dependent NADH oxidation at 340 nm. The ability of NQO1 to scavenge O(2)(*)(-) was also examined using cell sonicates prepared from isogenic cell lines containing no NQO1 activity (NQO1(-)) or very high levels of NQO1 activity (NQO1(+)). We demonstrated that addition of NAD(P)H and cell sonicate from NQO1(+) but not NQO1(-) cells resulted in an increased level of O(2)(*)(-) scavenging could be inhibited by 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. NQO1 can generate hydroquinones that are redox active, and the O(2)(*)(-) scavenging activity of NQO1 may allow protection against O(2)(*)(-) at the site of hydroquinone generation. In addition, the O(2)(*)(-) scavenging activity of NQO1 may provide an additional level of protection against O(2)(*)(-) induced toxicity.