Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity.
The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe2+-H2O2 or Fe3+-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)+, only Fe2+-H2O2 also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe2+ iron (concentration range 3-180 microM) or H2O2 (concentration range 50-1000 microM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)+, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)+/ADP-ribose(P) ratio was about 4 at any Fe2+ or H2O2 concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)+/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)+ production of Fe2+-H2O2-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where "conventional" oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.
Tavazzi
B
B
Department of Experimental Medicine and Biochemical Sciences, University of Rome, Tor Vergata, Italy. tavazzi@med.uniroma2.it
Di Pierro
D
D
Amorini
A M
AM
Fazzina
G
G
Galvano
M
M
Lupi
A
A
Giardina
B
B
Lazzarino
G
G
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D005609:Free Radicals; D006868:Hydrolysis; D066298:In Vitro Techniques; D008297:Male; D008954:Models, Biological; D015428:Myocardial Reperfusion Injury / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009536:Niacinamide / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*
D005609:Free Radicals; D017382:Reactive Oxygen Species; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D009249:NADP