Aromatic hydroxylation in PBN spin trapping by hydroxyl radicals and cytochrome P-450.
Phenyl N-tert-butylnitrone (PBN) is widely used as a spin trapping agent, but is not useful detecting hydroxyl radicals because the resulting spin adduct is unstable. However, hydroxyl radicals could attack the phenyl ring to form stable phenolic products with no electron paramagnetic resonance signal, and this possibility was investigated in the present studies. When PBN was added to a Fenton reaction system composed of 25 mM H(2)O(2) and 0.1 mM FeSO(4), 4-hydroxyPBN was the primary product detected, and benzoic acid was a minor product. When the Fe(2+) concentration was increased to 1.0 mM, 4-hydroxyPBN concentrations increased dramatically, and smaller amounts of benzoic acid and 2-hydroxyPBN were also formed. Although PBN is extensively metabolized after administration to animals, its metabolites have not been identified. When PBN was incubated with rat liver microsomes and a reduced nicotinamide adenine dinculeotide phosphate (NADPH)-generating system, 4-hydroxyPBN was the only metabolite detected. When PBN was given to rats, both free and conjugated 4-hydroxyPBN were readily detected in liver extracts, bile, urine, and plasma. Because 4-hydroxyPBN is the major metabolite of PBN and circulates in body fluids, it may contribute to the pharmacological properties of PBN. But 4-hydroxyPBN formation cannot be used to demonstrate hydroxyl radical formation in vivo because of its enzymatic formation.
Reinke
L A
LA
Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Moore
D R
DR
Sang
H
H
Janzen
E G
EG
Kotake
Y
Y
D000818:Animals; D001711:Biotransformation; D003497:Cyclic N-Oxides; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006861:Hydrogen Peroxide; D017665:Hydroxyl Radical / Q000378:metabolism*; D006900:Hydroxylation; D007501:Iron; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D009589:Nitrogen Oxides / Q000737:chemistry / Q000493:pharmacokinetics*; D051381:Rats; D017208:Rats, Wistar; D013113:Spin Labels
D003497:Cyclic N-Oxides; C045076:Fenton's reagent; D009589:Nitrogen Oxides; D013113:Spin Labels; D017665:Hydroxyl Radical; C029217:phenyl-N-tert-butylnitrone; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D007501:Iron