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 PEN was added to a Fenton reaction system composed of 25 mM H2O2 and 0.1 mM FeSO4, 4-hydroxyPBN was the primary product detected, and benzoic acid was a minor product. When the Fe2+ concentration was increased to 1.0 mM, 4-hydroxyPBN concentrations increased dramatically, and smaller amounts of benzoic acid and 2-hydroxpPBN were also formed. Although PEN is extensively metabolized after administration to animals, its metabolites have not been identified. When PEN was incubated with rat liver microsomes and a reduced nicotinamide adenine dinculeotide phosphate (NADPH)-generating system, 4-hydroxyPBN was the only metabolite detected. When PEN 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 PEN and circulates in body fluids, it may contribute to the pharmacological properties of PEN. But 4-hydroxyPBN formation cannot be used to demonstrate hydroxyl radical formation in vivo because of its enzymatic formation. (C) 2000 Elsevier Science Inc.