title: Phenylbutazone Oxidation via Cu,Zn-SOD Peroxidase Activity: An EPR Study.
abstract: We investigated the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD)-peroxidase activity on the oxidation of the nonsteroidal anti-inflammatory drug phenylbutazone (PBZ). We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical intermediates of PBZ, UV-vis spectrophotometry to monitor PBZ oxidation, oxygen analysis to determine the involvement of C-centered radicals, and LC/MS to determine the resulting metabolites. Using EPR spectroscopy and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3(•-) (DMPO/(•)OH) was attenuated with increasing PBZ concentrations. The resulting PBZ radical, which was assigned as a carbon-centered radical based on computer simulation of hyperfine splitting constants, was trapped by both DMPO and MNP spin traps. Similar to Cu,Zn-SOD-peroxidase activity, an identical PBZ carbon-centered radical was also detected with the presence of both myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). Oxygen analysis revealed depletion in oxygen levels when PBZ was oxidized by SOD peroxidase-activity, further supporting carbon radical formation. In addition, UV-vis spectra showed that the λmax for PBZ (λ = 260 nm) declined in intensity and shifted to a new peak that was similar to the spectrum for 4-hydroxy-PBZ when oxidized by Cu,Zn-SOD-peroxidase activity. LC/MS evidence supported the formation of 4-hydroxy-PBZ when compared to that of a standard, and 4-hydroperoxy-PBZ was also detected in significant yield. These findings together indicate that the carbonate radical, a product of SOD peroxidase activity, appears to play a role in PBZ metabolism. Interestingly, these results are similar to findings from heme peroxidase enzymes, and the context of this metabolic pathway is discussed in terms of a mechanism for PBZ-induced toxicity.
authors.affiliation: §Pharmacology and Toxicology Department, Faculty of Pharmacy, Taibah University, Madinah, Saudi Arabia.
authors.forename: Naif
authors.initials: N
authors.lastname: Aljuhani
authors.forename: Randy M
authors.initials: RM
authors.lastname: Whittal
authors.forename: Saifur R
authors.initials: SR
authors.lastname: Khan
authors.forename: Arno G
authors.initials: AG
authors.lastname: Siraki
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis / Q000737:chemistry; D010653:Phenylbutazone / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013482:Superoxide Dismutase / Q000378:metabolism*
chemical_list: D003497:Cyclic N-Oxides; D005609:Free Radicals; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D013482:Superoxide Dismutase; D010653:Phenylbutazone; D010100:Oxygen