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 (PI3Z). 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 spintrapping with 5,5-dirnethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3 center dot- (DMPO/(OH)-O-center dot) 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 IVINP spin traps. Similar to Cu,ZnSOD-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 Amax for PBZ = 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.