Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells

Abstract

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H₂O₂) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 μM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 μM H₂O₂ or a flux of H₂O₂ generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H₂O₂-induced toxicity was ameliorated at high edaravone concentrations (100-200 μM). In contrast, low concentrations of edaravone (1-10 μM) exacerbated the H₂O₂-induced toxicity. However, the effect of edaravone at low concentration (0-10 μM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Keywords: Antioxidant; Edaravone; Electron spin resonance; Free radicals; HL-60 cells; Myeloperoxidase.