A comparison of four assays detecting oxidizing species

Quin2, a fluorescent calcium probe, has a low affinity for calcium in comparison to its affinities for transition metal ions. Chelation of ferric ion with quin2 strongly enhanced the formation of oxidizing species in the presence of bolus H₂O₂ as detected with four assays, electron spin resonance with the spin-trap DMPO, the deoxyribose assay, the DMSO assay, and plasmid DNA strand breakage. In comparison, Fe(III)-EDTA reacted with bolus H₂O₂ only as detected with electron spin resonance and the deoxyribose assay, but not as detected with the two latter assays. The addition of reductants, like ascorbate or superoxide generated by hypoxanthine/xanthine oxidase, to Fe(III)-EDTA in the presence of H₂O₂ produced plasmid DNA strand breakage and strong reactivity in both the DMSO and the deoxyribose assays. Our findings suggest that the main oxidizing species produced in Fenton-type reactions is hydroxyl radical. However, the reaction between Fe(III)-EDTA and bolus H₂O₂ appears to be exceptional and dominated by a nonhydroxyl radical species.