Oxygen-derived free radical and active oxygen complex formation from cobalt(II) chelates in vitro

The electron paramagnetic resonance (EPR) spin trapping technique was used to study the generation of oxygen free radicals from the reaction of hydrogen peroxide with various Co(II) complexes in pH 7.4 phosphate buffer. The 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap was used in these experiments to detect superoxide and hydroxyl free radicals. Superoxide radical was generated from the reaction of H2O2 with Co(II), but was inhibited when Co(II) was chelated with adenosine 5'-diphosphate or citrate. Visible absorbance spectra revealed no change in the final oxidation state of the cobalt ion in these samples. The EDTA complex also prevented detectable free-radical formation when H2O2 was added, but visible absorbance data indicated oxidation of the Co(II) to Co(III) in this case. The amount of DMPO/.OOH adduct detected by EPR was greatly enhanced when H2O2 reacted with the nitrilotriacetate complex relative to Co(II) alone, and in addition, concurrent formation of the DMPO/.OH adduct due to slow oxidation of Co(II) was observed. The hydroxyl radical adduct formation was suppressed by ethanol, but not DMSO, indicating that free hydroxyl radical was not formed. The deferoxamine nitroxide radical was exclusively formed when H2O2 was added to the Co(II) complex of this ligand, most probably in a site-specific manner. In the presence of ethylenediamine, Co(II) bound molecular O2 and directly oxidized DMPO to its DMPO/.OH adduct without first forming free superoxide, hydroxyl radical, or hydrogen peroxide. An experiment using 17O-enriched water revealed that the Co(II)-ethylenediamine complex caused the DMPO to react with solvent water to form the DMPO/.OH adduct. The relevance of these results to toxicological studies of cobalt is discussed.