DIRECT-DETECTION OF OTOTOXICANT-INDUCED REACTIVE OXYGEN SPECIES GENERATION IN COCHLEAR EXPLANTS

The proposal that free-radical generation contributes to the ototoxici ties of several chemical agents was studied utilizing electron paramag netic resonance (EPR) spectrometry to detect directly ototoxicant-indu ced reactive oxygen species formation in cochlear tissue. Guinea pig c ochlear explants in chelexed artificial perilymph (AP; 200 mu l) were exposed to an ototoxicant or AP for 10 min. Ototoxic agents included g entamicin sulfate (4.0 mM), kanamycin monosulfate (4.0 mM), ethacrynic acid (0.5 mM), furosemide (0.3 mM), cisplatin (0.1 mM), trimethyltin chloride (0.1 mM), and quinine HCl (3.0 mM). Following incubation, 20 mu l of AP/ototoxicant mixture was replaced by the filtered spin trap, 5,5-dimethylpyrroline-N-oxide (DMPO). After 10 min, the EPR spectrum of the mixture was obtained. Four line EPR spectra of relative intensi ties 1:2:2:1, associated with hydroxyl radical (OH)/DMPO adduct format ion, were evidenced by reaction mixtures containing cochlear explants exposed to each ototoxicant. Cisplatin, quinine and the loop diuretics produced weak OH-associated EPR signals in the absence of a cochlear explant, which were amplified in its presence. Deferoxamine quenched a ll OH spectral peaks. Peroxide levels, assayed in parallel experiments , were diminished by each ototoxicant relative to those seen following AP exposure, suggesting possible H2O2 conversion to OH. These data su pport the proposal that various ototoxic agents are capable of reactiv e oxygen species generation or promotion in cochlear tissues.