Ototoxic Aminoglycosides Inhibit NADH Metabolism and Increase Reactive Oxygen Species Formation in Cochlear Cells

Abstract

Despite causing permanent hearing loss by damaging inner ear sensory cells, aminoglycosides (AGs) remain one of the most widely used classes of antibiotics in the world. Although the mechanisms of cochlear sensory cell damage are not fully known, reactive oxygen species (ROS) may be involved. Mitochondrial-specific ROS formation was evaluated in acutely-cultured murine cochlear explants exposed to gentamicin (GM), a representative ototoxic AG antibiotic. Superoxide (O2.-) and hydrogen peroxide (H2O2) ROS were measured using MitoSox Red and Dihydrorhodamine 123, respectively. Acute GM did not significantly increase O2.-formation but did increase H2O2 formation in all cell types within one hour of exposure. At the same point, GM significantly increased manganese superoxide dismutase (mnSOD) levels while significantly decreasing copper/zinc superoxide dismutase (cuznSOD) in cochlear sensory cells. This suggests 1) a rapid conversion of highly reactive O2.-to H2O2 during the acute stage of ototoxic antibiotic exposure and 2) that the endogenous antioxidant system is significantly altered by ototoxic antibiotics. Fluorescence intensity-based measurements of nicotinamide adenine dinucleotide (NADH) were used to determine if increased ROS production was correlated with acute GM-induced changes in mitochondrial metabolism. Acute exposure also triggered a redistribution of NADH in the seven distinct NADH lifetime pools identified in cochlear cells. This project provides a base for understanding the mechanisms of mitochondrial ROS production in cochlear cells exposed to ototoxic antibiotics. Understanding the nature of ototoxic antibiotic-induced changes in mitochondrial metabolism are critical for developing hearing loss treatment and prevention strategies. Supported by the National Institute on Deafness and Other Communication Disorders (NIDCD,RO3DC012109), and COBRe (8P20GM103471-09) to HJS and a Ferlic Undergraduate Research Scholarship to DD.