The Role of Complex I in Mitochondrial Reactive Oxygen Species Formation in Cochlear Sensory and Supporting Cells during Ototoxic Aminoglycoside Exposure

Abstract

Aminoglycosides (AGs) are the most widely used class of antibiotics in the world despite causing permanent hearing loss by damaging inner ear sensory cells. Although the mechanisms of cochlear sensory cell damage are not fully known, reactive oxygen species (ROS) are clearly involved. During normal mitochondrial metabolism low levels of ROS, primarily superoxide, are produced at complexes I and III in the electron transport chain. These levels can increase when mitochondrial dysfunction occurs. Complex I-specific ROS formation was evaluated in acutely-cultured murine cochlear explants exposed to gentamicin (GM, 300 μg/ml), a representative ototoxic AG antibiotic. Mitochondrial membrane potential and pro-apoptotic signaling were measured using Tetramethylrhodamine and apoptosis-inducing factor (AIF) labeling, respectively. Fluorescence intensity-based measurements of nicotinamide adenine dinucleotide (NADH) were used to detect changes mitochondrial metabolism. Relative amounts of superoxide and hydrogen peroxide produced during acute GM exposure were measured using MitoSox Red and Dihydrorhodamine 123, respectively. GM increased NADH fluorescence intensity in low- and high-frequency sensory cells. The complex I inhibitor rotenone (250 nM) significantly increased superoxide, not hydrogen peroxide, in low- and high-frequency sensory cells (p < 0.01). GM significantly increased superoxide and hydrogen peroxide formation in low- and high-frequency sensory cells (p < 0.05). Rotenone increased GM-induced superoxide formation but decreased GM-induced hydrogen peroxide formation. This effect was greatest in high-frequency cells indicating fundamental differences in ROS formation in high- and low-frequency sensory cells exposed to ototoxic antibiotics. This project provides a base for understanding the underlying mechanisms of mitochondrial ROS production in cochlear cells during exposures to ototoxic antibiotics. 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.