Cellular and subcellular localization of tritiated gentamicin in the guinea pig cochlea following combined treatment with ethacrynic acid

Guinea pigs (GPs) receiving one intra-muscular injection of gentamicin (GM) (150 mg/kg) in which 2 mg of tritiated GM (2 mCi) were incorporated, followed 1.5 h later by an intra-cardiac injection of ethacrynic acid (EA) (30 mg/kg) were sacrificed 25 min, 1, 4 and 24 h after the EA injection. Other GPs were treated with one injection of GM or EA alone and sacrificed 24 h later. Cochlear function was monitored by recording VIIIth nerve compound action potential (CAP) responses to clicks at 70 dB peak-equivalent Sound Pressure Level (pe SPL) and CAP audiograms. At 24 h thresholds were significantly elevated for high frequencies only in GPs treated with the GM/EA combination. GM was revealed in the cochlea and kidney by autoradiography using light and electron microscopy. In the kidney GM was already detected in the proximal tubule cells at 25 min and at 24 h. In the cochlea GM was systematically not observed at 25 min. At 1 h a weak labelling was detected in vessels of the stria vascularis and in sensory cells at the base of the cochlea. At 4 h the labelling disappeared in stria vascularis but increased in the hair cells. At 24 h GM labelling was found exclusively in hair cells, particularly outer hair cells, with a gradient from base to apex and from first to 3rd row, this distribution pattern correlating well with the pattern of threshold changes prominent at high frequencies. Inside the hair cells, labelling was diffuse in the cytoplasm at an early stage but at 24 h it was clearly localized in the hair bundle area, the cuticular plate, particularly dense on the lysosomes concentrated below the cuticular plate, and over the nucleus. This specific cellular and intracellular localization of GM, and its progressive accumulation inside the hair cell body, along with the development of functional changes, tend to indicate that the toxicity results from specific intracellular effects of the aminoglycoside molecule.