No abstract

We have demonstrated recently that incubation of the aminoglycoside gentamicin with an hepatic post-mitochondrial fraction produces a compound toxic to sensory cells from the inner ear in short-term culture; in contrast, the parent aminoglycoside was non-toxic in vitro (Huang MY and Schacht J, Biochem Pharmacol 40: R11-R14, 1990). In the present study, we investigated the subcellular distribution of the enzymatic activity and the nature of the metabolite. Isolated outer hair cells from the guinea pig cochlea were used to assay for cytotoxicity. The enzyme(s) responsible for this novel reaction of aminoglycosides was exclusively localized to the cytosolic fraction of guinea pig liver. No activity was detected in nuclear, lysosomal/mitochondrial or microsomal preparations. Furthermore, the toxin-forming enzymatic activity was associated with the high molecular weight fraction of the cytosol and did not require low molecular weight components. Filtration of the toxin through molecular weight cut-off membranes showed a molecular size of approximately 500. This evidence is consistent with the toxin being a gentamicin derivative.

We have previously postulated an enzymatic transformation of gentamicin (to a metabolite or an ‘activated’ molecule) as part of its ototoxic action. Here we test with eight aminoglycosides whether the proposed mechanism applies to these antibiotics as a group. Drugs were activated by incubation with a subcellular fraction from liver, and cytotoxicity was tested in a bioassay using isolated outer hair cells from guinea pig. None of the aminoglycosides compromised the viability of the cells when assayed directly, i.e. without a preceding activation. In contrast, all clinically ototoxic aminoglycosides tested were significantly cytotoxic following the incubation. Neamine, considered to be non-ototoxic, did not yield a cytotoxin. A subcellular fraction from cochlear lateral wall tissues also converted gentamicin to a cytotoxin. The results support the hypothesis that activation of aminoglycosides precedes their toxic actions and demonstrate that the capability for activation is not confined to liver.

Ornithine decarboxylase activity was determined during the development of the peripheral auditory system in the murine otocyst with the goal of understanding the role of this enzyme in the morphological and functional maturation of the inner ear. At gestational days 11 and 12 enzyme activity was more than 10-fold higher than adult levels. A sharp decline occurred between day 12 and 13 after which activity rose to a peak around day 15. Activity then dropped continuously until near-adult levels were reached at birth. A lower specific activity of ODC but a similar time-course was seen in otocysts explanted at gestational day 13 and subsequently cultured for 6 days. For two stages of development, enzyme activity and binding of 3H-alpha-difluoromethylornithine were compared. The four-fold difference in enzymatic activity on gestational days 15 and 17 was paralleled by a similar difference in binding. Ornithine decarboxylase activity during inner ear development therefore seems primarily regulated at the level of protein synthesis. Ornithine decarboxylase activity correlates with major inductive events in the morphogenesis of the cartilagenous otic capsule that serves as a template for the formation of the bony labyrinth. The pattern of activity may reflect the changes in the head mesenchyme that is recruited by the otocyst to aggregate and form its protective otic capsule.