title: Formation of reactive oxygen species following bioactivation of gentamicin.
abstract: The present study investigated the ability of gentamicin to catalyze free radical reactions and probed the underlying mechanisms by hydroethidine imaging, oxygen consumption, and reduction of cytochrome c. In Epstein-Barr virus-transformed lymphoblastoid cells, a respiratory burst was induced by phorbol ester and detected by hydroethidine, a fluorescent indicator of superoxide radical. The addition of gentamicin increased the fluorescence two-fold while gentamicin did not produce fluorescence in the absence of phorbol ester. In membrane preparations, gentamicin did not enhance NADPH consumption ruling out a direct activation of NADPH oxidase. The formation of reactive oxygen species by gentamicin was additionally supported by experiments that showed gentamicin increased oxygen consumption two-fold in intact cells and a cell-free system. In addition, generation of superoxide was indicated by the gentamicin-stimulated reduction of cytochrome c. The stimulation by gentamicin depended upon the presence of iron (FeII/FeIII) and of arachidonic acid as an electron donor. These results support the hypothesis that an iron-gentamicin complex can increase reactive oxygen species in nonenzymatic and in biological systems. The requirement for a reductive activation in intact cells (e.g., by a respiratory burst) is interpreted as the conversion of an inactive FeIII-gentamicin to a redox-active FeII-gentamicin complex.
authors.affiliation: Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan, Ann Arbor 48109-0506, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Sha
authors.forename: J
authors.initials: J
authors.lastname: Schacht
mesh_terms: D000704:Analysis of Variance; D001711:Biotransformation; D002384:Catalysis; D002461:Cell Line, Transformed; D002474:Cell-Free System; D003574:Cytochrome c Group / Q000378:metabolism; D005456:Fluorescent Dyes; D005609:Free Radicals; D005839:Gentamicins / Q000493:pharmacokinetics*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000187:drug effects*; D010617:Phenanthridines; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D005609:Free Radicals; D005839:Gentamicins; D010617:Phenanthridines; D017382:Reactive Oxygen Species; C058428:hydroethidine; D009249:NADP