Formation of reactive oxygen species following bioactivation of gentamicin.
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.
Sha
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Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan, Ann Arbor 48109-0506, USA.
Schacht
J
J
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*
D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D005609:Free Radicals; D005839:Gentamicins; D010617:Phenanthridines; D017382:Reactive Oxygen Species; C058428:hydroethidine; D009249:NADP