title: Evaluation of apoptotic markers in HEI-OC1 cells treated with gentamicin with and without the mitochondria-targeted antioxidant mitoquinone.
abstract: OBJECTIVE
  Mitoquinone (MitoQ) attenuates aminoglycoside (AG)-induced upregulation of the proapoptotic molecules Bak and harakiri (Hrk) and decreases the percentage of apoptotic House Ear Institute Organ of Corti 1 (HEI-OC1) cells.
  
  
  BACKGROUND
  The primary mechanism of AG ototoxicity is the formation of reactive oxygen species, which leads to hair cell death via apoptotic and nonapoptotic pathways. Antioxidants have been shown to protect against AG ototoxicity. Mitoquinone is a mitochondria-targeted derivative of the antioxidant ubiquinone. Thus, MitoQ may be more effective in preventing AG ototoxicity compared with untargeted antioxidants.
  
  
  METHODS
  Ribonucleic acid from untreated HEI-OC1 cells and cells exposed to gentamicin with and without preincubation with MitoQ, idebenone (IDB, an untargeted ubiquinone), or decylTPP (positive control) were used to assess gene expression of Bak and Hrk using real-time polymerase chain reaction. Protein expression of Bak and Hrk was determined by Western blotting. Annexin V assay using flow cytometry was performed to assess the percentage of apoptotic HEI-OC1 cells treated with gentamicin with and without preincubation with MitoQ, decylTPP, or IDB.
  
  
  RESULTS
  Preincubation of HEI-OC1 cells with MitoQ significantly decreased the gentamicin-induced upregulation of Bak gene (p = 0.03) but not preincubation with IDB (p = 0.87). Harakiri levels were very low that relative quantification could not be carried out. Protein levels of Bak and Hrk were not different between treatments. Annexin V assay showed that gentamicin increased the percentage of apoptotic cells (p < 0.05) compared with control. However, the percentages of apoptotic cells in gentamicin-treated and cells pretreated with the antioxidants MitoQ or IDB were not different.
  
  
  CONCLUSIONS
  Mitoquinone attenuated the gentamicin-induced upregulation of the Bak gene but not its product, the proapoptotic molecule Bak, and MitoQ did not significantly decrease the gentamicin-induced cell apoptosis in vitro. Further in vivo studies are needed to assess the clinical significance of these findings.
authors: Department of Otolaryngology-Head and Neck Surgery, University of Florida College of Medicine, Gainesville, Florida, U.S.A.
authors: Armon
authors: A
authors: Jadidian
authors: Patrick J
authors: PJ
authors: Antonelli
authors: Carolyn P
authors: CP
authors: Ojano-Dirain
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002460:Cell Line; D005839:Gentamicins / Q000494:pharmacology*; D006198:Hair Cells, Auditory / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000975:Antioxidants; D051017:Apoptosis Regulatory Proteins; D005839:Gentamicins; C484586:HRK protein, human; D009943:Organophosphorus Compounds; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D014451:Ubiquinone; C429014:mitoquinone; C036619:idebenone