title: Mitochondrial DNA damage induced by 7-ketocholesterol in human retinal pigment epithelial cells in vitro.
abstract: OBJECTIVE
  To assess oxysterol-induced mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in cultured human retinal pigment epithelial cells (ARPE- 19).
  
  
  METHODS
  ARPE-19 cultures were exposed for 6 and 24 hours to 40 microg/mL 7-ketocholesterol (7kCh), and total DNA was extracted. Long-extension polymerase chain reaction was performed to amplify the full-length mtDNA genome. The products were separated by electrophoresis on a 0.8% agarose gel stained with ethidium bromide. Superoxide and reactive oxygen/nitrogen species (ROS/RNS; hydrogen peroxide, peroxynitrite anions, and peroxyl radicals) were measured with an amine-reactive green-dye assay and 2',7'-dicholorodihydrofluorescein diacetate (H(2)DCFDA) dye assay, respectively. The changes in mitochondrial membrane potential (DeltaPsim) were measured with a cationic (green) dye assay. Western blot analysis was used to assess porins, a structural protein of the mitochondrial membranes.
  
  
  RESULTS
  The 7kCh-treated cultures showed significant increase in ROS/RNS production (P < 0.001) compared with untreated controls, but the superoxide levels were unchanged. The 7kCh-treated ARPE-19 cultures had diminished levels of the full-length 16.2-kb mtDNA band, a 2.2-fold decrease of the DeltaPsim compared with control cultures (P < 0.001), and decreased levels of porins.
  
  
  CONCLUSIONS
  7kCh causes significant damage to the full-length intact mtDNA and mitochondrial dysfunction in ARPE-19 cells. These observations suggest that the mitochondria and its DNA may be targets for oxysterol-induced oxidative stress and may play a role in the pathogenesis of retinal diseases.
authors.affiliation: Department of Ophthalmology, University of California, Irvine, California, USA.
authors.forename: Ana L
authors.initials: AL
authors.lastname: Gramajo
authors.forename: Leandro C
authors.initials: LC
authors.lastname: Zacharias
authors.forename: Aneesh
authors.initials: A
authors.lastname: Neekhra
authors.forename: Saurabh
authors.initials: S
authors.lastname: Luthra
authors.forename: Shari R
authors.initials: SR
authors.lastname: Atilano
authors.forename: Marilyn
authors.initials: M
authors.lastname: Chwa
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Brown
authors.forename: Baruch D
authors.initials: BD
authors.lastname: Kuppermann
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Kenney
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA / Q000302:isolation & purification; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007653:Ketocholesterols / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016133:Polymerase Chain Reaction; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D007653:Ketocholesterols; D017382:Reactive Oxygen Species; D013481:Superoxides; D004247:DNA; C003001:7-ketocholesterol