Mitochondrial DNA damage induced by 7-ketocholesterol in human retinal pigment epithelial cells in vitro.
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.
Gramajo
Ana L
AL
Department of Ophthalmology, University of California, Irvine, California, USA.
Zacharias
Leandro C
LC
Neekhra
Aneesh
A
Luthra
Saurabh
S
Atilano
Shari R
SR
Chwa
Marilyn
M
Brown
Donald J
DJ
Kuppermann
Baruch D
BD
Kenney
M Cristina
MC
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
D004272:DNA, Mitochondrial; D007653:Ketocholesterols; D017382:Reactive Oxygen Species; D013481:Superoxides; D004247:DNA; C003001:7-ketocholesterol