Journal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
Volume 51, Issue 2, Pages 1164-1170Publisher
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.09-3443
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Funding
- Pan-American Association of Ophthalmology Foundation
- Discovery Eye Foundation
- Iris and B. Gerald Cantor Foundation
- Gilbert Foundation
- Lincy Foundation
- Ko Family Foundation
- Research to Prevent Blindness Foundation
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PURPOSE. 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 mu g/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 (Delta Psi m) 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 Delta Psi m 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. (Invest Ophthalmol Vis Sci. 2010;51:1164-1170) DOI:10.1167/iovs.09-3443
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