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Ana L. Gramajo, Leandro C. Zacharias, Aneesh Neekhra, Saurabh Luthra, Shari R. Atilano, Marilyn Chwa, Donald J. Brown, Baruch D. Kuppermann, M. Cristina Kenney; Mitochondrial DNA Damage Induced by 7-Ketocholesterol in Human Retinal Pigment Epithelial Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2010;51(2):1164-1170. doi: https://doi.org/10.1167/iovs.09-3443.
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To assess oxysterol-induced mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in cultured human retinal pigment epithelial cells (ARPE- 19).
ARPE-19 cultures were exposed for 6 and 24 hours to 40 μg/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 (H2DCFDA) dye assay, respectively. The changes in mitochondrial membrane potential (ΔΨm) were measured with a cationic (green) dye assay. Western blot analysis was used to assess porins, a structural protein of the mitochondrial membranes.
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 ΔΨm compared with control cultures (P < 0.001), and decreased levels of porins.
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.
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