Mitochondrial DNA damage induced by 7-ketocholesterol in human retinal pigment epithelial cells in vitro

Abstract

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 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.