Mitochondrial DNA Damage Is 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 40µg/ml of 7-ketocholesterol (7kCh) and total DNA was extracted. Long-extension-polymerase chain reaction (LX-PCR) 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 mtDNA to nuclear DNA (nDNA) ratios were calculated by comparing PCR amplified mtND2 and r18S gene products. 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.

Results: : The 7-kCh treated cultures showed significant increase in ROS/RNS production (p<0.001) compared to untreated controls but the superoxide levels were unchanged. The ARPE-19 cultures had diminished levels of the full-length 16.2 kb mtDNA band, a 2.2-fold decrease of the ΔΨm compared to 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 might play a role in the pathogenesis of retinal diseases.

Keywords: retinal culture • drug toxicity/drug effects • mitochondria