6533b7d9fe1ef96bd126bec9

RESEARCH PRODUCT

Assessment of the toxicity of cholesterol oxides on retinal pigment epithelial cultured cells

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International audience; Purpose: Among the retinal lipid deposits found in AMD (age related macular degeneration), esterified and unesterified cholesterol have been identified. Cholesterol that accumulates in Bruch's membrane could be oxidized and lead to cholesterol oxides known as oxysterols. These compounds have cytotoxic activities on many vascular cell types, and we conducted this study to evaluate the cytotoxics effects of oxysterols on retinal pigment epithelial (RPE) cells. Methods: Human RPE cells (ARPE–19) were cultured and treated by 7–ketocholesterol (7–keto), 7–betahydroxycholesterol (7–beta) and 25–hydroxycholesterol (25–OH). ARPE–19 cells viability was determined with MTT analysis after incubation with the different oxysterols. To specify the mechanisms toxicity, flow cytometry analysis was performed with propidium iodide that measure membrane permeability, acridine orange was used to evaluate lysosomal degradation and DiOC6(3) to assess mitochondrial transmembrane potential. Apoptosis cellular death characteristics were harvested with Hoechst 33342 nuclear staining, DNA gel electrophoresis and measurement of caspases activity by Fluorochrome–Labeled Inhibitors of Caspases (FLICA). Results : 7–beta and 7–keto had cytotoxics effects on ARPE–19 cells. MTT analysis showed a decrease of cellular viability and propidium iodide analysis revealed an increase in membrane permeability. 25–hydroxy had no toxics effects on ARPE–19 cells. Nuclear morphology of cells treated with oxysterols was not typical of an apoptosis nuclear morphology, there was no DNA internucleosomal fragmentation and no activation of caspases was identified by FLICA analysis. Conclusions: 7–keto and 7–beta induce cellular death without typical characteristics of apoptosis. However it seems that mitochondria and lysosoma play a role in the ARPE–19 death after exposure to 7–keto and 7–beta. The ARPE–19 death is probably mediated by an alternative independent caspases pathway.