Oxysterol-induced toxicity in R28 and ARPE-19 cells.
Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0-50 microg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe-based assay (2',7'-dichlorodihydrofluorescein diacetate [H2DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 micro/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol-treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.
Ong
John M
JM
Molecular Eye Research Laboratory, Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA. ongj@cshs.org
Aoki
Annette M
AM
Seigel
Gail M
GM
Sacerio
Ingrid
I
Castellon
Raquel
R
Nesburn
Anthony B
AB
Kenney
M Cristina
MC
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006888:Hydroxycholesterols / Q000633:toxicity*; D006914:Hydroxysteroids / Q000633:toxicity; D007653:Ketocholesterols / Q000633:toxicity*; D007700:Kinetics; D051381:Rats; D012160:Retina
D006888:Hydroxycholesterols; D006914:Hydroxysteroids; D007653:Ketocholesterols; C007997:25-hydroxycholesterol; C003001:7-ketocholesterol