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Bruce Pfeffer, Libin Xu, Ned Porter, Steven Fliesler; In Vitro Toxicity of 7-Dehydrocholesterol-Derived Oxysterols Depends on Retinal Cell Type. Invest. Ophthalmol. Vis. Sci. 2013;54(15):712.
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7-dehydrocholesterol (7DHC), an immediate precursor to cholesterol, accumulates in tissues of humans afflicted with Smith-Lemli-Opitz Syndrome (SLOS), and gives rise via in situ oxidation to a variety of oxysterols, some of which are cytotoxic (J Lipid Res 51:3259, 2010). Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS (Biochim Biophys Acta 1821:877, 2012). Selective loss of photoreceptors and attenuated electroretinogram responses characterize the retinal degeneration in this model, while the RPE layer remains intact, and Müller cells (MC) persist and become hypertrophic. Here, we evaluated the relative in vitro susceptibility of pure cultures of retina-derived cells to the cytotoxic effects of two 7DHC-derived oxysterols: 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD) and 7-ketocholesterol (7kCHOL).
Cultures of 661W cells (mouse cone-derived cell line), normal diploid monkey RPE cells, and rMC-1 cells (rat MC line), either subconfluent or confluent, were incubated with either EPCD or 7kCHOL at 0.1 - 45 µM. After 24 h, fluorescent cell viability assays were carried out: Calcein AM (CaAM) to assess metabolic capacity, and Sytox Orange (SO; binds to DNA) to assess loss of cell membrane integrity. Results were correlated with phase-contrast images of the cultures. Cells treated with staurosporine (Stsp) or cumene hydroperoxide (CuOOH) served as positive controls for cell death, while those treated with vehicle alone served as negative controls.
Cytotoxic potency of oxysterols was higher for subconfluent, vs. confluent, cells. By the CaAM assay, 45 µM 7kCHOL was required to significantly decrease RPE cell viability, while 23 and 30 µM sufficed to achieve comparable effects in 661W and rMC-1cells, respectively. Cytotoxicity of EPCD was much greater, giving a maximal SO readout at <3 µM for confluent 661W cells, vs. 10 µM for rMC-1 and 20 µM for RPE cells, respectively. RPE were resistant to doses of Stsp and CuOOH that were fully toxic to 661W cells.
Our results are consistent with the hypothesis that retinal degeneration in the SLOS rat model is due to the selective susceptibility of photoreceptor cells to the cytotoxicity of 7DHC-derived oxysterols, compared to other retinal cells.
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