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Bruce A. Pfeffer, Priyanka H. Patel, Libin Xu, Ned A. Porter, Steven J. Fliesler; 7-Dehydrocholesterol-derived Oxysterols are Differentially Cytotoxic to 661W Photoreceptor Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4300.
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© ARVO (1962-2015); The Authors (2016-present)
7-dehydrocholesterol (7DHC), an immediate precursor of cholesterol (CHOL), accumulates as the dominant sterol in tissues and bodily fluids of humans afflicted with Smith-Lemli-Opitz Syndrome (SLOS), a recessive metabolic disease. 7DHC is ca. 200-fold more labile to peroxidation than is CHOL, and gives rise to a variety of oxysterol products, some of which are cytotoxic (Korade et al., JLR 2010). Retinal degeneration (involving progressive photoreceptor death), along with 7DHC and 7DHC-derived oxysterol formation, occur in the AY9944-induced rat model of SLOS (Fliesler et al., Arch Ophthalmol. 2004; Xu et al., ARVO 2011). Here, we evaluated the relative susceptibility of cultured 661W cells (a photoreceptor-derived cell line) to the cytotoxic effects of 7DHC-derived oxysterols.
661W cells were incubated with 7-ketocholesterol (7kCHOL), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4β-hydroxy-7DHC (4HDHC), 5,9-endoperoxy-cholest-7-en-3β,6β-diol (EPCD), or CHOL, each at 0.1 to 30 µM, solvated with hydroxypropyl-β-cyclodextrin (HPCD, 0.96 mM final conc’n), in DMEM/F12 culture media containing 0.2% (v/v) serum (1.9 µM CHOL). After 48 h, cellular metabolic competence was assessed by resazurin reduction (RzR) assay, while cell death was assayed by Sytox Orange (SO) binding; SO values were normalized to RzR values (the latter proportional to cell number). Cell morphology was monitored by indirect photomicroscopy.
At 30 µM, both 7kCHOL and DHCEO were markedly cytotoxic, causing cell rounding/blebbing and detachment, precluding quantitative SO measurements, whereas 30 µM 4HDHC was moderately well-tolerated. CHOL was not soluble at 30 µM in HPCD/medium. At 3 and 10 µM, the order of cytotoxicity was: EPCD >> 7kCHOL > DHCEO > 4HDHC >> CHOL (CHOL was comparable to medium alone, +/- HPCD). EPCD exhibited toxicity even at 1 µM.
661W cells exhibit differential cytotoxicity to 7DHC-derived oxysterols. Our results are consistent with the hypothesis that such oxysterols are involved in the progressive photoreceptor degeneration observed in the SLOS rat model, and potentially in SLOS as well.
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