title: Differential cytotoxic effects of 7-dehydrocholesterol-derived oxysterols on cultured retina-derived cells: Dependence on sterol structure, cell type, and density.
abstract: Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4β-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL >> DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that retinal degeneration as well as to SLOS.
authors: Research Service, VA Western New York Healthcare System, Buffalo, NY, USA; SUNY Eye Institute, Buffalo, NY, USA; Departments of Ophthalmology and Biochemistry, University at Buffalo, The State University of New York (SUNY), Buffalo, NY, USA.
authors: Bruce A
authors: BA
authors: Pfeffer
authors: Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA.
authors: Libin
authors: L
authors: Xu
authors: Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA.
authors: Ned A
authors: NA
authors: Porter
authors: Research Service, VA Western New York Healthcare System, Buffalo, NY, USA; SUNY Eye Institute, Buffalo, NY, USA; Departments of Ophthalmology and Biochemistry, University at Buffalo, The State University of New York (SUNY), Buffalo, NY, USA.
authors: Sriganesh Ramachandra
authors: SR
authors: Rao
authors: Research Service, VA Western New York Healthcare System, Buffalo, NY, USA; SUNY Eye Institute, Buffalo, NY, USA; Departments of Ophthalmology and Biochemistry, University at Buffalo, The State University of New York (SUNY), Buffalo, NY, USA. Electronic address: fliesler@buffalo.edu.
authors: Steven J
authors: SJ
authors: Fliesler
mesh_terms: D000818:Animals; D002452:Cell Count; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003684:Dehydrocholesterols / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D063928:Ependymoglial Cells / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects*; D008253:Macaca mulatta; D023421:Models, Animal; D000072376:Oxysterols / Q000494:pharmacology*; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects*; D051381:Rats; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology
chemical_list: D003684:Dehydrocholesterols; D000072376:Oxysterols; C016705:7-dehydrocholesterol
keywords: Cell culture; Cell viability assay; Müller cell; Oxysterol; Photoreceptor; Retinal pigment epithelium