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J. W. Lee, J. Sanchez, R. Riddick, J.-D. Huang, S. J. Fliesler, I. R. Rodriguez; Potentiation of Visible Light-Induced Photodamage in Cultured RPE Cells by Treatment With AY9944, A 7-Dehydrocholesterol Reductase Inhibitor. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1843.
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To determine if accumulation of 7-dehydrocholesterol (7DHC) sensitizes RPE cells to visible light-induced photodamage. Also, to identify 7DHC-derived cytotoxic compounds generated by photo-oxidation.
ARPE19 cells were treated with various concentrations of AY9944 in serum-free medium. 7DHC levels were determined by HPLC 24 and 48 h post-treatment. AY9944-treated cells were exposed to white light (570~650 lux) for 5-7 days at 37°C or kept in complete darkness (controls). Cell morphology was monitored by light microscopy. Apoptosis was detected using a TUNEL assay (Roche). 7-dehydrocholesterol reductase (DHCR7) mRNA expression was measured by qRT-PCR. Cell pellets and conditioned media were collected and aqueous- and lipid-soluble extracts were tested for cytotoxicity using a cellular dehydrogenase cell viability assay. The cytotoxic lipid extract was subfractionated by HPLC. The toxic subfractions were analyzed by LCMS.
ARPE19 cells contain levels of DHCR7 mRNA comparable to those in human brain. AY9944 (3 µM) treatment alone increased the levels of 7DHC by 50-fold relative to untreated controls in 48 h without compromising cell viability or morphology. AY9944-treated cells exposed to constant visible light died in 5-7 days, while AY9944-treated cells kept in the dark remained healthy. Lipid extracts from the dead cells caused cell death when given to fresh ARPE19 cells. Identification of the cytotoxic components by LCMS is in progress.
7DHC accumulation potentiates visible light-induced photodamage of cultured RPE cells. These findings may have important implications to retinal degeneration in patients with Smith-Lemli-Opitz syndrome (SLOS), where DHCR7 is defective. Finding ways to block the formation of cytotoxic 7DHC by-products may provide a useful therapeutic intervention in SLOS, particularly with respect to retinal photodamage.
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