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M.E. Verdugo, D. Simic, A.B. Jakowski, D.J. Baltrukonis, C.J. Somps, M.W. Tengowski; Investigating Cytoskeletal Alterations as a Potential Marker of Retinal and Lens Drug Related Toxicity . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3653.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Actin filaments play a critical role in the normal physiology of lenticular and retinal tissue of the eye. Disruption of actin is associated with retinal pathology and lens cataract. The purpose of this study is to investigate changes in the structure of actin filaments as a potential marker of drug–induced retinal and lens toxicity. Methods: Lens Epithelial Cells (LEP) (human cell line (SRA 01/04) and rat primary (RLE)) and Retinal Pigment Epithelial cells (RPE) (rat cell line (RPE–J), human cell line (ARPE–19) and monkey primary retinal epithelial cells) were grown to 80% confluency. Cells were treated for 24h with positive control Cytochalasin D or with increasing concentrations of Tamoxifen, and compared to untreated controls. Viability was assessed using the wst–1 test in cells treated with increasing concentrations of Tamoxifen. Cells were treated using non– cytotoxic doses of the compound, fixed, stained for actin with Rhodamine Phalloidin, and counterstained for nuclei with TOTO–3. Confocal imaging was preformed using a Leica SP Laser Scanning Microscope at 40X resolution. Results: Cytochalasin D used at a non–cytotoxic concentration of 2uM caused a considerable amount of F–actin depolymerization. Based on the Wst–1 viability assay Tamoxifen is cytotoxic at concentrations ≥ 50uM in all cells tested. Tamoxifen induced concentration dependant changes of F–Actin in SRA 01/04, RLE, RPE–J, ARPE–19 and Monkey primary retinal epithelial cells at non–cytotoxic drug levels. In lens cells, reduced actin fluorescence was observed at 25uM of the drug, while in retinal cells actin is affected at doses as low as 1uM. The effects ranged between cell types and were expressed as loss/reduction of stress fibers, focal adhesion points and submembranous actin or simple breakage and subsequent clumping of actin fibers. Conclusions: Tamoxifen, a known in vivo lens and retinal toxicant, caused a reduction in F–actin fluorescence at non–cytotoxic concentrations in all cells tested. Drug–induced cytoskeletal alterations may be useful as a potential marker of retinal and lens toxicity.
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