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R.E. Neal, C. Lin, J.S. Zigler, Jr; Pb Exposure Induces Cataract Formation in Cultured Rat Lens and Alters Cytoskeletal Protein Profiles . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3491.
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Purpose: To examine the effect of Pb on cultured lens cytoskeletal protein expression and to determine if Pb can induce lens opacity in vitro. Methods: Lenses for Sprague-Dawley rats (75-100 g) were cultured in modified TC-199 medium containing 2.5 µM Pb for 4-10 days. Upon culture termination, whole lenses were solubilized with 9M urea and stored at –70C until electrophoresis. Using 2D minigels with Colloidal Blue or silver staining, alterations in the abundance of selected cytoskeletal proteins and crystallin degradation products were analyzed with Phoretix image analysis software. Proteins were identified via MALDI-TOF-MS following spot excision and tryptic digestion. A minimum of 20% protein coverage with 4 peptides identified was used for spot identification. Results: Pb exposure induced isolated ocular opacities which progressed to nuclear cataract by culture day 12. Over 40 spots in the molecular weight range of 40-97 kDa and numerous crystallin degradation products in the range of 14-20 kDa were visualized on the 2D gels. We have identified 12 protein spot chains present in the control cultured lenses which includes HSC 70, filensin, phakinin, actin, α-enolase and tubulin. Commitant with the cataract formation observed following Pb exposure, increased degradation of crystallins including α-crystallin and decreased abundance of specific cytoskeletal proteins including phakinin and filensin was observed. Conclusions: Physiologically relevant dosages of Pb can induce lenticular opacity in vitro and degradation of crystallins and cytoskeletal proteins. This suggests alterations in Ca-dependent proteases occur with Pb exposure.
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