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O. Ringen, A. Oscoz, K. Haug, M. Moe, C. Zetterstrom, A. Collins, B. Nicolaissen; Human Lens Epithelium in Cataract: Qualitative and Quantitative Evaluation of DNA Damage, Apoptosis and Evaluation of a System for Intervention Assays. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2622.
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© ARVO (1962-2015); The Authors (2016-present)
Cell damage including apoptosis as well as UV- and oxidative induced DNA damage are pathogenetic factors in the development of human cataract. The main objective was to focus on apoptosis, characterise DNA damage and in addition to explore the usefulness of an ex vivo system for intervention studies.
The anterior lens capsule with the monolayered anterior lens epithelial cells was obtained during cataract surgery (n=48). Consecutive samples were immediately analyzed by the comet assay, others fixed and processed for immunohistochemistry or TEM. A third group was cultured in DMEM-F12 with 15% FBS for 2 weeks prior to examination by light microscopy and TEM.
In fresh lens epithelial cells we observed relatively high levels of formamidopyrimidine DNA glycosylase (FPG) -sensitive sites compared with endonuclease III - sensitive sites. Generally there were very low levels of strand breaks. Immunohistochemical staining for PCNA was prominently positive whereas Ki67, Caspase 3 and TUNEL were negative.Light microscopy and TEM of cultured epithelium showed a basically retained morphology.
For the first time we demonstrate the DNA damage profile using the comet assay in human lens epithelial cells from patients with cataract. The very low levels of strand breaks reflect the excellent quality and ease of handling of these cells. Most striking are high levels of FPG-sensitive sites compared with EndoIII-sensitive sites, which might indicate a preponderance of damage caused by singlet oxygen - a well-characterised mode of action of light in the presence of a variety of photosensitisers, which leads to 8-oxoguanine as the predominant product. Our results further indicate a high level of DNA repair, a neglectable level of apoptotic activity, and also the usefulness of the ex vivo system in assays aimed at exploring the activity of biosubstances in prophylaxis of DNA damage and as effectors of DNA repair.
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