Abstract
Abstract: :
Purpose: To develop an experimental system for continuous evaluation of oxidative stress–induced damage in cultured human epithelial cells (HLEC) and intact mouse lenses. Especially we want to analyze effects of glucose overexposure in order to elucidate the mechanism behind diabetic cataract. Methods: Confluent HLEC and intact mouse lenses were exposed to 50 mM glucose during 24 hours prior to analysis. The use of cell permeable fluorogenic substrates allowed us to follow, in real time, biochemical intracellular changes. For this propose 5 µg/ml rhodamine 123 (R123) and 25 µM monochlorobimane (MCB) were used for analysis of alterations in mitochondrial membrane potential and reduced glutathione (GSH) levels respectively. The production of superoxide was monitored via 5 µM hydroethidium (HET), peroxides were asseyed with 20 µM 2',7'–dichlorofluorescein–diacetate (DCFH–DA). 50 µM Suc–Leu–Leu–Tyr–Val–7–amido–4–methylcoumarin (LLVY–AMC) was used for detection of proteolytic activity. Histological sections of cultured mouse lenses were examined under a light microscope. Results: Histological sections showed fiber cell swelling, globulization and liquefaction of equatorial cortex. The epithelial layer in the germinative zone shows an increase in cell height and signs of degeneration. However the lenses were still clear before fixation. In the presence of 50 mM glucose proteolytic activities were 1,7 and 3,8 fold higher in intact mouse lens and HLEC, respectively. The level of GSH was significantly increased by 18,1 % during high glucose. There was also a significant decrease by 16,9% in peroxide levels in HLEC but an increase by 31,6% in the intact mouse lenses. There were almost no changes in superoxide levels and mitochondrial membrane potential in comparison with the control. Conclusions: High glucose induced an adaptive antioxidant response in HLEC. In the formation of diabetic cataract it is possible that activation of protease systems are due to oxidative stress that might originate from changes in the intracellular levels of glucose or/and its metabolites.
Keywords: cataract • oxidation/oxidative or free radical damage • proteolysis