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J.M. Flynn, P.R. Cammarata; 17ß–Estradiol Attenuates Mitochondrial Depolarization in Polyol–Stressed Cultured Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3865.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Lens epithelium cultured in high extracellular galactose converts the aldose sugar to its respective sugar alcohol, galactitol (GalOH), via aldose reductase (AR). This study determined the consequence of GalOH accumulation on mitochondrial membrane potential (ΔΨm) and if estrogen (17ß–E2) or the AR inhibitor (ARI), Sorbinil administered prior to and concomitant with galactose exposure might prevent or delay mitochondrial depolarization. Methods: Normal secondary cultures of bovine lens epithelial cells (BLECs) and a virally–transformed human lens epithelial cell line (HLE–B3) were maintained in 40 mM galactose (Gal). Endogenous accumulation of reactive oxygen species (ROS) was assessed by loading cells with DCFH–DA which upon oxidation in the presence of ROS becomes the fluorescent compound DCF. In order to assess ΔΨm, the potentiometric fluorescent dye, JC–1 was used in conjunction with confocal microscopy. Red fluorescence indicates polarization; green fluorescence is typical of mitochondrial depolarization. 17ß–E2 as well as its isomer, 17α–E2 (which exhibits marginal binding affinity for estrogen receptor) as well as the ARI, Sorbinil were administered to the cell cultures to determine their comparative protective effects. Eight random confocal images per treatment were evaluated for green/red fluorescence ratio in order to determine the extent of mitochondrial membrane depolarization after 3 days of Gal ± estradiol or ARI. Results: BLECs more so than HLE–B3 cells accumulate high intracellular levels of GalOH with exposure to ambient Gal. BLECs were significantly depolarized while HLE–B3 cells showed little to no depolarization over the same course of Gal exposure. Intracellular accumulation of ROS exhibited marginal increase over control cells in BLECs and was similar to levels seen with estradiol or ARI administration. The addition of either 17α–E2 or 17ß–E2 to BLECs over a dose range of 0.01 µM to 1.0 µM completely prevented mitochondrial membrane potential depolarization, as did the addition of Sorbinil. Conclusions: This study confirms that polyol accumulation promotes mitochondrial depolarization and that the decrease in ΔΨm is prevented by prior treatment and co–administration of estradiol or ARI with Gal. Estradiol is likely not acting as an ARI nor can the Gal–induced depolarization be attributed to intracellular ROS production. While the precise mode of targeted mitochondrial protection has yet to be determined, results with 17α–E2 suggests that the stabilization of ΔΨm against polyol accumulation is mediated via an estrogen receptor–independent mechanism.
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