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Oxidative Damage to Human Lens Epithelial Cells in Culture: Estrogen Protection of Mitochondrial Potential, ATP, and Cell Viability

¹From the Departments of Pharmacology and Neuroscience and ³Pathology and Anatomy, the ⁵Division of Cell Biology and Genetics, and the ⁶North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas; the ²Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, Florida; and ⁴MitoKor, San Diego, California.

PURPOSE. Epidemiologic studies demonstrate a higher incidence of cataracts in estrogen-deprived postmenopausal women, but the mechanism for the increased risk of cataracts is unclear. An elevated level of H₂O₂ in aqueous humor and whole lenses has been associated with cataractogenesis. In the present study, for the first time, the protective effect of estrogens against oxidative stress were tested in cultured human lens epithelial cells (HLECs).

METHODS. To investigate the involvement of 17ß-estradiol (17ß-E₂) in protection against oxidative stress, HLECs were exposed to insult with H₂O₂ at a physiological level (100 µM) over a time course of several hours, with and without pretreatment with 17ß-E₂. Cell viability was measured by calcein AM assay, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to determine intracellular reactive oxygen species (ROS). Intracellular adenosine triphosphate (ATP) level was quantified with a luciferin- and luciferase-based assay and mitochondrial potential (_m) was monitored by a fluorescence resonance energy-transfer technique.

RESULTS. H₂O₂ caused a dose-dependent decrease in mitochondrial membrane potential, intracellular ATP levels, and cell viability. Dose-dependent increases in cell viability and intracellular ATP level were observed with pretreatment of 17ß-E₂ for 2 hours before oxidative insult. At 1 nM, 17ß-E₂ increased cell viability from 39% ± 4% to 75% ± 3%, and at 100 nM or higher, it increased survival to greater than 95%. The level of intracellular ATP approached normal with 17ß-E₂ at 100 nM or higher. Pretreatment with 17ß-E₂ did not diminish intracellular ROS accumulation after exposure to H₂O₂. Moreover, two nonfeminizing estrogens, 17-E₂ and ent-E₂, both of which do not bind to either estrogen receptor or ß, were as effective as 17ß-E₂ in the recovery of cell viability. The estrogen receptor antagonist, ICI 182,780, did not block protection by 17ß-E₂. Both 17ß- and 17-E₂ moderated the collapse of _m in response to either H₂O₂ or excessive Ca²⁺ loading.

CONCLUSIONS. The present study indicates that both 17- and 17ß-E₂ can preserve mitochondrial function, cell viability, and ATP levels in human lens cells during oxidative stress. Although the precise mechanism responsible for protection by the estradiols against oxidative stress remains to be determined, the ability of nonfeminizing estrogens, which do not bind to estrogen receptors, to protect against H₂O₂ toxicity indicates that this conservation is not likely to be mediated through classic estrogen receptors.

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