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 H(2)O(2) 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 17beta-estradiol (17beta-E(2)) in protection against oxidative stress, HLECs were exposed to insult with H(2)O(2) at a physiological level (100 microM) over a time course of several hours, with and without pretreatment with 17beta-E(2). 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 (deltapsi(m)) was monitored by a fluorescence resonance energy-transfer technique. RESULTS: H(2)O(2) 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 17beta-E(2) for 2 hours before oxidative insult. At 1 nM, 17beta-E(2) 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 17beta-E(2) at 100 nM or higher. Pretreatment with 17beta-E(2) did not diminish intracellular ROS accumulation after exposure to H(2)O(2). Moreover, two nonfeminizing estrogens, 17alpha-E(2) and ent-E(2), both of which do not bind to either estrogen receptor alpha or beta, were as effective as 17beta-E(2) in the recovery of cell viability. The estrogen receptor antagonist, ICI 182,780, did not block protection by 17beta-E(2). Both 17beta- and 17alpha-E(2) moderated the collapse of deltapsi(m) in response to either H(2)O(2) or excessive Ca(2+) loading. CONCLUSIONS: The present study indicates that both 17alpha- and 17beta-E(2) 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 H(2)O(2) toxicity indicates that this conservation is not likely to be mediated through classic estrogen receptors.
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 H(2)O(2) 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 17beta-estradiol (17beta-E(2)) in protection against oxidative stress, HLECs were exposed to insult with H(2)O(2) at a physiological level (100 microM) over a time course of several hours, with and without pretreatment with 17beta-E(2). 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 (deltapsi(m)) was monitored by a fluorescence resonance energy-transfer technique. RESULTS:H(2)O(2) 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 17beta-E(2) for 2 hours before oxidative insult. At 1 nM, 17beta-E(2) 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 17beta-E(2) at 100 nM or higher. Pretreatment with 17beta-E(2) did not diminish intracellular ROS accumulation after exposure to H(2)O(2). Moreover, two nonfeminizing estrogens, 17alpha-E(2) and ent-E(2), both of which do not bind to either estrogen receptor alpha or beta, were as effective as 17beta-E(2) in the recovery of cell viability. The estrogen receptor antagonist, ICI 182,780, did not block protection by 17beta-E(2). Both 17beta- and 17alpha-E(2) moderated the collapse of deltapsi(m) in response to either H(2)O(2) or excessive Ca(2+) loading. CONCLUSIONS: The present study indicates that both 17alpha- and 17beta-E(2) 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 H(2)O(2)toxicity indicates that this conservation is not likely to be mediated through classic estrogen receptors.