Effects of 17β-estradiol on proliferation, cell viability and intracellular redox status in native human lens epithelial cells

Abstract

Purpose: The purpose of this study was to examine the effects of 17β-estradiol on proliferation, cell death and redox status in cultured human lens epithelial cells (HLECs).

Methods: HLECs were exposed to 17β-estradiol after which cell viability was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and the number of mitotic and apoptotic cell nuclei was determined after staining with Hoechst 33342. Apoptosis was also determined by measuring caspase-3 activity and propidium iodide was used to determine the proportion of non-viable cells. Pro- and antioxidative effects of 17β-estradiol was investigated by measuring peroxides, superoxides and glutathione, using dichlorofluorescein diacetate (DCFH-DA), dihydroethidium (HET), and monochlorobimane (MCB), respectively. Effects on mitochondrial membrane potential were determined using 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The ability of 17β-estradiol to prevent reactive oxygen species (ROS)-production in HLECs after exposure to 25 µM H₂O₂ for 24h was also measured.

Results: This study demonstrates increased mitotic activity in HLECs exposed to physiologic concentrations of 17β-estradiol (1 nM). Pharmacological concentrations of 17β-estradiol caused increased number of apoptotic cell nuclei and caspase-3 activation. Physiologic concentrations of 17β-estradiol (0.1-10 nM) stabilized the mitochondrial membrane potential. Similar or slightly higher concentrations of 17β-estradiol (0.01-1 µM) protected against H₂O₂-induced oxidative stress as evident by decreased levels of peroxides and superoxides.

Conclusions: The present study demonstrates mitogenic and anti-oxidative effects of 17β-estradiol at physiologic concentrations, whereas pharmacological levels induced oxidative stress and acted pro-apoptotic in cultured lens cells.

Figure 1

Effects on proliferation and cell death in human lens epithelial cells (HLECs) exposed to 17β-estradiol at different concentrations for 24 h. A: The change in number of viable cells (% of control) was determined by the MTT colorimetric assay. B: Difference in the number of mitotic cells (% of control) as evident after staining with Hoechst 33342. C, D: HLECs stained with PI after exposure to 10 µM 17β-estradiol and corresponding control. E: Differences in the number of dead cells (% of control) determined by labeling with PI. Mean±SEM is shown. Experiments were performed three times in triplicates (n=3) and one representative experimental run for each method is shown. *p<0.05 as compared to control without 17β-estradiol (0) exposure. Scale bar=100 µm.

Figure 2

Apoptosis in human lens epithelial cells (HLECs) exposed to 17β-estradiol at different concentrations for 24 h. A: Increase in Caspase-3 activity after 17β-estradiol exposure. B: Increased number of apoptotic cells (% of control) as evident by staining with Hoechst 33342. Mean±SEM is shown. Experiments were performed three times in triplicates (n=3) and one representative experimental run for each method is shown. *p<0.05 as compared to control without 17β-estradiol (0) exposure. C, D: HLECs stained with Hoechst after exposure to 10 µM 17β-estradiol and corresponding control. Insets show mitotic and apoptotic nucleus respectively. Scale bar=50 µm.

Figure 3

Effects on reactive oxygen species levels and mitochondrial membrane potential in human lens epithelial cells (HLECs), exposed to 17β-estradiol at different concentrations for 24 h. A: An increase in peroxide levels was observed at the highest concentration used, 10 µM. B: Elevated levels of superoxides was evident at 10 µM 17β-estradiol. C: The levels of reduced glutathione (GSH) decreased at 10 µM 17β-estradiol. D: An increase in mitochondrial membrane potential (JC-1) at lower concentrations, 0.1–10 nM, of 17β-estradiol. Changes are expressed as the ratio of red signal and green signal. Mean±SEM is shown. Experiments were performed three times in triplicates (n=3) and one representative experimental run for each method is shown. *p<0.05 as compared to control without 17β-estradiol (0) exposure.

Figure 4

Antioxidative effects of 17β-estradiol against H₂O₂ exposure. Human lens epithelial cells (HLECs) were preincubated with 17β-estradiol for 4 h and then simultaneously exposed to 25 µM H₂O₂ and 17β-estradiol for 24 h. A: A decrease in peroxide levels at low concentrations of 17β-estradiol (0.01–1 µM) was demonstrated. B: Superoxide levels were decreased at 0.01–1 µM 17β-estradiol. Mean±SEM is shown. Experiments were performed three times in triplicates (n=3) and one representative experimental run for each method is shown. *p<0.05 as compared to control without 17β-estradiol (0) exposure.