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Effects of dexamethasone on human lens epithelial cells in culture.

Petersen A, Carlsson T, Karlsson JO, Jonhede S, Zetterberg M - Mol. Vis. (2008)

Bottom Line: No effect on superoxide production by dexamethasone was seen.Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms.The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, the Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden. anne.petersen@gu.se

ABSTRACT

Purpose: Treatment with glucocorticoids is a well known risk factor for cataract development, although the pathogenic mechanism has not been elucidated. The aim of the study was to investigate the effects of glucocorticoids in cultured human lens epithelial cells.

Methods: Human lens epithelial cells (HLECs) were exposed to dexamethasone for 24 h. The number of viable cells was determined using the 3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide (MTT) assay, and proliferation was quantified using Ki-67. Apoptosis was investigated by measuring caspase-3 activity and by evaluating nuclear morphology of cells stained with Hoechst 33342. Mitochondria depolarization was measured using the potential-sensitive color, JC-1. Cells were assayed for changes in superoxide production using dihydroethidium (HET), for alterations in peroxide production using dichlorofluorescein diacetate (DCFH-DA), and for glutathione (GSH) variations using monochlorobimane (MCB). Caspase-3 activity was also measured in HLECs simultaneously exposed to dexamethasone and the glucocorticoid antagonist, RU486.

Results: Low doses of dexamethasone (0.1 microM) resulted in increased proliferation of HLECs. Apoptosis was increased in HLECs exposed to 1 microM, 10 microM, and 100 microM of dexamethasone as revealed by nuclear morphology studies. Apoptosis was also confirmed by measuring caspase-3 activation. No effect on superoxide production by dexamethasone was seen. There were no effects on GSH levels or mitochondrial depolarization either. Only the highest concentration of dexamethasone (100 microM) caused an increase in peroxide production. In HLECs incubated with the glucocorticoid antagonist, RU486, apoptosis was induced at a lower concentration of dexamethasone (0.1 microM) than with dexamethasone alone.

Conclusions: Low doses of dexamethasone cause a moderate increase in proliferation of cultured HLECs. Slightly higher but still physiologically relevant concentrations of dexamethasone result in a dose-dependent increase in apoptosis. Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms. The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor. Effects on proliferation and/or dysregulation of apoptosis in lens epithelial cells may be an important factor in human steroid-induced posterior subcapsular cataract.

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A: Superoxide production in human lens epithelial cells exposed to dexamethasone. Dexamethasone exposure to human lens epithelial cells for 24 h had no effect on superoxide production. Superoxide production was measured over time and expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). The experiment was repeated twice with similar results. B: Peroxide production in human lens epithelial cells exposed to dexamethasone is also demonstrated. Exposure of human lens epithelial cells to dexamethasone for 24 h resulted in increased peroxide production only at the highest concentration of 100 µM used. The experiment was repeated twice with similar results. Peroxide production is expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). Mean±SEM from three separate culture wells are shown; the asterisk indicates a p<0.05. C: Glutathione in human lens epithelial cells exposed to dexamethasone. Dexamethasone did not affect the GSH level in human lens epithelial cells after incubation with dexamethasone for 24 h. One representative experiment of three is shown. The level of GSH was measured after 2 h and expressed as relative fluorescence units and gram protein (RFU x g-1). Mean ±SEM from 3 separate culture wells is shown.
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f4: A: Superoxide production in human lens epithelial cells exposed to dexamethasone. Dexamethasone exposure to human lens epithelial cells for 24 h had no effect on superoxide production. Superoxide production was measured over time and expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). The experiment was repeated twice with similar results. B: Peroxide production in human lens epithelial cells exposed to dexamethasone is also demonstrated. Exposure of human lens epithelial cells to dexamethasone for 24 h resulted in increased peroxide production only at the highest concentration of 100 µM used. The experiment was repeated twice with similar results. Peroxide production is expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). Mean±SEM from three separate culture wells are shown; the asterisk indicates a p<0.05. C: Glutathione in human lens epithelial cells exposed to dexamethasone. Dexamethasone did not affect the GSH level in human lens epithelial cells after incubation with dexamethasone for 24 h. One representative experiment of three is shown. The level of GSH was measured after 2 h and expressed as relative fluorescence units and gram protein (RFU x g-1). Mean ±SEM from 3 separate culture wells is shown.

Mentions: Superoxide production in HLECs was not significantly changed after exposure to dexamethasone (Figure 4A) for 24 h. Cells treated with 100 µM dexamethasone showed increased peroxide production, which was not present at lower concentrations (Figure 4B). No effect of dexamethasone on GSH levels was evident in HLECs after 24 h exposure (Figure 4C).


Effects of dexamethasone on human lens epithelial cells in culture.

Petersen A, Carlsson T, Karlsson JO, Jonhede S, Zetterberg M - Mol. Vis. (2008)

A: Superoxide production in human lens epithelial cells exposed to dexamethasone. Dexamethasone exposure to human lens epithelial cells for 24 h had no effect on superoxide production. Superoxide production was measured over time and expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). The experiment was repeated twice with similar results. B: Peroxide production in human lens epithelial cells exposed to dexamethasone is also demonstrated. Exposure of human lens epithelial cells to dexamethasone for 24 h resulted in increased peroxide production only at the highest concentration of 100 µM used. The experiment was repeated twice with similar results. Peroxide production is expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). Mean±SEM from three separate culture wells are shown; the asterisk indicates a p<0.05. C: Glutathione in human lens epithelial cells exposed to dexamethasone. Dexamethasone did not affect the GSH level in human lens epithelial cells after incubation with dexamethasone for 24 h. One representative experiment of three is shown. The level of GSH was measured after 2 h and expressed as relative fluorescence units and gram protein (RFU x g-1). Mean ±SEM from 3 separate culture wells is shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480483&req=5

f4: A: Superoxide production in human lens epithelial cells exposed to dexamethasone. Dexamethasone exposure to human lens epithelial cells for 24 h had no effect on superoxide production. Superoxide production was measured over time and expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). The experiment was repeated twice with similar results. B: Peroxide production in human lens epithelial cells exposed to dexamethasone is also demonstrated. Exposure of human lens epithelial cells to dexamethasone for 24 h resulted in increased peroxide production only at the highest concentration of 100 µM used. The experiment was repeated twice with similar results. Peroxide production is expressed as relative fluorescence units per second and gram protein (RFU s−1g−1). Mean±SEM from three separate culture wells are shown; the asterisk indicates a p<0.05. C: Glutathione in human lens epithelial cells exposed to dexamethasone. Dexamethasone did not affect the GSH level in human lens epithelial cells after incubation with dexamethasone for 24 h. One representative experiment of three is shown. The level of GSH was measured after 2 h and expressed as relative fluorescence units and gram protein (RFU x g-1). Mean ±SEM from 3 separate culture wells is shown.
Mentions: Superoxide production in HLECs was not significantly changed after exposure to dexamethasone (Figure 4A) for 24 h. Cells treated with 100 µM dexamethasone showed increased peroxide production, which was not present at lower concentrations (Figure 4B). No effect of dexamethasone on GSH levels was evident in HLECs after 24 h exposure (Figure 4C).

Bottom Line: No effect on superoxide production by dexamethasone was seen.Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms.The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, the Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden. anne.petersen@gu.se

ABSTRACT

Purpose: Treatment with glucocorticoids is a well known risk factor for cataract development, although the pathogenic mechanism has not been elucidated. The aim of the study was to investigate the effects of glucocorticoids in cultured human lens epithelial cells.

Methods: Human lens epithelial cells (HLECs) were exposed to dexamethasone for 24 h. The number of viable cells was determined using the 3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide (MTT) assay, and proliferation was quantified using Ki-67. Apoptosis was investigated by measuring caspase-3 activity and by evaluating nuclear morphology of cells stained with Hoechst 33342. Mitochondria depolarization was measured using the potential-sensitive color, JC-1. Cells were assayed for changes in superoxide production using dihydroethidium (HET), for alterations in peroxide production using dichlorofluorescein diacetate (DCFH-DA), and for glutathione (GSH) variations using monochlorobimane (MCB). Caspase-3 activity was also measured in HLECs simultaneously exposed to dexamethasone and the glucocorticoid antagonist, RU486.

Results: Low doses of dexamethasone (0.1 microM) resulted in increased proliferation of HLECs. Apoptosis was increased in HLECs exposed to 1 microM, 10 microM, and 100 microM of dexamethasone as revealed by nuclear morphology studies. Apoptosis was also confirmed by measuring caspase-3 activation. No effect on superoxide production by dexamethasone was seen. There were no effects on GSH levels or mitochondrial depolarization either. Only the highest concentration of dexamethasone (100 microM) caused an increase in peroxide production. In HLECs incubated with the glucocorticoid antagonist, RU486, apoptosis was induced at a lower concentration of dexamethasone (0.1 microM) than with dexamethasone alone.

Conclusions: Low doses of dexamethasone cause a moderate increase in proliferation of cultured HLECs. Slightly higher but still physiologically relevant concentrations of dexamethasone result in a dose-dependent increase in apoptosis. Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms. The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor. Effects on proliferation and/or dysregulation of apoptosis in lens epithelial cells may be an important factor in human steroid-induced posterior subcapsular cataract.

Show MeSH
Related in: MedlinePlus