Effects of melatonin and its receptor antagonist on retinal pigment epithelial cells against hydrogen peroxide damage

Abstract

Purpose: Recently, we reported finding that circulating melatonin levels in age-related macular degeneration patients were significantly lower than those in age-matched controls. The purpose of this study was to investigate the hypothesis that melatonin deficiency may play a role in the oxidative damage of the retinal pigment epithelium (RPE) by testing the protective effect of melatonin and its receptor antagonist on RPE cells exposed to H(2)O(2) damage.

Methods: Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H(2)O(2). Cell viability was measured using the microculture tetrazoline test (MTT) assay. Cells were pretreated with or without melatonin for 24 h. Luzindole (50 μM), a melatonin membrane-receptor antagonist, was added to the culture 1 h before melatonin to distinguish direct antioxidant effects from indirect receptor-dependent effects. All tests were performed in triplicate.

Results: H(2)O(2) at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H(2)O(2). Cell viability of RPE cells pretreated with 10(-10), 10(-8), 10(-6), and 10(-4) M melatonin for 24 h was 130%, 160%, 187%, and 230% of cells treated with H(2)O(2) alone (all p<0.05). Using cells cultured without H(2)O(2) as the control, cell viability of cells treated with H(2)O(2) after pretreatment with 10(-10)-10(-4) M melatonin was still significantly lower than that of the controls, suggesting that melatonin significantly decreased but did not completely abolish the in vitro cytotoxic effects of H(2)O(2). Luzindole completely blocked melatonin's protective effects at low concentrations of melatonin (10(-10)-10(-8) M) but not at high concentrations (10(-6)-10(-4) M).

Conclusions: Melatonin has a partial protective effect on RPE cells against H(2)O(2) damage across a wide range of concentrations (10(-10)-10(-4) M). This protective effect occurs through the activation of melatonin membrane receptors at low concentrations (10(-10)-10(-8) M) and through both the direct antioxidant and indirect receptor activation effects at high concentrations (10(-6)-10(-4) M).

Figure 1

Melatonin protected the retinal pigment epithelium (RPE) cells against H₂O₂ damage, especially in high concentrations (10⁻⁴ M) and luzindole decreased the protective effects of melatonin. Phase-contrast microscopic images of the effects of melatonin and its membrane-receptor antagonist (luzindole) on retinal pigment epithelial cells against H₂O₂ damage. A-F: The RPE cells are from the ARPE-19 cell line (an immortal RPE cell line from a 19-year-old donor). G-L: The RPE cells are from the primary culture (PC) from the donor eye. ARPE-19 and PC cells were cultured with or without H₂O₂, melatonin, and luzindole, as follows: without H₂O₂, melatonin, and luzindole(A and G); with H₂O₂ at 0.5 mM concentrations for 24 h (B and H); with H₂O₂ and a pretreatment of melatonin at 10⁻¹⁰ M (C and I) or at 10⁻⁴ M (D and J) for 24 h; or with luzindole (50 μM, 1 h), followed by melatonin at 10⁻¹⁰ M (E and K) or at 10⁻⁴ M for 24 h (F and L). H₂O₂ was then added and the ARPE-19 and PC cells cultured for 24 h.

Figure 2

H₂O₂ dose-dependently decreased cell viability of retinal pigment epithelial cells as tested by microculture tetrazoline test. Retinal pigment epithelial (RPE) cells were plated in 96-well plates and treated with various concentrations of H₂O₂ for 24 h. Cell viability was evaluated by the microculture tetrazoline test (MTT) test and expressed as percentage of that in cells without H₂O₂ (mean±standard deviation [SD] in triplicate tests). A: H₂O₂ showed dose-dependent cytotoxic effects on ARPE-19 cells (cells from an immortal RPE cell line from a 19-year-old donor) at concentrations from 0.25 mM to 1.00 mM (p<0.05, compared to cells cultured without H₂O₂. B: Studies of primary cultures of human RPE cells isolated from donor eye showed similar results. *p<0.05, compared with the controls (cells cultured with H₂O₂ alone).

Figure 3

The ARPE19 cells (an immortal retinal pigment epithelial cell line from a 19-year-old donor) were treated with melatonin (M) at different concentrations. After 1 h, 24 h, and 48 h culture, 0.5 mM H₂O₂ (H) was added and cultured for 24 h. Cells cultured with H₂O₂ alone were used as the controls. Cell viability was evaluated by the microculture tetrazoline test and expressed as percentages of controls (mean±SD in triplicate tests). Error bars represent SD A: Pretreatment with low concentrations of melatonin at 10⁻¹⁰ M (M-10) for 1 h, 24 h, and 48 h significantly protected cells against H₂O₂. B: Pretreatment with high concentrations of melatonin at 10⁻⁶ M (M-6) obtained similar results. The difference between cells cultured with and without melatonin at both high and low concentrations was statistically significant at all three different pretreatment periods. *p<0.05, compared with the controls.

Figure 4

Melatonin dose-dependently protected retinal pigment epithelial cells against H₂O₂ damage as tested by microculture tetrazoline test. Retinal pigment epithelial (RPE) cells were pretreated with melatonin (M) at concentrations of 10⁻¹⁰ M (M-10), 10⁻⁸ M (M-8), 10⁻⁶ M (M-6), and 10⁻⁴ M (M-4). After 24 h, 0.5 mM H₂O₂ (H) was added and cultured for 24 h. Cells treated with H₂O₂ alone were used as the controls (H). Cell viability was evaluated by the microculture tetrazoline test and expressed as percentages of the controls (mean±standard deviation [SD] in triplicate tests). Error bars represent SD. Pretreatment with melatonin showed dose-dependent protective effects on ARPE-19 cells (an immortal RPE cell line from a 19-year-old donor) against H₂O₂ damage (A). Studies in primary culture of human RPE cells isolated from the donor eye showed similar results (B). *p<0.05, compared with the controls (cells treated with H₂O₂ alone).

Figure 5

Melatonin dose-dependently protected retinal pigment epithelial cells against H₂O₂ damage as tested by microculture tetrazoline test, compared with cells not treated with H₂O₂. Retinal pigment epithelial (RPE) cells were pretreated with melatonin (M) at concentrations of 10⁻¹⁰ M (M-10), 10⁻⁸ M (M-8), 10⁻⁶ M (M-6), and 10⁻⁴ M (M-4). After 24 h, 0.5 mM H₂O₂ (H) was added and cultured for 24 h. Cells not treated with H₂O₂ were used as negative controls (0). Cell viability was evaluated by the microculture tetrazoline test and expressed as percentages of negative controls (mean±standard deviation [SD] in triplicate tests). Error bars represent SD A: Cell viability of cell treated with melatonin and H₂O₂ still significantly lower than that in cells cultured without H₂O₂ in the ARPE-19 cells (an immortal RPE cell line from a 19-year-old donor). B: The same was true in the primary-culture RPE cells. * p<0.05, compared with the negative controls (cells treated without H₂O₂).

Figure 6

Luzindole decreased protective effects of melatonin on retinal pigment epithelial cells against H₂O₂ damage as tested by microculture tetrazoline test. Cultured retinal pigment epithelial (RPE) cells were treated with or without 50 μM luzindole (L). One hour later melatonin (M) was added to the culture medium at concentrations of 10⁻¹⁰ M (M-10), 10⁻⁸ M (M-8), 10⁻⁶ M (M-6), and 10⁻⁴ M (M-4). After 24 h, 0.5 mM H₂O₂ (H) was added and cultures were incubated for 24 h. Cell viability was evaluated by the microculture tetrazoline test and expressed as percentages of cells cultured with H₂O₂ alone (mean±standard deviation [SD] in triplicate tests). Error bars represent SD A: Luzindole significantly decreased melatonin-induced protective effects at 10⁻¹⁰ to 10⁻⁴ M in ARPE-19 cells (an immortal RPE cell line from a 19-year-old donor). B: The same was true in the primary-culture RPE cells. * p<0.05, comparison between cells treated with and without luzindole.