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, 9 (3), 287

Light Action Spectrum on Oxidative Stress and Mitochondrial Damage in A2E-loaded Retinal Pigment Epithelium Cells

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Light Action Spectrum on Oxidative Stress and Mitochondrial Damage in A2E-loaded Retinal Pigment Epithelium Cells

Mélanie Marie et al. Cell Death Dis.

Abstract

Aims: Blue light is an identified risk factor for age-related macular degeneration (AMD). We investigated oxidative stress markers and mitochondrial changes in A2E-loaded retinal pigment epithelium cells under the blue-green part of the solar spectrum that reaches the retina to better understand the mechanisms underlying light-elicited toxicity.

Results: Primary retinal pigment epithelium cells were loaded with a retinal photosensitizer, AE2, to mimic aging. Using a custom-made illumination device that delivers 10 nm-wide light bands, we demonstrated that A2E-loaded RPE cells generated high levels of both hydrogen peroxide (H2O2) and superoxide anion (O2•-) when exposed to blue-violet light. In addition, they exhibited perinuclear clustering of mitochondria with a decrease of both their mitochondrial membrane potential and their respiratory activities. The increase of oxidative stress resulted in increased levels of the oxidized form of glutathione and decreased superoxide dismutase (SOD) and catalase activities. Furthermore, mRNA expression levels of the main antioxidant enzymes (SOD2, catalase, and GPX1) also decreased.

Conclusions: Using an innovative illumination device, we measured the precise action spectrum of the oxidative stress mechanisms on A2E-loaded retinal pigment epithelium cells. We defined 415-455 nm blue-violet light, within the solar spectrum reaching the retina, to be the spectral band that generates the highest amount of reactive oxygen species and produces the highest level of mitochondrial dysfunction, explaining its toxic effect. This study further highlights the need to filter these wavelengths from the eyes of AMD patients.

Conflict of interest statement

Patents by S.P., C.B., T.V., and J.S. C.B. and T.V. are Essilor employees. S.P. received honoraria for participating to a meeting organized by Essilor. M.M., K.B., C.A., P.G., D.P., S.F., and G.L. declare no competing interests.

Figures

Fig. 1
Fig. 1. ROS levels in light-exposed RPE cells.
Hydrogen peroxide (H2O2, b, n = 3–5) and superoxide anion (O2, c, n = 4–5) levels were measured in RPE cells loaded with 20 µM A2E and exposed to light (a, n = 16) for 15 h. Differences between samples and dark controls were considered to be significant when p < 0.05 (▲/*), p < 0.01 (▲▲/**), or p < 0.001 (▲▲▲/***). Triangles (▲) refer to the difference with the untreated dark control and stars (*) to A2E-treated dark control. Each 10 nm spectral band is designated by its central wavelength
Fig. 2
Fig. 2. Impact of light on mitochondria in A2E-loaded RPE cells.
a Confocal images of RPE cells exposed to A2E and light (440 nm and 630 nm) showing changes in mitochondrial distribution at 440 nm. Mitochondria were immunostained with an anti-ATP synthase antibody, tight junctions with an anti-ZO-1 antibody, nuclei were counter-stained with DAPI and images were acquired using confocal microscopy. Scale bar represents 10 µm. Quantification of the cell area b, the number of mitochondria per cell c and the cell mitochondrial fluorescence intensity d using the ‘Cell’ module of Imaris software. n = 3. Differences between samples and dark controls were considered to be significant when p < 0.05 (▲/*), p < 0.01 (▲▲/**), or p < 0.001 (▲▲▲/***). Triangles (▲) refer to the difference with the untreated dark control and stars (*) to A2E-treated dark control. Each 10 nm spectral band is designated by its central wavelength
Fig. 3
Fig. 3. Mitochondrial respiration in light-exposed RPE cells.
Representative results of mitochondrial oxygen consumption measured with an oxygraph: the oxygen concentration in a closed chamber is represented by the blue line and oxygen flux by the red line a. Maximum respiration rate (b) and activities of mitochondrial Complex I and II c, d were determined after the addition of various exogenous substrates and drugs immediately after light exposure. n = 4–5. Differences between samples and dark controls were considered to be significant when p < 0.05 (▲/*), p < 0.01 (▲▲/**), or p < 0.001 (▲▲▲/***). Triangles (▲) refer to the difference with the untreated dark control and stars (*) to A2E-treated dark control. Each 10 nm spectral band is designated by its central wavelength
Fig. 4
Fig. 4. Mitochondrial membrane potential in light-exposed RPE cells.
a The mitochondrial membrane potential was measured by quantifying dye aggregates on a microplate reader in RPE cells exposed to light for 15 h b. Scale bar represents 20 µm. TL: transmitted light. n = 3. Differences between samples and dark controls were considered to be significant when p < 0.05 (▲/*), p < 0.01 (▲▲/**), or p < 0.001 (▲▲▲/***). Triangles (▲) refer to the difference with the untreated dark control and stars (*) to A2E-treated dark control. Each 10 nm spectral band is designated by its central wavelength
Fig. 5
Fig. 5. mRNA expression levels of oxidative stress defense proteins in light-exposed RPE cells.
We measured the mRNA expression levels for SOD1 a, SOD2 b, catalase c, and GPX1 d proteins in RPE cells after 15 h of light exposure in the presence or absence of A2E (20 µM). All values were normalized to the expression level of ribosomal 18S RNA under the same experimental conditions and then normalized to the expression levels of RPE cells maintained in darkness without A2E. n = 3. Differences between samples and dark controls were considered to be significant when p < 0.05 (▲/*), p < 0.01 (▲▲/**), or p < 0.001 (▲▲▲/***). Triangles (▲) refer to the difference with the untreated dark control and stars (*) to A2E-treated dark control. Each 10 nm spectral band is designated by its central wavelength
Fig. 6
Fig. 6. Catalase and superoxide dismutase (SOD) activities in light-exposed RPE cells.
SOD (a and b, n = 5) and catalase (c, n = 5) enzymatic activities were measured in RPE cells after 15 h of light exposure in the presence or absence of A2E (20 µM). Differences between samples and dark controls were considered to be significant when p < 0.05 (▲/*), p < 0.01 (▲▲/**), or p < 0.001 (▲▲▲/***). Triangles (▲) refer to the difference with the untreated dark control and stars (*) to A2E-treated dark control. Each 10 nm spectral band is designated by its central wavelength
Fig. 7
Fig. 7. Total, oxidized, and reduced glutathione levels in light-exposed RPE cells.
Oxidized (GSSG, a) and total glutathione levels b were measured in A2E-loaded cells after a 15-h light exposure. Glutathione ratios (GSH/GSSG, c) were calculated according to the manufacturer’s instructions. n = 3. Differences between samples and dark controls were considered to be significant when p < 0.05 (▲/*), p < 0.01 (▲▲/**), or p < 0.001 (▲▲▲/***). Triangles (▲) refer to the difference with the untreated dark control and stars (*) to A2E-treated dark control. Each 10 nm spectral band is designated by its central wavelength

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