The influence of visible light exposure on cultured RGC-5 cells

Abstract

Purpose: To determine the effects of visible light on normal or metabolically compromised cultured rat RGC-5 cells.

Methods: Cultured RGC-5 cells were exposed to different durations as well as intensities of optical radiation, filtered to exclude wavelengths below 400 nm. Some cells were also subjected to metabolic compromise by depriving them of serum (serum deprivation; SD). Treated cells were assayed for cell viability using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, for DNA breakdown by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-linked nick end labeling (TUNEL), apoptotic protein activation by immunoblotting, and the production of reactive oxygen species (ROS) with dihydroethidium. A subset of cells was treated with 100 pM rotenone as an alternative means to induce metabolic stress; this was to determine that the influence of light on compromised cells was not specific to serum-deprivation alone.

Results: Exposure to the light for 48 h activated both caspase-3 and Bcl-associated X-protein (Bax) in cultured RGC-5 cells. Furthermore, light (1000 or 4000 lux), SD, and rotenone caused minor but significant decreases in cellular MTT reduction. SD and light also led to cellular DNA breakdown, although only light caused ROS production. Light (48 h) significantly exacerbated the effect of SD on MTT reduction and DNA cleavage. Furthermore, the antioxidant, trolox, significantly blunted the detrimental influence of light on cell viability, increase in TUNEL-positive cells, and the generation of ROS.

Conclusions: Exposure to light was slightly, but significantly, harmful to healthy RGC-5 cells alone, but was much more toxic to those cells that were energetically compromised. Continuous light exposure can therefore detrimentally affect metabolically stressed RGC-5 cells. This may have implications for some ocular retinopathies such as glaucoma.

Figure 1

Spectral irradiance profile of the fluorescent light source used. The filtration profile of the Lee 2C ultraviolet (UV) filter is also shown alongside; it is evident that light filtration maintains illuminance of the cells within the visible region of the electromagnetic spectrum.

Figure 2

The effect of serum deprivation and filtered light on viability of cultured RGC-5 cells. Cells were incubated in normal culture conditions in the presence (red bars) or absence (empty bars) of 10% (v/v) serum and in the presence or absence of light of intensity 1000 lux, as shown for A 24 h or B 48 h. After treatment, the MTT reduction assay was used to determine the level of cell survival, compared with controls. Serum deprivation caused a small but significant reduction in culture viability, as compared with cells incubated with serum in the dark. This effect was independent of the time of incubation (24 or 48 h). Furthermore, filtered light itself caused a significant reduction in cell viability, but only after 48 h. The toxic effects of both the light treatment and the serum deprivation were additive after 48 h. Statistical analysis using the one-way ANOVA followed by a post-hoc Bonferroni test and comparing with controls incubated in the dark for the same length of time in the presence of serum (n=10), showed significance levels denoted as follows: *p <0.05, ** p <0.01, ***p <0.001.

Figure 3

The appearance of DNA damage after light exposure. These experiments were conducted in normal culture medium (A, C) and medium deprived of serum (B, D). Cultures were exposed to light, 1000 lux, for 48 h (C, D) or maintained in the dark (A, B) and processed for the localization of breakdown of DNA (TUNEL). It can be seen that light enhanced the numbers of TUNEL-positive cells (arrows) and this was greatest in serum-free conditions. The scale bar represents a distance of 20 μm. Quantification of several experiments for each condition shown in A-D is shown in E (black bars represent cells incubated with serum, while empty bars indicate cells incubated without serum). Statistical analysis using the one-way ANOVA followed by a post-hoc Bonferroni test and comparing with controls incubated in the dark for the same length of time in the presence of serum (n=10), showed significance levels denoted as follows: *p <0.05, ***p <0.001.

Figure 4

Dihydroethidium labeling of RGC-5 cells. Dihydroethidium labeling of RGC-5 cells showed that only filtered light (1000 lux; C) and not serum deprivation (B) caused the detectable production of reactive oxygen species (red fluorescence, as marked by the arrows), as compared with control cells incubated in the dark in the presence of 10% (v/v) serum (A), after 48 h. This effect did not appear to be obviously enhanced for cells incubated in serum free medium and exposed to light (D). The scale bar represents a distance of 20 μm.

Figure 5

Changes in cellular pro-apoptotic proteins. RGC-5 cells incubated in filtered light for 48 h showed an increased production of pro-apoptotic Bax and cleaved (17 kD) caspase-3 proteins compared with cells grown in the dark (A). Quantification by densitometric analysis of protein levels showed the effect to be significant in both cases (*p<0.05, by one-way ANOVA followed by a post-hoc Bonferroni test; n=4). All extracts were prepared, in these experiments, from cells incubated in the presence of serum (black bars represent cells incubated in the dark and unfilled bars in the light).

Figure 6

The counteracting effect of trolox. Cell viability assays with 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showed that 10 µM trolox significantly blunted the detrimental effect of light (1000 lux, 48 h) in serum-free medium (white, unfilled bars) compared with cells incubated in normal medium in the dark (black bars; Figure 6E). Moreover, analysis of DNA breakdown by TUNEL (Figure 6A and 6B) or ROS formation (Figure 6C and 6D) showed that the detrimental effect of light (Figure 6A and 6C) was counteracted by inclusion in the medium of 10 µM trolox (B, D). Black arrows indicate TUNEL-positive cells. White arrows mark red fluorescence in cells staining strongly for ROS (DHE labeling). The scale bar represents a distance of 20 μm. Statistical significance, as indicated (*p <0.05), was determined by one-way ANOVA followed by a post-hoc Bonferroni test which either compared cells in the light (without serum) to controls, or compared cells in the light (without serum) with or without 10 μM trolox, as indicated in the figure. Note that trolox had no significant effect on the viability measurement of cells incubated in the dark, in medium containing serum.