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

Wood JP, Lascaratos G, Bron AJ, Osborne NN - Mol. Vis. (2007)

Bottom Line: Furthermore, light (1000 or 4000 lux), SD, and rotenone caused minor but significant decreases in cellular MTT reduction.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.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom.

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.

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Related in: MedlinePlus

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.
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f3: 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.

Mentions: Both filtered light at 1000 lux (Figure 3C, E) and serum deprivation (Figure 3B, E) caused a significant increase in the amount of cultured RGC-5 cell nuclei that labeled positively for TUNEL after 48 h. When both light treatment and serum deprivation were concurrent for 48 h, the effect was to significantly enhance the influence of either treatment alone (Figure 3D, E).


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

Wood JP, Lascaratos G, Bron AJ, Osborne NN - Mol. Vis. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254956&req=5

f3: 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.
Mentions: Both filtered light at 1000 lux (Figure 3C, E) and serum deprivation (Figure 3B, E) caused a significant increase in the amount of cultured RGC-5 cell nuclei that labeled positively for TUNEL after 48 h. When both light treatment and serum deprivation were concurrent for 48 h, the effect was to significantly enhance the influence of either treatment alone (Figure 3D, E).

Bottom Line: Furthermore, light (1000 or 4000 lux), SD, and rotenone caused minor but significant decreases in cellular MTT reduction.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.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus