Light-induced Cell Damage Research Articles

Sulforaphane inhibits blue light–induced inflammation and apoptosis by upregulating the SIRT1/PGC-1α/Nrf2 pathway and autophagy in retinal pigment epithelial cells

The present study elucidated mechanisms through which sulforaphane (SFN) protects retinal pigment epithelial (RPE) cells from blue light–induced impairment. SFN could activate the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increase the expression of the heme oxygenease-1 (HO-1) gene and production of glutathione. SFN reduced blue light–induced oxidative stress, and effectively activated cytoprotective components including Nrf-2, HO-1, thioredoxin-1, and glutathione. The protective effect of SFN on blue light–induced injury was blocked by the Nrf2 inhibitor ML385, suggesting that the SFN-induced Nrf2 pathway is involved in the cytoprotective effect of SFN. SFN inhibited intercellular adhesion molecule-1 expression induced by TNF-α or blue light, suggesting the anti-inflammatory activity of SFN. The inhibitory effect of SFN was associated with the blocking of NF-κB p65 nuclear translocation in blue light–exposed RPE cells. SFN protected RPE cells from blue light–induced interruption of the mitochondrial membrane potential and reduction of the Bcl-2/Bax ratio and cleaved caspase-3 and PARP-1 expression, suggesting the antiapoptotic activity of SFN. SFN alone or together with blue light exposure increased the expression of the autophagy-related proteins LC3BII and p62. An autophagy inhibitor, 3-MA, inhibited the protective effect of SFN on blue light–induced cell damage. SFN increased sirtuin-1 (SIRT1) expression; however, treatment with blue light induced peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) expression. Our study results demonstrated that SFN exerts its protective effect under blue light exposure by maintaining the Nrf2-related redox state and upregulating SIRT1 and PGC-1α expression and autophagy.

Solanum melongena L. Extract Protects Retinal Pigment Epithelial Cells from Blue Light-Induced Phototoxicity in In Vitro and In Vivo Models.

N-retinylidene-N-retinylethanolamine (A2E) accumulation in the retina is a prominent marker of retinal degenerative diseases. Blue light exposure is considered as an important factor contributing to dry age-related macular degeneration (AMD). Eggplant and its constituents have been shown to confer health benefits, but their therapeutic effects on dry AMD remain incompletely understood. In this study, we showed that an extract of Solanum melongena L. (EPX) protected A2E-laden ARPE-19 cells against blue light-induced cell death via attenuating reactive oxygen species. Transcriptomic analysis demonstrated that blue light modulated the expression of genes associated with stress response, inflammation, and cell death, and EPX suppressed the inflammatory pathway induced by blue light in A2E-laden ARPE-19 cells by inhibiting the nuclear translocation of nuclear factor kappa B and transcription of pro-inflammatory genes (CXCL8 and IL1B). The degradation of intracellular A2E was considered the major mechanism underlying the protective effect of EPX. Moreover, chlorogenic acid isolated from EPX exerted protective effects against blue light-induced cell damage in A2E-laden ARPE-19 cells. In vivo, EPX administration in BALB/c mice reduced the fundus damage and degeneration of the retinal layer in a blue light-induced retinal damage model. Collectively, our findings suggest the potential role of Solanum melongena L. extract for AMD treatment.

Astaxanthin analogs, adonixanthin and lycopene, activate Nrf2 to prevent light-induced photoreceptor degeneration

Carotenoids, in particular astaxanthin, possess potent antioxidant capabilities. Astaxanthin also induces NF-E2-related factor 2 (Nrf2), which plays a major regulatory role in the antioxidative response. However, little is known whether the carotenoid, by-products of astaxanthin, activate Nrf2. Toward this end, we screened eight astaxanthin analogs for Nrf2 activation in murine photoreceptor cell line, 661 W, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In addition, we monitored cell death in 661 W cells pretreated with astaxanthin analogs or only pretreated for 6 h with astaxanthin analogs and then exposed to light. Furthermore, we quantified the reactive oxygen species (ROS) production. Cell death was quantified after light exposure by nuclear staining. Nrf2-controlled genes Ho-1, Nqo-1, and Gclm by qRT-PCR and Nrf2 in the nucleus were upregulated in 661 W cells exposed astaxanthin, adonixanthin, echinenone, and lycopene. Moreover, astaxanthin, adonixanthin, echinenone, β-carotene, adonirubin, and lycopene, but not canthaxanthin, suppressed ROS production and protected cells against light-induced damage. Moreover, pretreatment with adonixanthin or lycopene only before light exposure protected against light-induced cell damage and Nrf2 silencing canceled these effects. These findings indicate that the more potent astaxanthin analogs, adonixanthin and lycopene, protect against light-induced cell damage through not only an anti-oxidative response but also through Nrf2 activation.

Surpassing light-induced cell damage in vitro with novel cell culture media

Light is extensively used to study cells in real time (live cell imaging), separate cells using fluorescence activated cell sorting (FACS) and control cellular functions with light sensitive proteins (Optogenetics). However, photo-sensitive molecules inside cells and in standard cell culture media generate toxic by-products that interfere with cellular functions and cell viability when exposed to light. Here we show that primary cells from the rat central nervous system respond differently to photo-toxicity, in that astrocytes and microglia undergo morphological changes, while in developing neurons and oligodendrocyte progenitor cells (OPCs) it induces cellular death. To prevent photo-toxicity and to allow for long-term photo-stimulation without causing cellular damage, we formulated new photo-inert media called MEMO and NEUMO, and an antioxidant rich and serum free supplement called SOS. These new media reduced the detrimental effects caused by light and allowed cells to endure up to twenty times more light exposure without adverse effects, thus bypassing the optical constraints previously limiting experiments.

Light-induced cell damage in live-cell super-resolution microscopy.

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm−2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm−2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light

Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm2). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light.

The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB). Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8) cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation.

Visible light induced cell damage of Gram positive bacteria by N-doped TiO2 mesoporous thin films

Highly ordered N-doped titania mesoporous inorganic–organic hybrid thin films were prepared according to a refined recipe by an evaporative induced self-assembly route using Pluronic P123 as the template. Films were characterized by a grazing incident small angle X-ray scattering, atomic force microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and X-ray diffraction. Visible light induced bactericidal activity was performed on Gram-positive bacteria (Bacillus amyloliquifacience (NCIM 2829)). Bacterial cell death was confirmed by cell viability assay and inhibition of bacterial growth against reactive oxygen species induction is reported.

Effects of Combined Ketamine/Xylazine Anesthesia on Light Induced Retinal Degeneration in Rats

ObjectivesTo explore the effect of ketamine-xylazine anesthesia on light-induced retinal degeneration in rats.MethodsRats were anesthetized with ketamine and xylazine (100 and 5 mg, respectively) for 1 h, followed by a recovery phase of 2 h before exposure to 16,000 lux of environmental illumination for 2 h. Functional assessment by electroretinography (ERG) and morphological assessment by in vivo imaging (optical coherence tomography), histology (hematoxylin/eosin staining, TUNEL assay) and immunohistochemistry (GFAP and rhodopsin staining) were performed at baseline (ERG), 36 h, 7 d and 14 d post-treatment. Non-anesthetized animals treated with light damage served as controls.ResultsKetamine-xylazine pre-treatment preserved retinal function and protected against light-induced retinal degeneration. In vivo retinal imaging demonstrated a significant increase of outer nuclear layer (ONL) thickness in the non-anesthetized group at 36 h (p<0.01) and significant reduction one week (p<0.01) after light damage. In contrast, ketamine-xylazine pre-treated animals showed no significant alteration of total retinal or ONL thickness at either time point (p>0.05), indicating a stabilizing and/or protective effect with regard to phototoxicity. Histology confirmed light-induced photoreceptor cell death and Müller cells gliosis in non-anesthetized rats, especially in the superior hemiretina, while ketamine-xylazine treated rats showed reduced photoreceptor cell death (TUNEL staining: p<0.001 after 7 d), thicker ONL and longer IS/OS. Fourteen days after light damage, a reduction of standard flash induced a-wave amplitudes and a-wave slopes (p = 0.01) and significant alterations in parameters of the scotopic sensitivity function (e.g. Vmax of the Naka Rushton fit p = 0.03) were observed in non-treated vs. ketamine-xylazine treated animals.ConclusionsOur results suggest that pre-treatment with ketamine-xylazine anesthesia protects retinas against light damage, reducing photoreceptor cell death. These data support the notion that anesthesia with ketamine-xylazine provides neuroprotective effects in light-induced cell damage.

Effects of Light Exposure and Use of Intraocular Lens on Retinal Pigment Epithelial Cells In Vitro

To investigate the effect of a blue light-filtering intraocular lens (IOL) and a UV-absorbing IOL on light-induced damage to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore N-retinylidene-N-retinylethanolamine (A2E), A2E-laden RPE cells were exposed to white light which was filtered by either a blue light-filtering IOL or a UV-absorbing IOL. After 30 min of illumination the cell viability and the level of reactive oxygen species (ROS), free glutathione (GSH), vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were determined. In the absence of an IOL, the white light exposure decreased cell viability to 37.2% of the nonirradiated control. The UV-absorbing IOL tended to reduce light-induced cell death; however, the decrease was not significant. The blue light-filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 79.5% of the nonirradiated control. The presence of the blue light-filtering IOL significantly increased GSH and PEDF levels, and decreased ROS and VEGF levels. This study suggests that a blue light-filtering IOL may be more protective against A2E-induced light damage and inhibit more light-induced ROS and VEGF production than a conventional UV-absorbing IOL.

Protektive Wirkung Blaulicht absorbierender IOLs auf das menschliche retinale Pigmentepithel

Primary human RPE cells were exposed to white light and either a SN60AT or SA60AT IOL was placed in the light beam. After 15-60 min of irradiation, viability, induction of apoptosis and cell death were determined in primary human RPE cells. Expression of vascular endothelial growth factor A (VEGF-A) and the anti-apoptotic XIAP protein and their mRNA were determined by RT-PCR, Western blot analysis and ELISA. Light exposure decreased cell viability depending on the duration of irradiation. Light-induced cell death and apoptosis as well as decrease of XIAP expression and cellular viability were significantly reduced by both the SN60AT and SA60AT IOL. In addition, these protective effects regarding light-induced cell damage were significantly stronger in the presence of the blue light-filtering SN60AT IOL compared to the SA60AT IOL. Both UV-filtering and blue light-absorbing IOLs reduce light-induced RPE damage. The blue light-absorbing IOL further reduced damage compared to the conventional IOL, which supports the hypothesis of possibly also preventing retinal damage in clinical use.

Cytoprotective effects of a blue light–filtering intraocular lens on human retinal pigment epithelium by reducing phototoxic effects on vascular endothelial growth factor-α, Bax, and Bcl-2 expression

To compare the possible protective effects of the ultraviolet (UV)-filtering and blue light-filtering SN60AT intraocular lens (IOL) and the untinted UV-filtering SA60AT IOL with regard to light-induced stress on human retinal pigment epithelium (RPE). Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Primary human RPE cells were exposed to white light, and a tinted or untinted IOL was placed in the light beam. After 15 to 60 minutes of irradiation, cell viability was determined by a colorimetric test (tetrazolium dye-reduction assay) and a microscopic live/dead assay. The expression of vascular endothelial growth factor-alpha (VEGF-alpha), Bax, and Bcl-2 and their mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Without an IOL, white-light exposure decreased cell viability compared with the decrease with the nonirradiated control in a time-dependent manner. Light-induced cell death was significantly reduced by both the tinted IOL and untinted IOL. The combined UV and blue-light filtering attenuated light-induced cell damage significantly more than UV filtering alone. Results of RT-PCR and Western blotting showed a significant time-dependent decrease in Bcl-2 and increase in Bax and VEGF-alpha that were significantly less with the tinted IOL than with the untinted IOL. Both IOLs reduced light-induced RPE damage. The UV- and blue light-filtering IOL reduced damage more than the conventional IOL. This supports the hypothesis that blue light-filtering IOLs may prevent retinal damage in clinical use.

Quantifying UVA Light Induced Cell Damage via Time-Lapse OIR Microscopy

Real-time OIR microscopy of living cells provides a means for monitoring cellular activity such as macromolecular streaming, cell motility and other structural movement [1]. If treatment-induced damage results in changes in cellular activity, then viewing time-lapse movies provides a qualitative assessment of that damage. An image analysis program, developed by the authors, was used to quantify the changes that were observed. The program uses procedures incorporated into the MaxIm DL software development library [2]. One algorithm computes the root-mean-square intensity (RMS I) associated with each OIR image. Although this value does not quantify movement, it does provide a measure of the overall image intensity. A second algorithm calculates the RMS value associated with the intensity difference computed on a pixel-by-pixel basis from adjacent OIR images (RMS dI/dt), providing a measure of the rate of change in cellular activity.

Effects of yellow intraocular lenses on light-induced upregulation of vascular endothelial growth factor

To investigate the protective effect of a blue-light filtering intraocular lens (yellow IOL) (YA60BB, Hoya) and an ultraviolet (UV)-absorbing IOL (VA60BB, Hoya) on light-induced phototoxicity to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore A2E and on the production of vascular endothelial growth factor (VEGF) after light exposure. University of Tokyo, Tokyo, Japan. The A2E-laden ARPE-19 cells were exposed to white light and a UV-absorbing IOL or a blue-light filtering IOL was placed over the light beam. After 48 hours of irradiation, the viability of the cells was determined with WST-1 (a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay, and the secreted protein level of VEGF was determined by enzyme-linked immunosorbent assay. Without an IOL, the white-light exposure decreased cell viability to 28% of the nonirradiated control. Although the UV-absorbing IOL tended to reduce light-induced cell death, the decrease was not significant. However, the presence of the blue-light filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 42%. The secreted VEGF protein level increased 3.2-fold after the A2E-laden RPE cells were exposed to white light. In the presence of the UV-absorbing IOL, the VEGF protein level decreased, but not significantly. The presence of the blue-light filtering IOL significantly attenuated the upregulated VEGF expression compared to upregulation without an IOL. This study supports the theory that a blue-light filtering IOL may be more protective against A2E-induced photochemical damage and inhibit more light-induced VEGF production than a conventional UV-absorbing IOL.

EU reduces threshold for canthaxanthin in animal feeds

Carotenoids, in particular astaxanthin, possess potent antioxidant capabilities. Astaxanthin also induces NF-E2-related factor 2 (Nrf2), which plays a major regulatory role in the antioxidative response. However, little is known whether the carotenoid, by-products of astaxanthin, activate Nrf2. Toward this end, we screened eight astaxanthin analogs for Nrf2 activation in murine photoreceptor cell line, 661 W, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In addition, we monitored cell death in 661 W cells pretreated with astaxanthin analogs or only pretreated for 6 h with astaxanthin analogs and then exposed to light. Furthermore, we quantified the reactive oxygen species (ROS) production. Cell death was quantified after light exposure by nuclear staining. Nrf2-controlled genes Ho-1, Nqo-1, and Gclm by qRT-PCR and Nrf2 in the nucleus were upregulated in 661 W cells exposed astaxanthin, adonixanthin, echinenone, and lycopene. Moreover, astaxanthin, adonixanthin, echinenone, β-carotene, adonirubin, and lycopene, but not canthaxanthin, suppressed ROS production and protected cells against light-induced damage. Moreover, pretreatment with adonixanthin or lycopene only before light exposure protected against light-induced cell damage and Nrf2 silencing canceled these effects. These findings indicate that the more potent astaxanthin analogs, adonixanthin and lycopene, protect against light-induced cell damage through not only an anti-oxidative response but also through Nrf2 activation.