Editor's Highlight: Periodic Exposure to Smartphone-Mimic Low-Luminance Blue Light Induces Retina Damage Through Bcl-2/BAX-Dependent Apoptosis

Toxicological Sciences, May 2017

Blue light-induced phototoxicity plays an important role in retinal degeneration and might cause damage as a consequence of smartphone dependency. Here, we investigated the effects of periodic exposure to blue light-emitting diode in a cell model and a rat retinal damage model. Retinal pigment epithelium (RPE) cells were subjected to blue light in vitro and the effects of blue light on activation of key apoptotic pathways were examined by measuring the levels of Bcl-2, Bax, Fas ligand (FasL), Fas-associated protein with death domain (FADD), and caspase-3 protein. Blue light treatment of RPE cells increased Bax, cleaved caspase-3, FasL, and FADD expression, inhibited Bcl-2 and Bcl-xL accumulation, and inhibited Bcl-2/Bax association. A rat model of retinal damage was developed with or without continuous or periodic exposure to blue light for 28 days. In this rat model of retinal damage, periodic blue light exposure caused fundus damage, decreased total retinal thickness, caused atrophy of photoreceptors, and injured neuron transduction in the retina.

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Editor's Highlight: Periodic Exposure to Smartphone-Mimic Low-Luminance Blue Light Induces Retina Damage Through Bcl-2/BAX-Dependent Apoptosis

TOXICOLOGICAL SCIENCES Periodic Exposure to Smartphone-Mimic Low-Luminance Blue Light Induces Retina Damage Through Bcl-2/BAX-Dependent Apoptosis Cheng-Hui Lin 0 2 Man-Ru Wu 2 Ching-Hao Li 1 4 Hui-Wen Cheng 2 Shih-Hsuan Huang 2 Chi-Hao Tsai 3 Fan-Li Lin 0 4 Jau-Der Ho kj Jaw-Jou Kang 3 George Hsiao 0 4 Yu-Wen Cheng 2 0 Department of Pharmacology, School of Medicine, College of Medicine 1 Department of Physiology, School of Medicine, College of Medicine 2 School of Pharmacy, College of Pharmacy 3 Institute of Toxicology, College of Medicine, National Taiwan University 4 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University Blue light-induced phototoxicity plays an important role in retinal degeneration and might cause damage as a consequence of smartphone dependency. Here, we investigated the effects of periodic exposure to blue light-emitting diode in a cell model and a rat retinal damage model. Retinal pigment epithelium (RPE) cells were subjected to blue light in vitro and the effects of blue light on activation of key apoptotic pathways were examined by measuring the levels of Bcl-2, Bax, Fas ligand (FasL), Fas-associated protein with death domain (FADD), and caspase-3 protein. Blue light treatment of RPE cells increased Bax, cleaved caspase-3, FasL, and FADD expression, inhibited Bcl-2 and Bcl-xL accumulation, and inhibited Bcl-2/Bax association. A rat model of retinal damage was developed with or without continuous or periodic exposure to blue light for 28 days. In this rat model of retinal damage, periodic blue light exposure caused fundus damage, decreased total retinal thickness, caused atrophy of photoreceptors, and injured neuron transduction in the retina. VC The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: blue light; age-related macular degeneration; apoptosis; Bax; Bcl-2; FasL; caspase-3 - The widespread use of smartphones is causing people to spend more time on their handsets, leading to smartphone dependency and even addiction. People who overuse smartphones might suffer from computer vision syndrome such as head and neck illnesses and especially eye fatigue (Rosenfield, 2011) . The light emitted by smartphones includes short-wavelength blue visible light (from 450 to 495 nm), which causes retinal injury and may be a risk factor for eye diseases, such as age-related macular degeneration (AMD) (Age-Related Eye Disease Study Research Group, 2000; Wielgus et al., 2010) . The number of AMD patients is forecast to increase steadily until 2020, due to aging and other risk factors (Owen et al., 2012). As a result, shortwavelength blue visible light-induced ophthalmological chronic diseases are becoming a considerable health concern. Age-related macular degeneration is a multifactorial chronic disease that can take two forms: dry-AMD (atrophic) and wet-AMD (exudative or neovascular). Macular degeneration is initiated by reactive oxygen species (ROS), which induce immunogenic inflammation through different mechanisms, such as Fas ligand/receptor interactions and complement cascade activation (Perez and Caspi, 2015) . Such ROS-induced inflammatory response causes death of retinal pigment epithelium (RPE) cells and eventually leads to geographic atrophy (GA) (Kim et al., 2014). Lightinduced phototoxicity can be classified into three categories: photomechanical damage, photothermal damage, and photochemical damage (Wu et al., 2006) . In particular, the formation of free radicals can cause extensive photochemical damage to retinal tissue. Mitogen-activated protein kinase (MAPK) pathway is a key upstream signaling cascade that controls several processes, such as mitogen activation, inflammation, and stress-related death receptor regulation (Raman et al., 2007; Weston and Davis, 2007) . Mitogen-activated protein kinase pathway activation plays an important role in cell survival/apoptosis through phosphorylation of pro- and antiapoptotic proteins. Three major MAPKs have been identified: p38 MAP, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated protein kinase (ERK). Blue light at 430 nm promotes phosphorylation of JNK and p38 in A2E-laden RPE cells (Westlund et al., 2009). In the visible spectrum (400–760 nm), short-wavelength blue light is considered high-energy radiation. Compared to white or green light, blue light has been shown to generate more ROS in primary retinal cell cultures via inactivation of ERK 1/2, activation of nuclear factor-kappa B (NF-jB) and p38 MAP kinase, aggregation of S-opsin, and cleavage of caspase-3 (Kuse et al., 2014) . Moreover, blue light has been suggested to decrease antioxidant enzymes, such as superoxide dismutase and catalase (Tokarz et al., 2013) stimulate RPE cell apoptosis by mediating Bax/Bcl-2 protein expression (Kernt et al., 2009) , and lead to photoreceptor death (Luthra et al., 2006) . Studies have shown that a short exposure to blue light has a deleterious impact on retinal morphology, including outer nuclear layer deformation, loss of outer/ inner photoreceptor segments, and pigment epithelium degeneration in an albino rat model (Grimm et al., 2001; Meng et al., 2013) . Furthermore, retinal cells displayed increased terminal deoxynucleotidyl transferase dUTP nick end labeling, as well as increased the levels of apoptosis-inducing factors and necrosispromoting receptor-interacting protein (RIP) kinases (Jaadane et al., 2015). Shang et al. (2015) exposed Sprague-Dawley rats with white and blue light-emitting diode (LED) light (750 lux) for 28 days and found the increment of free radical production in LED-exposed group. Outer nuclear layer thickness was decreased in the white and blue LED group after 28 days exposure. Albino Wistar and pigmented Long Evans rats were exposed to LEDs with 500–6000 lux (cold white, blue, and green) for 1–28 days and found blue component of the white-LED decreased photoreceptor layer thickness and caused retinal toxicity (Krigel et al., 2016) . However, few studies investigate long-term and low luminance light-induced phototoxicity in retina. In this study, we investigated the long-term blue LED light exposure induced retinopathy in a rat model. Although there are some reports describing the molecular mechanisms involved in blue light-induced phototoxicity, the widespread use of smartphones calls for a better understanding of the long-term effects of blue light exposure on the eyes. There was an undergoing clinical trial conducted by Assuta Medical Center (ClinicalTrials.gov Identifier: NCT02839395, Assuta Medical Center, Tel Aviv, Israel) which investigated the relationship between smartphone use and retinal degeneration. There was a case reported that a 22-year old woman used smartphone at night before going to sleep presenting impaired vision in the right eye for 12 months. Following enquiry revealed that transient vision loss was specifically related to using her smartphone (Alim-Marvasti et al., 2016) . Also, O’Hagan et al. (2016) revealed the blue light hazard from computer screens, laptop screens, tablet computer screens, and smartphone screens. All screens were set to maximum brightness. The luminance ranged from 43 to 409 cd/m2 and the blue light weighted radiance ranged from 0.034 to 0.380 W/m2/sr1 (O’Hagan et al., 2016) . Thereafter, the correlation between smartphone usage and retinal degeneration of dry-form AMD is becoming a global healthy issue in recent years and is worthy to investigate. To study the phototoxic effects of shortwavelength blue visible light, we placed a blue LED electronic system inside a cell culture incubator. To determine the mechanisms involved in blue light-induced RPE cell apoptosis, we analyzed oxidative stress, regulation of the MAPK and PI3K signaling pathways, protein expression in mitochondriadependent apoptosis pathways, activity of the death receptor pathway, and activation of the caspase cascade. We found that long-term blue light exposure could induce retinal damage in rats and that the damage to RPE and neuron cells was irreversible. Moreover, Bcl-2, Bax, and Fas ligand were all involved in blue light-induced apoptosis. MATERIALS AND METHODS Chemicals and products. SP600125, LY294002, PD98059, and rapamycin were purchased from Calbiochem (San Diego, California). All other chemicals were from Sigma-Aldrich (St. Louis, Missouri). In situ cell death detection kit (Cat. No. 11-684-817910) were purchased from Roche (Mannhem, Germany). Retinal pigment epithelium cell culture. Human RPE cells were obtained from the American Type Culture Collection (ARPE-19, ATCC CRL-2302, Manassas, Virginia). The RPE cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% heatinactivated fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37 C. To maintain ARPE-19 cell, cells were grown on 10-cm dishes (100 mm Cell Culture Dish, Orange Scientific, Braine-l’Alleud, Belgium), and the medium was replaced every 3–4 days. On passaging with 0.05% trypsin–EDTA (Gibco), cells were replated at a 1:3 to a 1:5 ratio. Before testing, ARPE-19 cells were seeded onto 48-well plates (48 Well Cell Culture Plate, Flat Bottom, Orange Scientific, Braine-l’Alleud, Belgium) for the cell viability analysis at a density of 1 105 cells/ml and 6-cm dishes (60 mm Cell Culture Dish, Orange Scientific, Braine-l’Alleud, Belgium) for Western blotting and mRNA analysis at a density of 2 105 cells/ml for 24 h incubation. For analysis of signaling pathways, RPE cells were preincubated with the appropriate concentration of inhibitor for 1 h. In vitro exposure protocols. In in vitro studies, we placed two electric LED plates (LED plate, ZAMI studio, Changhua, Taiwan) inserted total number of 360 blue or red LEDs (12V blue/red LED, ZAMI studio, Changhua, Taiwan) in a cell culture incubator (Figure 1A). The luminance was measure by a light meter (LM81LX, Lutron Electronic Enterprise, Taipei, Taiwan). Blue and red LED emitting lights peaked at 460 nm, 80 lux (Figure 1C) and 620 nm, 80 lux (Figure 1D) for the indicated time (0–48 h) depending on the experimental designs, respectively. Cell viability determination. Cell viability was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Carmichael et al., 1987) . The RPE cells were exposed to blue light for different times, 50 ll/well MTT (0.5 mg/ml) was added and cells were incubated at 37 C for another 30 min. Following, medium was removed, and the formazan product was dissolved in 200 ll DMSO. Absorption was measured at 570 nm using an enzyme-linked immunosorbent assay plate reader (MRX-TC; Dynex Technologies, Chantilly, Virginia). Values were corrected for background absorbance by subtracting the appropriate blanks. Data are from at least nine independent assays. Western blot analysis. After periodic exposure to blue light, cells were washed three times with ice-cold Phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer (1% Nonidet P-40, 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM Na3VO4, 1 mM EGTA, and protease/phosphatase inhibitor cocktail). Whole cell lysates were obtained by centrifugation at 10 000 g. Protein concentration was determined using Braford reagent (Bio-Rad, Hercules, California). Whole lysate supernatant was mixed with 4 sample loading buffer (250 mM Tris–HCl, 8% SDS, 40% glycerin, b-mercaptoethanol, 0.5% bromophenol blue, pH 6.8), separated by 10% SDS-PAGE under reducing conditions, and transferred to a polyvinylidene difluoride (PVDF) membrane following the manufacturer’s protocol (PerkinElmer Life Sciences, Boston, Massachusetts). The PVDF membrane was blocked with 5% nonfat milk for 2 h and incubated in Tris–buffered saline with Tween-20 (TBST) and antibodies specific for phospho-Akt, Akt, phospho-JNK, JNK, phospho-p38, and p38 (all Santa Cruz Biotechnology, Dallas, Texas); target of rapamycin (mTOR), phospho-mTOR, phospho-p70S6 kinase, p70S6 kinase, Bcl-2, Bax, Bcl-xL, Bid, caspase-3, cleaved caspase-3, caspase-8, Fasassociated protein with death domain (FADD), b-Actin, and poly(ADP-ribose) polymerase (PARP) (all Cell Signaling, Danvers, Massachusetts). Blots were incubated with HRP-conjugated secondary antibodies (1:10 000 in TBST; Cayman Chemical, Ann Arbor, Michigan) for 2 h at room temperature, followed by chemiluminescent ECL detection according to the manufacturer’s protocol (Millipore, Billerica, Massachusetts). b-Actin was used as an internal control. Immunoprecipitation. Whole cell lysates (4 mg/ml) were precleared by incubation with 20 ll protein A magnetic beads (Millipore) for 2 h at 4 C. Supernatants were collected, 4 lg/ml polyclonal rabbit anti-Bcl-2 antibody (Genetex, Inc., Irvine, California) was added, and samples were rotated overnight at 4 C. Protein A magnetic beads (20–40 ll) were then added and samples were rotated for a further 2 h at 4 C. Bead-conjugated immune complexes were collected by centrifugation and washed five times with RIPA buffer before adding 50 ll of 2 SDS sample buffer and heating at 95 C for 5 min. The associated proteins were separated by 10% SDS-PAGE and immunodetection was performed as described above. RNA isolation and reverse-transcriptase polymerase chain reaction. Total RNA was isolated by Tripure reagent (Roche, Indianapolis, Indiana) following the manufacturer’s protocol. To analyze mRNA expression of apoptosis-related genes, reverse-transcriptase polymerase chain reaction (RT-PCR) was performed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal control. First-strand cDNA was synthesized from 6 lg total RNA at 42 C for 50 min. cDNA was amplified in a DNA thermal cycler (MJ Research, Watertown, Massachusetts) using the following program: denaturation for 5 min at 95 C, followed by 30 cycles of denaturation for 30 s at 95 C, annealing for 30 s at 54 C, and extension for 40 s at 72 C; with a final extension step of 5 min at 72 C. PCR primers were as follows: Bcl-2 sense: 50CCATTTGGTGTTCGGAGTTTA-30, antisense: 50-TTCGCAGAAGT CCTGTGATGT-30; Bax sense: 50-CCAGGGTGGTTGGGTGAGACT-30, antisense: 50-TGGGAGGTCAGCAGGGTAGAT-30; Caspase-3 sense: 50-GAACTGGACTGTGGCATTGAG-30, antisense: 50-CAAAGCGACT GGATGAACCA-30; Caspase-9 sense: 50-CAGTAACCCCGAGCCAGAT -30, antisense: 50-GAAACAGCATTAGCGACCCT-30; GAPDH sense: 50-GGGTGTCGCTGTTGAA-30, antisense: 50-CTGAGCTGAACGGGAA G-30. The PCR products were separated by electrophoresis on 1.5% agarose gels (PB1200, BIOMAN SCIENTIFIC, Taipei, Taiwan) and visualized by ethidium bromide staining. Immunofluorescence. Cells were cultured directly on glass coverslips, washed with PBS, fixed with 4% paraformaldehyde in PBS for 15min, permeabilized with 0.2% Triton X-100 in PBS for 15 min, blocked with 5% FBS in PBS for 30 min. We used anticleaved caspase-3 antibody (Cell Signaling, Danvers, Massachusetts) as a first antibody in a ratio of 1:250 in the immunofluorescence staining. After incubation for 24 h with glass coverslips, we used rabbit IgG antibody (Dylight 488) (GTX213110-04, Genetex, Inc.) as a secondary antibody in a ratio of 1:200. Nuclei were stained with 4’-6-diamidino-2-phenylindole (DAPI; AAT Bioquest, Sunnyvale, California) and cells were observed using a Laser CS SP5 confocal spectral microscope imaging system (Leica, Teban Gardens, Singapore). Animal experiments. Male Brown Norway (BN) rats (300–350 g body weight) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and kept for 28 days under controlled conditions consisting of 12 h/12 h light/dark cycles, at 26 6 1 C, 35%–47% relative humidity, and ad libitum water and food. Untreated rats kept in darkness served as controls. The animal protocol detailed below was reviewed and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (approval number: LAC-2015-0088). All experimental procedures involving the use of animals complied with the Association for Research in Vision and Ophthalmology (ARVO) statements for the use of animals in ophthalmic and vision experimental research. In vivo exposure protocols. We placed six electric LED plates (LED plate, ZAMI studio, Changhua, Taiwan) inserted total number of 820 blue LEDs in an animal exposing chamber. The BN rats were divided into four groups and subjected them to periodic BLL exposure (460 nm, 150 lux) for 0 h (Group A), 0.5 h (Group B, 9:00 a.m. to 9:30 a.m.), 1 h (Group C, 9:30 a.m. to 10:30 a.m.), and 3 h (Group D, 10:30 a.m. to 1:30 p.m.) without pupils dilated per day in a dark room (n ¼ 4 for each group). Rats in Group A as a control group were kept in the animal colony in a dark room during the exposing experiment. All rats were placed back in the animal colonies in a cyclic light/dark (250 lux, 12 h/12 h) environment after the experiment finished. Fundus angiography analysis. Rats were anesthetized with an intramuscular injection of ketamine (80 mg/kg) and xylazine (20 mg/kg). Before optical examination, both pupils were dilated with 0.125% atropine sulfate (Sinphar, Yilan, Taiwan). Vibrissae were trimmed with scissors to prevent them from disturbing the experiments. Rats were held on the microscope stage on their sides. Image focusing was achieved by moving the rats and the stage. Rat angle and position were adjusted to study different parts of the fundus. The eyes were covered with 2% Methocel gel (OmniVision, SA, Neuhausen, Switzerland), and fundus images and fluorescein angiography images were captured using a Micron III retinal imaging microscope (Phoenix Research Laboratories, Tempe, Arizona). For fluorescein angiography experiments, 10 mg/kg sodium fluorescein was injected intravenously and images were captured after 10 s. The drug was completely excreted through urine within 24 h. To determine leakage areas and photoreceptor nuclei, data were quantified using Image-Pro software (Media Cybernetics, Bethesda, Maryland). Spectral-domain optical coherence tomography. The 830-nm spectral-domain optical coherence tomography (SD-OCT) system (Phoenix Research Laboratories) is customized for retinal imaging of rats; it contains a scanning head, an OCT engine, and a computer with software to detect and photograph the retina. Scan length, location, and rotation could be adjusted within the entire scan region. Following manufacturer’s instructions, we used the A-scan mode to focus on the rat’s retina at a 90 angle. After the retina was positioned in the center of the field, OCT data were collected from inner retina images (Lin et al., 2015) . Electroretinographic recording. We connected a MP36 4-channel data acquisition system (BIOPAC Systems, Pershore, UK) to a photic stimulator (model ps33-plus; GRASS technologies, West Warwick, Rhode Island) to build electroretinographic (ERG) recordings. Prior to experiments, rats were kept in the dark overnight and were prepared for recording under dim red light using LED illumination. Following anesthesia, pupils were dilated with 0.125% atropine sulfate for 10 min. Lens and cornea were covered with 2% Methocel gel before recording via DTL fiber electrodes to increase electronic conductibility. ERG signals were amplified (DC to 300 Hz) and digitized at 1 kHz with a resolution of 2 lV. Immunohistochemistry. Following 28 days of experimentation, rats were sacrificed with a ketamine/xylazine overdose, eyes were removed carefully and the anterior parts and lens were discarded. We fixed the posterior parts and the lens using modified Davidson’s fixative overnight. After dehydration in ethanol and embedding in paraffin, radial 5-lm sections were collected for hematoxylin and eosin staining. Data and statistical analysis. InSight XL software (Phoenix Research Laboratories) developed by Voxleron LLC (Pleasanton, California) was used for SD-OCT data analysis. Given that the retina consists of different layers, the main purpose of this analysis was to determine thickness of the retinal neuron layer. In order to investigate the retinal damage induced by periodic blue light exposure, we applied the distinction in three layers proposed previously (Lin et al., 2015) . After SD-OCT, InSight XL software was used to determine the thickness of the three layers. Data represent mean 6 standard deviation (SD) (n ¼ 4). In addition, the TUNEL-positive cells were scanned and analyzed with the TissueFAXSi-plus imaging system (TissueGnostics, Vienna, Austria). We analyzed the images for the numbers of TUNELpositive cells with TissueQuest/HistoQuset software (TissueGnostics). Statistically significant differences between groups were determined using one-way ANOVA with post hoc Tukey HSD. P < .05 was considered statistically significant. RESULTS Blue Light-Emitting Diode Light Exposure is Cytotoxic to RPE Cells Through Expression of Bcl-2 and BAX To investigate the effect of blue LED light (BLL) in inducing RPE cell apoptosis. Next, the MTT assay was used to assess viability of cultured RPE cells in response to varying periods of blue and red LED light (RLL) exposure. Significant loss of viability was observed already after 6 h in the BLL but not in the RLL group (Figure 1B) and the cell morphology were shown in Supplementary Figure 1A and B. Reverse-transcriptase polymerase chain reaction (RT-PCR) revealed a significant dosedependent decrease in Bcl-2 mRNA and increase in Bax mRNA expression after 12 h BLL treatment (Figure 1E and F), while other apoptosis related genes, such as caspase-3 and caspase-9 remained unchanged (Figure 1E and G). Blue, but Not Red, Light-Emitting Diode Light Increases Cell Apoptosis by Modulating Bax/Bcl-2 and Death Pathways Having observed the negative effect of BLL on RPE cell viability and Bcl-2 expression, we examined other apoptotic markers. Laser scanning microscopy revealed increased cleaved-caspase3 staining as well as pyknotic and fragmented nuclei after 48 h of exposure to BLL (Figure 2A). Next, we detected elements of the Bax/Bcl-2 pathway, such as Bax, Bcl-2, Bcl-xL, caspase-3, cleaved caspase-3, PARP and cleaved PARP, after BLL and RLL treatment for 6, 12, 24, and 48 h (Figure 2B). Bax protein levels increased significantly after 48 h BLL exposure (Figure 2D). In contrast, Bcl-2 and Bcl-xL protein levels decreased significantly (Figure 2E and F), beginning as early as 24 h after BLL exposure (Bcl-2; Figure 2E). Caspase-3 and PARP protein levels remained unchanged (Supplementary Figure 2A and B), as did also all other markers after RLL exposure. We then detected elements of the death pathway, such as Fas ligand (FasL), FADD, caspase8, and Bid, after BLL and RLL treatment for 6, 12, 24, and 48 h (Figure 2C). FasL and FADD protein levels increased significantly after 48 h BLL exposure, starting as early as 12 h (Figure 2G and H). Pro caspase-8 protein levels showed a significant difference after 48 h of exposure, while Bid remained unchanged (Supplementary Figure 2C and D). To investigate the endpoint of apoptosis and the effect of BLL and RLL in inducing RPE cell apoptosis, we performed TUNEL assay by using an in situ cell death detection kit. Data showed that only the condition of 48 h BLL induced large numbers of apoptotic cells in the RPE in in vitro study (as marked arrows indicated) (Supplementary Figure 3). Blue Light-Emitting Diode Light Decreases Bcl-2 and Bax Protein Protein Interactions We evaluated interactions among Bcl-2, Bax, and Bcl-xL by immunoprecipitation with anti-Bcl-2 antibodies, followed by immunoblotting of the resolved complexes with anti-Bax and anti-Bcl-xL antibodies. We found that Bcl-2/Bax, Bcl-2/Bcl-2, and Bcl-2/Bcl-xL associations in RPE cells changed upon BLL exposure (Figure 3A and B). Bcl-2/Bcl-2 protein interaction was significantly higher after 24 h RLL compared to 24 h BLL exposure (Figure 3C). Bcl-2/Bcl-xL protein interaction was significantly higher compared to 0 h RLL exposure (Figure 3C). These data suggest BLL might decrease Bcl-2/Bax interaction and Bcl-2/Bcl2 dimerization, thus inducing RPE cell apoptosis through the Bax/Bcl-2 pathway. Blue Light-Emitting Diode Light Accelerates Bax Protein Accumulation by Stimulating c-Jun NH2-Terminal Kinase, p38, and mTOR/p70S6K Pathways Next, we found that blue light-induced Bax protein accumulation was completely inhibited by treating cells with LY294002 (20 lM, PI-3K inhibitor), rapamycin (20 nM, mTOR inhibitor), and SP600125 (20 lM, JNK inhibitor), but was unaffected by addition of PD98059 (20 lM, p42/p44 ERK inhibitor) (Figure 3D). Moreover, Bcl-2 protein accumulated after treatment with LY294002, rapamycin, and SP600125 (Figure 3D). We also assessed the activation states of ERK, JNK, p38 Akt, mTOR, and p70S6K in BLL or RLL-exposed RPE cells by immunoblotting with antibodies against the phosphorylated forms of these enzymes (Figure 4A and B). The ERK and JNK phosphorylation was significantly induced 60–180 min after exposure to blue light, but not red light (Figure 4C). p70S6K and mTOR phosphorylation were also induced by BLL exposure; however, mTOR and p70S6K phosphorylation decreased 180–360 min after RLL exposure (Figure 4D). Taken together, our results indicate that BLL but not RLL exposure could activate JNK, p38, and mTOR/p70S6K pathways, induce Bax protein accumulation, and cause RPE cell apoptosis. Periodic Blue Light-Emitting Diode Light Exposure Causes Fundus Damage in a Rat Animal Model To mimic the use of smartphones, we divided rats into four groups and subjected them to periodic BLL exposure (460 nm, 150 lux) for 0 h (Group A), 0.5 h (Group B), 1 h (Group C), and 3 h (Group D) per day for 28 days (Figure 5A). We did not observe any damage in the bright-field images of Groups B and C, but Group D presented clear retinal damage from day 14 to day 28 (Figure 5Ch and Cl). Following intravenous injection of sodium fluorescein in the tail, fluorescein angiography showed dye leakage at day 14 and day 28 in Group C (Figure 5Dg and Dk), which was even more pronounced in Group D (Figure 5Dh and Dl). The leakage area was 1.01 6 0.07-fold on day 14 and 1.06 6 0.52-fold on day 28 (Group A), 1.37 6 0.26-fold on day 14 and 2.06 6 0.38-fold on day 28 (Group B) 3.02 6 0.52-fold on day 14 and 4.29 6 0.89-fold on day 28 (Group C), 2.59 6 1.16-fold on day 14 and 19.92 6 3.88-fold on day 28 (Group D) (Figure 5B). Compared with the control group, there were significant differences in Groups C and D between day 14 and day 28. In the animal model of BLL exposure to BN rats for 28 days in in vivo study, we found there were TUNEL-positive cells in the tissue section of retina after 28 days BLL exposure (Supplementary Figure 4). These data suggest that continuous and periodic BLL exposure could lead to progressive retinal damage in rats. Periodic Blue Light-Emitting Diode Light Exposure Reduces Total Retinal Thickness We used SD-OCT scan technology for animals to further investigate the pathophysiology and structure of the rat retina and in particular the effects of BLL on retinal neural layers in rats. As described previously (Lin et al., 2015) , we defined and separated the rat retina into three layers: nerve fiber layer (NFL) to inner plexiform layer (IPL), IPL to inner segment/outer segment (IS/ OS), and IS/OS to RPE (Supplementary Figure 5). A morphological deformation could be observed in the photoreceptor outersegment of Group C after 1 h periodic BLL exposure for 28 days (Figure 6Ak, arrow). Moreover, daily exposure to 3 h blue light could cause serious retinal damage in IPL-IS/OS and IS/OS-RPE layers after exposure for 14 and 28 days. We found significant interneuron layer loss in the inner nuclear and outer plexiform layers after 3 h daily exposure for 14 days. The choroid surface became extremely rough and interrupted, which could cause the disordered arrangement of the RPE layer (Figure 6Ah). The inner and outer segments of rods and cones were barely observed in Group D and neuronal loss in the interneuron layer was even more pronounced. In addition, the arrangement of RPE cells became more abnormal and interrupted after 3 h of periodic BLL exposure (Figure 6Al). We found that 3 h periodic BLL exposure could significantly decrease the thickness of RPE, IS/OS, and IPL-IS/OS layers (Figure 6B–E). Periodic blue light exposure during 28 days could cause retinal neuron cell damage, RPE cell deformation, and overall retinal thinning in rats. Periodic Blue Light-Emitting Diode Light Exposure Causes Atrophy of Photoreceptors and Damages Retinal Neuron Transduction Next, we evaluated retinal neuron transduction by means of an electroretinogram (ERG) examination. The ERG measures the activity of neuron cells in the retina in response to light stimulation; the responses result mainly from changes in sodium and potassium ions. Fluorescein angiography and SD-OCT failed to prove any prominent damage could be caused by 0.5 and 1 h periodic BLL exposures (Figs. 5D and 6A). The functional ERG test showed that after 28 days periodic 0.5, 1, and 3 h BLL exposure, a- and b-wave amplitudes decreased significantly compared to day 0 (Figure 7C and D) which hinted that BLL could induce neuron cell apoptosis. Immunohistochemistry showed extensive breakdown of the outer segment of photoreceptor and RPE cell arrangement in Groups B and C (Figure 7Bb and Bc). After 28 days of periodic 3 h BLL exposure, photoreceptor nuclei counts were 1169.40 6 152.73 (Group A), 1044.40 6 203.69 (Group B), 815.60 6 196.20 (Group C), and 232.50 6 94.90 (Group D) (Figure 7E). In summary, these values suggest that periodic BLL exposure could induce retinal damage in the retina via RPE cell damage and photoreceptor deformation. The signal and apoptotic interactions were elucidated in Figure 8. DISCUSSION Age-related macular degeneration is a worldwide ocular disease with increasing prevalence in industrialized countries. Advanced dry-AMD, also called GA, accounts for 85%–90% of AMD cases and is characterized by RPE regression and damage, followed by degeneration of the relevant photoreceptors (cones and rods) and thinning of the retina. The etiology of dry-AMD remains elusive due to a multifactorial progression affected by environmental factors, continuous and prolonged oxidative stress, and chronic inflammation (Zarrouk et al., 2014) . Longterm use of smartphones has been reported to cause vision blurring, vision disturbance, and inflammation (Balik et al., 2005) . Owing to large number of user, smartphone-emitted short-wavelength blue visible light has become a global concern. Initial pathogenesis involving degeneration and dysfunction of RPE often leads to AMD. Given the tight interactions between RPE and photoreceptors, gradual RPE damage and cell death lead to secondary degeneration of photoreceptors (Liang and Godley, 2003) . As opposed to long-wavelength red/near-infrared light (630–1000 nm), which provides clinical therapeutic treatment for stroke and macular degeneration, short-wavelength blue light negatively affects retinal mitochondrial functions and causes uveal melanoma (Fitzgerald et al., 2013; Logan et al., 2015) . Blue light in the range from 425 to 475 nm has been shown to cause damage to RPE, retinal ganglion cells, and other epithelia (Wood et al., 2008; Roehlecke et al., 2009) . Illumination with blue light for 15–60 min could inhibit Bcl-2 while increasing Bax mRNA expression in a time-dependent manner in primary human RPE cells (Kernt et al., 2009) . Although high intensity light sources with 1000 lux or above were applied in most previous studies, they cannot truly replicate long-term smartphone usage. In the present study, we were the first to expose RPE cells to short-wavelength blue LED light (BLL) of 460 nm with low luminance (80 lux) in order to mimic chronic progressive RPE damage. Compared with the same luminance using red LED light (RLL), BLL exposure for 48 h significantly decreased cell viability. Bcl-2 mRNA expression decreased after 12 h BLL exposure (Figure 1E). In contrast, Bax mRNA expression rose with increasing BLL exposure times. An increase in light intensity failed to produce any significant differences compared with the control group. These findings suggest that low luminance BLL can damage RPE cells and cause cell apoptosis via Bcl-2/Bax mRNA modulation. There is a strong correlation between blue light and Bcl-2/ Bax. In this study, we found that prolonged exposure to BLL increased Bax, cleaved caspase-3, and cleaved PARP protein levels, while production of Bcl-2 and Bcl-xL decreased (Figure 2B). Moreover, we observed cells that showed signs of apoptosis, such as shrinkage necrosis, blebbing, and navicular shapes in the nucleus (Figure 2Ad; Supplementary Figure 6). Immunofluorescence revealed the presence of cleaved caspase3 in the cytoplasm following exposure to BLL, indicating a clear commitment to apoptosis and necrosis (Figure 2Ae and Ak). The association of Bcl-2/Bax plays an important role in cell apoptosis, because anti-apoptotic effectors could seclude and bind to proapoptotic proteins to prevent activation of the downstream mitochondrial death cascade (Dlugosz et al., 2006; Mikhailov et al., 2001) . Treatment with 60 J/m2 UVC has been shown to increase BAX levels and induce apoptosis in MCF7 cells (Bose et al., 2013). Here, we found that 460 nm BLL could decrease Bcl2/Bax interaction and Bcl-2/Bcl-2 dimerization, while 620 nm RLL increased the latter (Figure 3C). This is consistent with previous observations whereby 2.16 J/cm2 UVA significantly reduced the Bcl-2/Bax mRNA ratio (Mujtaba et al., 2013) . Besides the intrinsic Bax/Bcl-2 pathway, cell apoptosis is initiated also through the extrinsic Fas/FasL pathway, which triggers cell surface death receptors such as FADD. Both pathways converge to activate the downstream caspase cascade (Green and Llambi, 2015) . Light at 3000 lux has been reported to stimulate angiotensin II type 1 receptor signaling, part of the renin—angiotensin system, potentially upregulating FasL through induction of cfos and activator protein-1 (AP-1) (Narimatsu et al., 2014). Clinical tests revealed higher soluble FasL in the serum of AMD patients, serving as a potential biomarker of disease progression (Jiang et al., 2008) . Here, we found that 460 nm BLL increased FasL, FADD, caspase-8, and Bid protein levels, in line with previous studies. It should be noted that FADD and Bax protein levels rose also after RLL exposure for 6 or 12 h, hence their role requires further elucidation (Figure 2B and C). Taken together, our results indicate that BLL promotes Bax but prevents Bcl-2 protein accumulation, thus controlling RPE cell apoptosis via Bax/Bcl-2 and Fas/FasL pathways. Regulation of MAPK and PI3K pathways plays a vital role in differentiation, survival, proliferation, and programmed cell death (Jha et al., 2015; Kim and Choi, 2010) . Studies have shown that high intensity blue LED light generated ROS, activated AP-1, hemeoxygenase-1, nuclear factor erythroid 2-like 2, NF-jB, JNK, and p38 signaling in 661W and RGC-5 cell models (Huang et al., 2014; Zhuang et al., 2016) . Among these, p38 acts as a stressactivated protein kinase responding to cytotoxic stress and cytokines in many chronic diseases, such as Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease (Kim and Choi, 2010; Correa and Eales, 2012) . In addition, activation of ERK and PI3K/Akt pathways contributes to neuroprotection, migration, and plasticity (Kitagishi et al., 2012). Proapoptotic protein Bad could be inactivated via ERK and Akt phosphorylation (Horbinski and Chu, 2005) . To identify the mechanisms involved in BLL-induced upregulation of Bax and downregulation of Bcl-2, RPE cells were exposed to blue and red LED light individually for different periods, and phosphorylation of MAPK and PI3K pathway elements was assessed (Figure 4A and B). Results coincided in part with previous studies showing reduced Akt phosphorylation and upregulation of JNK and p38 after BLL exposure. According to our long-term low luminance light exposure in vitro model, phosphorylation of ERK and p70 were upregulated following exposure to both blue and red LED light, warranting further investigation. mTOR is a major negative regulator of autophagy (Zhang et al., 2015) . Here, mTOR phosphorylation decreased after 30 min RLL exposure, suggesting a protective effect on RPE. Instead, phosphorylation of mTOR and p70 increased after 30 min BLL exposure. Interestingly, pretreatment with specific signaling pathway inhibitors (LY294002 and rapamycin) prior to BLL exposure completely inhibited Bax protein expression, while restoring Bcl-2 levels (Figure 3D). Taken together, these data suggest that longterm exposure to BLL with low luminance could directly induce RPE cell apoptosis through Akt repression and p38/JNK activation. To investigate the phototoxic effect of BLL and mimic retinal damage, we created an animal model for BLL (460 nm, 150 lux) exposure using BN rats. BLL phototoxicity, a likely cause of macular degenerations and photoreceptor damage, has been studied in other models (Geiger et al., 2015; Jaadane et al., 2015) . After 3 days of white light exposure (453 J/cm2), the thickness of the outer nuclear layer decreased in a time-dependent manner and the sequestosome 1 protein (p62) involved in autophagy and stress response could be found in the ganglion cell layer and the outer and inner nuclear layers (Giansanti et al., 2013; Kitaoka et al., 2013) . Although Geiger et al. reported extensive damage from the inner retina to the subretina, massive vascular leakage, and blood–retinal barrier disruption after 48 h BLL treatment in R91W; Nrl / transgenic mice, the exact mechanism is still unknown. Instead of high intensity BLL exposure or transgenic animals, here we exposed normal BN rats to low luminance periodic BLL (460 nm, 150 lux) for 28 days, thus mimicking chronic BLL-induced retinal damage. We designed the periodic exposure time from 0.5 to 3 h per day and found there were obvious leakages in Groups C and D (Figure 5Dg and Dh). In addition, SD-OCT revealed that thickness of RPE-IS/OS and IS/OS-IPL layers decreased after 28 day of exposure (Figure 6Ah and Al). These data were consistent with previous studies. Immunohistochemistry revealed the morphological change in RPE-IS/OS after periodic BLL exposure for 28 days. The outer nuclear layer became disorganized and the OS showed partial breakdown after 0.5 to 1 h BLL (Figure 7Bb and Bc). More strikingly, 3 h periodic BLL exposure for 28 days could injure the RPE cell structures leading to photoreceptor nuclei descending into a much thinner IS/OS layer (Figure 7Bd), causing retinal damage. In conclusion, we report that, compared with 620 nm RLL, periodic 460 nm BLL low luminance (80 lux) exposure induces the Bax/Bcl-2 and Fas/FasL pathways and caspase cascades in RPE cells. Periodic BLL (460 nm, 80 lux) increases phosphorylation of p38, JNK, and mTOR, further affecting the accumulation of Bax and Bcl-2 (in vitro). Through fluorescein angiography, SDOCT, and ERG examination, periodic BLL (460 nm, 150 lux) has been documented to cause retinal damage in the RPE, IS/OS, and outer nuclear layers, in addition to photoreceptor nuclei descending into the IS/OS layer in a BN rat animal model (in vivo). Assuta Medical Center have conducted a clinical trial investigating the relationship between smartphone use and retinal degeneration, which emphasizing that the impact of longterm smartphone usage has a chance to lead retinopathy. Moreover, a 40-year old woman reported visual impairment on waking and further enquiry revealed the onset of symptoms corresponded to the acquisition of her smartphone. ERG examination revealed that visual sensitivity was reduced after smartphone viewing (Alim-Marvasti et al., 2016) . Herein, in both models, we found low-luminance blue light caused retinal damage and apoptosis. We have demonstrated the phototoxic effect of periodic BLL and created a smartphone-mimic retinal damage animal model, which could help elucidate dry-AMD disease. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. AUTHORS’ CONTRIBUTIONS Y.-W.C. and G.H. conceived and designed the experiments, interpreted the data, and drafted the manuscript and revised it critically; C.-H.L. wrote the main manuscript text, prepared Figures 1–7 and statistically analyzed the data; M.-R.W., C.-H.L., H.-W.C., S.-H.H., C.H.T., F.-L.L., J.-D.H., and J.-J.K. participated in a fraction of the experiments; C.-H.L. contributed to the manuscript preparation. FUNDING This study was supported in part by grants (NSC102-2320-B038-018-MY3, NSC102-2628-B-038-009-MY3 and MOST1042811-B-038-024) from the Ministry of Science and Technology, Taiwan. Age-Related Eye Disease Study Research Group ( 2000 ). Risk factors associated with age-related macular degeneration. 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Lin, Cheng-Hui, Wu, Man-Ru, Li, Ching-Hao, Cheng, Hui-Wen, Huang, Shih-Hsuan, Tsai, Chi-Hao, Lin, Fan-Li, Ho, Jau-Der, Kang, Jaw-Jou, Hsiao, George, Cheng, Yu-Wen. Editor's Highlight: Periodic Exposure to Smartphone-Mimic Low-Luminance Blue Light Induces Retina Damage Through Bcl-2/BAX-Dependent Apoptosis, Toxicological Sciences, 2017, 196-210, DOI: 10.1093/toxsci/kfx030