Editor's Highlight: Periodic Exposure to Smartphone-Mimic Low-Luminance Blue Light Induces Retina Damage Through Bcl-2/BAX-Dependent Apoptosis
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
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
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
(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,
. 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
et al., 2013)
stimulate RPE cell apoptosis by mediating Bax/Bcl-2
(Kernt et al., 2009)
, and lead to photoreceptor
(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.,
. 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
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
(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
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
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,
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
(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.
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
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
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
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
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
(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.
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.,
. Owing to large number of user, smartphone-emitted
short-wavelength blue visible light has become a global
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
. 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.,
. Blue light in the range from 425 to 475 nm has been
shown to cause damage to RPE, retinal ganglion cells, and other
(Wood et al., 2008; Roehlecke et al., 2009)
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
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)
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,
. 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
(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
(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
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
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
(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 are available at Toxicological Sciences online.
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.
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
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