The Protective Effects of a Dietary Carotenoid, Astaxanthin, Against Light-Induced Retinal Damage

Abstract

Dietary carotenoids exhibit various biological activities, including antioxidative activity. In particular, astaxanthin, a type of carotenoid, is well known as a powerful antioxidant. We investigated whether astaxanthin would protect against light-induced retinal damage. In an in vivo study, ddY male mice were exposed to white light at 8,000 lux for 3 h to induce retinal damage. Five days after light exposure, retinal damage was evaluated by measuring electroretinogram (ERG) amplitude and outer nuclear layer (ONL) thickness. Furthermore, expression of apoptotic cells, 8-hydroxy-deoxyguanosine (8-OHdG), was measured. In an in vitro study, retinal damage was induced by white light exposure at 2,500 lux for 24 h, and propidium iodide (PI)-positive cells was measured and intracellular reactive oxygen species (ROS) activity was examined. Astaxanthin at 100 mg/kg inhibited the retinal dysfunction in terms of ERG and ONL loss and reduced the expression of apoptotic and 8-OHdG-positive cells induced by light exposure. Furthermore, astaxanthin protected against increases of PI-positive cells and intracellular reactive oxygen species (ROS) activity in 661W cells. These findings suggest that astaxanthin has protective effects against light-induced retinal damage via the mechanism of its antioxidative effect.

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209
J Pharmacol Sci 123, 209 – 218 (2013) Journal of Pharmacological Sciences
© The Japanese Pharmacological Society
Full Paper
Introduction
Age-related macular degeneration (AMD) is the leading
cause of adult vision loss in the Western world. AMD
occurs as a result of photoreceptor degeneration, causing
a decline not of only quality of vision (QOV), but also of
quality of life (QOL). AMD mainly injures the macula,
which detects photoreceptors. Two types of AMD are
known to exist: dry AMD, in which age causes macular
degeneration and failing visual acuity, and wet AMD, in
which the macula is impaired by angiogenesis. Recently,
angiogenic inhibitors, such as ranibizumab and pegaptanib,
have been used for useful medical treatments.
Almost all retinitis pigmentosa (RP) patients have
genetic factors; this disorder occurs at a rate of one
in 4,000 – 8,000 people. Night blindness occurs first,
and constriction of the visual field presents gradually.
Currently, treatment approaches to improve retinal func-
tion and block the progression of RP do not exist; there
are only supportive cares, such as light-resistant eye-
glasses, vitamin A, cardiovascular agents, and vitamin
B12 (1). Therefore, there is a need for elucidation of
the pathophysiology and therapeutic medicine.
Previous reports have suggested that excessive light
exposure is one of the factors of AMD occlusion and that
it promotes AMD progression (2). In addition, it has been
reported that genetic aberrance causing retinal degenera-
tion elevates photoreceptor fragility against light in RP
(3). Finally, in both AMD and RP, excessive light expo-
sure evokes photoreceptor apoptosis in the same manner
(4, 5). As such, a light-induced photoreceptor degenera-
tion model has been widely used to clarify the mechanism
of photoreceptor injury in AMD and RP. ROS are pro-
duced easily in the retina, as the retina is routinely
The Protective Effects of a Dietary Carotenoid, Astaxanthin,
Against Light-Induced Retinal Damage
Tomohiro Otsuka1, Masamitsu Shimazawa1, Tomohiro Nakanishi1, Yuta Ohno1, Yuki Inoue1,
Kazuhiro Tsuruma1, Takashi Ishibashi2, and Hideaki Hara1,*
1Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu 501-1196, Japan
2Biotechnology Business Section, Merchandise Business Department, Nippon Oil Corporation, Tokyo 100-8162, Japan
Received April 4, 2013; Accepted September 1, 2013
Abstract. Dietary carotenoids exhibit various biological activities, including antioxidative
activity. In particular, astaxanthin, a type of carotenoid, is well known as a powerful antioxidant.
We investigated whether astaxanthin would protect against light-induced retinal damage. In an in
vivo study, ddY male mice were exposed to white light at 8,000 lux for 3 h to induce retinal
damage. Five days after light exposure, retinal damage was evaluated by measuring electroretino-
gram (ERG) amplitude and outer nuclear layer (ONL) thickness. Furthermore, expression of
apoptotic cells, 8-hydroxy-deoxyguanosine (8-OHdG), was measured. In an in vitro study, retinal
damage was induced by white light exposure at 2,500 lux for 24 h, and propidium iodide (PI)-
positive cells was measured and intracellular reactive oxygen species (ROS) activity was examined.
Astaxanthin at 100 mg/kg inhibited the retinal dysfunction in terms of ERG and ONL loss and
reduced the expression of apoptotic and 8-OHdG-positive cells induced by light exposure.
Furthermore, astaxanthin protected against increases of PI-positive cells and intracellular reactive
oxygen species (ROS) activity in 661W cells. These findings suggest that astaxanthin has protec-
tive effects against light-induced retinal damage via the mechanism of its antioxidative effect.
Keywords: astaxanthin, photoreceptor, oxidative stress, age-related macular degeneration,
retinitis pigmentosa
*Corresponding author. hidehara@gifu-pu.ac.jp
Published online in J-STAGE on October 22, 2013
doi: 10.1254/jphs.13066FP

210 T Otsuka et al
exposed to light (6, 7). It is known that ROS is produced
by light exposure in the retina, and it evokes photoreceptor
degeneration (8, 9); thus, antioxidants such as dimethyl-
thiourea (10), phenyl-N-tert-butyl nitrone (11), and
2,2,6,6-tetramethyl-4-piperidinol-1-oxyl (12) have been
reported to be effective in animal experiments.
Astaxanthin, a carotenoid, is present in many well-
known seafoods such as salmon, trout, red sea-bream,
shrimp, lobster, and fish eggs. Astaxanthin exhibits
various pharmacological activities, including antioxida-
tive (13 – 17), antitumor (18), anti-inflammatory (19),
antidiabetic (20), hepatoprotective (21), and immuno-
modulatory effects (18, 22). Moreover, one of the char-
acteristics of astaxanthin is its high degree of safety (23).
In the retina, astaxanthin has protective effects against
choroidal neovascularization (24), and our laboratory
previously reported that N-methyl-d-aspartate (NMDA)-
induced retinal ganglion cell death was inhibited by
astaxanthin (25). However, the effect of astaxanthin
against the light-induced photoreceptor degeneration
model, which is the model of dry AMD and RP, has not
been clarified yet. The aim of this study was to elucidate
the protective effects of astaxanthin against light-induced
photoreceptor degeneration.
Materials and Methods
Animals
All experiments were performed in accordance with
the ARVO Statement for the Use of Animals in Ophthalmic
and Vision Research, and they were approved and
monitored by the Institutional Animal Care and Use
Committee of Gifu Pharmaceutical University. Male
albino ddY mice (Japan SLC, Hamamatsu), aged 8 – 10
weeks, were used in this study. They were kept under
controlled lighting conditions (12:12 h light/dark).
Exposure to light
After dark adaptation for 24 h, the pupils were dilated
with 1% cyclopentolate hydrochloride eye drops (Santen
Pharmaceuticals Co., Ltd., Osaka) 30 min before expo-
sure to light. The non-anesthetized mice were exposed
to 8,000 lux of white fluorescent light (Toshiba, Tokyo)
for 3 h in cages with reflective interiors. The temperature
during the exposure to light was maintained at 25°C ±
1.5°C. After the exposure to light, all the mice were
placed in the dark for 24 h and then returned to the
normal light/dark cycle.
Astaxanthin (Asahi Kasei Pharma Corp., Tokyo) at
100 mg/kg was dissolved in olive oil just before use
and was administered orally eight times (at 6 h before
and at 0, 6, 12, 24, 36, 48, and 72 h after light irradiation)
for histological analysis, six times (at 6 h before and at 0,
6, 12, 24, and 36 h after light irradiation) for terminal
deoxynucleotidyl transferase-mediated dUTP nick-end
labeling (TUNEL) staining analysis, four times (at 6 h
before and at 0, 6, and 12 h after light irradiation) for
immunostaining analysis, or two times (at 6 h before
and just before light irradiation) with a volume of 0.05
mL / 10 g body weight.
Electroretinogram
ERG readings were recorded 5 days after the light
exposure. Thirty-two mice in total were used in this
experiment. The mice were maintained in a completely
dark room for 24 h, after which they were intraperitone-
ally anesthetized with a mixture of ketamine (120 mg/kg)
(Daiichi-Sankyo, Tokyo) and xylazine (6 mg/kg) (Bayer
Health Care, Tokyo). The pupils were dilated with 1%
tropicamide and 2.5% phenylephrine (Santen Pharma-
ceuticals). Flash ERG was recorded in the left eyes of
the dark-adapted mice by placing a golden-ring electrode
(Mayo, Aichi) in contact with the cornea and a reference
electrode (Nihon Kohden, Tokyo) through the tongue.
A neutral electrode (Nihon Kohden) was inserted
subcutaneously near the tail. All procedures were per-
formed under dim red light. The amplitude of the a-wave
was measured from the baseline to the maximum a-wave
peak, and the b-wave was measured from the maximum
a-wave peak to the maximum b-wave peak. The a-wave
shows the function of the photoreceptors, and the b-wave
reflects bipolar cell and Müller cell function.
Histological analysis
Fifty-seven mice in total were used in this experiment.
The mice were euthanized by cervical spine fracture
dislocation. Each eye was enucleated and kept immersed
for at least 24 h at 4°C in a fixative solution containing
4% paraformaldehyde. Six paraffin-embedded sections
(thickness, 5 mm) cut from the optic disc of each eye
were prepared in the standard manner, and stained with
hematoxylin and eosin. The damage induced by light
exposure was then evaluated, with the six sections from
each eye used for morphometric analysis, as described
below. Light-microscopy images were photographed,
and the thickness of the outer nuclear layer (ONL) from
the optic disc was measured at 240-mm intervals on the
photographs. Data from three sections (selected ran-
domly from the six sections) were averaged for each eye.
TUNEL staining
TUNEL staining was performed according to the
manufacturer’s protocols (In Situ Cell Death Detection
kit; Roche Biochemicals, Mannheim, Germany) to detect
the retinal cell death induced by exposure to light.
Twenty-eight mice in total were used in this experiment.

211Protective Effects of Astaxanthin
The eyes were enucleated, fixed overnight in 4% para-
formaldehyde, and immersed for 2 days in 25% sucrose
with 0.01 M phosphate-buffered saline (PBS). The eyes
were then embedded in a supporting medium for frozen-
tissue specimens (OCT compound; Tissue-Tek, Miles
Laboratories, Naperville, IL, USA). Retinal sections
(10-mm thick) were cut on a cryostat at −25°C and stored
at −80°C until staining. After being washed with PBS
twice, sections were incubated with methanol containing
3% H2O2 for 10 min, 0.1% sodium citrate aqueous
containing 0.1% Triton X-100 for 10 min, and TUNEL
reaction mixture, 10% terminal deoxyribonucleotidyl
transferase (TdT) enzyme solution diluted in fluorescein-
dUTP mixture solution, at 37°C for 1 h. The sections
were washed in PBS for 5 min three times at room
temperature. Two eye sections per eye (two images per
one section between 285 – 715 mm from the optic disc)
were photographed and counted for TUNEL-positive
cells in the ONL. The average of the four images was
used as the data per eye.
Immunohistochemistry
Light exposure was performed as described in the
exposure to light section. Thirty mice in total were used
in this experiment. The eyes were enucleated, fixed
overnight in 4% paraformaldehyde, and immersed for
two days in 25% sucrose with PBS. The eyes were then
embedded in a supporting medium for frozen tissue
specimens (optimum cutting temperature compound,
Tissue-Tek) and kept at −80°C. Retinal sections were
cut at 10-mm thickness on a cryostat at −20°C and stored
at −80°C until staining. Immunohistochemical staining
was performed in accordance with the following proto-
col. Briefly, tissue sections were washed in 0.01 M PBS
for 30 min, followed by preincubation with 10% normal
goat serum in 0.01 M PBS for 1 h. Then, they were
incubated overnight at 4°C with 8-OHdG monoclonal
antibody diluted 1:20 in a solution of 10% goat serum
in 0.01 M PBS containing 0.3% (v/v) Triton X-100.
After washing with 0.01 M PBS, the sections were
incubated for 30 min at room temperature with a mixture
of an Alexa Fluor 488 labeled F(ab’)2 fragment of
goat anti-rabbit IgG (H+L) (1:1000 dilution) (A11070;
Invitrogen, Carlsbad, CA, USA). We confirmed the
staining by comparison with the negative control.
Immuno fluorescence images were taken using a micro-
scope (BX50; Olympus, Tokyo) with a cooled charge-
coupled device camera (DP30BP, Olympus) at 1360 ×
1024 pixels via MetaMorph software (Molecular
Devices, Sunnyvale, CA, USA), and the 8-OHdG-
positive cells were counted in the ONL at a distance of
285 – 715 mm from the optic disc on the images. Three
eye sections per eye (two images per one section) were
photographed and the average of the six images was
used as the data per eye.
RNA isolation
To examine the changes in gene expressions of endo-
genous antioxidant after light exposure, non-treated
and light-exposed retinas were obtained. Thirty mice in
total were used in this experiment. Mice were euthanized
by cervical-spine dislocation, and the eyeballs were
quickly removed. The retinas were carefully separated
from the eyeballs and rapidly frozen in liquid nitrogen.
RNA was isolated from retinas with the aid of a
NucleoSpin® RNA II (Macherey-Nagal GmbH & Co.
KG, Düren, Germany).
Real-time RT-PCR
RNA concentrations were determined spectrophoto-
metrically at 260 nm. RT-PCR was performed using the
Thermal Cycler Dice Real Time System (Takara, Shiga).
The reverse transcription reaction was performed at 50°C
for 15 min using a PrimeScript RT reagent kit (Perfect
Real Time; Takara) and Thermal Cycler Dice Real
Time System (Takara). The target cDNA was amplified
by 40 cycles of PCR using SYBR Premix Ex Taq
(Takara) and a TP 8000 Thermal Cycler Dice Real
Time system (Takara). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as the reference
standard, and relative levels of Sod (superoxide dis-
mutase) 1, MT (metallothionein)-II, MT-III compared to
that of GAPDH were calculated. The following primers
were used: Sod1, 5′-AGCATTCCATCATTGGCCGTA-
3′ (forward) and 5′-TACTGCGCAATCCCAATCACTC-
3′ (reverse); MT-II, 5′-CCTGTGCCTCCGATGGAT-3′
(forward) and 5′-ACTTGTCGGAAGCCTCTTTG-3′
(reverse); MT-III, 5′-CTGAGACCTGCCCCTGTC-3′
(forward) and 5′-TTCTCGGCCTCTGCCTTG-3′ (reverse);
GAPDH, 5′-TGTGTCCGTCGTGGATCTGA-3′ (forward),
5′-TTGCTGTTGAAGTCGCAGGAG-3′ (reverse).
Chemicals and reagents
Dulbecco’s modified Eagle’s medium (DMEM) was
purchased from Sigma-Aldrich (St. Louis, MO, USA).
N-Acetyl-l-cysteine (NAC) was purchased from Wako
(Osaka). Fetal bovine serum (FBS) was purchased from
Valeant (Costa Mesa, CA, USA). Dimethyl sulfoxide
(DMSO) and olive oil were purchased from Nacarai
Tesque, Inc. (Kyoto). Penicillin and streptomycin were
purchased from Meiji Seika Kaisha, Ltd (Tokyo).
Hoechst 33342 and 5-(and-6)-chloromethyl-2′,7′-
dichlorodihydrofluorescein diacetate acetyl ester (CM-
H2DCFDA) were purchased from Molecular Probes
(Eugene, OR, USA). Astaxanthin was the free form
derived from Paracoccus carotinifacien. The purity of

212 T Otsuka et al
the astaxanthin and carotenoid were 60% and 99%,
respectively.
Cell culture
The mouse retinal cone-cell line 661W, a transformed
mouse cone cell line derived from mouse retinal tumors,
was provided by Dr. Muayyad R. Al-Ubaidi (University
of Oklahoma Health Sciences Center, Oklahoma City,
OK, USA). The cells were maintained in DMEM con-
taining 10% FBS, 100 U/ml penicillin, and 100 mg/ml
streptomycin. Cultures were maintained at 37°C in a
humidified atmosphere of 95% air and 5% CO2. The
661W cells were passaged by trypsinization every 3 – 4
days.
Exposure of mouse retinal cone-cell line 661W cells to
white light
The 661W cells were seeded in 96-well plates, at
1 × 103 cells per well, and then incubated for 24 h. The
entire medium was then replaced with fresh medium
containing 1% FBS. Astaxanthin was added, and 24 h
following treatment, the cells were exposed to 2,500 lux
of white light (C-FPS115D; Nikon, Tokyo) for 24 h at
37°C. NAC was added 1 h before white light irradiation.
Nuclear staining assays were performed immediately
after light exposure.
Nuclear staining assays
At the end of the culture period, Hoechst 33342
(lex = 360 nm, lem > 490 nm) and PI (lex = 535 nm,
lem > 617 nm) were added to the culture medium for
15 min at final concentrations of 8.1 and 1.5 mM, respec-
tively. Hoechst 33342 freely enters living cells and stains
the nuclei of viable cells, as well as those that have
suffered apoptosis or necrosis. Propidium iodide is a
membrane-impermeable dye that is generally excluded
from viable cells. Images were collected using an
Olympus IX70 inverted epifluorescence microscope
(Olympus). We counted the total number of cells and
calculated the percentage of PI-positive cells as a mea-
sure of dead cells.
Reactive oxygen species detection
Intracellular radical activation within 661W cells was
measured using CM-H2DCFDA. CM-H2DCFDA that
is taken up into the cell is converted to dichlorodihydro-
fluorescein (DCFH) by an intracellular esterase. Non-
fluorescent DCFH was oxidized by the ROS to fluores-
cent DCFH. At the end of the light exposure period,
CM-H2DCFDA was added to the culture medium and
incubated at 37°C for 1 h, at a final concentration of 10
mM. The 96-well plate was loaded onto a plate in a
fluorescence spectrophotometer, and the reaction was
carried out at 37°C. Cell fluorescence was determined
by Hoechst 33342 staining and was used to calculate
ROS production per cell (26).
Statistical analyses
Data were presented as the mean ± S.E.M. Statistical
comparisons were made with Dunnett’s test or Student’s
t-test, using statistical analysis software (StatView
version 5.0; SAS Institute, Cary, NC, USA). P < 0.05
was considered to indicate statistical significance.
Results
Retinal dysfunction and histological damages after light
exposure in mice
The mice were exposed to 8,000-lux light for 3 h,
and retinal functions were recorded by ERG 5 days
after light exposure and evaluated by both a- and b-wave
amplitudes (n = 10 or 11). A-wave represents photore-
ceptor function, and b-wave represents secondary
neurons, such as Müller cell and bipolar cell functions.
In the light-exposed group, the a- and b-wave amplitudes
had significantly decreased when compared 5 days after
exposure to light, at −0.02 log cds/m2, with the non-
treated retinas. Astaxanthin (100 mg/kg, p.o.) inhibited
the reduction of these amplitudes by maximums of 47%
and 61%, respectively (Fig. 1).
The effects of astaxanthin on light-induced retinal
damage were further examined by histological analysis
(n = 18 – 20). Light damage mainly occurred in the
ONL, which includes the rods and cones. ONL thickness
was significantly decreased at 5 days after light exposure
compared with the non-exposed retinas. Astaxanthin
(100 mg/kg, p.o.) prevented the reduction of ONL thick-
ness by a maximum of 57% (Fig. 2).
Apoptosis after light exposure
TUNEL staining is used to detect apoptosis. To
investigate light-induced apoptotic cell death and the
effect of astaxanthin on cell death, we performed TUNEL
staining (n = 7 – 11) and assessed the numbers of
TUNEL-positive cells (Fig. 3). Several studies (27) per-
formed TUNEL staining at 48 h after light exposure
and we followed these time courses. TUNEL-positive
cells appeared in the ONL. At the same time, TUNEL-
positive cells were not observed in any retinal area in the
normal retina. Quantitative analysis (Fig. 3D) showed
that light irradiation to mouse retina significantly
increased the number of TUNEL-positive cells in
ONL compared with the normal retina. Astaxanthin
(100 mg/kg, p.o.) significantly reduced the number of
TUNEL-positive cells compared with the vehicle-treated
group, and the inhibition was approximately 28%.

213Protective Effects of Astaxanthin
Fig. 2. Effects of oral administration of
astaxanthin on retinal damage induced by
exposure to light in mice. A) Nontreated,
B) light exposure (8,000 lux, 3 h) plus
vehicle-treated, and C) light exposure plus
astaxanthin-treated (100 mg/kg, p.o.) retinal
cross-sections at 5 days after light expo-
sure in mice. D) Measurement of thickness
in the outer nuclear layer at 5 days after
light exposure. Data are shown as the
mean ± S.E.M. (n = 18 – 20). *P < 0.05,
**P < 0.01 vs. normal group; #P < 0.05,
##P < 0.01 vs. vehicle group (t-test). The
horizontal scale bar represents 50 mm.
Fig. 1. Measurement of dark-adapted
ERG amplitudes at 5 days after exposure
to light in the mouse retina. A) Typical
traces of dark-adapted ERG responses
measured at 5 days after exposure to
light. Stimulus flashes were used from
0.98 log cds/m2. B and C) Amplitudes of
a- and b-waves of light exposure (8,000
lux), plus the vehicle-treated group vs.
light exposure, plus the astaxanthin-treated
group (100 mg/kg, p.o.). Data are shown
as the mean ± S.E.M. (n = 10 or 11).
*P < 0.05, **P < 0.01 vs. normal group;
#P < 0.05, ##P < 0.01 vs. vehicle group
(t-test).

214 T Otsuka et al
Fig. 4. Effects of oral administration of
astaxanthin on expression of 8-OHdG-positive
cells induced by exposure to light in mice. A)
Nontreated, B) light exposure (8,000 lux, 3 h)
plus vehicle-treated, and C) light exposure
plus astaxanthin-treated (100 mg/kg, p.o.)
retinal cross-sections at 24 h after light expo-
sure in mice. D) Measurement of 8-OHdG-
positive cells in the outer nuclear layer. Data
are shown as the mean ± S.E.M. (n = 10).
**P < 0.01 vs. normal group, #P < 0.05 vs.
vehicle-treated group (t-test). Scale bars: 50 mm
(low magnification), 10 mm (small square; high
magnification).
Fig. 3. Effects of oral administration of astaxan-
thin on expression of TUNEL-positive cells in-
duced by exposure to light in mice. A) Nontreated,
B) light exposure (8,000 lux, 3 h) plus vehicle-
treated, and C) light exposure plus astaxanthin-
treated (100 mg/kg, p.o.) retinal cross-sections at
48 h after light exposure in mice. D) Measurement
of TUNEL-positive cells in the outer nuclear
layer. Data are shown as the mean ± S.E.M.
(n = 7 – 11). **P < 0.01 vs. normal group, #P < 0.05
vs. vehicle-treated group (t-test). Scale bar: 50 mm.

215Protective Effects of Astaxanthin
Oxidative stress after light exposure in mice
To clarify whether the protective effect of astaxanthin
is related to its antioxidative effect, immunostaining was
performed (n = 10). Because 8-hydroxy-2-deoxiguanosine
(8-OHdG) was a marker of DNA oxidative damage, we
evaluated the numbers of these cells in the ONL. At
24 h after light exposure, 8-OHdG-positive cells were
observed in the ONL; no 8-OHdG-positive cells were
observed in the normal retinas (Fig. 4). Treatment with
astaxanthin significantly decreased the number of
8-OHdG-positive cells; the inhibition was approximately
23%.
Expression of antioxidant genes
To clarify changes in mRNA levels of endogenous
antioxidant, we investigated mouse retinas after 6 h of
light exposure by real-time RT-PCR (Fig. 5, n = 10). We
were referred to the previous report (28) to determine
the timing of sampling retinas. The mRNA expression
of Sod1, MT-II, and MT-III were elevated significantly
after light exposure (20.7-, 1.7-, 1.2-fold increase, re-
spectively). Astaxanthin (100 mg/kg, p.o.) had no effect
against Sod1, MT-II, or MT-III compared to vehicle-
treated retinas.
Effects of astaxanthin against light-induced 661W cell
death
We examined the effect of astaxanthin on light-induced
photoreceptor degeneration. Representative photographs
of Hoechst 33342 and PI staining are shown in Fig. 4A.
Hoechst 33342 stains all cell nuclei (live and dead cells),
while PI stains only dead cells. Pretreatment with astax-
anthin at concentrations of 1 – 100 nM protected the
cells from light-induced cell death in a concentration-
dependent manner. NAC at 1 mM inhibited cell death
(Fig. 5B).
Effects of astaxanthin on the intracellular oxidation of
DCFH induced by various types of ROS
CM-H2DCFDA, a ROS-sensitive probe, was used as
a radical scavenging capacity assay. Light irradiation
increased during ROS production, and astaxanthin
(100 nM) significantly reduced the production and
astaxanthin at 10 nM had a tendency to reduce it, but
not significantly (Fig. 6).
Fig. 5. Effects of astaxanthin on 661W cell damage induced by light exposure. A – C) Representative fluorescence microscopy
of Hoechst 33342 staining, moments after 24-h light exposure. A) Non-treated cells showed normal nuclear morphology. B)
Light-induced degeneration occurred. C) Pretreatment with 100 nM astaxanthin. D) PI-positive cells were assessed by immersing
them in Hoechst 33342 and PI complex. Astaxanthin and Trolox significantly inhibited the light-induced retinal cell damage.
*P < 0.05, **P < 0.01 vs. vehicle; ##P < 0.01 vs. control; data are expressed as the mean ± S.E.M. (n = 12).
Fig. 6. Effects of astaxanthin on light-induced ROS production in
661W cells. Cellular radical intensity was quantified by fluorescence
microscopy of the CM-H2DCFDA probe. Light exposure causes
ROS production, which was partly prevented by the astaxanthin and
NAC treatment. **P < 0.01 vs. vehicle, ##P < 0.01 vs. control; data
are expressed as the mean ± S.E.M. (n = 6).

216 T Otsuka et al
Discussion
In the previous report from our laboratory, astaxanthin
prevented retinal damage induced by NMDA intravitreal
injection in vivo and RGC-5 degeneration induced by
serum deprivation and addition of H2O2 in vitro (25).
This report suggests that astaxanthin has a protective
effect against glaucoma via prevention of oxidative
stress. We hypothesized that astaxanthin shows protec-
tion against retinal diseases related with oxidative stress,
but the effects of light-induced retinal degeneration,
which is the model of dry AMD and RP, is still unknown
and there are no reports about this so far. In the present
study, we demonstrated that astaxanthin protected against
light-induced retinal damage, and inhibited oxidative
stress of the photoreceptors.
Previous reports have studied the concentrations of
free astaxanthin in plasma after single-dose oral gavage
with free astaxanthin (29, 30). According to astaxanthin
(500 mg/kg, p.o.) treatment in emulsion in mice, Cmax
was approximately 400 nM (29). In this study, astaxanthin
was orally administered four times a day, at a dose of
100 mg/kg, indicating that a total of about 400 mg/kg
astaxanthin was administered per day. This finding
indicates that the maximal plasma concentration of
astaxanthin in this study could be at least 100 nM. This
concentration corresponded to the in vitro assay, wherein
astaxanthin reduced intracellular ROS activity and
661W-induced cell damage at concentrations of 1 – 100
nM (Figs. 5 and 6).
Some reports have indicated that dietary astaxanthin
treatment is safe in animals (31, 32) and humans (33, 34).
In this study, astaxanthin caused no apparent abnormality,
and there were no body weight changes in the light-
exposure group compared with the vehicle-treated group.
This observation suggests that the present study provides
evidence that eight times treatment with astaxanthin at
100 mg/kg within four days causes no ill effects, in
agreement with other reports published thus far.
Previous reports have indicated that light exposure
induces Sod1 (8) and that the retinas of Sod-1–knockout
mice are likely to be impaired by light exposure (9),
suggesting that light exposure induces ROS in the retina
and evokes retinal degeneration. ROS is related to the
regulation of many principal cell functions such as acti-
vation of transcription factor (35), gene expression (36),
and cell proliferation (37). On the other hand, excessive
production of ROS induces cell death in various cells
(37). In this study, 8-OHdG-positive cells appeared in
the ONL layer, but astaxanthin reduced their number
(Fig. 4). 8-OHdG is a biomarker of DNA oxidative stress
(38), indicating that astaxanthin protected the retinal
cells via reducing oxidative stress. Visible-light exposure
to the retina evokes the photooxidation of N-retinylidene-
N-retinylethanolamine (A2E) and all-trans-retinal dimer,
which are the efficient producers of singlet oxygen (O2·)
(39). O2·, an ROS, is O2 in an excited electronic state
which provokes the peroxidation of lipids, cellular
membrane damage, and DNA damage when it changes
to its ground state (40). O2· is also related to retinal cell
death (41). Carotenoids have been known to detoxify O2·
catalytically; a single beta carotene molecule can remove
250 – 1000 molecules of O2· (42). Astaxanthin, one of
the carotenoids, is well known to possess the capacity to
detoxify O2· catalytically (43). Therefore, the findings of
this study indicate that O2· produced by light irradiation
might have been prevented by the existence of astaxan-
thin, oxidative stress was weakened, and retinal cell
death was reduced.
Astaxanthin (100 mg/kg, p.o.) significantly prevented
retinal damages induced by light irradiation (Figs. 1 – 4).
The levels of these inhibitory effects are different in
these evaluations. In retinal function and histological
analysis, approximately 50% damage was prevented
(Figs. 1 and 2). However, astaxanthin prevented retinal
damage by only 28% (Fig. 3). These findings suggest
that photoreceptor damage is related to not only cellular
apoptosis, but also necroptosis (44) or autophagy (45),
and they are not strictly separated, for example, apoptosis
and autophagy (46). There is a possibility that the protec-
tive effect of astaxanthin administration may also involve
in these pathways of cell death. Additionally, the ratio
of the protective effect against apoptosis is not consistent
with the inhibitory effect of 8-OHdG (Figs. 3 and 4).
Several factors are involved in light-induced retinal
apoptosis via oxidative stress, for example, DNA damage,
lipid peroxidation (12), and inflammation (19). Astaxan-
thin might have protective effect against not only DNA
oxidative stress or apoptotic degeneration, but also
inflammation or lipid peroxidation or other effects.
However, further investigations are needed to solve
these issues.
We clarified that astaxanthin has protective effect
against oxidative stress. However, whether endogenous
antioxidant is influenced by astaxanthin is not apparent.
Therefore, we investigated this by measuring the mRNA
expression of three antioxidants. It has been reported that
Sod1 is involved in the change of superoxide anion into
oxygen and hydrogen peroxide and induced in the retina
after light exposure (47). MT is expressed in the tissues
and central nervous system, and it has a high content of
sulfhydryls, which targets it to scavenge superoxide
anion and hydroxyl radicals with an affinity more than
300-fold higher that of reduced glutathione (48). In a
previous report, Sod1, MT-II, and MT-III mRNAs were
found to be increased in the retina 6 h after light exposure

217Protective Effects of Astaxanthin
(28), and our data was consistent with this. However,
astaxanthin did not influence Sod1, MT-II, and MT-III
mRNA. Therefore our observations suggest that astaxan-
thin does not interact with endogenous antioxidants.
One of the constructive characteristics of astaxanthin
is that it has polar ionone rings at the ends and a non-
polar zone of conjugated carbon–carbon bonds in the
middle. It has been suggested that the inner membrane
and surface are protected from ROS because the polar
end groups overlap the polar boundary zones of the
bi-layer membrane, and the nonpolar middle chain fits
the membrane’s nonpolar interior (49, 50). In this study,
astaxanthin actually reduced intracellular ROS produc-
tion (Fig. 6). These findings suggest that astaxanthin
might inhibit intracellular DNA degeneration caused
by ROS more strongly than similar carotenoids and that
it exhibits protective effects against light-induced photo-
receptor degeneration.
In conclusion, these findings suggest that dietary
astaxanthin treatment is effective against light-induced
retinal cell death, indicating that astaxanthin intake leads
to the prevention and inhibition of the progression of RP
and dry AMD.
References
1 Hamel C. Retinitis pigmentosa. Orphanet J Rare Dis. 2006;1:40.
2 Hirakawa M, Tanaka M, Tanaka Y, Okubo A, Koriyama C, Tsuji
M, et al. Age-related maculopathy and sunlight exposure
evaluated by objective measurement. Br J Ophthalmol. 2008;
92:630–634.
3 Paskowitz DM, LaVail MM, Duncan JL. Light and inherited
retinal degeneration. Br J Ophthalmol. 2006;90:1060–1066.
4 Noell WK, Walker VS, Kang BS, Berman S. Retinal damage by
light in rats. Invest Ophthalmol. 1966;5:450–473.
5 Nickells RW, Zack DJ. Apoptosis in ocular disease: a molecular
overview. Ophthalmic Genet. 1996;17:145–165.
6 Yang JH, Basinger SF, Gross RL, Wu SM. Blue light-induced
generation of reactive oxygen species in photoreceptor ellipsoids
requires mitochondrial electron transport. Invest Ophthalmol Vis
Sci. 2003;44:1312–1319.
7 Godley BF, Shamsi FA, Liang FQ, Jarrett SG, Davies S, Boulton
M. Blue light induces mitochondrial DNA damage and free
radical production in epithelial cells. J Biol Chem. 2005;280:
21061–21066.
8 Yamashita H, Horie K, Yamamoto T, Nagano T, Hirano T. Light-
induced retinal damage in mice. Hydrogen peroxide production
and superoxide dismutase activity in retina. Retina. 1992;12:
59–66.
9 Hashizume K, Hirasawa M, Imamura Y, Noda S, Shimizu T,
Shinoda K, et al. Retinal dysfunction and progressive retinal
cell death in SOD1-deficient mice. Am J Pathol. 2008;172:
1325–1331.
10 Organisciak DT, Darrow RA, Barsalou L, Darrow RM, Lininger
LA. Light-induced damage in the retina: differential effects of
dimethylthiourea on photoreceptor survival, apoptosis and DNA
oxidation. Photochem Photobiol. 1999;70:261–268.
11 Ranchon I, Chen S, Alvarez K, Anderson RE. Systemic admin-
istration of phenyl-N-tert-butylnitrone protects the retina from
light damage. Invest Ophthalmol Vis Sci. 2001;42:1375–1379.
12 Tanito M, Li F, Elliott MH, Dittmar M, Anderson RE. Protective
effect of TEMPOL derivatives against light-induced retinal dam-
age in rats. Invest Ophthalmol Vis Sci. 2007;48:1900–1905.
13 Kurashige M, Okimasu E, Inoue M, Utsumi K. Inhibition of
oxidative injury of biological membranes by astaxanthin. Physiol
Chem Phys Med NMR. 1990;22:27–38.
14 O’Connor I, O’Brien N. Modulation of UVA light-induced
oxidative stress by beta-carotene, lutein and astaxanthin in
cultured fibroblasts. J Dermatol Sci. 1998;16:226–230.
15 Iwamoto T, Hosoda K, Hirano R, Kurata H, Matsumoto A,
Miki W, et al. Inhibition of low-density lipoprotein oxidation by
astaxanthin. J Atheroscler Thromb. 2000;7:216–222.
16 Kobayashi M. In vivo antioxidant role of astaxanthin under
oxidative stress in the green alga Haematococcus pluvialis.
Appl Microbiol Biotechnol. 2000;54:550–555.
17 Aoi W, Naito Y, Sakuma K, Kuchide M, Tokuda H, Maoka T,
et al. Astaxanthin limits exercise-induced skeletal and cardiac
muscle damage in mice. Antioxid Redox Signal. 2003;5:
139–144.
18 Chew BP, Park JS, Wong MW, Wong TS. A comparison of the
anticancer activities of dietary beta-carotene, canthaxanthin and
astaxanthin in mice in vivo. Anticancer Res. 1999;19:1849–
1853.
19 Ohgami K, Shiratori K, Kotake S, Nishida T, Mizuki N, Yazawa
K, et al. Effects of astaxanthin on lipopolysaccharide-induced
inflammation in vitro and in vivo. Invest Ophthalmol Vis Sci.
2003;44:2694–2701.
20 Uchiyama K, Naito Y, Hasegawa G, Nakamura N, Takahashi J,
Yoshikawa T. Astaxanthin protects beta-cells against glucose
toxicity in diabetic db/db mice. Redox Rep. 2002;7:290–293.
21 Kang JO, Kim SJ, Kim H. Effect of astaxanthin on the hepato-
toxicity, lipid peroxidation and antioxidative enzymes in the liver
of CCl4-treated rats. Methods Find Exp Clin Pharmacol. 2001;
23:79–84.
22 Okai Y, Higashi-Okai K. Possible immunomodulating activities
of carotenoids in in vitro cell culture experiments. Int J Immuno-
pharmacol. 1996;18:753–758.
23 Miyawaki H, Takahashi J, Tsukahara H, Takehara I. Effects of
astaxanthin on human blood rheology. J Clin Biochem Nutr.
2008;43:69–74.
24 Izumi-Nagai K, Nagai N, Ohgami K, Satofuka S, Ozawa Y,
Tsubota K, et al. Inhibition of choroidal neovascularization with
an anti-inflammatory carotenoid astaxanthin. Invest Ophthalmol
Vis Sci. 2008;49:1679–1685.
25 Nakajima Y, Inokuchi Y, Shimazawa M, Otsubo K, Ishibashi T,
Hara H. Astaxanthin, a dietary carotenoid, protects retinal cells
against oxidative stress in-vitro and in mice in-vivo. J Pharm
Pharmacol. 2008;60:1365–1374.
26 Matsunaga N, Imai S, Inokuchi Y, Shimazawa M, Yokota S,
Araki Y, et al. Bilberry and its main constituents have neuro-
protective effects against retinal neuronal damage in vitro and
in vivo. Mol Nutr Food Res. 2009;53:869–877.
27 Imai S, Inokuchi Y, Nakamura S, Tsuruma K, Shimazawa M,
Hara H. Systemic administration of a free radical scavenger,
edaravone, protects against light-induced photoreceptor degen-
eration in the mouse retina. Eur J Pharmacol. 2010;642:77–85.
28 Tsuruma K, Shimazaki H, Ohno Y, Inoue Y, Honda A, Imai S,

218 T Otsuka et al
et al. Metallothionein-III deficiency exacerbates light-induced
retinal degeneration. Invest Ophthalmol Vis Sci. 2012;53:7896–
7903.
29 Showalter LA, Weinman SA, Osterlie M, Lockwood SF. Plasma
appearance and tissue accumulation of non-esterified, free
astaxanthin in C57BL/6 mice after oral dosing of a disodium
disuccinate diester of astaxanthin (Heptax). Comp Biochem
Physiol C Toxicol Pharmacol. 2004;137:227–236.
30 Mercke Odeberg J, Lignell A, Pettersson A, Hoglund P. Oral
bioavailability of the antioxidant astaxanthin in humans is
enhanced by incorporation of lipid based formulations. Eur J
Pharm Sci. 2003;19:299–304.
31 Stewart JS, Lignell A, Pettersson A, Elfving E, Soni MG. Safety
assessment of astaxanthin-rich microalgae biomass: Acute and
subchronic toxicity studies in rats. Food Chem Toxicol. 2008;
46:3030–3036.
32 Ono A, Sekita K, Saitoh M, Umemura T, Ogawa Y, Furuya T,
et al. [A 13-week subchronic oral toxicity study of haematococcus
color in F344 rats]. Kokuritsu Iyakuhin Shokuhin Eisei
Kenkyusho Hokoku. 1999;91–98. (text in Japanese)
33 Spiller GA, Dewell A. Safety of an astaxanthin-rich Haemato-
coccus pluvialis algal extract: a randomized clinical trial. J Med
Food. 2003;6:51–56.
34 Satoh A, Tsuji S, Okada Y, Murakami N, Urami M, Nakagawa
K, et al. Preliminary clinical evaluation of toxicity and efficacy
of a new astaxanthin-rich Haematococcus pluvialis extract. J
Clin Biochem Nutr. 2009;44:280–284.
35 Schreck R, Rieber P, Baeuerle PA. Reactive oxygen inter-
mediates as apparently widely used messengers in the activation
of the NF-kappa B transcription factor and HIV-1. Embo J.
1991;10:2247–2258.
36 Lo YY, Cruz TF. Involvement of reactive oxygen species in
cytokine and growth factor induction of c-fos expression in
chondrocytes. J Biol Chem. 1995;270:11727–11730.
37 Murrell GA, Francis MJ, Bromley L. Modulation of fibroblast
proliferation by oxygen free radicals. Biochem J. 1990;265:
659–665.
38 Shigenaga MK, Ames BN. Assays for 8-hydroxy-2’-deoxy-
guanosine: a biomarker of in vivo oxidative DNA damage. Free
Radic Biol Med. 1991;10:211–216.
39 Wu Y, Yanase E, Feng X, Siegel MM, Sparrow JR. Structural
characterization of bisretinoid A2E photocleavage products and
implications for age-related macular degeneration. Proc Natl
Acad Sci U S A. 2010;107:7275–7280.
40 Fukuzawa K, Inokami Y, Tokumura A, Terao J, Suzuki A. Rate
constants for quenching singlet oxygen and activities for
inhibiting lipid peroxidation of carotenoids and alpha-tocopherol
in liposomes. Lipids. 1998;33:751–756.
41 Sparrow JR, Zhou J, Ben-Shabat S, Vollmer H, Itagaki Y,
Nakanishi K. Involvement of oxidative mechanisms in blue-
light-induced damage to A2E-laden RPE. Invest Ophthalmol Vis
Sci. 2002;43:1222–1227.
42 Johnson EA, Schroeder WA. Microbial carotenoids. Adv Bio-
chem Eng Biotechnol. 1996;53:119–178.
43 Hama S, Takahashi K, Inai Y, Shiota K, Sakamoto R, Yamada A,
et al. Protective effects of topical application of a poorly soluble
antioxidant astaxanthin liposomal formulation on ultraviolet-
induced skin damage. J Pharm Sci. 2012;101:2909–2916.
44 Chang Q, Peter ME, Grassi MA. Fas ligand-Fas signaling partici-
pates in light-induced apoptotic death in photoreceptor cells.
Invest Ophthalmol Vis Sci. 2012;53:3703–3716.
45 Wenzel A, Grimm C, Samardzija M, Reme CE. Molecular
mechanisms of light-induced photoreceptor apoptosis and neuro-
protection for retinal degeneration. Prog Retin Eye Res. 2005;
24:275–306.
46 Bursch W. The autophagosomal-lysosomal compartment in pro-
grammed cell death. Cell Death Differ. 2001;8:569–581.
47 Yamamoto M, Lidia K, Gong H, Onitsuka S, Kotani T, Ohira
A. Changes in manganese superoxide dismutase expression after
exposure of the retina to intense light. Histochem J. 1999;31:
81–87.
48 Thornalley PJ, Vasak M. Possible role for metallothionein in
protection against radiation-induced oxidative stress. Kinetics
and mechanism of its reaction with superoxide and hydroxyl
radicals. Biochim Biophys Acta. 1985;827:36–44.
49 Pashkow FJ, Watumull DG, Campbell CL. Astaxanthin: a novel
potential treatment for oxidative stress and inflammation in
cardiovascular disease. Am J Cardiol. 2008;101:58D–68D.
50 Kidd P. Astaxanthin, cell membrane nutrient with diverse clinical
benefits and anti-aging potential. Altern Med Rev. 2011;16:
355–364.