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

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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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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.
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... ARMD is characterized by retinal pigment epithelium (RPE) cells within the damaged macula, leading to foveal photoreceptors loss [47]. AST intake leads to prevention and inhibition of dry ARMD progression [48]. A randomized controlled trial (RCT) reported that supplementation with carotenoids and antioxidants (including vitamin C (180 mg), lutein (10 mg), zeaxanthin (1 mg), and AST (4 mg) selectively improved central retinal dysfunctions (0°-5°). ...
... Numerous animal studies have further confirmed the protective effects of AST on the eye and its effectiveness in the prevention of ocular diseases, ranging from the ocular surface to the fundus, including DES, phototoxic keratitis [44], cataract [51], diabetic retinopathy [11], ARMD [48,52], retinal ischemia injury [53], and other ocular diseases. All the details are presented in Table 2. ...
... Tomohiro et al. reported that AST protected the photoreceptor cells at a dose of 100 mg/kg and inhibited retinal dysfunction in terms of electroretinogram (ERG) and outer nuclear layer (ONL) loss and reduced the expression of apoptotic and 8-OHdG-positive cells, induced by light exposure via its antioxidative effect mechanism. Moreover, AST protected against increases of PI-positive cells and intracellular ROS activity in retinal core cells [48,52]. Pathological neovascularization of the retina is another important cause of choroidal neovascularization (CNV) and AMD [57]. ...
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Astaxanthin (AST) is a naturally occurring carotenoid that has strong antioxidant, anti-inflammatory, and antiapoptosis effects and is used for the prevention of cancer. There is growing evidence that AST has multiple protective effects against various eye diseases. This article reviews the function and the potential mechanism of AST in dry eye syndrome, keratitis, cataract, diabetic retinopathy, age-related macular degeneration, high intraocular pressure, and other ocular diseases. It provides a theoretical basis for the clinical application of AST as a potential nutraceutical.
... Astaxanthin (AST) is one of the most common carotenoids and is found in shellfish and the red pigment of salmon (13). It has various pharmacological properties such as antioxidant, antitumor, anti-inflammatory, antidiabetic, hepatoprotective, and immunomodulatory effects (13)(14)(15). Moreover, one of the characteristic features is that it is highly safe (14). ...
... It has various pharmacological properties such as antioxidant, antitumor, anti-inflammatory, antidiabetic, hepatoprotective, and immunomodulatory effects (13)(14)(15). Moreover, one of the characteristic features is that it is highly safe (14). It has been shown that astaxanthin induces reactive oxygen radicals and reduces inflammation by inhibiting nuclear factor kappa B (NF-kB) activation (16). ...
... It is known that there is an increase in apoptosis and loss of immune system cells in the process of sepsis (18). In the study of Otsuka et al. (14), apoptosis was evaluated by TUNEL method in retinal cells damaged by excessive light after the administration of astaxanthin and it was found that apoptosis was less than 28%. In the study of Zhang et al. (21), they induced subarachnoid hemorrhage and administrated intracerebroventricular astaxanthin and they evaluated the effects of astaxanthin on early brain injury. ...
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... In AMD experimental models, the increase of 8-OHdG in the retinal tissue was induced by the sub-retinal injection of human lipid hydroperoxide (HpODE), a product derived from the sub-macular region of elderly and AMD patients [213]. It may also be induced genetically [214], using H2O2 in cultured ARPE19 cells [215], induced by light [216][217] or the exposure to chronic cigarette smoke [218]. In regard to smoking, one of the main modifiable AMD factors, it has been demonstrated that exposure of ARPE-19 cells to a cigarette smoke concentrate (CSC) not only enhanced ROS levels but also induced 8-OHdG DNA lesions [219]. ...
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... Previous studies have demonstrated the efficacy of various compounds in vivo via an oral route in an acute light-induced retinal degeneration model. Astaxantin that was dissolved in olive oil and orally administrated at 100 mg/kg to ddY mice partially preserve photoreceptors before the induction of retinal degeneration with white light (3 h at 8000 Lux) [52]. In another work, NSP-116 suspended in water with 0.5% sodium carboxymethyl cellulose (CMC) was administered orally at 30 mg/kg to ddY mice 30 min before inducing retinal degeneration with white light (8000 Lux for 3 h). ...
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Citation: Vincent, M.; Simon, L.; Brabet, P.; Legrand, P.; Dorandeu, C.; Him, J.L.K.; Durand, T.; Crauste, C.; Begu, S. Formulation and Evaluation of SNEDDS Loaded with Original Lipophenol for the Oral Route to Prevent Dry AMD and Stragardt's Disease.
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Oxidative stress induced by hyperglycemia possibly causes the dysfunction of pancreatic β-cells and various forms of tissue damage in patients with diabetes mellitus. Astaxanthin, a carotenoid of marine microalgae, is reported as a strong anti-oxidant inhibiting lipid peroxidation and scavenging reactive oxygen species. The aim of the present study was to examine whether astaxanthin can elicit beneficial effects on the progressive destruction of pancreatic β-cells in db/db mice - a well-known obese model of type 2 diabetes. We used diabetic C57BL/KsJ-db/db mice and db/m for the control. Astaxanthin treatment was started at 6 weeks of age and its effects were evaluated at 10, 14, and 18 weeks of age by non-fasting blood glucose levels, intraperitoneal glucose tolerance test including insulin secretion, and β-cell histology. The non-fasting blood glucose level in db/db mice was significantly higher than that of db/m mice, and the higher level of blood glucose in db/db mice was significantly decreased after treatment with astaxanthin. The ability of islet cells to secrete insulin, as determined by the intraperitoneal glucose tolerance test, was preserved in the astaxanthin-treated group. Histology of the pancreas revealed no significant differences in the β-cell mass between astaxanthin-treated and -untreated db/db mice. In conclusion, these results indicate that astaxanthin can exert beneficial effects in diabetes, with preservation of β-cell function. This finding suggests that anti-oxidants may be potentially useful for reducing glucose toxicity.
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purpose. To investigate whether photoreceptor ellipsoids generate reactive oxygen species (rOx) after blue light illumination. methods. Cultured salamander photoreceptors were exposed to blue light (480 ± 10 nm; 10 mW/cm²). The light-induced catalytic redox activity in the culture was monitored with the use of 3,3′-diaminobenzidine (DAB). Tetramethylrhodamine ethyl ester (TMRE) and 2′,7′-dichlorodihydro-fluorescein acetate (DHF-DA) were used as probes to measure the mitochondrial membrane potential and intracellular rOx, respectively. results. A significant deposit of DAB polymers was found in the culture after exposure to blue light. Basal levels of rOx were observed in photoreceptor ellipsoids when cells were stained with DHF-DA. This staining colocalized with TMRE. After exposure to blue light, a sharp increase of rOx immediately occurred in the ellipsoids of most photoreceptors. When the light intensity was reduced, the response kinetics of rOx generation were slowed down; however, comparable amounts of rOx were generated after a standard time of exposure to light. The production of rOx in photoreceptors was markedly decreased when an antioxidant mixture was included in the medium during exposure to light. Rotenone or antimycin A, the respiratory electron transport blockers at complex I and III, respectively, significantly suppressed the light-evoked generation of rOx. conclusions. A robust amount of rOx is produced in the ellipsoid when photoreceptors are exposed to blue light. This light-induced effect is antioxidant sensitive and strongly coupled to mitochondrial electron transport. The cumulative effect of light on rOx generation over time may implicate a role for mitochondria in light-induced oxidative damage of photoreceptors.
Article
Purpose: Retinal photoreceptor damage is a common feature of ophthalmic disorders, such as age-related macular degeneration and retinitis pigmentosa. Oxidative stress has a key role in these diseases. Metallothioneins (MTs) are a family of cysteine-rich proteins, and various physiologic functions have been reported, including protection against metal toxicity and antioxidative potency. We investigated the functional role of MT-III in light-induced retinal damage. Methods: The expression of retinal MT-I, -II, and -III mRNA was evaluated by real-time reverse-transcription PCR in retina exposed to light. Retinal damage in MT-deficient mice was induced by exposure to white light at 16,000 lux for 3 hours after dark adaptation. Photoreceptor damage was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) 5 days after light exposure and by electroretinogram recording. In an in vitro experiment, the MT-III siRNAs were tested for their effects on light-induced mouse photoreceptor cell (661W) damage. Results: The mRNAs of the MTs were increased significantly in murine retina after light exposure. The ONL in the MT-III-deficient mice was remarkably thinner compared to light-exposed wild-type (WT) mice, and a- and b-wave amplitudes were decreased; the damage induced in MT-I/-II-deficient mice was comparable to that observed in WT mice. MT-III knockdown by siRNA in 661W exacerbated the cell damage and increased the production of reactive oxygen species in response to light exposure. Conclusions: These findings suggested that MT-III can help protect against light-induced retinal damage compared to MT-I/II. Some of these effects may be exerted by its antioxidative potency.
Chapter
Carotenoids occur universally in photosynthetic organisms but sporadically in nonphotosynthetic bacteria and eukaryotes. The primordial carotenogenic organisms were cyanobacteria, and eubacteria that carried out anoxygenic photosynthesis. The phylogeny of carotenogenic organisms is evaluated to describe groups of organism which could serve as sources of carotenoids. Terrestrial plants, green algae, and red algae acquired stable endosymbionts (probably cyanobacteria) and have a predictable complement of carotenoids compared to prokaryotes, other algae, and higher fungi which have a more diverse array of pigments. Although carotenoids are not synthesized by animals, they are becoming known for their important role in protecting against damage by singlet oxygen and preventing chronic, diseases in humans. The growth of aquaculture during the past decade as well as the biological roles of carotenoids in human disease will increase the demand for carotenoids. Microbial synthesis offers a promising method for production of carotenoids.
Article
The 1O2 quenching rate constants (k Q ) of α-tocopherol (α-Toc) and carotenoids such as β-carotene, astaxanthin, canthaxanthin, and lycopene in liposomes were determined in light of the localization of their active sites in membranes and the micropolarity of the membrane regions, and compared with those in ethanol solution. The activities of α-Toc and carotenoids in inhibiting 1O2-dependent lipid peroxidation (reciprocal of the concentration required for 50% inhibition of lipid peroxidation: [IC50]−1) were also measured in liposomes and ethanol solution and compared with their k Q values. The k Q and [IC50]−1 values were also compared in two photosensitizing systems containing Rose bengal (RB) and pyrenedodecanoic acid (PDA), respectively, which generate 1O2 at different sites in membranes. The k Q values of α-Toc were 2.9×108M−1s−1 in ethanol solution and 1.4×107 M−1s−1 (RB system) or 2.5×106 M−1s−1 (PDA system) in liposomes. The relative [IC50]−1 value of α-Toc in liposomes was also five times higher in the RB system than in the PDA-system. In consideration of the local concentration of the OH-group of α-Toc in membranes, the k Q value of α-Toc in liposomes was recalculated as 3.3×106 M−1s−1 in both the RB and PDA systems. The k Q values of all the carotenoids tested in two photosensitizing systems were almost the same. The k Q value of α-Toc in liposomes was 88 times less than in ethanol solution, but those of carotenoids in liposomes were 600–1200 times less than those in ethanol solution. The [IC50]−1 value of α-Toc in liposomes was 19 times less than that in ethanol solution, whereas those of carotenoids in liposomes were 60–170 times less those in ethanol solution. There were no great differences (less than twice) in the k q and [IC50]−1 values of any carotenoids. The k Q values of all carotenoids were 40–80 times higher than that of α-Toc in ethanol solution but only six times higher that of α-Toc in liposomes. The [IC50]−1 values of carotenoid were also higher than that of α-Toc in ethanol solution than in liposomes, and these correlated well with the k Q values.
Article
Astaxanthin (Asx) would be expected to prevent ultraviolet (UV)-induced skin damage, as it is regarded as a potent antioxidative carotenoid in biological membranes. However, it is difficult to administer Asx topically to skin because of its poor water solubility. In this study, we attempted to solve this problem by preparing liposomes containing Asx (Asx-lipo), which were dispersible in the water phase, and therefore, suitable for topical application to the skin. Asx-lipo was shown to have potent scavenging ability against chemiluminescence-dependent singlet oxygen production in the water phase. When Asx-lipo was applied to skin before UV exposure, UV-induced skin thickening was prevented. Interestingly, collagen reduction induced by UV exposure was also prevented by preadministration of Asx-lipo. In addition, topical administration of Asx-lipo containing cationic lipid inhibited melanin production in skin exposed to UV. Consequently, we succeeded in preventing UV-induced skin damage using a topical application of a liposomal formulation containing Asx.
Article
We investigated the function of Fas in photoreceptors. Postmortem human eyes and mouse-derived photoreceptor cells (661W) were examined for Fas expression by in situ hybridization and immunofluorescence. 661W cells were treated with FasL or Fas agonistic antibody, or exposed to light with/without pharmacological manipulation of Fas signaling, followed by apoptosis detection by TUNEL, immunofluorescence and fluorescence activated cell scanning (FACS). Fractionated cellular extracts were used to detect protein expression or protein phosphorylation after immunoprecipitation by Western blot. Fas was expressed in the photoreceptor layer of human retina. Fas and a cleaved form of FasL were found on the cell surface of 661W cells. Treatment with FasL or Fas agonistic antibody induced apoptosis in 661W cells. Blocking the activity of FasL or administration of caspase-8 inhibitor z-IETD inhibited light-induced apoptosis. However, it simultaneously caused induction of necroptosis, which could be blocked by the receptor-interacting protein 1 (RIP1) inhibitor, necrostatin-1. Light exposure in the presence of z-IETD caused hyper-phosphorylation of RIP1. Light exposure did not elevate the expression of Fas, FasL, or the Fas-associated death domain adaptor protein (FADD). Cells or conditioned medium after light exposure induced apoptosis in dark-adapted cells, which could be attenuated by blockade of Fas. Fas has a pro-apoptotic role in photoreceptors. Under light stress, soluble and membrane-bound FasL can bind to Fas, inducing apoptosis via a paracrine mechanism. Although blocking Fas signaling inhibits apoptosis, it does not improve the overall photoreceptor survival due to a compensatory activation of necroptosis. Hence, prevention of photoreceptor loss from retinal photo-oxidative stress should target Fas and RIP1.