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Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress

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Abstract

Mitochondria combine the production of energy with an efficient chain of reduction-oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.
1
Astaxanthin protects mitochondrial redox state and
functional integrity against oxidative stress.
Alexander M Wolf
a
, Sadamitsu Asoh
a
, Hidenori Hiranuma
a
, Ikuroh Ohsawa
a,b
,
Kumiko Iio
c
, Akira Satou
c
, Masaharu Ishikura
c
, Shigeo Ohta
a,*
a
Department of Biochemistry and Cell Biology,
b
The Center of Molecular Hydrogen Medicine
Institute of Development and Aging Sciences, Nippon Medical School,
1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan
c
Life Science Institute, Yamaha Motor Co. Ltd.
3001-10 Kuno, Fukuroi, Shizuoka, 437-0061 Japan
*Corresponding author. Fax: +81-44-733-9268 Tel: +81-44-733-9267
E-mail address: ohta@nms.ac.jp (S. Ohta)
Department of Biochemistry and Cell Biology, Institute of Development and Aging
Sciences, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki,
Kanagawa, 211-8533 Japan
2
Abstract
Mitochondria combine the production of energy with an efficient chain of
reduction-oxidation (redox) reactions, but also with the unavoidable production of
reactive oxygen species (ROS). Oxidative stress leading to mitochondrial dysfunction
is a critical factor in many diseases, such as cancer and neurodegenerative and
lifestyle related diseases. Effective antioxidants thus offer great therapeutic and
preventive promise. Investigating the efficacy of antioxidants, we found that a
carotenoid, astaxanthin (AX) decreased physiologically occurring oxidative stress and
protected cultured cells against strong oxidative stress induced with a respiratory
inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane
potential and stimulated respiration. Investigating how AX stimulates and interacts
with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably
expressed in the cytosol and mitochondrial matrix to measure the redox state in the
respective compartments. AX at nanomolar concentrations was effective in
maintaining mitochondria in a reduced state. Additionally, AX improved the ability
of mitochondria to remain in a reduced state under oxidative challenge. Taken
together, these results suggest that AX is effective in improving mitochondrial
function through retaining mitochondria in the reduced state.
Keywords: Oxidative stress; Mitochondrial membrane potential; Oxygen
consumption; Redox-sensitive GFP; Astaxanthin; Metabolic syndrome
3
1. Introduction
Oxidative stress is involved in the pathogenesis of atherosclerosis [1,2], cancer
[3], diabetes [4,5], neurodegenerative [6-8] and other diseases, as well as in the aging
process itself. Antioxidant treatment has therefore great promise in alleviating some
of the detrimental effects of oxidative stress [9], and several types of antioxidants
stimulate lipid oxidation, which may have merit in improving metabolic syndrome
[10-12]. A huge selection of both natural and synthetic antioxidants is available and
can be ingested easily with, in the case of food-derived antioxidants, little concern
about adverse side effects. This attractiveness has on one hand led a multi-billion
dollar supplement industry often based on anecdotal evidence [13,14] and, on the
other hand, to large scale clinical trials to determine efficacy, often with disappointing
results [15,16]. Selecting the most promising substances is therefore important to
avoid costly failures [17].
Cellular reduction-oxidation (redox) state is directly affected in conditions of
oxidative stress, and depletion of endogenous antioxidants plays a critical role in
disease progression [18]. Genetically encoded indicators can be targeted
to specific
organelles of interest and expressed
in a wide variety of cells and organisms [19]. The
indicator used in this work, redox-sensitive green fluorescent proteins (roGFP1),
allows the real-time visualization of the oxidation
state of the indicator [20,21]. In
roGFP1
(GFP with mutations C48S, S147C, and Q204C), two surface-exposed
cysteines are placed at
positions 147 and 204 on adjacent β-strands close to the
chromophore.
Disulfide formation between the cysteine residues promotes protonation
of the chromophore and increases the excitation spectrum peak
near 400 nm at the
4
expense of the peak near 490 nm. The ratios
of fluorescence from excitation at 400
and 490 nm indicate the
extent of oxidation and thus the redox potential while
cancelling
out the amount of indicator and the absolute optical sensitivity [20,21]. In
contrast to its close cousin roGFP2, roGFP1 also offers the advantage to be insensitive
to variations in pH [21].
Astaxanthin (AX), a red-orange carotenoid pigment, is a powerful antioxidant
that occurs naturally in a wide variety of living organisms and has positive effects on
cancer, diabetes, the immune system, and ocular health [22]. Health benefits such as
cardiovascular disease prevention, immune system boosting, bioactivity against
Helicobacter pylori, and cataract prevention, have been associated with AX [23]. Oral
supplementation of a synthetic AX derivative reduced lipid peroxidation levels and
provided significant cardioprotection, consistent with its lipophilic nature and in vitro
antioxidant properties [24]. However, the concentrations of AX used in these studies
were generally much higher than what can be achieved using supplementation [25] or
dietary intervention [26]. We investigated how AX could exert an antioxidant effect in
a concentration range chosen close to what can be achieved using supplements and/or
diet.
2. Materials and methods
2.1. Cell culture
HeLa human cervical cancer cells were maintained in DMEM/F12 medium
(Invitrogen Japan K.K.) supplemented with 10% fetal bovine serum (FBS).
5
Undifferentiated PC12 rat pheochromocytoma cells were cultured in DMEM
supplemented with 10% FBS and 5% heat-inactivated horse serum. Jurkat
immortalized T lymphocyte cells were cultivated in RPMI1640 (Invitrogen)
containing 10% FBS. All media contained 100 Units/ml penicillin and 100 µg/ml
streptomycin, and all cell types were maintained at 37°C in a humidified atmosphere
of 5% CO
2
and 95% air.
2.2. Transfection
HeLa cells were transfected with the mammalian expression vector pEGFP-N1
(Clontech, Takara Bio Inc., CA) containing roGFP1 (for expression in the cytosol) or
roGFP1 with a mitochondrial targeting sequence (pyruvate dehydrogenase E1 subunit
leader sequence, for expression in mitochondria) using the Effectene Transfection
Reagent (Qiagen Japan) according to the manufacturer’s protocol. Stable transfectants
were selected using 400 µg/ml Geneticin (Invitrogen) for more than 4 weeks, and
cloned. Expression of roGFP1 was confirmed by its fluorescence.
2.3. Cell viability
Antimycin A (Sigma) was dissolved to 50 mg/ml in DMSO. The medium was
changed to DMEM supplemented with 1.0% FBS over night, and the PC12 cells then
treated with AX (Sigma) at the indicated concentrations for 6 and 24 h in DMEM
supplemented with 1.0% FBS. After AX treatment, 20 µg/ml antimycin A was added
(final DMSO concentration: 0.1%) and the cells incubated for 20 h. The number of
6
live/dead cells was counted with a fluorescence microscope (Nikon AZ100, Tokyo)
using the LIVE/DEAD cell viability kit for mammalian cells (Invitrogen) and the
survival rate expressed as live cells/(live cells + dead cells).
2.4 Fluorescence microspcopy
Fluorescence images were recorded using a multi-dimensional imaging
workstation (AS MDW, Leica Microsystems, Wetzlar, Germany) consisting of a
tunable light source (Polychrome IV monochromator, Till Photonics, Gräfelfing,
Germany), an inverted epifluorescence microscope (DM IRE2, Leica Microsystems)
contained in a climate chamber maintained at 37°C, and a cooled CCD camera
(CoolSnap HQ, Roper Scientific, Princeton, NJ). A 0.35X demagnifiying lens (Leica
Microsystems) was inserted between the microscope and the CCD camera. The
components were controlled by custom-made software written in C (Bloodshed
Dev-C++) and LabVIEW (National Instruments, Austin, TX).
2.5 Redox-sensitive fluorescent protein fluorescence recording
HeLa cells expressing roGFP1 were washed with PBS and fluorescence
recorded. Hydrogen peroxide was diluted from a 30% (w/v) stock solution with PBS
(37 °C) to 500 µM, and added to reach the indicated concentration. Dual excitation
ratio imaging was performed using a 20X objective (N.A. 0.5) and 800 ms exposure
time at 410 nm and 490 nm excitation wavelength. A 500 nm short pass excitation
filter (E500SP), 515 nm extended range dichroic mirror (515DCXR) and 520-600 nm
7
band pass emission filter (HQ560/80, all from Chroma Technology Corporation,
Rockingham, VT) was used for both wavelengths.
Fluorescence data was analyzed using MATLAB (MathWorks, Version 7
Release 14, Natick, MA). After subtracting background fluorescence and CCD dark
field, areas corresponding to cells were automatically selected using the criteria of
more than 10 connected pixels with fluorescence intensity above both 3% of the
maximum fluorescence in the field of view and more than 8 standard deviations above
the background noise/intensity in the image obtained with 410 nm excitation
wavelength. Such regions were marked, and visually confirmed to correspond to cells
expressing fluorescent protein. The fluorescence ratio was formed by dividing the
fluorescence integral of such regions at 410 nm and 490 nm excitation. When
following the time course of the fluorescence ratio of individual cells, in order to be
counted, cells had to fulfil the above criteria at every recorded time point.
2.6 Mitochondrial membrane potential
A 0.5 mM stock solution of JC-1 (Invitrogen) was prepared in equal volumes
of ethanol and DMSO. HeLa cells were cultured in DMEM/F12 + 10% FBS with or
without 800 nM AX (control: DMSO) for 6 h, 1 day and 2 days, then stained with 250
nM JC-1 in culture medium for 60 min, washed with PBS and fluorescence recorded
immediately, all at 37°C. Dual excitation ratio imaging was performed using a 20X
objective (N.A. 0.5) and 200 ms exposure time at 520 nm and 570 nm excitation
wavelength. A 580 nm short pass excitation filter (E580SP), 585 nm extended range
dichroic mirror (585DCXR) and 590-670 nm band pass emission filter (HQ635/60, all
8
from Chroma Technology Corporation) was used for both excitation wavelengths.
Fluorescence data was analyzed using MATLAB (MathWorks, Version 7 Release 14).
After subtracting background (dark field) fluorescence, areas corresponding to cells
were automatically selected using the criteria of more than 10 connected pixels with
fluorescence intensity above both 3 % of the maximum fluorescence in the field of
view and more than 8 standard deviations above the background noise/intensity in the
image obtained with 520 nm excitation wavelength. The fluorescence ratio was
formed by dividing the fluorescence integral of all such regions at 570 nm and 520 nm
excitation. The field of view usually contained about 100 to 200 cells.
2.7 Superoxide measurement
HeLa cells were cultured in DMEM/F12 + 10% FBS with or without 800 nM
AX (control: DMSO) for 6 h, 1 day and 2 days, then exposed to 30 µg/ml antimycin A
for 15 min, then 250 nM MitoSOX Red (Invitrogen) and 500 nM Hoechst34580
(Invitrogen) added and incubated for 60 min, and fluorescence recorded using a 20X
(N.A. 0.5) objective. Hoechst34580 fluorescence was excited at 395 nm in
combination with a 500 nm short pass excitation filter (E500SP), 515 nm extended
range dichroic mirror (515DCXR) and 520-600 nm band pass emission filter
(HQ560/80). Ethidium (MitoSOX Red oxidation product) fluorescence was excited at
520 nm and recorded using a 580 nm short pass excitation filter (E580SP), 585 nm
extended range dichroic mirror (585DCXR) and 590-670 nm band pass emission filter
(HQ635/60). Fluorescence data was analyzed using MATLAB (MathWorks, Version 7
Release 14). After subtracting background (dark field) fluorescence, cell nuclei were
9
automatically detected using the criteria of more than 25 connected pixels with
fluorescence intensity above both 20 % of the maximum fluorescence in the field of
view and more than 10 standard deviations above the background noise/intensity in
the Hoechst 34580 image. Mean ethidium fluorescence intensity in the area above the
nucleus was used as the measure of superoxide production.
2.8 Flow cytometry
Jurkat cells were cultivated in medium in the presence or absence (DMSO) of
AX (800 nM) for 6 h, 1 day and 2 days without medium change. There was no
difference among the growth rates of these cell cultures. For detection of superoxide
anion, cells were incubated with antimycin A (30 µg/ml) for 10 min. Then,
MitoSOX Red (250 nM) was added. After 1 h, the cells were analyzed by flow
cytometry (Cell Lab Quanta system, Beckman Coulter Inc., Chaska, MN). For
detection of hydroxyl peroxides, cells were incubated with H
2
DCFDA (20 µ M,
Invitrogen). After 15 min, antimycin A (30 µg/ml) was added. One hour later, the
cells were analyzed by flow cytometry as above. Ten thousand cells of normal size
(assessed by forward scattering) were analyzed under each condition.
2.9 Physiologically occurring (basal) oxidative stress
Cells were cultured in DMEM/F12 + 10%FBS with or without 800 nM AX
(control: DMSO) for 2 days, then incubated with 20 µM H
2
DCFDA and 500 nM
Hoechst34580 in medium for 60 minutes, washed with PBS and the
10
dichlorofluorescein (DCF) fluorescence of individual cells measured immediately
afterwards using a 20X objective (N.A. 0.5). Fluorescence was recorded using the
BGR triple band pass filter set (Leica Microsystems). Hoechst34580 was excited at
403 nm and DCF at 496 nm wavelength. Fluorescence data was analyzed using
MATLAB (MathWorks, Version 7 Release 14). After subtracting background
(darkfield) fluorescence, cell nuclei were automatically detected using the criteria of
more than 25 connected pixels with fluorescence intensity above both 5 % of the
maximum fluorescence in the field of view and more than 10 standard deviations
above the background noise/intensity in the Hoechst 34580 image. Mean DCF
fluorescence intensity in the area above the nucleus was used as the measure of
oxidative stress.
2.10 Oxygen consumption
HeLa cells were cultured in DMEM/F12 + 10% FBS with or without 800
nM AX (control: DMSO) for 6 h, 1 day and 2 days, trypsinized and resuspended in
DMEM/F12. Oxygen consumption was measured using the Oxygen Meter Model 781
and the Mitocell MT200 closed respiratory chamber (Strathkelvin Instruments, North
Lanarkshire, UK), continuously stirred at 37 °C. The oxygen respiration rate was
calculated in the following three conditions: basal rate (no additions), state 4 (after
addition of 2 µM oligomycin (Sigma)), uncoupled (after addition of 80 µM DNP
(Sigma)) using the Strathkelvin 949 Oxygen System software. Cell concentration was
determined using a hemocytometer.
11
2.11 Confocal microscopy
To confirm targeting of roGFP1 to the mitochondrial matrix, HeLa cells
expressing the mitochondrial targeting vector were cultured in a glass bottom dish and
stained with 40 nM tetramethyl rhodamine methyl ester (TMRM, Molecular Probes,
Invitrogen) for 20 minutes. Fluorescence was observed using a FluoView FV300
confocal microscope (Olympus, Tokyo, Japan) with a 60X (N.A. 1.4) oil immersion
objective. roGFP1 was excited at 488 nm and emission collected above 510 nm, while
TMRM was excited at 543 nm and emission collected above 570 nm.
2.12 Statistical calculations
Statistical significance was determined using the unpaired two-tailed student’s
t-test in Excel (Microsoft, Redmond, WA) or one-way analysis of variance (ANOVA)
followed by a Tukey-Kramer multiple comparison test in Matlab (MathWorks,
Version 7 Release 14). Error bars indicate the standard deviation of three or more
measurements. The number of asterisks indicates statistical significance. Specifically,
* indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001.
3. Results
3.1. AX improves cell survival under oxidative stress
To test whether AX protects cells against oxidative stress, we exposed rat
12
adrenal pheochromocytoma (PC12) cells, a neuronal model cell line shown to be
sensitive to oxidative stress [27], to antimycin A. Antimycin A, an inhibitor of
complex III of the electron transport chain, induces oxidative stress by increasing
mitochondrial superoxide production [28] (see also Fig. 2B & 2E). Preincubation with
AX for 6 h did not significantly increase PC12 cell survival, but when cells were
exposed to AX for 24 hours, a significant increase in the number of surviving cells
was observe with more than 200 nM AX, demonstrating that AX was able to protect
PC12 cells against oxidative stress at relatively low concentrations (Fig. 1). The effect
was concentration dependent, with 400 nM AX being more protective than 100 nM
AX (p < 0.01).
3.2. AX reduces basal oxidative stress levels but not acute oxidative stress
To determine the mechanism by which AX protects against oxidative stress,
we determined whether AX was able to directly scavenge superoxide radical. Human
cervial cancer (HeLa) and T lymphocyte (Jurkat) cells were exposed to antimycin A as
an acute stress model, and oxidation of the superoxide-sensitive probe MitoSOX Red
quantified as described in Materials and methods. Preincubation of cells with 800 nM
AX did not significantly reduce the amount of superoxide detected (Fig. 2A),
suggesting that AX did not scavenge excess amounts of superoxide under this
unphysiological condition. Similarly, Jurkat cells were treated with antimycin A
generated superoxide anion (as assessed by MitoSOX Red) and reactive peroxides (as
assessed by H
2
DCFDA) to a great extent (Fig. 2B & 2E). In these flow cytometry
experiments, the population of cells with an intensity of 1000 or more (marked with *
13
in Fig. 2B and 2C) was between 2 and 4 % of the cells analyzed. Quantitative analysis
revealed no significant difference in the fluorescence intensity of oxidized MitoSOX
product (Fig. 2C) or DCF (Fig. 2F) between AX-treated or non-treated cells, even
when cells were treated with AX for 2 days. Post-addition of AX did not change
fluorescent patterns of the antimycin A-treated cells without AX-treatment (data not
shown), indicating that AX does not interfere with fluorescence of oxidized MitoSOX
and DCF.
However, when the basal level of oxidative stress, that is the physiologically
occurring oxidative stress, in HeLa cells was determined using the oxidant sensitive
probe 2’,7’-dichlorodihydrofluorescein, AX significantly reduced the amount of
fluorescent oxidation product (DCF) produced (Fig. 2D), indicating that AX is able to
reduce endogenous oxidative stress. This signifies that there is a low but detectable
amount of endogenous oxidative stress under normal culture conditions, which AX
can reduce.
3.3. AX helps maintaining the mitochondrial membrane potential
We proceeded to determine whether such endogenous oxidants affect cellular
function and whether AX could influence it. The mitochondrial membrane potential
was quantified using the aggregate-forming probe JC-1 [29]. Using dual excitation
ratio imaging, we observed a significantly higher mitochondrial membrane potential
when AX was present in culture after 2 days (Fig. 3A). For shorter incubation times,
the difference was not significant. Interestingly, rather than increasing the
mitochondrial membrane potential, AX seemed to slow down a gradual loss of
14
membrane potential that may occur with time in culture. While a significant loss in
the membrane potential occurred from 6 hours to 2 days in culture without AX (Fig.
3B, p < 0.001), no significant difference/loss could be detected in the presence of AX.
3.4. AX effect on respiratory control
Speculating that a decrease in mitochondrial membrane could also affect
mitochondrial respiration, we measured the oxygen consumption of intact HeLa cells
cultured under the same conditions. Measuring first baseline oxygen consumption (no
additions), then the mitochondrial state 4 consumption (by blocking complex V with
oligomycin) and then maximal oxygen consumption by uncoupling mitochondria with
DNP (80 µM) (Fig. 4A), we could not observe significant changes in the absolute
consumption rates (Fig. 4B), but the ratio of baseline to uncoupled oxygen
consumption was significantly higher in the presence of AX, i.e. mitochondria were
more active in the presence of AX (Fig.4C). In addition, the relative reduction in
oxygen consumption upon addition of oligomycin was increased in the presence of
AX (Fig. 4C). Together, these findings suggest that AX stimulates respiration
probably by maintaining a higher membrane potential (Fig. 3).
3.5. AX improves the mitochondrial redox state
Due to these positive but rather subtle effects of AX on mitochondrial
function, we looked for a sensitive method to detect relatively mild oxidative stress at
the organelle level. We chose redox-sensitive GFP as a promising method, since it is
15
ratiometric and can be targeted to various cellular compartments [20,21,30] .
Furthermore, it has been shown to be a quantitative sensor for the redox potential of
the cellular glutathione redox buffer [31]. HeLa cells were stably tranfected with the
expression vector for roGFP1 targeted to mitochondria as described above and the
colocalization of roGFP1 with mitochondria confirmed using confocal microscopy.
RoGFP1 showed almost perfect overlap with the mitochondrial marker dye TMRM
(Fig. 5A). Cells expressing mitochondrial roGFP1 showed no abnormal morphology
(Fig. 5A&B) and mitochondria exhibited the characteristic tubular shape and
movement presumably along microtubules (Suppl. Movie). The mitochondrial redox
state of individual cells was measured using dual excitation imaging (Fig. 5C).
Mitochondrial roGFP1 (basal redox state) was more reduced in cells cultured with AX
for 6 h, 1 day and 2 days (Fig. 5D&E). Mitochondrial ability to maintain the reducing
environment was then tested by exposing cells to 100 µM H
2
O
2
, which quickly
oxidized mitochondrial roGFP1, but significantly less so when AX was present (Fig.
5D&E). To exclude the possibility that the more reduced state after addition of H
2
O
2
is
simply a consequence of the more reduced (basal) state prior to addition of H
2
O
2
, we
also compared the change in fluorescence ratio induced by H
2
O
2
. This difference was
also significantly smaller in the presence of AX (Fig. 5E). Whether this antioxidant
effect of AX was limited to mitochondria or also extended to the cytosol was tested
using cells expressing roGFP1 in the cytosol (Fig.6A). The basal redox state and
resistance to oxidative challenge with H
2
O
2
was tested in the same manner as for
mitochondria, but no effect of AX on the redox state of the cytosol could be detected
(Fig. 6B).
16
4. Discussion
Above results demonstrate that, under basal conditions, AX had a small but
significant positive effect on mitochondrial function (higher membrane potential,
higher respiratory control). This is reassuring, since endogenous oxidative stress,
though clearly present [32], should be rather mild in the absence of external stress
inducing agents. Since mitochondria are a major source of ROS in the cell,
accumulation of AX in the mitochondrial membrane would potentiate its antioxidant
effects. There is a report showing AX protects mesangial cells from
hyperglycemia-induced oxidative signaling [33]. The fact that AX was able to
maintain mitochondria but not the cytosol in a reduced state (Fig. 5) indicates that the
effect of AX is, at least during mild, endogenous oxidative stress, concentrated on
mitochondria. The effect of AX became more pronounced with incubation time.
Interestingly, such a time dependency was also observed in an investigation of the
antitumour activity of AX [34]. AX, at a total serum concentration of approximately
1.2 µM suppressed tumour cell growth when mice were fed AX before inoculation,
but was ineffective when the AX-supplemented diet was started at the same time as
tumor inoculation [34].
Cell types differ in their dependence on mitochondria-generated ATP as a
source of energy and in their sensitivity to oxidative stress. Mitochondria isolated
from PC12 cells had oxygen consumption rates similar to isolated rat liver
mitochondria (data not shown) [35]. The dopaminergic, neuron-like cell line PC12 is
very sensitive to oxidative stress, and oxidative stress-induced cytotoxicity in PC12
cells is frequently employed as an in vitro model of Parkinson’s disease [27,36]. AX
17
protected PC12 cells against antimycin A-induced cell death. HeLa cells, in contrast,
can rely on glycolysis as the sole energy supply and survive even in the absence of a
functional electron transport chain [37]. Accordingly, blocking electron transport with
antimycin A, though clearly inducing oxidative stress, does not necessarily lead to
HeLa cell death [38,39]. That AX was protective in both cells lines indicates that the
effect of AX should not be cell type or cancer specific.
Although direct superoxide scavenging by a highly water-dispersible
carotenoid phospholipid has been reported at high concentrations [40], AX may not
exert its strong effect by scavenging superoxide. In fact, under conditions of acute
oxidative stress when large amounts of ROS are produced, AX showed no detectable
effect, indicating that the effects of AX are not due direct scavenging of ROS such as
superoxide or peroxides (Fig. 2A). This is to be expected since superoxide is a
charged radical unable to cross cellular membranes and carotenoids are extremely
lipophilic. On the other hand, since AX reduced the endogenous oxidative stress level
(Fig. 2D), it is reasonable to assume it protects cells against oxidative damage in the
lipd phase (Fig. 1). Thus, even though it may not svavenge ROS directly, AX has the
potential to protect cells against damage mediated by oxidative stress. Since ROS
including superoxide and hydrogen peroxide play significant roles in the signal
transduction at low concentrations[41], this characteristic of AX suggests little
possibilities of negative side effects of AX consumption.
The increase in mitochondrial oxygen consumption of AX-treated cells is
probably in part a consequence of the change in mitochondrial membrane
potential[41]. The relatively higher reduction in oxygen consumption upon addition of
oligomycin in the presence of AX would be expected as mitochondrial membrane
18
potential is higher in the presence of AX, while in the absence of AX, oligomycin is
unable to increase membrane potential to the same level. Such an inhibition of
respiration upon inactivation of complex V is referred to as “respiratory control ratio”,
an important measure of mitochondrial health when investigating isolated
mitochondria [41], even though it should be pointed out that we used intact cells,
where respiration during stimulation of complex V with ADP is not accessible, and
the ratios are not comparable. Physiological effects of AX have been shown to include
stimulation of β-oxidation for fatty acids [42,43]. This would be of considerable
benefit in reducing obesity and metabolic syndrome in affluent societies. Much of the
benefit of one of the most effective drugs for diabetes, metformin, has been attributed
to activation of AMP-dependent kinase, which helps by reducing gluconeogenesis and
driving oxidation of fat in muscle mitochondria [44]. The AX concentration upon
which a significant benefit could be observed (200nM, Fig. 1) is also well within the
range that can be achieved with supplementation. A single oral dose of 10 mg AX
resulted in a peak plasma concentration of ~130 nM, whereas a single dose of 100 mg
AX resulted in a peak plasma concentration of ~470 nM, with half lives in the order
of days [25]. Similarly, daily consumption of 250 g either wild or farmed salmon
(farmed salmon is fed synthetic AX to give it its natural coloring) lead to a plateau of
~50 nM AX in plasma after about 6 days [26].
Redox-sensitive GFP targeted to mitochondria could detect changes in the
mitochondrial redox state even after short incubation with AX, where conventional
methods failed. This indicates that roGFP is a powerful tool to measure oxidative
stress and redox balance at the organelle level. Targeting roGFP to mitochondria
allowed us to detect changes in the mitochondrial redox state that were too small to
19
affect the cytosol. Redox-sensitive GFP possibly interacts with a number of cellular
enzymes and redox couples, but it is still unclear which determine the roGFP1 redox
state to what extent. The reduction of roGFP1 is likely enzyme-dependent, as
reductase and dehydrogenase inhibitors reduced or prevented reduction of the close
cousin roGFP2 in the cytosol [20]. Conversely, oxidation of roGFP proceeded
considerably faster when, compared to the isolated protein, it was expressed in cells,
suggesting that the oxidation is also catalyzed by intracellular enzymes. In our hands,
the response of roGFP1 to hydrogen peroxide was also significantly faster than
previously reported [20]. RoGFP oxidation was observed immediately, and the
maximum response in the cytosol was reached within five minutes (Fig. 6B).
The high sensitivity of roGFP to changes in organelle redox state suggest that
it is a powerful tool to investigate the biochemistry of oxidative stress and redox
balance at the organelle level. Accurate measurement of oxidative stress is an ongoing
challenge, and despite the availability and ongoing development of a wide range of
probes and methods, their correct use is often complicated (for example [45]) and/or
cumbersome (for example [46]). The most widely employed method to “quantify”
ROS, the conversion of dichlorodihydrofluorescein (H
2
DCF) to DCF, requires a
catalyst for H
2
DCF to be oxidized by hydrogen peroxide and reacts indiscriminately
with a variety of oxidizing factors, including light and itself [47,48].
RoGFP1 is a relatively new non-destructive and ratiometric sensor for cellular
redox state [20,21] permitting a wide range of measurements previously impossible or
extremely labour-intensive and therefore open to errors and artefacts. Experiments to
establish the sensitivity of roGFP1 to various stressors and antioxidants are currently
under way in our laboratory, but in our opinion, roGFP offers great promise for
20
pin-point detection of oxidative stress. Targeting roGFP1 to subcellular structures
allowed us to observe oxidative stress restricted to mitochondria without affecting the
cytosol, demonstrating that changes in redox state and oxidative stress can be
confined to cellular compartments. To understand cellular redox states, it might
therefore be important to determine compartmental redox states independently.
Furthermore, transgenic animals expressing roGFPs should be a valuable tool to test
the efficacy of treatments aimed at reducing oxidative stress.
Acknowledgments
We express our deep gratitude to Prof. Jim Remington and his team for kindly
providing the roGFP1 containing plasmids. Alexander Wolf was supported by a
Postdoctoral Fellowship and grant from the Japan Society for the Promotion of
Science.
List of Abbreviations
AX, astaxanthin; CCD, charge coupled device; DCF, 2’,7’-dichlorofluorescein;
DMEM/F12, Dulbecco’s Modified Eagle Medium / F-12 nutrient mixture; DNP,
2,4-dinitrophenol; FBS, fetal bovine serum; GFP, green fluorescent protein;
H
2
DCFDA, 2’,7’-dichlorodihydrofluorescein diacetate; HNE, 4-hydroxy-2-nonenal;
JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; N.A.,
numerical aperture; PUFA, polyunsaturated fatty acid; roGFP, redox-sensitive green
fluorescent protein; TMRM, tetramethyl rhodamine methyl ester;
21
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Figure legends
Fig. 1. Astaxanthin effect on PC12 cell survival under oxidative stress. Cells were
cultured in the presence of 0, 100, 200 and 400 nM astaxanthin (AX) for 6 hours or 24
hours and then exposed to 30 µg/ml antimycin A as described under cell viability in
Materials and methods. No effect was detected when cells were preincubated with AX
for 6 hours. When preincubated with AX for 24 hours, cells were protected against
cell death induced by oxidative stress, significantly for 200 and 400 nM AX (p < 0.01).
Data are means ± SD from 3 fields containing 100-200 cells each.
29
Fig. 2. Superoxide and oxidative stress. (A) PC12 cells were preincubated with
astaxanthin (AX) for indicated periods, followed by exposure to antimycin A to
induce superoxide. Superoxide, as measured using MitoSOX Red, was not changed
significantly by culturing cells with AX. Data were obtained as the means ± SD by
quantifying signal intensities of 70 100 cells. (B) Superoxide production is
induced by incubating Jurkat cells with 30 µg/ml antimycin A. (C) Jurkat cells
cultivated in the presence or absence (DMSO) of AX (800 nM) for 6 h, 1 day, and 2
days were treated with antimycin A (30 µ g/ml). No significant difference in the
amount of oxidized MitoSOX Red was observed, indicating that, under conditions of
oxidative stress and at a concentration of 800 nM, AX is unable to scavenge
significant amounts of superoxide (D) Basal oxidative stress level in cells, as
measured by incubating cells with H
2
DCFDA. AX significantly decreased the amount
of DCF produced in HeLa cells incubated with AX for 2 days (n = 383 cells, control:
30
n = 403 cells; from 3 independent experiments each condition). *** p < 0.001. (E)
Antimycin A (30 µg/ml) also induced acute oxidative stress as assessed using
H
2
DCFDA. The amount of DCF produced was increased more than tenfold. (F)
Similar to C, AX did not reduce acute oxidative stress. No significant difference in the
amount of DCF was observed.
31
Fig. 3. Mitochondrial membrane potential (A) Representative transmitted light (upper
row) and membrane potential (lower row) images of Hela cells cultured for 2 days in
the presence (right) or absence (left) of 800 nM astaxanthin (AX). The membrane
potential image was created by color coding the dual excitation fluorescence ratio.
32
Asterisks (pink) mark the approximate positions of cell nuclei. (B) Average JC-1 dual
excitation fluorescence ratio from 3 independent experiments, with fluorescence
recorded at 2 locations for each experiment. Membrane potential was significantly
higher (p < 0.001) in the presence of AX after 2 days in culture, but rather than
increasing the mitochondrial membrane potential, AX seemed to prevent a loss of
membrane potential that occurred with increasing time in culture.
33
Fig. 4. Oxygen consumption profiles. (A) Representative traces of the oxygen
consumption of intact HeLa cells cultured with (800 nM, red trace) or without (black
trace) AX. Oligomycin and DNP were added at the indicated (arrows) time points.
(B) Average oxygen consumption per cell under baseline conditions (BL; dark gray
34
bars), in the presence of 2 µM oligomycin (+OM; black bars) and when uncoupled
with 80 µM DNP (+DNP, light gray bars). Data are the average ± SD of three
measurements of cells cultured for 6 hours (6H), 1 day (1D), or 2 days (2D) in the
presence or absence of 800 nM astaxanthin (AX) (control: DMSO). (C) AX increased
the ratio of baseline divided by uncoupled oxygen consumption and decreased the
ratio of oxygen consumption in the presence of oligomycin divided by baseline
oxygen consumption. Data are mean ± SD. ** p < 0.01.
35
Fig. 5. Mitochondrial redox state measured using redox sensitive GFP. (A) Confocal
and transmitted light images of a HeLa cell expressing roGFP1 targeted to
mitochondria. Overlap of TMRM and roGFP1 fluorescence confirms mitochondrial
localization of roGFP1. Scale bar: 20 µm. (B) Epifluorescence image of HeLa cells
expressing roGFP1 targeted to mitochondria. Regions automatically selected for
analysis are marked in pink. Scale bar: 20 µm. (C) Representative traces of the
36
roGFP1 redox state time course (dual excitation fluorescence ratio). Four individual
cells show the hetereogeneity of mitochondrial redox state between cells. After the
indicated periods, 100 µM hydrogen peroxide was added (arrow). (D) Average time
course indicating mitochondrial redox state of cells cultured with or without 800 nM
astaxanthin (AX) for 6 h (6H)(+AX: n = 35 cells, control: n = 34 cells), 1 day
(1D)(+AX: n = 41 cells, control: n = 32 cells), or 2 days (2D)(+AX: n = 32 cells,
control: n = 27 cells). Average baseline as well as redox state after addition of 100
µM hydrogen peroxide (arrow) was more reduced when AX was present. (E) Average
baseline fluorescence ratio, ratio after addition of hydrogen peroxide, and amount of
oxidation (ratio difference) induced by hydrogen peroxide, was significantly lower, i.e.
reduced, when AX was present. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p <
0.001.
37
Fig. 6. Effect of astaxanthin on the cytosol redox state. (A) Epifluorescence image of
HeLa cells expressing roGFP1 in the cytosol. Regions automatically selected for
analysis as individual cells are marked in pink. Scale: 20 µm. (B) Timecourse of
cytosol redox state (410/490 nm dual excitation fluorescence ratio) upon addition of
100 µM H
2
O
2
(arrow). No difference was detected, neither in the basal redox state nor
in the change or state after addition of H
2
O
2
, between cells cultured for 2 days in the
presence (open diamonds, n = 73 cells) or absence (filled diamonds, n = 90 cells) of
800 nM astaxanthin.
Supplementary Movie. Cells expressing roGFP1 targeted to mitochondria were
cultured in the presence (800 nM, upper row) or absence (DMSO, lower row) of AX
for one day (left column) or two days (right column) and observed for 5 minutes. One
image was taken every two seconds using a 63X immersion objective and 410 nm
wavelength excitation.
... possibly, astaxanthin accumulate in the mitochondrial membrane and possibly enhance FATox through protecting CPT1 (Aoi et al., 2008;Kidd, 2011;Wolf et al., 2010). Another mechanism which is stated is AMP-activated protein kinase (AMPK) is upregulated following astaxanthin consumption (Aoi et al., 2018). ...
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Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an antioxidant and a redox modulator. We have generated dopaminergic PC12 cell lines in which levels of GSH can be inducibly down-regulated via doxycycline induction of antisense messages against both the heavy and light subunits of γ-glutamyl-cysteine synthetase, the rate-limiting enzyme in glutathione synthesis. Down-regulation of glutamyl-cysteine synthetase results in reduction in mitochondrial GSH levels, increased oxidative stress, and decreased mitochondrial function. Interestingly, decreases in mitochondrial activities in GSH-depleted PC12 cells appears to be because of a selective inhibition of complex I activity as a result of thiol oxidation. These results suggest that the early observed GSH losses in the SN may be directly responsible for the noted decreases in complex I activity and the subsequent mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with PD.
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