From the Departments of Chemistry and
¶ Biochemistry, University of Otago, Box 56, Dunedin, New
Zealand
Received for publication, October 5, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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With the recognition of the central
role of mitochondria in apoptosis, there is a need to develop specific
tools to manipulate mitochondrial function within cells. Here we report
on the development of a novel antioxidant that selectively blocks
mitochondrial oxidative damage, enabling the roles of mitochondrial
oxidative stress in different types of cell death to be inferred. This
antioxidant, named mitoQ, is a ubiquinone derivative targeted to
mitochondria by covalent attachment to a lipophilic
triphenylphosphonium cation through an aliphatic carbon chain. Due to
the large mitochondrial membrane potential, the cation was accumulated
within mitochondria inside cells, where the ubiquinone moiety inserted
into the lipid bilayer and was reduced by the respiratory chain. The
ubiquinol derivative thus formed was an effective antioxidant that
prevented lipid peroxidation and protected mitochondria from oxidative
damage. After detoxifying a reactive oxygen species, the ubiquinol
moiety was regenerated by the respiratory chain enabling its
antioxidant activity to be recycled. In cell culture studies, the
mitochondrially localized antioxidant protected mammalian cells from
hydrogen peroxide-induced apoptosis but not from apoptosis induced by
staurosporine or tumor necrosis factor- The mitochondrial respiratory chain is a major source of
superoxide and, therefore, mitochondria accumulate oxidative damage more rapidly than the rest of the cell, contributing to mitochondrial dysfunction and cell death in degenerative diseases and in aging (1-5). Mitochondria are also central to activating apoptosis and
oxidative damage can lead to cell death, however, the significance of
mitochondrial oxidative damage for cell death is unclear (6-8). One
approach to this problem is to selectively target antioxidants to
mitochondria (9-11). This should allow the relative importance of
mitochondrial and cytoplasmic oxidative stress for cell death to be
distinguished, and also enable the contribution of mitochondrial damage
to aging, diabetes, and cancer to be investigated in cell and animal models.
Derivatives of ubiquinol are promising antioxidants to target to
mitochondria (11, 12). In mammals ubiquinone comprises a
2,3-dimethoxy-5-methylbenzoquinone core with a hydrophobic 45- to
50-carbon chain at the 6 position (13, 14). Mitochondrial ubiquinone is
a respiratory chain component buried within the lipid core of the inner
membrane where it accepts two electrons from complexes I or II becoming
reduced to ubiquinol, which then donates electrons to complex III (14).
The ubiquinone pool in vivo is largely reduced and ubiquinol
is an effective antioxidant, as well as being a mobile electron carrier
(15-18). Ubiquinol acts as an antioxidant by donating a hydrogen atom
from one of its hydroxyl groups to a lipid peroxyl radical, thereby
decreasing lipid peroxidation within the mitochondrial inner membrane
(18-20). The ubisemiquinone radicals thus formed disproportionate to
ubiquinone and ubiquinol (21), or react with oxygen to form
superoxide and ubiquinone thereby transferring the radical to the
aqueous phase for detoxification by superoxide dismutase and
peroxidases (17, 20). The respiratory chain then recycles ubiquinone
back to ubiquinol to restore its antioxidant function. Vitamin E is another important antioxidant within the mitochondrial inner membrane, and the tocopheroxyl radical thus formed is regenerated to active vitamin E by reaction with ubiquinol or ubisemiquinone (15, 17, 20, 22,
23). Therefore, in vivo ubiquinol probably acts as an
antioxidant by direct reaction with peroxyl radicals and by
regenerating vitamin E (16, 17, 20).
The low solubility of ubiquinone in water makes it difficult to use
in vitro, and animals must be fed ubiquinone-enriched diets
for several weeks to increase levels in subsequently isolated mitochondria (11, 14). Therefore, to manipulate mitochondrial ubiquinone content in vitro we synthesized a ubiquinone
analog selectively targeted to mitochondria by addition of a
lipophilic triphenylphosphonium cation. Such lipophilic cations
easily permeate lipid bilayers and accumulate in mitochondria within
cells, driven by the large mitochondrial membrane potential (9,
10, 24). Here we report on the antioxidant and antiapoptotic properties of this mitochondrially targeted ubiquinone derivative.
Chemical Syntheses--
To synthesize 11-bromoundecanoic
peroxide (1) 11-bromoundecanoic acid (4.00 g, 15.1 mmol) and
SOCl2 (1.6 ml, 21.5 mmol) were heated at 90 °C for 15 min (25). Excess SOCl2 was removed by distillation under
reduced pressure (15 mm Hg, 90 °C) and the residue
(IR,1 1799 cm-1)
was dissolved in diethyl ether (20 ml) and cooled to 0 °C. Hydrogen peroxide (30%, 1.8 ml) was added, followed by dropwise addition of
pyridine (1.4 ml) over 45 min, then diethyl ether (10 ml) was added and
after 1 h at room temperature the product was diluted with diethyl
ether (150 ml), washed with H2O (2 × 70 ml), 1.2 M HCl (2 × 70 ml), H2O (70 ml), 0.5 M NaHCO3 (2 × 70 ml), and H2O
(70 ml). After drying over MgSO4 the solvent was removed at room temperature under reduced pressure, giving crude 1 as a
white solid (3.51 g), which was used without delay. IR (Nujol mull) 1810, 1782 cm-1.
6-(10-Bromodecyl)ubiquinone (2) was synthesized by stirring
crude 1 (3.51 g, 12.5 mmol),
2,3-dimethoxy-5-methyl-1,4-benzoquinone (1.31 g, 7.19 mmol, Aldrich),
and acetic acid (60 ml) for 20 h at 100 °C. After cooling to
room temperature, the reaction was diluted with diethyl ether (600 ml),
washed with H2O (2 × 400 ml), 1 M HCl
(2 × 450 ml), 0.5 M NaHCO3 (2 × 450 ml), and H2O (2 × 400 ml), and dried over
MgSO4. Removal of the solvent under reduced pressure gave a
reddish solid (4.31 g). Column chromatography on silica gel, eluting
with CH2Cl2, gave 2 as a red oil (809 mg, 28%) and unreacted 2,3-dimethoxy-5-methyl-1,4-benzoquinone (300 mg, 1.6 mmol, 13%). 2: TLC: RF
(CH2Cl2, diethyl ether 20:1) 0.46; IR (film)
2928, 2854, 1650, 1611, 1456, 1288 cm-1;
To form the quinol, 6-(10-bromodecyl)-ubiquinol (3),
NaBH4 (295 mg, 7.80 mmol) was added to 2 (649 mg, 1.62 mmol) in methanol (6 ml) and stirred under argon for 10 min
(Scheme 1). Excess NaBH4 was
quenched with 5% HCl (2 ml), diluted with diethyl ether (40 ml),
washed with 1.2 M HCl (40 ml), saturated NaCl (2 × 40 ml), and dried over MgSO4. Removal of the solvent under
reduced pressure gave 3 as a yellow oily solid (541 mg,
83%). 1H NMR (299.9 MHz) 5.31 (s, 1H, -OH),
5.26 (s, 1H, -OH), 3.89 (s, 6H, 2×
-OCH3), 3.41 (t, J = 6.8 Hz, 2H,
-CH2-Br), 2.59 (t, J = 7.7 Hz, 2H ubquinol-CH2-), 2.15 (s,
3H, CH3), 1.85 (quin,
J = 7.4 Hz, 2H,
-CH2-CH2-Br), 1.44-1.21 (m, 14H,
-(CH2)7-) ppm.
To synthesize 10-(6'-ubiquinolyl)decyltriphenylphosphonium bromide
(4), triphenylphosphine (387 mg, 1.48 mmol), 3 (541 mg, 1.34 mmol), and ethanol (95%, 2.5 ml) were sealed under argon
in a 15-ml Kimax tube and stirred in the dark for 88 h at 85 °C. Removal of the solvent under reduced pressure gave an oily orange residue, which was dissolved in CH2Cl2
(2 ml). Addition of diethyl ether (20 ml) gave a suspension, and after
5 min the supernatant was decanted. The residue was dissolved in
CH2Cl2 (2 ml) followed by addition of diethyl
ether (20 ml), and the supernatant was decanted. The
CH2Cl2/diethyl ether extraction was repeated
twice more, and residual solvent was removed under reduced pressure,
giving crude 4 as a cream solid (507 mg). 1H NMR
(299.9 MHz) 7.9-7.6 (m, 15H, -P+
Ph3), 3.89 (s, 6H, 2×
-OCH3), 3.91-3.77 (m, 2H, -CH2-P+Ph3), 2.57 (t,
J = 7.8 Hz, 2H ubquinol-CH2-),
2.14 (s, 3H, CH3), 1.6-1.2 (m, 16H,
-(CH2)8-) ppm. 31P NMR
(121.4 MHz) 25.1 ppm.
Crude 4 (200 mg) was oxidized to
10-(6'-ubiquinonyl)decyltriphenyl-phosphonium bromide (5) by
stirring in CDCl3 at room temperature under an oxygen
atmosphere. The solvent was removed under reduced pressure, the residue
was dissolved in CH2Cl2 (5 ml), diethyl ether
(15 ml) was added, and the resultant suspension was stirred for 5 min. The supernatant was decanted, and the
CH2Cl2/diethyl ether precipitation was repeated
twice more. Residual solvent was removed under reduced pressure, giving
crude 5 as a brown sticky solid (173 mg). IR (film) 3357, 2927, 2857, 1650, 1609, 1438, 1266, 1113 cm-1.
1H NMR (299.9 MHz) 7.9-7.6 (m,
15H-P+Ph3), 3.98 (s, 6H, 2×
-OCH3), 3.93-3.8 (m, 2H,
-CH2-P+Ph3), 2.42 (t, J = 7.4 Hz, 2H,
ubiquinone-CH2-), 2.00 (s, 3H,
CH3), 1.6-1.2 (m, 16H,
(CH2)8-) ppm; 13C NMR
(75.4 MHz) 184.8 (C=O), 184.2 (C=O) 144.3 (2C, ring), 143.1 (ring), 138.8 (ring). 135.0 (d,
J = 2.4 Hz, -P+Ph3
para), 133.8 (d, J = 85.0 Hz,
-P+Ph3 ortho/meta), 130.5 (d,
J = 13.3 Hz, P+Ph3
ortho/meta), 118.6 (d, J = 85.0 Hz,
P+Ph3 ipso); 30.4 (d,
J = 15.8 Hz,
-CH2-CH2-CH2-P+Ph3),
29.8 (-CH2-), 29.3 (
To synthesize 3H-enriched
10-(6'-ubiquinolyl)decyltriphenylphosphonium bromide,
triphenylphosphine (4.09 mg; 15.6 µmol), 3 (6.3 mg; 15.6 µmol), and 250 µl of ethanol containing
[3H]triphenylphosphine (74 µCi, Moravek Biochemicals,
Brea, CA, 1 Ci/mmol) were sealed under argon in a Kimax tube and
stirred in the dark for 55 h at 80 °C. After cooling the
product was precipitated by addition of diethyl ether, and the orange
solid was dissolved in a few drops of CH2Cl2
and precipitated with diethyl ether. This was repeated four times to
remove unreacted triphenylphosphine and 3. Two separate
syntheses of 3H-enriched
10-(6'-ubiquinonyl)decyltriphenylphosphonium bromide were carried out
giving products of 2.6 and 2.46 mCi/mmol, respectively, which gave the
same results in experiments with isolated mitochondria, and their UV
absorption spectra were as expected for a mixture of 4 and
5. TLC followed by scintillation counting of sectioned
plates and comparison with the RF values of the
unlabeled compounds confirmed radiopurity.
Characterization of Products--
Stock solutions containing a
mixture of 4 and 5 in ethanol were stored at
-80 °C, and their concentrations were confirmed by 31P
NMR. Fully oxidized solutions were generated by incubation in basic
95% ethanol on ice (13) or with beef heart mitochondrial membranes at
room temperature. Both procedures gave an extinction coefficient of
10,400 M-1 cm-1 at 275 nm for the
quinone, with shoulders at 263 and 268 nm corresponding to the
triphenylphosphonium moiety (26, 27) and a broad shoulder at 290 nm due
to the quinone (13) (Fig. 1A). Reduction with NaBH4 gave the quinol, which had local maxima at 290 nm
( Mitochondrial Preparations and Incubations--
Rat liver
mitochondria were prepared by homogenization followed by differential
centrifugation (29). Beef heart mitochondria were isolated by standard
procedures, and membrane fragments were prepared by sonication followed
by centrifugation (30). Protein concentrations were determined by the
biuret assay using bovine serum albumin as a standard (31). Endogenous
ubiquinone was removed from lyophilized beef heart mitochondria by
pentane extraction, and complete extraction was confirmed by the
inability of these mitochondria to oxidize NADH in the absence of added
Q1 (13).
For respiration measurements, rat liver mitochondria (2 mg of
protein/ml) were suspended in KCl medium (120 mM KCl, 10 mM Hepes, 1 mM EGTA, pH 7.2) at 25 °C
supplemented with respiratory substrates and 1 mM phosphate
in a 3-ml oxygen electrode (Rank Brothers, Bottisham, Cambridge, UK).
After measuring the rate of coupled respiration 200 µM
ADP was added, the rate of phosphorylating respiration was measured and
then FCCP (300 nM) was added and the rate of uncoupled
respiration determined. To measure membrane potential, rat liver
mitochondria (2 mg of protein/ml) were incubated for 3 min in 0.5 ml of
KCl medium supplemented with nigericin (1 µg/ml), 5 mM
each of glutamate and malate, 1 µM TPMP, and 100 nCi/ml
[3H]TPMP. After incubation the mitochondria were pelleted
by centrifugation, the radioactivity in the pellet and supernatant were
measured by scintillation counting, and the membrane potential was
calculated using the Nernst equation, assuming a mitochondrial volume
of 0.5 µl/mg of protein and that 60% of the intramitochondrial TPMP was membrane-bound (32, 33). The uptake of [3H]mitoQ by
rat liver mitochondria was measured under the same conditions. Scanning
spectra and kinetic measurements were made with an Aminco DW-2000 dual
beam spectrophotometer using matched 1-ml quartz cuvettes at 20 °C.
Beef heart mitochondrial membranes and freeze-thawed yeast mitochondria
were incubated in 50 mM potassium phosphate, pH 7.2. Rat
liver mitochondria were incubated in KCl medium.
Oxidative Damage Assays--
To measure thiobarbituric acid
reactive species (TBARS), rat liver mitochondria (2 mg of protein/ml)
were incubated at 37 °C with shaking for 45 min in 100 mM KCl, 10 mM Tris-HCl, pH 7.6. Then 0.8-ml
aliquots were mixed with 400 µl of 0.5% thiobarbituric acid in 35%
HClO4, heated at 100 °C for 15 min, diluted with 3 ml of
water, and extracted into 3 ml of n-butanol. TBARS were determined fluorometrically (
To measure cis-parinaric acid oxidation, mitochondria (2 mg
of protein/ml) were suspended in a 3-ml fluorimeter cuvette in 100 mM KCl and 10 mM Tris-HCl, pH 7.6, at
37 °C. The oxidation of cis-parinaric acid (Molecular
Probes) was monitored fluorometrically ( Yeast Experiments--
The Saccharomyces cerevisiae
strains used were: CY4- Mammalian Cell Culture--
Human osteosarcoma 143B cells were
cultured at 37 °C under humidified 95% air/5% CO2 in
DMEM supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml), and 10% fetal calf serum. For toxicity studies, cells were
grown to confluence in 24-well tissue culture dishes and incubated for
24 h with DMEM/serum containing the compound. The supernatants
were then harvested, and the amount of LDH released was assayed and
compared with that present in untreated wells lysed with 0.1% Triton.
For uptake studies cells were suspended in 0.5 ml of DMEM supplemented
with 10 mM HEPES and 5 µM
[3H]mitoQ. After incubation, cells were pelleted by
centrifugation, and the radioactivity in the pellet was quantitated by
scintillation counting. For digitonin fractionation, cells were
incubated as above, and then 500 µl of the cell suspension was mixed
rapidly with 1.2 ml of ice-cold 250 mM sucrose, 20 mM MOPS pH 6.7, 3 mM EDTA, and 1 mg of
digitonin, then 1 ml was layered onto 350 µl of oil (66% silicone
oil/34% dioctyl pthalate) over 100 µl of 500 mM sucrose,
0.1% Triton and separated into mitochondrial and cytoplasmic fractions
by centrifugation. The two fractions were assayed for citrate synthase
and LDH activity or for content of radioactivity by scintillation
counting (39). The Jurkat human T lymphocyte line was grown at 37 °C
under humidified 95% air/5% CO2 in RPMI 1640 supplemented
with penicillin (100 units/ml), streptomycin (100 mg/ml), and 10%
fetal calf serum. Apoptosis was induced by addition of hydrogen
peroxide (8). Caspase activation in lysed cell pellets was measured
fluorometrically by the cleavage of the peptide DEVD labeled with AMC
(DEVD-AMC) and calibrated using an AMC standard curve (40). The
proportion of cells undergoing apoptosis was quantitated by annexin
V-fluorescein isothiocyanate staining followed by detection of
annexin-positive cells using a Becton Dickinson FACScan flow cytometer.
General Procedures--
Column chromatography was on Silica Gel
type 60, 200-400 mesh, 40-63 µm (Merck). TLC was on Silica Gel 60F
254 (Merck) or on C-18 silica (Whatman). Nuclear magnetic resonance
spectra were acquired on Varian 500 MHz or Varian 300 MHz instruments
in CDCl3. Chemical shifts are in Redox Activity of Mitoquinone and Mitoquinol--
The
mitochondrially targeted quinol,
10-(6'-ubiquinolyl)decyltriphenylphosphonium (4), and
quinone, 10-(6'-ubiquinonyl)decyltriphenylphosphonium (5),
were synthesized as shown in Scheme 1. Here they are called mitoquinol
(reduced) and mitoquinone (oxidized), respectively, and mitoQ refers to
a mixture of redox forms. As shown in Table I, mitoQ was intermediate in
hydrophobicity between the simple phosphonium salt TPMP and the
ubiquinone precursor bromodecyl ubiquinone (2). The
distinctive absorption spectra of mitoquinone and mitoquinol are shown
in Fig. 1A.
Electron Transfer between Mitochondria and mitoQ--
To determine
whether the respiratory chain could reduce mitoquinone, we incubated it
with beef heart mitochondrial membranes and recorded its spectrum (Fig.
1B, t = 0). Addition of the respiratory substrate succinate reduced mitoquinone to mitoquinol (Fig.
1B). Mitoquinone-mitoquinol interconversion was then
measured continuously by monitoring mitoquinone at 275 nm (Fig.
2). Mitoquinone was reduced by beef heart
mitochondrial membranes and succinate, and this reduction was blocked
by the complex II inhibitor malonate (Fig. 2A). Chemically
reduced mitoquinol was also oxidized by membranes, and this oxidation
was blocked by the complex III inhibitor myxothiazol (Fig.
2B). Mitoquinol and mitochondrial membranes reduced
ferricytochrome c and myxothiazol inhibited this reduction by 60-70% (data not shown). Rat liver mitochondria respiring on succinate or glutamate/malate reduced mitoquinone, and this activity was blocked by the respiratory inhibitors malonate (Fig. 2C)
or rotenone (Fig. 2D), respectively. Dissipation of the
membrane potential with the uncoupler FCCP also eliminated the
reduction of mitoquinone by preventing its uptake into mitochondria
(Fig. 2, C and D).
Reduction of Mitoquinone by Respiratory Complexes--
To
distinguish between mitoQ reactions with respiratory complexes and the
endogenous ubiquinone/ubiquinol pool, we extracted beef heart
mitochondrial membranes with pentane to remove endogenous ubiquinone.
These mitochondria still oxidized mitoquinol and, in the presence of
succinate, reduced mitoquinone by a malonate-sensitive pathway (Fig.
3A). This strategy was
extended to investigate yeast that entirely lacked endogenous
ubiquinone due to inactivation of ubiquinone biosynthesis. These yeast
did not grow on the nonfermentable carbon source YPEG until the
short-chain ubiquinone analog Q2 was added, but addition of
mitoQ did not lead to cell growth (Fig. 3B). When
respiratory chain activity was measured in mitochondria isolated from
these yeast, there was no electron flux from NADH or succinate to
cytochrome c until Q2 was added (Fig. 3,
C and D). MitoQ did stimulate
myxothiazol-sensitive cytochrome c reduction, but less so
than Q2 (Fig. 3, C and D). When
ubiquinone reduction was analyzed directly, mitochondria lacking
ubiquinone reduced both Q2 and mitoquinone, however, the
rate for mitoquinone was slower than for Q2 (Fig.
3E). The rate of reduction of mitoquinone by mitochondria
was similar in the presence or absence of endogenous ubiquinone (Fig.
3, E and F). We conclude that mitoQ can be
reduced and oxidized by the mitochondrial respiratory chain and that
this is primarily through the active sites of respiratory complexes rather than via the endogenous ubiquinone pool (14, 28).
Uptake of mitoQ by Isolated Mitochondria--
Tritiated mitoQ was
taken up rapidly by energized mitochondria, and addition of the
uncoupler FCCP caused its immediate efflux (Fig.
4A). This FCCP-sensitive
accumulation of mitoQ was substantial up to 20 µM mitoQ
(Fig. 4B). The mitoQ accumulation ratios were measured
relative to the simple lipophilic cation TPMP over a range of membrane
potentials (Fig. 4C). At low membrane potential, the mitoQ
accumulation ratio was greater than for TPMP, but at high potentials
the maximum accumulation ratio for mitoQ (~500-600) was slightly
less than that for TPMP (~1300), suggesting that the greater
hydrophobicity of mitoQ decreases its uptake slightly relative to
TPMP.
Location of mitoQ within
Mitochondria--
Alkyltriphenylphosphonium cations equilibrate
between the bulk phase and a potential energy well on the membrane
surface where they adsorb as a monolayer close to the carbonyl groups
of the phospholipid fatty acids (41-43). This adsorption is described by Uptake of mitoQ by Mitochondria within Cells--
Tritiated mitoQ
incubated with a suspension of 143B osteosarcoma cells was taken up
over 20-40 min, and this uptake was decreased by disrupting the
mitochondrial membrane potential (Fig.
5A). To determine the location
of mitoQ within cells, we disrupted the plasma membrane of cells with
the detergent digitonin and pelleted the mitochondria by centrifugation
through oil (Fig. 5, B and C). This separated
cells into mitochondrially enriched and cytosolic fractions, as
confirmed by the distribution of the mitochondrial and cytosolic marker
enzymes citrate synthase and lactate dehydrogenase (Fig.
5B). About half the mitoQ within the cell was found in
mitochondrial fraction, similar to the proportion of mitoQ uptake
sensitive to FCCP (Fig. 5C). Therefore, substantial amounts
of mitoQ are taken up by both isolated mitochondria and mitochondria
within cells, driven by the mitochondrial membrane potential.
Low Toxicity of mitoQ to Mitochondria and Cells--
The toxicity
of mitoQ was investigated in mitochondria and cells. Up to 10 µM mitoQ had little effect on the membrane potential of
isolated mitochondria but at 25 µM and above the
potential decreased (Fig. 6A).
For mitochondria respiring on either succinate or glutamate/malate, 10 µM mitoQ had little effect on uncoupled or
phosphorylating respiration, but was inhibitory at 25-50
µM (Fig. 6, B and C). MitoQ
stimulated coupled respiration by increasing the proton leak though the
inner membrane; this effect was minimal for succinate at 10 µM but was noticeable at 10 µM for
glutamate/malate (Fig. 6C). MitoQ toxicity to human 143B
cells was determined from the release of lactate dehydrogenase into the
culture medium over 24 h (Fig. 6D). MitoQ up to 10 µM did not affect cell viability, and concentrations of
25-50 µM were required for substantial cell death (Fig.
6D). In summary, mitoQ concentrations up to 10 µM do not disrupt mitochondrial or cell function and,
therefore, concentrations of 1-5 µM were used in
subsequent experiments.
Antioxidant Properties of mitoQ--
To investigate the
antioxidant efficacy of mitoQ, we incubated mitochondria with
cis-parinaric acid (Fig.
7A). This fatty acid
fluoresces within a lipid environment, and its conjugated double bond
fluorophore is susceptible to oxidation. Consequently, the
disappearance of fluorescence is a measure of lipid peroxidation (34).
In the presence of mitochondria, cis-parinaric acid
fluoresced due to its insertion into lipid bilayers (Fig.
7A). MitoQ prevented the oxidation of
cis-parinaric acid by hydrogen peroxide and ferrous iron,
demonstrating that mitoquinol is an antioxidant (Fig. 7A). To quantitate the antioxidant efficacy of mitoQ, mitochondria were
incubated with ferrous iron, and the accumulation of MDA was measured
as a marker of lipid peroxidation (Fig. 7B). This oxidative
damage also disrupted mitochondrial function as indicated by a decrease
in the membrane potential (Fig. 7C). Incubation with mitoQ
prevented both the accumulation of MDA and the disruption to
mitochondrial function caused by oxidative stress (Fig. 7, B
and C). To determine whether mitoquinone or mitoquinol was
the effective antioxidant, we oxidized mitoQ to mitoquinone and
prevented its reduction by the respiratory chain by including malonate
and rotenone in the incubation. Under these conditions, mitoquinone did
not block lipid peroxidation (Fig. 7D). In contrast, when mitoQ was reduced to mitoquinol by the respiratory chain, oxidative damage was prevented (Fig. 7D). The simple lipophilic cation
TPMP did not prevent lipid peroxidation (Fig. 7D).
Therefore, the antioxidant activity of mitoQ is due to its ubiquinol
moiety.
Recycling of mitoQ by the Respiratory Chain--
To determine
whether mitoquinol was recycled by the respiratory chain after
detoxifying a reactive oxygen species, we studied its interaction with
peroxynitrite, a biologically significant oxidant formed from nitric
oxide and superoxide (45-47). Because the half-life of peroxynitrite
is only 1-2 s, mitoquinol regeneration from mitoquinone can be studied
after all the added peroxynitrite has decomposed (45). Mitoquinol was
rapidly oxidized to mitoquinone by peroxynitrite, however, mitoquinone
was only detected when its reduction by the respiratory chain was
prevented by malonate (Fig.
8A, upper trace).
Continuous monitoring of the mitoquinone concentration showed that
peroxynitrite rapidly oxidized the mitoquinol to mitoquinone, which was
then reduced to mitoquinol by the respiratory chain (Fig.
8B). When malonate was present, this reduction by the
respiratory chain was prevented (Fig. 8B). We conclude that mitoQ is an effective antioxidant that can be recycled to its active
form by the respiratory chain after detoxifying a reactive oxygen
species.
Prevention of Apoptosis by mitoQ--
A range of stimuli induce
apoptosis by releasing cytochrome c from mitochondria into
the cytoplasm, where it activates caspases. These include the oxidant
hydrogen peroxide (8), the protein kinase C inhibitor staurosporine
(40), and tumor necrosis factor-
Addition of hydrogen peroxide to Jurkat cells led to caspase activation
and induction of apoptotic cell death 4-6 h later (Fig.
9). Preincubation with 1 µM
mitoQ completely blocked caspase activation (Fig. 9, A and
B) and substantially decreased apoptotic cell death, as
determined by the proportion of annexin-positive cells (Fig. 9,
C and D). To determine whether the mitochondrial localization of mitoQ was required for this protective effect, we
compared mitoQ with Q1, a quinol antioxidant that
distributes evenly throughout the cell, and found that Q1
did not block caspase activation (Fig. 9B). In contrast to
the situation with hydrogen peroxide, mitoQ did not prevent apoptosis
in Jurkat cells treated with staurosporine (40) or in WEHI 164 cells
treated with tumor necrosis factor- Conclusion--
To provide new approaches to investigate the role
of mitochondrial oxidative damage in cell death, we synthesized a
mitochondrially targeted antioxidant, mitoQ, comprising a ubiquinone
attached to a triphenylphosphonium lipophilic cation. The ubiquinone
moiety was found to cycle between its oxidized (mitoquinone) and
reduced (mitoquinol) forms by exchanging electrons with the respiratory chain. Mitoquinol was an effective antioxidant protecting mitochondria from oxidative damage and was rapidly regenerated by the respiratory chain after detoxifying a reactive oxygen species. As anticipated, the
triphenylphosphonium cation led to the rapid and reversible accumulation of mitoQ by isolated mitochondria and by mitochondria within cells. Therefore, mitoQ is a mitochondrial-specific antioxidant.
We then used mitoQ to help elucidate the role of mitochondrial
oxidative damage in apoptotic cell death. As a first step we showed
that mitoQ prevented apoptosis caused by hydrogen peroxide but not that
caused by staurosporine or tumor necrosis factor-. This was compared with
untargeted ubiquinone analogs, which were ineffective in preventing
apoptosis. These results suggest that mitochondrial oxidative stress
may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis
factor-
. We have shown that selectively manipulating mitochondrial
antioxidant status with targeted and recyclable antioxidants is a
feasible approach to investigate the role of mitochondrial oxidative
damage in apoptotic cell death. This approach will have further
applications in investigating mitochondrial dysfunction in a range of
experimental models.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
max
(ethanol): 278 nm; 1H NMR (299.9 MHz) 3.99 (s, 6H, 2 x-OCH3), 3.41 (t, J = 6.8 Hz,
2H, -CH2-Br), 2.45 (t, J = 7.7 Hz, 2H, ubquinone-CH2-), 2.02, (s, 3H,
-CH3). 1.89 (quin, J = 7.4 Hz,
2H, -CH2 -CH2-Br), 1.42-1.28
(m, 14H, -(CH2)7-) ppm;
13C NMR (125.7 MHz) 184.7 (C = O), 184.2 (C = O), 144.3 (2C, ring), 143.1 (ring),
138.7 (ring), 61.2 (2× -OCH3), 34.0 (-CH2-), 32.8 (-CH2-),
29.8 (-CH2-), 29.4 (2×
-CH2-), 29.3 (-CH2-),
28.7 (2× -CH2-), 28.2 (-CH2-), 26.4 (-CH2-),
11.9 (-CH3) ppm. Anal. calcd. for
C19H29O4Br: C, 56.86; H, 7.28; found: C, 56.49, H, 7.34%; mass spectrum: calcd. for
C19H29O4Br 400/402; found
400/402.
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Scheme 1.
Synthesis of mitoquinol (4) and mitoquinone
(5). Numbers refer to percentage yields for syntheses.
CH2-), 29.2 (2×
-CH2-), 29.1 (-CH2-),
28.7 (-CH2-), 26.4 (-CH2-), 22.9 (d, J = 48.5 Hz,
-CH2-P+Ph3), 22.7 (d,
J = 4.9 Hz,
-CH2-CH2-P+Ph3),
11.9 (-CH3) ppm. 31P NMR (121.4 MHz)
25.1 ppm. Anal. calcd. for
C37H44O4PBr: C, 66.97; H 6.68;
found: C, 66.69; H, 6.99; mass spectrum: calcd. for
C37H44O4P 583.2977; found
583.2972.
= 1800 M-1 cm-1) and at
268 nm (
= 3000 M-1 cm-1)
(27). The
ox-red at 275 nm in 50 mM
sodium phosphate, pH 7.2, was 7000 M-1
cm-1. The quinone extinction coefficient (10,400 M-1 cm-1 at 275 nm) was slightly
lower than that reported for other quinones (12,250 M-1 cm-1) in aqueous buffer (28).
This difference was not due to an intermolecular interaction between
the phosphonium and the quinone, because the absorbances of
2 and the simple phosphonium methyltriphenylphosphonium
(TPMP) were additive when 50 µM of each were mixed
together in either ethanol or aqueous buffer. To prepare mitoquinol an
ethanolic solution was diluted in ~0.5-1 ml of water and a few
grains of NaBH4 were added. After incubation on ice in the
dark for 5 min, excess NaBH4 was quenched with 5% HBr (0.2 ml) and the quinol was extracted into CH2Cl2
(3 × 0.5 ml). The extract was then washed with water and 2 M NaCl, then the CH2Cl2 was removed
under a stream of nitrogen. The pale yellow solid residue was dissolved
in acidified 96% ethanol. The yield was typically 70-80% and, as the
quinol slowly oxidized in air, it was freshly prepared and stored on
ice under argon in the dark. To determine partition coefficients,
compounds were added to 2 ml each of 1-octanol-saturated PBS and
PBS-saturated 1-octanol then shaken at 37 °C for 30 min in the dark.
After separation by centrifugation, the amounts in each phase were
determined by absorption relative to standard curves in
1-octanol-saturated PBS or PBS-saturated 1-octanol.
excite = 515 nm;
emission = 553 nm) and expressed as nanomoles of MDA by
comparison with standard solutions of 1,1,3,3-tetraethoxypropane
processed as above. Prior to analyzing samples, their mitoQ contents
were brought to the same concentration to eliminate differences in MDA
formation during heating and processing. To measure the membrane
potential after exposure to oxidative damage, mitochondria were
pelleted by centrifugation and resuspended in KCl medium, and their
membrane potentials were determined as described above.
excite = 324 nm;
emission = 413 nm). A control experiment showed that
titration of cis-parinaric acid into a mitochondrial
suspension initially increased the fluorescence as it partitioned into
membranes, but the increase became nonlinear after about 3 µM and declined above 10 µM due to self
quenching (34). Therefore, 3 µM cis-parinaric acid was used for all experiments. Peroxynitrite was synthesized from
acidified H2O2 and NaNO2 in a
simple flow reactor as described previously (35), concentrated by
freeze fractionation and stock solutions in 1.5 M NaOH
quantitated [
302 = 1.67 mM-1.cm-1 (36)].
COQ3 (MATa ura3-52 leu2-3
leu2-112 trp1-1 ade2-1 his3-11 can1-100 coq3::HIS3),
kindly supplied by Prof. Ian W. Dawes, University of New South Wales,
Australia (37) and CEN.PK2-1C-
COQ3 (CEN.PK2-1C coq3::LEU2) kindly supplied by Prof. Cathy Clarke,
UCLA. Both
COQ3 yeast strains were auxotrophic for ubiquinone when
grown on nonfermentable medium. Yeasts were grown in Erlenmeyer flasks at 28 °C under air with shaking at 200 rpm. For growth analysis, cultures in YPD (1% bacto yeast extract, 2% bactopeptone, 2%
dextrose) were diluted into YPEG (1% bacto yeast extract, 2%
bactopeptone, 3% ethanol, 3% glycerol) to an
A600 of 0.1 and then grown in the dark while the
A600 was monitored. For studies on yeast
mitochondria, mitochondria were prepared from lactate-grown yeast of
the CY4-
COQ3 and CY4 wild type strains (38). Briefly, lactate-grown
yeast were isolated by centrifugation, the cell wall was removed by digestion with Zymolyase, spheroplasts were homogenized, and
mitochondria were isolated by differential centrifugation. Mitochondria
were stored at -80 °C in 0.6 M sorbitol, 20 mM HEPES, pH 7.4, supplemented with 10 mg/ml fatty
acid-free bovine serum albumin. For spectrophotometric assays, yeast
mitochondria were washed in 0.6 M sorbitol, 20 mM HEPES and freeze-thawed in 50 mM potassium
phosphate, pH 7.2.
units (ppm) downfield
from tetramethylsilane for 1H NMR and 13C NMR
and 85% phosphoric acid for 31P NMR. In some cases the
total integral ratios in the 1H NMR were not precisely as
expected; however, all other structural data are fully consistent with
the proposed structures. Infrared absorption spectra were acquired
using a PerkinElmer 1600 FTIR spectrometer. Phosphonium salts were
examined by applying a concentrated deuterochloroform solution to NaCl
discs followed by evaporation of the solvent in a nitrogen stream.
Other samples were examined neat or as Nujol mulls between NaCl discs.
Mass spectra were obtained from the Chemistry Department, University of
Canterbury. Data are presented as m/z values for
the parent molecular ion. Fluorescence measurements were made using a
PerkinElmer MPF-3L fluorescence spectrophotometer.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
The octanol/PBS partition coefficients were determined at 37 °C and
are means ± S.E.M. of three separate determinations
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Fig. 1.
Absorption spectra of mitoquinol and
mitoquinone. A, mitoQ (50 µM) was
incubated with beef heart mitochondrial membranes (20 µg of
protein/ml) for 1 h to oxidize it to mitoquinone. Reduction with
NaBH4 (~250 µg) gave mitoquinol. B, 50 µM mitoQ was incubated with beef heart mitochondrial
membranes, and the spectrum of mitoquinone was recorded
(t = 0). Then antimycin (5 µM) and
succinate (5 mM) were added, and further spectra were
acquired at 5-min intervals (t = 5-25).
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Fig. 2.
Reduction of mitoquinone and oxidation of
mitoquinol by mitochondria. A, beef heart mitochondrial
membranes (20 µg of protein/ml) were incubated with rotenone (8 µg/ml) and mitoQ (50 µM). A275
was monitored continuously and succinate (5 mM) and
malonate (20 mM) were added where indicated. B,
mitochondrial membranes were incubated as above with mitoquinol (50 µM), rotenone (8 µg/ml), and malonate (20 mM). Myxothiazol (10 µM) was added where
indicated. When this experiment was repeated in the presence of
ferricytochrome c (50 µM) and KCN (200 µM) the ferricytochrome c was reduced and this
reduction was decreased by 60-70% by myxothiazol (data not shown).
C and D, rat liver mitochondria (100 µg of
protein/ml) were incubated in KCl, medium, and
A275 was monitored. For the experiments shown in
C, rotenone (8 µg/ml) and succinate (5 mM)
were present and mitoQ (20 µM) was added where indicated.
This experiment was repeated in the presence of malonate (20 mM) or FCCP (300 nM: not shown but identical to
the experiment in the presence of malonate). For the experiments shown
in D glutamate and malate (5 mM of each) were
present and mitoQ (20 µM) was added where indicated. This
experiment was repeated in the presence of rotenone (8 µg/ml) or FCCP
(300 nM; not shown but identical to the experiment in the
presence of rotenone).
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Fig. 3.
Reduction of mitoquinone by
ubiquinone-depleted mitochondria. A, pentane-extracted
beef heart mitochondrial membranes (50 µg of protein/ml) were
incubated with mitoQ (50 µM), and
A275 was monitored continuously. Succinate (5 mM) and malonate (20 mM) were added where
indicated. B, CY4. Coq3 yeast were grown in YPEG medium
with either no additions (open triangles) or supplemented
with 50 µM Q2 (open squares) or 50 µM mitoQ (filled squares), and growth was
analyzed by measuring A600 over time. Addition
of mitoQ up to 250 µM or daily additions of 5 µM mitoQ did not lead to cell growth (data not shown).
Results show a typical experiment that was repeated several times and
similar results were obtained when the Cen
Coq3 yeast strain was
used. C-E, mitochondria from the CY4.
Coq3 yeast strain
were freeze-thawed and suspended at 50 µg of protein/ml and
ferricytochrome c reduction measured at 550 nm.
C, 125 µM NADH, 50 µM
ferricytochrome c, and 2 mM KCN were present
with or without myxothiazol (myx; 10 µM). 10 µM Q2 or 25 µM mitoQ were added
as indicated. D, 10 mM succinate was present;
myxothiazol (myx; 10 µM), 10 µM
Q2, or 25 µM mitoQ were added as indicated;
and ferricytochrome c reduction was measured at 550 nm.
E, 10 µM Q2 or 25 µM
mitoQ were added to freeze-thawed mitochondria respiring on 10 mM succinate, and ubiquinone reduction was monitored
continuously at 275 nm. The inhibition of mitoQ reduction by malonate
(20 mM) is shown. F, the experiments shown in
E were repeated using normal mitochondria isolated from the
wild type CY4 strain.
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Fig. 4.
Uptake of mitoQ by energized
mitochondria. A, rat liver mitochondria were incubated
in KCl medium supplemented with 10 mM succinate, rotenone
(8 µg/ml), nigericin (1 µg/ml), 10 µM mitoQ, and 2.5 nCi of [3H]mitoQ/ml. Mitochondrial mitoQ uptake was
determined at various times (filled squares). Where
indicated, 300 nM FCCP was added after 2.5-min mitoQ
accumulation (open circles). This gave the same final mitoQ
accumulation as when FCCP was present from the start of the incubation
(data not shown). Data are means of duplicate determinations. In all
cases the range is smaller than the symbols. This
shows a typical experiment, which was repeated on two separate
mitochondrial preparations. B, the mitoQ accumulation ratio
(µl/mg of protein) was determined by incubating mitochondria for 5 min with various concentrations of mitoQ in the absence (closed
bars) or presence (open bars) of 300 nM
FCCP. Data are means ± range of duplicate determinations and are
typical of experiments repeated on two mitochondrial preparations.
C, the mitoQ and TPMP accumulation ratios were determined in
parallel by carrying out incubations in the presence of 5 µM mitoQ and 1 µM TPMP supplemented with
either [3H]mitoQ or [3H] TPMP. A range of
different membrane potentials was established by including FCCP (300 nM) or different concentrations of malonate (0-16
mM) in the incubations. mitoQ and TPMP accumulation ratios
were determined in duplicate for each condition and are plotted against
each other ± range. This figure combines data from two separate
mitochondrial preparations. The membrane potential at the
top of the panel was calculated from the TPMP accumulation
ratio. D, mitochondria were incubated for 5 min in the
presence of 300 nM FCCP and various concentrations of mitoQ
(filled squares) or TPMP (open circles), and data
are means of duplicate determinations. In the inset in
D the amount of TPMP or mitoQ bound (nmol/mg of protein) is
plotted against the concentration of TPMP or mitoQ added to the
incubation (µM). In the main panel the
reciprocal of the amount of TPMP or mitoQ bound is plotted against the
reciprocal of the free concentration. For mitoQ the slope was 0.013, giving a bound-to-free ratio of 77 µl/mg of protein, whereas for TPMP
the slope was 0.034, giving a bound-to-free ratio of 29 µl/mg of
protein.
, the ratio of the surface density of adsorbed ions to their volume density in the bulk phase (42). We measured the nonspecific adsorption of mitoQ and TPMP to de-energized mitochondria over a range
of concentrations and determined the ratio of adsorbed to free cations
from double-reciprocal plots (Fig. 4D). From these and the
surface area of rat liver mitochondria (155 and 520 cm2/mg
of protein for the outer and inner membranes, respectively (44)) we
calculated
= 22 × 10
6 cm for TPMP and
= 57 × 10
6 cm for mitoQ, values which are
comparable to those of tetraphenylphosphonium adsorption to simple
phospholipid bilayers (
= 4.2 × 10
6 cm
(42)). An earlier study showed that the greater adsorption of
decyltriphenyl phosphonium relative to TPMP was due to the increased
entropy change for insertion of the alkyl group into the lipid bilayer,
whereas the enthalpy change for cation adsorption was unaffected (42).
This suggests that mitoQ adsorbs to membranes about three times more
strongly than TPMP because of insertion of its hydrophobic side chain
into the lipid bilayer (41). We conclude that mitoQ is taken up into
energized mitochondria and is then largely adsorbed to the matrix face
of the inner membrane with the phosphonium cation at the level of the
fatty acid carbonyls while the hydrophobic side chain inserts into the
lipid interior of the membrane.
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Fig. 5.
Uptake of mitoQ by cells. A,
143B cells (5 × 106) were incubated in 500 µl of
DMEM/HEPES supplemented with 5 µM [3H]mitoQ
(filled squares). These incubations were repeated in the
presence of 4 µM FCCP (open triangle) or with
a mixture of FCCP, valinomycin, ouabain, and oligomycin (open
circles). Data are means ± range of two determinations, and
this is a typical experiment that was repeated on three separate cell
incubations. B, cells were incubated as above for 60 min
with 5 µM mitoQ, and then intact cells were pelleted
through oil (no additions) or the cells were treated with
digitonin and the mitochondria pelleted through oil (plus
digitonin). Citrate synthase (open bars) and lactate
dehydrogenase (closed bars) activities were then measured in
the pellet and supernatant fractions, and the proportion of the total
activity that was found in the pellet was calculated. Data are
means ± S.D. of determinations on three separate cell
preparations. C, cells were incubated as above with 5 µM [3H]mitoQ in the presence or absence of
4 µM FCCP, and then intact cells were pelleted through
oil (cell) or the cell suspension was treated with digitonin
and the mitochondria were pelleted through oil (mito). Data
are means ± S.D. of experiments on four independent cell
preparations.
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Fig. 6.
Effect of mitoQ on mitochondrial and cell
function. A, the membrane potential of mitochondria
respiring on succinate was measured in the presence of mitoQ. Data are
expressed as a percentage of the membrane potential in the absence of
mitoQ and are the means ± S.E. of measurements on three separate
mitochondrial preparations. B and C, rates of
coupled (closed bars), phosphorylating (open
bars), and uncoupled (stippled bars) respiration were
measured for mitochondria respiring on succinate (B) or
glutamate and malate (C). Data are a percentage of the
corresponding respiration rates in the absence of mitoQ and are the
means ± S.E. of determinations on three separate mitochondrial
preparations. In D mitoQ was incubated with 143B cells for
24 h, and LDH release into the culture medium was measured and
expressed as a percentage of the total amount of LDH present in
untreated wells. Data are means ± S.D. of three independent
experiments.
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Fig. 7.
Antioxidant efficacy of mitoQ.
A, rat liver mitochondria were incubated with 10 mM succinate, 8 µg/ml rotenone, 50 µM
ferrous sulfate, 100 µM ascorbic acid, and 1 mM hydrogen peroxide in the presence or absence of 5 µM mitoQ. Where indicated, 3 µM
cis-parinaric acid was added and the fluorescence measured.
Data are from a typical experiment repeated on three separate
mitochondrial preparations. TPMP (5 µM) did not block
cis-parinaric acid oxidation (data not shown). B,
mitochondria were preincubated with 10 mM succinate and
mitoQ for 5 min, and then a sample was taken for TBARS analysis (zero
time). Ferrous sulfate (100 µM) and 300 µM
ascorbic acid were then added, and 40 min later MDA formation was
quantitated. Data are means ± range of duplicate determinations
and are typical of experiments repeated on three separate mitochondrial
preparations. C, mitochondria were incubated as described
for B, isolated by centrifugation, and their membrane
potential determined from the uptake of TPMP while respiring on
glutamate/malate. Data are means ± range of a typical experiment
repeated on three separate mitochondrial preparations. D,
mitochondria were incubated as in B supplemented with 8 µg/ml rotenone. Succinate (5 mM) and mitoQ or TPMP (5 µM) were added to some incubations, whereas for the other
incubations malonate (20 mM) was present and mitoQ was
oxidized completely to mitoquinone before addition by incubation at
basic pH. After preincubation for 5 min, ferrous sulfate (50 µM) was added, and 40 min later MDA formation was
quantitated. Data are means ± range of duplicate
determinations.
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Fig. 8.
Regeneration of mitoquinol after oxidation by
peroxynitrite. A, beef heart mitochondrial membranes
(100 µg of protein/ml) were incubated in 50 mM potassium
phosphate, pH 7.4, at 20 °C with 50 µM mitoQ and 10 mM succinate. The lower trace
( peroxynitrite) shows that this treatment reduced all of
the mitoQ to mitoquinol. Then malonate (20 mM) was added to
prevent further reduction, and finally, peroxynitrite (200 µM) was added and the spectrum of mitoquinone was
acquired (+peroxynitrite). When peroxynitrite was added
without malonate the spectrum obtained was similar to that of
mitoquinol (
peroxynitrite). B, beef heart
mitochondrial membranes were incubated with 50 µM mitoQ
and succinate (5 mM) as above, and the mitoquinone
concentration monitored continuously at 275 nm. Where indicated
peroxynitrite (500 µM) was added alone
(
malonate), or in the presence of 20 mM
malonate (+malonate; dashed line).
(48). The mechanisms by which these
stimuli cause cytochrome c release from mitochondria are
unclear, but some or all may involve increased mitochondrial oxidative
stress. Of particular interest is whether cytochrome c
release induced by hydrogen peroxide is caused directly by
mitochondrial oxidative damage or is a secondary consequence of
cytoplasmic redox changes (8). A mitochondrially targeted antioxidant
can help elucidate the role of mitochondrial oxidative damage in
apoptosis, because it would only block those apoptotic signals that
require mitochondrial oxidative stress. Therefore, we investigated the
effect of mitoQ on apoptotic cell death.
(49) (data not shown). We
conclude that mitoQ blocks apoptosis induced by hydrogen peroxide.
Because mitochondrial localization of the antioxidant is required to
prevent apoptosis, mitochondrial oxidative stress may be a critical
step in hydrogen peroxide-induced apoptosis but not for apoptosis
following treatment with staurosporine or tumor necrosis
factor-
.
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Fig. 9.
Prevention of apoptosis by mitoQ.
A, Jurkat cells (5 × 106) in 5 ml of
medium were preincubated for 30 min with no additions (open
circles) or with 1 µM mitoQ (filled
circles), then 150 µM hydrogen peroxide was added
and cells were harvested at various times and their caspase activity
was measured as the rate of DEVD-AMC cleavage. Without hydrogen
peroxide there was no caspase activation either in the absence
(open squares) or presence of 1 µM mitoQ
(filled squares). Preincubation with 1 µM TPMP
did not decrease caspase activation by hydrogen peroxide (filled
triangle). The figure shows a typical experiment repeated on five
separate cell preparations. B, Jurkat cells incubated with 1 µM mitoQ or Q1 were treated with 150 µM hydrogen peroxide and 6 h later their caspase
activity compared with cells treated with hydrogen peroxide alone. This
figure shows typical experimental traces for caspase assays.
C and D, Jurkat cells were treated with 150 µM hydrogen peroxide in the presence or absence of 1 µM mitoQ, cells were harvested at various times and
annexin V binding analyzed by flow cytometry. C shows
results from a typical experiment to measure the annexin V binding of
cells harvested 6 h after treatment with 150 µM
hydrogen peroxide in the presence or absence of 1 µM
mitoQ. D shows measurements of the proportion of annexin
V-positive cells at various times after addition of 150 µM hydrogen peroxide in the absence (open
circles) or presence (filled circles) of 1 µM mitoQ. Cells incubated in the presence (filled
squares) or absence (open squares) of 1 µM mitoQ without hydrogen peroxide treatment are also
shown. Data are means ± range of duplicate determinations, and
the experiment was repeated on two cell preparations with similar
results.
. This suggests
that mitochondrial oxidative damage plays an important role in hydrogen
peroxide-induced apoptosis but is not required for apoptosis induced by
staurosporine or tumor necrosis factor-
. Further work using these
and other mitochondrially targeted compounds to dissect out the role of
mitochondrial oxidative changes in hydrogen peroxide-induced apoptosis
is ongoing. The findings reported here demonstrate that mitochondrially
targeted antioxidants such as mitoQ can be used to investigate the role
of mitochondrial oxidative stress in cell death. This strategy also has
potential for unraveling the contribution of oxidative stress to other
pathologies involving mitochondrial dysfunction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. Cathy Clarke, UCLA and Prof. Ian Dawes, University of New South Wales, Sydney for supplying yeast strains.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants (to M. P. M. and R. A. J. S.) from the Health Research Council of New Zealand and from the Marsden Fund, administered by the Royal Society of New Zealand.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A Foundation for Research, Science and Technology Bright Futures Scholar.
A New Zealand Science and Technology postdoctoral research fellow.
** A Health Research Council repatriation fellow.
To whom correspondence should be addressed: Medical Research
Council-Dunn Human Nutrition Unit, Wellcome Trust/MRC Bldg, Hills Rd.
Cambridge CB2 2XY, UK. Tel.: 44-1223-252703; Fax: 44-1223-252705; E-mail: michael.murphy@stonebow.otago.ac.nz.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M009093200
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ABBREVIATIONS |
---|
The abbreviations used are: IR, infrared; AMC, aminomethylcoumarin; DMEM, Dulbecco's modified Eagle's medium; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; LDH, lactate dehydrogenase; MDA, malondialdehyde; mitoquinol, 10-(6'-ubiquinolyl)decyltriphenylphosphonium; mitoquinone, 10-(6'-ubiquinonyl)decyltriphenylphosphonium; mitoQ, mixture of mitoquinol and mitoquinone; Q1, ubiquinone-1; Q2, ubiquinone-2; TBARS, thiobarbituric acid-reactive species; TPMP, methyltriphenylphosphonium cation; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid.
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