X-Message-Number: 26522
Date: Mon, 4 Jul 2005 20:57:23 -0700 (PDT)
From: Doug Skrecky <>
Subject: MitoQ might be an ideal antioxidant

[MitoQ is a ubiquinone derivative that is dramatically superior at
protecting mitochondria from free radicals. As such, this might be
an ideal antioxidant for testing the mitochondrial variant of the
free radical theory of aging.]

FASEB J. 2005 Jul;19(9):1088-95
Targeting an antioxidant to mitochondria decreases cardiac
ischemia-reperfusion injury.
    Mitochondrial oxidative damage contributes to a wide range of
pathologies, including cardiovascular disorders and
neurodegenerative diseases. Therefore, protecting mitochondria from
oxidative damage should be an effective therapeutic strategy.
However, conventional antioxidants have limited efficacy due to the
difficulty of delivering them to mitochondria in situ. To overcome
this problem, we developed mitochondria-targeted antioxidants,
typified by MitoQ, which comprises a lipophilic triphenylphosphonium
(TPP) cation covalently attached to a ubiquinol antioxidant. Driven
by the large mitochondrial membrane potential, the TPP cation
concentrates MitoQ several hundred-fold within mitochondria,
selectively preventing mitochondrial oxidative damage. To test
whether MitoQ was active in vivo, we chose a clinically relevant
form of mitochondrial oxidative damage: cardiac ischemia-reperfusion
injury. Feeding MitoQ to rats significantly decreased heart
dysfunction, cell death, and mitochondrial damage after
ischemia-reperfusion. This protection was due to the antioxidant
activity of MitoQ within mitochondria, as an untargeted antioxidant
was ineffective and accumulation of the TPP cation alone gave no
protection. Therefore, targeting antioxidants to mitochondria in vivo
is a promising new therapeutic strategy in the wide range of human
diseases such as Parkinson's disease, diabetes, and Friedreich's
ataxia where mitochondrial oxidative damage underlies the pathology.

J Biol Chem. 2005 Jun 3;280(22):21295-312. Epub 2005 Mar 23.
Interactions of mitochondria-targeted and untargeted ubiquinones
with the mitochondrial respiratory chain and reactive oxygen
species. Implications for the use of exogenous ubiquinones as
therapies and experimental tools.
    Antioxidants, such as ubiquinones, are widely used in
mitochondrial studies as both potential therapies and useful
research tools. However, the effects of exogenous ubiquinones can
be difficult to interpret because they can also be pro-oxidants or
electron carriers that facilitate respiration. Recently we
developed a mitochondria-targeted ubiquinone (MitoQ10) that
accumulates within mitochondria. MitoQ10 has been used to prevent
mitochondrial oxidative damage and to infer the involvement of
mitochondrial reactive oxygen species in signaling pathways.
However, uncertainties remain about the mitochondrial reduction of
MitoQ10, its oxidation by the respiratory chain, and its pro-oxidant
potential. Therefore, we compared MitoQ analogs of varying alkyl
chain lengths (MitoQn, n = 3-15) with untargeted exogenous
ubiquinones. We found that MitoQ10 could not restore respiration in
ubiquinone-deficient mitochondria because oxidation of MitoQ analogs
by complex III was minimal. Complex II and glycerol 3-phosphate
dehydrogenase reduced MitoQ analogs, and the rate depended on chain
length. Because of its rapid reduction and negligible oxidation,
MitoQ10 is a more effective antioxidant against lipid peroxidation,
peroxynitrite and superoxide. Paradoxically, exogenous ubiquinols
also autoxidize to generate superoxide, but this requires their
deprotonation in the aqueous phase. Consequently, in the presence of
phospholipid bilayers, the rate of autoxidation is proportional to
ubiquinol hydrophilicity. Superoxide production by MitoQ10 was
insufficient to damage aconitase but did lead to hydrogen peroxide
production and nitric oxide consumption, both of which may affect
cell signaling pathways. Our results comprehensively describe the
interaction of exogenous ubiquinones with mitochondria and have
implications for their rational design and use as therapies and as
research tools to probe mitochondrial function.


Am J Physiol Cell Physiol. 2005 Jun;288(6):C1440-50.
Epub 2005 Jan 12
Inhibition of complex I of the electron transport chain causes
O2-. -mediated mitochondrial outgrowth.
    Recent evidence indicates that oxidative stress is central to
the pathogenesis of a wide variety of degenerative diseases, aging,
and cancer. Oxidative stress occurs when the delicate balance
between production and detoxification of reactive oxygen species
is disturbed. Mammalian cells respond to this condition in several
ways, among which is a change in mitochondrial morphology. In the
present study, we have used rotenone, an inhibitor of complex I of
the respiratory chain, which is thought to increase mitochondrial
O(2)(-)* production, and mitoquinone (MitoQ), a
mitochondria-targeted antioxidant, to investigate the relationship
between mitochondrial O(2)(-)* production and morphology in human
skin fibroblasts. Video-rate confocal microscopy of cells pulse
loaded with the mitochondria-specific cation rhodamine 123,
followed by automated analysis of mitochondrial morphology,
revealed that chronic rotenone treatment (100 nM, 72 h)
significantly increased mitochondrial length and branching without
changing the number of mitochondria per cell. In addition, this
treatment caused a twofold increase in lipid peroxidation as
determined with C11-BODIPY(581/591). Finally, digital imaging
microscopy of cells loaded with hydroethidine, which is oxidized by
O(2)(-)* to yield fluorescent ethidium, revealed that chronic
rotenone treatment caused a twofold increase in the rate of O(2)(-)*
production. MitoQ (10 nM, 72 h) did not interfere with
rotenone-induced ethidium formation but abolished rotenone-induced
outgrowth and lipid peroxidation. These findings show that increased
mitochondrial O(2)(-)* production as a consequence of, for instance,
complex I inhibition leads to mitochondrial outgrowth and that MitoQ
acts downstream of this O(2)(-)* to prevent alterations in
mitochondrial morphology.

FEBS Lett. 2005 May 9;579(12):2669-74. Epub 2005 Apr 14.
A targeted antioxidant reveals the importance of mitochondrial
reactive oxygen species in the hypoxic signaling of HIF-1alpha.
    Exposure to limiting oxygen in cells and tissues induce the
stabilization and transcriptional activation of the
hypoxia-inducible factor 1 alpha (HIF-1alpha) protein, a key
regulator of the hypoxic response. Reactive oxygen species (ROS)
generation has been implicated in the stabilization of HIF-1alpha
during this response, but this is still a matter of some debate.
In this study we utilize a mitochondria-targeted antioxidant,
mitoubiquinone (MitoQ), and examine its effects on the hypoxic
stabilization of HIF-1alpha. Our results show that under conditions
of reduced oxygen (3% O(2)), MitoQ ablated the hypoxic induction of
ROS generation and destabilized HIF-1alpha protein. This in turn
led to an abrogation of HIF-1 transcriptional activity. Normoxic
stabilization of HIF-1alpha, on the other hand, was unchanged in
the presence of MitoQ suggesting that ROS were not involved. This
study strongly suggests that mitochondrial ROS contribute to the
hypoxic stabilization of HIF-1alpha.

Free Radic Biol Med. 2005 Mar 1;38(5):644-54.
Mitochondrial redox state regulates transcription of the
nuclear-encoded mitochondrial protein manganese superoxide dismutase:
a proposed adaptive response to mitochondrial redox imbalance.
    Overexpression of human manganese superoxide dismutase (MnSOD)
in mouse NIH/3T3 cells using an inducible retroviral system led to
alterations in the mitochondrial redox state since levels of reactive
oxygen species rapidly increased after induction of human MnSOD
(Antioxid. Redox Signal.6:489-500; 2004). Alterations in exogenous
human MnSOD led to large increases in levels of endogenous mouse
MnSOD (sod2) and thioredoxin 2 (txn2) mRNAs, but smaller increases in
MnSOD and thioredoxin 2 protein expression. Tight regulation of
mitochondrial protein levels seems to be necessary for optimal
cellular function, since mitochondrial antioxidant protein levels did
not increase to the same extent as antioxidant protein mRNA levels.
We hypothesize that these changes in antioxidant proteins are
adaptations to the altered mitochondrial redox state elicited by
MnSOD overexpression. The mitochondrial-specific antioxidant MitoQ
reversed cell growth inhibition, and greatly decreased levels of
endogenous sod2 and txn2 transcripts following induction of exogenous
MnSOD. Elevated levels of mouse sod2 transcripts resulted from
transcriptional activation of the endogenous sod2 gene since
actinomycin D prevented transcription of this gene. Therefore, the
mitochondrial redox state appears to modulate a nuclear-driven
biochemical event, i.e., transcriptional activation of a nuclear gene
encoding a protein targeted to mitochondria.

J Biol Chem. 2004 Sep 3;279(36):37575-87. Epub 2004 Jun 25.
Supplementation of endothelial cells with mitochondria-targeted
antioxidants inhibit peroxide-induced mitochondrial iron uptake,
oxidative damage, and apoptosis.
    The mitochondria-targeted drugs mitoquinone (Mito-Q) and
mitovitamin E (MitoVit-E) are a new class of antioxidants containing
the triphenylphosphonium cation moiety that facilitates drug
accumulation in mitochondria. In this study, Mito-Q (ubiquinone
attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E
attached to a triphenylphosphonium cation) were used. The aim of
this study was to test the hypothesis that mitochondria-targeted
antioxidants inhibit peroxide-induced oxidative stress and apoptosis
in bovine aortic endothelial cells (BAEC) through enhanced scavenging
of mitochondrial reactive oxygen species, thereby blocking reactive
oxygen species-induced transferrin receptor (TfR)-mediated iron
uptake into mitochondria. Glucose/glucose oxidase-induced oxidative
stress in BAECs was monitored by oxidation of
dichlorodihydrofluorescein that was catalyzed by both intracellular
H(2)O(2) and transferrin iron transported into cells. Pretreatment of
BAECs with Mito-Q (1 microM) and MitoVit-E (1 microM) but not
untargeted antioxidants (e.g. vitamin E) significantly abrogated
H(2)O(2)- and lipid peroxide-induced 2',7'-dichlorofluorescein
fluorescence and protein oxidation. Mitochondria-targeted
antioxidants inhibit cytochrome c release, caspase-3 activation, and
DNA fragmentation. Mito-Q and MitoVit-E inhibited H(2)O(2)- and lipid
peroxide-induced inactivation of complex I and aconitase, TfR
overexpression, and mitochondrial uptake of (55)Fe, while restoring
the mitochondrial membrane potential and proteasomal activity. We
conclude that Mito-Q or MitoVit-E supplementation of endothelial
cells mitigates peroxide-mediated oxidant stress and maintains
proteasomal function, resulting in the overall inhibition of
TfR-dependent iron uptake and apoptosis.

FEBS Lett. 2004 Jul 30;571(1-3):9-16.
Fine-tuning the hydrophobicity of a mitochondria-targeted
antioxidant.
    The mitochondria-targeted antioxidant MitoQ comprises a
ubiquinol moiety covalently attached through an aliphatic carbon
chain to the lipophilic triphenylphosphonium cation. This cation
drives the membrane potential-dependent accumulation of MitoQ into
mitochondria, enabling the ubiquinol antioxidant to prevent
mitochondrial oxidative damage far more effectively than
untargeted antioxidants. We sought to fine-tune the hydrophobicity
of MitoQ so as to control the extent of its membrane binding and
penetration into the phospholipid bilayer, and thereby regulate
its partitioning between the membrane and aqueous phases within
mitochondria and cells. To do this, MitoQ variants with 3, 5, 10
and 15 carbon aliphatic chains were synthesised. These molecules
had a wide range of hydrophobicities with octan-1-ol/phosphate
buffered saline partition coefficients from 2.8 to 20000. All
MitoQ variants were accumulated into mitochondria driven by the
membrane potential, but their binding to phospholipid bilayers
varied from negligible for MitoQ3 to essentially total for MitoQ15.
Despite the span of hydrophobicites, all MitoQ variants were
effective antioxidants. Therefore, it is possible to fine-tune the
degree of membrane association of MitoQ and other mitochondria
targeted compounds, without losing antioxidant efficacy. This
indicates how the uptake and distribution of mitochondria-targeted
compounds within mitochondria and cells can be controlled, thereby
facilitating investigations of mitochondrial oxidative damage.

FASEB J. 2003 Oct;17(13):1972-4. Epub 2003 Aug 15.
Mitochondria-targeted antioxidants protect Friedreich Ataxia
fibroblasts from endogenous oxidative stress more effectively
than untargeted antioxidants.
    Friedreich Ataxia (FRDA), the most common inherited ataxia,
arises from defective expression of the mitochondrial protein
frataxin, which leads to increased mitochondrial oxidative damage.
Therefore, antioxidants targeted to mitochondria should be
particularly effective at slowing disease progression. To test
this hypothesis, we compared the efficacy of mitochondria-targeted
and untargeted antioxidants derived from coenzyme Q10 and from
vitamin E at preventing cell death due to endogenous oxidative
stress in cultured fibroblasts from FRDA patients in which
glutathione synthesis was blocked. The mitochondria-targeted
antioxidant MitoQ was several hundredfold more potent than the
untargeted analog idebenone. The mitochondria-targeted antioxidant
MitoVit E was 350-fold more potent than the water soluble analog
Trolox. This is the first demonstration that mitochondria-targeted
antioxidants prevent cell death that arises in response to
endogenous oxidative damage. Targeted antioxidants may have
therapeutic potential in FRDA and in other disorders involving
mitochondrial oxidative damage.


Aging Cell. 2003 Apr;2(2):141-3.
MitoQ counteracts telomere shortening and elongates lifespan of
fibroblasts under mild oxidative stress.

J Biol Chem. 2001 Feb 16;276(7):4588-96. Epub 2000 Nov 22.
Selective targeting of a redox-active ubiquinone to mitochondria
within cells: antioxidant and antiapoptotic properties.
    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-alpha. 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-alpha. 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.

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