Báo cáo khoa học: Is there more to aging than mitochondrial DNA and reactive oxygen species? pot

REVIEW ARTICLE

Is there more to aging than mitochondrial DNA and
reactive oxygen species?
Mikhail F. Alexeyev1,2
1 Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL, USA
2 Institute of Molecular Biology and Genetics, Kyiv, Ukraine

Keywords
antioxidants; lifespan extension;
mitochondria; mitochondrial DNA
degradation; mitochondrial DNA mutations;
mitochondrial DNA repair; mitochondrial
theory of aging; oxidative damage
Correspondence
M. Alexeyev, University of South Alabama,
Department Cell Biology and Neuroscience,
307 University Blvd., MSB1201, Mobile, AL
36688, USA
Fax: +1 251 460 6771
Tel: +1 251 460 6789
E-mail:

With the aging of the population, we are seeing a global increase in the
prevalence of age-related disorders, especially in developed countries.
Chronic diseases disproportionately affect the older segment of the population, contributing to disability, a diminished quality of life and an increase
in healthcare costs. Increased life expectancy reflects the success of contemporary medicine, which must now respond to the challenges created by this
achievement, including the growing burden of chronic illnesses, injuries and
disabilities. A well-developed theoretical framework is required to understand the molecular basis of aging. Such a framework is a prerequisite for
the development of clinical interventions that will constitute an efficient
response to the challenge of age-related health issues. This review critically

analyzes the experimental evidence that supports and refutes the Free Radical ⁄ Mitochondrial Theory of Aging, which has dominated the field of
aging research for almost half a century.

(Received 12 July 2009, revised 3 August
2009, accepted 11 August 2009)
doi:10.1111/j.1742-4658.2009.07269.x

Introduction
Aging is a multifactorial phenomenon characterized by
a time-dependent decline in physiological function [1].
This decline is believed to be associated with an accumulation of defects in metabolic pathways. More than
50 years ago, Harman first proposed the Free Radical
Theory of Aging [2], which, over the years, has been
refined to include not only free radicals, but also other
reactive species such as hydrogen peroxide (H2O2) and
singlet oxygen. In 1972, Harman identified mitochondria as both the main source of reactive oxygen species
(ROS) and a major target for their damaging effects
[3]. This development has identified mitochondrion as

a biological clock, but because the mitochondrion has
a complex biochemical composition, a question about
the molecular identity of this clock remained open.
RNA, proteins and other cellular macromolecules with
relatively short half-lifes are poor candidates for the
progressive accumulation of damage over a lifetime, as
would be expected of such ‘tally keepers’. For this reason, even early studies on the molecular mechanisms
of aging have focused on DNA [4,5].
In mammalian cells, mitochondria are the only
organelles, besides the nucleus, that contain their own
genome, which led Miquel [6] to postulate that aging is

Abbreviations
BER, base excision repair; ESCODD, European Standards Committee on Oxidative DNA Damage; ETC, electron transport chain; GPx,
glutathione peroxidase; H2O2, hydrogen peroxide; mtDNA, mitochondrial DNA; MTA, mitochondrial theory of aging; nDNA, nuclear DNA;
8-oxodG, 7,8-dihydro-8-oxo-2¢-deoxyguanosine; Polg, DNA polymerase c; Prx, peroxiredoxin; RET, reverse electron transfer; ROS, reactive
oxygen species; Sod, superoxide dismutase.

5768

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS


M. F. Alexeyev

caused by accumulation of damage to the mitochondrial DNA (mtDNA). This narrowed the focus of
the theory and resulted in the Mitochondrial Theory
of Aging (MTA). Several lines of evidence indirectly
implicate mtDNA in longevity. The Framingham
Longevity Study of Coronary Heart Disease found that
longevity is more strongly associated with age of maternal death than with age of paternal death, suggesting
the cytosolic (mitochondrial) inheritance [7]. In addition, certain mtDNA polymorphisms have been associated with longevity. For example, male Italian
centenarians have an increased incidence of mtDNA
haplogroup J [8], while French centenarians have an
increased incidence of a G to A transition at mt9055
[9]. In a Japanese population, longevity was associated
with mtDNA haplogroups D4a, D4b2b and D5 [10,11].
However, a study of an Irish population failed to link
longevity to any particular mitochondrial haplotype,
indicating that factors other than mtDNA polymorphism also may play a role in aging [12]. Finally, Castri
et al. have found that while mtDNA variants can be

linked to both increased and decreased longevity, the
time period in which a person was born has a much
greater impact on longevity than the presence or
absence of a particular polymorphism [13].
Environmental genotoxins may facilitate preferential
mtDNA mutagenesis. Mitochondria accumulate high
levels of lipophilic carcinogens, such as polycyclic aromatic hydrocarbons [14,15]. When cells are exposed to
some of these compounds, mtDNA is damaged preferentially [16]. Other mutagenic chemicals have also been
shown to preferentially target mtDNA [15,17–21].
Therefore, it is conceivable that lifelong exposure to
certain environmental toxins could result in a preferential accumulation of mtDNA damage, leading to
aging. However, aging can occur in the absence of
detectable exposure to environmental toxins, which
suggests that a role of these toxins in natural aging is
limited. At present, after many years of refinement,
there is no universally accepted definition of the MTA.
Nonetheless, most investigators agree that it contains
the following components.
l
Mitochondria are a major source of ROS in the
cell.
l
Mitochondrially produced ROS inflict oxidative
damage on mtDNA.
l Oxidative mtDNA damage results in mutations that
lead to defective electron transport chain (ETC) components.
l
Incorporation of defective subunits into the ETC
causes a further increase in ROS production, leading
to a ‘vicious cycle’ of ROS production and mtDNA

mutations.

mtDNA + ROS = Aging?

l
mtDNA mutations, ROS production and cellular
damage by ROS eventually reach levels that are
incompatible with life.
Despite its intellectual appeal, the MTA was not
well received initially [22], but until recently it has
enjoyed almost universal acceptance. However, recent
years have seen an abundance of experimental evidence
that contradicts the MTA in its present form. This
article critically reviews the evidence in support of, and
against, the MTA, by addressing each of the components listed above, in turn.

Mitochondria are a major source of
ROS in the cell
The premise that mitochondria produce substantial
amounts of ROS appears to be valid and is rarely disputed. Some researchers in the field have taken this
argument further, however, claiming that mitochondria
are the primary source of ROS in cells. This is based,
at least in part, on early estimates of mitochondrial
production of H2O2 under nonphysiological conditions
[23]. It is important to note in this regard that cells
possess multiple enzyme systems capable of generating
ROS, and the relative contribution of each system,
which will probably depend on the cell type and physiological state, has not yet been determined. Therefore,
it is impossible to state, a priori, that mitochondria are
the main source of ROS in every cell type and under

all physiological conditions [24].
Mitochondria possess at least nine enzyme systems
that are capable of producing ROS under favorable
conditions [25]. However, in the context of aging, only
ROS production by ETC complexes I and III is usually
considered. This is mostly because early studies established that 1–2% of oxygen consumed by mitochondria
can be converted to H2O2. Considering the constitutive
nature of respiration, such a leak corresponds to a large
quantity of ROS, establishes mitochondrial ETC as a
major cellular source of ROS and establishes ROS as
compulsory by-products of respiration [23]. These findings, however, were subsequently challenged by Hansford et al. [26] who found that active H2O2 production,
which is an indirect measure of superoxide (OÀ ) gener2
ation, requires both a high fractional reduction of
complex I, as determined by the NADH ⁄ (NADH +
NAD+) ratio and a high membrane potential (DW).
The authors state that these conditions are achieved
only with supraphysiological concentrations of the
complex II substrate succinate. With physiological
concentrations of the NAD+-linked substrates that are
the main source of reduced equivalents for oxidative
phosphorylation, H2O2-formation rates are much

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5769


mtDNA + ROS = Aging?

M. F. Alexeyev

lower, at less than 0.1% of the respiratory chain electron flux. Staniek and Nohl [27,28] also reported that
when mitochondria use complex I and complex II substrates for respiration, detectable H2O2 is generated
only in the presence of the complex III inhibitor antimycin. They suggest that the rates of mitochondrial
H2O2 production reported by other studies were artificially high because of experimental design flaws, and
point out that because mitochondrial OÀ formation
2
under homeostatic conditions has not yet been demonstrated in situ, conclusions drawn from isolated mitochondria should not be overinterpreted [28].
St Pierre et al. capitalized on these findings and used
an improved experimental design to show that mitochondria do not release measurable amounts of OÀ or
2
H2O2 when respiring on complex I or complex II substrates, but release significant amounts of OÀ from
2
complex I when respiring on palmitoyl carnitine [29].
However, even at saturating concentrations of palmitoyl
carnitine, only 0.15% of the electron flow is estimated
to give rise to H2O2. These results were obtained under
resting conditions with a respiration rate of 200 nmol of
electrons per min, per mg of mitochondrial protein.
Under physiological conditions, the rate is predicted to
be even lower because the partial pressure of oxygen,
the concentration of palmitoyl carnitine and the
mitochondrial membrane potential are all lower. The
authors conclude [29] that under physiological conditions ROS are produced by ETC in quantities that can
be efficiently scavenged by mitochondrial antioxidant
systems. They proposed that as long as cells have normal levels of antioxidants, an electron leak from the
ETC should not result in significant oxidative damage
to mitochondrial components, including mtDNA. This
conclusion is consistent with observations from transgenic animal models showing that overexpression of
ROS-scavenging enzymes generally does not extend life

span and can even be detrimental (discussed later).
The highest production of ROS by mitochondria
in vitro was observed under conditions of reverse electron transfer (RET) from complex II through complex
I, towards NAD+. This flow is thermodynamically
unfavorable and must be coupled to the expenditure of
the energy of membrane potential. This energy is maximal when ADP supply is limited (state 4 respiration),
or when electron flow through complex III is blocked
by antimycin. Under these conditions, the dependence
of ROS production on the membrane potential is so
great that a 10% drop in membrane potential results in
a 90% reduction in ROS production ([30,31]; reviewed
in [25]). Although the feasibility of RET in vivo remains
to be fully elucidated, this possibility cannot be completely excluded [32,33]. Nonetheless, even if RET
5770

occurs physiologically, current evidence suggests that it
may occur only intermittently, under a narrow set of
conditions. While it is plausible that RET may generate
significant quantities of ROS in mitochondria under
certain circumstances, it is currently unclear whether or
not it can lead to a lifelong accumulation of mtDNA
mutations, as specified by the MTA.

Mitochondrially produced ROS inflict
oxidative damage on mtDNA
In vitro, DNA damage by ROS exposure is well documented [34–40], but in vivo, mitochondria possess multiple and redundant ROS scavenging systems. mtDNA
damage by ROS requires oxidative stress, an imbalance between ROS production and ROS neutralization. The mitochondrial pathways for ROS generation
and scavenging are briefly considered here.
Mitochondrial ROS generation
The proximal ROS generated by electron leak from

the ETC is OÀ (Fig. 1 and Eqn 1), which is charged,
2
comparatively unstable and has relatively low reactivity. The negative charge has been proposed to render
OÀ impermeable to membranes [41], and this hypothe2
sis is supported by results obtained from studies using
thylakoid and phospholipid liposome membranes
[42–44]. The permeability of the mitochondrial inner
membrane to OÀ is one of the factors that determines
2
the accessibility of the agent to mtDNA. Therefore,
$ 50% of OÀ generated at complex III has no access
2
to mtDNA, while all OÀ generated at complex I has
2
unimpeded access to it [41]. Although OÀ permeates
2
erythrocyte ghost membranes through an anion channel [45], no evidence exists for a similar channel in the
inner mitochondrial membrane, which is probably
impermeable to this species.

Fig. 1. A major pathway for the detoxification of ROS in the mitochondrial matrix. OÀ is formed by the reduction of O2 with elec2
trons leaked from the ETC. OÀ is efficiently converted to H2O2 by
2
mitochondrial superoxide dismutase (Sod2). H2O2 is then detoxified
to H2O either by mitochondrial glutathione peroxidase (GPx1) with
concomitant oxidation of glutathione (GSH), or by peroxiredoxins III
and V (PrxIII and PrxV). GSH, reduced glutathione; GSSG, oxidized
glutathione.

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

M. F. Alexeyev

In fact, however, the membrane permeability of OÀ
2
may be of little consequence because it is unable to
react directly with DNA [46–50]. Reaction of OÀ with
2
nonradicals is spin forbidden. In biological systems,
this means that the main reactions of OÀ are with
2
itself (dismutation) or with another biological radical,
such as nitric oxide.
One important feature of OÀ production by mito2
chondria is that it can be self-limiting through the
inactivation of mitochondrial aconitase. This inactivation can reduce NADH formation by the citric acid
cycle and, consequently, electron flow through the
ETC. The net effect would be a lowering of the
steady-state levels of reduction of complexes I and III,
which would diminish OÀ production [51,52].
2

mtDNA + ROS = Aging?

The family of mammalian Prxs has at least six members, of which PrxIII and PrxV are mitochondrial.
PrxIII is found only in mitochondria and is about
30-fold more abundant than GPx1 in HeLa cell
mitochondria [61]. PrxV is expressed as a long and
short forms, which are found in the mitochondrion

and in peroxisomes, respectively [62–64]. Catalase has
been reported in rat cardiac mitochondria [65], but this
was not confirmed in a follow-up study [66]. Therefore,
GPx1, and PrxIII and V are the main, and probably
only, contributors to H2O2 detoxication in the mitochondrial matrix (Fig. 1).
O2 ỵ e ! O
2
2O ỵ 2Hỵ ! H2 O2 ỵ O2
2

The O generated by the ETC is quickly converted to
2
H2O2 (Fig. 1 and Eqn 2), which is the principal cellular
mediator of oxidative stress. This conversion occurs
either spontaneously, with a second-order rate constant
of approximately 105 m)1s)1, or enzymatically, catalyzed by superoxide dismutases, with a first-order rate
constant of 109 m)1s)1 [53]. Mitochondria possess two
superoxide dismutases: Sod1 (Cu ⁄ ZnSod) in the intermembrane space; and Sod2 (MnSod) in the matrix.
Intriguingly, Sod1 appears to exist in an inactive,
reduced form that can be activated by ETC-generated
OÀ [54]. The relative stability and membrane perme2
ability of H2O2 allows it free access to mtDNA, yet, like
OÀ , it is also unable to react directly with DNA [46–
2
50]. However, in the presence of redox-active metal ions,
such as Fe2+, H2O2 can undergo Fenton chemistry
(Eqn 3), generating the extremely reactive hydroxyl radical •OH that efficiently damages DNA [34,35]. To prevent the potentially devastating consequences of the
Fenton reaction, H2O2 is detoxified in the mitochondrial
matrix by glutathione peroxidase 1 (GPx1; Fig. 1 and
Eqn 4) and peroxiredoxins III and V (PrxIII and PrxV;

Fig. 1 and Eqns 5 and 6, respectively; [55]). At least
seven GPx enzymes have been described to date in
mammalian cells [56], and two – GPx1 and GPx4
(PHGPx4) – are ubiquitously expressed [56–58]. GPx1 is
found in both the cytosol and the mitochondrial matrix,
and its preferred substrate is H2O2. GPx4 is most efficient at reducing lipid hydroperoxides. In addition to
direct inactivation of ROS, GPx enzymes indirectly protect the cell from damage by the OÀ , by preventing per2
oxide-mediated inactivation of Sod1 [59]. Interestingly,
Sod itself protects GPx from inactivation by OÀ [60].
2
Thus, Sod and GPx may participate in a crossprotection that prevents their inactivation by ROS.

2ị

Fe2ỵ ỵ H2 O2 ! Fe3ỵ ỵ  OH ỵ OH

3ị

H2 O2 þ 2GSH ! GS À SG þ 2H2 O

Mitochondrial ROS neutralization

1ị

4ị

H2 O2 ỵ 2 Pr xIII(SH)2 ! 2H2 O ỵ Pr xIII(SH)
S S(SH) Pr xIII

5ị

H2 O2 ỵ Pr xV(SH)2 ! 2H2 O ỵ Pr xV(S S)

6ị

7,8-Dihydro-8-oxo-2Â-deoxyguanosine as a marker of
oxidative mtDNA damage
The main pyrimidine product of oxidative DNA base
damage is thymine glycol [67] and the main purine product is 7,8-dihydro-8-oxo-2¢-deoxyguanosine (8-oxodG)
[68–70]. The former has low mutagenicity, while the latter, upon replication, can cause characteristic G:T transversions at a relatively low frequency [71]. Initial studies
revealed that mtDNA accumulates approximately 15
times more 8-oxodG than nuclear DNA (nDNA), thus
establishing extensive mtDNA damage by ROS under
physiological conditions [72]. These studies also suggested potential causes for the increased sensitivity of
mtDNA to oxidative stress, which include the proximity
to the source of ROS, the lack of protective histones
and relatively inefficient mtDNA repair. Each of these
causes is examined in more detail below.
Proximity of mtDNA to the ETC and steady-state
oxidative damage
The hypothesis that mtDNA is at greater risk to oxidative damage than nDNA because it is close to the source
of ROS was logical, especially when early reports suggested that mtDNA contained higher levels of oxidative

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5771


mtDNA + ROS = Aging?

M. F. Alexeyev

lesions than nDNA [72]. However, revision of the initial
data no longer supports this conclusion [73–75]. In any
case, oxidative damage resulting from proximity to the
ETC is only possible if protection by antioxidant
defenses and DNA repair are inadequate.
Lack of histones in mitochondria and susceptibility of
mtDNA to oxidative stress
Histone proteins are reported to protect DNA from a
variety of potentially dangerous reactive species, such as
•
OH [76–78]. Mitochondria lack histones, and this is
cited as a possible reason for the higher susceptibility of
mtDNA to ROS damage. However, nucleosome packaging does not protect DNA from the damage caused
by charge transfer through base pair stacks [37,79].
Electron transfer occurs easily from histones to DNA,
leading to DNA damage [80]. In addition, damage
induced by Cu2+ ⁄ H2O2 is enhanced in nucleosomal
DNA compared with naked DNA [37], and some DNA–
peptide interactions can increase metal ⁄ H2O2-induced
DNA breakage [81]. Therefore, histones are protective
under some, but not all, conditions. In addition, a
recent study demonstrated that protein components of
mitochondrial nucleoids show the same protection as
histones, under conditions in which histones protect
against oxidative stress [82]. This is in agreement with
a report that mitochondrial transcription factor A
(a DNA-binding protein and a major component of
mitochondrial nucleoids) is present in mitochondria in

quantities sufficient to completely cover mtDNA [83].
Repair of oxidative base lesions in mitochondria
The discovery that mitochondria are unable to repair
UV-induced pyrimidine dimers [84,85] and some types
of alkylating damage [18], demonstrated that they contain a reduced complement of DNA-repair pathways.
However, Anderson and Friedberg [86] found uracilDNA glycosylase activity in mitochondrial extracts,
suggesting at least the presence of the base excision
repair (BER) pathway. This was confirmed by mitochondrial repair of O6-ethyl-2¢-deoxyguanosine, which
is also processed by BER in the nucleus [87,88]. Subsequently, repair of a variety of mtDNA lesions, including those arising from oxidative damage, was
demonstrated [89–98]. Recently, long-patch BER of
oxidative DNA lesions [99–101], and mismatch repair
[102], have been discovered in mammalian mitochondria, so to date, no specific defect in the mitochondrial,
as compared to nuclear, repair of oxidative damage
has been reported. BER, with its single-nucleotide and
long-patch subpathways, is the main pathway for
5772

repairing oxidative base lesions in both the nucleus
and mitochondria, and 8-oxodG, the most prominent
oxidative base lesion, is repaired more efficiently in
mitochondria than in the nucleus [103].
Accumulation of oxidative damage in mtDNA
compared with nDNA
The report that mtDNA has a greater 8-oxodG content
than nDNA was quickly followed by the report of an
age-dependent increase of this lesion in cellular DNA
[104]. However, a decade later, the same group reduced
the estimates of cellular 8-oxodG by an order of magnitude, after finding that the isolation procedure used in
earlier studies resulted in the artificial oxidation of
DNA [105]. Nevertheless, the steady-state level of 8-oxodG in the DNA of old rats was almost three times

higher than that of young animals [105], and 8-oxodG
became widely accepted as a marker of oxidative DNA
damage. Reported values for the baseline 8-oxodG content of mtDNA span almost five orders of magnitude,
however, and the lowest reported values are not significantly different from those reported for nDNA [106]. A
series of carefully designed studies established that the
endogenous oxidative damage of mtDNA is not greater
than that of nDNA [73–75], and one study showed that
some oxidative lesions (including 8-hydroxyguanine,
Fapy-adenine, 8-hydroxyadenine, 5,6-dihydroxyuracil,
5-hydroxyuracil, 5-hydroxycytosine and 5-hydroxymethyluracil) are found less often in mtDNA [73].
Yakes and Van Houten [107] reported that the
mtDNA of mouse embryonic fibroblasts exposed to
H2O2 had more polymerase-blocking lesions than
nDNA. These lesions are predominantly strand breaks
that are generated, either directly or indirectly, through
the action of mitochondrial apurinic and apyrimidinic
endonuclease at abasic sites, or through the action of
bifunctional glycosylases on oxidatively damaged
DNA bases. In any case, this apparent increase in the
susceptibility of mtDNA to oxidative damage may in
fact be part of a mitochondria-specific mechanism that
protects mtDNA integrity through the degradation of
severely damaged mtDNA molecules (discussed later).

Oxidative mtDNA damage results in
mutations that lead to defective ETC
components
The mitochondrial genome accumulates mutations
approximately one order of magnitude faster than
nDNA [108–110]. This could be caused by a variety of

factors, including an intrinsically lower fidelity of replication by mitochondria-specific DNA polymerase c

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS


M. F. Alexeyev

(Polg), a lower efficiency of mtDNA repair, or chronic
exposure of mtDNA to noxious factors, such as ROS.
In reality, explanations other than ROS exposure and
the limited repertoire of mtDNA repair pathways are
rarely considered. As described above, the BER pathway that repairs oxidative DNA lesions in the nucleus
is present in mitochondria, and at least one oxidative
DNA lesion – 8-oxodG – is repaired more efficiently
in mitochondria than in the nucleus. In addition, the
exact in vivo rate of ROS production by mitochondria
is unknown, which complicates the evaluation of their
contribution to mtDNA mutagenesis.
Even with these uncertainties, a simple assumption
is that the accumulation of mutations in mtDNA
should be directly proportional to the rate of ROS
production, and inversely dependent on the level of antioxidants and the efficiency of mtDNA repair. That
said, it is important to note that mtDNA mutagenesis
is a stochastic process, and as long as ROS are produced, there is a finite probability of ROS-mediated
mtDNA mutagenesis. To make the MTA plausible,
mutagenesis has to occur at a certain threshold rate,
but the question is how much ROS imbalance, defined
as a prevalence of ROS production over the combined
defenses of antioxidants and mtDNA repair, is
required to sustain this rate. A second, equally important, question is whether this level of ROS imbalance

is physiologically attainable. To our knowledge, these
questions have not yet been addressed. In the absence
of direct information on whether in vivo attainable
levels of ROS production and oxidative stress could
theoretically be the cause of the mtDNA mutationmediated aging, we will next consider existing indirect
evidence from mtDNA damage and repair systems.
8-oxodG as a major source of mtDNA mutations
DNA oxidation mainly results in the base lesions thymine glycol and 8-oxodG [67–70]. The former has low
mutagenicity, but the latter can result in G:T transversions because unrepaired 8-oxodG can pair with either
C or A with almost equal efficiency. Based on the
MTA, one might expect that G:T transversions would
account for a significant fraction of pathogenic
mtDNA mutations. However, when we analyzed 188
pathogenic mtDNA point mutations [111], we found
that even though 8-oxodG is widely regarded as the
prime lesion that results from oxidative insult to DNA,
G:T transversions accounted for only 5.9% of the
mutations. Even taking into account the potentially
mutagenic 8-oxo deoxyguanosine triphosphate (8-oxodGTP), which results from oxidation of the cytosolic
and matrix dGTP pools and causes T:G transversions,

mtDNA + ROS = Aging?

the cumulative impact of both types of mutations was
still only 8.5% [112]. For comparison, 82% (or almost
10 times as many) pathogenic mtDNA point mutations
were consistent with deamination of adenine and cytosine. The unexpectedly low number of mutations that
potentially resulted from 8-oxodG could be explained
by efficient mitochondrial BER of 8-oxodG [103].
These results argue against 8-oxodG as the prime mutagenic lesion, so the key factors responsible for the accumulation of point mutations in mtDNA in response to

oxidative stress remain to be defined. Oxidative DNA
damage can produce a range of base lesions whose
mutagenic potential has not been fully elucidated [113],
and one or few of these may be responsible for the bulk
of ROS-mediated mtDNA mutagenesis. Alternatively,
the paucity of experimental data on the relationship
between oxidative stress and mtDNA mutagenesis
leaves open the possibility that factors other than oxidative stress are primarily responsible for the accumulation of mutations in mtDNA.
A unique mitochondrial mechanism for
maintaining mtDNA integrity
Unlike the nuclear genome, the mitochondrial genome
is redundant, consisting of hundreds to thousands
copies per somatic cell. Therefore, a ‘repair or die’
constraint is not imposed on mtDNA. A cell can lose a
substantial fraction of its mtDNA molecules without
detriment. The lost mtDNA molecules can then be
replenished by replication. Furthermore, because replication of mtDNA is not linked to the cell cycle, it can
occur throughout it [114]. Rat mtDNA turns over continuously in vivo, with a half-life of 9.4–31 days, depending on the organ [115]. Cells can survive both a gradual
loss of mtDNA through chronic treatment with ethidium bromide [116], or the acute destruction of a fraction
[117] or even loss of all of their mtDNA [118] by mitochondrially targeted restriction endonucleases. Therefore, an early hypothesis for how cells cope with the
inability of mitochondria to repair UV-induced damage
was that mitochondria do not repair DNA and damaged
mtDNA is simply turned over [84,85]. However, the lack
of experimental support for this hypothesis, and the
discovery of mitochondrial repair of oxidative and alkylating DNA damage [92,98,119], which contradicts the
notion mandatory degradation of damaged mtDNA,
prevented the model of mtDNA turnover as a mechanism for protecting the integrity of the mitochondrial
genome from becoming established. Subsequent
evidence has caused renewed interest in this model.
Ethanol has been reported to induce mtDNA loss in

yeast [120]. This observation was followed by studies

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5773


mtDNA + ROS = Aging?

M. F. Alexeyev

revealing that the intragastric administration of ethanol to mice induced oxidative stress which was accompanied by a reversible loss of mtDNA [121]. The loss
of mtDNA was approximately 50% in all organs studied. It could be partially prevented by the antioxidants
melatonin, vitamin E and coenzymeQ, and was followed by adaptive mtDNA resynthesis [122]. Lipopolysaccharide, a known inducer of in vivo oxidative stress,
also caused mtDNA depletion [123]. Angiotensin II
induced mitochondrial ROS production and decreased
skeletal muscle mtDNA content in mice [124]. Finally,
H2O2-induced oxidative stress in hamster fibroblasts
was accompanied by Ca2+-dependent degradation of
mtDNA [125]. Taken together, these findings establish
a link between oxidative stress and mtDNA degradation, yet they do not address the possibility of a
relationship between mtDNA degradation and the
maintenance of mtDNA integrity.
Rotenone inhibits the ETC complex I, resulting in
the release of OÀ on the matrix side of the mito2
chondrial inner membrane [29,41]. However, exposing
human colon carcinoma cells or mouse embryonic fibroblasts to sublethal concentrations of rotenone for
30 days did not result in a significant increase in the
rate of mtDNA mutagenesis [126]. Similarly, repeated
treatment of HCT116 colon cancer cells with H2O2

failed to induce significant mtDNA mutagenesis.
Instead, H2O2 treatment induced alkali-labile lesions
(predominantly DNA-strand breaks, as well as abasic
sites and other lesions that are converted to strand
breaks under alkaline conditions). Alkali-labile lesions
were generated at a rate at least 10 times higher
than the rate at which mutagenic bases were produced. Consistent with the notion that irreparable
mtDNA molecules are degraded, the inhibition of
BER by BER inhibitor methoxyamine, enhanced
mtDNA degradation in response to both oxidative
and alkylating damage [126]. The elimination of
damaged mtDNA was preceded by the accumulation
of linear mtDNA molecules, which may represent
degradation intermediates, because, unlike undamaged circular molecules, they are susceptible to exonucleolytic degradation.
The high rate of alkali-labile lesions in mtDNA
induced by ROS suggests a mechanism by which
mitochondria may maintain integrity of their genetic
information (Fig. 2). In this model, the oxidative
stress-mediated generation of a single, mutagenic lesion
in mtDNA, is accompanied by the generation of as
many as 10 strand breaks, which leads to degradation
of the entire molecule. Components of the mitochondrial BER pathway, such as lesion-specific DNA
glycosylases and apurinic and apyrimidinic endonucle5774

Damage

mtDNA
AP site
APE

R
e
p
a
i
r

Base damage
GlycosylaseII
or
Glycosylase I + APE

Single-strand
breaks

Double-strand
breaks

Degradation
Fig. 2. Potential interactions between mtDNA repair and degradation pathways. ROS induce both single-strand and double-strand
breaks in mtDNA, as well as abasic (AP) sites and base damage.
Both base damage and AP sites are converted to single-strand
breaks, which in turn are either repaired by BER, or converted to
double-strand breaks. Formation of double-strand breaks is a commitment step leading to degradation. Glycosylase I and glycosylase
II are monofunctional and bifunctional DNA glycosylases. A bifunctional DNA glycosylase also possesses AP-lyase activity (which
makes an incision at an abasic site). AP site, abasic site; APE,
apurinic ⁄ apyrimidinic endonuclease APE ⁄ Ref1; SSB and DSB,
single-strand break and double-strand break, respectively.

ase, may aid in the generation of abasic sites and single-strand breaks. This model provides a mechanistic

explanation for the observations made by Suter and
Richter [127], who found that the 8-oxodG content of
circular mtDNA is low and does not increase in
response to oxidative insult. However, fragmented
mtDNA had a very high 8-oxodG content, which
increased further after oxidative stress. The model is
consistent with the observations of Yakes and van
Houten [107], who found that oxidative stress promoted
a higher incidence of polymerase-blocking strand
breaks and abasic sites in mtDNA than in nDNA. Ikeda and Ozaki [128] found that mitochondrial endonuclease G is more active on oxidatively modified DNA
in vitro than on undamaged DNA, identifying a candidate enzyme that may be involved in the degradation
of oxidatively damaged mtDNA. Finally, the mechanism of strand breaks in mtDNA after oxidative stress,
as a means of protecting the integrity of the genetic
information, concurs with evolutionary theory. It suggests that, in combination with the high redundancy of

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS


M. F. Alexeyev

mtDNA, this unique mechanism may have evolved in
response to the exposure of mtDNA to the elevated
levels of ROS.

The ‘vicious cycle’
Polgexo)/) mice and the existence of the ‘vicious
cycle’
The main premise of the ‘vicious cycle’ hypothesis is
the existence of a feed-forward cycle of ROS production and mtDNA mutations. This notion appears to
have some footing in observations made with pathogenic mtDNA mutations. Thus, an increased oxidative

burden, presumably caused by the ETC defect, was
demonstrated in cells harboring some of these mutations [129–136]. Three caveats, however, suggest caution in extending these observations to aging. First, not
all diseases caused by mtDNA mutations are associated
with increased oxidative stress, so only some mtDNA
mutations induce increased ROS production. Second,
there is no evidence of increased rates of mtDNA
mutagenesis or accelerated aging in patients with mitochondrial disease, even when the disease is associated
with increased ROS production. Third, pathogenic
mtDNA mutations usually have a ‘threshold’ level of
mutant mtDNA, below which no diseased phenotype is
observed [137]. This threshold is variable, but is usually
quite high, around 70–90% [138]. In practical terms,
this means that a substantial fraction of the copies of a
particular gene must be mutated before a diseased phenotype is manifested. The threshold phenomenon can
be mediated, at least in part, by intramitochondrial and
intermitochondrial complementation [139–141]. However, the combined mtDNA mutation load in aged
human tissues is usually less than one mutation per
mitochondrial genome [126,142]. Taken together with
the random nature of aging-associated mtDNA mutations, these observations suggest that the observed
burden of scattered mutations, or even mutations in a
particular gene, some of which will be synonymous or
functionally neutral, is probably too low to cause a
noticeable increase in ROS production in aged tissues.
The phenotype of Polgexo) ⁄ ) mice appears to support
this conclusion. These mice accumulate elevated levels
of mtDNA mutations and, in accordance with the
MTA, exhibit accelerated aging [143,144]. However,
these mice do not support the ‘vicious cycle’ hypothesis,
because aging in this model is not accompanied by
increased ROS production, even though mitochondrial

function is severely impaired and the mutational burden is at least 10 times higher than that observed in
normal aging [143,145].

mtDNA + ROS = Aging?

ROS production by isolated mitochondria and
the ‘vicious cycle’ hypothesis
Measurements of ROS production by mitochondria
isolated from young and old human subjects have been
used to test the ‘vicious cycle’ hypothesis. Increased
ROS production by mitochondria from old tissue
would support the existence of the cycle, and some
studies have indeed found increased ROS production
by mitochondria in aged tissues [146–149], while others
did not. Rasmussen et al. [150,151] assayed 13 different
enzyme activities using optimized preparation techniques, and found that the central bioenergetic systems,
including pyruvate dehydrogenase, the tricarboxylic
acid cycle, the ETC and ATP synthesis, appeared unaltered with age. Maklashina and Ackrell [152], critically
examined the literature on the role of ETC dysfunction
in aging and concluded that the experimental evidence
in support of the model of age-related inactivation of
the respiratory chain can be challenged based on the
impurity of the mitochondrial preparations and the
inadequacy of assay procedures in the published
reports. In these author’s opinion, the experimental
evidence does not, in fact, support the MTA [152].
Another uncertainty in the interpretation of studies
with isolated mitochondria is whether these experiments can faithfully reproduce in vivo conditions. At
least some tissues have distinct mitochondrial subpopulations, such as the subsarcolemmal and interfibrillar
mitochondria in skeletal muscle, and the aging process

may differentially affect our ability to isolate these
subpopulations. This may, in turn, lead to differences
in observed ROS levels without actual, in vivo, changes
in ROS production. The mechanical stability of mitochondria from aged tissues may also be altered, leading to increased damage of these mitochondria during
isolation [153]. Finally, even when increased mitochondrial ROS production in older tissues can be demonstrated, it is unclear whether this increase is caused by
increased mutational burden in mtDNA, which would
be expected under the ‘vicious cycle’ hypothesis.
mtDNA content of 8-oxodG in young and old
tissue and the ‘vicious cycle’ hypothesis
The simplest oxidative DNA lesion to detect is
8-oxodG, so it is widely used as a marker of oxidative
stress. An increased 8-oxodG content in the mtDNA
from older subjects might provide evidence for
increased mitochondrial ROS production with aging,
validating the ‘vicious cycle’ hypothesis, assuming that
antioxidant defenses and 8-oxodG repair do not
decrease with age and that output of ROS from other

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5775


mtDNA + ROS = Aging?

M. F. Alexeyev

sources does not increase over time. Decreased antioxidant defenses or 8-oxodG repair, or increased ROS
production by non-ETC sources, could all account
for increased 8-oxodG content in the mtDNA of older

subjects, independently of the status of mitochondrial
ROS. Therefore, although many studies have reported
an increased 8-oxodG content in the mtDNA from
older subjects [154–159], the results cannot be interpreted as supporting the MTA, because these assumptions were not validated. Moreover, some investigations
did not detect an increase in the 8-oxodG content in the
mtDNA of older subjects [73], or even in aged Ogg1) ⁄ )
Csb) ⁄ ) knockout mice deficient in 8-oxodG repair [160].
The latter study illustrates the need for caution in the
interpretation of 8-oxodG measurements because it
found no increase in the 8-oxodG content of mtDNA in
wild-type mice compared with Ogg1) ⁄ )Csb) ⁄ ) knockout mice, contradicting a previous report that found an
approximately 20-fold increase in the 8-oxodG content
of the mtDNA of OGG) ⁄ ) mice [161].

Evidence from animal models
The predictions of the MTA have been extensively
tested in both vertebrate and invertebrate animal models. These studies were reviewed in depth by van Remmen et al. [162,163], who concluded that ‘the majority
of the initial pioneering studies in mice to test the mitochondrial theory of aging have yielded results that
either do not support the theory or remain inconclusive. An exception is a single study involving the overexpression of catalase in mitochondria’ [162]. As the
reviews cited above provide a comprehensive analysis
of both vertebrate and invertebrate studies, we consider
here only arguments not raised previously and studies
published too recently to be covered by these reviews.
mitoCAT mice and the MTA
A study on catalase overexpression in mouse mitochondria is cited as the only one which appears to support
the MTA [162]. In this work, the human catalase gene
with 11 amino acid C-terminal truncation was targeted
to the mitochondria of transgenic animals [164]. Two
founder lines were established, 4033 and 4403. The
expression of the transgene was mosaic, with hearts

showing the highest level of expression, but with only 10
to 50% of cardiac myocytes positive for catalase expression by immunocytochemistry analysis. Moreover, in
the founder 4403, only the heart, out of five organs
tested (brain, liver, kidney, heart and skeletal muscle),
showed increased catalase activity in the mitochondria.
Even then, the specific activity of catalase in the hearts
5776

of 4403 mice was approximately 10 times lower than in
the hearts of another founder line, 4033. Despite this
difference, there were similar median lifetime extensions
of 17% and 21% for the founder lines 4403 and 4033,
respectively. These observations call for caution in the
interpretation of a link between catalase overexpression
and lifetime extension in this study. Addressing the following additional questions may clarify whether there is
an actual causal relationship.
First, does catalase activity, especially in the founder
4403, substantially contribute to H2O2 metabolism in
mitochondria? Catalase has a low affinity for H2O2
(Km > 30 mm [165,166]) and can be inhibited (reversibly and irreversibly) by this substrate [167]. By contrast, GPx1 and PrxIII and V, which normally
detoxify H2O2 in mitochondria, have about 1 000-fold
lower Km values [63,168,169] and therefore are better
suited for the elimination of low (physiological) concentrations of H2O2. Clearly, analysis of the relative
contribution of each H2O2 scavenging system, similar
to that performed by Antunes et al. [170], would have
been extremely helpful. In that study, the authors conclude that the relative contributions of GPx1 and catalase to H2O2 detoxification are determined, among
other factors, by their relative abundance, and that
catalase does not contribute significantly to H2O2
detoxification in mitochondria under their experimental conditions. However, this situation can change
upon overexpression of catalase [170]. Unfortunately,

the study of Antunes et al. did not take into account
the contributions of PrxIII and PrxV, one of which
(PrxIII) is 30 times more abundant than GPx1 in the
mitochondria of HeLa cells [61]. It is of note that the
overexpression of GPx1, which is better suited for the
detoxification of low levels of H2O2, not only failed to
extend the life span in mice [171,172], but resulted in
the development of insulin resistance and obesity [171],
metabolic problems often linked to aging [173,174].
Another issue that should be resolved is whether
properties of catalase, other than peroxisomal targeting, were affected by the C-terminal truncation. Catalase is a bifunctional enzyme, exhibiting both
peroxidatic and catalatic activities [170]. Neither the
effect of truncation or addition of the mitochondrial
targeting sequence on the kinetic properties of the
enzyme, nor the ratio of peroxidatic to catalatic activity in the truncated enzyme, were reported.
Finally, the possibility that life span extension of
transgenic animals was mediated by the oxidation of
low-molecular-weight substrates in the mitochondrial
matrix by the peroxidatic activity of catalase, rather
than by reduction of the steady-state H2O2 level was
not addressed in this study.

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS


M. F. Alexeyev

Catalase overexpression can be detrimental, according to some indications. Overexpression of catalase in
the mitochondrial or cytosolic compartments increases
the sensitivity of HepG2 cells to tumor necrosis factora-induced apoptosis [175]. The mosaic pattern of transgene expression in this study might be the result of

selection against the detrimental effects of catalase
overexpression. Overall, a causal link between
increased H2O2 neutralization and life span extension,
as reported by the authors, is intriguing, but requires
some additional experimental evidence.
Apoptosis and premature aging in mice
As described earlier, accelerated aging in Polgexo) ⁄ )
knock-in mice is not accompanied by increased ROS
production [143,145]. In explanation, an increased sensitivity to apoptosis because of an increased mtDNA
mutation burden was proposed to be responsible for
accelerated aging [143]. This notion, however, is contradicted by observations made in two long-living
mouse models – aMUPA and Ames dwarf mice – which
also show increased apoptosis [176,177]. Moreover, life
span extension in GPx4+ ⁄ ) mice is associated with
increased susceptibility to apoptosis [178]. Also, it has
been suggested that in the Polgexo) ⁄ ) mice the lack of
evidence of oxidative damage to cellular components,
including mtDNA, could be caused by the loss of cells,
containing such damage, by apoptosis. This hypothesis
may provide a plausible mechanistic explanation for
some aging-related phenomena, such as sarcopenia
[179,180], but it necessarily assumes that any increase in
oxidative damage to cellular components triggers apoptosis (otherwise, intermediate levels of oxidative damage
would persist and therefore would be measurable). This
assumption contradicts our current knowledge of the
effects of oxidative stress in cellular systems and therefore is unlikely to be valid. Moreover, alternative mechanisms for sarcopenia were proposed (e.g. through the
reduction in both estrogen and androgen production
[181], impaired glucose and ⁄ or fatty acid metabolism,
nitrogen imbalance, decreased muscle protein synthesis
and reduced physical activity [182]). Therefore, the link

between apoptosis and aging, whether normal or in
Polgexo) ⁄ ) mice, remains unclear.
Evidence against the MTA from invertebrates
Genetic studies in invertebrates have provided a significant body of evidence that is inconsistent with predictions of the MTA. Research in Drosophila showed that
overexpression of antioxidant enzymes does not necessarily extend life span [183], and can even be detrimen-

mtDNA + ROS = Aging?

tal [184]. Other studies have shown that the beneficial
effect of antioxidant enzyme expression on life span is
restricted to short-lived strains [185]. Recently, Yang
et al. [186] reported the effects of knockdown of sod-1
and sod-2, which encode superoxide dismutases, in
long-lived mutants of C. elegans. Disruption of sod-1
or sod-2 expression failed to shorten the life span,
although it produced numerous phenotypes, including
increased sensitivity to paraquat and increased oxidative damage to proteins in wild-type worms, but not in
long-lived daf-2 mutants. In fact, sod-1 knockdown
increased the life span of daf-2 mutants, and sod-2
knockdown extended the life span of clk-1 mutants.
The authors concluded that increased OÀ detoxifica2
tion and low oxidative damage are not crucial for the
longevity of the mutants examined, with the possible
exception of daf-2, where the results were inconclusive.
Similarly, Honda et al. [187] found that in the longlived daf-2 mutant, knockout of the genes for two
MnSod isoforms, sod-2 and sod-3, increased the sensitivity to oxidative stress, but did not shorten the life
span. Finally, Van Raamsdonk and Hekimi [188] examined the effect of eliminating each of five C. elegans
Sod isoforms, either individually or in groups of three,
which simultaneously eliminated either all cytosolic or
all mitochondrial isoforms of Sod. None of the deletion

mutants showed a decreased life span compared with
wild-type worms, despite a clear increase in sensitivity
to paraquat- or juglone-induced oxidative stress. Even
mutants lacking combinations of two or three sod genes
survived for at least as long as wild-type worms. Examination of gene expression in these mutants revealed
mild compensatory up-regulation of other sod genes.
Worms with mutation in sod-2 were found to be longlived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on
known pathways of life span extension revealed a clear
interaction with genes that affect mitochondrial function. For example, a sod-2 deletion markedly increased
the life span of clk-1 worms, while it clearly decreased
the life span of isp-1 worms. Sod2 is mitochondrially
localized, and sod-2 mutant worms exhibit phenotypes
that are characteristic of long-lived mitochondrial
mutants, including slow development, low brood size
and slow defecation. This suggests that deletion of
sod-2 extends life span through a similar mechanism, a
conclusion that is supported by the decreased oxygen
consumption seen in sod-2 mutant worms. Therefore, in
agreement with previous studies, this study also showed
that increased oxidative stress caused by deletion of
sod genes does not result in decreased life span in
C. elegans, and that the deletion of sod-2 extends worm
life span by altering mitochondrial function [188].

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5777


mtDNA + ROS = Aging?

M. F. Alexeyev

Effect of antioxidants on life span
One of the predictions made by the MTA is that reduction in oxidative stress should extend the life span, and
this prediction has been extensively tested in various
experimental systems. In general, both overexpression
of enzymatic antioxidants [162,163] and lifelong administration of nonenzymatic antioxidants [189–192] have
failed to provide consistent and reproducible life span
extension. Howes [193] reviewed the results of antioxidant studies conducted on more than 550 000 humans
and concluded that ‘not only have antioxidants failed
to stop disease and aging but also they may cause harm
and mortality, which precipitated the stoppage of several large studies’. Recently, however, new antioxidants
have claimed to break this trend [194,195]. ‘Skulachev
ions’ are of particular interest because of reports of
extraordinary effects, such as restoration of eyesight to
experimental animals [196], life span extension in various experimental systems [197] and the ability to cure a
spectrum of age-related maladies [198,199]. Also, unlike
previously tested antioxidants, they appear to exert
their effects by accumulating in mitochondria [200].
Still, the greatest challenge of similar studies so far has
been their reproducibility and therefore it would be
interesting to see how well these results can be recapitulated by other laboratories.

Limitations of experimental techniques
Assays for antioxidant and DNA-repair enzymes
Assays for the activity of antioxidant and DNA-repair
enzymes have been used to support the hypothesis of
increased oxidative stress in aging. However, reports
on age-related changes in the activity of these enzymes

are often contradictory. For example, both an increase
and a decrease in the activity of 8-oxoguanine DNA
glycosylase, which is responsible for the removal of
8-oxodG from mtDNA, were associated with aging in
the mitochondria [201,202]. Opposing trends in the
activity of Sod2 and GPx have also been reported with
aging [203–207].
Even more controversial is the interpretation of both
increases and decreases in enzyme activity, as support
for the MTA. Decreased activity of antioxidant and
DNA-repair enzymes in aged tissues is usually interpreted as causing increased oxidative stress. However,
increased activity of these enzymes is interpreted as an
adaptation to an age-related increase in oxidative
stress. This is usually stated, however, as being unable
to fully compensate for elevated stress, thus enabling
the ‘vicious cycle’. While both explanations are plausi5778

ble, the mere fact of interpreting the opposite trends in
favor of the MTA suggests that the usefulness of these
assays is limited to making inferences about specific
underlying mechanisms of oxidative stress. The existence of such stress, however, must be established
using independent techniques.
Measurement of protein nitration and protein
carbonyls
Protein oxidation may be one of the most physiologically important form of oxidative damage. Some
30–40% of proteins exhibit oxidative modification as a
part of normal aging [208]. The build up of oxidized
proteins in cells can lead to failures in protein maintenance, loss of protein ⁄ enzyme function and several deleterious alterations, such as protein fragmentation and
aggregation, which cause cellular toxicity [209–211].
An increase in the mitochondrial content of protein

oxidation products, such as nitrotyrosine and protein
carbonyls, is often used as a reporter for mitochondrial
oxidative stress. However, the steady-state mitochondrial content of modified proteins that is reported by
most assays is a product of two opposing processes:
protein oxidation and degradation of oxidized proteins
by the mitochondrial Lon protease. Therefore, it is
important to take into consideration that an increased
mitochondrial content of protein carbonyls and protein
nitration products does not necessarily reflect increased
oxidative stress, but can also be a result of decreased
turnover of damaged proteins [212]. Because cellular
levels of oxidized proteins are dependent upon so
many variables, mechanisms responsible for the accumulation of oxidatively modified proteins in one study
may be very different from those involved in another
study [209].
8-oxodG as a reliable marker of oxidative DNA
damage
Discrepancies in the reported baseline levels of 8-oxodG content have prompted the establishment of the
European Standards Committee on Oxidative DNA
Damage (ESCODD), whose 27 member laboratories
critically examined different approaches to measuring
products of DNA base oxidation, in particular 8-oxodG. Several techniques have been evaluated by
ESCODD, including HPLC with electrochemical detection (HPLC-ECD), gas chromatography followed by
mass spectrometry (GC-MS) and HPLC followed by
tandem mass spectrometry-mass spectrometry (HPLCMS ⁄ MS). Laboratories that employed HPLC-ECD
were able to detect induction of 8-oxodG with similar

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

M. F. Alexeyev

efficiencies and similar dose–response profiles, while
GC-MS and HPLC-MS ⁄ MS, which were employed by
three different laboratories, failed to detect a dose–
response. The median value for 8-oxodG in untreated
cells was determined to be 4.01 per 106 guanines. However, a difference of approximately 10-fold was
observed between the highest and lowest background
values of mtDNA damage, and in one instance, different laboratories observed different trends in 8-oxodG
content on the same set of DNA samples [213]. The
final ESCODD report [214] indicated that even when
using highly standardized DNA isolation and processing protocols, and the same DNA samples, the levels of
detected 8-oxodG can vary between different laboratories by as much as 6–10-fold. Also, a consistent difference of 5–12-fold was seen between the 8-oxodG levels
detected by enzymatic and HPLC-based methods,
either because of over-reporting by HPLC-based methods or under-reporting by enzymatic methods [214].
This high variability suggests that any quantitative data
on the 8-oxodG content in mtDNA should be interpreted with caution.

Concluding remarks
Six years ago Jacobs [215] pointed out that the ‘mitochondrial theory of aging has been neither proven nor
disproven. It has not been tested’. Today, the situation
is somewhat different. Two groups who attempted to
test this theory directly by generating knock-in mice
with an elevated rate of mtDNA mutagenesis reported
mixed results. The elevated rate of mtDNA mutagenesis indeed resulted in accelerated aging [143,144], yet
no evidence was seen for enhanced ROS production as
a result of increased levels of mtDNA mutations
[143,145].
Further evidence against the MTA was provided by
Vermulst et al. [216], using a novel random mutation

capture assay to quantify mutation burden in
Polgexo+ ⁄ + and Polgexo+ ⁄ ) mice. The authors reported
that although the mutation burden in young Polgexo+ ⁄ )
mice is approximately 30 times higher than in old
Polgexo+ ⁄ + littermates, the life spans of these two genotypes are not statistically different. This strongly argues
against a causal role for mtDNA mutations in natural
aging. The Free Radical Theory of Aging, which is a
more general version of the MTA [3], suggests that mitochondrion, rather than mtDNA, are both the principal
target of ROS and a ‘biological clock’. It allows for a
wide spectrum of both ROS sources and molecular targets of ROS, including mtDNA. While not supported
by direct evidence, it has not been refuted and awaits
substantial improvements in our understanding of, and

mtDNA + ROS = Aging?

ability to manipulate, mitochondria and mitochondrial
ROS in order to be tested directly. Some of the arguments in favor of the Free Radical Theory of Aging are
essentially the same as those for the MTA, so some of
the criticisms presented in this review are applicable to
both theories. In summary, most experimental evidence
to date does not support the MTA. However, it is very
likely that mitochondrial ROS and mitochondrial dysfunction, mediated by damage to various mitochondrial
components, including mtDNA, play an important, but
not necessarily causal, role in the aging process.

Acknowledgement
This work was supported by National Institutes of
Health (grant numbers PO1 HL06629907 and
R21RR02396101).

References
1 Mandavilli BS, Santos JH & Van Houten B (2002)
Mitochondrial DNA repair and aging. Mutat Res 509,
127–151.
2 Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J Gerontol 11, 298–300.
3 Harman D (1972) The biologic clock: the mitochondria? J Am Geriatr Soc 20, 145–147.
4 Price GB, Modak SP & Makinodan T (1971)
Age-associated changes in the DNA of mouse tissue.
Science 171, 917–920.
5 Wheeler KT & Lett JT (1974) On the possibility that
DNA repair is related to age in non-dividing cells. Proc
Natl Acad Sci USA 71, 1862–1865.
6 Miquel J, Economos AC, Fleming J & Johnson JE Jr
(1980) Mitochondrial role in cell aging. Exp Gerontol
15, 575–591.
7 Brand FN, Kiely DK, Kannel WB & Myers RH
(1992) Family patterns of coronary heart disease
mortality: the Framingham Longevity Study. J Clin
Epidemiol 45, 169–174.
8 De Benedictis G, Rose G, Carrieri G, De Luca M,
Falcone E, Passarino G, Bonafe M, Monti D, Baggio
G, Bertolini S et al. (1999) Mitochondrial DNA inherited variants are associated with successful aging and
longevity in humans. FASEB J 13, 1532–1536.
9 Ivanova R, Lepage V, Charron D & Schachter F
(1998) Mitochondrial genotype associated with French
Caucasian centenarians. Gerontology 44, 349.
10 Bilal E, Rabadan R, Alexe G, Fuku N, Ueno H,
Nishigaki Y, Fujita Y, Ito M, Arai Y, Hirose N et al.
(2008) Mitochondrial DNA haplogroup D4a is a marker
for extreme longevity in Japan. PLoS ONE 3, e2421.

11 Alexe G, Fuku N, Bilal E, Ueno H, Nishigaki Y,
Fujita Y, Ito M, Arai Y, Hirose N, Bhanot G et al.

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5779


mtDNA + ROS = Aging?

12

13

14

15

16

17

18

19

20

21

22

23

24

25

M. F. Alexeyev

(2007) Enrichment of longevity phenotype in mtDNA
haplogroups D4b2b, D4a, and D5 in the Japanese population. Hum Genet 121, 347–356.
Ross OA, McCormack R, Curran MD, Duguid RA,
Barnett YA, Rea IM & Middleton D (2001)
Mitochondrial DNA polymorphism: its role in longevity of the Irish population. Exp Gerontol 36,
1161–1178.
Castri L, Melendez-Obando M, Villegas-Palma R, Barrantes R, Raventos H, Pereira R, Luiselli D, Pettener
D & Madrigal L (2008) Mitochondrial Polymorphisms
Are Associated Both with Increased and Decreased
Longevity. Hum Hered 67, 147–153.
Allen JA & Coombs MM (1980) Covalent binding of
polycyclic aromatic compounds to mitochondrial and
nuclear DNA. Nature 287, 244–245.
Wunderlich V, Schutt M, Bottger M & Graffi A (1970)
Preferential alkylation of mitochondrial deoxyribonucleic acid by N-methyl-N-nitrosourea. Biochem J 118,
99–109.
Backer JM & Weinstein IB (1980) Mitochondrial DNA
is a major cellular target for a dihydrodiol-epoxide
derivative of benzo[a]pyrene. Science 209, 297–299.
Rossi SC, Gorman N & Wetterhahn KE (1988) Mitochondrial reduction of the carcinogen chromate: formation of chromium(V). Chem Res Toxicol 1, 101–107.

Miyaki M, Yatagai K & Ono T (1977) Strand breaks
of mammalian mitochondrial DNA induced by carcinogens. Chem Biol Interact 17, 321–329.
Neubert D, Hopfenmuller W & Fuchs G (1981) Manifestation of carcinogenesis as a stochastic process on
the basis of an altered mitochondrial genome. Arch
Toxicol 48, 89–125.
Niranjan BG, Bhat NK & Avadhani NG (1982) Preferential attack of mitochondrial DNA by aflatoxin B1
during hepatocarcinogenesis. Science 215, 73–75.
Tomasi A, Albano E, Banni S, Botti B, Corongiu F,
Dessi MA, Iannone A, Vannini V & Dianzani MU
(1987) Free-radical metabolism of carbon tetrachloride
in rat liver mitochondria. A study of the mechanism of
activation. Biochem J 246, 313–317.
Harman D & Harman H (2003) ‘‘I thought, thought,
thought for four months in vain and suddenly the idea
came’’ – an interview with Denham and Helen Harman. Interview by K. Kitani and G.O. Ivy. Biogerontology 4, 401–412.
Boveris A, Oshino N & Chance B (1972) The cellular
production of hydrogen peroxide. Biochem J 128, 617–
630.
Nohl H, Gille L & Staniek K (2005) Intracellular generation of reactive oxygen species by mitochondria.
Biochem Pharmacol 69, 719–723.
Andreyev AY, Kushnareva YE & Starkov AA (2005)
Mitochondrial metabolism of reactive oxygen species.
Biochemistry (Mosc) 70, 200–214.

5780

26 Hansford RG, Hogue BA & Mildaziene V (1997)
Dependence of H2O2 formation by rat heart mitochondria on substrate availability and donor age. J Bioenerg Biomembr 29, 89–95.
27 Staniek K & Nohl H (1999) H(2)O(2) detection from
intact mitochondria as a measure for one-electron

reduction of dioxygen requires a non-invasive assay
system. Biochim Biophys Acta 1413, 70–80.
28 Staniek K & Nohl H (2000) Are mitochondria a permanent source of reactive oxygen species? Biochim Biophys Acta 1460, 268–275.
29 St-Pierre J, Buckingham JA, Roebuck SJ & Brand MD
(2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain.
J Biol Chem 277, 44784–44790.
30 Starkov AA & Fiskum G (2003) Regulation of brain
mitochondrial H2O2 production by membrane potential and NAD(P)H redox state. J Neurochem 86, 1101–
1107.
31 Korshunov SS, Skulachev VP & Starkov AA (1997)
High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria.
FEBS Lett 416, 15–18.
32 Muller FL, Liu Y, Abdul-Ghani MA, Lustgarten MS,
Bhattacharya A, Jang YC & Van Remmen H (2008)
High rates of superoxide production in skeletal-muscle
mitochondria respiring on both complex I- and
complex II-linked substrates. Biochem J 409, 491–499.
33 Starkov AA (2008) The role of mitochondria in reactive oxygen species metabolism and signaling. Ann NY
Acad Sci 1147, 37–52.
34 Henle ES, Luo Y, Gassmann W & Linn S (1996) Oxidative damage to DNA constituents by iron-mediated
fenton reactions. The deoxyguanosine family. J Biol
Chem 271, 21177–21186.
35 Henle ES, Luo Y & Linn S (1996) Fe2+, Fe3+, and
oxygen react with DNA-derived radicals formed during
iron-mediated Fenton reactions. Biochemistry 35,
12212–12219.
36 Jornot L, Petersen H & Junod AF (1998) Hydrogen
peroxide-induced DNA damage is independent of
nuclear calcium but dependent on redox-active ions.
Biochem J 335, 85–94.

37 Liang Q & Dedon PC (2001) Cu(II) ⁄ H2O2-induced
DNA damage is enhanced by packaging of DNA as a
nucleosome. Chem Res Toxicol 14, 416–422.
38 Moraes EC, Keyse SM & Tyrrell RM (1990) Mutagenesis by hydrogen peroxide treatment of mammalian
cells: a molecular analysis. Carcinogenesis 11, 283–293.
39 Rodriguez H, Drouin R, Holmquist GP, O’Connor
TR, Boiteux S, Laval J, Doroshow JH & Akman SA
(1995) Mapping of copper ⁄ hydrogen peroxide-induced
DNA damage at nucleotide resolution in human
genomic DNA by ligation-mediated polymerase chain
reaction. J Biol Chem 270, 17633–17640.

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS


M. F. Alexeyev

40 Tkeshelashvili LK, McBride T, Spence K & Loeb LA
(1991) Mutation spectrum of copper-induced DNA
damage. J Biol Chem 266, 6401–6406.
41 Muller FL, Liu Y & Van Remmen H (2004) Complex III releases superoxide to both sides of the inner
mitochondrial membrane. J Biol Chem 279, 49064–
49073.
42 Takahashi MA & Asada K (1983) Superoxide anion
permeability of phospholipid membranes and chloroplast thylakoids. Arch Biochem Biophys 226, 558–566.
43 Frimer AA, Strul G, Buch J & Gottlieb HE (1996) Can
superoxide organic chemistry be observed within the
liposomal bilayer? Free Radic Biol Med 20, 843–852.
44 Mao GD & Poznansky MJ (1992) Electron spin resonance study on the permeability of superoxide radicals
in lipid bilayers and biological membranes. FEBS Lett

305, 233–236.
45 Lynch RE & Fridovich I (1978) Permeation of the
erythrocyte stroma by superoxide radical. J Biol Chem
253, 4697–4699.
46 Halliwell B & Aruoma OI (1991) DNA damage by
oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett 281, 9–19.
47 Lesko SA, Lorentzen RJ & Ts’o PO (1980) Role of
superoxide in deoxyribonucleic acid strand scission.
Biochemistry 19, 3023–3028.
48 Rowley DA & Halliwell B (1983) DNA damage by
superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin. Biochim Biophys Acta 761, 86–93.
49 Blakely WF, Fuciarelli AF, Wegher BJ & Dizdaroglu
M (1990) Hydrogen peroxide-induced base damage in
deoxyribonucleic acid. Radiat Res 121, 338–343.
50 Brawn K & Fridovich I (1981) DNA strand scission by
enzymically generated oxygen radicals. Arch Biochem
Biophys 206, 414–419.
51 Gardner PR (1997) Superoxide-driven aconitase FE-S
center cycling. Biosci Rep 17, 33–42.
52 Gardner PR & Fridovich I (1991) Superoxide sensitivity of the Escherichia coli aconitase. J Biol Chem 266,
19328–19333.
53 Liochev SI & Fridovich I (2007) The effects of superoxide dismutase on H2O2 formation. Free Radic Biol
Med 42, 1465–1469.
54 Inarrea P, Moini H, Han D, Rettori D, Aguilo I, Alava MA, Iturralde M & Cadenas E (2007) Mitochondrial
respiratory chain and thioredoxin reductase regulate
intermembrane Cu,Zn-superoxide dismutase activity:
implications for mitochondrial energy metabolism and
apoptosis. Biochem J 405, 173–179.
55 Rhee SG, Yang KS, Kang SW, Woo HA & Chang TS
(2005) Controlled elimination of intracellular H(2)O(2):

regulation of peroxiredoxin, catalase, and glutathione
peroxidase via post-translational modification. Antioxid
Redox Signal 7, 619–626.

mtDNA + ROS = Aging?

56 Margis R, Dunand C, Teixeira FK & Margis-Pinheiro
M (2008) Glutathione peroxidase family – an evolutionary overview. FEBS J 275, 3959–3970.
57 Knopp EA, Arndt TL, Eng KL, Caldwell M, LeBoeuf
RC, Deeb SS & O’Brien KD (1999) Murine phospholipid hydroperoxide glutathione peroxidase: cDNA
sequence, tissue expression, and mapping. Mamm
Genome 10, 601–605.
58 de Haan JB, Bladier C, Griffiths P, Kelner M, O’Shea
RD, Cheung NS, Bronson RT, Silvestro MJ, Wild S,
Zheng SS et al. (1998) Mice with a homozygous null
mutation for the most abundant glutathione peroxidase, Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen
peroxide. J Biol Chem 273, 22528–22536.
59 Hodgson EK & Fridovich I (1975) The interaction of
bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation.
Biochemistry 14, 5299–5303.
60 Blum J & Fridovich I (1985) Inactivation of glutathione peroxidase by superoxide radical. Arch Biochem
Biophys 240, 500–508.
61 Chang TS, Cho CS, Park S, Yu S, Kang SW & Rhee
SG (2004) Peroxiredoxin III, a mitochondrion-specific
peroxidase, regulates apoptotic signaling by mitochondria. J Biol Chem 279, 41975–41984.
62 Yamashita H, Avraham S, Jiang S, London R, Van
Veldhoven PP, Subramani S, Rogers RA & Avraham
H (1999) Characterization of human and murine
PMP20 peroxisomal proteins that exhibit antioxidant
activity in vitro. J Biol Chem 274, 29897–29904.

63 Seo MS, Kang SW, Kim K, Baines IC, Lee TH &
Rhee SG (2000) Identification of a new type of
mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. J Biol Chem
275, 20346–20354.
64 Knoops B, Clippe A, Bogard C, Arsalane K, Wattiez
R, Hermans C, Duconseille E, Falmagne P & Bernard
A (1999) Cloning and characterization of AOEB166, a
novel mammalian antioxidant enzyme of the peroxiredoxin family. J Biol Chem 274, 30451–30458.
65 Radi R, Turrens JF, Chang LY, Bush KM, Crapo JD
& Freeman BA (1991) Detection of catalase in rat
heart mitochondria. J Biol Chem 266, 22028–22034.
66 Zhou Z & Kang YJ (2000) Cellular and subcellular
localization of catalase in the heart of transgenic mice.
J Histochem Cytochem 48, 585–594.
67 Wang D, Kreutzer DA & Essigmann JM (1998) Mutagenicity and repair of oxidative DNA damage: insights
from studies using defined lesions. Mutat Res 400, 99–
115.
68 Bohr VA (2002) Repair of oxidative DNA damage in
nuclear and mitochondrial DNA, and some changes
with aging in mammalian cells. Free Radic Biol Med
32, 804–812.

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5781


mtDNA + ROS = Aging?

M. F. Alexeyev

69 Christmann M, Tomicic MT, Roos WP & Kaina B
(2003) Mechanisms of human DNA repair: an update.
Toxicology 193, 3–34.
70 De Bont R & van Larebeke N (2004) Endogenous
DNA damage in humans: a review of quantitative
data. Mutagenesis 19, 169–185.
71 Hanes JW, Thal DM & Johnson KA (2006) Incorporation and replication of 8-Oxo-deoxyguanosine by the
human mitochondrial DNA polymerase. J Biol Chem
281, 36241–36248.
72 Richter C, Park JW & Ames BN (1988) Normal
oxidative damage to mitochondrial and nuclear DNA
is extensive. Proc Natl Acad Sci USA 85, 6465–6467.
73 Anson RM, Senturker S, Dizdaroglu M & Bohr VA
(1999) Measurement of oxidatively induced base lesions
in liver from Wistar rats of different ages. Free Radic
Biol Med 27, 456–462.
74 Anson RM, Hudson E & Bohr VA (2000) Mitochondrial endogenous oxidative damage has been overestimated. FASEB J 14, 355–360.
75 Lim KS, Jeyaseelan K, Whiteman M, Jenner A &
Halliwell B (2005) Oxidative damage in mitochondrial
DNA is not extensive. Ann NY Acad Sci 1042, 210–220.
76 Enright HU, Miller WJ & Hebbel RP (1992) Nucleosomal histone protein protects DNA from iron-mediated
damage. Nucleic Acids Res 20, 3341–3346.
77 Ljungman M & Hanawalt PC (1992) Efficient protection against oxidative DNA damage in chromatin. Mol
Carcinog 5, 264–269.
78 Enright H, Miller WJ, Hays R, Floyd RA & Hebbel
RP (1996) Preferential targeting of oxidative base damage to internucleosomal DNA. Carcinogenesis 17,
1175–1177.
79 Nunez ME, Noyes KT & Barton JK (2002) Oxidative
charge transport through DNA in nucleosome core

particles. Chem Biol 9, 403–415.
80 Cullis PM, Jones GD, Symons MC & Lea JS (1987)
Electron transfer from protein to DNA in irradiated
chromatin. Nature 330, 773–774.
81 Liang R, Senturker S, Shi X, Bal W, Dizdaroglu M &
Kasprzak KS (1999) Effects of Ni(II) and Cu(II) on
DNA interaction with the N-terminal sequence of
human protamine P2: enhancement of binding and
mediation of oxidative DNA strand scission and base
damage. Carcinogenesis 20, 893–898.
82 Guliaeva NA, Kuznetsova EA & Gaziev AI (2006)
Proteins associated with mitochondrial DNA protect it
against the action of X-rays and hydrogen peroxide.
Biofizika 51, 692–697.
83 Alam TI, Kanki T, Muta T, Ukaji K, Abe Y, Nakayama H, Takio K, Hamasaki N & Kang D (2003)
Human mitochondrial DNA is packaged with TFAM.
Nucleic Acids Res 31, 1640–1645.
84 Clayton DA, Doda JN & Friedberg EC (1974) The
absence of a pyrimidine dimer repair mechanism in

5782

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

mammalian mitochondria. Proc Natl Acad Sci USA
71, 2777–2781.
Clayton DA, Doda JN & Friedberg EC (1975)
Absence of a pyrimidine dimer repair mechanism for
mitochondrial DNA in mouse and human cells. Basic
Life Sci 5B, 589–591.
Anderson CT & Friedberg EC (1980) The presence of
nuclear and mitochondrial uracil-DNA glycosylase in

extracts of human KB cells. Nucleic Acids Res 8, 875–
888.
Myers KA, Saffhill R & O’Connor PJ (1988) Repair of
alkylated purines in the hepatic DNA of mitochondria
and nuclei in the rat. Carcinogenesis 9, 285–292.
Satoh MS, Huh N, Rajewsky MF & Kuroki T (1988)
Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo. J Biol Chem 263, 6854–6856.
LeDoux SP & Wilson GL (2001) Base excision repair
of mitochondrial DNA damage in mammalian cells.
Prog Nucleic Acid Res Mol Biol 68, 273–284.
LeDoux SP, Driggers WJ, Hollensworth BS & Wilson
GL (1999) Repair of alkylation and oxidative damage
in mitochondrial DNA. Mutat Res 434, 149–159.
LeDoux SP, Patton NJ, Avery LJ & Wilson GL (1993)
Repair of N-methylpurines in the mitochondrial DNA
of xeroderma pigmentosum complementation group D
cells. Carcinogenesis 14, 913–917.
Driggers WJ, LeDoux SP & Wilson GL (1993) Repair
of oxidative damage within the mitochondrial DNA of
RINr 38 cells. J Biol Chem 268, 22042–22045.
Shen CC, Wertelecki W, Driggers WJ, LeDoux SP &
Wilson GL (1995) Repair of mitochondrial DNA damage induced by bleomycin in human cells. Mutat Res
337, 19–23.
Driggers WJ, Grishko VI, LeDoux SP & Wilson GL
(1996) Defective repair of oxidative damage in the
mitochondrial DNA of a xeroderma pigmentosum
group A cell line. Cancer Res 56, 1262–1266.
Druzhyna N, Nair RG, LeDoux SP & Wilson GL
(1998) Defective repair of oxidative damage in mitochondrial DNA in Down’s syndrome. Mutat Res 409,
81–89.

Grishko VI, Druzhyna N, LeDoux SP & Wilson GL
(1999) Nitric oxide-induced damage to mtDNA and its
subsequent repair. Nucleic Acids Res 27, 4510–4516.
LeDoux SP, Wilson GL, Beecham EJ, Stevnsner T,
Wassermann K & Bohr VA (1992) Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells. Carcinogenesis 13, 1967–1973.
Pettepher CC, LeDoux SP, Bohr VA & Wilson GL
(1991) Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the
nitrosourea streptozotocin. J Biol Chem 266, 3113–3117.
Akbari M, Visnes T, Krokan HE & Otterlei M (2008)
Mitochondrial base excision repair of uracil and AP

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS


M. F. Alexeyev

100

101

102

103

104

105

106
107

108

109

110
111

112

113

sites takes place by single-nucleotide insertion and
long-patch DNA synthesis. DNA repair 7, 605–616.
Liu P, Qian L, Sung JS, de Souza-Pinto NC, Zheng L,
Bogenhagen DF, Bohr VA, Wilson DM III, Shen B &
Demple B (2008) Removal of Oxidative DNA Damage
via FEN1-Dependent Long-Patch Base Excision
Repair in Human Cell Mitochondria. Mol Cell Biol 16,
4975–4987.
Szczesny B, Tann AW, Longley MJ, Copeland WC &
Mitra S (2008) Long patch base excision repair in
mammalian mitochondrial genomes. J Biol Chem 283,
26349–26356.
de Souza-Pinto NC, Mason PA, Hashiguchi K,
Weissman L, Tian J, Guay D, Lebel M, Stevnsner TV,
Rasmussen LJ & Bohr VA (2009) Novel DNA
mismatch-repair activity involving YB-1 in human
mitochondria. DNA repair 8, 704–719.
Thorslund T, Sunesen M, Bohr VA & Stevnsner T

(2002) Repair of 8-oxoG is slower in endogenous
nuclear genes than in mitochondrial DNA and is
without strand bias. DNA repair 1, 261–273.
Fraga CG, Shigenaga MK, Park JW, Degan P & Ames
BN (1990) Oxidative damage to DNA during aging:
8-hydroxy-2¢-deoxyguanosine in rat organ DNA and
urine. Proc Natl Acad Sci USA 87, 4533–4537.
Helbock HJ, Beckman KB, Shigenaga MK, Walter
PB, Woodall AA, Yeo HC & Ames BN (1998) DNA
oxidation matters: the HPLC-electrochemical detection
assay of 8-oxo-deoxyguanosine and 8-oxo-guanine.
Proc Natl Acad Sci USA 95, 288–293.
Beckman KB & Ames BN (1999) Endogenous oxidative damage of mtDNA. Mutat Res 424, 51–58.
Yakes FM & Van Houten B (1997) Mitochondrial
DNA damage is more extensive and persists longer
than nuclear DNA damage in human cells following
oxidative stress. Proc Natl Acad Sci USA 94, 514–519.
Brown WM, George M Jr & Wilson AC (1979) Rapid
evolution of animal mitochondrial DNA. Proc Natl
Acad Sci USA 76, 1967–1971.
Tatarenkov A & Avise JC (2007) Rapid concerted
evolution in animal mitochondrial DNA. Proc Biol Sci
274, 1795–1798.
Ballard JW & Whitlock MC (2004) The incomplete
natural history of mitochondria. Mol Ecol 13, 729–744.
Brandon MC, Lott MT, Nguyen KC, Spolim S, Navathe SB, Baldi P & Wallace DC (2005) MITOMAP: a
human mitochondrial genome database – 2004 update.
Nucleic Acids Res 33, D611–D613.
Alexeyev M, Ledoux SP & Wilson GL (2006) Mitochondrial DNA and aging. In Handbook of Models for
Human Aging (Conn PM ed), pp. 507–520. Academic

Press, Burlington, MA.
Evans MD, Dizdaroglu M & Cooke MS (2004) Oxidative DNA damage and disease: induction, repair and
significance. Mutat Res 567, 1–61.

mtDNA + ROS = Aging?

114 Clay Montier LL, Deng JJ & Bai Y (2009) Number
matters: control of mammalian mitochondrial DNA
copy number. J Genet Genomics 36, 125–131.
115 Gross NJ & Rabinowitz M (1969) Synthesis of new
strands of mitochondrial and nuclear deoxyribonucleic
acid by semiconservative replication. J Biol Chem 244,
1563–1566.
116 King MP & Attardi G (1989) Human cells lacking
mtDNA: repopulation with exogenous mitochondria
by complementation. Science 246, 500–503.
117 Alexeyev MF, Venediktova N, Pastukh V, Shokolenko
I, Bonilla G & Wilson GL (2008) Selective elimination
of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes.
Gene Ther 15, 516–523.
118 Kukat A, Kukat C, Brocher J, Schafer I, Krohne G,
Trounce IA, Villani G & Seibel P (2008) Generation of
{rho}0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses. Nucleic
Acids Res.
119 Driggers WJ, Holmquist GP, LeDoux SP & Wilson
GL (1997) Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress
in a region of rat mtDNA. Nucleic Acids Res 25, 4362–
4369.
120 Ibeas JI & Jimenez J (1997) Mitochondrial DNA loss
caused by ethanol in Saccharomyces flor yeasts. Appl

Environ Microbiol 63, 7–12.
121 Mansouri A, Gaou I, De Kerguenec C, Amsellem S,
Haouzi D, Berson A, Moreau A, Feldmann G, Letteron P, Pessayre D et al. (1999) An alcoholic binge
causes massive degradation of hepatic mitochondrial
DNA in mice. Gastroenterology 117, 181–190.
122 Mansouri A, Demeilliers C, Amsellem S, Pessayre D &
Fromenty B (2001) Acute ethanol administration oxidatively damages and depletes mitochondrial dna in
mouse liver, brain, heart, and skeletal muscles: protective effects of antioxidants. J Pharmacol Exp Ther 298,
737–743.
123 Suliman HB, Carraway MS & Piantadosi CA (2003)
Postlipopolysaccharide oxidative damage of mitochondrial DNA. Am J Respir Crit Care Med 167,
570–579.
124 Mitsuishi M, Miyashita K, Muraki A & Itoh H (2008)
Angiotensin II Reduces Mitochondrial Content in
Skeletal Muscle and Affects Glycemic Control. Diabetes 58, 710–717.
125 Crawford DR, Abramova NE & Davies KJ (1998)
Oxidative stress causes a general, calcium-dependent
degradation of mitochondrial polynucleotides. Free
Radic Biol Med 25, 1106–1111.
126 Shokolenko I, Venediktova N, Bochkareva A, Wilson
GL & Alexeyev MF (2009) Oxidative stress induces
degradation of mitochondrial DNA. Nucleic Acids Res
37, 2539–2548.

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5783


mtDNA + ROS = Aging?

M. F. Alexeyev

127 Suter M & Richter C (1999) Fragmented mitochondrial
DNA is the predominant carrier of oxidized DNA
bases. Biochemistry 38, 459–464.
128 Ikeda S & Ozaki K (1997) Action of mitochondrial
endonuclease G on DNA damaged by L-ascorbic acid,
peplomycin, and cis-diamminedichloroplatinum (II).
Biochem Biophys Res Commun 235, 291–294.
129 Geromel V, Kadhom N, Cebalos-Picot I, Ouari O,
Polidori A, Munnich A, Rotig A & Rustin P (2001)
Superoxide-induced massive apoptosis in cultured skin
fibroblasts harboring the neurogenic ataxia retinitis
pigmentosa (NARP) mutation in the ATPase-6 gene of
the mitochondrial DNA. Hum Mol Genet 10, 1221–
1228.
130 Ohkoshi N, Mizusawa H, Shiraiwa N, Shoji S, Harada
K & Yoshizawa K (1995) Superoxide dismutases of
muscle in mitochondrial encephalomyopathies. Muscle
Nerve 18, 1265–1271.
131 Filosto M, Tonin P, Vattemi G, Spagnolo M, Rizzuto
N & Tomelleri G (2002) Antioxidant agents have a different expression pattern in muscle fibers of patients
with mitochondrial diseases. Acta Neuropathol (Berl)
103, 215–220.
132 Lu CY, Wang EK, Lee HC, Tsay HJ & Wei YH
(2003) Increased expression of manganese-superoxide
dismutase in fibroblasts of patients with CPEO syndrome. Mol Genet Metab 80, 321–329.
133 Kunishige M, Mitsui T, Akaike M, Kawajiri M, Shono
M, Kawai H & Matsumoto T (2003) Overexpressions

of myoglobin and antioxidant enzymes in ragged-red
fibers of skeletal muscle from patients with mitochondrial encephalomyopathy. Muscle Nerve 28, 484–
492.
134 Geromel V, Rotig A, Munnich A & Rustin P (2002)
Coenzyme Q10 depletion is comparatively less detrimental to human cultured skin fibroblasts than respiratory chain complex deficiencies. Free Radic Res 36,
375–379.
135 Mattiazzi M, Vijayvergiya C, Gajewski CD, DeVivo
DC, Lenaz G, Wiedmann M & Manfredi G (2004) The
mtDNA T8993G (NARP) mutation results in an
impairment of oxidative phosphorylation that can be
improved by antioxidants. Hum Mol Genet 13, 869–
879.
136 Indo HP, Davidson M, Yen HC, Suenaga S, Tomita K,
Nishii T, Higuchi M, Koga Y, Ozawa T & Majima HJ
(2006) Evidence of ROS generation by mitochondria
in cells with impaired electron transport chain and
mitochondrial DNA damage. Mitochondrion 7, 106–118.
137 Rossignol R, Faustin B, Rocher C, Malgat M, Mazat
JP & Letellier T (2003) Mitochondrial threshold
effects. Biochem J 370, 751–762.
138 Sacconi S, Salviati L, Nishigaki Y, Walker WF, Hernandez-Rosa E, Trevisson E, Delplace S, Desnuelle C,
Shanske S, Hirano M et al. (2008) A Functionally

5784

139

140

141

142

143

144

145

146

147

148

149

150

151

Dominant Mitochondrial DNA Mutation. Hum Mol
Genet 17, 1814–1820.
Legros F, Malka F, Frachon P, Lombes A & Rojo M
(2004) Organization and dynamics of human mitochondrial DNA. J Cell Sci 117, 2653–2662.
Nakada K, Sato A & Hayashi JI (2009) Mitochondrial
functional complementation in mitochondrial DNAbased diseases. Int J Biochem Cell Biol 41, 1907–1913.
Ono T, Isobe K, Nakada K & Hayashi JI (2001)
Human cells are protected from mitochondrial dysfunction by complementation of DNA products in
fused mitochondria. Nat Genet 28, 272–275.

Khrapko K, Kraytsberg Y, de Grey AD, Vijg J &
Schon EA (2006) Does premature aging of the mtDNA
mutator mouse prove that mtDNA mutations are
involved in natural aging? Aging Cell 5, 279–282.
Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K,
Wohlgemuth SE, Hofer T, Seo AY, Sullivan R,
Jobling WA et al. (2005) Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian
aging. Science 309, 481–484.
Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink
JN, Rovio AT, Bruder CE, Bohlooly YM, Gidlof S,
Oldfors A, Wibom R et al. (2004) Premature ageing in
mice expressing defective mitochondrial DNA polymerase. Nature 429, 417–423.
Trifunovic A, Hansson A, Wredenberg A, Rovio AT,
Dufour E, Khvorostov I, Spelbrink JN, Wibom R,
Jacobs HT & Larsson NG (2005) Somatic mtDNA
mutations cause aging phenotypes without affecting
reactive oxygen species production. Proc Natl Acad Sci
USA 102, 17993–17998.
Sawada M & Carlson JC (1987) Changes in superoxide
radical and lipid peroxide formation in the brain, heart
and liver during the lifetime of the rat. Mech Ageing
Dev 41, 125–137.
Sohal RS & Sohal BH (1991) Hydrogen peroxide
release by mitochondria increases during aging. Mech
Ageing Dev 57, 187–202.
Sohal RS & Dubey A (1994) Mitochondrial oxidative
damage, hydrogen peroxide release, and aging. Free
Radic Biol Med 16, 621–626.
Capel F, Rimbert V, Lioger D, Diot A, Rousset P,
Mirand PP, Boirie Y, Morio B & Mosoni L (2005)

Due to reverse electron transfer, mitochondrial H2O2
release increases with age in human vastus lateralis
muscle although oxidative capacity is preserved. Mech
Ageing Dev 126, 505–511.
Rasmussen UF, Krustrup P, Kjaer M & Rasmussen
HN (2003) Human skeletal muscle mitochondrial
metabolism in youth and senescence: no signs of functional changes in ATP formation and mitochondrial
oxidative capacity. Pflugers Arch 446, 270–278.
Rasmussen UF, Krustrup P, Kjaer M & Rasmussen
HN (2003) Experimental evidence against the

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS


M. F. Alexeyev

152

153

154

155

156

157

158

159

160

161

162

mitochondrial theory of aging. A study of isolated
human skeletal muscle mitochondria. Exp Gerontol 38,
877–886.
Maklashina E & Ackrell BA (2004) Is defective
electron transport at the hub of aging? Aging Cell 3,
21–27.
Van Remmen H & Jones DP (2009) Current thoughts
on the role of mitochondria and free radicals in the
biology of aging. J Gerontol A Biol Sci Med Sci 64,
171–174.
Ames BN, Shigenaga MK & Hagen TM (1993) Oxidants, antioxidants, and the degenerative diseases of
aging. Proc Natl Acad Sci USA 90, 7915–7922.
Van Remmen H, Ikeno Y, Hamilton M, Pahlavani M,
Wolf N, Thorpe SR, Alderson NL, Baynes JW,
Epstein CJ, Huang TT et al. (2003) Life-long reduction
in MnSOD activity results in increased DNA damage
and higher incidence of cancer but does not accelerate
aging. Physiol Genomics 16, 29–37.
Van Remmen H, Qi W, Sabia M, Freeman G, Estlack
L, Yang H, Mao Guo Z, Huang TT, Strong R, Lee S
et al. (2004) Multiple deficiencies in antioxidant
enzymes in mice result in a compound increase in

sensitivity to oxidative stress. Free Radic Biol Med 36,
1625–1634.
de la Asuncion JG, Millan A, Pla R, Bruseghini L,
Esteras A, Pallardo FV, Sastre J & Vina J (1996)
Mitochondrial glutathione oxidation correlates with
age-associated oxidative damage to mitochondrial
DNA. FASEB J 10, 333–338.
Mecocci P, MacGarvey U, Kaufman AE, Koontz D,
Shoffner JM, Wallace DC & Beal MF (1993) Oxidative
damage to mitochondrial DNA shows marked agedependent increases in human brain. Ann Neurol 34,
609–616.
Pallardo FV, Asensi M, Garcia de la Asuncion J,
Anton V, Lloret A, Sastre J & Vina J (1998) Late
onset administration of oral antioxidants prevents agerelated loss of motor co-ordination and brain mitochondrial DNA damage. Free Radic Res 29, 617–623.
Trapp C, McCullough AK & Epe B (2007) The
basal levels of 8-oxoG and other oxidative modifications in intact mitochondrial DNA are low even in
repair-deficient (Ogg1() ⁄ )) ⁄ Csb() ⁄ ))) mice. Mutat
Res 625, 155–163.
de Souza-Pinto NC, Eide L, Hogue BA, Thybo T,
Stevnsner T, Seeberg E, Klungland A & Bohr VA
(2001) Repair of 8-oxodeoxyguanosine lesions in mitochondrial dna depends on the oxoguanine dna glycosylase (OGG1) gene and 8-oxoguanine accumulates in
the mitochondrial dna of OGG1-defective mice. Cancer
Res 61, 5378–5381.
Jang YC & Remmen VH (2009) The mitochondrial
theory of aging: insight from transgenic and knockout
mouse models. Exp Gerontol 44, 256–260.

mtDNA + ROS = Aging?

163 Muller FL, Lustgarten MS, Jang Y, Richardson A &

Van Remmen H (2007) Trends in oxidative aging theories. Free Radic Biol Med 43, 477–503.
164 Schriner SE, Linford NJ, Martin GM, Treuting P,
Ogburn CE, Emond M, Coskun PE, Ladiges W, Wolf
N, Van Remmen H et al. (2005) Extension of murine
life span by overexpression of catalase targeted to
mitochondria. Science 308, 1909–1911.
165 van Eyk AD, Litthauer D & Oelofsen W (1992) The
isolation and partial characterization of catalase and a
peroxidase active fraction from human white adipose
tissue. Int J Biochem 24, 1101–1109.
166 Ogura Y & Yamazaki I (1983) Steady-state kinetics of
the catalase reaction in the presence of cyanide. J Biochem 94, 403–408.
167 Lardinois OM, Mestdagh MM & Rouxhet PG (1996)
Reversible inhibition and irreversible inactivation of
catalase in presence of hydrogen peroxide. Biochim
Biophys Acta 1295, 222–238.
168 Chae HZ, Kim HJ, Kang SW & Rhee SG (1999) Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of
thioredoxin. Diabetes Res Clin Pract 45, 101–112.
169 Carmagnol F, Sinet PM & Jerome H (1983) Seleniumdependent and non-selenium-dependent glutathione
peroxidases in human tissue extracts. Biochim Biophys
Acta 759, 49–57.
170 Antunes F, Han D & Cadenas E (2002) Relative contributions of heart mitochondria glutathione peroxidase and catalase to H(2)O(2) detoxification in in vivo
conditions. Free Radic Biol Med 33, 1260–1267.
171 McClung JP, Roneker CA, Mu W, Lisk DJ, Langlais
P, Liu F & Lei XG (2004) Development of insulin
resistance and obesity in mice overexpressing cellular
glutathione peroxidase. Proc Natl Acad Sci USA 101,
8852–8857.
172 Ho YS, Magnenat JL, Bronson RT, Cao J, Gargano
M, Sugawara M & Funk CD (1997) Mice deficient in

cellular glutathione peroxidase develop normally and
show no increased sensitivity to hyperoxia. J Biol
Chem 272, 16644–16651.
173 Wheatcroft SB, Kearney MT, Shah AM, Ezzat VA,
Miell JR, Modo M, Williams SC, Cawthorn WP,
Medina-Gomez G, Vidal-Puig A et al. (2007) IGFbinding protein-2 protects against the development
of obesity and insulin resistance. Diabetes 56, 285–
294.
174 Mathews ST, Rakhade S, Zhou X, Parker GC, Coscina DV & Grunberger G (2006) Fetuin-null mice are
protected against obesity and insulin resistance associated with aging. Biochem Biophys Res Commun 350,
437–443.
175 Bai J & Cederbaum AI (2000) Overexpression of catalase in the mitochondrial or cytosolic compartment
increases sensitivity of HepG2 cells to tumor necrosis

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5785


mtDNA + ROS = Aging?

176

177

178

179

180

181

182

183

184

185

186

187

188

M. F. Alexeyev

factor-alpha-induced apoptosis. J Biol Chem 275,
19241–19249.
Tirosh O, Schwartz B, Zusman I, Kossoy G, Yahav S
& Miskin R (2004) Long-lived alpha MUPA transgenic
mice exhibit increased mitochondrion-mediated apoptotic capacity. Ann NY Acad Sci 1019, 439–442.
Kennedy MA, Rakoczy SG & Brown-Borg HM (2003)
Long-living Ames dwarf mouse hepatocytes readily
undergo apoptosis. Exp Gerontol 38, 997–1008.
Ran Q, Liang H, Ikeno Y, Qi W, Prolla TA, Roberts
LJ II, Wolf N, Van Remmen H & Richardson A
(2007) Reduction in glutathione peroxidase 4 increases

life span through increased sensitivity to apoptosis.
J Gerontol A Biol Sci Med Sci 62, 932–942.
Marzetti E, Anne Lees H, Eva Wohlgemuth S &
Leeuwenburgh C (2009) Sarcopenia of aging: underlying cellular mechanisms and protection by calorie
restriction. Biofactors 35, 28–35.
Barreiro E, Coronell C, Lavina B, Ramirez-Sarmiento A, Orozco-Levi M & Gea J (2006) Aging, sex
differences, and oxidative stress in human respiratory
and limb muscles. Free Radic Biol Med 41, 797–
809.
Kamel HK, Maas D & Duthie EH Jr (2002) Role of
hormones in the pathogenesis and management of
sarcopenia. Drugs Aging 19, 865–877.
Carmeli E, Coleman R & Reznick AZ (2002) The
biochemistry of aging muscle. Exp Gerontol 37,
477–489.
Seto NO, Hayashi S & Tener GM (1990) Overexpression of Cu-Zn superoxide dismutase in Drosophila
does not affect life-span. Proc Natl Acad Sci USA 87,
4270–4274.
Reveillaud I, Niedzwiecki A, Bensch KG & Fleming
JE (1991) Expression of bovine superoxide dismutase
in Drosophila melanogaster augments resistance of
oxidative stress. Mol Cell Biol 11, 632–640.
Orr WC, Mockett RJ, Benes JJ & Sohal RS (2003)
Effects of overexpression of copper-zinc and manganese superoxide dismutases, catalase, and thioredoxin
reductase genes on longevity in Drosophila melanogaster. J Biol Chem 278, 26418–26422.
Yang W, Li J & Hekimi S (2007) A Measurable
Increase in Oxidative Damage Due to Reduction in
Superoxide Detoxification Fails to Shorten the Life
Span of Long-Lived Mitochondrial Mutants of Caenorhabditis elegans. Genetics 177, 2063–2074.
Honda Y, Tanaka M & Honda S (2008) Modulation

of longevity and diapause by redox regulation mechanisms under the insulin-like signaling control in Caenorhabditis elegans. Exp Gerontol 43, 520–529.
Van Raamsdonk JM & Hekimi S (2009) Deletion of
the mitochondrial superoxide dismutase sod-2 extends
lifespan in Caenorhabditis elegans. PLoS Genet 5,
e1000361.

5786

189 Selman C, McLaren JS, Meyer C, Duncan JS, Redman
P, Collins AR, Duthie GG & Speakman JR (2006)
Life-long vitamin C supplementation in combination
with cold exposure does not affect oxidative damage or
lifespan in mice, but decreases expression of antioxidant protection genes. Mech Ageing Dev 127, 897–904.
190 Bayne AC & Sohal RS (2002) Effects of superoxide
dismutase ⁄ catalase mimetics on life span and oxidative
stress resistance in the housefly, Musca domestica. Free
Radic Biol Med 32, 1229–1234.
191 Bass TM, Weinkove D, Houthoofd K, Gems D &
Partridge L (2007) Effects of resveratrol on lifespan in
Drosophila melanogaster and Caenorhabditis elegans.
Mech Ageing Dev 128, 546–552.
192 Keaney M & Gems D (2003) No increase in lifespan in
Caenorhabditis elegans upon treatment with the superoxide dismutase mimetic EUK-8. Free Radic Biol Med
34, 277–282.
193 Howes RM (2006) The free radical fantasy: a panoply
of paradoxes. Ann NY Acad Sci 1067, 22–26.
194 Kim J, Takahashi M, Shimizu T, Shirasawa T, Kajita
M, Kanayama A & Miyamoto Y (2008) Effects of a
potent antioxidant, platinum nanoparticle, on the lifespan of Caenorhabditis elegans. Mech Ageing Dev 129,
322–331.

195 Skulachev VP, Anisimov VN, Antonenko YN,
Bakeeva LE, Chernyak BV, Erichev VP, Filenko OF,
Kalinina NI, Kapelko VI, Kolosova NG et al. (2008)
An attempt to prevent senescence: a mitochondrial
approach. Biochim Biophys Acta 1787, 437–461.
196 Neroev VV, Archipova MM, Bakeeva LE, Fursova A,
Grigorian EN, Grishanova AY, Iomdina EN, Ivashchenko Zh N, Katargina LA, Khoroshilova-Maslova
IP et al. (2008) Mitochondria-targeted plastoquinone
derivatives as tools to interrupt execution of the aging
program. 4. Age-related eye disease. SkQ1 returns
vision to blind animals. Biochemistry (Mosc) 73, 1317–
1328.
197 Anisimov VN, Bakeeva LE, Egormin PA, Filenko OF,
Isakova EF, Manskikh VN, Mikhelson VM, Panteleeva AA, Pasyukova EG, Pilipenko DI et al. (2008)
Mitochondria-targeted plastoquinone derivatives as
tools to interrupt execution of the aging program. 5.
SkQ1 prolongs lifespan and prevents development of
traits of senescence. Biochemistry (Mosc) 73, 1329–
1342.
198 Agapova LS, Chernyak BV, Domnina LV, Dugina
VB, Efimenko AY, Fetisova EK, Ivanova OY,
Kalinina NI, Khromova NV, Kopnin BP et al. (2008)
Mitochondria-targeted plastoquinone derivatives as
tools to interrupt execution of the aging program. 3.
Inhibitory effect of SkQ1 on tumor development from
p53-deficient cells. Biochemistry (Mosc) 73, 1300–1316.
199 Bakeeva LE, Barskov IV, Egorov MV, Isaev NK,
Kapelko VI, Kazachenko AV, Kirpatovsky VI,

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

M. F. Alexeyev

200

201

202

203

204

205

Kozlovsky SV, Lakomkin VL, Levina SB et al. (2008)
Mitochondria-targeted plastoquinone derivatives as
tools to interrupt execution of the aging program. 2.
Treatment of some ROS- and age-related diseases
(heart arrhythmia, heart infarctions, kidney ischemia,
and stroke). Biochemistry (Mosc) 73, 1288–1299.
Antonenko YN, Avetisyan AV, Bakeeva LE, Chernyak
BV, Chertkov VA, Domnina LV, Ivanova OY,
Izyumov DS, Khailova LS, Klishin SS et al. (2008)
Mitochondria-targeted plastoquinone derivatives as
tools to interrupt execution of the aging program. 1.
Cationic plastoquinone derivatives: synthesis and in
vitro studies. Biochemistry (Mosc) 73, 1273–1287.
Shen GP, Galick H, Inoue M & Wallace SS (2003)

Decline of nuclear and mitochondrial oxidative base
excision repair activity in late passage human diploid
fibroblasts. DNA repair 2, 673–693.
Ishchenko A, Sinitsyna O, Krysanova Z, Vasyunina
EA, Saparbaev M, Sidorkina O & Nevinsky GA
(2003) Age-dependent increase of 8-oxoguanine-, hypoxanthine-, and uracil- DNA glycosylase activities in
liver extracts from OXYS rats with inherited overgeneration of free radicals and Wistar rats. Med Sci Monit
9, BR16–BR24.
Mauriz JL, Molpeceres V, Garcia-Mediavilla MV,
Gonzalez P, Barrio JP & Gonzalez-Gallego J (2007)
Melatonin prevents oxidative stress and changes in
antioxidant enzyme expression and activity in the liver
of aging rats. J Pineal Res 42, 222–230.
Lu CY, Lee HC, Fahn HJ & Wei YH (1999) Oxidative damage elicited by imbalance of free radical
scavenging enzymes is associated with large-scale
mtDNA deletions in aging human skin. Mutat Res
423, 11–21.
Wei YH, Wu SB, Ma YS & Lee HC (2009) Respiratory function decline and DNA mutation in mitochondria, oxidative stress and altered gene expression
during aging. Chang Gung Med J 32, 113–132.

mtDNA + ROS = Aging?

206 Gianni P, Jan KJ, Douglas MJ, Stuart PM &
Tarnopolsky MA (2004) Oxidative stress and the
mitochondrial theory of aging in human skeletal
muscle. Exp Gerontol 39, 1391–1400.
207 Judge S, Jang YM, Smith A, Hagen T & Leeuwenburgh C (2005) Age-associated increases in oxidative
stress and antioxidant enzyme activities in cardiac interfibrillar mitochondria: implications for the mitochondrial theory of aging. FASEB J 19, 419–421.
208 Sohal RS (2002) Role of oxidative stress and protein
oxidation in the aging process. Free Radic Biol Med

33, 37–44.
209 Stadtman ER (2006) Protein oxidation and aging. Free
Radic Res 40, 1250–1258.
210 Friguet B (2006) Oxidized protein degradation and
repair in ageing and oxidative stress. FEBS Lett 580,
2910–2916.
211 Giulivi C & Davies KJ (2001) Mechanism of the formation and proteolytic release of H2O2-induced dityrosine and tyrosine oxidation products in hemoglobin
and red blood cells. J Biol Chem 276, 24129–24136.
212 Bota DA, Van Remmen H & Davies KJ (2002) Modulation of Lon protease activity and aconitase turnover
during aging and oxidative stress. FEBS Lett 532, 103–
106.
213 ESCODD (2003) Measurement of DNA oxidation in
human cells by chromatographic and enzymic methods.
Free Radic Biol Med 34, 1089–1099.
214 Gedik CM & Collins A (2005) Establishing the background level of base oxidation in human lymphocyte
DNA: results of an interlaboratory validation study.
FASEB J 19, 82–84.
215 Jacobs HT (2003) The mitochondrial theory of aging:
dead or alive? Aging Cell 2, 11–17.
216 Vermulst M, Bielas JH, Kujoth GC, Ladiges WC,
Rabinovitch PS, Prolla TA & Loeb LA (2007) Mitochondrial point mutations do not limit the natural
lifespan of mice. Nat Genet.

FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS

5787