REVIEW ARTICLE
Is there more to aging than mitochondrial DNA and
reactive oxygen species?
Mikhail F. Alexeyev
1,2
1 Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL, USA
2 Institute of Molecular Biology and Genetics, Kyiv, Ukraine
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 accu-
mulation 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 (H
2
O
2
) and
singlet oxygen. In 1972, Harman identified mitochon-
dria 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 rea-

stand 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 Radi-
cal ⁄ Mitochondrial Theory of Aging, which has dominated the field of
aging research for almost half a century.
Abbreviations
BER, base excision repair; ESCODD, European Standards Committee on Oxidative DNA Damage; ETC, electron transport chain; GPx,
glutathione peroxidase; H
2
O
2
, 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
caused by accumulation of damage to the mitochon-
drial 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 mater-
nal death than with age of paternal death, suggesting
the cytosolic (mitochondrial) inheritance [7]. In addi-
tion, certain mtDNA polymorphisms have been associ-
ated 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

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) com-
ponents.
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.
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 compo-
nents 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 dis-
puted. Some researchers in the field have taken this

compulsory by-products of respiration [23]. These find-
ings, however, were subsequently challenged by Hans-
ford et al. [26] who found that active H
2
O
2
production,
which is an indirect measure of superoxide (O
À
2
) gener-
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, H
2
O
2
-formation rates are much
M. F. Alexeyev mtDNA + ROS = Aging?
FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS 5769

2
O
2
when respiring on complex I or complex II sub-
strates, but release significant amounts of O
À
2
from
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 H
2
O
2
. 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 condi-
tions ROS are produced by ETC in quantities that can
be efficiently scavenged by mitochondrial antioxidant
systems. They proposed that as long as cells have nor-
mal 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 trans-

mented [34–40], but in vivo, mitochondria possess mul-
tiple and redundant ROS scavenging systems. mtDNA
damage by ROS requires oxidative stress, an imbal-
ance between ROS production and ROS neutraliza-
tion. 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 O
À
2
(Fig. 1 and Eqn 1), which is charged,
comparatively unstable and has relatively low reactiv-
ity. The negative charge has been proposed to render
O
À
2
impermeable to membranes [41], and this hypothe-
sis is supported by results obtained from studies using
thylakoid and phospholipid liposome membranes
[42–44]. The permeability of the mitochondrial inner
membrane to O
À
2
is one of the factors that determines
the accessibility of the agent to mtDNA. Therefore,
$ 50% of O
À
2
generated at complex III has no access

2
is then detoxified
to H
2
O 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.
mtDNA + ROS = Aging? M. F. Alexeyev
5770 FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS
In fact, however, the membrane permeability of O
À
2
may be of little consequence because it is unable to
react directly with DNA [46–50]. Reaction of O
À
2
with
nonradicals is spin forbidden. In biological systems,
this means that the main reactions of O
À
2
are with
itself (dismutation) or with another biological radical,
such as nitric oxide.
One important feature of O
À
2
production by mito-
chondria is that it can be self-limiting through the

9
m
)1
s
)1
[53]. Mitochondria possess two
superoxide dismutases: Sod1 (Cu ⁄ ZnSod) in the inter-
membrane space; and Sod2 (MnSod) in the matrix.
Intriguingly, Sod1 appears to exist in an inactive,
reduced form that can be activated by ETC-generated
O
À
2
[54]. The relative stability and membrane perme-
ability of H
2
O
2
allows it free access to mtDNA, yet, like
O
À
2
, it is also unable to react directly with DNA [46–
50]. However, in the presence of redox-active metal ions,
such as Fe
2+
,H
2
O
2

Sod itself protects GPx from inactivation by O
À
2
[60].
Thus, Sod and GPx may participate in a cross-
protection that prevents their inactivation by ROS.
The family of mammalian Prxs has at least six mem-
bers, 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 H
2
O
2
detoxification in the mito-
chondrial matrix (Fig. 1).
O
2
þ e
À
! O
À
2
ð1Þ

2
O ð4Þ
H
2
O
2
þ 2 Pr xIII(SH)
2
! 2H
2
O þ Pr xIII(SH)
ÀS À S(SH) Pr xIII
ð5Þ
H
2
O
2
þ Pr xV(SH)
2
! 2H
2
O þ Pr xV(S À S) ð6Þ
7,8-Dihydro-8-oxo-2¢-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 prod-
uct is 7,8-dihydro-8-oxo-2¢-deoxyguanosine (8-oxodG)
[68–70]. The former has low mutagenicity, while the lat-
ter, upon replication, can cause characteristic G:T trans-
versions at a relatively low frequency [71]. Initial studies

aging 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 Cu
2+
⁄ H
2
O
2
is enhanced in nucleosomal
DNA compared with naked DNA [37], and some DNA–
peptide interactions can increase metal ⁄ H
2
O
2
-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

tude, 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-ox-
odG 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 con-
tent of mtDNA span almost five orders of magnitude,
however, and the lowest reported values are not signifi-
cantly 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-hydroxym-
ethyluracil) are found less often in mtDNA [73].
Yakes and Van Houten [107] reported that the
mtDNA of mouse embryonic fibroblasts exposed to
H
2
O
2
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

tioxidants 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 pro-
duced, 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 impor-
tant, 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 mutation-
mediated 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 thy-
mine glycol and 8-oxodG [67–70]. The former has low
mutagenicity, but the latter can result in G:T transver-
sions 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,

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 repli-
cation of mtDNA is not linked to the cell cycle, it can
occur throughout it [114]. Rat mtDNA turns over con-
tinuously in vivo, with a half-life of 9.4–31 days, depend-
ing on the organ [115]. Cells can survive both a gradual
loss of mtDNA through chronic treatment with ethidi-
um bromide [116], or the acute destruction of a fraction
[117] or even loss of all of their mtDNA [118] by mito-
chondrially targeted restriction endonucleases. There-
fore, 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 alky-
lating DNA damage [92,98,119], which contradicts the
notion mandatory degradation of damaged mtDNA,
prevented the model of mtDNA turnover as a mecha-
nism 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
M. F. Alexeyev mtDNA + ROS = Aging?
FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS 5773
revealing that the intragastric administration of etha-
nol to mice induced oxidative stress which was accom-

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 H
2
O
2
failed to induce significant mtDNA mutagenesis.
Instead, H
2
O
2
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 pro-
duced. 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 undam-
aged circular molecules, they are susceptible to exo-
nucleolytic 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

APE
mtDNA
GlycosylaseII
or
Glycosylase I + APE
R
e
p
a
i
r
Damage
Single-strand
breaks
Double-strand
breaks
Degradation
Fig. 2. Potential interactions between mtDNA repair and degrada-
tion 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 com-
mitment step leading to degradation. Glycosylase I and glycosylase
II are monofunctional and bifunctional DNA glycosylases. A bifunc-
tional 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.
mtDNA + ROS = Aging? M. F. Alexeyev

particular gene must be mutated before a diseased phe-
notype is manifested. The threshold phenomenon can
be mediated, at least in part, by intramitochondrial and
intermitochondrial complementation [139–141]. How-
ever, 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 muta-
tions, 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 Polg
exo) ⁄ )
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 bur-
den is at least 10 times higher than that observed in
normal aging [143,145].
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

isolation [153]. Finally, even when increased mitochon-
drial ROS production in older tissues can be demon-
strated, 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
M. F. Alexeyev mtDNA + ROS = Aging?
FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS 5775
sources does not increase over time. Decreased antioxi-
dant 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 inter-
preted as supporting the MTA, because these assump-
tions 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
) ⁄ )

mitoCAT mice and the MTA
A study on catalase overexpression in mouse mitochon-
dria 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 expres-
sion 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
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 fol-
lowing additional questions may clarify whether there is
an actual causal relationship.
First, does catalase activity, especially in the founder
4403, substantially contribute to H
2
O
2
metabolism in

clude that the relative contributions of GPx1 and cata-
lase to H
2
O
2
detoxification are determined, among
other factors, by their relative abundance, and that
catalase does not contribute significantly to H
2
O
2
detoxification in mitochondria under their experimen-
tal 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 H
2
O
2
, 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 target-
ing, were affected by the C-terminal truncation. Cata-

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 Polg
exo) ⁄ )
knock-in mice is not accompanied by increased ROS
production [143,145]. In explanation, an increased sensi-
tivity to apoptosis because of an increased mtDNA
mutation burden was proposed to be responsible for
accelerated aging [143]. This notion, however, is con-
tradicted 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 Polg
exo) ⁄ )
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 apop-
tosis (otherwise, intermediate levels of oxidative damage
would persist and therefore would be measurable). This

knockdown extended the life span of clk-1 mutants.
The authors concluded that increased O
À
2
detoxifica-
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 long-
lived daf-2 mutant, knockout of the genes for two
MnSod isoforms, sod-2 and sod-3, increased the sensi-
tivity to oxidative stress, but did not shorten the life
span. Finally, Van Raamsdonk and Hekimi [188] exam-
ined 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. Exam-
ination 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 long-
lived despite a significant increase in oxidatively dam-
aged proteins. Testing the effect of sod-2 deletion on
known pathways of life span extension revealed a clear
interaction with genes that affect mitochondrial func-
tion. For example, a sod-2 deletion markedly increased

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 vari-
ous 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 recapitu-
lated 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 inter-
preted as causing increased oxidative stress. However,

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 accu-
mulation 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-ox-
odG 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-ox-
odG. Several techniques have been evaluated by
ESCODD, including HPLC with electrochemical detec-
tion (HPLC-ECD), gas chromatography followed by
mass spectrometry (GC-MS) and HPLC followed by
tandem mass spectrometry-mass spectrometry (HPLC-
MS ⁄ MS). Laboratories that employed HPLC-ECD
were able to detect induction of 8-oxodG with similar
mtDNA + ROS = Aging? M. F. Alexeyev
5778 FEBS Journal 276 (2009) 5768–5787 ª 2009 The Author Journal compilation ª 2009 FEBS
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

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
Polg
exo+ ⁄ +
and Polg
exo+ ⁄ )
mice. The authors reported
that although the mutation burden in young Polg
exo+ ⁄ )
mice is approximately 30 times higher than in old
Polg
exo+ ⁄ +
littermates, the life spans of these two geno-
types 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 mito-
chondrion, 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 tar-
gets of ROS, including mtDNA. While not supported
by direct evidence, it has not been refuted and awaits
substantial improvements in our understanding of, and
ability to manipulate, mitochondria and mitochondrial
ROS in order to be tested directly. Some of the argu-
ments in favor of the Free Radical Theory of Aging are
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