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

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 Keywords antioxidants; lifes

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

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 (H2O2) 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-son, 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

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: malexeye@jaguar1.usouthal.edu

(Received 12 July 2009, revised 3 August

2009, accepted 11 August 2009)

doi:10.1111/j.1742-4658.2009.07269.x

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 popula-tion, contributing to disability, a diminished quality of life and an increase

in healthcare costs Increased life expectancy reflects the success of contem-porary 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 under-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; 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.

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 patermater-nal 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

[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

polymor-phism 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

aro-matic hydrocarbons [14,15] When cells are exposed to

some of these compounds, mtDNA is damaged

prefer-entially [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

preferen-tial 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)

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 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 physi-ological 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 estab-lished 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 find-ings, however, were subsequently challenged by Hans-ford et al [26] who found that active H2O2production, which is an indirect measure of superoxide (O2 ) 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, H2O2-formation rates are much

lower, at less than 0.1% of the respiratory chain

elec-tron flux Staniek and Nohl [27,28] also reported that

when mitochondria use complex I and complex II

sub-strates for respiration, detectable H2O2 is generated

only in the presence of the complex III inhibitor

anti-mycin They suggest that the rates of mitochondrial

H2O2 production reported by other studies were

artifi-cially high because of experimental design flaws, and

point out that because mitochondrial O

2 formation under homeostatic conditions has not yet been

demon-strated in situ, conclusions drawn from isolated

mito-chondria should not be overinterpreted [28]

St Pierre et al capitalized on these findings and used

an improved experimental design to show that

mito-chondria do not release measurable amounts of O2 or

H2O2 when respiring on complex I or complex II

sub-strates, but release significant amounts of O2 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 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

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-genic 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

elec-tron 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

maxi-mal 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

com-pletely excluded [32,33] Nonetheless, even if RET

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 docu-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

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

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 O2 is one of the factors that determines the accessibility of the agent to mtDNA Therefore,

 50% of O

2 generated at complex III has no access

to mtDNA, while all O2 generated at complex I has unimpeded access to it [41] Although O2 permeates erythrocyte ghost membranes through an anion chan-nel [45], no evidence exists for a similar chanchan-nel in the inner mitochondrial membrane, which is probably impermeable to this species

Fig 1 A major pathway for the detoxification of ROS in the mito-chondrial matrix O 

2 is formed by the reduction of O 2 with elec-trons leaked from the ETC O 

2 is efficiently converted to H2O2by mitochondrial superoxide dismutase (Sod2) H2O2is 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.

In fact, however, the membrane permeability of O2

may be of little consequence because it is unable to

react directly with DNA [46–50] Reaction of O2 with

nonradicals is spin forbidden In biological systems,

this means that the main reactions of O2 are with

itself (dismutation) or with another biological radical,

such as nitric oxide

One important feature of O

2 production by mito-chondria is that it can be self-limiting through the

inactivation of mitochondrial aconitase This

inactiva-tion can reduce NADH formainactiva-tion 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 O2 production [51,52]

Mitochondrial ROS neutralization

The O2 generated by the ETC is quickly converted to

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 105m)1s)1, or enzymatically,

cata-lyzed by superoxide dismutases, with a first-order rate

constant of 109m)1s)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

O2 [54] The relative stability and membrane

perme-ability of H2O2allows it free access to mtDNA, yet, like

O2 , it is also unable to react directly with DNA [46–

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

rad-ical•OH that efficiently damages DNA [34,35] To

pre-vent the potentially devastating consequences of the

Fenton reaction, H2O2is 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

effi-cient at reducing lipid hydroperoxides In addition to

direct inactivation of ROS, GPx enzymes indirectly

pro-tect the cell from damage by the O2 , by preventing

per-oxide-mediated inactivation of Sod1 [59] Interestingly,

Sod itself protects GPx from inactivation by O2 [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 H2O2 detoxification in the mito-chondrial matrix (Fig 1)

O2þ e! O2 ð1Þ 2O2 þ 2Hþ! H2O2þ O2 ð2Þ

Fe2þþ H2O2! Fe3þþOHþ OH ð3Þ

H2O2þ 2GSH ! GS  SG þ 2H2O ð4Þ

H2O2þ 2 Pr xIII(SH)2! 2H2Oþ Pr xIII(SH)

S  S(SH) Pr xIII ð5Þ

H2O2þ Pr xV(SH)2! 2H2Oþ Pr xV(S  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 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 sug-gested 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 oxida-tive damage than nDNA because it is close to the source

of ROS was logical, especially when early reports sug-gested that mtDNA contained higher levels of oxidative

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

pack-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 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

con-tain a reduced complement of DNA-repair pathways

However, Anderson and Friedberg [86] found

uracil-DNA glycosylase activity in mitochondrial extracts,

suggesting at least the presence of the base excision

repair (BER) pathway This was confirmed by

mito-chondrial repair of O6-ethyl-2¢-deoxyguanosine, which

is also processed by BER in the nucleus [87,88]

Subse-quently, repair of a variety of mtDNA lesions,

includ-ing 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

mitochon-dria, 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

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 magni-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

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 repli-cation by mitochondria-specific DNA polymerase c

(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

path-way 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

an-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,

G:T transversions accounted for only 5.9% of the

mutations Even taking into account the potentially

mutagenic 8-oxo deoxyguanosine triphosphate

(8-oxo-dGTP), which results from oxidation of the cytosolic

and matrix dGTP pools and causes T:G transversions,

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 cyto-sine 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 muta-genic lesion, so the key factors responsible for the accu-mulation 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 oxi-dative stress are primarily responsible for the accumula-tion of mutaaccumula-tions 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 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

revealing that the intragastric administration of

etha-nol to mice induced oxidative stress which was

accom-panied by a reversible loss of mtDNA [121] The loss

of mtDNA was approximately 50% in all organs

stud-ied It could be partially prevented by the antioxidants

melatonin, vitamin E and coenzymeQ, and was

fol-lowed by adaptive mtDNA resynthesis [122]

Lipopoly-saccharide, 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

degrada-tion, 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 O2 on the matrix side of the

mito-chondrial inner membrane [29,41] However, exposing

human colon carcinoma cells or mouse embryonic

fi-broblasts 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

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

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

mitochon-drial BER pathway, such as lesion-specific DNA

glycosylases and apurinic and apyrimidinic

endonucle-ase, may aid in the generation of abasic sites and sin-gle-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

Ike-da and Ozaki [128] found that mitochondrial endonu-clease G is more active on oxidatively modified DNA

in vitro than on undamaged DNA, identifying a candi-date enzyme that may be involved in the degradation

of oxidatively damaged mtDNA Finally, the mecha-nism of strand breaks in mtDNA after oxidative stress,

as a means of protecting the integrity of the genetic information, concurs with evolutionary theory It sug-gests that, in combination with the high redundancy of

Base damage

AP site 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, 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

produc-tion and mtDNA mutaproduc-tions This noproduc-tion appears to

have some footing in observations made with

patho-genic mtDNA mutations Thus, an increased oxidative

burden, presumably caused by the ETC defect, was

demonstrated in cells harboring some of these

muta-tions [129–136] Three caveats, however, suggest

cau-tion in extending these observacau-tions 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

mito-chondrial 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

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 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

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 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 tech-niques, and found that the central bioenergetic systems, including pyruvate dehydrogenase, the tricarboxylic acid cycle, the ETC and ATP synthesis, appeared unal-tered 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 experi-ments can faithfully reproduce in vivo conditions At least some tissues have distinct mitochondrial subpop-ulations, 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 mito-chondria from aged tissues may also be altered, lead-ing to increased damage of these mitochondria durlead-ing 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

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 investigaassump-tions

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) ⁄ )

knock-out 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

mod-els These studies were reviewed in depth by van

Rem-men et al [162,163], who concluded that ‘the majority

of the initial pioneering studies in mice to test the

mito-chondrial theory of aging have yielded results that

either do not support the theory or remain

inconclu-sive An exception is a single study involving the

over-expression 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

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 H2O2 metabolism in mitochondria? Catalase has a low affinity for H2O2 (Km> 30 mm [165,166]) and can be inhibited (revers-ibly and irrevers(revers-ibly) by this substrate [167] By con-trast, 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) con-centrations 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 con-clude that the relative contributions of GPx1 and cata-lase 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 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 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 target-ing, were affected by the C-terminal truncation Cata-lase 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 activ-ity 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

Catalase overexpression can be detrimental,

accord-ing to some indications Overexpression of catalase in

the mitochondrial or cytosolic compartments increases

the sensitivity of HepG2 cells to tumor necrosis

factor-a-induced apoptosis [175] The mosaic pattern of

trans-gene 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

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 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

apop-tosis (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

there-fore is unlikely to be valid Moreover, alternative

mech-anisms 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

signifi-cant body of evidence that is inconsistent with

predic-tions of the MTA Research in Drosophila showed that

overexpression of antioxidant enzymes does not

neces-sarily extend life span [183], and can even be

detrimen-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 oxida-tive 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 O

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 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]