REVIEW ARTICLE Is there more to aging than mitochondrial DNA and reactive oxygen species? Mikhail F Alexeyev1,2 Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL, USA 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: malexeye@jaguar1.usouthal.edu 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 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 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 not release measurable amounts of OÀ or H2O2 when respiring on complex I or complex II substrates, but release significant amounts of OÀ 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 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 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 and Eqn 1), which is charged, 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 the accessibility of the agent to mtDNA Therefore, $ 50% of OÀ generated at complex III has no access to mtDNA, while all OÀ generated at complex I has unimpeded access to it [41] Although OÀ permeates 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 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 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 may be of little consequence because it is unable to react directly with DNA [46–50] Reaction of OÀ with nonradicals is spin forbidden In biological systems, this means that the main reactions of OÀ are with 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] 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 detoxification in the mitochondrial matrix (Fig 1) O2 ỵ e ! O 2O ỵ 2Hỵ ! H2 O2 ỵ O2 The OÀ generated by the ETC is quickly converted to H2O2 (Fig 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– 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 (GPx1; Fig and Eqn 4) and peroxiredoxins III and V (PrxIII and PrxV; Fig and Eqns 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] 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 ỵ 2GSH ! GS SG ỵ 2H2 O Mitochondrial ROS neutralization 1ị 4ị H2 O2 ỵ Pr xIII(SH)2 ! 2H2 O þ Pr xIII(SH) ÀS À S(SH) Pr xIII ð5Þ 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 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 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 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 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 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 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 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 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