Mitochondria, oxidative DNA damage, and aging

Protection from reactive oxygen species (ROS) and from mitochondrial oxidative damage is well known to be necessary to longevity. The relevance of mitochondrial DNA (mtDNA) to aging is suggested by the fact that the two most commonly measured forms of mtDNA damage, deletions and the oxidatively induced lesion 8-oxo-dG, increase with age. The rate of increase is species-specific and correlates with maximum lifespan. It is less clear that failure or inadequacies in the protection from reactive oxygen species (ROS) and from mitochondrial oxidative damage are sufficient to explain senescence. DNA containing 8-oxo-dG is repaired by mitochondria, and the high ratio of mitochondrial to nuclear levels of 8-oxo-dG previously reported are now suspected to be due to methodological difficulties. Furthermore, MnSOD −/+ mice incur higher than wild type levels of oxidative damage, but do not display an aging phenotype. Together, these findings suggest that oxidative damage to mitochondria is lower than previously thought, and that higher levels can be tolerated without physiological consequence. A great deal of work remains before it will be known whether mitochondrial oxidative damage is a “clock” which controls the rate of aging. The increased level of 8-oxo-dG seen with age in isolated mitochondria needs explanation. It could be that a subset of cells lose the ability to protect or repair mitochondria, resulting in their incurring disproportionate levels of damage. Such an uneven distribution could exceed the reserve capacity of these cells and have serious physiological consequences. Measurements of damage need to focus more on distribution, both within tissues and within cells. In addition, study must be given to the incidence and repair of other DNA lesions, and to the possibility that repair varies from species to species, tissue to tissue, and young to old.     

Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys

While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging.     

Oxidative stress, mitochondria and mtDNA-mutator mice

The oxidative stress theory of aging, an expansion of the mitochondrial theory of aging, is based around the idea of a vicious cycle, in which somatic mutations of mitochondrial DNA (mtDNA) provoke respiratory chain dysfunction leading to enhanced ROS production and in turn to the accumulation of further mtDNA mutations. Mitochondrial dysfunction and mtDNA mutations are amplified during the course of aging. Recently, results obtained from mtDNA-mutator mice further strengthen the role of mitochondria in the aging process. However, lack of increased oxidative stress in the mtDNA-mutator mice raises doubts in the direct connection of mtDNA mutations with increased ROS production, challenging the oxidative stress theory of aging. The purpose of this short review is to highlight several studies that provide direct evidence that accelerated aging is linked to mtDNA mutations, without an increase in oxidative damage.     

Perspectives on the mitochondrial etiology of replicative aging in yeast

In a now classical paper, Denham Harman suggested that free radicals produced during mitochondrial respiration cause cumulative oxidative damage, resulting in aging and age-related disorders and pathologies. Proponents of this hypothesis have focused their attention, not surprisingly, on mitochondria arguing that these organelles may serve as the biological clock for aging. Indeed, work on many models, including filamentous fungi, nematodes, and mammals have revealed that age-dependent reorganizations of the mitochondrial DNA (mtDNA) may play a central role in the aging of these organisms. Furthermore, genetic alterations of mitochondrial function may either shorten or extend life span. In this paper, we focus on the role of mitochondria in the replicative aging of yeast mother cells, whether this role of mitochondria is really a linked to altered ROS production and/or respiration, and highlight some important questions that remain to be answered.     

Melatonin protects against MPTP/MPP+ -induced mitochondrial DNA oxidative damage in vivo and in vitro

The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine ion (MPP(+)) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8-hydroxyguanine (8-oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose-dependently prevented MPTP-induced DNA oxidative damage. In SH-SY5Y cells, MPP(+) (1 mm) increased the immunoreactivity of 8-oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 microm) preincubation significantly attenuated MPP(+)-induced mtDNA oxidative damage. Furthermore, MPP(+) time-dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Psim) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP(+) caused cell death in 49% of the control. However, melatonin prevented MPP(+)-induced mtOFR generation and Psim collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP(+)-induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Psim collapse.     

Impaired base excision repair and accumulation of oxidative base lesions in CD4+ T cells of HIV-infected patients

Several studies have reported enhanced oxidative stress in patients with HIV infection. An important pathophysiologic consequence of increased oxidative stress is endogenous DNA damage, and the base excision repair pathway is the most important mechanism to withstand such deleterious effects. To investigate the role of base excision repair in HIV infection, we examined 7,8-dihydro-8-oxoguanine (8-oxoG) levels as a marker of oxidative DNA damage and DNA glycosylase activities in CD4(+) and CD8(+) T cells of HIV-infected patients and controls. These results showed that the HIV-infected patients, particularly those with advanced disease, had increased levels of 8-oxoG in CD4(+) T cells and marked declines in DNA glycosylase activity for the repair of oxidative base lesions in these cells. In contrast, CD8(+) T cells from HIV-infected patients, with 8-oxoG levels similar to those in healthy controls, showed enhanced capacity to repair oxidative DNA damage. Finally, highly active antiretroviral therapy induced increased glycosylase activity in CD4(+) T cells and normalized 8-oxoG levels. This imbalance between the accumulation of oxidative DNA damage and the capacity to repair such lesions in CD4(+) T cells may represent a previously unrecognized mechanism involved in the numerical and functional impairment of CD4(+) T cells in patients with HIV infection.     

Oxidative damage to mitochondria and aging

Oxidative damage has been implicated to be a major factor in the decline in physiologic function that occurs during the aging process. Because mitochondria are a primary site of generation of reactive oxygen species, they have become a major focus of research in this area. Increased oxidative damage to mitochondrial proteins, lipid and DNA has been reported to occur with age in several tissues in a variety of organisms. Decreased activity of electron transport chain complexes and increased release of reactive oxygen species from the mitochondria with age suggest that alterations in mitochondrial function occur with age as a consequence of increased oxidative damage. In addition, age-related alterations in the mitochondrial pathway of apoptosis, which could have profound affects on the physiological function of a tissue, could arise from oxidative damage to mitochondria. Alterations in mitochondrial turnover with age could also contribute to an increase in the number of dysfunctional mitochondria with age.     

Decline in skeletal muscle mitochondrial function with aging in humans

Cumulative mtDNA damage occurs in aging animals, and mtDNA mutations are reported to accelerate aging in mice. We determined whether aging results in increased DNA oxidative damage and reduced mtDNA abundance and mitochondrial function in skeletal muscle of human subjects. Studies performed in 146 healthy men and women aged 18-89 yr demonstrated that mtDNA and mRNA abundance and mitochondrial ATP production all declined with advancing age. Abundance of mtDNA was positively related to mitochondrial ATP production rate, which in turn, was closely associated with aerobic capacity and glucose tolerance. The content of several mitochondrial proteins was reduced in older muscles, whereas the level of the oxidative DNA lesion, 8-oxo-deoxyguanosine, was increased, supporting the oxidative damage theory of aging. These results demonstrate that age-related muscle mitochondrial dysfunction is related to reduced mtDNA and muscle functional changes that are common in the elderly.     

Mitochondrial alterations, cellular response to oxidative stress and defective degradation of proteins in aging

Respiratory function decline and increase ofoxidative stress in mitochondria have beenproposed as important contributors to humanaging. A wide spectrum of alterations in agedindividuals and senescent cells are similar andare correlated to cellular response tosublethal dose of oxidative stress. Thesealterations and responses include: (1) declinein mitochondrial respiratory function; (2)increase in the rate of production of reactiveoxygen species (ROS); (3) accumulation ofmitochondrial DNA (mtDNA) mutations; (4)increase in the levels of oxidative damage toDNA, protein, and lipids; and (5) decrease inthe capacities of degradation of oxidativelydamaged proteins and other macromolecules. Responses to oxidative stress and theirsubsequent interactions in tissues result inthe deleterious effect of ROS on the cellularfunction, which culminate in aging anddegenerative diseases. In this review, wefocus on the roles that ROS play in age-relatedoxidative damage to mtDNA and proteins andoxidative stress responses at the molecular andcellular levels. The alterations of geneexpression profiles elicited by oxidativestress in aging animals are discussed. Wesuggest that the increase in mitochondrialproduction of ROS and decline in the cellularcapacity to cope with oxidative stress andsubsequent accumulation of mtDNA mutations andoxidized proteins play an important role in theaging process.     

Effect of estrogens on base excision repair in brain and liver mitochondria of aged female rats

Changes in the endocrine system have been suggested to act as signaling factors in the regulation of age-related events. Among the different hormones that have been linked to the aging process, estrogens have been widely investigated. They have been associated with inflammatory and oxidative processes and several investigations have established a relationship between the protective effects of estrogens and the mitochondrial function. Mitochondrial DNA is subjected to continuous oxidative attack by free radicals, and the base excision repair (BER) pathway is the main DNA repair route present in mitochondria. We have investigated the effect of estrogen levels on some of the key enzymes of BER in brain and liver mitochondria. In both tissues, depletion of estrogens led to an increased mitochondrial AP endonuclease (mtAPE1) activity, while restoration of estrogen levels by exogenous supplementation resulted in restitution of control APE1 activity only in liver. Moreover, in hepatic mitochondria, changes in estrogen levels affected the processing of oxidative lesions but not deaminations. Our results suggest that changes in mtAPE1 activity are related to specific translocation of the enzyme from the cytosol into the mitochondria probably due to oxidative stress changes as a consequence of changes in estrogen levels.     

增龄大鼠心肌线粒体DNA损伤及DNA修复酶γ表达的变化

目的:探讨大鼠增龄过程中心肌组织DNA损伤和DNA修复酶表达的变化。方法:从不同月龄的大鼠心肌组织中提取DNA、RNA及蛋白。采用定量多聚酶链反应(Q-PCR)检测心肌DNA损伤;用RT-PCR和Western blot检测DNA修复酶γ(Polγ)表达的变化。用ELISA法检测DNA内8羟基脱氧鸟苷(8-OHdG)的水平。结果:随着鼠龄的增长,大鼠心肌组织中核DNA(nDNA)损伤不明显,线粒体DNA(mtDNA)损伤严重,DNA内8-OHdG的水平增加,Polγ的表达下降。结论:随着鼠龄的增长,大鼠心肌组织中DNA修复酶Polγ的表达下降,mtDNA损伤增加。DNA损伤与DNA修复能力下降间会引起恶性循环,最终可导致DNA损伤的增加加快心脏衰老。     

Mitochondrial DNA repair and association with aging – An update

Mitochondrial DNA is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to play a critical role in the aging process and to be particularly deleterious in post-mitotic cells. Thus, DNA repair is an important mechanism for maintenance of genomic integrity. Despite the importance of mitochondria in the aging process, it was thought for many years that mitochondria lacked an enzymatic DNA repair system comparable to that in the nuclear compartment. However, it is now well established that DNA repair actively takes place in mitochondria. Oxidative DNA damage processing, base excision repair mechanisms were the first to be described in these organelles, and consequently the best understood. However, new proteins and novel DNA repair pathways, thought to be exclusively present in the nucleus, have recently been described also to be present in mitochondria. Here we review the main mitochondrial DNA repair pathways and their association with the aging process.     

The role of mitochondrial DNA mutations and free radicals in disease and ageing

Considerable efforts have been made to understand the role of oxidative stress in age‐related diseases and ageing. The mitochondrial free radical theory of ageing, which proposes that damage to mitochondrial DNA (mtDNA) and other macromolecules caused by the production of reactive oxygen species (ROS) during cellular respiration drives ageing, has for a long time been the central hypothesis in the field. However, in contrast with this theory, evidence from an increasing number of experimental studies has suggested that mtDNA mutations may be generated by replication errors rather than by accumulated oxidative damage. Furthermore, interventions to modulate ROS levels in humans and animal models have not produced consistent results in terms of delaying disease progression and extending lifespan. A number of recent experimental findings strongly question the mitochondrial free radical theory of ageing, leading to the emergence of new theories of how age‐associated mitochondrial dysfunction may lead to ageing. These new hypotheses are mainly based on the underlying notion that, despite their deleterious role, ROS are essential signalling molecules that mediate stress responses in general and the stress response to age‐dependent damage in particular. This novel view of ROS roles has a clear impact on the interpretation of studies in which antioxidants have been used to treat human age‐related diseases commonly linked to oxidative stress.     

Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress

A significant amount of reactive oxygen species (ROS) is generated during mitochondrial oxidative phosphorylation. Several studies have suggested that mtDNA may accumulate more oxidative DNA damage relative to nuclear DNA. This study used quantitative PCR to examine the formation and repair of hydrogen peroxide-induced DNA damage in a 16.2-kb mitochondrial fragment and a 17.7-kb fragment flanking the β-globin gene. Simian virus 40-transformed fibroblasts treated with 200 μM hydrogen peroxide for 15 or 60 min exhibited 3-fold more damage to the mitochondrial genome compared with the nuclear fragment. Following a 60-min treatment, damage to the nuclear fragment was completely repaired within 1.5 hr, whereas no DNA repair in the mitochondrion was observed. Mitochondrial function, as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, also showed a sharp decline. These cells displayed arrested-cell growth, large increases in p21 protein levels, and morphological changes consistent with apoptosis. In contrast, when hydrogen peroxide treatments were limited to 15 min, mtDNA damage was repaired with similar kinetics as the nuclear fragment, mitochondrial function was restored, and cells resumed division within 12 hr. These results indicate that mtDNA is a critical cellular target for ROS. A model is presented in which chronic ROS exposure, found in several degenerative diseases associated with aging, leads to decreased mitochondrial function, increased mitochondrial-generated ROS, and persistent mitochondrial DNA damage. Thus persistent mitochondrial DNA damage may serve as a useful biomarker for ROS-associated diseases.     

Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production

The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.     

Effects of aging and methionine restriction applied at old age on ROS generation and oxidative damage in rat liver mitochondria

It is known that a global decrease in food ingestion (dietary restriction, DR) lowers mitochondrial ROS generation (mitROS) and oxidative stress in young immature rats. This seems to be caused by the decreased methionine ingestion of DR animals. This is interesting since isocaloric methionine restriction in the diet (MetR) also increases, like DR, rodent maximum longevity. However, it is not known if old rats maintain the capacity to lower mitROS generation and oxidative stress in response to MetR similarly to young immature animals, and whether MetR implemented at old age can reverse aging-related variations in oxidative stress. In this investigation the effects of aging and 7 weeks of MetR were investigated in liver mitochondria of Wistar rats. MetR implemented at old age decreased mitROS generation, percent free radical leak at the respiratory chain and mtDNA oxidative damage without changing oxygen consumption. Protein oxidation, lipoxidation and glycoxidation increased with age, and MetR in old rats partially or totally reversed these age-related increases. Aging increased the amount of SIRT1, and MetR decreased SIRT1 and TFAM and increased complex IV. No changes were observed in the protein amounts of PGC1, Nrf2, MnSOD, AIF, complexes I, II and III, and in the extent of genomic DNA methylation. In conclusion, treating old rats with isocaloric short-term MetR lowers mitROS production and free radical leak and oxidative damage to mtDNA, and reverses aging-related increases in protein modification. Aged rats maintain the capacity to lower mitochondrial ROS generation and oxidative stress in response to a short-term exposure to restriction of a single dietary substance: methionine.     

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G.C-->T.A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1(-/-) null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.     

Mitochondrial-nuclear Cross-talk in the Aging and Failing Heart

Hypothesis Damage to heart mitochondrial structure and function occur with aging, and in heart failure (HF). However, the extent of mitochondrial dysfunction, the expression of mitochondrial and nuclear genes, and their cross-talk is not known. Observations Several observations have suggested that somatic mutations in mitochondrial DNA (mtDNA), induced by reactive oxygen species (ROS), appear to be the primary cause of energy decline, and that the generation of ROS is mainly the product of the mitochondrial respiratory chain. The free radical theory of aging, that could also be applied to HF, and in particular the targeting of mtDNA is supported by a plurality of observations from both animal and clinical studies showing decreased mitochondrial function, increased ROS levels and mtDNA mutations in the aging heart. Discussion Aging and HF with their increased ROS-induced defects in mtDNA, including base modifications and frequency of mtDNA deletions, might be expected to cause increased errors or mutations in mtDNA-encoded enzyme subunits, resulting in impaired oxidative phosphorylation and defective electron transport chain (ETC) activity which in turn creates more ROS. These events in both the aging and failing heart involve substantial nuclear–mitochondrial interaction, which is further illustrated in the progression of myocardial apoptosis. In this review the cross-talk between the nucleus and the mitochondrial organelle will be examined based on a number of animal and clinical studies, including our own.