Oxidative Stress, Mitochondrial DNA Mutation, and Impairment of Antioxidant Enzymes in Aging1

mtDNA MUTATION IN AGING

In the past 15 years, a large number of large-scale deletions, point mutations, and tandem duplications of mtDNA have been
found in various tissues of aged individuals (Table I⇓) (12, 16, 17). Each mammalian cell contains several hundred to more than one thousand mitochondria, each of which carries two to 10 copies
of mtDNA (18). The mutant mtDNA(s) usually co-exist with the wild-type mtDNA within a cell (a condition termed heteroplasmy), and the
degree of heteroplasmy often varies in different tissues of the same individual (19, 20). The random segregation of mtDNA during cell division often results in the mosaic pattern of distribution of mutant mtDNA
molecules in the affected tissues. It has been established that many of these mtDNA mutations start to occur after the mid-thirties
and they accumulate with age in postmitotic tissues of the human body (16, 21–32). Some of these aging-associated mtDNA mutations were originally observed in the affected tissues of patients with mitochondrial
diseases. Among them, the most common one is the 4,977-bp deletion with a 13-bp direct repeat flanking the 5`− and 3`−end
breakpoints at nucleotide position (np) 8,470/8,482 and np 13,447/13,459, respectively (33, 34). This mtDNA deletion was first observed in the muscle of patients with chronic progressive external ophthalmoplegia, Kearns-Sayre
syndrome, and Pearsons’ syndrome (33, 35). Multiple large-scale deletions of mtDNA can be easily detected, but at different levels in a number of tissues of elderly
subjects (16, 26, 31). Two point mutations at np 8,344 and np 3,243 of mtDNA, which are respectively associated with myoclonic epilepsy and ragged-red
fibers (MERRF) and mitochondrial encephalomyopathy, lactic acidosis and stroke like episodes (MELAS) syndromes (36), have been also found to accumulate in the muscle of aged individuals (37–39). Recently, it was reported that a T414G transversion in the D-loop of mtDNA is accumulated at 25%–50% of the total mtDNA
in the skin fibroblasts from elderly subjects (40). This mutation is located at the control region of mtDNA and may impair the replication and transcription of mtDNA in tissue
cells of the elderly human subjects. Additionally, we found that six different tandem duplications occur in the D-loop region
of mtDNA from the brain, muscle, liver, and skin tissues of normal subjects and that the incidence and abundance of mtDNAs
with these tandem duplications increased with age (16, 29). It is noteworthy that there is a high degree of heteroplasmy in the D-loop region of mtDNA in the human brain, and the
sequence variations are increased in aged individuals (41).

It is important to note that the proportions of mtDNA with these mutations in aging human tissues rarely exceed 1% (16, 26, 28, 29, 31, 32). It has been questioned as to how can such low levels of mtDNA mutation cause significant effects on the bioenergetic function
of mitochondria in aging. One of the possibilities is that the mutated mtDNA molecules in aging tissues may be underestimated
due to the limitation of available analytic methods. To tackle this question, a comprehensive detection system was designed
for screening of all possible large-scale deletions of mtDNA in human tissues (42). By using 180 kinds of PCR primer pairs, this system has made it possible to detect all the deletions in mtDNA over 500
bp. Hayakawa et al. (42) applied this system to analyze mtDNAs from normal hearts of human subjects of different ages. They found that mtDNA molecules
in the heart muscle of elderly subjects were extensively fragmented into minicircles with different sizes. As a result, the
total amount of mtDNA mutations, including point mutations and length mutations, may reach such a high level that it could
cause significant impairment on mitochondrial respiration and oxidative phosphorylation (43). The other possibility is that the mutated mtDNA molecules may be distributed unevenly among the cells of affected tissues
(a mosaic pattern of mtDNA segregation). It is equally plausible that mutated mtDNA molecules are distributed in the affected
muscle fibers in a segmental manner. Indeed, the respiratory function of skeletal muscle mitochondria in the fiber segments
harboring high proportions of mutated mtDNAs is severely impaired (44). These observations are supported by the recent reports that mtDNA rearrangements are extensive and the levels of full-length
mtDNA are reduced in the cytochrome c oxidase-negative fibers of the skeletal muscle of aged individuals (45, 46).

Functional Decline of Mitochondria in Aging

It is generally accepted that accumulation of mutated mtDNA is a contributory factor for the age-dependent decline of the
respiratory function, especially in postmitotic cells (7, 12, 19). The five enzyme complexes in the respiratory chain are composed of more than 100 polypeptides, most of which are encoded
by genes in the nuclear genome. However, 13 polypeptides involved in the respiration and oxidative phosphorylation are encoded
by mtDNA. The respiratory function and number of mitochondria are delicately regulated by a coordinated expression of mitochondrial
proteins encoded by the nuclear and mitochondrial genomes (47, 48). It was recently demonstrated that mitochondrial biogenesis and respiration of the human cells are vigorously controlled
by the nuclear respiratory factors NRF-1 and NRF-2, mitochondrial transcription factor A (mtTFA), and the thermogenic coactivator
PGC-1 (a 90-kDa nuclear protein) (48, 49). Thus, any molecular defect that leads to altered expression of the mtDNA-encoded genes or impairment in the biogenesis
of mitochondria would cause a deficiency in energy metabolism of the affected tissue cells.

There are three lines of research that lead to the same conclusion that bioenergetic function of mitochondria declines with
age. First, it was found that cytochrome c oxidase-negative fibers in human heart, ocular muscle, and diaphragm are increased with age (50–52). Second, it was demonstrated in late 1980s that the respiratory function of isolated mitochondria and electron transfer
activities of respiratory enzyme complexes gradually decline with age in human liver (10) and skeletal muscle (11, 53), respectively. Because the age-dependent decline of the glutamate-malate-supported respiration was found to be more dramatic
than that of the succinate-supported respiration, it was suggested that mutation(s) in the seven genes of NADH dehydrogenase
encoded by mtDNA may be involved in this aging-associated respiratory function decline. Beside this, it was observed that
the extent of mtDNA mutation strongly correlates with the progressive decrease of cytochrome c oxidase activity in aging human muscle (52). These findings have been confirmed in later studies on various human tissues (11, 54, 55).

On the other hand, a reduction in the steady-state levels of mtRNA was reported in Drosophila melanogaster (56) and in the aging tissues of the human and rats (57–60). The decrease in the levels of mitochondrial transcripts may result in the decline of protein biosynthesis and mitochondrial
respiration. The age-related decrease in the transcripts of mtDNA may result from a decline in the efficiency of mitochondrial
transcription or reduction in the copy number of mtDNA in tissue cells. Indeed, it was demonstrated that the efficiency of
mitochondrial transcription is decreased in aged tissues of the rat (57, 58). Oxidative stress may cause incomplete processing of the transcripts of the mitochondrial genome encoding ATPase subunits
6 and 8 plus the adjacent gene for cytochrome c oxidase subunit III (61). However, the mtDNA copy number was found to increase with age in D. melanogaster (62) and in various tissues of the rat (63) and humans (59, 60, 64, 65), respectively. It was argued that the age-related increase in mtDNA content is not correlated with the lower level of transcripts
and a decline in mitochondrial function. This can be rationalized by the fact that mutation and oxidative damage of mtDNA
are increased with age in various tissues of the human (12, 16, 28, 29, 66–68) and animals (63). These observations suggest that both the decrease in the efficiency of mitochondrial transcription and decline in the quality
of mtDNA contribute to the reduced steady-state levels of mitochondrial transcripts in aging tissues.

Last, it was found that the mitochondrial membrane potential, the driving force for oxidative phosphorylation, is decreased
in tissue cells of old animals (44, 69–71). This was supported by the recent work of Harper and co-workers (72), who demonstrated quantitatively that the proton leakage of the respiratory chain (for heat production) is increased and
ATP synthesis is decreased with age in the liver mitochondria of the mouse. Taken together, data accumulated from the above
three lines of research strongly support the view that the bioenergetic function of mitochondria declines with age in the
human and animals.

Eenhanced Oxidative Stress and Damage in Aging Tissues

Because mitochondria are not only the main producer of ATP but are also the major intracellular source of ROS in the human
cell, it is expected that mitochondrial disorders be primarily manifested in the organs or tissues that have a high demand
for energy. We demonstrated in early 1990s that oxidative damage and mutation of mtDNA are increased with age more conspicuously
in those tissues with higher energy demand (16, 28). Under normal physiological conditions, ROS and free radicals (e.g., ubisemiquinone and flavosemiquinone) are generated
and maintained at a relatively high steady-state level in mitochondria of tissue cells (73, 74). Respiratory enzyme Complex I and protonmotive Q cycle operating in Complex III are the major sites that generate ROS in
the respiratory chain (74, 75). It was estimated that one normal rat liver mitochondrion can produce about 3 × 10⁷ superoxide anions in a day (7).

It has been well documented that the rate of production of superoxide anions and hydrogen peroxide in mitochondria is increased
with age in animal tissues (76–78). It was found that the increase in hydrogen peroxide production of D. melanogaster under oxidative stress is related to the oxidative damage to mtDNA and membrane lipids of mitochondria (79). Sohal et al. (80) further demonstrated that the average lifespan of dipteran flies is inversely correlated with the rate of production of
superoxide anions and hydrogen peroxide in mitochondria and with the level of protein carbonyls in the tissue cells. Moreover,
the age-related increase in the rate of generation of hydrogen peroxide in mitochondria was observed to decrease 40% in the
fruit flies overexpressing Cu/ZnSOD and catalase as compared with the wild-type flies (81). Therefore, the rate and amount of hydrogen peroxide generated by mitochondria is an important determinant of the oxidative
damage sustained by mitochondria. Richter et al. (82) first demonstrated that oxidative damage to mtDNA is much more extensive than that to nuclear DNA. The specific content
of 8-hydroxy 2`-deoxyguanosine (8-OHdG), an index of oxidative damage to DNA, of mtDNA was about 16 times higher than that
of nuclear DNA in the liver of 3-month-old rats. Furthermore, the 8-OHdG level in liver mtDNA of the 24-month-old rat was
three times higher than that of the 3-month-old rat. Moreover, the levels of oxidative stress and proteins with oxidative
modification and lipid peroxides in mitochondria have been shown to increase with age (83, 84). In addition, the 8-OHdG contents in the mtDNA of human diaphragm, heart muscle, and brain tissues were found to increase
in an age-dependent manner (66, 85). These observations are consistent with the report that mitochondrial glutathione is markedly oxidized with aging in the
rat and mouse (86). The ratio between the oxidized and reduced glutathione rises with age in the liver, kidney, and brain of these animals.
In the same study, the 8-OHdG content of mtDNA was also found to increase with age of the rat and mouse. Moreover, these investigators
showed that oral administration of antioxidants protected the animals from glutathione oxidation and mtDNA damage. Several
recent studies also demonstrated that human cells harboring mutated mtDNA and/or defective mitochondria had lower respiratory
function and exhibited higher rate of production of superoxide anions, hydroxyl radicals, and hydrogen peroxide (14, 71, 87). Laderman et al. (88) investigated the respiratory function of a series of cybrids constructed by fusion of mtDNA-less human osteosarcoma cells
with human fibroblasts from donors of different ages. They showed that both growth potential and oxygen consumption rate of
the cybrids with respiratory deficiency are significantly lower for those made from the fibroblasts of older donors. They
suggested that accumulation of age-dependent mtDNA mutations resulting from damage by free radicals and ROS might be responsible
for the age-dependent decline of respiratory function of the cybrids.

On the other hand, age-related increase in mitochondria may provide an extra source of oxidants. Overproliferation of abnormal
mitochondria generally occurs in the muscle of aged individuals and of patients with mitochondrial myopathy. An increase in
mtDNA copy number has been observed in aging human tissues (59, 60, 64, 65). In addition, both mitochondrial mass and mtDNA copy number are increased during in vivo aging process and in vitro cellular replicative senescence (89, 90). In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in the mitochondrial
mass and the mtDNA copy number of human cells (91). Moreover, we found that ROS production was elevated in the cells harboring higher density of mitochondria (90, 92). Therefore, these observations suggest that the age-dependent increase in the production of superoxide anions and hydrogen
peroxide from the defective mitochondria is one of the factors involved in the decline of respiratory function during the
aging process.

These findings and other evidence have been accumulated to support the notion that oxidative stress is an important contributor
to the decline of mitochondrial respiratory function during aging (12). Mitochondrial function is sharply declined in cultured human cells after treatment with hydrogen peroxide (93). Beside exogenous oxidative insults, endogenous oxidative stress elicited by electron leakage of the mitochondrial electron
transport chain was suggested to cause a loss of mitochondrial function (94). Moreover, it was recently shown that an increase in mitochondrial ROS production contributes to the decline in the activities
of NADH dehydrogenase and succinate dehydrogenase in skeletal muscle and heart of the MnSOD-deficient mice (95). This is the first direct proof that mitochondrial respiratory function is impaired under oxidative stress. These observations
suggest a close relationship between oxidative stress, indicated by glutathione oxidation and oxidative damage to mtDNA, and
mitochondrial dysfunction in human and animal cells during the aging process (Fig. 1⇓). We have viewed this mitochondrial role in aging in the scenario of a “vicious cycle” (12, 67, 92). However, it remains unclear whether the decline in mitochondrial respiratory function during aging mainly results from
oxidative stress or is a consequence of synergistic effects of many factors associated with aging

The Impairment of Antioxidant Enzymes in Aging

It is generally accepted that the activities and capacities of antioxidant systems of tissue cells are declined with age,
leading to the gradual loss of prooxidant/antioxidant balance and accumulation of oxidative damage in the aging process. Although
many investigators have studied aged-related changes in antioxidant defenses, the results are controversial (96). It is worth mentioning that the activity of MnSOD located in the mitochondria is most significantly elevated during aging
in various tissues of the human and animals. Recently, it was found that human cells overexpressing MnSOD resulted in cell
cycle arrest, decrease in mitochondrial mass, accumulation of intracellular hydrogen peroxide, and induction of mRNA levels
of matrix-degrading metalloprotease-1, which plays a major role in the process of carcinogenesis and aging (97). This indicates that superoxide anions produced by mitochondria may be scavenged by MnSOD, but hydrogen peroxide thus accumulated
in mitochondria may increase oxidative stress during the aging process. Moreover, the recently developed MnSOD knockout mice
exhibited dilated cardiomyopathy, neonatal lethality, severe defect in mitochondrial function, increased cortical damage in
ischemic injury, and enhanced oxidative damage to mitochondria (98). The proper level of expression of MnSOD is important for cells to cope with oxygen radical-mediated chemical toxicity.
On the other hand, alterations in the levels of Cu/ZnSOD, CAT, and GPx are also important contributory factors in the aging
process. It has been observed that the fruit flies with homozygous mutations in either Cu/ZnSOD or CAT gene exhibit increased
sensitivity to oxidative stress and have reduced viability and shorter lifespan (99, 100). Flies overexpressing Cu/ZnSOD alone or in combination with the overexpression of CAT exhibited higher resistance to oxidative
stress, had significantly less oxidative damage to proteins, and lived longer (81, 101). The aforementioned results indicate that a balance between the free radical scavenging enzymes is important for the cellular
resistance to oxidative stress.

Furthermore, caloric restriction was demonstrated to extend the average and the maximum lifespan, and to concurrently decrease
the age-related accumulation of 8-OHdG in various tissues of the mouse (77, 102). It was recently found that the activities of antioxidant enzymes such as CAT were increased and the age-related deterioration
of respiratory enzymes was retarded in the liver of the rats fed on a restricted diet with 60% calorie of the ad libitum diet (103). These observations imply that oxidative damage to DNA and other cellular components elicited by oxidative stress and free
radicals generated by aerobic metabolism indeed play an important role in aging (104).

Common Cause of Oxidative Damage and mtDNA Mutation

Although the proportion of the mutant mtDNA was found to correlate with the 8-OHdG content of mtDNA (85, 104), it is poorly understood as to how oxidative stress or ROS causes mtDNA mutations. Human mtDNA is a naked compact DNA molecule
without protection by histones or DNA-binding proteins, and it is replicated rapidly by a unique D-loop mechanism without
proofreading (105). The presence of the D-loop during replication of mtDNA elicit many large-scale deletions that have occurred in the large
arc between the replication origins of the heavy and light strands of mtDNA (19). Moreover, mtDNA is attached to the mitochondrial inner membrane in which a considerable amount of ROS is continually produced
by the respiratory chain (106, 107). These characteristics have rendered mtDNA vulnerable to attack by ROS and free radicals generated by the electron leakage
of the respiratory chain of mitochondria, particularly in the aging tissue cells (12, 19, 82, 108).

It is possible that exposure of mtDNA to elevated oxidative stress (increased ROS levels) causes base modifications at some
nucleotides in mtDNA, which are more prone to large-scale deletions (Fig. 1⇑). Adachi and co-workers (109) demonstrated that ROS may cause large-scale deletion of mtDNA in animals. They detected a 4-kb deletion of mtDNA in the
heart of the Balb/c mice that had received chronic intraperitoneal injection of doxorubicin, which is known to induce cardiomyopathy
and elicit profound lipid peroxidation of heart mitochondria. Moreover, they found that administration of coenzyme Q₁₀ (a free radical scavenger) to the mice could effectively prevent the mtDNA deletion and could decrease the lipid peroxides
content of the heart mitochondria. This and a later study from our laboratory (104) provided important data to support the notion that ROS and free radicals are involved in the mechanism underlying large-scale
deletion of mtDNA. Ishii et al. (110) reported that a mutation in cytochrome b gene of Caenorhabditis elegans resulted in enhanced oxidative stress and displayed a phenotype of oxygen hypersensitivity and shortened lifespan. This observation
indicates that a defect in mitochondrial respiratory system can lead to an increase of oxidative damage and premature aging
or death. In the development of animal models for studies of mitochondrial diseases, Wallace and co-workers (95) succeeded in knocking-out sod-2 and ant1 genes, respectively. They found that sod-2 knockout mice displayed elevated oxidative stress and enhanced oxidative damage to proteins and DNA in brain and heart tissues
(95). Moreover, the MnSOD-deficient mice exhibited declined electron transport activities in respiratory enzyme Complexes I and
II, a phenotype similar to that commonly found in patients with mitochondrial myopathy. In another model, they demonstrated
that the mitochondria from skeletal muscle, heart, and brain of the mice lacking adenine nucleotide translocase (ANT) produced
markedly increased amounts of ROS (14). Most importantly, a host of deletions and rearrangements of mtDNA were induced in the heart muscle of ant1 knockout mice. Although it remains to be established as to how mtDNA mutations are initiated or promoted by ROS and free
radicals, these molecular biological and biochemical studies have provided strong evidence to support that oxidative damage
is generally accompanied by defects in mitochondrial respiration and oxidative phosphorylation.

Possible Mechanisms of mtDNA Mutation

Although mtDNA mutations have been detected in a wide range of aging human tissues, it is still poorly understood as to how
they occur. Sequence analysis of the reported deletions of human mtDNA revealed that they occurred more frequently between
the origins of replication of the H and L strands (111). These deletions cause a loss or truncation of many genes encoding tRNAs and mRNAs that are essential for the proper functioning
of mitochondria (15, 105). The breakpoints of many of these mtDNA deletions are flanked by direct repeat sequences. Slipped mispairing during DNA
replication between these direct repeat sequences (34), homologous replication (112), and topoisomerase II cleavage (113) have been suggested to be the possible mechanisms for mtDNA deletions. Presumably due to their stem-loop structures, the
mitochondrial tRNA genes have been found to be hot spots for point mutations of mtDNA (20). Indeed, 15 different point mutations have been found in the tRNA^Leu(UUR) gene (113). On the other hand, the start sites and the insertion sites of the tandem duplications in the D-loop region of mtDNA have
been found to be localized in the regions containing either a poly C run or a direct repeat sequence (16). Moreover, certain regions of human mtDNA have been demonstrated to be particularly sensitive to oxidative insult of ROS
and are prone to mutation (114). The putative hot spots for oxidative modification and mutation of mtDNA could be near or at the unusual structures including
bent, antibent, and non-B DNA sequences in human mtDNA. These observations suggest that the unusual structure and/or nucleotide
sequences of some segments of human mtDNA are the important factors involved in aging-associated mtDNA mutations (114).

In addition, it was hypothesized that genotoxic intermediates of lipid peroxidation may play a role in eliciting age-associated
DNA mutation (107). Recently, Esteve et al. (115) demonstrated that oxidative damage to mtDNA is directly related to the GSSG/GSH ratio in fibroblasts, an index of oxidative
stress. Because mtDNA is attached to the ROS-generating sites in the mitochondrial inner membrane, it should be more susceptible
to oxidative damage, strand breakage, and point mutation. Furthermore, ROS-induced mutagenesis has been observed to be DNA
polymerase specific (116). Thus, it is possible that the frequency of occurrence and the type of mtDNA mutation are determined, at least in part,
by the interaction between mitochondrial DNA polymerase and the DNA molecules that bear the ROS-induced oxidative damage during
DNA replication.

It was demonstrated that treatment of human skin fibroblasts with a sublethal level of oxidative stress results in the formation
and accumulation of the common 4977-bp deletion in mtDNA (117). Moreover, an environmental insult such as UV irradiation was reported to induce large-scale deletions in human skin (30, 31, 118) and in cultured human skin fibroblast (119). Fahn et al. (120) found that the frequency of occurrence and abundance of mtDNA deletions in the lung of smokers were higher than those of
the nonsmokers. Moreover, the proportions of deleted mtDNAs were increased as a function of smoking index in the lung of smokers.
Furthermore, Mansouri et al. (121) demonstrated that multiple mtDNA deletions are accumulated in the liver biopsies of alcoholic patients, especially those
with microvesicular steatosis. Recently, we found that betel quid chewing enhances the occurrence and accumulation of mtDNA
mutations in human oral tissue (122). These observations suggest that ROS and free radicals generated by the environmental insults (e.g., UV irradiation, cigarette
smoke, and air pollutants) and xenobiotics (e.g., drugs and betal quid), and alcohol drinking may induce the occurrence and
accumulation of mtDNA mutations in human tissues during the aging process. Moreover, exogenous oxidative stress may also impair
the replication of mtDNA and result in a decrease of mtDNA copy number (mtDNA depletion). Indeed, Attardi and co-workers (88) found that the mtDNA/nuclear DNA ratios of the cybrids made from fibroblasts of old donors was significantly lower than
those of the young donors. These molecular changes in the quality and quantity of mtDNA may affect, in a synergistic manner,
the bioenergetic function of mitochondria in the aging process (123).

DNA Repair Systems in Mitochondria

As mentioned above, oxidative damage to mtDNA occurs at a frequency approximately 20 times greater than that for nuclear DNA
(20, 82). It is expected that mitochondria would need efficient DNA repair mechanisms to remove oxidative damage for the damage to
their own DNA. The base excision repair (BER) is an important mechanism for the removal of oxidative DNA damage. Recent studies
revealed that mitochondria contain the BER system such as uracil- or 8-OHdG DNA glycosylase (124, 125), apurinic/apyrimidinic (AP) endonucleases (126–128), and 8-OHdGTPase (129). In the process of DNA repair by BER, DNA glycosylase recognizes a damaged base and then cleaves the N-glycosyl bond between
the sugar and the base to generate an AP site. Some glycosylases have an associated AP lyase activity that cleaves the DNA
phosphate backbone, whereas others rely on AP endonucleases for strand cleavage. A phosphodiesterase then excises the unsaturated
sugar derivatives at the 3` end of the DNA, and the gap of one nucleotide thus generated is bridged by a DNA polymerase and
the ends are sealed by a DNA ligase. The 8-OHdG residues in oxidatively modified mtDNA molecules may be removed by mitochondrial
8-OHdGTPase. However, this enzyme does not remove the other modified nucleobases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine.
Recombinational DNA repair has also been reported to exist in mammalian mitochondria (130, 131). However, UV damage cannot be repaired in mitochondria because nucleotide excision repair (NER) exists in the nucleus but
does not appear to be present in mitochondria (132–134). Hirano et al. (135) examined the age-related change of the DNA repair activity for 8-OHdG in TIG-3 cells and found that the activity of 8-OHdG
repair is indeed changed with age in cultured human fibroblasts. Driggers and co-workers (136) also reported that repair of oxidative damage to mtDNA is defective in a human cell line. However, Souza-Pinto et al. (128) demonstrated that 8-OHdG glycosylase/AP lyase activity was increased with age in rat heart and liver mitochondria. By contrast,
the activity of endonuclease G, which is the major mitochondrial endonuclease, was found to decrease with age in the heart
mitochondria of the rat (128). Thus, the DNA repair system in human mitochondria is not fully competent in the repair of various types of damage caused
by ROS and free radicals. Even though the activities of some of the endonucleases capable of repairing oxidative damage are
upregulated in the aging tissues, some others are unchanged (e.g., uracil DNA glycosylase) or even declined (e.g., endonuclease
G). Because successful repair of damaged DNA relies on the coordinated action of many repair enzyme systems, age-dependent
decline or imbalance of the activities of the aforementioned DNA repair enzymes will result in the compromise of the overall
capacity of repair for the damaged DNA molecules in the aging process. This may account for the ever-increasing accumulation
of oxidative damage and mutation to DNA in mitochondria of the aging tissues.

Mitochondrial Role in Apoptosis During Aging

Aging is characterized by an increased production of ROS in somatic tissues (4), and an increase in the production of ROS may promote the induction of apoptosis. Several lines of evidence suggest that
aging is accompanied by alterations in the apoptotic process. It has been demonstrated that overproduction of oxidants can
induce oxidative stress and cell death (4, 9). Mitochondria are the major intracellular source of ROS (4, 12, 73). Moreover, age-related increase in mitochondrial mass of tissue cells may generate higher levels of ROS (90, 92). Thus, the high levels of oxidants produced from mitochondria can induce apoptosis by changing cellular redox potentials,
depleting reduced glutathione, reducing ATP levels, and decreasing reducing equivalents such as NADH and NADPH. These changes
can facilitate lipid peroxidation and the opening of permeability transition pores, leading to the subsequent release of cytochrome
c. Interestingly, it was observed that there is enhanced activation of the mitochondrial permeability transition pore (PTP)
in the brain and liver of aging mice (137). This leads to a lower threshold for the release of apoptogenic proteins into the cytosol. Moreover, aged cells have been
associated with increased mitochondrial oxidant production and elevated intracellular Ca²⁺ levels (138, 139). These events contribute to the enhanced activation of PTP and the release of proapoptotic proteins from the intermembrane
space of mitochondria. Recently, Kokoszka et al. (140) demonstrated that chronic oxidative stress in the mice with a partial deficiency in MnSOD resulted in an increased sensitization
of the mitochondrial PTP and premature induction of apoptosis. Therefore, respiratory functional decline, mitochondrial ROS
production, oxidative stress, and the susceptibility to apoptosis are central events of the aging process.

Mitochondrial oxidative stress and a decline in mitochondrial energy production may lead to activation of apoptotic pathways.
It has been shown that apoptosis is enhanced in the liver, brain, heart, skeletal muscle, splenocytes, thymocytes, and oocytes
of aged animals (141–149). This loss in cells via apoptosis may be mediated by mitochondrial dysfunction caused by chronic exposure to oxidants, which
increased the likelihood of the opening of mitochondrial permeability transition pores.

Interestingly, a reduction in the total number of myocytes has been associated with an accelerated decline in cardiac function
in the aging heart (144, 145). In addition to necrosis, apoptosis may be a major factor contributing to the age-associated loss of cardiac myocytes. Moreover,
it was observed that apoptosis is more prevalent during the late stages of aging (143). Recently, it was shown that cytosolic cytochrome c content was significantly elevated in the heart cells of 16- and 24-month-old male Fischer 344 rats when compared with that
of the 6-month-old rats (146). Moreover, mitochondrial Bcl-2, an antiapoptotic protein, was found to show a strong tendency to decrease with age, but
mitochondrial Bax, a proapoptotic protein, remained unchanged (146). Because Bcl-2 can prevent cytochrome c release, these observations have provided a potential mechanism underlying the increase in apoptosis observed in the aging
heart.

Recently, it was found that splenocytes and thymocytes undergo apoptosis with aging in rats and the apoptosis was associated
with enhanced expression of p53, Bax, and caspase-3 (148). Moreover, it was also demonstrated that there are differential expressions of Bcl-2 family proteins and caspase family
proteins during aging in rat brain (150, 151). Bcl-2 protein level reached the highest level on embryonic day 19 (E19) and decreased after birth. Bax, Bak, and Bad levels
were high from E19 to 2 weeks, and decreased significantly at 4 weeks, but Bcl-x levels remained high from early stage to
96 weeks of age (150). Moreover, the expression profile of caspase-3 and -7 is similar to that of Bax, Bak, and Bad, whereas that of caspase-8
and -10 is similar to that of Bcl-2. The constitutive expression of caspases 6 and 9 and Apaf-1 is similar to that of Bcl-x
(151). However, the biological significance of the alterations of these enzymes and proteins involved in the execution and regulation
of apoptosis during aging remains to be defined.

On the other hand, it was demonstrated that aging attenuates apoptosis in the colonic mucosa of Fischer 344 rats (152). Moreover, it was also shown that the livers of old rats are resistant to apoptosis in response to a moderate dose of genotoxic
stress compared with the younger rat (153). These observations suggest that the age-related downregulation of apoptosis might be involved in the increase of DNA damage
and mutations with age, and could partially explain the increase in the incidence of cancer during aging. However, it remains
to be answered as to whether these results are tissue specific and characteristic of mitotic but not postmitotic tissues.
Whether the alterations in the sensitivity to proapoptotic conditions observed during aging are part of the consequences of
aging, and whether apoptosis per se participates in the normal aging process warrant further investigation.

Concluding Remarks

Although the classical role of mitochondria in the generation of ATP by aerobic metabolism has been established for more than
half a century, the other face of mitochondria in the participation of human aging and disease was not recognized until about
15 years ago. It has been appreciated only very recently that mitochondria are not only the major metabolic energy supplier,
but are also the main intracellular source and target of ROS and free radicals generated by the respiratory chain. In the
mammalian cell, the proper assembly and functioning of mitochondria are effected through the coordination between gene products
encoded by the nuclear and mitochondrial genomes (47). Communication between the nucleus and mitochondria is essential for delicate regulation of synthesis of proteins in the
cytoplasm and their subsequent import into mitochondria. ROS and some metabolites that regulate the activation of specific
transcription factors, which may exert their functions in the nucleus, have been proposed to be among the signals for communication
between mitochondria and the nucleus (49). Beside the effects of the nuclear genome on the expression of mitochondrial genes, the mitochondrial genome can also affect
the expression of some nuclear DNA-encoded mitochondrial proteins (154, 155). Oxygen tension in tissue cells, exercise, and hormone levels have been shown to be able to regulate the mRNA levels of
cytochrome c oxidase subunits in the mammal (156, 157). Therefore, mitochondrion may act as a sensor in regulating energy metabolism and release of ROS in response to extracellular
stimuli. Under normal physiological conditions, the ROS generated by the respiratory chain can be scavenged by enzymatic and
nonenzymatic antioxidant systems to prevent deleterious oxidative damage to the cell. However, as a result of aging-associated
increase of ROS generation in the respiratory chain and decrease in the intracellular concentrations of antioxidants and activities
of free radical scavenging enzymes, an elevation of ROS and oxidative stress is inevitable, and oxidative damage and apoptosis
might just occur in the cell (158). As the major intracellular source of ROS, mitochondria and their constituents including mtDNA are particularly vulnerable
to oxidative damage. Experimental data from this and other laboratories have provided ample evidence to support the notion
that mutation and oxidative damage to mtDNA and mitochondrial respiratory function decline in tissue cells are important contributors
to human aging (Fig. 1⇑) (12, 92). Although a causal relationship between oxidative modification and mutation of mtDNA, mitochondrial dysfunction, and aging
has emerged, the detailed mechanisms by which these molecular and biochemical events cause human aging remain to be established.
Understanding of the age-related changes in the structure and function of mitochondria in the aging process is critical for
the elucidation of the molecular basis of aging and for the better management of aging and age-related diseases in the new
millennium.