More than 100 different types of oxidative DNA lesions that occur in vivo have been identified (10). Of the many types of oxidative damage that have been identified, 7,8-dihydro-8-oxoguanosine (oxo8dG) has been the most extensively studied lesion because it represents approximately 5% of the total oxidized bases that are known to occur in the DNA and therefore is present in quantities that are sufficient to be readily detected (7). Oxo8dG is formed by the attack at carbon 8 of the purine ring by a hydroxyl radical, resulting in a hydroxyl moiety replacing the hydrogen atom (Fig. 1) (9). Oxo8dG can also be produced by a variety of chemical agents (e.g., 2-nitropropane, potassium bromate, and ciprofibrate) and γ-irradiation in vivo.
Oxo8dG in DNA is considered a potentially important factor in carcinogenesis because oxo8dG preferentially base pairs with adenine rather than cytosine, generating a G to T transversion. If this transversion is not repaired, a mutation will occur that can lead to induction of carcinogenesis. Thus, increases in the level of 8oxodG can have important implications for mutagenesis and the induction of tumors.
In addition to guanine, adenine and the pyrimidines can also be modified by interaction with hydroxyl radicals (14). For example, interaction at the carbon 8 position of adenine results in the formation of 8-oxo-2-deoxyadenosine (oxo8dA). In mammalian cells, oxo8dA has been associated with A to G and A to C transitions. However, oxo8dA does not block DNA synthesis and is not mutagenic in bacteria. Therefore, because of the relatively benign nature of oxo8dA, interest in this lesion has been limited. Oxidation of cytidine can generate cytidine glycol, a highly unstable species that decomposes rapidly to form 5,6-dihydro-5,6-dihydroxy-2-deoxyuridine (uridine glycol), which has been detected in vivo. Uridine glycol has been shown to induce a C to T transition. When uridine glycol is dehydrated it forms 5-hydroxy-2-deoxycytidine (5-OhdC), and 5-OhdC also has been shown to generate a C to T transition. Another lesion, thymidine glycol, is the major thymine derived adduct detected after oxidation or irradiation of DNA in vitro and in vivo. Thymidine glycol is known to induce a T to C transition. In summary, a variety of oxidative modifications to DNA occur that can lead to misreading of DNA templates if not corrected and to formation of mutations. DNA mutations can result in production of proteins that may or may not show a changed function. Therefore, oxidative damage to DNA can contribute to both carcinogenesis and age-related alterations in biological functions resulting from altered or inactive proteins.
OXIDATIVE STRESS HYPOTHESIS OF AGING
Oxidative stress and damage to cellular components from reactive oxygen species (ROS) have been proposed to play an integral role in the age-related deterioration in biochemical and physiologic processes and the incidence of age-related disease. This concept was established more than a half century ago as the basis of the free radical theory of aging proposed by Denham Harman (5). The free radical theory points to a causal role for free radicals produced as a by product of aerobic metabolism in generating random oxidative damage that can accumulate over time and contribute to aging and age-associated diseases. To date, the premise of this theory remains one of the most popular explanations for how aging occurs at the biochemical level. The original theory has recently been modified to include reactive oxygen species such as peroxides, which are not true free radicals, but which also contribute to oxidative damage within a cell. The current theory, the oxidative stress hypothesis of aging, emphasizes that an imbalance exists between cellular antioxidants and pro-oxidants in resulting in a chronic state of oxidative stress and a steady-state accumulation of oxidative damage in a variety of macromolecules (12).
Evidence in support of this theory and for a causal role for oxidative stress or damage in aging has been generated by numerous studies over the past 20 yrs showing a strong correlation between increasing age and the accumulation of oxidative damage to a variety of biomolecules (2). Most of the early studies on aging and oxidative stress examined the accumulation of oxidative damage to lipid with age; however, in recent years, a number of studies have looked at alterations in oxidative damage to protein and DNA. Further support for a role of oxidative damage in aging comes from the fact that increased survival in rodents after dietary restriction is correlated to a reduction oxidative damage in a lipids, proteins, and DNA (15). In addition, some age-related pathologies have been shown to be associated with increased levels of oxidative damage to cellular components (1).
AGING AND OXIDATIVE DAMAGE TO DNA
Shown in Table 1 is a summary of studies that have measured the level of oxo8dG in nuclear DNA as a marker of DNA oxidative damage with age in rats. In the first published study on DNA oxidation with age in 1990, Fraga et al. observed a significant (approximately twofold) increase in oxo8dG levels in nuclear DNA isolated from liver, kidney, and intestine of male Fischer 344 (F344) rats between 2 and 24 months of age (see Table 1). However, no significant change in the levels of oxo8dG was found in nuclear DNA isolated from brain and testis in this study. Subsequent studies also reported tissue-specific alteration in DNA oxidative damage with age, that is, increases in some tissues and not in others. The effect of age on oxo8dG levels in other models is shown in Table 2. It is evident from the studies listed in Tables 1 and 2 that although many studies have reported a significant increase in oxidative damage to nuclear DNA with age, some investigators have been unable to detect a significant increase in DNA oxidation in rodent tissues with age. A reasonable explanation for the discrepancy in the results is the possibility of methodological problems that exist in the measurement of DNA oxidation, such as artifactual oxidation during the isolation of the DNA. In fact, the accurate measurement of oxidative damage to DNA has been subject to recent controversy because of the high probability of introducing oxidative damage to the DNA during the isolation procedure. Traditionally, DNA isolation was performed using phenol extraction. Using phenol to extract the DNA creates a potential problem because phenol is also a strong oxidizing agent and thus could introduce artifactual damage to DNA during the extraction process (3). Recently, DNA isolation using sodium iodide (NaI) has provided an important alternative to isolation with phenol. DNA isolated using NaI has very low levels of oxo8dG compared with DNA isolated using phenol. In our laboratory, we found that isolating the DNA using NaI greatly minimized the artifactual oxidation of DNA, allowing us to achieve reliable and accurate measurement of DNA oxidative damage (oxo8dG) (3). Using this method, we have measured the levels of oxo8dG increase with age in nuclear DNA isolated from a wide variety of tissues in F344 rats and two strains of mice (Tables 1–3) (4).
The data in Table 3 are the results from a study by our laboratory in which we measured the effect of age on DNA oxidation in male F344 rats and two strains of mice in DNA isolated using the NaI method (4). We observed a significant increase in oxo8dG levels in nDNA in every tissue and strain of rodent studied. In male F344 rats, oxo8dG levels increased in nuclear DNA isolated from liver, skeletal muscle, brain, kidney, and heart. We also found that the increase in oxo8dG levels varies considerably from tissue to tissue and between mice and rats. For example, the age-related increase in oxo8dG from 6 to 24 months of age ranged from nearly 90% in liver to over 350% in heart, and the age-related increase in DNA oxidation was greater in all tissues of F344 rats than in the same tissues in the mouse strains studied. Levels of oxo8dG were also increased with age in several tissues from young and old male B6D2F1 mice and C57BL/6 mice of both sexes. We found a similar tissue-to-tissue variation in the mice as we found in the rat, that is, the age-related increase in oxo8dG levels for male B6D2F1 mice and C57BL/6 mice were lowest in liver and kidney an highest in brain and heart. A possible reason for the higher level of oxo8dG in brain and heart is the higher metabolic activity of these tissues compared with other tissues that could translate to greater potential for exposure of the nDNA in these tissues to oxidative stress. In addition, because brain and heart are postmitotic tissues, cell turnover would be reduced compared with other tissues. Thus, we consistently found an increase in oxidative damage to DNA using this method, and these data strongly suggest that increased DNA oxidative damage is linked to aging as suggested by the oxidative stress theory.
AGING AND OXIDATIVE DAMAGE TO MITOCHONDRIAL DNA
Mitochondria are the primary site for superoxide anion generation and mitochondrial DNA is located in the mitochondrial matrix, associated with the inner mitochondrial membrane. Thus, it is reasonable that mitochondrial DNA may be particularly vulnerable to oxidative damage, and in fact, mtDNA has been shown to have a higher level of oxidative damage relative to nuclear DNA (3). Listed in Table 4 are several studies that have measured the levels of oxo8dG with age in mitochondrial DNA. Most studies measuring levels of oxo8dG in mitochondrial DNA have reported an increase; however, like the studies in nuclear DNA, there are some studies that failed to find an increase in oxo8dG with age in mitochondrial DNA. There are several possible factors that could contribute to the disparate results. First, the measurement of oxo8dG in mitochondrial DNA is more difficult than in nuclear DNA because of the relatively small amount of mitochondrial DNA compared with nuclear DNA. There are further complications because the mitochondria must be isolated from the tissue before isolation of the mtDNA, and the same problems exist concerning oxidation of DNA during isolation. Finally, contamination of mitochondrial DNA by nuclear DNA can lead to an underestimation of the amount of oxo8dG present in the mitochondrial DNA.
Our laboratory has measured oxo8dG levels in mitochondrial DNA isolated from liver, heart, and brain using NaI to isolate mitochondrial DNA (3). Mitochondria were pooled from several animals to increase the yield of mitochondrial DNA. We found significantly higher levels of oxo8dG in mitochondrial DNA than in nuclear DNA for all three of these tissues. Interestingly, we also found that the level of oxidative damage in mitochondrial DNA varies significantly from tissue to tissue. For example, the level of oxo8dG in mitochondrial DNA from liver was 40% to 50% less than that found in mitochondrial DNA isolated from either brain or heart. The levels of oxo8dG in mitochondrial DNA from brain and heart were 23- and 16-fold higher, respectively, than nuclear DNA levels. Table 3 shows the increase we observed in oxo8dG levels in liver and brain mitochondrial DNA with age in mice and rats. The level of oxo8dG in mitochondrial DNA from male F344 liver increased 71% between 6 and 24 months of age. Oxo8dG levels increased 51% in mitochondrial DNA isolated from the livers of old B6D2F1 mice compared with young B6D2F1 mice. The levels of oxo8dG in mitochondrial DNA also increased significantly with age in liver (126%) and brain (63%) from C57Bl6 mice.
EFFECT OF DIETARY RESTRICTION
Dietary restriction is known to extend the lifespan of rodents and to decrease the incidence of various age-related diseases (8). It has been proposed that the antiaging action of dietary restriction occurs through a reduction in oxidative damage to the various macromolecules, including DNA. If the age-related increase in DNA oxidation is important in aging, dietary restriction should reduce it. We have measured the effect of dietary restriction on age-related changes in nuclear and mitochondrial DNA in male F344 rats and in male B6D2F1 mice (4). The results of this study are shown in Table 5. We found that the level of oxo8dG in nDNA from the brain, heart, and skeletal muscle of dietary restricted 24-month-old rats were significantly lower than those of 24-month-old rats fed ad libitum. However, dietary restriction had no significant effect on the levels of oxo8dG found in rat liver or kidney nDNA. In contrast, dietary restriction completely prevented the age-related increase in oxo8dG levels in rat liver mitochondrial DNA. In male B6D2F1 mice, dietary restriction significantly reduced the levels of oxo8dG in nDNA from all tissues studied, for example, liver, kidney, brain, heart, and kidney. In addition, dietary restriction prevented the age-related increase in oxo8dG levels in liver mitochondrial DNA of the B6D2F1 mice. Our data suggest that dietary restriction has a greater effect on the age-related accumulation of DNA oxidative damage in mitochondrial DNA as compared with nuclear DNA. Also, the fact that dietary restriction has a greater effect in brain and heart compared with liver and kidney suggests that oxidative damage to some tissues may be more closely related to the aging phenotype.
EXERCISE AND DNA OXIDATION
Exercise increases the demand for energy and energy production several fold. During maximal muscle contraction in humans, oxygen consumption in resting local muscle fibers can increase severalfold higher than the increase in whole body oxygen consumption. The increase in electron flux in an effort to produce increased energy in the form of ATP has significant potential to increase local production of ROS and causes increased oxidative stress to the tissue. An increase in ROS production occurs in muscle after exercise, and there may be an increased susceptibility with age to the increased ROS production and oxidative stress imposed by exercise.
Little is known with respect to DNA damage specifically in muscle after exercise and even less about the effect of age on this process. Although some studies have observed DNA damage in response to exercise, (6) there are also studies that found the level of DNA damage unaffected (13). Overall, the intensity of the exercise seems to be a key factor in determining whether DNA damage is altered following exercise. Thus, studies showing exercise induced DNA damage usually involve vigorous exercise conditions and possibly also involved muscle damage, whereas the studies that failed to find DNA damage involved moderate or long-term exercise conditions. Surprisingly, in a recent study, 8 wk of wheel training in rats led to an attenuation of the age-related increase in oxo8dG levels in skeletal muscle (11). The effect of age on exercise-induced damage is an important area for future study.
Supported by Merit Review and Environmental Hazards Center grants from the Department of Veteran Affairs, NIH Grants 1PO1-AG14674 (AR) and 1PO1-AG20591 (HVR, AR), and a grant from the American Cancer Society (HVR).
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Keywords:©2003 The American College of Sports Medicine
mitochondrial DNA; nuclear DNA; exercise; dietary restriction; oxidative stress