The aging paradox: free radical theory of agingBadithe T. Ashok, Rashid Ali*
Department of Biochemistry, J.N. Medical College, Aligarh Muslim University, Aligarh 202 002 (U.P.) IndiaReceived 17 August 1998; received in revised form 13 January 1999; accepted 13 January 1999
There are more than 300 theories to explain the aging phenomenon. Many of them originatefrom the study of changes that accumulate with time. Among all the theories, the free radicaltheory of aging, postulated first by Harman, is the most popular and widely tested, and is based onthe chemical nature and ubiquitous presence of free radicals. This review aims to recapitulatevarious studies on the role of free radicals in DNA damageboth nuclear as well asmitochondrialthe oxidative stress they impose on cells, the role of antioxidants, the presence ofautoantibodies, and their overall impact on the aging process. 1999 Elsevier Science Inc. Allrights reserved.
Keywords: Aging; Autoantibodies; DNA damage; Free radicals; Reactive oxygen species
Aging is the accumulation of changes responsible for the sequential alterations thataccompany advancing age and the associated progressive increases in the chance of diseaseand death. These changes may be attributed to disease, environment, immune dysfunction,and to an inborn processthe aging process. This produces aging changes at an apparentlyunalterable and exponentially increasing rate with advancing age. The contribution of theaging process to changes occurring with age are small early in life but rapidly increase withage because of the exponential nature of the process (Harman, 1991).
Many theories have been propounded to account for the aging phenomenon (Warner et al.,1987; Medvedev, 1990), but no single theory is generally acceptable. In fact, it is doubtful
* Corresponding author. Tel.: 191-571-400535; fax: 191-571-400678.
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that a single theory can explain all the mechanisms of aging (Schneider, 1987). Among themany theories advanced, the free radical theory of aging shows promise of application today.This theory was first proposed by Harman (1956), based on a premise that a single commonprocess, modifiable by genetics and environmental factors, was responsible for the aging anddeath of all living beings (Harman, 1981). The theory postulates that aging is caused by freeradical reactions may be involved in production of the aging changes associated with theenvironment, disease, and the intrinsic aging process. This theory is based on the chemicalnature of free radical reactions and their ubiquitous and prominent presence in living beings(Holmes et al., 1992).
2. Materials and methods
2.1. Caloric restriction and life span
McCay et al. (1943) first reported an important relationship between life span and dietarycaloric intake in rats and mice. They observed that animals fed calorically restricted dietslived about one-third longer than animals fed ad libitum. A new hypothesis for the molecularmechanisms of caloric restriction effects based on the rate of DNA damage was proposed(Simic and Bergtold, 1991) and was extended further to the mechanisms of aging. It wasproposed that a high metabolic rate and dietary caloric intake would generate higher yieldsof O2, H2O2, and zOH (Szatrowski and Nathan, 1991).
Species with a high metabolic rate have a high rate of oxidative DNA damage (Ames,1989), with humans having the lowest rate of DNA damage. The studies showed that theyields of biomarkers were not proportional to specific metabolic rate, indicating the partic-ipation of other parameters like the efficacy of the antioxidant defense system. Dietarycaloric intake was found to play a major role in the rate of oxidative DNA damage (Simicand Bergtold, 1991). The effect of caloric intake on DNA damage is, however, not as largecompared with the effect of specific metabolic rate of different species.
For mice and rats, a 40% reduction in dietary caloric intake extended maximum life spanby one third (Weindruch and Walford, 1988). Dietary caloric intake may also affect thefidelity of DNA repair and general free radical damage observed in organisms (Turturro andHart, 1991). Therefore, a reduced rate of oxidative DNA damage, increased fidelity of DNArepair, and an overall decrease in free radical damage constitute a molecular basis forextension of maximum life span in organisms subjected to caloric restriction (HaleyZitlinand Richardson, 1993).
2.2. DNA damage and aging
The most reactive of all oxygen free radicals (OFR) is the zOH, which reacts rapidly withdeoxyribose and DNA bases (von Sonntag, 1987). Two products of zOH damage to DNAserve as suitable biomarkersthymine glycol and 8-hydroxydeoxyguanine (8-OHdG).These modified bases are eliminated by DNA repair enzymes. An equilibrium level of theseproducts on DNA, however, is always present (Fraga et al., 1990). It is now possible to
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measure less than nM levels of products generated by oxidative damage to DNA (Dizdaro-glu, 1991). The measurements can be made either directly on DNA or on excised damagedproducts found in urine (Ames, 1989). We have recently demonstrated age-related increasesin oxidative lesions in DNA isolates from aged population (approximate age 70 years) byusing a monoclonal antibody against reactive-oxygen-species-modified DNA (ROS-DNA)(Ashok et al., 1997), while DNA isolates from a younger population (,50 years) were notbound by the monoclonal antibody. This antibody was also used to detect oxidative lesionsin DNA from cancer and systemic lupus erythematosus patients (Ashok and Ali, 1998a;Ahmad et al., 1998). The yield of urinary biomarkers of oxidative DNA damage is relatedto specific metabolic rate (Ames, 1989) and more justifiably to maximum life span (Simicand Bergtold, 1991). Formation, measurement, and application of these products in aging andcarcinogenesis have been excellently reviewed (Loft and Poulsen, 1996).
Cancer and aging, the two biological consequences with complex etiology, also may be afunction of other types of endogenous DNA damage. Despite enzymatic repair and otherdefenses, continuous DNA damage progressively alters genomic sequence, as indicated byaging (King et al., 1994) and leads to the development of cancer by activation of oncogenesor inactivation of suppressor genes. Consequently, the probability of mutation and concom-itant development of tumors as well as reduction of maximum life span increases with therate of oxidative DNA damage.
The apparent relationship between DNA damage and maximum life span suggests twomajor conclusions: 1) Agents or processes that increase the rate of DNA damage anddiminish DNA repair may accelerate the rate of aging, thereby decreasing life span (WeirichSchwaiger, 1994); and 2) The rate of damage apparently may be decreased by lowering thespecific metabolic rate, decreasing dietary caloric intake, inhibiting damaging species, orincreasing the fidelity of DNA repair (Higami et al., 1994).
Cutler (1985a) proposed that longer-lived species had more protective mechanisms re-sulting from genetic changes occurring in regulatory genes. The data showed that humans didhave higher levels of superoxide dismutase relative to the amount of oxygen used. Anotherassay showed that the rate of auto-oxidation of brain tissue was least in humans. It would beappropriate to assume that these tissues would have a lower level of oxidative damage. Cutler(1992) has indeed demonstrated a lower steady state level for 8-OHdG in longer-livedspecies compared with short-lived ones, which could be due to the fact that high levels ofantioxidants have been found in human tissue. The comparative levels of antioxidants, DNAdamage, and urine levels of oxidized nucleosides, together, support the importance ofoxidative damage as a cause of aging.
2.3. Genetic stability and oxidative stress in aging
Despite the vast complexity of the aging process, relatively few mechanisms might governthe aging rate (Cutler, 1992). This concept forms the basis of the longevity determinanthypothesis of aging and depends on the following: 1) if cell dysdifferentiation represents aprimary aging process; 2) if reactive oxygen species (ROS) cause dysdifferentiation; and 3)if mechanisms acting to stabilize the proper state of differentiation, such as antioxidants,represent a class of longevity determinants.
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The longevity determinant gene hypothesis predicts that aging is a result of normalbiological processes necessary for life which have long-term negative or aging effects on theorganism. These normal biological processes are broadly classified as: 1) developmentallylinked biosenescent processes; and 2) the continually acting biosenescent processes. Thelongevity of a species is, therefore, related to how efficiently the aging effect of thesecommon problems are reduced.
ROS generated during normal oxidative metabolism interacts with the cells geneticapparatus and alters the proper state of differentiation. These changes, occurring duringnormal aging, are termed dysdifferentiation, leading to aging and cancer (Cutler, 1991a).Much evidence supports the involvement of ROS in dysdifferentiation (Table 1). Dysdif-ferentiation may lead to an increase in improper gene expression with age, as in the case ofc-myc expression, and may be caused by genetic alterations. Such alterations may accumu-late with time in normal tissue, increasing the probability steadily with age of the transfor-mation of every cell in the tissue (Tlsty et al., 1995).
2.4. Oxidative mitochondrial DNA damage and aging
Harman (1972) first proposed the role of free radicals in mitochondrial damage andconsequent senescence. It has been shown that aging indeed results in a decrease in thenumber of mitochondria, and that the organelles in older cells undergo biochemical alter-ations. A reduction in oxygen in the respiratory chain of the inner mitochondrial membraneis accompanied by the formation of superoxide radicals depending on the metabolic state ofthe organelles (Flohe, 1982). Several studies have suggested that mtDNA may accumulatemore oxidative DNA damage relative to nuclear DNA, which may serve as a usefulbiomarker for ROS-associated diseases (Yakes and Van Houten, 1997). Table 2 shows aseries of biosenescent hypotheses on the role of mitochondria and their damage in aging.
It has been proposed that aging is caused by injury to mitochondrial DNA (mtDNA) andlipid peroxidation by free radicals from the inner mitochondrial membrane (Bagchi et al.,1995; Richter, 1995). Fast-replicating cells do not suffer free radical attack because of theirlower levels of oxygen utilization. Irreversibly differentiated cells, such as neurons, however,suffer mitochondrial aging owing to high levels of oxygen utilization. Mitochondria fromshort-lived species generate larger amounts of ROS than long-lived species. Damage tomtDNA, therefore, would block mitochondrial turnover and replication, with concomitant
Table 1Reactive oxygen species as a causative factor in cell dysdifferentiation
1. Low concentration of mutagenic/carcinogenic agents induce improper gene regulation, some by inductionof transposable elements involved in control of gene expression.
2. ROS reacts with chromatin, modify DNA bases and cause single-strand breaks.3. ROS induce chromosomal aberrations, and longer lived species have a slower rate of accumulation of
chromosomal aberrations.4. Longer-lived species show a slower rate in accumulation of lipofuscin age pigments.5. Aging rate is proportional to metabolic rate (cal/g/day) for many different mammalian species.
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peroxidative membrane breakdown and replication, autophagic digestion of organelles, anddecline in production of ATP and protein synthesis.
Another major modified lesion present in mtDNA of rat liver is 8-OHdG. It is 16 timesthe level found in nuclear DNA (Richter, 1988), thus showing a high degree of oxidativedamage to mtDNA. Studies suggest the lack of adequate repair mechanisms in mtDNA(Fukanaga and Yieding, 1979), specifically the repair of strand breaks. The potential forintrinsic mitochondrial mutagenesis clearly exists.
Studies on differentiated cells of Drosophila muscle and mammalian hepatocytes andneurons indicate a senescent change in the amount and/or structure of mitochondrial popu-lations (Miquel and Fleming, 1986). The activity of cytochrome oxidase, often used as amarker enzyme for the mitochondrial inner membrane, shows a senescent decline in mito-chondria of liver and striated muscle (Miquel and Fleming, 1986). A senescent decline inextractable mtDNA was also observed in Drosophila (Massie et al., 1975) and liver of Fisherrats (Stocco and Hutson, 1978). Changes in mtDNA structure, characterized by an increasein dimeric catenated forms and appearance of DNA with altered ethidium bromide interca-lation, in aging mice and rats have been reported (Murray and Balcavage, 1982). Also, anage-dependant increase in frequency of circular dimers was seen in mtDNA of brain, heart,kidney, and liver of rats and mice (Piko et al., 1984). Alterations of the mitochondria andmtDNA may play a central role in aging and age-related degenerative diseases (Wei, 1998).
2.5. Antioxidants and aging
An attractive explanation of the causes of aging is the damage to biosystems caused byfree radical processes (Harman, 1991). It has been postulated that if free radical reactionswere the major cause of aging, a reduction in their levels by antioxidants or antioxienzymes(e.g., catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx))should, in principle, retard aging with a concomitant increase in maximum life span.However, levels of antioxidants and antioxienzymes in different mammalian species appear
Table 2Mutations of mitochondria DNA and its role in aginga
Probable cause of aging Reference
Free radical damage to mitochondria andtheir DNA.
Free radical or lipid peroxide inducedinactivation of mitochondrial DNA(mtDNA) of fixed mitotic cells.
Miquel et al. (1980)
Irreversible injury to mtDNA. Fleming et al. (1982)Changes in the inner membrane due to free
radical effects on both nuclear andmtDNA.
Intrinsic mitochondrial mutagenesis interminally differentiated cells.
Miquel and Fleming (1984)
Nuclear accumulation of mtDNA fragments. Richter (1988)a Adapted from Miquel (1991).
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to be constant. This relationship has been explained by considering the concentrations ofantioxidants and antioxienzymes relative to specific metabolic rate rather than absoluteconcentrations (Cutler, 1985b).
Cells are well-protected by a series of antioxidants such as vitamin E, C, quinones,glutathione, etc., and antioxidant enzymes like SOD, CAT, and GPx to counter oxidativeDNA damage. The levels and activities of SOD and GPx are much higher in long-livingspecies than in short-living ones (Cutler, 1991b). This is very clear from the rate ofauto-oxidation in brain homogenates assayed by the level of malonaldehyde accumulation(Cutler, 1985b), which has been found to be lowest in humans and highest in mice.Conflicting reports exist with increased, decreased, or unchanged activities of SOD, CAT,and GPx (Sohal et al., 1990; Benzi et al., 1989). The activity of SOD was found to be stablein subjects less than 65 years of age and slightly decreased in the elderly (Guemouri et al.,1991). Similarly, GPx activities increased in young adults, became stable in adults under 65years, and declined in older persons (Artur et al., 1992). Variations in CAT were found tobe similar to those in SOD and in agreement with earlier reports (Schafer and Thorling,1990).
Administration of large amounts of antioxidants (vitamins A and E) and nutrients in-creases the average life expectancy of animals (Duthie et al., 1996). Selenium is an importanttrace element for GPx activity and acts as a protective agent against the toxic effect ofhydroperoxides. Deficient levels of selenium have been implicated in carcinogenesis, hepaticlesions, and coronary heart disease. Selenium supplementation has been reported to increaselongevity, with populations having an abundance of selenium having low ischemic heartdeath rates and vice versa (Simonoff et al., 1992).
Vitamins A and E also play an important role in premature infants, parenteral nutrition,elderly people, and cancer patients. Vitamin E deficiency leads to lowered erythrocyte lifespan, neurological dysfunction, and certain forms of cancer (Trickler and Shklar, 1987),cardiovascular diseases (Gey et al., 1987), and impaired immune response. High levels ofvitamin E are associated with lower incidence of infections in healthy adults over 60 yearsof age (Chevance et al., 1985). These studies suggest that elderly individuals might benefitfrom a supplementation of vitamin E.
Vitamin A is required for growth, reproduction, vision, and immune processes, and for itsinhibitory effect on carcinogenesis. Low serum retinol levels have been reported to beassociated with an increase in risk of cancer (Kark et al., 1981). Simonoff et al., (1992) havereported the protective effect of vitamin A and b-carotene against cancer. Supplementationof vitamin A would be beneficial, but care should be exercised to prevent toxic hypervita-minosis A.
2.6. Protein modification in aging
The ROS attack on proteins, direct or indirect (through lipid peroxidation), results instimulation or inhibition of enzymatic activity. Damage to membrane transport proteins leadsto alteration in intercellular calcium and potassium levels affecting cellular metabolism. Insome cases, proteinase attack on altered protein is enhanced. Signal transfer in cells is also
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modified as a consequence of ROS-induced changes in receptor proteins and gap junctionproteins (Klaunig et al., 1998; Stadtman and Berlett, 1998).
There are contradictory reports on the decline in catalytic activity of enzymes and theiraccumulation in aging. Moreover, the evidence that the accumulated inactive molecules areoxidized is lacking. The mechanisms of degradation of oxidized proteins have also not beenclearly established. It is, therefore, premature to relate changes in the activity of individualproteolyic systems to the accumulation of oxidized proteins in aging. (See review by Deanet al., 1997.)
The protein oxidative damage thus can result in the modification in structure, enzymaticactivity, and signaling pathways. The basic questions of whether protein oxidation is primaryor secondary in aging and is critical in the progression of aging require additional scientificinput.
2.7. Autoantibodies in aging
Healthy, aged people have an increased frequency of autoantibodies (Hijmans et al., 1984;Manoussakis et al., 1990). The phenomenon indicates immunoregulatory perturbationsduring senescence whose nature is, however, obscure (Talor and Rose, 1991; Hartwig, 1992).Much less is known about the pathological and physiological significance of age-relatedautoantibodies. They have been associated with shortened survival in humans and sponta-neous development of organ-specific autoimmune lesions in aged nonautoimmune mice(Mariotti et al., 1992).
Of significant interest is the presence of antinuclear antibodies in the aged population(6080 years) (Moulias et al., 1984). Reports show about 1037% of antinuclear antibodiesprevalence in the elderly compared with 3.8% in the young population (Talor and Rose,1991). Xavier et al. (1995) have also reported high levels of antinuclear antibodies in theaged, and, particularly in high titers, these antibodies assume a persistent character. Studiesalso indicate the presence of anti-DNA antibodies, characteristically found in the autoim-mune disease systemic lupus erythematosus, in persons over 70 years of age (Schuller et al.,1981; Xavier et al., 1995), and anti-ssDNA antibodies (Kasjanov et al., 1984). Recent studiesby us also support and reinforce these observations. In addition to the presence of high levelsof anti-DNA antibodies in the aged, of particular interest was the presence of higher levelsof antibodies binding to ROS-DNA (Ashok and Ali, 1998b). This study reiterates that olderpeople are subjected to tremendous oxidative stress leading to DNA damage, resulting in theinduction of antibodies against ROS-DNA. The antibodies to ROS-DNA may, therefore, becrossreactive to native DNA. It has been unambiguously shown earlier that ROS modifica-tion of DNA drastically alters its immunogenicity, inducing high-titer antibodies that cross-react with native DNA (Ashok and Ali, 1999).
It is also likely that the high frequency of autoantibodies observed in the elderly is at leastpartly associated with diseases appearing with age and partly with immunological senes-cence. Another hypothesis is based on the function of the thymus (Rose, 1994). Because thethymus involutes asymmetrically, a clonal imbalance is generated with age as the proportionof autoantigen-specific helper/inducer T cells increases relative to the number of autoantigen-specific regulatory T cells. This leads to increased levels of autoantibodies with age. In
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addition, nonspecific immunoregulatory mechanisms also increase with age. As the thymiccortex atrophies, the response to foreign antigens declines, whereas the response to self-antigens rises, generating the age-related increase in autoantibodies.
A sizeable majority of published reports support the free radical theory of aging. Focus isnow shifting toward the mutations of mtDNA, as it is susceptible to severe free radicaldamage. However, many more comprehensive studies are required to understand the mo-lecular mechanisms underlying the damage to the genome by free radicals and how theycontribute to aging.
The Department of Biotechnology, Government is gratefully acknowledged for a researchgrant (BT/TF/09/36/90) to R.A. B.T.A. is a recipient of NET Fellowship of the UniversityGrants Commission, Government of India.
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