Relationship between Antioxidants, Prooxidants, and the Aging Processa R. S. SOHAL AND W. C . ORR Department of Biological Sciences Southern Methodist University Dallas, Texas 75275 A key assumption of the free radical hypothesis of aging is that the endogenous antioxidant defenses of aerobic cells are deficient and the residual prooxidants exert a certain level of oxidative stress even under normal physiological conditions. The hypothesis would thus predict that, other things being equal, the level of oxidative stress would be directly related to the rate of senescence. Such predictions, however, are likely to become confounded if senescence is found, in reality, to be due t o the involvement of several independently variable causal factors, as is widely conjectured at present. From an investigative point of view a multifactorial aging process will pose a very daunting problem because of the difficulty in apportioning relative causality to various individual factors. Notwithstanding, to simplify the strategy for identifying the possible causal factors involved in the aging process, it can be reasoned that any causal hypothesis of aging should attempt to explain: (1) individual aging, that is, loss of homeostatic ability in individual animals as a result of senescence, and ( 2 ) variations in life spans especially among phylogenetically closely related species as well as intraspecies variations in longevity. With these caveats in mind, the objective of this article is to provide a synthetic overview of our studies dealing with the possible association between antioxidants, prooxidants, and the aging process. METABOLIC RATE AND AGING It has been amply demonstrated in mammalian species that the rate of energy production is inversely proportional to the maximum life-span potential of the species (MLSP), albeit with some deviations from this generalization, most notably in However, the strongest studies in support of the view that the rate of metabolism is linked to the rate of aging is provided by experimental evidence in poikilotherms and hibernating mammals. Life spans of insects, such as Drosophila rnelanogaster and the housefly Musca dornestica, were shown to be much longer towards the lower than towards the upper limits of tolerated ambient temperature..'-' Life spans of Turkish hamsters were reported to be lengthened in proportion to the time spent in hibernatiom6 As variations in ambient temperatures do not elicit responses of a similar magnitude in various biochemical processes, objections have been raised regarding the mechanisms that may be operative in influencing life spans at different temperature^.^,^ To avoid such complications, the effect of metabolic rate on life span was studied in this laboratory, employing experimental regimens that affected metabolic rate by modulating the level of physical activity.*p9Environmental aThis research is currently supported by grants R 0 1 AG7657 and R01 AG8459 from the National Institutes of Health-National Institute on Aging. 14
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conditions, such as overcrowding, low female-to-male ratio, and large housing containers, which increased the level of physical activity (measured by Radar Doppler), invariably decreased longevity and vice versa.s~x~y If the houseflies are confined individually in small vials where they are able to walk but are unable to fly, the average and maximum life span is prolonged more than twofold that of houseflies kept as a group in a large cage, where they can fly and interact with each other (FIG. 1). This and other experimental data leave little doubt that metabolic rate influences the rate of aging because the maximum life span is prolonged. The inverse relationship behveen metabolic rate and life span was originally encapsulated by Pearlio in the rate ofriving theory. It is unfortunate that some authors have mistakenly attacked Pearl's inference, because the metabolic potential or the total amount of energy consumed during life differs under different environmental conditions or in different strains and species. The main point of Pearl's theory is that for a given genotype the rate of aging is dependent on the rate of metabolism. All the available experimental evidence conforms with this simple relationship. Much criticism of the rate of living theory seems to be based on misinterpretation of the postulate.s The mechanism by which metabolic rate affects life span is controversial, but
FIGURE 1. Survivorship curves of male houseflies kept under conditions of relatively low (LA) and high (HA) levels of physical activity. LA flies were housed in small vials where they were unable to fly, whereas HA flies were housed in 1-cubic-foot cages where they could fly.
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should rationally involve several basic processes. As also recently pointed out by Masoro and McCarter,Ii the rate of metabolism can be viewed in the context of processes related to fuel consumption. Although our laboratory has concentrated on the products of oxygen metabolism, utilization of carbohydrates, fats, and proteins also involves the generation of potentially deleterious products. For example, carbohydrate metabolism give rise to glycation reactions, free fatty acids can affect cell membrane functions, and amino acids may have nephrotoxic and neurotoxic effects." Partially reduced oxygen species (ROS) are widely speculated to play a role in senescence ever since they were shown to be the basis for oxygen toxicity. It has been proposed that because of the inadequacy of antioxidant defenses, cells are under a certain level of oxidative stress even under normal physiological conditions.'* The main evidence in support of this view is that oxidatively damaged macromolecules can b e demonstrated in normal healthy organisms.i3 On the assumption that the rates of oxygen consumption and ROS generation are correlated, it is widely postulated that animals with relatively higher metabolic rates are also under relatively higher levels of oxidative stress because of elevated levels of ROS. This
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inference is supported by our finding that the rate of alkane exhalation by flies is decreased at lower ambient temperatures, which have a corresponding effect on the rate of oxygen con~umption.'~ OXIDATIVE STRESS AND AGING A crucial question concerning the relationship between ROS and aging is whether the level of oxidative stress remains relatively constant or increases as a function of aging. In vivo studies in rats and houseflies (FIG.2) have shown that the rate of alkane exhalation increases with age.14 As alkanes are products of lipid peroxidation, this is interpreted to indicate that the in vivo level of oxidative stress increases during senescence. In the housefly15 and Drosophila l 6 we found an agerelated increase in the ratio of GSSG/GSH. An age-related decline in the ratios of NADPH/NADP+ and NADH/NAD+was shown in rat skeletal muscle17 and housefly15 homogenates. Such data tend to support the interpretation that the level of oxidative stress increases during the aging process. The next pertinent question concerns the mechanism by which the level of oxidative stress increases during aging, that is, is it due to a decline in antioxidant defenses or an increase in the rate of prooxidant generation? A perusal of the literature indicates a chaotic lack of agreement.18J9Activities of the same enzymes in the same tissue of the same species are reported to go up or down or remain unchanged during aging. A study of the heart, brain and liver of Sprague-Dawley rats in this laboratory indicated the lack of a consistent pattern.20 Interestingly, catalase and glutathione peroxidase exhibited a mutually contradictory pattern which may be taken to mean that the ability to remove HzOZis not notably compromised as a result of aging (TABLE 1, FIG.3). An age-related decline in superoxide dismutase and catalase activity was observed in the houseflyz1 but not in Drosophila16 where superoxide dismutase activity increased during aging whereas catalase activity deTABLE1. Units of Catalase Activity in 3- and 18-Month-Old Rats Age (mo) 3 18
Liver 56.5 +. 3.1 48.6 2 0.9
Heart 2.6 +. 0.5 2.4 f 0.09
Brain 0.163 +. 0.002 0.219 f 0.006
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FIGURE 3. Comparison of the activities of superoxide dismutase (a), glutathione peroxidase (b), and glutathione reductase (c) in the homogenates of liver, heart, and brain between 3- and 18-month-old Sprague-Dawley rats.
creased only after the average life span of the population had been achieved (FIG.4). As the decline in antioxidant defenses did not precede the dying phase of the flies, there is no compelling argument to believe that decreased antioxidant defenses causally contribute to the death of the flies. Thus, in these two dipteran species, the pattern of antioxidant defenses during aging was quite different. Overall, the lack of
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a uniform age-related pattern in antioxidant defenses tends to suggest that senescence of individual animals cannot be explained on the basis of a decline in such defenses. For the sake of debate, let us examine the possible significance of the reported age-related declines in antioxidant defenses. In virtually all cases, the age-associated declines of individual antioxidant enzymes are less than 50%, most often around 10-25%. Do declines of such magnitude have functional consequences in terms of the aging process? Studies on SOD and catalase hypomorphic mutants in Drosophilu rnelanogaster indicate no decrease in life span in flies with even less than 50% of SOD or catalase activity.22-25 Such data cast doubt on the role of the decline in antioxidant defenses being a causal factor in senescence. This should not be taken to mean that antioxidant defenses are not vital for survival, but rather that the observed decline in antioxidant defenses is not likely to have a significant impact on the age-related loss of functional capacity. One of the fascinating initial questions about the free radical hypothesis was whether the species-specific differences in life spans within a phylogenetic group, say primates or mammals, correspond to variations in antioxidant defenses. Tolmasoff et al.26 initially reported a correlation between SOD divided by metabolic rate and maximum life span of the species (MLSP) in a sample of 13 mammals. However, this approach has been criticized as being inappropriate. For example, Sullivanz7pointed out that because the metabolic rate itself is inversely correlated with MLSP, any unrelated parameter such as hemoglobin content/unit blood, which is similar in various mammalian species, would, using this approach, also be found to be correlated with MLSP. Studies in this laboratory have revealed no consistent pattern with regard to the activities of SOD, catalase, and glutathione peroxidase and the concentration of glutathione in the heart, brain, and liver of six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig, and cow.28In each organ, some antioxidant defenses were positively correlated with MLSP and some were negatively correlated with MLSP, indicating a possible compensatoty balance among the various components of the antioxidant defenses. No overall relation between antioxidant defenses and MLSP was detectable (FIG. 5). Thus, according to current information, antioxidant defenses neither seem to decline with age nor correspond to MLSP, thus weakening the possibility of a direct involvement in aging. Nevertheless, the possibility that antioxidant defenses may play a role in aging or in determining the level of metabolic potential cannot as yet be totally ruled out. One approach to help resolve this issue is the use of transgenics to study the effects of overexpression of antioxidant enzymes on life span and metabolic potential. Studies by Seto et al.22
Catalase
.
.9=0 Glutathione Red
.c6H
~eurv,vorship
FIGURE 4. Age-related profile of antioxidant defenses and survivorship curve of male Drosophila melanogaster (Orgeon R). A decline in antioxidant defenses does not precede the beginning of the dying phase of the population.
SOHAL & ORR. ANTIOXIDANTS, PROOXIDANTS, AND AGING
cow rabbit
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e 10
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~
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,
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FIGURE 5. Comparison of the activities of superoxide dismutase, catalase, and glutathione peroxidase, and concentration of glutathione in the heart of six different mammalian species with a maximum life span potential (MLSP) ranging between 3.5 and 30 years.
indicated that overexpression of SOD in transgenic Drosophilu does not extend life span. In contrast, Reveillaud et uLZ9reported a small increase in the mean but not maximum life span of Drosophilu. Recent studies in this laboratory have shown that overexpression of catalase does not extend the life span of Drosophilu. Because the activities of SOD and catalase act in concert to eliminate Oz- and H202, it is possible that for transformants with increased levels of both SOD and catalase, life spans may be altered. Such studies are currently being conducted in our laboratory. PROOXIDANTS AND AGING The alternate possibility, namely, that age-related increases in the level of oxidative stress may be due to enhancement of the rate of ROS generation, was examined in this laboratory. In the housefly, the rate of antimycin A-resistant mitochondrial respiration and mitochondrial Oz- and HZO2generation was found to increase with age.") Housefly mitochondria produced copious amounts of H 2 0 2(1-2 nmol/min/mg protein) even without the use of respiratory chain blockers such as
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FIGURE 6. Effect of age on the release of H2Oz by mitochondria from the flight muscles of the housefly. The incubation mixture included a-glycerophosphate as a substrate, p-hydroxyphenylacetate and horseradish peroxidase, but no respiratory inhibitors. (Reproduced, with permission, from Sohal and Sohal.3") -
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;
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AGE (days)
antimycin A31 (FIG.6). The activity of several oxidoreductases in the electron transport chain, such as NADH-ferricytochrome c reductase, NADH-cytochrome c reductase, and succinate-ubiquinone reductase, was also increased with age which suggested age-related alterations in the inner mitochondrial membrane, possibly involving cross-linking and increased electron flow between the respiratory comp l e ~ e s It . ~was ~ shown by Miki et ~ 1and. Girotti ~ ~ et that oxidative damage to membranes leads to cross-linking of membrane proteins. We reasoned that the age-related increase in 0 2 - and H202 generation by mitochondria may be due to oxidant damage of the inner mitochondrial membrane caused by locally generated ROS. Such oxidative damage may lead to cross-linking and other changes in membrane structural organization, resulting in an increased rate of ROS generat i ~ n . ~This ' hypothesis was tested using two approaches. First, it was found that experimental damage to mitochondria by exposure to tert-butyl hydroperoxide or ADP-iron ascorbate or AAPH (2,2-azobis (2-aminopropane) dihydrochloride, all of which induce lipid peroxidation, resulted in an increased production of H z 0 2by the treated mitochondria (FIG.7). Secondly, exposure of mitochondria to glutaraldehyde, a well known intermolecular cross-linker of proteins and lipids, also was found to greatly increase the rate of H202 generation by m i t ~ c h o n d r i a The . ~ ~ response to glutaraldehyde was much more pronounced in young flies than in old flies, which
FIGURE 7. Effect of 2,2-azobis (2aminoprophane) dihydrochloride (AAPH), a known inducer of lipid peroxidation, on mitochondrial release of H202. Mitochondria were exposed to different concentrations of AAPH for 20 minutes, centrifuged, washed twice in buffer, and then incubated. (Reproduced, with permission, from Sohal and S ~ h a l . ~ ' )
N 0 N
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would be expected if it is assumed that mitochondria in young flies are less damaged and exhibit less molecular cross-linking than do those in old flies. It therefore seems that self-inflicted mitochondrial damage and intermolecular cross-linking may provide the mechanism for increased production of H202. An age-associated increase in mitochondrial 02-and H202generation was also observed in the heart, liver, and brain of Sprague-Dawley rats.20 However, the magnitude of increase was less in the rat than in the housefly. Increased 02-and H 2 0 2production in aged rat hearts was also reported by Noh1 and H e g n e ~ - . ~ ~ As mentioned, any causal hypothesis of aging should explain intra- as well as interspecies variations in life spans. The hypothesis that the rate of mitochondrial 02-and H202generation is associated with the rate of aging was tested by comparing houseflies of similar chronological but different physiological ages. In one study, the physiological age (defined as metabolic age or “nearness to death”) of houseflies was altered experimentally by adjusting the environment to eliminate flight activity. This resulted in a twofold extension of their average and maximum life spans. In a second study, flies of different physiological ages were selected in the same population based on their ability to fly. All houseflies undergo a gradual age-related decline in flight 150
E
i p 3 . N
4
rat
r = -0.91
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FIGURE 8. Comparison of the rate of HzOz released by liver mitochondria from sixdifferent mammalian species with varying maximum life span potential (MLSP). (Reproduced, with permission, from Sohal ei al.”)
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ability, resulting in total inability to fly a few days prior to death. (Such flies are not in the process of dying, because they are able to successfully fertilize young virgin females.) Thus, in a population of aging flies, those undergoing accelerated senescence can be identified by their inability to fly. Such flies have been termed “crawlers,” and their cohorts, still capable of flight, are termed “fliers.”3s In both studies, the rates of mitochondrial 0 2 - and H202 production were found to be associated with physiological rather than chronological age.30.36At the same chronological age, flies kept under conditions of low physical activity had lower rates of 0 2 and H 2 0 2generation than did those kept under high activity conditions. Similarly, mitochondria from “fliers” had a lower rate of 02-and Hz02 generation than did those from “crawlers” of the same age.36 Thus, rates of 0 2 - and Hz02generation were associated with life expectancy of flies. Comparisons were made among six different mammals, namely, mouse, rat, guinea pig, rabbit, pig, and cow, to ascertain if variations in niaximum life span potentials of species (MLSP) were associated with the mitochondrial rate of 02-and
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H202 generation. The rates of 0 2 - and Hz02 generation by liver mitochondria were inversely correlated with MLSP.37Up to a sixfold difference was observed in the rate of 02-generation, whereas a 42-fold difference was observed in the rate of H2OZ3* generation between different species (FIG.8). Although this comparison involved a limited number of species, the results show that the rate of 0 2 - and H202generation is a better correlate of the rate of aging than are antioxidant defenses. It is also worth pointing out that under identical conditions of assay, mitochondria from different mammalian species produce widely different amounts of 0 2 - and H202 which would indicate differences in the structural organization of the ubiquinone-cytochrome b region of the electron transport chain. Overall, the results of our studies suggest that rates of 0 2 - and H202 may be a causal factor in aging. At present, the nature of the mechanism by which oxidative stress may influence the rate of the aging process is obscure. The two alternative views are: (1) Oxyradicals cause damage at various loci within cells, and due to the inadequacy of repair and regenerative mechanisms, such damage accumulates and underlies the aging process.*2Differences in aging rates are envisioned to be related to the efficiency of antioxidant defenses and/or repair mechanisms. (2) The second view, favored by US,^^,^ is that oxidative stress influences the rate of aging by modulating gene expression. It is reasoned that as age-related changes in various species follow a similar predictable pattern, any putative causal factor would be expected to act through effects on gene expression rather than simply by infliction of damage which would tend to be random. Furthermore, evidence for extensive structural damage during aging at the cellular level is lacking. Such damage that is observed is believed to be incidental to oxidant generation rather than causal to the aging process.
SUMMARY It is argued that reduced oxygen species may be one of the causal factors underlying the aging process. Experimental studies strongly support the view that the rate of metabolism is inversely associated with the rate of aging. It is pointed out that Pearl’s rate of living theory is widely misunderstood, because of the mistaken belief that it advocates a h e d metabolic potential for different species or genotypes within a species. The in vivo level of oxidative stress tends to increase with age in insects and mammals as indicated by increased exhalation of alkanes. A search for the causes of this increase revealed that an age-associated decline in antioxidant defenses is neither widespread nor very impressive in magnitude. A comparison of antioxidant defenses (activities of SOD, catalase, and glutathione) in six different mammalian species did not suggest a clear association between these defenses and maximum life span potential of the species. In contrast, mitochondria1 rates of 02-and H202were found to increase with age in insects and mammals, and the MLSP of six mammals was found to be inversely correlated with liver mitochondrial rates of 02-and H202 generation. It seems that the age-related increase in oxidative stress is mainly due to the enhanced rate of 0 2 - and H202 generation. It is hypothesized that variations in the rates of aging in different species, that are otherwise closely related phylogenetically, may be in part due to differences in rates of 0 2 - and H202 production. Overall, the rates of oxidant generation are a better correlate of the rates of aging than are the levels of antioxidant defenses.
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REFERENCES G. A. 1977. Life table modifications and life prolongation. In Handbook of the 1. SACHER, Biology of Aging. C. E. Finch & L. Haflick, eds.: 582-638. Van Nostrand. New York. R. G. 1984. Antioxidants, aging and longevity. In Free Radicals in Biology. W. A. 2. CUTLER, Pryor, ed. 6 371428. Academic Press. New York. J., P. R. LUNDREN, K. G. BENSCH & H. ATLAN.1976. Effects of temperature on 3. MIQUEL, the life span, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Dev. 5: 347-370. R. S. 1986. The rate of living theory: A contemporary interpretation. In Compara4. SOHAL, tive Biology of Aging in Insects. K. G. Collatz & R. S. Sohal, eds.: 23-44. SpringerVerlag. Heidelberg. 5. FARMER,K. J. & R. S. SOHAL.1987. Effect of ambient temperature on free radical generation, antioxidant defenses and life span in the adult housefly, Musca dornestica. Exp. Gerontol. 22: 59-65. C. P., R. C. O'BRIEN,G. C. GREENE & E. D. PAPAFRANGOS. 1981. Hibernation 6. LYMAN, and longevity in the Turkish hamster Mesocricetus brandti. Science 212: 668-670. 7. SOHAL,R. S. 1976. Metabolic rate and life span. In Interdisciplinary Topics in Gerontology. H. P. von Hahn, ed. 9 2540. S. Karger. Basel. S. S. & R. S. SOHAL.1973. Mating behavior, physical activity and aging in the 8. RAGLAND, housefly, Musca dornestica. Exp. Gerontol. 8 135-145. 9. SOHAL,'R.S. & P. B. BuCHAN.1981. Relationship between physical activity and life span in the adult housefly, Musca dornestica. Exp. Gerontol. 1 6 157-162. 10. PEARL,R. 1928. The Rate of Living. A. A. Knopf. New York. E. J. & R. J. M. MCCARTER. 1991. Aging as a consequence of fuel utilization. 11. MASOKO, Aging 3: 117-128. D. 1981. The aging process. Proc. Natl. Acad. Sci. IJSA 7 8 7124-7128. 12. HARMAN, R. E. & K. J. A. DAVIES.1991. Protein, lipid and DNA repair systems in 13. PACIFICI, oxidative stress: The free-radical theory of aging revisited. Gerontology 37: 166-180. QL H. SIES.1985. Effect of age and ambient 14. SOHAL,R. S., A. MULLER,B. KOLETZKO temperature on n-pentane production in adult housefly, Musca dornestica. Mech. Ageing Dev. 2 9 317-326. 1987. Age-related changes in the redox status of 15. SOHAL,R. S., P. L. TOY& K. J. FARMER. the housefly, Musca dornestica. Arch. Gerontol. Geriat. 6 95-100. & W. C. ORR. 1990. Effect of age on superoxide dismutase, 16. SOHAL,R. S., L. ARNOLD catalase, glutathione reductase, inorganic peroxides, TBA-reactive material, GSH/ GSSG NADPH/NADP+ and NADH/NAD+ in Drosophila melanogaster. Mech. Ageing Dev. 5 6 223-235. & A. GAFNI.1985. Age-related changes in the redox status of rat 17. NOY,N., H. SCHWARTZ muscle cells and their role in enzyme-aging. Mech. Ageing Dev. 2 9 63-69. 18. SOHAL,R. S. 1987. The free radical theory of aging: A critque In Review of Biological Research in Aging. M. Rothstein, ed. 3: 385-415. Alan R. Liss. New York. G., E. XIA& A. RICHARDSON. 1990. Effect of age on the expression of antioxidant 19. LO, enzymes in male Fisher F34 rats. Mech. Ageing Dev. 53: 49-60. & B. H. SOHAL.1990. Age-related changes in antioxidant 20. SOHAL,R. S., L. A. ARNOLD enzymes and prooxidant generation in tissues of the rat with special reference to parameters in two insect species. Free Rad. Biol. Med. 1 0 495-500. R. S., K. J. FARMER, R. G. ALLEN& N. R. COHEN.1984. Effect of age on oxygen 21. SOHAL, consumption, superoxide dismutase, catalase, glutathione, inorganic peroxides and chloroform-soluble antioxidants in the adult male housefly, Musca dornestica. Mech. Ageing Dev. 2 4 185-195. & G. M. TENER.1990. Overexpression of Cu-Zn superoxide 22. SETO,N. 0. L., S. HAYASHI dismutase in Drosophila does not affect life-span. Proc. Natl. Acad. Sci. USA 87: 42704274. W. J . & G. C. BEWLEY.1989. The genetics of catalase in Drosophila rnelano23. MACKAY, gasrer: Isolation and characterization of acatalasemic mutants. Genetics 122: 643-652.
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24. MACKAY, W. J., W. C. ORR& G. C. BEWLEY.1990. Genetic and molecular analysis of antioxidant enzymes in Drosophila melanogaster: A correlation between catalase activity levels, life span, and spontaneous mutation rate. In Molecular Biology of Aging. M. Clegg & S. O’Brien, eds.: 157-170. Alan R. Liss. New York. D. MICHAUD,M. CHARBONNEAU & A. HILLIER. 1989. J. P., S. D. CAMPBELL, 25. PHILLIPS, Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity. Proc. Natl. Acad. Sci. USA 8 6 2761-2765. 26. TOLMASOFF, J. M., T. ONO& R. G. CUTLER.1980. Superoxide dismutase: Correlation with life span and specific metabolic rate in primate species. Proc. Natl. Acad. Sci. USA 77: 2777-2781. J. L. 1982. Superoxide dismutase: Correlation with lifespan and metabolic rate 27. SULLIVAN, in primate species. Gerontology 2 8 242-244. 28. SOHAL,R. S., B. H. SOHAL& U. T. BRUNK.1990. Relationship between antioxidant defenses and longevity in different mammalian species. Mech. Ageing Dev. 53: 217227. 29. REVEILLAUD, I., A. NIEDZWIECKI, K. G. BENSCH& J. E. FLEMING. 1991. Expression of bovine superoxide dismutase in Drosophila melanogasrer augments resistance to oxidative stress. Mol. Cell. Biol. 11: 632-640. 30. FARMER,K. J. & R. S. SOHAL.1989. Relationship between superoxide anion radical generation and aging in the housefly, Musca domestica. Free Rad. Biol. Med. 7: 23-29. 31. SOHAL,R. S. & B. H. SOHAL.1990. Hydrogen peroxide release by mitochondria increases during aging. Mech. Ageing Dev. 5 6 223-235. & E. NIKI. 1987. Free-radical chain 32. MIKI, M., H. TAMAI,M. MINO, Y. YAMAMOTO oxidation of rat red blood cells by molecular oxygen and its inhibition by a-tocopherol. Arch. Biochem. Biophys. 2 5 8 373-380. 33. GIROTU,A. W., J. P. THOMAS& J. E. JORDAN.1986. Xanthine oxidase-catalyzed cross-linking of cell membrane proteins. Arch. Biochem. Biophys. 251: 639-653. 34. NOHL,H. & D. HEGNER.1978. Do mitochondria produce oxygen radicals in vivo? Eur. J. Biochem. 82: 563-567. 35. SOHAL,R. S., P. L. TOY& K. J. FARMER.1986. Relationship between life expectancy, endogenous antioxidants and products of oxygen free radical reactions in the housefly, Musca domestica. Mech. Aging Dev. 3 6 71-77. 36. SOHAL,R. S. 1992. Hydrogen peroxide production by mitochondria may be a biomarker of aging. Mech. Ageing Dev. 60:189-198. 37. SOHAL,R. S., I. SVENSSON, B. H. %HAL & U. T. BRUNK.1989. Superoxide anion radical production in different animal species. Mech. Ageing Dev. 4 9 129-135. 38. SOHAL,R. S., I. SVENSSON & U. T. BRUNK.1990. Hydrogen peroxide production by liver mitochondria in different species. Mech. Ageing Dev. 53: 209-215. 39. SOHAL,R. S. & R. G. ALLEN. 1986. Relationship between oxygen metabolism, aging and development. Adv. Free Rad. Biol. Med. 2 117-160. 1990. Oxidative stress as a causal factor in differentiation and 40. SOHAL,R. S. & R. G. ALLEN. aging: A unifying hypothesis. Exp. Gerontol. 2 5 499-522.
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conditions, such as overcrowding, low female-to-male ratio, and large housing containers, which increased the level of physical activity (measured by Radar Doppler), invariably decreased longevity and vice versa.s~x~y If the houseflies are confined individually in small vials where they are able to walk but are unable to fly, the average and maximum life span is prolonged more than twofold that of houseflies kept as a group in a large cage, where they can fly and interact with each other (FIG. 1). This and other experimental data leave little doubt that metabolic rate influences the rate of aging because the maximum life span is prolonged. The inverse relationship behveen metabolic rate and life span was originally encapsulated by Pearlio in the rate ofriving theory. It is unfortunate that some authors have mistakenly attacked Pearl's inference, because the metabolic potential or the total amount of energy consumed during life differs under different environmental conditions or in different strains and species. The main point of Pearl's theory is that for a given genotype the rate of aging is dependent on the rate of metabolism. All the available experimental evidence conforms with this simple relationship. Much criticism of the rate of living theory seems to be based on misinterpretation of the postulate.s The mechanism by which metabolic rate affects life span is controversial, but
FIGURE 1. Survivorship curves of male houseflies kept under conditions of relatively low (LA) and high (HA) levels of physical activity. LA flies were housed in small vials where they were unable to fly, whereas HA flies were housed in 1-cubic-foot cages where they could fly.
Q
+
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20
40
60
LA HA
80
AGE (days)
should rationally involve several basic processes. As also recently pointed out by Masoro and McCarter,Ii the rate of metabolism can be viewed in the context of processes related to fuel consumption. Although our laboratory has concentrated on the products of oxygen metabolism, utilization of carbohydrates, fats, and proteins also involves the generation of potentially deleterious products. For example, carbohydrate metabolism give rise to glycation reactions, free fatty acids can affect cell membrane functions, and amino acids may have nephrotoxic and neurotoxic effects." Partially reduced oxygen species (ROS) are widely speculated to play a role in senescence ever since they were shown to be the basis for oxygen toxicity. It has been proposed that because of the inadequacy of antioxidant defenses, cells are under a certain level of oxidative stress even under normal physiological conditions.'* The main evidence in support of this view is that oxidatively damaged macromolecules can b e demonstrated in normal healthy organisms.i3 On the assumption that the rates of oxygen consumption and ROS generation are correlated, it is widely postulated that animals with relatively higher metabolic rates are also under relatively higher levels of oxidative stress because of elevated levels of ROS. This
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1 c
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FIGURE 2. In vivo n-pentane exhalation by male houseflies at different ages.
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I
I
inference is supported by our finding that the rate of alkane exhalation by flies is decreased at lower ambient temperatures, which have a corresponding effect on the rate of oxygen con~umption.'~ OXIDATIVE STRESS AND AGING A crucial question concerning the relationship between ROS and aging is whether the level of oxidative stress remains relatively constant or increases as a function of aging. In vivo studies in rats and houseflies (FIG.2) have shown that the rate of alkane exhalation increases with age.14 As alkanes are products of lipid peroxidation, this is interpreted to indicate that the in vivo level of oxidative stress increases during senescence. In the housefly15 and Drosophila l 6 we found an agerelated increase in the ratio of GSSG/GSH. An age-related decline in the ratios of NADPH/NADP+ and NADH/NAD+was shown in rat skeletal muscle17 and housefly15 homogenates. Such data tend to support the interpretation that the level of oxidative stress increases during the aging process. The next pertinent question concerns the mechanism by which the level of oxidative stress increases during aging, that is, is it due to a decline in antioxidant defenses or an increase in the rate of prooxidant generation? A perusal of the literature indicates a chaotic lack of agreement.18J9Activities of the same enzymes in the same tissue of the same species are reported to go up or down or remain unchanged during aging. A study of the heart, brain and liver of Sprague-Dawley rats in this laboratory indicated the lack of a consistent pattern.20 Interestingly, catalase and glutathione peroxidase exhibited a mutually contradictory pattern which may be taken to mean that the ability to remove HzOZis not notably compromised as a result of aging (TABLE 1, FIG.3). An age-related decline in superoxide dismutase and catalase activity was observed in the houseflyz1 but not in Drosophila16 where superoxide dismutase activity increased during aging whereas catalase activity deTABLE1. Units of Catalase Activity in 3- and 18-Month-Old Rats Age (mo) 3 18
Liver 56.5 +. 3.1 48.6 2 0.9
Heart 2.6 +. 0.5 2.4 f 0.09
Brain 0.163 +. 0.002 0.219 f 0.006
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FIGURE 3. Comparison of the activities of superoxide dismutase (a), glutathione peroxidase (b), and glutathione reductase (c) in the homogenates of liver, heart, and brain between 3- and 18-month-old Sprague-Dawley rats.
creased only after the average life span of the population had been achieved (FIG.4). As the decline in antioxidant defenses did not precede the dying phase of the flies, there is no compelling argument to believe that decreased antioxidant defenses causally contribute to the death of the flies. Thus, in these two dipteran species, the pattern of antioxidant defenses during aging was quite different. Overall, the lack of
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a uniform age-related pattern in antioxidant defenses tends to suggest that senescence of individual animals cannot be explained on the basis of a decline in such defenses. For the sake of debate, let us examine the possible significance of the reported age-related declines in antioxidant defenses. In virtually all cases, the age-associated declines of individual antioxidant enzymes are less than 50%, most often around 10-25%. Do declines of such magnitude have functional consequences in terms of the aging process? Studies on SOD and catalase hypomorphic mutants in Drosophilu rnelanogaster indicate no decrease in life span in flies with even less than 50% of SOD or catalase activity.22-25 Such data cast doubt on the role of the decline in antioxidant defenses being a causal factor in senescence. This should not be taken to mean that antioxidant defenses are not vital for survival, but rather that the observed decline in antioxidant defenses is not likely to have a significant impact on the age-related loss of functional capacity. One of the fascinating initial questions about the free radical hypothesis was whether the species-specific differences in life spans within a phylogenetic group, say primates or mammals, correspond to variations in antioxidant defenses. Tolmasoff et al.26 initially reported a correlation between SOD divided by metabolic rate and maximum life span of the species (MLSP) in a sample of 13 mammals. However, this approach has been criticized as being inappropriate. For example, Sullivanz7pointed out that because the metabolic rate itself is inversely correlated with MLSP, any unrelated parameter such as hemoglobin content/unit blood, which is similar in various mammalian species, would, using this approach, also be found to be correlated with MLSP. Studies in this laboratory have revealed no consistent pattern with regard to the activities of SOD, catalase, and glutathione peroxidase and the concentration of glutathione in the heart, brain, and liver of six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig, and cow.28In each organ, some antioxidant defenses were positively correlated with MLSP and some were negatively correlated with MLSP, indicating a possible compensatoty balance among the various components of the antioxidant defenses. No overall relation between antioxidant defenses and MLSP was detectable (FIG. 5). Thus, according to current information, antioxidant defenses neither seem to decline with age nor correspond to MLSP, thus weakening the possibility of a direct involvement in aging. Nevertheless, the possibility that antioxidant defenses may play a role in aging or in determining the level of metabolic potential cannot as yet be totally ruled out. One approach to help resolve this issue is the use of transgenics to study the effects of overexpression of antioxidant enzymes on life span and metabolic potential. Studies by Seto et al.22
Catalase
.
.9=0 Glutathione Red
.c6H
~eurv,vorship
FIGURE 4. Age-related profile of antioxidant defenses and survivorship curve of male Drosophila melanogaster (Orgeon R). A decline in antioxidant defenses does not precede the beginning of the dying phase of the population.
SOHAL & ORR. ANTIOXIDANTS, PROOXIDANTS, AND AGING
cow rabbit
" 1
e 10
L, mw
guinea pig
0
79
30
20 MLSP (years)
o
0
rabbit mouse guinea 10 pig 20
30
MLSP (years)
2.0
41
-
5
5
1.5-
E
P
5 5
rabbit
1.0-
4
0.5-
E
.. 0
m
10
-
,
20 MLSP (years)
~
30
,
0.0
I ~
0
10 20 MLSP (Years)
30
FIGURE 5. Comparison of the activities of superoxide dismutase, catalase, and glutathione peroxidase, and concentration of glutathione in the heart of six different mammalian species with a maximum life span potential (MLSP) ranging between 3.5 and 30 years.
indicated that overexpression of SOD in transgenic Drosophilu does not extend life span. In contrast, Reveillaud et uLZ9reported a small increase in the mean but not maximum life span of Drosophilu. Recent studies in this laboratory have shown that overexpression of catalase does not extend the life span of Drosophilu. Because the activities of SOD and catalase act in concert to eliminate Oz- and H202, it is possible that for transformants with increased levels of both SOD and catalase, life spans may be altered. Such studies are currently being conducted in our laboratory. PROOXIDANTS AND AGING The alternate possibility, namely, that age-related increases in the level of oxidative stress may be due to enhancement of the rate of ROS generation, was examined in this laboratory. In the housefly, the rate of antimycin A-resistant mitochondrial respiration and mitochondrial Oz- and HZO2generation was found to increase with age.") Housefly mitochondria produced copious amounts of H 2 0 2(1-2 nmol/min/mg protein) even without the use of respiratory chain blockers such as
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FIGURE 6. Effect of age on the release of H2Oz by mitochondria from the flight muscles of the housefly. The incubation mixture included a-glycerophosphate as a substrate, p-hydroxyphenylacetate and horseradish peroxidase, but no respiratory inhibitors. (Reproduced, with permission, from Sohal and Sohal.3") -
I
0
;
i
12
16
AGE (days)
antimycin A31 (FIG.6). The activity of several oxidoreductases in the electron transport chain, such as NADH-ferricytochrome c reductase, NADH-cytochrome c reductase, and succinate-ubiquinone reductase, was also increased with age which suggested age-related alterations in the inner mitochondrial membrane, possibly involving cross-linking and increased electron flow between the respiratory comp l e ~ e s It . ~was ~ shown by Miki et ~ 1and. Girotti ~ ~ et that oxidative damage to membranes leads to cross-linking of membrane proteins. We reasoned that the age-related increase in 0 2 - and H202 generation by mitochondria may be due to oxidant damage of the inner mitochondrial membrane caused by locally generated ROS. Such oxidative damage may lead to cross-linking and other changes in membrane structural organization, resulting in an increased rate of ROS generat i ~ n . ~This ' hypothesis was tested using two approaches. First, it was found that experimental damage to mitochondria by exposure to tert-butyl hydroperoxide or ADP-iron ascorbate or AAPH (2,2-azobis (2-aminopropane) dihydrochloride, all of which induce lipid peroxidation, resulted in an increased production of H z 0 2by the treated mitochondria (FIG.7). Secondly, exposure of mitochondria to glutaraldehyde, a well known intermolecular cross-linker of proteins and lipids, also was found to greatly increase the rate of H202 generation by m i t ~ c h o n d r i a The . ~ ~ response to glutaraldehyde was much more pronounced in young flies than in old flies, which
FIGURE 7. Effect of 2,2-azobis (2aminoprophane) dihydrochloride (AAPH), a known inducer of lipid peroxidation, on mitochondrial release of H202. Mitochondria were exposed to different concentrations of AAPH for 20 minutes, centrifuged, washed twice in buffer, and then incubated. (Reproduced, with permission, from Sohal and S ~ h a l . ~ ' )
N 0 N
I
AAPH (mM)
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would be expected if it is assumed that mitochondria in young flies are less damaged and exhibit less molecular cross-linking than do those in old flies. It therefore seems that self-inflicted mitochondrial damage and intermolecular cross-linking may provide the mechanism for increased production of H202. An age-associated increase in mitochondrial 02-and H202generation was also observed in the heart, liver, and brain of Sprague-Dawley rats.20 However, the magnitude of increase was less in the rat than in the housefly. Increased 02-and H 2 0 2production in aged rat hearts was also reported by Noh1 and H e g n e ~ - . ~ ~ As mentioned, any causal hypothesis of aging should explain intra- as well as interspecies variations in life spans. The hypothesis that the rate of mitochondrial 02-and H202generation is associated with the rate of aging was tested by comparing houseflies of similar chronological but different physiological ages. In one study, the physiological age (defined as metabolic age or “nearness to death”) of houseflies was altered experimentally by adjusting the environment to eliminate flight activity. This resulted in a twofold extension of their average and maximum life spans. In a second study, flies of different physiological ages were selected in the same population based on their ability to fly. All houseflies undergo a gradual age-related decline in flight 150
E
i p 3 . N
4
rat
r = -0.91
100
FIGURE 8. Comparison of the rate of HzOz released by liver mitochondria from sixdifferent mammalian species with varying maximum life span potential (MLSP). (Reproduced, with permission, from Sohal ei al.”)
E
0
2 -
50
zn
0 10
20 MLSP (years)
30
ability, resulting in total inability to fly a few days prior to death. (Such flies are not in the process of dying, because they are able to successfully fertilize young virgin females.) Thus, in a population of aging flies, those undergoing accelerated senescence can be identified by their inability to fly. Such flies have been termed “crawlers,” and their cohorts, still capable of flight, are termed “fliers.”3s In both studies, the rates of mitochondrial 0 2 - and H202 production were found to be associated with physiological rather than chronological age.30.36At the same chronological age, flies kept under conditions of low physical activity had lower rates of 0 2 and H 2 0 2generation than did those kept under high activity conditions. Similarly, mitochondria from “fliers” had a lower rate of 02-and Hz02 generation than did those from “crawlers” of the same age.36 Thus, rates of 0 2 - and Hz02generation were associated with life expectancy of flies. Comparisons were made among six different mammals, namely, mouse, rat, guinea pig, rabbit, pig, and cow, to ascertain if variations in niaximum life span potentials of species (MLSP) were associated with the mitochondrial rate of 02-and
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H202 generation. The rates of 0 2 - and Hz02 generation by liver mitochondria were inversely correlated with MLSP.37Up to a sixfold difference was observed in the rate of 02-generation, whereas a 42-fold difference was observed in the rate of H2OZ3* generation between different species (FIG.8). Although this comparison involved a limited number of species, the results show that the rate of 0 2 - and H202generation is a better correlate of the rate of aging than are antioxidant defenses. It is also worth pointing out that under identical conditions of assay, mitochondria from different mammalian species produce widely different amounts of 0 2 - and H202 which would indicate differences in the structural organization of the ubiquinone-cytochrome b region of the electron transport chain. Overall, the results of our studies suggest that rates of 0 2 - and H202 may be a causal factor in aging. At present, the nature of the mechanism by which oxidative stress may influence the rate of the aging process is obscure. The two alternative views are: (1) Oxyradicals cause damage at various loci within cells, and due to the inadequacy of repair and regenerative mechanisms, such damage accumulates and underlies the aging process.*2Differences in aging rates are envisioned to be related to the efficiency of antioxidant defenses and/or repair mechanisms. (2) The second view, favored by US,^^,^ is that oxidative stress influences the rate of aging by modulating gene expression. It is reasoned that as age-related changes in various species follow a similar predictable pattern, any putative causal factor would be expected to act through effects on gene expression rather than simply by infliction of damage which would tend to be random. Furthermore, evidence for extensive structural damage during aging at the cellular level is lacking. Such damage that is observed is believed to be incidental to oxidant generation rather than causal to the aging process.
SUMMARY It is argued that reduced oxygen species may be one of the causal factors underlying the aging process. Experimental studies strongly support the view that the rate of metabolism is inversely associated with the rate of aging. It is pointed out that Pearl’s rate of living theory is widely misunderstood, because of the mistaken belief that it advocates a h e d metabolic potential for different species or genotypes within a species. The in vivo level of oxidative stress tends to increase with age in insects and mammals as indicated by increased exhalation of alkanes. A search for the causes of this increase revealed that an age-associated decline in antioxidant defenses is neither widespread nor very impressive in magnitude. A comparison of antioxidant defenses (activities of SOD, catalase, and glutathione) in six different mammalian species did not suggest a clear association between these defenses and maximum life span potential of the species. In contrast, mitochondria1 rates of 02-and H202were found to increase with age in insects and mammals, and the MLSP of six mammals was found to be inversely correlated with liver mitochondrial rates of 02-and H202 generation. It seems that the age-related increase in oxidative stress is mainly due to the enhanced rate of 0 2 - and H202 generation. It is hypothesized that variations in the rates of aging in different species, that are otherwise closely related phylogenetically, may be in part due to differences in rates of 0 2 - and H202 production. Overall, the rates of oxidant generation are a better correlate of the rates of aging than are the levels of antioxidant defenses.
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24. MACKAY, W. J., W. C. ORR& G. C. BEWLEY.1990. Genetic and molecular analysis of antioxidant enzymes in Drosophila melanogaster: A correlation between catalase activity levels, life span, and spontaneous mutation rate. In Molecular Biology of Aging. M. Clegg & S. O’Brien, eds.: 157-170. Alan R. Liss. New York. D. MICHAUD,M. CHARBONNEAU & A. HILLIER. 1989. J. P., S. D. CAMPBELL, 25. PHILLIPS, Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity. Proc. Natl. Acad. Sci. USA 8 6 2761-2765. 26. TOLMASOFF, J. M., T. ONO& R. G. CUTLER.1980. Superoxide dismutase: Correlation with life span and specific metabolic rate in primate species. Proc. Natl. Acad. Sci. USA 77: 2777-2781. J. L. 1982. Superoxide dismutase: Correlation with lifespan and metabolic rate 27. SULLIVAN, in primate species. Gerontology 2 8 242-244. 28. SOHAL,R. S., B. H. SOHAL& U. T. BRUNK.1990. Relationship between antioxidant defenses and longevity in different mammalian species. Mech. Ageing Dev. 53: 217227. 29. REVEILLAUD, I., A. NIEDZWIECKI, K. G. BENSCH& J. E. FLEMING. 1991. Expression of bovine superoxide dismutase in Drosophila melanogasrer augments resistance to oxidative stress. Mol. Cell. Biol. 11: 632-640. 30. FARMER,K. J. & R. S. SOHAL.1989. Relationship between superoxide anion radical generation and aging in the housefly, Musca domestica. Free Rad. Biol. Med. 7: 23-29. 31. SOHAL,R. S. & B. H. SOHAL.1990. Hydrogen peroxide release by mitochondria increases during aging. Mech. Ageing Dev. 5 6 223-235. & E. NIKI. 1987. Free-radical chain 32. MIKI, M., H. TAMAI,M. MINO, Y. YAMAMOTO oxidation of rat red blood cells by molecular oxygen and its inhibition by a-tocopherol. Arch. Biochem. Biophys. 2 5 8 373-380. 33. GIROTU,A. W., J. P. THOMAS& J. E. JORDAN.1986. Xanthine oxidase-catalyzed cross-linking of cell membrane proteins. Arch. Biochem. Biophys. 251: 639-653. 34. NOHL,H. & D. HEGNER.1978. Do mitochondria produce oxygen radicals in vivo? Eur. J. Biochem. 82: 563-567. 35. SOHAL,R. S., P. L. TOY& K. J. FARMER.1986. Relationship between life expectancy, endogenous antioxidants and products of oxygen free radical reactions in the housefly, Musca domestica. Mech. Aging Dev. 3 6 71-77. 36. SOHAL,R. S. 1992. Hydrogen peroxide production by mitochondria may be a biomarker of aging. Mech. Ageing Dev. 60:189-198. 37. SOHAL,R. S., I. SVENSSON, B. H. %HAL & U. T. BRUNK.1989. Superoxide anion radical production in different animal species. Mech. Ageing Dev. 4 9 129-135. 38. SOHAL,R. S., I. SVENSSON & U. T. BRUNK.1990. Hydrogen peroxide production by liver mitochondria in different species. Mech. Ageing Dev. 53: 209-215. 39. SOHAL,R. S. & R. G. ALLEN. 1986. Relationship between oxygen metabolism, aging and development. Adv. Free Rad. Biol. Med. 2 117-160. 1990. Oxidative stress as a causal factor in differentiation and 40. SOHAL,R. S. & R. G. ALLEN. aging: A unifying hypothesis. Exp. Gerontol. 2 5 499-522.
