Mechanisms of aging: an appraisal of the oxidative stress hypothesis

The main purpose of this article is to provide a critical overview of the currently available evidence bearing on the validity of the oxidative stress hypothesis of aging, which postulates that senescence-associated attenuations in physiological functions are caused by molecular oxidative damage. Several lines of correlative evidence support the predictions of the hypothesis, e.g., macromolecular oxidative damage increases with age and tends to be associated with life expectancy of organisms. Nevertheless, a direct link between oxidative stress and aging has not as yet been established. Single gene mutations have been reported to extend the life spans of lower organisms, such as nematodes and insects; however, such prolongations of chronological clock time survival are usually associated with decreases in the rate of metabolism and reproductive output without affecting the metabolic potential, i.e., the total amount of energy consumed during life. Studies on genetic manipulations of the aging process have often been conducted on relatively short-lived strains that are physiologically weak, whereby life-span extensions can not be unambiguously assigned to a slowing effect on the rate of aging. It is concluded that although there is considerable evidence implicating oxidative stress in the aging process, additional evidence is needed to clearly define the nature of the involvement.     

Prevention of flight activity prolongs the life span of the housefly, Musca domestica, and attenuates the age-associated oxidative damamge to specific mitochondrial proteins

The purpose of this study was to explore the mechanisms by which oxidative stress affects the aging process. The hypothesis that the rate of accumulation of oxidative damage to specific mitochondrial proteins is linked to the life expectancy of animals was tested in the housefly. The rate of oxygen consumption and life expectancy of the flies were experimentally altered by confining the flies in small jars, where they were unable to fly. Prevention of flight activity decreased the rate of oxygen utilization of flies and almost tripled their life span as compared to those permitted to fly. Rate of mitochondrial H(2)O(2) generation at various ages was lower in the low activity flies than in the high activity flies. Oxidative damage to mitochondrial proteins, adenine nucelotide translocase, and aconitase, detected as carbonyl modifications, was attenuated; and the loss in their functional activity occurring with age was retarded in the long-lived low activity flies as compared to the short-lived high activity flies. The two proteins were previously identified to be the only mitochondrial proteins exhibiting age-related increases in carbonylation. Results support the hypothesis that accrual of oxidative damage to specific protein targets and the consequent loss of their function may constitute a mechanism by which oxidative stress controls the aging process.     

Endogenous mutagens and the causes of aging and cancer

A very large oxidative damage rate to DNA occurs as part of normal metabolism. In each rat cell the steady-state level is estimated to be about 10⁶ oxidative adducts and about 10⁵ new adducts are formed daily. It is argued that this endogenous DNA damage is a major contributor to aging and the degenerative diseases of aging, such as cancer. The oxidative damage rate in mammalian species with a high metabolic rate, short life span, and high age-specific cancer rate is much higher than the rate in humans, a long-lived creature with a lower metabolic rate and a lower age-specific cancer rate. It is argured that deficiency of micronutrients, such as dietary antioxidants or folate, is a major contributor to human cancer and degenerative diseases.

Understanding the role of mitogenesis in mutagenesis is critical for clarifying the mechanisms of carcinogenesis and interpreting high-dose animal cancer tests. High-dose animal cancer tests have been done mainly on synthetic industrial chemicals, yet almost all of the chemicals humans are exposed to are natural. About half of natural chemicals tested in high-dose animal cancer tests are rodent carcinogens, a finding that is consistent with the view that high-dose tests frequently increase mitogenesis rates. Animals have numerous defenses against toxins that make them very well buffered against low doses of almost all toxins, whether synthetic or natural.     

Methionine oxidation and aging

It is well established that many amino acid residues of proteins are susceptible to oxidation by various forms of reactive oxygen species (ROS), and that oxidatively modified proteins accumulate during aging, oxidative stress, and in a number of age-related diseases. Methionine residues and cysteine residues of proteins are particularly sensitive to oxidation by ROS. However, unlike oxidation of other amino acid residues, the oxidation of these sulfur amino acids is reversible. Oxidation of methionine residues leads to the formation of both R- and S-stereoisomers of methionine sulfoxide (MetO) and most cells contain stereospecific methionine sulfoxide reductases (Msr's) that catalyze the thioredoxin-dependent reduction of MetO residues back to methionine residues. We summarize here results of studies, by many workers, showing that the MetO content of proteins increases with age in a number of different aging models, including replicative senescence and erythrocyte aging, but not in mouse tissues during aging. The change in levels of MetO may reflect alterations in any one or more of many different mechanisms, including (i) an increase in the rate of ROS generation; (ii) a decrease in the antioxidant capacity; (iii) a decrease in proteolytic activities that preferentially degrade oxidized proteins; or (iv) a decrease in the ability to convert MetO residues back to Met residues, due either to a direct loss of Msr enzyme levels or indirectly to a loss in the availability of the reducing equivalents (thioredoxin, thioredoxin reductase, NADPH generation) involved. The importance of Msr activity is highlighted by the fact that aging is associated with a loss of Msr activities in a number of animal tissues, and mutations in mice leading to a decrease in the Msr levels lead to a decrease in the maximum life span, whereas overexpression of Msr leads to a dramatic increase in the maximum life span.     

An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations

Aging is an inherently complex process that is manifested within an organism at genetic, molecular, cellular, organ, and system levels. Although the fundamental mechanisms are still poorly understood, a growing body of evidence points toward reactive oxygen species (ROS) as one of the primary determinants of aging. The "oxidative stress theory" holds that a progressive and irreversible accumulation of oxidative damage caused by ROS impacts on critical aspects of the aging process and contributes to impaired physiological function, increased incidence of disease, and a reduction in life span. While compelling correlative data have been generated to support the oxidative stress theory, a direct cause-and-effect relationship between the accumulation of oxidatively mediated damage and aging has not been strongly established. The goal of this minireview is to broadly describe mechanisms of in vivo ROS generation, examine the potential impact of ROS and oxidative damage on cellular function, and evaluate how these responses change with aging in physiologically relevant situations. In addition, the mounting genetic evidence that links oxidative stress to aging is discussed, as well as the potential challenges and benefits associated with the development of anti-aging interventions and therapies.     

Testing the 'free radical theory of aging' hypothesis: physiological differences in long-lived and short-lived colubrid snakes

We test the 'free radical theory of aging' using six species of colubrid snakes (numerous, widely distributed, non-venomous snakes of the family Colubridae) that exhibit long (> 15 years) or short (< 10 years) lifespans. Because the 'rate of living theory' predicts metabolic rates to be correlated with rates of aging and oxidative damage results from normal metabolic processes we sought to answer whether physiological parameters and locomotor performance (which is a good predictor of survival in juvenile snakes) mirrored the evolution of lifespans in these colubrid snakes. We measured whole animal metabolic rate (oxygen consumption Vo2), locomotor performance, cellular metabolic rate (mitochondrial oxygen consumption), and oxidative stress potential (hydrogen peroxide production by mitochondria). Longer-lived colubrid snakes have greater locomotor performance and reduced hydrogen peroxide production than short-lived species, while whole animal metabolic rates and mitochondrial efficiency did not differ with lifespan. We present the first measures testing the 'free radical theory of aging' using reptilian species as model organisms. Using reptiles with different lifespans as model organisms should provide greater insight into mechanisms of aging.     

Oxidative Damage in Rat Brain During Aging: Interplay Between Energy and Metabolic Key Target Proteins

Aging is characterized by a gradual and continuous loss of physiological functions and responses particularly marked in the central nervous system. Reactive oxygen species (ROS) can react with all major biological macromolecules such as carbohydrates, nucleic acids, lipids, and proteins. Since proteins are the major components of biological systems and regulate multiple cellular pathways, oxidative damage of key proteins are considered to be the principal molecular mechanisms leading to age-related deficits. Recent evidences support the notion that a decrease of energy metabolism in the brain contribute to neuronal loss and cognitive decline associated with aging. In the present study we identified selective protein targets which are oxidized in aged rats compared with adult rats. Most of the oxidatively modified proteins we found in the present study are key proteins involved in energy metabolism and ATP production. Oxidative modification of these proteins was associated with decreased enzyme activities. In addition, we also found decreased levels of thiol reducing system. Our study demonstrated that oxidative damage to specific proteins impairs energy metabolism and ATP production thus contributing to shift neuronal cells towards a more oxidized environment which ultimately might compromise multiple neuronal functions. These results further confirm that increased protein oxidation coupled with decreased reducing systems are characteristic hallmarks of aging and aging-related degenerative processes.     

Assessing metabolic activity in aging Caenorhabditis elegans: concepts and controversies

It is widely believed that normal by-products of oxidative metabolism and the subsequent molecular damage inflicted by them couple the aging process to metabolic rate. Accordingly, high metabolic rates would be expected to accelerate aging, and life-extending interventions are often assumed to act by attenuating metabolic rate. Notorious examples in Caenorhabditis elegans are food restriction, mutation in the Clock genes and several genes of the insulin-like signalling pathway. Here we discuss how metabolic rate can be accurately measured and normalized, and how to deal with differences in body size. These issues are illustrated using experimental data of the long-lived mutant strains clk-1(e2519) and daf-2(e1370). Appropriate analysis shows that metabolic rates in wildtype and in the clk-1 mutant are very similar. In contrast, the metabolic rate profiles point to a metabolic shift toward enhanced efficiency of oxidative phosphorylation in the daf-2 worms.     

Update on the oxidative stress theory of aging: Does oxidative stress play a role in aging or healthy aging?

The oxidative stress theory of aging predicts that manipulations that alter oxidative stress/damage will alter aging. The gold standard for determining whether aging is altered is life span, i.e., does altering oxidative stress/damage change life span? Mice with genetic manipulations in their antioxidant defense system designed to directly address this prediction have, with few exceptions, shown no change in life span. However, when these transgenic/knockout mice are tested using models that develop various types of age-related pathology, they show alterations in progression and/or severity of pathology as predicted by the oxidative stress theory: increased oxidative stress accelerates pathology and reduced oxidative stress retards pathology. These contradictory observations might mean that (a) oxidative stress plays a very limited, if any, role in aging but a major role in health span and/or (b) the role that oxidative stress plays in aging depends on environment. In environments with minimal stress, as expected under optimal husbandry, oxidative damage plays little role in aging. However, under chronic stress, including pathological phenotypes that diminish optimal health, oxidative stress/damage plays a major role in aging. Under these conditions, enhanced antioxidant defenses exert an “antiaging” action, leading to changes in life span, age-related pathology, and physiological function as predicted by the oxidative stress theory of aging.     

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N ^ε-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.     

Importance of individuality in oxidative stress and aging

Tight linkage between aging and oxidative stress is indicated by the observations that reactive oxygen species generated under various conditions of oxidative stress are able to oxidize nucleic acids, proteins, and lipids and that aging is associated with the accumulation of oxidized forms of cellular constituents, and also by the fact that there is an inverse relationship between the maximum life span of organisms and the age-related accumulation of oxidative damage. Nevertheless, validity of the oxidative stress hypothesis of aging is questioned by (i) the failure to establish a causal relationship between aging and oxidative damage and (ii) lack of a consistent correlation between the accumulation of oxidative damage and aging. The present discussion is focused on the complexity of the aging process and suggests that discrepancies between various studies in this area are likely due to the fact that aging is not a single process and that the lack of consistent experimental results is partly explained by individual variations. Even so, there is overwhelming support for a dominant role of oxidative stress in the aging of some individuals.     

Repeated intraspecific divergence in life span and aging of African annual fishes along an aridity gradient

Life span and aging are substantially modified by natural selection. Across species, higher extrinsic (environmentally related) mortality (and hence shorter life expectancy) selects for the evolution of more rapid aging. However, among populations within species, high extrinsic mortality can lead to extended life span and slower aging as a consequence of condition‐dependent survival. Using within‐species contrasts of eight natural populations of Nothobranchius fishes in common garden experiments, we demonstrate that populations originating from dry regions (with short life expectancy) had shorter intrinsic life spans and a greater increase in mortality with age, more pronounced cellular and physiological deterioration (oxidative damage, tumor load), and a faster decline in fertility than populations from wetter regions. This parallel intraspecific divergence in life span and aging was not associated with divergence in early life history (rapid growth, maturation) or pace‐of‐life syndrome (high metabolic rates, active behavior). Variability across four study species suggests that a combination of different aging and life‐history traits conformed with or contradicted the predictions for each species. These findings demonstrate that variation in life span and functional decline among natural populations are linked, genetically underpinned, and can evolve relatively rapidly.     

Early Life Hormetic Treatments Decrease Irradiation-Induced Oxidative Damage,Increase Longevity,and Enhance Sexual Performance during Old Age in the Caribbean Fruit Fly

Early life events can have dramatic consequences on performance later in life. Exposure to stressors at a young age affects development, the rate of aging, risk of disease, and overall lifespan. In spite of this, mild stress exposure early in life can have beneficial effects on performance later in life. These positive effects of mild stress are referred to as physiological conditioning hormesis. In our current study we used anoxia conditioning hormesis as a pretreatment to reduce oxidative stress and improve organismal performance, lifespan, and healthspan of Caribbean fruit flies. We used gamma irradiation to induce mild oxidative damage in a low-dose experiment, and massive oxidative damage in a separate high-dose experiment, in pharate adult fruit flies just prior to adult emergence. Irradiation-induced oxidative stress leads to reduced adult emergence, flight ability, mating performance, and lifespan. We used a hormetic approach, one hour of exposure to anoxia plus irradiation in anoxia, to lower post-irradiation oxidative damage. We have previously shown that this anoxic-conditioning treatment elevates total antioxidant capacity and lowers post-irradiation oxidative damage to lipids and proteins. In this study, conditioned flies had lower mortality rates and longer lifespan compared to those irradiated without hormetic conditioning. As a metric of healthspan, we tracked mating both at a young age (10 d) and old age (30 d). We found that anoxia-conditioned male flies were more competitive at young ages when compared to unconditioned irradiation stressed male flies, and that the positive effects of anoxic conditioning hormesis on mating success were even more pronounced in older males. Our data shows that physiological conditioning hormesis at a young age, not only improves immediate metrics of organismal performance (emergence, flight, mating), but the beneficial effects also carry into old age by reducing late life oxidative damage and improving lifespan and healthspan.     

Ginkgo biloba extract EGb 761 protects against mitochondrial aging in the brain and in the liver.

According to the free radical theory of aging, oxygen-derived free radicals causes the age-associated impairment at the cellular and tissue levels. The mitochondrial theory of aging points to mitochondria, and specially mitochondrial DNA, as the major targets of free radical attack upon aging. Thus, oxidative damage to mtDNA accumulate with age in human and rodent tissues and also is inversely related to maximum life span of mammals. Mitochondrial deficits, such as a decrease in mitochondrial membrane potential, occur upon aging due to oxidative damage. The age-related mitochondrial oxidative stress may be prevented by late onset administration of certain antioxidants, such as Ginkgo biloba extract EGb 761. These antioxidants may also delay the physiological impairment associated with aging.     

The soma-germline communication: implications for somatic and reproductive aging

Aging is characterized by a functional decline in most physiological processes, including alterations in cellular metabolism and defense mechanisms. Increasing evidence suggests that caloric restriction extends longevity and retards age-related diseases at least in part by reducing metabolic rate and oxidative stress in a variety of species, including yeast, worms, flies, and mice. Moreover, recent studies in invertebrates – worms and flies, highlight the intricate interrelation between reproductive longevity and somatic aging (known as disposable soma theory of aging), which appears to be conserved in vertebrates. This review is specifically focused on how the reproductive system modulates somatic aging and vice versa in genetic model systems. Since many signaling pathways governing the aging process are evolutionarily conserved, similar mechanisms may be involved in controlling soma and reproductive aging in vertebrates.     

The role of sirtuins in modulating redox stressors

For much of the time since their discovery, the sirtuin family of deacetylase enzymes has been associated with extension of life span. This longevity-promoting capacity in numerous model systems has enabled the sirtuins to gain "celebrity status" in the field of aging research. However, the mechanisms underpinning these changes remain incompletely defined. A general phenotype long associated with aging is the dysregulation of biological systems, which partly occurs via the accumulation of damage over time. One of the major sources of this damage is oxidative stress, which can harm both biological structures and the mechanisms with which they are repaired. It is now becoming clear that the beneficial life-span effects of sirtuins, along with many of their other functions, are closely linked to their ability to regulate systems that control the redox environment. Here we investigate the links between sirtuins and their oxidative/redox environment and review the control mechanisms that are regulated by the activity of sirtuin deacetylase proteins.     

Insights from comparative analyses of aging in birds and mammals

Many laboratory models used in aging research are inappropriate for understanding senescence in mammals, including humans, because of fundamental differences in life history, maintenance in artificial environments, and selection for early aging and high reproductive rate. Comparative studies of senescence in birds and mammals reveal a broad range in rates of aging among a variety of taxa with similar physiology and patterns of development. These comparisons suggest that senescence is a shared property of all vertebrates with determinate growth, that the rate of senescence has been modified by evolution in response to the potential life span allowed by extrinsic mortality factors, and that most variation among species in the rate of senescence is independent of commonly ascribed causes of aging, such as oxidative damage. Individuals of potentially long‐lived species, particularly birds, appear to maintain high condition to near the end of life. Because most individuals in natural populations of such species die of aging‐related causes, these populations likely harbor little genetic variation for mechanisms that could extend life further, or these mechanisms are very costly. This, and the apparent evolutionary conservatism in the rate of increase in mortality with age, suggests that variation in the rate of senescence reflects fundamental changes in organism structure, likely associated with the rate of development, rather than physiological or biochemical processes influenced by a few genes. Understanding these evolved differences between long‐lived and short‐lived organisms would seem to be an essential foundation for designing therapeutic interventions with respect to human aging and longevity.     

The rate of free radical production as a determinant of the rate of aging: evidence from the comparative approach

The relationship of oxidative stress with maximum life span (MLSP) in different vertebrate species is reviewed. In all animal groups the endogenous levels of enzymatic and non-enzymatic antioxidants in tissues negatively correlate with MLSP and the most longevous animals studied in each group, pigeon or man, show the minimum levels of antioxidants. A possible evolutionary reason for this is that longevous animals produce oxygen radicals at a low rate. This has been analysed at the place where more than 90% of oxygen is consumed in the cell, the mitochondria. All available work agrees that, across species, the longer the life span, the lower the rate of mitochondrial oxygen radical production. This is true even in animal groups that do not conform to the rate of living theory of aging, such as birds. Birds have low rates of mitochondrial oxygen radical production, frequently due to a low free radical leak in their respiratory chain. Possibly the low rate of mitochondrial oxygen radical production of longevous species can decrease oxidative damage at targets important for aging (like mitochondrial DNA) that are situated near the places of free radical generation. A low rate of free radical production can contribute to a low aging rate both in animals that conform to the rate of living (metabolic) theory of aging and in animals with exceptional longevities, like birds and primates. Available research indicates there are at least two main characteristics of longevous species: a high rate of DNA repair together with a low rate of free radical production near DNA. Simultaneous consideration of these two characteristics can explain part of the quantitative differences in longevity between animal species. Accepted: 12 December 1997     

Regulation of Redox Signaling by Selenoproteins

The unique chemistry of oxygen has been both a resource and threat for life on Earth for at least the last 2.4 billion years. Reduction of oxygen to water allows extraction of more metabolic energy from organic fuels than is possible through anaerobic glycolysis. On the other hand, partially reduced oxygen can react indiscriminately with biomolecules to cause genetic damage, disease, and even death. Organisms in all three superkingdoms of life have developed elaborate mechanisms to protect against such oxidative damage and to exploit reactive oxygen species as sensors and signals in myriad processes. The sulfur amino acids, cysteine and methionine, are the main targets of reactive oxygen species in proteins. Oxidative modifications to cysteine and methionine can have profound effects on a protein’s activity, structure, stability, and subcellular localization. Non-reversible oxidative modifications (oxidative damage) may contribute to molecular, cellular, and organismal aging and serve as signals for repair, removal, or programmed cell death. Reversible oxidation events can function as transient signals of physiological status, extracellular environment, nutrient availability, metabolic state, cell cycle phase, immune function, or sensory stimuli. Because of its chemical similarity to sulfur and stronger nucleophilicity and acidity, selenium is an extremely efficient catalyst of reactions between sulfur and oxygen. Most of the biological activity of selenium is due to selenoproteins containing selenocysteine, the 21st genetically encoded protein amino acid. The most abundant selenoproteins in mammals are the glutathione peroxidases (five to six genes) that reduce hydrogen peroxide and lipid hydroperoxides at the expense of glutathione and serve to limit the strength and duration of reactive oxygen signals. Thioredoxin reductases (three genes) use nicotinamide adenine dinucleotide phosphate to reduce oxidized thioredoxin and its homologs, which regulate a plethora of redox signaling events. Methionine sulfoxide reductase B1 reduces methionine sulfoxide back to methionine using thioredoxin as a reductant. Several selenoproteins in the endoplasmic reticulum are involved in the regulation of protein disulfide formation and unfolded protein response signaling, although their precise biological activities have not been determined. The most widely distributed selenoprotein family in Nature is represented by the highly conserved thioredoxin-like selenoprotein W and its homologs that have not yet been assigned specific biological functions. Recent evidence suggests selenoprotein W and the six other small thioredoxin-like mammalian selenoproteins may serve to transduce hydrogen peroxide signals into regulatory disulfide bonds in specific target proteins.