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INVITED REVIEW
NEW INVESTIGATOR AWARD IN REGULATORY AND INTEGRATIVE PHYSIOLOGY OF THE WATER AND ELECTROLYTE HOMEOSTASIS SECTION, 2006
Department of Integrative Physiology and Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, Iowa
Submitted 15 May 2006 ; accepted in final form 16 August 2006
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ABSTRACT |
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 TOP ABSTRACT GRANTREFERENCES |
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oxidative damage; antioxidants; free radicals; reactive oxygen species; hyperthermia; caloric restriction
"If I'd known I was going to live this long, I would have taken better care of myself."
— Eubie Blake, American ragtime musician, who lived well into his 90s
HEIGHTENED INTEREST IN THE aging process, both in scientific and public settings, has been stimulated by a number of factors. One key observation, the impressive increase in average life expectancy in humans over recent centuries, lends some import to the pronouncement made by renowned jazz musician Eubie Blake as he neared 100 years of age. In addition, the growing percentage of elderly making up the population base in most developed countries (212) and the large health care expenditures that are committed to the elderly (42) have stimulated both scientific inquiry and heightened public awareness on issues related to aging.
Harman defines aging as the progressive "... accumulation of diverse deleterious changes in cells and tissues with advancing age that increase the risk of disease and death." (95). This definition illustrates two widely recognized and equally important aspects of the aging process: 1) aging is characterized as a progressive decline in biological functions with time, and 2) aging results in a decreased resistance to multiple forms of stress, as well as an increased susceptibility to numerous diseases.
Even with a well-described definition and a familiar set of characteristics, aging remains one of the most poorly understood of all biological phenomena, due in large part to its inherently complex and integrative nature, as well as the difficulty in dissociating the effects of normal aging from those manifested as a consequence of age-associated disease conditions. As a result, while disciplines ranging from physiology and genetics to epidemiology and demography have developed a large number of theories that attempt to explain why we age (152), definitive mechanisms to explain the process across species and systems remain equivocal.
One of the prevalent theories in the current literature revolves around free radicals, which are molecules containing unpaired, highly reactive electrons, as causal agents in the process of aging. In the 1950s, Harman proposed the "free radical theory," postulating that damage to cellular macromolecules via free radical production in aerobic organisms is a major determinant of life span (94). It was subsequently discovered that reactive oxygen species (ROS), some of which are not free radicals (because they do not have an unpaired electron in their outer shell), contribute to the accumulation of oxidative damage to cellular constituents. Thus, a more modern version of this tenet is the "oxidative stress theory" of aging, which holds that increases in ROS accompany aging, leading to functional alterations, pathological conditions, and even death (91). Oxidative stress in a physiological setting can be defined as an excessive bioavailability of ROS, which is the net result of an imbalance between production and destruction of ROS (with the latter being influenced by antioxidant defenses). Over the past two decades, many reviews have been published that contain extensive information regarding the oxidative stress theory of aging (7, 12, 21, 33, 57, 96, 142). However, despite a large body of evidence supporting the notion that ROS are produced in cells and can manifest damage, a causal link between ROS and aging has still not been clearly established.
In recent years, several related theories containing an ROS component have also been proposed (283). One that has been extensively studied is the mitochondrial theory of aging, which hypothesizes that mitochondria are the critical component in control of aging. It is proposed that electrons leaking from the electron transport chain (ETC) produce ROS and that these molecules can then damage ETC components and mitochondrial DNA, leading to further increases in intracellular ROS levels and a decline in mitochondrial function (276). In support of a mitochondrial theory of aging, evidence suggests that mitochondrial DNA damage is increased with aging (90, 93). Another consideration is the cellular senescence theory of aging, which emphasizes the importance of cellular signal responses to stress and damage. These signaling responses subsequently stimulate pathways related to cell senescence and death (20). At the cellular level, ROS have been found to modulate various signals leading to accelerated mitogenesis and premature cellular senescence (106). An additional theory that has gained more attention in recent years is the molecular inflammatory theory of aging, whereby the activation of redox-sensitive transcriptional factors by age-related oxidative stress causes the upregulation of proinflammatory gene expression. As a result, various proinflammatory molecules are generated, leading to inflammation processes in various tissues and organs. This inflammatory cascade is exaggerated during aging and has been linked with many age-associated pathologies, such as cancer, various cardiovascular diseases, arthritis, and several neurodegenerative diseases (47). Interestingly, a common phenomenon in aging-related pathologies is the discovery of ROS as a potential unifying mechanism contributing to many of these diseases (72, 150, 176, 205).
Despite much investigation in recent years, no single theory has been completely successful in explaining the aging process. In fact, while many investigators focus on a limited number of genes in selected model organisms, current evidence has led to the suggestion that it is impossible for aging to be accounted for by a single theory (127). The purpose of this minireview is to discuss recent evidence that links oxidative stress to biological aging at molecular, cellular, and organismal levels. A primary focus will be to provide an integrated view of the involvement of oxidative stress in normal aging processes. The authors recognize that the role of oxidative stress in numerous age-related pathologies is another important aspect of the aging process; however, providing a detailed assessment of this broad topic is outside the scope of this article and many reviews are currently available in this particular area (153, 187, 214, 249, 284).
Basics of ROS
Sources of ROS. ROS are metabolites of molecular oxygen (O2) that have higher reactivity than O2. ROS can include unstable oxygen radicals such as superoxide radical (O2·–) and hydroxyl radical (HO·), and nonradical molecules like hydrogen peroxide (H2O2). These ROS, which are continually generated as byproducts of normal aerobic metabolism, can also be produced to a greater extent under stress and pathological conditions, as well as taken up from the external environment. Thus, all organisms living in an aerobic environment are exposed to ROS on a continual basis.
As noted in the previous section, one of the primary intracellular sites for in vivo ROS production is the mitochondion (19). This organelle generates ATP through a series of oxidative phosphorylation processes that ultimately involve a four-electron reduction of O2 to water. However, during this process, one- or two-electron reductions of O2 can occur, leading to the formation of O2·– or H2O2, and these species can be converted to other ROS. Additional examples of intracellular sources of ROS production include reactions involving peroxisomal oxidases (236), cytochrome P-450 enzymes (293), NAD(P)H oxidases (143), or xanthine-xanthine oxidase (220). A variety of exogenous stimuli, such as radiation (222), pathogen infections (238), and exposure to xenobiotics (196) can also cause in vivo ROS production. Additionally, several specific types of environmental stress can lead to the production of ROS, including heat stress (297), herbicide/insecticide contamination (3), environmental toxins (240), and ultraviolet light exposure (235). ROS generation via these various sources can be specific for particular tissues, cells, and organelles.
Oxidative damage to macromolecules by ROS. It has long been recognized that high levels of ROS can inflict direct damage to macromolecules, such as lipids, nucleic acids, and proteins (32). Due to the bis-allylic structures of polyunsaturated fatty acids, lipids are one of the most sensitive oxidation targets for ROS. Once lipid peroxidation is initiated, a propagation of chain reactions will take place until termination products are produced. Therefore, end products of lipid peroxidation, such as malondialdehyde (MDA), 4-hydroxy-2-nonenol (4-HNE), and F2-isoprostanes are accumulated in biological systems. DNA bases are also very susceptible to ROS oxidation, and the predominant detectable oxidation product of DNA bases in vivo is 8-hydroxy-2-deoxyguanosine. Oxidation of DNA bases can cause mutations and deletions in both nuclear and mitochondrial DNA. Mitochondrial DNA is especially prone to oxidative damage due to its proximity to a primary source of ROS and its deficient repair capacity compared with nuclear DNA. Almost all amino acid residues in a protein can be oxidized by ROS. Some widely studied oxidative products of amino acid residues include the formation of disulfide bonds at cysteine residues, carbonyl derivatives, and many others oxidized residues, such as methionine sulfoxide. These oxidative modifications lead to functional changes in various types of proteins, which can have substantial physiological impact. For instance, oxidative damage to enzymes can cause a modification of their activity, while oxidant-derived injury to structural proteins and chaperones produces protein aggregation. Similarly, redox modulation of transcription factors, as detailed in the next section, can produce an increase or decrease in their specific DNA binding activities, which stimulates gene expression changes that impact on cell survival, death, and senescence pathways (Fig. 1).
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ROS regulation of transcription factors can occur by direct modification of critical amino acid residues, primarily through the formation of disulfide bonds, at DNA-binding domains or via indirect phosphorylation/dephosphorylation as a result of changes in redox-modulated signaling pathways. The consequences of redox modulation are bidirectional, producing either activation or inactivation of affected transcription factors. Examples of aging-related transcription factors known to be redox-regulated include tumor suppressor p53, Forkhead transcription factors, activator protein-1 (AP-1), and NF-
B.
Tumor suppressor p53 is a 53-kD protein that controls cell cycle arrest, cell apoptosis, and senescence (97). Forkhead transcription factors encompass a large family of proteins characterized by a conserved DNA-binding domain termed "Forkhead box" (118). These proteins are ubiquitous in eukaryotic cells and thought to be critical in regulating cell cycle arrest, cellular stress responses, apoptosis, and longevity (86). AP-1 and NF-B are two of the early-response transcriptional factors that have been shown to suppress apoptosis and induce cellular transformation, proliferation, stress resistance, and inflammation (119, 208).
We have shown that the activation of AP-1 by in vivo adenovirus administration is redox-modulated and involves the participation of redox factor-1 (Ref-1). Ref-1 is a unique molecule that has two distinct enzymatic functions: it serves as both a DNA repair enzyme and a redox regulatory transcription factor (296). Under oxidative stress conditions, the Ref-1 molecule also undergoes major conformation changes while two critical cysteines, Cys65 and Cys93 at the NH2-terminal region of Ref-1, interact with conserved cysteine residues (e.g., Fos cys-154 and Jun cys-272) in the AP-1 DNA binding domain to stimulate the activation of AP-1 (287). Similarly, reduced cysteines in the zinc-finger domain of p53 are critical for its DNA binding ability. ROS inhibits site-specific p53 binding while Ref-1 can reactivate DNA binding of oxidized p53 in vitro and stimulate p53 transactivation in vivo (115).
Furthermore, it is thought that the phosphorylation of IB, the inhibitory subunit of NF-B, is the key step in NF-B redox activation. ROS-mediated phosphorylation of IB, leading to its ubiquitination and degradation, allows the NF-B complex to be translocated to the nucleus and act as a transcriptional activator (208). On the other hand, direct oxidation of critical cysteine residues in the p50 subunit of NF-B is believed to significantly decrease its DNA binding ability (209). Although the ROS-targeted protein kinases remain to be identified for the redox-regulation of Forkhead transcription factors, there are some data indicating that oxidative stress caused by H2O2, menadione, or heat shock stimulates the phosphorylation and translocation of Forkhead proteins and activation of Akt, a serine/threonine kinase also known as protein kinase B. This protein is directly responsible for the phosphorylation of Forkhead protein in the phosphatidylinositol 3-Akt signaling pathway (76). In addition, oxidative stress promotes the acetylation of Forkhead proteins at several critical lysine residues by acetylases, such as p300, and cAMP-response element-binding protein-binding protein (160, 204). The acetylation of Forkhead proteins results in an inhibition of the transactivation activity of Forkhead transcription factors.
Antioxidant systems. A number of sophisticated antioxidant systems exist in aerobic organisms, and they function to balance the cellular production of ROS that has been described in the previous sections. Endogenous antioxidant defenses include a network of compartmentalized antioxidant enzymes that are usually distributed within the cytoplasm and among various organelles in cells. A variety of small nonenzymatic molecules present in the internal milieu are also capable of scavenging ROS. In eukaryotic organisms, several ubiquitous primary antioxidant enzymes, such as SOD, catalase, and different forms of peroxidases work in a complex series of integrated reactions to convert ROS to more stable molecules, such as water and O2. Besides the primary antioxidant enzymes, a large number of secondary enzymes act in concert with small molecular-weight antioxidants to form redox cycles that provide necessary cofactors for primary antioxidant enzyme functions. Small molecular-weight antioxidants (e.g., GSH, NADPH, thioredoxin, vitamins E and C, and trace metals, such as selenium) can also function as direct scavengers of ROS. These enzymatic and nonenzymatic antioxidant systems are necessary for sustaining life by their ability to both maintain a delicate intracellular redox balance and reduce or prevent cellular damage caused by ROS (284).
Oxidative Stress in Cellular Senescence and Death
Cellular aging is characterized by an accumulation of damage that results in cell senescence and death. In recent years, pathways leading to cell senescence and death have been implicated as important factors contributing to organismal aging due to critical and irreplaceable cell loss. Therefore, this section will focus on the involvement of ROS and ROS-modified molecules in the activation of several signaling cascades related to cell death and senescence.
Oxidative stress and cellular responses. At the cellular level, oxidative stress generated by ROS and ROS-modified molecules can influence a wide range of cellular functions. The direct consequence of oxidative stress is damage to various intracellular constituents. For example, when lipid peroxidation occurs, changes in cellular membrane permeability and even membrane leakage can be manifested (233). Oxidative damage to both nuclear and mitochondrial DNA has detrimental effects, leading to uncontrolled cell proliferation or accelerated cell death (64). As would be expected, protein oxidation has many important physiological consequences that affect normal cellular functions (248). There is evidence that oxidative stress-mediated protein aggregation may be the primary cause of the neuronal death in several forms of aging-related neurodegenerative diseases (85). Furthermore, redox modification of transcriptional factors, as discussed in the previous section, leads to the activation or inactivation of signaling pathways that will subsequently produce changes in gene expression profiles (155), including those affecting cellular proliferation, differentiation, senescence, and death (Fig. 1). It is important to note that many oxidatively damaged macromolecules also act as regulatory molecules in cell-signaling pathways. For instance, several lipid peroxidation products have been implicated in the activation of stress-response signal transduction pathways (114, 140, 267), while DNA damage by oxidative stress triggers the activation of cell apoptosis and senescence cascades via p53 activation (87, 125).
Oxidative stress in apoptosis
Apoptosis, also known as programmed cell death, plays an important role in all stages of an organism's development. While there are controversies in the literature regarding the role of apoptosis in aging, age-associated increases in apoptosis have been observed in several physiological systems, including the human immune system, human hair follicle, and rat skeletal muscle (2, 5, 247).
Apoptotic cell death is executed via two major signaling pathways, the intrinsic and extrinsic pathways, in either caspase-dependent or caspase-independent manners (44). The intrinsic pathway involves the induction of various protein responses, such as posttranslational modifications, conformational changes and interorganelle translocation of specific proteins. These responses can produce an alteration in mitochondrial membrane potential and the release of apoptogenic factors, such as cytochrome c and apoptosis-inducing factor, from the mitochondria to the cytoplasm. A cascade of downstream signals, including caspases, is then stimulated to orchestrate apoptotic responses. In contrast, the induction of apoptosis by extrinsic pathways requires the binding of ligands to membrane receptors and recruitment of cytosolic adaptor proteins, which will, in turn, activate a series of initiator and effector caspases.
It has been clearly established that ROS and ROS-modulated molecules participate in both intrinsic and extrinsic apoptotic pathways (161). Some well-known exogenous ROS-generating stressors, such as radiation, proinflammatory cytokine treatment, growth factor withdrawal, and physiological challenges, such as heat stress, will stimulate apoptosis (8, 89, 124, 215). One example of oxidative stress involvement in extrinsic apoptotic signaling pathways is the redox activation of the MAPK cascade upon sustained oxidative stress. A novel protein in the mitogen-activated-kinase-kinase-kinase family, known as apoptosis signal-regulating kinase 1 (ASK1), has recently been identified as a critical redox sensor in the MAPK pathway (83, 262, 268). Thioredoxin, a small enzyme that participates in redox reactions, can have a negative regulatory influence on ASK1 and, subsequently, apoptosis (229, 298). Oxidative stress-induced apoptosis can also be reduced by a dominant-negative mutation of ASK1 (110). Another example involving the extrinsic apoptotic pathway is the downregulation of signaling pathways associated with growth factor receptor stimulation in response to oxidative stress (299).
In the intrinsic apoptotic pathway, it has been shown that proteins in the mitochondrial permeability transition pore complex, which controls mitochondrial membrane potential, are the direct targets of ROS (135). These proteins include the adenine nucleotide translocator in the inner membrane (82), the voltage-dependent anion channel in the outer membrane (151), and cyclophilin D at the matrix (11). Prooxidants capable of induction of mitochondrial permeability potential include not only chemicals, such as t-butyl hydroperoxide and diamide (207) but also lipid peroxidation products such as 4-hydroxynonenal (217). Moreover, it has been increasingly recognized that oxidative damage to organelles, such as lysosomes and the endoplasmic reticulum, stimulates crosstalk between these organelles and mitochondria and induction of apoptosis via intrinsic signaling pathway (122, 256).
More importantly, recent studies on p66Shc redox protein may provide a link between oxidative stress-mediated apoptosis and biological aging (169). The p66Shc redox protein is the third isoform discovered in the Shc protein family, and this group of proteins was initially identified as signal transduction adapters involved in mitogenic signaling through Ras, a small GTP-binding protein. Evidence has suggested that p66Shc is an atypical signal transducer that can be regulated by oxidative stress and also plays a role in H2O2 generation (80, 168). While mice lacking p66Shc (p66Shc–/–) live 30% longer than the control animals, p66Shc–/– cells from knockout mice are resistant to ROS-induced apoptosis (168, 181). Several lines of evidence indicate that p66Shc potentially acts at sites upstream of the mitochondrial permeability transition pore in oxidative stress-mediated apoptosis (80, 192, 203).
Oxidative stress in autophagy. Autophagy is characterized by the sequestration of bulk proteins, membrane fragments, and organelles into autophagic vesicles and the subsequent infusion and degradation of these vesicles in lysosomes. Cellular autophagy was discovered in the early 1960s, but renewed interests were recently triggered by the first genetic evidence suggesting that autophagy is essential in the life span extension of the nematode Caenorhabditis elegans (166). Specific inhibition of autophagy by RNAi techniques abolished the life-extension effect in C. elegans that carried a mutated gene in the insulin-like signaling pathway. While it has often been regarded as a housekeeping system for cells, autophagy is also a major pathway for the degradation and recycling of damaged cellular components to ensure cell survival under stress conditions, such as nutrition deprivation (178). However, recently it has been suggested that autophagy is not only important for cellular survival but can also induce cell death. For instance, there is evidence indicating that autophagy is directly involved in both cytokine- and chemical-induced cell death (216, 286).
It appears that the dual roles played by autophagy, involving cell survival as well as cell death, are both important during aging process. On one hand, reports have shown that autophagic function declines with age in in vivo and in vitro settings (52, 158, 260). In support of these observations, cells from old rodents subjected to caloric restriction (CR), a life-extension intervention, have similar levels of autophagic function as their young counterparts (24, 55). Conversely, excessive activation of autophagy, leading to cell death, was observed in neurons with increased protein aggregation, suggesting that autophagy may play an important role in aging-related neurodegenerative diseases (71, 292).
Although the regulation of autophagy is not yet completely understood, ROS have been implicated in the process. There is some evidence suggesting that aging-related increases in ROS production can result in elevated oxidative damage to proteins, including lysosomal proteins and proteins in autophagic pathways (38). However, more direct evidence for ROS involvement in autophagy comes from a recent report showing that cellular autophagy induced by caspase inhibition can lead to catalase degradation, resulting in ROS accumulation, lipid peroxidation, and loss of membrane integrity (291).
Oxidative stress in cellular senescence. Cellular senescence was first observed in cultured primary cells that ceased proliferation after a finite number of divisions. This concept of cellular senescence, termed replicative senescence, was later identified in primary cells under acute stress conditions and is also known as premature senescence (263). Two tumor suppressor proteins that are involved in cell cycle regulation, p53 and Rb protein, play central roles in the molecular mechanisms of cellular senescence. Evidence has shown that both p53 and Rb protein are activated when cells are in a senescent state (131, 182), while inactivation of these two proteins prevents senescence of human lung fibroblasts (280) and allows senescent cells to resume proliferation (81, 228). It is now known that several factors contribute to the activation of p53 and Rb protein and initiate senescence processes. Telomere shortening, acting through a pathway involving p53, is one of the most well-documented triggers for cellular senescence (101).
Oxidative stress is another important cell senescence trigger. Exogenous treatment with hydrogen peroxide or inhibition of antioxidant enzymes initiates premature senescence in human fibroblasts (29). Similarly, senescence is routinely observed in cells grown in a high ambient oxygen concentration, while the proliferation life span of cells is extended when they were grown under physiologically relevant low oxygen tension (43, 195, 200). Based on a wide range of observations, it appears that oxidative stress participates in multiple steps of senescence signaling pathways, functioning at sites either upstream or downstream of p53 activation. For example, ROS can cause DNA damage and also accelerate telomere shortening (13, 273). In addition, ROS released by the activation of Ras oncogene can induce cell senescence via p53 activation (136), while the overexpression of Akt, an important cell-signaling molecule, led to an inhibition of Foxo3a transcription activity and an elevation of intracellular ROS that later induced a senescence-like cell growth arrest in a p53 dependent manner (172). Moreover, increased p53 activation can trigger a senescence response that is accompanied by increased levels of intracellular ROS (41), and p53-mediated cell fate also appears to correlate with the levels of intracellular ROS (149). Elevations in p53 are associated with high levels of ROS and cell apoptosis, while slight increases in p53 expression induce cell senescence that is reminiscent of a small increase in oxidative stress.
The relevance of cellular senescence to in vivo aging has not yet been clearly delineated. Whether the mechanisms underlining premature senescence reflect the mechanisms of organismal aging is also a controversy. However, a recent study indicated that cellular markers of senescence, such as telomere shortening, are exponentially increased with age in skin fibroblasts of primates (100). Furthermore, hematopoietic stem cells obtained from mice that develop ataxia telangiectasia syndrome, which is characterized by premature aging, neurodegenerative diseases, immunodeficiency and cancers, showed increased levels of ROS and signs of cellular premature senescence (14, 113). Therefore, developing a better understanding of the molecular mechanisms of cellular senescence may provide some insight into the biology of organismal aging and potential sites for therapeutic interventions involving senescence pathways.
Oxidative Stress in Normal Organismal Aging
One of the central themes of the oxidative stress hypothesis is that ROS are the primary causal factor underlying aging-associated declines in physiological function. Several lines of direct and indirect evidence generated over the past two decades have demonstrated a positive relationship between increased in vivo oxidative stress and biological aging. While the majority of these correlative studies have been supportive of the oxidative stress hypothesis of aging, one controversial aspect of the hypothesis has been the lack of data clearly demonstrating a cause-and-effect relationship between the accumulation of oxidation-mediated cellular damage and aging. In the following sections, we discuss evidence related to ROS accumulation and changes in cellular redox status with aging, the potential to modulate oxidative damage via antioxidant enzyme manipulation, and current models being utilized to test the oxidative stress hypothesis.
Increased in vivo ROS levels and a shift in redox status with age.
One significant challenge that investigators face in their attempt to directly assess oxidant levels in vivo are the low concentrations of ROS present within a cell and the extremely transient nature of these species. The only analytical approach that is currently available to directly detect radical species in biological systems is electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance) (132). The EPR approach takes advantage of the magnetic properties of unpaired electrons, and the energy states of these species produce characteristic "footprints" that are detectable on the electromagnetic spectrum. As summarized in a review by Tarpey et al. (257), some other techniques specific for individual types of ROS, such as the reaction of dihydroethidium with O2
– to produce a red fluorescence, have also been developed in recent years. Using these techniques, recent studies have shown that ROS levels are increased with age in major organ systems such as liver, heart, brain, and skeletal muscle (22, 23, 56, 84, 297).
Due to the technical difficulties of directly assessing oxidant levels in vivo, investigators have generally had to rely on more indirect measurements of oxidative stress. In particular, redox status is considered an important parameter for assessing the prooxidant environment in an in vivo system (213, 234). Several indicators of in vivo redox status are available, including the ratios of GSH to GSSG, NADPH to NAPD+, and NADH to NAD+, as well as the balance between reduced and oxidized thioredoxin. Among these redox pairs, the GSH-to-GSSG ratio is thought to be one of most abundant redox buffer systems in mammalian species (234). A decrease in this ratio, indicating a relative shift from a reduced to an oxidized form of GSH, suggests the presence of oxidative stress at the cellular or tissue level. Experimentally, a progressive change to a more prooxidant environment has been noted in many species during aging (58, 245, 294, 297). In this scenario, an age-related shift from a cellular environment that is in redox balance to one with an oxidative profile would likely result in a blunted ability to buffer ROS that are generated in both "normal" conditions and at times of challenge. As demonstrated in recent studies from our laboratory in which rodents were exposed to a hyperthermic challenge (297), a primary outcome of this diminished buffering capability would be an increase in oxidative stress and widespread cellular damage. Thus, a progressive shift in cellular redox status could potentially be one of the primary molecular mechanisms contributing to the aging process and accompanying functional declines.
Accumulation of oxidative damage with age. In addition to elevations in in vivo ROS levels and redox balance in aged organisms, one of the most common types of evidence presented by investigators is the strong correlation between aging and an increase in oxidative damage to tissues throughout the body in species ranging from C. elegans to humans (21, 33, 183, 184, 245). Studies of oxidant-associated damage during aging have been focused on oxidative modification of intracellular macromolecules, primarily lipids, proteins and DNA (1, 4, 25, 294, 297). In the case of DNA, oxidative damage to mitochondrial and nuclear nucleic acids is significantly increased in all major tissues in aged organisms, including mice (93), rats (93, 269), hamsters (255), and humans (79, 241). Substantially higher levels of lipid peroxidation products (e.g., MDA, 4-HNE, and F2-isoprostanes) have been observed in aged compared with young organisms in tissues, such as kidney (194, 279), brain (211, 223, 285), liver (194, 279), lung (139, 285), and muscle (117, 197). Moreover, investigators have discovered age-related oxidative modifications to a large variety of proteins, including changes in structural proteins (88), enzymes, and proteins important in signal transduction pathways (27). Many investigators believe this increase in oxidized protein is the consequence of increased ROS production and impairment in protein turnover (65, 73). Removal of damaged proteins is mainly achieved by proteolytic degradation pathways including proteasome proteases, lysosome proteases, and mitochondrial proteases. Age-associated impairment has generally been reported in the function of all these proteolytic pathways (40, 74). It is important to point out that the extent of this age-associated increase in oxidative damage to macromolecules varies greatly among different tissues, species, and detection methods.
Aberrant regulation of redox-sensitive signal pathways with age.
Although the original concept of the oxidative stress theory suggested that ROS-induced accumulation of random macromolecular damage results in functional alterations and pathological conditions in old organisms, studies over the past decade have demonstrated that aging is associated with the regulation of several known redox-sensitive signal transduction pathways. For instance, as shown in Fig. 2, our laboratory has demonstrated an aging-associated increase in the DNA binding activities of NF-B and AP-1 in livers of old animals (294). Similar observations were made in many species by other investigators (126, 142, 155, 297). In two of the predominant animal models of longevity, C. elegans and Drosophila, it has been found that extension of life span is dependent on the activation of members of the Forkhead transcription factor family (107, 278). Moreover, transgenic mice containing an endogenous "superactive" form of p53 have a shortened life span and show signs of accelerated aging (266). However, in another strain of "super" p53 transgenic mice, increased activation of p53 did not affect life span (77).
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Studies along these lines have pointed toward new areas of inquiry related to oxidative stress and aging that could provide more detailed insight into the role of redox-sensitive transcription factor activation in modulating gene expression. Moreover, while many studies have documented the accumulation of oxidative markers with advancing age, only a limited amount of research, in a diverse range of species, such as worms, flies, mice, rats, and humans, has been conducted with regard to potential changes in gene expression profiles (102, 137, 138, 224, 295, 300). Thus, it will be necessary to gain more detailed insight into the mechanisms governing age-related changes in cell-signaling pathways with oxidative stress to better understand the integrative processes of aging, as well as to more carefully assess genes and metabolic pathways that may be directly involved in life span extension.
Effects of antioxidant manipulation on aging. Although numerous studies have demonstrated a correlation between in vivo oxidative damage and aging, a more insightful test of the oxidative stress theory would be to assess the direct effects of antioxidants on aging processes. Two approaches have been widely applied in this area of study. The first, which involves the evaluation of changes in antioxidant profiles of older compared with young organisms, has proven difficult to generalize. On one hand, it is reasonable to postulate that the increase in oxidative stress and damage to cellular constituents associated with aging could be due to a decline in antioxidant defense systems. However, the pattern of aging-related changes in antioxidants in many tissues and species has been inconsistent. There are certainly studies supporting the notion that a decline in antioxidant defenses occurs with aging (91), but substantial data also exist indicating that there is no generalized decrease in antioxidant enzyme function (159, 221, 243). When viewed broadly over a range of different species, tissues, and conditions, the lack of a consistent decline in antioxidant enzyme activities suggests that antioxidant enzymes may not be a primary limiting factor governing the degree of cellular oxidative damage with aging.
The second approach has been to directly determine whether experimental interventions can ameliorate oxidative damage or slow the rate of aging. This remains an active area of inquiry, as investigators attempt to increase intracellular antioxidant defenses, either by dietary supplementation of antioxidants or by overexpressing genes encoding antioxidant enzymes (e.g., SOD, catalase).
Early attempts at antioxidant intervention as a means to delay aging were initiated soon after the free radical theory of aging was proposed, but these pursuits failed to extend life span in most cases (28, 261). In recent years, some impressive successes have been achieved in nonmammalian models using synthetic antioxidant enzyme mimetics, and these results have stimulated additional interest in aging research. In one of the first studies of this type, Melov et al. (167) showed that C. elegans treated with EUK-8 and EUK-134, antioxidant enzyme mimetics with both SOD and catalase activity, had significantly longer life spans than untreated nematodes. However, the same conclusions could not be drawn from subsequent studies involving house flies treated with similar agents (17). Moreover, Keaney et al. (121) recently found that administration of these antioxidant enzyme mimetics to C. elegans, while increasing cellular SOD activity in a dose-dependent manner, did not extend life span.
As advancements in genetic manipulation of animals have progressed, studies involving gene transfer of antioxidant enzymes, such as CuZnSOD, MnSOD, and catalase have been conducted in Drosophila. However, as noted by Sohal et al. (244), the results obtained from these studies have been difficult to reconcile. For example, overexpression of CuZnSOD and MnSOD by an inducible promoter in adult Drosophila was associated with extended life span (199, 251, 252), while overexpression of catalase in Drosophila had no effect on longevity (173, 191). Furthermore, other investigations using different strains of Drosophila and different promoters for antioxidant enzyme gene transfer have also yielded mixed results (18, 189). It is important to note that there can be substantial variation in both life span and effects of antioxidant enzymes in different fly strains. Moreover, Sohal et al. (243) have suggested that the magnitude of any extension of life span in these types of studies could be attributed to the utilization of relatively short-lived control flies in the different experimental designs. Thus, extrapolation of these findings in lower species such as Drosophila to more complex mammalian species should be carefully scrutinized.
The direct impact of antioxidant enzyme treatment on life span is even less clearly defined in mammalian models. Some studies have failed to demonstrate improvements in life span with antioxidant enzyme manipulation, while others showed positive effects on longevity in mouse models. For instance, transgenic mice that constitutively overexpress human CuZnSOD did not live longer than control animals (105), while heterozygous mice with reduced MnSOD activity have a life expectancy that is similar to wild-type mice, although these animals have increased oxidative damage to DNA (272). In contrast to these negative results, a recent study provides some of the first direct evidence in support of the oxidative stress theory of aging in mammals. Schriner et al. (237) showed that transgenic mice overexpressing human catalase in mitochondria had increases in both median and maximum life span by averages of 5 and 5.5 mo, respectively. It was also significant that this extension of life span was accompanied by reductions in selected markers of oxidative damage, such as attenuated H2O2 production and H2O2-sensitive aconitase inactivation in heart and skeletal muscle.
The expression of other antioxidant enzymes has also affected life span in a variety of animal models. In Drosophila, overexpression of glutamate-cysteine ligase, a rate-limiting enzyme for de novo GSH biosynthesis, extended mean and maximum life span by 50% (190). Similarly, overexpression of methionine sulfoxide reductase, a enzyme that catalyzes the reduction of methionine sulfoxide back to methionine, extended life span by 70% in Drosophila (227), while mice with reduced activity of methionine sulfoxide reductase have an increased sensitivity to oxidative stress (179). Furthermore, transgenic mice overexpressing human thioredoxin, a small protein with important redox regulatory properties, exhibited extended median and maximum life span values compared with their wild-type counterparts (171).
These controversial and sometimes contradictory outcomes from experiments involving the manipulation of antioxidants by either pharmacological or genetic approaches add further support for the postulate that aging is a complicated and multifaceted phenomenon that cannot be accounted for by a single theory. Future research should focus some efforts on delineating common pathways that contribute to the aging process, especially among longer-lived species. These types of studies will provide more mechanistic insight into potential therapeutic targets and approaches for modulating aging, age-related diseases, and longevity.
Oxidative stress in aging models. Many of the in vivo aging models currently being utilized by investigators involve animals with an altered life span that has been produced by either pharmacological intervention or gene manipulation. Such studies have provided significant insights into aging processes and, significantly, are now progressing to include higher mammalian species. There are generally two types of aging models: long-lived animals with retarded aging processes or short-lived animals with accelerated aging processes.
CR, an intervention involving a prolonged reduction in caloric intake while maintaining adequate nutrition, is known to increase life span and retard age-associated physiological deterioration in species ranging from yeast to mammals (111, 129, 156, 245, 282, 289). One potential mechanism contributing to these beneficial effects associated with CR is a reduction in oxidative stress (129, 245). Currently, several pieces of evidence support this postulation. For instance, CR can blunt the increase in lipid peroxidation, accumulation of oxidized proteins, and oxidative damage to DNA that occurs with aging (59, 159, 242). This reduction in oxidative damage by CR has been attributed to a decline in the rate of ROS generation and an enhanced ability to repair oxidative damage in CR animals (148, 231). Moreover, recent findings have demonstrated that CR can enhance some aspects of antioxidant enzyme function (6, 49, 92), although other studies that focused on the activities of individual antioxidant enzymes in a variety of tissues during aging or in response to CR have generated conflicting results (92, 159, 221, 245). In addition, our laboratory demonstrated that a long-term reduction in caloric intake also improves tolerance to heat stress and reduces heat-induced oxidative damage in aged rodents (92).
Information obtained from several long-lived animal models has been supportive of the oxidative stress theory. For instance, growth hormone deficiency leads to extended life span in several strains of mice, including Ames dwarf, Snell dwarf, and genetically modulated, growth-hormone receptor knock-out mice, and most of these strains have shown an increased resistance to oxidative stress (16). Specifically, Ames dwarf mice exhibit upregulation of antioxidant enzyme expression (35, 36, 98), reduced mitochondrial ROS production, and decreased oxidative damage (34, 230) in several tissue types. Importantly, these physiological changes are associated with life span increases of over 50% compared with their wild-type littermates. Another category of long-lived animals involves species defective in the insulin/IGF-I signaling pathway. Not surprisingly, life-extension in these animals is generally accompanied by increased antioxidant defenses and resistance to oxidative stress (15, 103, 144, 258, 271).
Studies by Nemoto and Finkel (181) have demonstrated that p66sch, a protein involved in the transduction of mitogenic and apoptotic signals, participates in the regulation of intracellular oxidant levels. In these experiments, immortalized fibroblasts from p66sch–/– mice subjected to the stress of serum deprivation had lowered levels of ROS than their wild-type counterparts as measured by a redox sensitive probe. Consistent with these findings, p66sch gene knock-out in mice increases life span by 30% and reduces in vivo oxidative damage and ROS production (181, 265).
Evidence from an animal model with a shortened life span has also been supportive of the oxidative stress theory. Senescence-accelerated mice have shown hastened declines in mitochondrial function (188), GSH-to-GSSG ratios (219), and antioxidant enzyme activities, along with increased lipid peroxidation (198) and enhanced sensitivity to oxidative stress (146) in multiple types of tissues.
Alternatives to the oxidative stress theory: evidence in animal models. While substantial evidence from various animal models provides a strong link between aging and oxidative stress, it is important to note that data from some aging models also suggest alternatives to the oxidative stress theory of aging. As an example, studies attempting to understand the effects of CR on aging have produced results implicating other causal factors for changes in aging processes, such as neuroendocrine alterations, a decrease in protein glycation, a lowering of body temperature and associated hypometabolic state, and alterations in gene expression (111, 156, 193). In addition, mice expressing a defective form of mitochondrial DNA polymerase, an enzyme important in repairing mitochondrial DNA damage, showed reduced mean and maximum life span and evidence of premature aging (264). However, no significant increases in markers of oxidative damage were found in these animals compared with their wild-type counterparts. Thus, results obtained from experiments performed in this animal model suggest that the accumulation of mitochondrial DNA damage that is independent of oxidative stress may also be important in the aging process.
Despite some contradictory results, animal models produced during the past decade have provided valuable information on biological systems that impact on aging. As more animal models are developed to study aging mechanisms, it will be important to carefully evaluate the relevance of these models to normal aging processes. For instance, a genetic manipulation that accelerates "aging" can yield important insight into the function of an intracellular signaling pathway on specific aging processes. However, it may be valid to question to what extent an accelerated aging model is relevant to normal biological aging. The same is true for life span extension models that are currently being utilized. Furthermore, the only intervention that has been consistently successful in extending life span in mammals is CR, and it is well documented that CR yields a vast array of physiological changes that impact on multiple systems. Therefore, one of the current challenges for researchers is to incorporate the information obtained from genetic manipulation studies into a broader context that focuses on integrated mechanisms of aging, with a particular eye on applications that are relevant to humans.
Oxidative stress in age-related stress tolerance. As noted in preceding sections, aging is linked to an increase in ROS generation and oxidative damage. In addition, many types of stressors, such as hyperthermia (297) and hypoxia (31), have been associated with an increased production of prooxidants and induction of oxidative stress. Recently, several studies have demonstrated that young organisms are capable of initiating an array of regulatory processes in response to oxidative stress, including the activation of stress-gene expression and modification of stress-responsive signal transcription pathways. In contrast, there is compelling evidence that these regulatory processes are altered in old organisms (39, 92, 108, 109, 128, 130, 142, 177, 270, 297). Therefore, stress-induced cellular injury appears to be exaggerated with advancing age. This failure to effectively respond to cellular challenge has been postulated to contribute to a reduction in stress tolerance and the development of various pathologies and diseases (155). Consistent with this view, Lithgow and colleagues (145, 162) have shown that extension of life span in C. elegans through genetic mutations was associated with an increase in resistance to a variety of insults. Moreover, Miller (170) has speculated that enhanced resistance to oxidative stress, through both antioxidant mechanisms and the engagement of multiple cellular signaling pathways associated with stress resistance, will be integral in explaining the mechanism of life span extension.
Studies from the authors' laboratory have addressed the issue of stress tolerance in detail in a series of experiments by investigating the effects of hyperthermia on old compared with young rodents (294, 296, 297). For instance, repeated heat challenge produces extensive injury in the liver of older rats, whereas young rats tolerate the stress very well. In the old cohort, this injury pattern is associated with increases in steady-state levels of ROS and substantial oxidative damage to hepatocellular macromolecules (e.g., lipids, DNA), along with alterations in intracellular redox status and aberrant activation of stress-response transcription factors (297). These data demonstrate that young animals can effectively cope with oxidative stress in response to environmental challenge. In contrast, in older animals, a decline in cellular redox potential, along with exaggerated ROS generation, leads to extensive oxidative damage and alterations in intracellular signal transduction. Factors such as these are likely to contribute to the cellular dysfunction and reductions in stress tolerance that are hallmarks of aging.
In subsequent experiments, in an effort to determine whether a therapeutic intervention that reduces the degree of oxidative damage can protect old animals from heat-stress induced liver injury, young and old rats were chronically administered a continuous, low-dose infusion of the synthetic catalytic ROS scavenger EUK-189 via an implanted osmotic pump and then were exposed to a heat stress protocol (294). Widespread oxidative injury to the liver (e.g., hepatocyte vacuolization, necrosis, monocyte infiltration) was present in old but not young vehicle-treated animals in response to hyperthermic challenge. However, SOD/catalase mimetic treatment markedly decreased the liver injury associated with heat stress in old animals. The reversal of histopathologic damage with EUK-189 in the old cohort was also associated with a striking reduction in hepatocellular lipid peroxidation (Fig. 3A) and an improvement in intracellular redox status, as evidenced by an increase in GSH-to-GSSG ratios within liver tissue (Fig. 3B).
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These results suggest that either a decline in redox potential or an exaggerated production of ROS will lead to extensive hepatocellular oxidative damage and alterations in intracellular signal transduction in older animals, which subsequently contribute to cellular and organ dysfunction and age-related reductions in stress tolerance. Based on these and other observations, it could be postulated that antioxidants would be therapeutically effective in an aged mammal exposed to a stressor that generates exaggerated oxidative injury. The findings from these studies also implicate an imbalance in intracellular redox status as having a direct causal role in the reduced stress tolerance that accompanies aging.
ROS, inflammation, and aging. Developing a clearer understanding of the basic mechanisms of biological aging certainly has long-term implications for improving both longevity and quality of life in humans. However, along with the desire to understand the "hows" of aging at a molecular level, there is also a compelling need to delineate why the aging process is accompanied by an increased incidence of various chronic diseases. This type of knowledge should allow scientists to develop more comprehensive therapeutic strategies to offset the increased vulnerability of aged individuals to a wide range of degenerative conditions.
As outlined in previous sections, the free radical theory of aging is currently among the most plausible explanations for the aging process in mammalian species. This theory serves not only to explain basic mechanisms of aging, but also the pathogenesis of a range of disease processes that consistently accompany aging, including atherosclerosis and other cardiovascular disorders, dementia, diabetes, arthritis, and osteoporosis (21, 51). In evaluating the free radical theory and its possible link to numerous age-related maladies, investigators have focused attention on the possibility that an increase in ROS, along with a concomitant disruption in redox balance, leads to a state of chronic inflammation (Fig. 4) (45, 47, 134, 232). Along these lines, Yu and colleagues (46) have proposed the "molecular inflammation hypothesis of aging" as a possible mechanistic link between biological aging and pathological conditions associated with aging.
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There is substantial evidence, in a range of systems and species, supporting the link between aging and chronic inflammation, as well as the integrated molecular and cellular signaling processes depicted in Fig. 4. Chronic inflammation is typically assessed by measuring various inflammatory biomarkers, and at the tissue level, the primary marker of chronic inflammation is the infiltration of macrophages, which is positively correlated to several age-associated diseases. For instance, investigators have observed activated macrophages in the brain of patients with various neurodegenerative diseases (163, 164), as well as in plaques obtained from patients with atherosclerosis (288). These activated macrophages generate reactive species, which can subsequently produce oxidative and nitrosative injury in specific tissues. In the systemic circulation, biomarkers include inflammatory cytokines and acute phase proteins such as C-reactive protein, and it is well-documented that circulating levels of these proinflammatory proteins are increased with advancing age. In humans, IL-1
, IL-6, and TNF-
are generally higher in the plasma of older individuals compared with their young counterparts (10, 37, 54, 61, 70, 202). However, it is important to note that this chronic inflammatory condition in aged populations is associated with substantially lower circulating levels of these inflammatory markers than would be generated during an acute inflammatory condition.
Evidence from both animal and human studies indicates that there are also positive correlations between chronic inflammation and the development of age-associated diseases (47, 232). Although many of these diseases involve a chronic inflammatory state, it is unclear whether inflammation is an integral component in the pathogenesis of a particular disease or an underlying secondary event. However, long-term inflammation can clearly influence the pathogenesis and progression of these diseases. Attempts to delineate basic mechanisms responsible for the pathogenesis of these disease conditions are currently being undertaken in a wide array of disciplines and will necessitate an integrative approach that includes an understanding of the entire inflammatory cascade. Relevant to this minireview, it appears that increased ROS levels, accompanying cellular redox imbalance, and related oxidative damage are key contributors to the pathogenesis of many age-related maladies. Therefore, knowledge gained concerning the role of oxidative stress in the pathogenesis of specific diseases and chronic inflammatory conditions may prove insightful to scientists pursuing more basic questions related to the causal agents of biological aging and issues of longevity in mammalian species.
Studies in human and nonhuman primates. A vast majority of studies addressing the role of oxidative stress in aging processes over the past half century have been performed in a wide range of animal species, and the results obtained have certainly shed a great deal of light on basic molecular mechanisms of aging. However, extrapolating findings from flies, worms, and rodents to humans, while useful as a potential means to optimize human health and longevity, should also be approached with caution. Given our complex genetic and physiological make-up, it is important to directly assess the role of oxidative stress in human aging processes.
At the present time, only limited experimental data are available regarding oxidation status in older humans, due in part to the difficulties of assessing aging per se compared with age-related disease effects. An increased presence of oxidative damage to mitochondrial DNA has been found in skeletal muscle and brain of older individuals (165, 241), and these observations are consistent with recent findings demonstrating a decline in mitochondrial function in older humans (48, 206). Some survey data evaluating plasma antioxidant levels and dietary intake of antioxidant nutrients in human populations as a function of age can be found in the literature (53, 75), although this type of information has only limited utility. However, as noted in a previous section, one of the more significant developments in this area over the past decade has been the accumulation of evidence to suggest that oxidative stress is a primary or secondary causal factor in many age-dependent human diseases (21, 26, 30, 50, 51, 69, 116, 250, 281).
Some potential insights into the impact of oxidative stress on aging processes in humans can also be drawn from the CR literature. Numerous studies have demonstrated that a decrease in caloric intake of 
40% throughout the life span of laboratory animals can eliminate or retard numerous age-associated chronic diseases, improve stress tolerance, and prolong both average and maximal life span. Several hypotheses have been proposed to explain the mechanisms by which CR functions to produce these beneficial effects, but a decrease in energy expenditure, along with concomitant reductions in ROS production and oxidative damage, appear to be key components in many studies (92, 129, 157, 245). Investigations involving nonhuman primates, which are ongoing, have reported beneficial effects of CR on overall health status. In rhesus monkeys, low calorie diets have impacted on several morphological and physiological parameters (112, 218, 226), including a reduction in skeletal muscle oxidative damage (226). However, the expression of genes involved in oxidative stress from skeletal muscle samples of monkeys was not affected by CR in a study by Kayo et al. (120). In addition, primate life span data are not yet available. In humans, experimental data on the effects of CR is quite limited. Some studies suggest that health benefits can be manifested from short-term reductions in caloric intake (68, 99, 274, 275), but there are currently no results from well-controlled studies involving long-term CR.
One emerging area of research that has important clinical ramifications is the potential role of ROS on vascular dysfunction that accompanies human aging (46, 154, 186, 201, 246, 290). Blood vessels are critically important to overall physiological homeostasis due to their role in supplying cells throughout the body with oxygen and nutrients. Thus, damage to these vessels can have a profound impact on organ function and contribute to a myriad of diseases that are typically associated with aging (e.g., diabetes, atherosclerosis, hypertension). Endothelial cells that line blood vessels, because of their location, are especially vulnerable to potentially damaging circulatory factors, such as oxidized macromolecules and proinflammatory cytokines, and endogenous ROS generation within endothelial cells can also lead to oxidative damage and cellular dysfunction. Yu and colleagues (46, 47, 290) have postulated that "vascular aging" may be a primary factor in the overall aging process; thus, alterations at cellular, tissue, and organ levels may be secondary to age-related vascular dysfunction. In such a scenario, therapeutic interventions aimed at reducing oxidative stress and accompanying damage to the vasculature could have beneficial effects on a range of age-associated pathologies.
Most antioxidant intervention studies have involved long-term treatments as a potential means to eliminate age-related oxidative damage. However, there is a developing literature focused on the use of acute or short-term administration of antioxidants as a method to determine the tonic influence of oxidative stress on altered physiological function in aged humans. Relevant examples can be found in studies that have examined the link between oxidative stress, endothelial dysfunction, and the potential development of cardiovascular diseases (e.g., atherosclerosis, hypertension). The endothelium functions to modulate both vascular tone and structure, primarily through the production of NO, which serves as a relaxing factor the vasculature. Endothelial dysfunction, which is increased with aging (60, 78, 254), can arise when oxidative stress reduces NO availability. Moreover, arterial endothelial dysfunction is a key component of many cardiovascular disorders (174, 225).
Based on the link established between oxidative stress and age-related vascular dysfunction, investigators have begun to view the vasculature as a key target for antioxidant therapeutic intervention with regard to aging and age-associated diseases. Initial studies in this area demonstrated that the acute administration of ascorbic acid (vitamin C) improved brachial artery blood flow in patients with coronary artery disease (141) or congestive heart failure (104). More recent studies in older humans have shown that vascular function (specifically, ischemia-induced increases in brachial artery blood flow) was reduced in old, compared with young, sedentary men, but acute ascorbic acid infusion restored arterial blood flow responses in the older group. However, ascorbic acid supplementation for a relatively short duration (30 days) did not have the same effect on age-related vascular responsiveness (62). These findings, along with supporting data from postmenopausal women treated acutely with ascorbic acid (175), suggest that the impairment in arterial vascular function that accompanies aging may be mediated by an increase in vascular oxidative stress. These results are some of the first evidence to implicate oxidative stress as an important contributor to vascular dysfunction with human aging. While it is unclear whether longer-term antioxidant treatments might prove effective in dealing with aging and conditions, such as vascular aging, provocative results from acute-treatment studies suggest that the vasculature should be viewed as a key target for therapeutic intervention (133, 180, 239).
Integrative Mechanisms
While the ultimate causes of aging are complex and multifaceted, it is clear that knowledge regarding the cellular, biochemical, and genetic changes that accompany aging is steadily growing. There is now strong correlative evidence implicating the formation of ROS and the accompanying increase in oxidative stress as key contributors to the process of aging in cells and tissues. This correlation is found in a wide range of species both short- and long-lived. While the oxidative stress theory remains a viable hypothesis, a direct casual link between oxidative stress and either aging pathologies or life span has not yet been definitively proven, which has hindered the widespread acceptance of this theory as the primary explanation for the aging process.
Therefore, at the current time, a more appropriate explanation would likely include an increase in ROS levels and subsequent oxidative stress/damage as key features of more complex physiological changes that contribute to aging. One possible integrative scenario by which ROS and oxidative stress could contribute to aging is proposed in Fig. 5. As illustrated in this figure, the aging process involves multifaceted changes that are affected by both exogenous conditions and endogenous factors at molecular, cellular, tissue, organ, and systemic levels. Aging effects are revealed first at molecular levels and include the accumulation of macromolecular damage and changes in signal transduction pathways. These alterations subsequently impact on cellular responses, such as organelle dysfunction, inflammation, cell proliferation, survival, and death. Eventually, dysfunction is manifested at systemic levels, which would likely include a decline in organ function, reduced stress tolerance, frailty, increased incidence of diseases, and death.
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Perspectives
While it remains unclear whether reactive species derived from oxygen molecules are the primary cause of aging in mammalian species, there is substantial evidence from a variety of species demonstrating that in vivo oxidative damage is highly correlated with biological aging. However, to date, interventions aimed at reducing oxidative damage in mammals, primarily through manipulations of antioxidant enzyme systems, have yielded disappointing results.
Another issue that must be carefully scrutinized as this field of research moves forward is whether insights gained from relatively short-lived mammalian species, such as mice with an average life span of 2 to 4 yr, are applicable to humans, who can live well beyond 100 years. If oxidative stress is a key component of biological aging, researchers must consider whether there are differences in ROS generation, cellular responses to these molecular species, or antioxidant defenses that explain the disparity in longevity of various mammalian species.
Despite these concerns, substantial progress has been made toward an integrative understanding of aging, and attempts to delineate mechanisms to explain the biology of aging should include ROS and oxidative stress as potential key participants. With the successes being achieved through genetic manipulations and the development of drug therapies that can modulate oxidative processes and antioxidant defe
P < 0.05 vs. old control and old heat + EUK groups; 
P < 0.05 vs. old control and old heat. Adapted from Zhang et al. (
