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

Invited Review:

An Integrated View of Oxidative Stress in Aging:

Basic Mechanisms, Functional Effects and Pathological Considerations

Kevin C. Kregel and Hannah J. Zhang

Department of Integrative Physiology and

Free Radical and Radiation Biology Program, Department of Radiation Oncology,

The University of Iowa, Iowa City, IA 52242

Abbreviated Title: Oxidative Stress in Aging

*Address Correspondence to:

Kevin C. Kregel, Ph.D.

Department of Integrative Physiology

532 FH

The University of Iowa

Iowa City, IA 52242

Phone: 319-335-7596; Fax: 319-335-6966

e-mail: [email protected]

of 73Articles in PresS. Am J Physiol Regul Integr Comp Physiol (August 17, 2006). doi:10.1152/ajpregu.00327.2006

Copyright © 2006 by the American Physiological Society.

mailto:[email protected]

Oxidative Stress in Aging page 2

ABSTRACT

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 towards 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 lifespan. 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 mini-review 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.

Keywords: Oxidative damage, antioxidants, free radicals, ROS, hyperthermia, caloric restriction

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INTRODUCTION

"If I’d known I was going to live this long, I would have taken better care of myself."

American ragtime musician Eubie Blake, who lived well into his nineties

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

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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 lifespan (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 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

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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 up-regulation of

pro-inflammatory gene expression. As a result, various pro-inflammatory 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 mini-

review 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).

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BASICS OF REACTIVE OXYGEN SPECIES

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 non-radical 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.

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Oxidative Damage to Macromolecules by ROS

It is 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

(8-OHdG). 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 next section, can produce an increase or decrease in their specific DNA binding

activities, which stimulates gene expression changes that impact on cell survive, death and

senescence pathways (Fig. 1).

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Redox Modulation of Transcriptional Factors by ROS

While ROS are known to function as harmful products of aerobic metabolism, especially

when present at high concentrations, low levels of these pro-oxidant molecules have more

recently been discovered to modulate transcription factor activation (66, 155). A growing

number of molecules, such as many kinases (147, 210), phosphatases (123), and transcription

factors (63, 67, 185, 253, 270, 296), in a wide range of signal transduction pathways, are thought

to be modulated by intracellular redox status. Moreover, a few transcription factors, such as the

small GTP-binding protein Rac, are known to activate ROS-generating enzymes (e.g., NADPH

oxidase), and produce ROS as a modulator of downstream molecules (9, 277).

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 bi-directional, 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 nuclear factor κB (NF-κB).

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-κB 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).

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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 N-

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 IκB, the inhibitory subunit of NF-

κB, is the key step in NF-κB redox activation. ROS-mediated phosphorylation of IκB, leading

to its ubiquitination and degradation, allows the NF-κB 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-κB 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 PI3-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 (CREB)-binding protein (160, 204). The

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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 of a network of compartmentalized

antioxidant enzymes that are usually distributed within the cytoplasm and among various

organelles in cells. A variety of small non-enzymatic molecules present in the internal milieu are

also capable of scavenging ROS. In eukaryotic organisms, several ubiquitous primary

antioxidant enzymes such as superoxide dismutase (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 - glutathione, NADPH, thioredoxin, vitamins E and C, and trace metals such

as selenium among them - can also function as direct scavengers of ROS. These enzymatic and

non-enzymatic antioxidant systems are necessary for sustaining life by their ability to both

maintaining a delicate intracellular redox balance and reduce or preventing cellular damage

caused by ROS (284).

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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-

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response signal transduction pathways (114, 140, 267), while DNA damage by oxidative stress

triggers the activation of cell apoptosis and senescence cascade 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 post-translational

modifications, conformational changes and inter-organelle 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, pro-inflammatory 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

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the redox activation of the mitogen-activated protein kinase (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

down-regulation 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). Pro-oxidants 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 biology of aging (169). 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

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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 lifespan 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, a life-extension

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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-relate 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

senescence 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).

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Oxidative stress is another important cell senescence trigger. Exogenous treatment with

hydrogen peroxide or inhibition of antioxidant enzymes initiated premature senescence in human

fibroblasts (29). Similarly, senescence is routinely observed in cells grown in a high ambient

oxygen concentration, while the proliferation lifespan of cells was 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 is 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

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signs of cellular premature senescence (14, 113). Therefore, developing a better understanding

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

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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 pro-oxidant environment in

an in vivo system (213, 234). Several indicators of in vivo redox status are available, including

the ratios of glutathione (GSH) to glutathione disulfide (GSSG), NADPH to NAPD+, and NADH

to NAD+, as well as the balance between reduced and oxidized thioredoxin. Among these redox

pairs, the GSH: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 glutathione, suggests the presence of oxidative stress at the cellular or tissue

level. Experimentally, a progressive change to a more pro-oxidant 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.

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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 to 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.

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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 Figure 2, our laboratory has demonstrated an aging-

associated increase in the DNA binding activities of NF-κB 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 lifespan 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 lifespan and show signs of accelerated aging (266).

However, in another strain of “super” p53 transgenic mice, increased activation of p53 did not

affect lifespan (77).

Investigators are now attempting to understand whether there is a direct causal link

between oxidative stress and the modulation of aging processes via specific signal transduction

pathways. As an example, mutations in the DAF-16 signaling pathway, a homologue of the

mammalian Forkhead protein pathway, have been shown to extend lifespan in C. elegans.

However, the manifestation of this result requires the expression of ctl-1, a gene coding for the

cytosolic form of catalase. Mutation of the ctl-1 gene, which led to a decrease in catalase

activity, abolished the life-extension effects in worms with daf-16 mutations (259). Furthermore,

Nemoto and Finkel (185) have identified a pathway for peroxide modulation of the Forkhead

protein in p66shc null mice fibroblasts and Migliaccio et al. (168) demonstrated that mice

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lacking p66shc exhibit an extended lifespan phenotype. Taken together, these results provide

additional support for the possibility that intracellular oxidants play a key role in modulating in

vivo aging processes.

Studies along these lines have pointed towards 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 in order 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 lifespan 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 to 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

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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 lifespan in

most cases (28, 261). In recent years, some impressive successes have been achieved in non-

mammalian 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 lifespans than untreated

nematodes. However, the same conclusions could not be drawn from subsequent studies

involving house flies treated with similar agents (17). Moreover, Kearney 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 lifespan.

As advancements in genetic manipulation of animals have progressed, studies involving

gene transfer of antioxidant enzymes such as CuZnSOD, MnSOD, and catalase have been

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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 lifespan

(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 lifespan and the effects of

antioxidant enzymes in different fly strains. Moreover, Sohal et al. (243) have suggested that the

magnitude of any extension of lifespan 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 lifespan is even less clearly defined

in mammalian models. Some studies have failed to demonstrate improvements in lifespan 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 even though 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 lifespan by averages of 5 and 5.5 months,

respectively. It was also significant that this extension of lifespan was accompanied by

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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 lifespan 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 lifespan 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 lifespan values compared to 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 lifespan 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

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two types of aging models - long-lived animals with retarded aging processes or short-lived

animals with accelerated aging processes.

Caloric restriction (CR), an intervention involving a prolonged reduction in caloric intake

while maintaining adequate nutrition, is known to increase lifespan 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 also 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 lifespan 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 up-regulation 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

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with lifespan increases of over 50% compared to their wild type littermates. Another category of

long-lived animals involves species defective in the insulin/insulin-like growth factor type 1

receptor (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 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 (185). Consistent with these findings, p66sch gene knock-

out in mice increases lifespan by 30% and reduces in vivo oxidative damage and ROS production

(181, 265).

Evidence from an animal model with a shortened lifespan has also been supportive of the

oxidative stress theory. Senescence-accelerated (SAM) mice have shown hastened declines in

mitochondrial function (188), GSH/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

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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 lifespan and evidence of premature aging (264). However, no significant

increases in markers of oxidative damage were found in these animals compared to 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 lifespan

extension models that are currently being utilized. Further, the only intervention that has been

consistently successful in extending lifespan 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.

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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 pro-oxidants 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 have shown that extension of lifespan in C. elegans through genetic mutations was

associated with an increase in resistance to a variety of insults (145, 162). 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 lifespan 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 to 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).

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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

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/GSSG ratios within liver tissue (Fig. 3B).

Cell signaling changes were also noted with EUK-189 treatment. AP-1, which is a

redox-sensitive early response transcription factor involved in the regulation of cellular stress

responses, was elevated in response to heat stress in old rats. With chronic antioxidant enzyme

mimetic treatment, DNA binding activity of AP-1 was reduced back to control levels (Fig. 3C).

Finally, plasma alanine aminotransferase (ALT), which is a systemic marker for hepatocellular

damage, was measured in animals after the heating protocol in order to assess in vivo functional

changes in relation to the alterations in oxidative stress and organ damage that were observed to

accompany the modulation in cellular redox status with EUK-189 treatment (Fig. 3D). ALT

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concentrations were markedly increased in older but not young animals following heating,

consistent with the histological and oxidative damage observed. Conversely, heat stressed old

animals that received chronic antioxidant mimetic treatment had circulating ALT levels that were

similar to those in young and old non-heated control animals.

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

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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.

An age-related disruption in intracellular redox balance appears to be a primary causal

factor in producing a chronic state of inflammation (Fig. 4). Besides impairing a cell’s ability to

effectively remove ROS, this redox imbalance leads to an activation of redox-sensitive

transcription factors and the subsequent generation of numerous pro-inflammatory mediators

(e.g., cytokines, chemokines, iNOS). These molecules, acting both systemically and at the tissue

level, can produce products that include both reactive oxygen and reactive nitrogen species (153,

187, 214, 249, 284), and it is postulated that the accumulation of these reactive species

contributes to the pathogenesis of age-related diseases (47, 232). In this scenario, a positive

feedback loop is also activated with the generation of these reactive species, which serves to

further augment the cascade of inflammatory processes and exacerbate inflammation-induced

cellular and tissue damage.

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-

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associated diseases. For instance, McGeer and McGeer 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 pro-

inflammatory proteins are increased with advancing age. In humans, IL-1β, IL-6, and TNFα are

generally higher in the plasma of older individuals compared to 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 mini-review, 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

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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 Non-Human 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 to 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 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 caloric restriction literature. Numerous studies have demonstrated

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that a decrease in caloric intake of approximately 40% throughout the lifespan of laboratory

animals can eliminate or retard numerous age-associated chronic diseases, improve stress

tolerance, and prolong both average and maximal lifespan. Several hypotheses have been

proposed to explain the mechanisms by which caloric restriction 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 non-human primates, which are ongoing, have reported

beneficial effects of caloric restriction 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

caloric restriction in a study by Kayo et al (120). In addition, primate lifespan data are not yet

available. In humans, experimental data on the effects of caloric restriction 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 caloric restriction.

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 pro-inflammatory cytokines,

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and endogenous ROS generation within endothelial cells can also lead to oxidative damage and

cellular dysfunction. Yu and Chung have postulated that ‘vascular aging’ may be a primary

factor in the overall aging process (46, 47, 290), and 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 nitric oxide (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 to young sedentary

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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 post-menopausal 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 lifespan have 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

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physiological changes that contribute to aging. One possible integrative scenario by which ROS

and oxidative stress could contribute to aging is proposed in Figure 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.

With this model has come a greater appreciation of the important roles played by ROS

and the profound physiological impact that an acute or chronic shift in cellular redox

environment can have on an organism, especially as it grows older. As reviewed in previous

sections, intracellular ROS production is increased with exposure to many environmental stress

conditions. While increased oxidative stress and other factors trigger an array of alterations at

molecular levels that contribute to aging, genetic factors, as well as lifestyle, can accelerate or

reverse the aging process by either sensitizing or preventing and repairing the defects. As the

molecular and cellular defects accumulate during the lifespan of an organism, the resulting

perturbation in redox balance and the endogenous generation of ROS will further influence the

regulation of a number of physiological functions (e.g., metabolism and stress tolerance) and,

ultimately, accelerate the aging process.

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CONCLUSIONS

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 lifespan of 2 to 4 years – 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 towards 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 defenses, it may be possible in the near future to more

clearly delineate the integrative mechanisms of aging that are common between a broad range of

species. By gaining more detailed knowledge of specific pathways affected by aging,

investigators will be provided with additional opportunities to impact both lifespan and age-

related diseases in humans.

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ACKNOWLEDGMENTS

We thank Steven Bloomer and Jodie Haak for thoughtful comments and Joan Seye for

assistance with manuscript preparation. The research performed in the authors’ laboratory was

supported by National Institutes of Health Grant AG-12350.

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FIGURE LEGENDS

Figure 1. ROS can play a role in cell signaling. Oxidative stress can activate numerous

intracellular signaling pathways via ROS-mediated modulation of various enzymes and critical

transcription factors. In one scenario, transcription factors activated in response to an increase in

ROS or oxidative damage travel from the cytoplasm to the nucleus within a cell and bind to

promoter regions of particular genes. As a result, these stress-activated pathways can have a

significant impact on gene expression within a cell, which will ultimately affect the fate of a cell

(e.g., apoptosis, proliferation, cytokines). The balance between ROS production, cellular

anitoxidant defenses, activation of stress-related signaling pathways and the production of

various gene products, as well as the effect of aging on these processes, will determine whether a

cell exposed to an increase in ROS will be destined for survival or death.

Figure 2. DNA binding activities of the early response transcriptional factors AP-1 and

NF-κB are higher in old rats. Liver samples were obtained from young and old rats in

euthermic control conditions. Nuclear extracts were analyzed by electrophoretic mobility shift

assay (EMSA) using AP-1-specific (panel A) or NFκB-specific (panel B) 32P-labeled

oligonucleotides. In both assays, DNA binding activities were greater in all old rats examined.

AP-1 and NF-κB DNA binding activities were supershifted by specific c-Jun or p50 antibodies

reacting with nuclear extracts from old control animals to verify that the bands produced in the

EMSA procedure represented the AP-1 and NF-κB complexes. Each nuclear extract (5 µg) was

incubated with 5 µl of antibody specific for AP-1 (c-Jun; panel A) or NF-κB (p50 and p65; panel

B) 2 h before the EMSA procedure. The experiments were repeated three times, and

representative images are presented. FP, free probe. From Zhang et al. (297).

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Figure 3. Impact of an environmental insult on age-related oxidative stress responses and

the effect of redox modulation with chronic antioxidant enzyme mimetic treatment. Liver

samples were obtained from young and old rats in euthermic control conditions and two hours

after a heat stress protocol. Prior to the heating protocol, some young and old rats were

chronically treated (4 weeks) with either the SOD/catalase mimetic EUK-189 (heat + EUK) or

vehicle (heat alone), both of which were delivered via an implanted mini-osmotic pump (n=5-9

rats/age group for each treatment). Panel A: EUK supplementation prevented heat-induced

hepatic oxidative damage. The lipid peroxidation marker malondialdehyde was elevated in old

rats after heat stress, but not young rats. Chronic supplementation with EUK significantly

reduced the oxidative damage associated with heat stress in old rats. Panel B: EUK

supplementation improved redox status in old rats. The ratio of GSH and GSSG was utilized to

evaluate cellular redox status. In control conditions, a more pro-oxidative environment was

present in the liver of old vs. young controls as indicated by the lower GSH/GSSG ratio in the

old animals. Following heat stress, there was an increase in oxidative stress in liver samples

from young rats, while the GSH/GSSG ratio remained low in the old rats. EUK supplementation

reduced the oxidative environment in the liver of both young and old rats after heat stress as

evidenced by an increase in the GSH/GSSG ratio. Panel C: EUK-189 supplementation

normalized DNA binding activity of the redox-sensitive transcription factor AP-1. Heat stress

produced an increase in AP-1 DNA binding activity in the liver of old rats, but EUK

supplementation resulted in a decrease in this activity back to control levels. Panel D: EUK-189

supplementation prevented hepatic alanine aminotransferase (ALT) release after heat stress.

ALT, which is a systemic marker for hepatocellular damage, was measured in plasma samples

from young and old rats. Heat stress produced an increase in ALT levels in old but not young

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rats. However, old rats supplemented with EUK and subsequently heat stressed had ALT levels

similar to control conditions. *, P<0.05 vs. young rats within a treatment group; †, 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. (294).

Figure 4. Relationship between ROS, inflammation and aging. An increase in ROS levels

and a redox imbalance stimulates an intracellular signaling cascade that can potentially stimulate

a state of chronic inflammation and contribute to both aging processes and the manifestation of

aging-associated diseases.

Figure 5. A schematic summary of proposed mechanisms by which ROS and oxidative

stress could contribute to the process of aging. A variety of exogenous and endogenous

factors can stimulate an increase in reactive oxygen species (ROS) production at the cellular

level. ROS can stimulate signal transduction pathways, resulting in changes in gene expression

that can modulate numerous responses that impact on cellular function and survival. In addition

to activating intracellular signaling pathways, elevations in ROS can produce oxidative damage

at molecular levels (DNA, proteins, lipids) if repair processes are insufficient. One result is

organelle damage, which can directly affect key cellular responses. In both scenarios – the

modulation of expression of various stress-response genes and the intracellular damage to

macromolecules – there are subsequent responses at cellular levels (e.g., inflammation,

proliferation, apoptosis, necrosis) that can stimulate additional ROS generation from endogenous

sources. ROS-induced changes at cellular levels can also lead to an integrated array of systemic

responses that can impact, with the passage of time, on aging processes, as well as organ

dysfunction, frailty, and age-related diseases.

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↑ ROS & oxidative damage

Kinase 1

∆ Gene Expression

Inactivated Transcription

Factors

Fig. 1

Kinase 2

Cell Survival

Cell Death & Senescence

Kinase 1-P

Kinase 2-P

Activated Transcription

Factors

Nucleus

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Figure 2

A.

p50p65

Young Old + –– +FP

B.AP-1

Young OldFP + c-Jun

NF-κκκκB

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Fig. 3

0

20

40

60

Control Heat Heat +EUK

GSH/GSSG ratio

*

B.

0.0

2.5

5.0


Lipid Peroxidation

†*

*

A.

old

0

125

250


Plasma ALT

†*

D.

§

C.

0

2

3


DNA Binding Activity of AP-1

young ‡‡

†

(µM/mg protein)

(U/L)(arbitrary densitometry units)

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ROS, Inflammation & Aging

Stimulus

Transcription factor activation

(e.g., AP-1, NF-kB)

↑ Pro-inflammatorymolecules

(e.g., cytokines, chemokines, iNOS)

Chronic inflammation(cellular and

tissue damage)

↑ ROS &Redox Imbalance

↑ Pro-inflammatorygenes

Aging &age-associated

diseases

Figure 4

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Fig. 5

Modulation ofsignal transduction

pathways

Accumulation ofmolecular injury:

DNA, protein, lipids

Changes ingene expression

Organelle damage(e.g., mitochondria,

peroxisomes)

Cellular responses:inflammation, survival,

proliferation, death

Exogenous Factorsenvironmentalconditions

stressconditionslifestyle

ROS repairdamageactiva

tionnorma

lization

Passage of Time

Endogenoussources of ROS

Systematic responses:aging, organ dysfunction,

frailty, diseases

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