Free Radical Biology & Medicine, Vol. 33, No. 1, pp. 37 44, 2002
Copyright © 2002 Elsevier Science Inc.
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PII S0891-5849(02)00856-0
Serial Review: Oxidatively Modified Proteins in Aging and Disease
Guest Editor: Earl Stadtman
ROLE OF OXIDATIVE STRESS AND PROTEIN OXIDATION IN THE
AGING PROCESS
RAJINDAR S. SOHAL
Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA, USA
(Received 28 December 2001; Revised 25 March 2002; Accepted 27 March 2002)
Abstract The hypothesis that the rate of oxygen consumption and the ensuing accrual of molecular oxidative damage
constitute a fundamental mechanism governing the rate of aging is supported by several lines of evidence: (i) life spans
of cold blooded animals and mammals with unstable basal metabolic rate (BMR) are extended and oxidative damage
(OxD) is attenuated by an experimental decrease in metabolic rate; (ii) single gene mutations in Drosophila and
Caenorhabditis elegans that extend life span almost invariably result in a generalized slowing of physiological activities,
albeit via different mechanisms, affecting a decrease in OxD; (iii) caloric restriction decreases body temperature and
OxD; and, (iv) results of studies on the effects of transgenic overexpressions of antioxidant enzymes are generally
supportive, but quite ambiguous. It is suggested that oxidative damage to proteins plays a crucial role in aging because
oxidized proteins lose catalytic function and are preferentially hydrolyzed. It is hypothesized that oxidative damage to
specific proteins constitutes one of the mechanisms linking oxidative stress/damage and age-associated losses in
physiological functions. © 2002 Elsevier Science Inc.
Keywords Oxidative stress, Protein oxidation, Free radical hypothesis of aging, Senescence, Mechanisms of aging,
Metabolic rate, Free radicals
INTRODUCTION
whereby the ability to maintain homeostasis is corre-
spondingly attenuated, leading eventually to the death of
The postreproductive phase of life of virtually all multi-
the organism. Although many hypotheses have been
cellular species is characterized by the progressive de-
advanced, the nature of the causal mechanisms that ini-
cline in the efficiency of various physiological functions,
tiate the deleterious alterations underlying this phenom-
enon, often referred to as  senescence or the  aging
This article is part of a series of reviews on  Oxidatively Modified
process, remains controversial. It has been advocated
Proteins in Aging and Disease. The full list of papers may be found on
previously that any causal hypothesis should explain: (i)
the homepage of the journal.
Rajindar S. Sohal is the current holder of the Timothy M. Chan the mechanistic basis of the age-associated losses in
Professorship in the Department of Molecular Pharmacology and Tox-
physiological capacity in individual organisms, (ii) vari-
icology at the University of Southern California. He was previously a
ations in the rates of progression of senescent alterations
University Distinguished Professor in the Department of Biological
Sciences at Southern Methodist University in Dallas, Texas. He was a among different individuals and species, and (iii) the
Senior Scholar in the Department of Zoology, University of Cam-
possibility that life span can be extended in some species
bridge, where he was a member of Wolfson College; a Guest Professor
by experimental manipulations such as caloric restriction
at University of Dusseldorf, Germany; and a Visiting Professor at
Linkoping University, Sweden, where he also received a Doctor of in rodents or decrease in metabolic rate in poikilotherms,
Medicine (Honoris causa). He received B.Sc. (Honors) and M.Sc.
and single gene mutations in Caenorhabditis elegans and
(honors) degrees in Zoology from Panjab University, Chandigarh,
Drosophila melanogaster [1]. A currently popular hy-
India, and Ph.D. degree in Zoology from Tulane University, New
Orleans. pothesis postulates that a progressive accumulation of
Address correspondence to: Dr. Rajindar S. Sohal, University of
macromolecular oxidative damage is the fundamental
Southern California, Department of Molecular Pharmacology and Tox-
underlying cause of senescence-associated deleterious
icology, 1985 Zonal Avenue, Los Angeles, CA 90033, USA; Tel: (323)
442-1860; Fax: (323) 442-2038; E-Mail: sohal@usc.edu. alterations. The nature of the evidence supporting the
37
38 R. S. SOHAL
role of molecular oxidative damage in general, and pro- aerobes entails the generation of reactive oxygen species
tein oxidation in particular, in the aging process is dis- (ROS) has provided a clear link between Pearl s rate of
cussed in this article. living hypothesis and the oxidative stress hypothesis of
The idea that the rate of energy utilization is a key aging [8,9].
determinant of the rate of aging was first proffered by
Pearl [2] on the basis of a series of studies on the effects
OXIDATIVE STRESS HYPOTHESIS OF AGING
of starvation on the survival of D. melanogaster and
growth of cantaloupe seedlings. His main conclusions
An initial question that is highly pertinent to the
were that duration of life is a function of two variables:
evaluation of oxidative stress hypothesis is whether the
(i) the genetically determined constitution of the individ-
existing evidence supports or refutes the various predic-
ual or  vitality, and (ii) the average rate of metabolism
tions of the hypothesis.
during life (pp. 139, 140). The second inference was
expressed by the phrase that  in general the duration of
Is organismic senescence due to accumulation of oxida-
life varies inversely as the rate of energy expenditure
tive damage? It has now been amply documented that
during life (p. 145), which has subsequently become
there is an age-associated increase in the steady state
known as the  rate of living hypothesis. The most
amounts of the products of free radical attacks on mac-
unambiguous support for the rate of living hypothesis
romolecules such as lipids, proteins, and DNA in various
has been provided by studies in poikilotherms, where
tissues [1,10 12]. In general, the oxidative damage,
lowering the rate of metabolism by virtually any nonle-
which has often been measured in tissue homogenates,
thal means, such as a decrease in the ambient tempera-
increases exponentially with age [10,13]. Tissues that are
ture, mutational inactivation of enzymes, decreased level
composed of long-lived, postmitotic cells, such as the
of physical activity, have all been found to result in
brain, heart, and skeletal muscle, tend to accrue relatively
increased life spans [3 5]. Among mammals, life spans
greater amounts of damage than those composed of
of certain species, such as Turkish hamster, can be ex-
short-lived nonmitotic cells [1,11,14]. Studies on possi-
tended experimentally by induced hibernation [6].
ble causes of age-associated increase in oxidative dam-
Critics of the  rate of living hypothesis have fre-
age have indicated that activities of some of the enzy-
quently cited the example of birds, which are both rela-
matic antioxidative defenses may decrease, while others
tively long-lived as well as having a high metabolic rate
increase or remain unaltered at different ages, suggesting
as compared to the mammals, to discredit the validity of
the absence of any generalizable pattern [15 17]. On the
this hypothesis. This argument, however, misrepresents
other hand, rates of mitochondrial O2 and H2O2 gener-
Pearl s point of view. Variations noted in the rate of
ation have been found to increase quite consistently with
growth of different seedlings and length of survival of
age [17 21]. Although the existing information on age-
individual flies in response to starvation indeed formed
related changes in the efficiency of degradation of oxi-
the basis of his first postulate, namely that different
dized molecules is relatively limited, a decrease in pro-
individuals have different sums of vitality. In fact, Pearl
teolytic activity with age has been noted in some, but not
explicitly stated that the relationship between duration of
in other tissues [22 25]. To conclude, the available data
life and rate of metabolism applied only to the same
tend to favor the view that the increased production of
individual. The notion that different species should ex-
ROS is the primary factor responsible for age-related
pend identical amounts of energy during life was thus
accrual of molecular oxidative damage. It is possible that
implicitly excluded. Furthermore, this notion also con-
elevation in ROS generation may secondarily damage
tradicts his explicitly stated postulate that the duration of
the proteosomal system [24,25].
life also depends on the genetically determined constitu-
tion of the individual. Thus, the genetic constitution
determines the total amount of energy consumed during Are interspecies variations in the rate of aging related to
life, or metabolic potential, while the rate at which that corresponding differences in rate of accrual of oxidative
energy is consumed, the metabolic rate, determines the damage? Interspecies comparisons among nonprimate
length of life. Subsequent studies have indicated that the mammalian species such as mouse, rat, rabbit, pig, and
total amount of energy consumed during life or meta- horse, among others, have indicated that maximum life
bolic potential, varies in different phylogenetic groups span (MLS) of the species is not correlated with the
[3,7]. However, within each group, there is a clearly activities of antioxidative enzymes, such as superoxide
demonstrable inverse relationship between metabolic dismutase, catalase, and glutathione peroxidase [26,27].
rate and species-specific life span [7]. Although the basis On the other hand, rates of mitochondrial O2 and H2O2
of such a relationship was unknown in Pearl s era, the generation are inversely related to MLS of the species
subsequent demonstration that oxygen consumption by [28 30]. Notably, the relationship between MLS of the
Oxidative stress and aging 39
nonprimate mammalian species and basal metabolic rate ization of the rate of oxygen consumption [36]. For
(BMR) is superimposable on that between their MLS and example, fats constitute 18% of the total body weight in
the rates of mitochondrial O2 /H2O2 generation [28]. AL and only 7% in CR rats, meaning that lean body mass
A similar inverse relationship also exists between MLS cannot be calculated on the basis of the assumption that
of different nonprimate mammalian species and the it is a fixed proportion of the body weight, as done by
steady state amounts of mitochondrial 8-hydroxydeox- McCarter and Palmer [34]. Notwithstanding this debate,
yguanosine, a product of DNA oxidation [31]. there is a large body of experimental data indicating that,
Different species of dipteran flies, that vary 2-fold in among rodents, decrease in the rate of oxygen consump-
average life span, also exhibited inverse relationships tion and body temperature are widely employed physio-
between average life span and both the rates of mito- logical mechanisms for adaptation to scarcity of food
chondrial O and H2O2 generation and the protein car- [38,39].
2
bonyl content of tissues [32]. Some phylogenetic groups, The rates of mitochondrial O2 and H2O2 generation
such as the birds and the primates, live relatively longer as well as steady state concentrations of the products of
than nonprimate mammalian species that have compara- free radical attacks on macromolecules, such as lipids,
ble metabolic rates. For instance, the pigeon and the rat proteins, and DNA, are lower in tissues of CR than in the
have similar metabolic rates but differ 7- to 8-fold in AL-fed animals [1,36]. In general, there are no consistent
MLS, meaning that they have different metabolic poten- or significant differences in activities of antioxidant en-
tials. Comparisons between these two species have indi- zymes between the two groups. In summary, the existing
cated that mitochondrial rates of O2 and H2O2 genera- information can be reasonably interpreted to suggest that
tion are considerably lower, whereas activities of some in the species that have variable basal metabolic rates,
antioxidative enzymes were higher in the pigeon than in life span can be experimentally extended by lowering
the rat [33,34]. In summary, MLS of the species, which metabolic activity via a variety of manipulations. Low-
have a similar metabolic potential, seem to be inversely ering of the metabolic rate demonstrably results in a
associated with rates of ROS generation and apparently corresponding attenuation of oxidative damage and an
unrelated to antioxidative defenses, whereas species that increase in life span.
differ in metabolic potential as well as metabolic rate,
MLS is negatively correlated with ROS generation and Can the rate of aging be altered by overexpression of
positively related to antioxidant defenses. antioxidative enzymes or single gene mutations? Results
of studies on overexpression of antioxidative genes have
Are experimental extensions in life span accompanied by been quite variable and somewhat ambiguous. Trans-
corresponding attenuations of oxidative damage? Avail- genic overexpression of Cu, ZnSOD has no life-prolong-
able evidence suggests that, almost invariably, experi- ing effect in mouse [40] and, according to most authors,
mental regimens that extend the life spans of poikilo- in D. melanogaster [41 43]. Overexpression of Mn-
therms also result in decreases in the rates of metabolism SOD [44], catalase [45], or a putative thioredoxin reduc-
and accumulation of oxidative damage [1]. For instance, tase [46] alone also have no effect on life span of
elimination of flying activity prolongs the life span of Drosophila, albeit in some cases the experimental ani-
flies up to 3-fold, while the rate of accumulation of mals exhibit relatively higher resistance to induced oxi-
protein and DNA oxidative damage is correspondingly dative stress. In another study, simultaneous overexpres-
diminished [13,55]. Among mammals, especially rodents sion of Cu, Zn-SOD and catalase was found to result in
such as the rat and the mouse, life span is extended if an increase of up to 34% in life span and an attenuation
caloric intake is decreased from the level consumed by of macromolecular oxidative damage in D. melanogaster
ad libitum (AL) fed animals. The body temperature of [47]. In a recent critique of the various transgenic studies
calorically restricted (CR) animals is daily transiently on aging in Drosophila, it was pointed out that the life
lowered up to 4°C in rat and up to 13°C in the mouse, span prolongation effects of overexpression of antioxi-
indicating that these species have unstableness in the rate dative genes may be limited to certain genetic back-
of basal metabolism, which is responsive to caloric in- grounds only [48]. In general, life spans of transgenic
take [reviewed in 1,36]. This issue has, however, become overexpressors of antioxidative enzymes may be ex-
controversial because direct comparisons of rates of ox- tended if the life spans of their respective controls are
ygen consumption per unit body mass were found not to relatively short. For instance, the longest reported exten-
exhibit any significant differences between AL and DR sion of life span in Cu-Zn SOD overexpressing D. mela-
groups [37]. In counterpoint, it has been argued that due nogaster (48%) was reported in a strain with an average
to the differences in the body composition and the rela- life span of 24 d [49], which is about one-third of the
tive organ size between the AL and CR animals, body length among healthy, relatively long-lived strains of this
weight can not be used in this model system for normal- species. In this particular study, the life span extensions
40 R. S. SOHAL
seemed to be inversely related to Cu, Zn-SOD activity as degradation [10,50,51]. Oxidative damage to a specific
well as the life spans of the respective controls. Thus, it protein, especially at the active site, can induce a pro-
is imperative that life span extensions of transgenic an- gressive loss of a particular biochemical function. Pio-
imals should be evaluated in the context of the life spans neering studies by Stadtman, Levine, and their associates
of the control strain(s) used vs. those of the robust have documented the relevance of protein oxidative
long-lived strains of the same species. In most instances, damage in the aging process and in the etiology of
life span extensions of the transgenic overexpressors certain pathological conditions [50,52]. Several types of
may merely be due to amelioration of a deficiency in an ROS-induced protein modifications have been demon-
unhealthy stock rather than retardation of the aging pro- strated [50,51,53], including the loss of sulfhyryl ( SH)
cess. groups, formation of carbonyls, disulphide crosslinks,
Recently, there has been a spate of reports about methionine sulfoxide, dityrosine cross-links, nitroty-
extensions of life spans in mutants of C. elegans, D. rosine, and glyoxidation and lipid peroxidation adducts,
melanogaster, and mice, which have been interpreted to among others.
suggest that the rate of aging is controlled by a limited Loss of protein SH groups can be induced by a wide
number of genes [59]. A common feature of virtually all array of ROS and is one of the most immediate responses
such life-prolonging mutations seems to be the loss of to an elevation in the level of oxidative stress [50].
specific function, which leads to the decline of the nor- Functional consequences of SH loss include protein
mal tempo of bioenergetic metabolism with ensuing hy- misfolding, catalytic inactivation, decreased antioxida-
pometabolic state [45,48]. Although various novel and tive capacity, and loss of certain specific functions, such
complicated mechanisms have been proposed by the as binding of heavy metals and sulfur-containing amino
authors to explain such life span extensions, direct evi- acids by albumin, among others [53]. Age-associated
dence has already been presented for a simpler interpre- losses in protein SH content have been reported in a
tation that these extensions occur due to a decrease in the variety of tissues and species, including homogenates of
rate of metabolism and consequently of ROS generation brain, heart, skeletal muscle, and kidney of rodents and
[4,5]. Metabolic defects resulting from a variety of mu- houseflies [20,54,55]. Caloric restriction attenuates SH
tations result in phenotypes, which have relatively slower loss, whereas hyperoxia has an opposite effect [54,56].
rates of physiological activities. Hypometabolic state is Another oxidative modification in proteins is the for-
known to increase resistance to various stresses and to mation of dityrosine crosslinks, which apparently arises
enhance antioxidative defenses. These responses are the following reaction between two tyrosyl radicals, gener-
effects of hypometabolism rather than the cause of ex- ated by peroxidases and other heme proteins. Dityrosine
tended life spans [48]. cross-linking of proteins has been found to increase with
To summarize, the view that oxidative stress is causal age in mouse skeletal muscle and heart, but not in the
to the induction of senescence is presently supported brain or liver; caloric restriction attenuates these in-
primarily by correlative evidence, such as that amounts creases [57].
of macromolecular oxidative damage and rates of gen- Addition of carbonyl-containing adducts to the side
eration of mitochondrial ROS increase with age and are chains of amino acid residues, such as lysine, arginine,
inversely related to the MLS of the species. Extension of proline, and threonine, is arguably the most well charac-
life span following overexpression of antioxidative en- terized, age-associated, post-translational structural alter-
zymes seems to be inversely related to life span of the ation in proteins [10,50,51]. Carbonylation can be caused
respective controls. Prolongation of life span in single by a variety of ROS; however, site-specific metal-cata-
gene mutants is almost invariably associated with atten- lyzed oxidation, involving the formation of hydroxyl/
uations in metabolic rate and/or fecundity. Unfortu- ferryl radical via Fenton-type scission of H2O2, seems to
nately, most of the authors have either ignored the role of be the most plausible in vivo mechanism [58]. A series of
rate of metabolism or have used unsuitable procedures studies by Stadtman and his associates have demon-
for its measurement [4] strated that amounts of protein carbonyl content in-
creases with age in several different mammalian tissues
[24,58].
PROTEIN OXIDATIVE DAMAGE AND AGING
A relationship between protein carbonylation and the
Among the various types of macromolecular oxida- rate of aging or life expectancy of animals has also been
tive damage that accrues during aging, oxidative modi- demonstrated in a number of organs and species. For
fications of intracellular proteins have been suggested to instance, the level of protein carbonyls in cultured human
play a key role in the causation of senescence-associated fibroblasts increases exponentially with age of the donors
losses in physiological functions because oxidized pro- [58]. Similarly, in the housefly, protein carbonyl content
teins often lose catalytic function and undergo selective in whole body homogenates increased with age in an
Oxidative stress and aging 41
exponential fashion [35]. In flies whose life span was the tissues of shorter-lived species like mice and rats
extended around 2- to 3-fold by elimination of flying [52]. To conclude, it seems that a combination of
activity, the accrual of protein carbonyls with age was several factors determines the steady state amounts of
slower than in flies that were permitted to fly and had a protein oxidation products, Nevertheless, rates of ox-
relatively shorter life span [35]. In another experiment idant generation may be the most dominant factor,
using flying ability as a criterion, aged flies were divided since variations in rates of ROS generation are most
into two groups, the  crawlers and the  fliers, which, closely associated with differences in oxidative dam-
respectively, have relatively short and long life expect- age and MLS of different species [31,32].
ancies. At comparable ages, the crawlers were found to
contain a higher concentration of protein carbonyls than
SELECTIVITY OF PROTEIN OXIDATIVE DAMAGE
the fliers, suggesting that onset of senescence is associ-
ated with more rapid accrual of protein oxidative damage Although there is strong evidence indicating that the
[35]. Overall, the findings in insects that protein carbon- steady state amounts of the products of free radical attack
yls increase with age and their steady state concentration on macromolecules, such as protein carbonyls, increase
is inversely related to life expectancy have been con- in tissue homogenates during aging, such evidence does
firmed in mammals [58]. not elucidate the specific mechanisms that cause losses in
Another approach to examine the relationship be- particular cellular functions. It was originally thought
tween life expectancy and protein carbonylation in- that free radical attacks on proteins and other macromol-
volved a comparison between ad libitum (AL) fed and ecules occur randomly because such interactions are
calorically restricted (CR) mice, which had a 35% uncatalyzed events. However, it is well documented that
longer life span than the AL mice. The age-related activities of most enzymes do not decline during aging
accrual of protein carbonyl content in homogenates of [63,64]. To understand the basis of this apparent discrep-
various tissues was considerably slower in the CR than ancy and to identify the possible mechanisms underlying
the AL mice [17,54,56]. Similarly, whole body ho- biochemical losses during aging, we hypothesized that
mogenates of relatively long-lived transgenic D. age-associated oxidative damage to proteins and the con-
melagonaster, overexpressing Cu, Zn-SOD and cata- sequent loss of their function was a selective rather than
lase, accrued protein carbonyls at a slower rate than a random phenomenon. This hypothesis was initially
the control [42]. tested in mitochondria of the flight muscles of houseflies
Variations in the rate of accrual of oxidatively and D. melanogaster by using Western blot analysis for
modified proteins in vivo have been variously hypoth- the detection of specific proteins exhibiting carbonyla-
esized to be due to corresponding differences in rates tion [60,61,65].
of oxidant generation, antioxidative defenses, repair It was found that only aconitase and adenine nucle-
and degradative capacity, or susceptibility to oxidative otide translocase (ANT) exhibited a detectable age-
modifications [61,62]. For instance, exposure of flies associated increase in carbonylation and a correspond-
to hyperoxia or relatively higher physical activity, ing loss in functional activity, suggesting that protein
which increases oxidant generation, also increase the carbonylation during aging is selective [60,61,65].
rate of accumulation of protein carbonyls [35]. Evi- Both aconitase and ANT were also found to be par-
dence supporting the modulatory role of antioxidative ticularly sensitive to carbonylation under conditions of
defenses in the accumulation of protein carbonyl is oxidative stress, induced by exposure to hyperoxia.
based on studies in transgenic Drosophila overex- Studies on rodent plasma also indicate that only a
pressing Cu, Zn-SOD and catalase [42]. Decreases in small fraction of proteins exhibit discernible carbony-
the ability to degrade carbonylated proteins is corre- lation at any age [66], suggesting that the specificity of
lated in some tissues with age-related increases in the protein oxidative damage during aging is not limited
amount of carbonylated proteins [50,51]. The tissues to insects. ROS probably act in a random fashion;
of the aged animals also seem to be more susceptible however, the sensitivities and proximities of potential
to sustain protein oxidation in response to experimen- targets differ. The factors that affect selectivity of
tally induced oxidative stress than those of the young oxidative damage to proteins include the presence of a
animals. For instance, aged, live houseflies, when ex- metal-binding site, molecular conformation, rate of
posed to x-rays, exhibited a higher net gain of protein proteolysis, and relative abundance of amino acid res-
carbonyls than the younger flies [52]. Comparisons idues susceptible to metal-catalyzed oxidation, among
among different species with varying longevities also others [65,66]. Selectivity of protein carbonylation
indicate that tissues of relatively longer-lived species, was further indicated by the findings that molecular
such as the pigeon and the cow, are less susceptible to mass of cytochrome c in flies remained unchanged
radiographically induced protein carbonylation than during aging as well as in response to hyperoxia [67].
42 R. S. SOHAL
Similarly, malate dehydrogenase, which copurifies specific protein targets may be an important mecha-
with aconitase, did not exhibit any discernible car- nism linking oxidative stress to age-associated physi-
bonylation in the aged flies [68]. Such findings are ological losses.
thus inconsistent with the previously prevalent view
that protein carbonylation during aging is a general
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