Am J Physiol Regul Integr Comp Physiol 301: R843–R863, 2011.
First published July 20, 2011; doi:10.1152/ajpregu.00034.2011.
Review
Molecular and structural antioxidant defenses against oxidative stress
in animals
Reinald Pamplona1 and David Costantini2
1
Department of Experimental Medicine, University of Lleida Biomedical Research Institute of Lleida, Lleida, Spain;
and 2Institute for Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, United Kingdom
Submitted 14 January 2011; accepted in final form 18 July 2011
free radicals; maximum life span; mitochondria; molecular evolution; oxidative
damage; redox reactions; redox signaling
THE INCREASE IN OXYGEN CONCENTRATION in the Earth’s atmosphere represented an important selective pressure for living
organisms and contributed toward setting up the pace of
evolutionary changes in physiological and metabolic systems
(69, 119, 129). Despite molecular mechanisms evolving toward the use of oxygen for efficient energy production have
been a relevant evolutionary innovation, the reduction-oxidation (or redox) reactions (redox reactions involve the transfer
of electrons or hydrogen atoms from one reactant to another)
associated with its use have been responsible for the production
of free radicals and, more generally, of reactive species (RS).
These products of oxygen metabolism may have damaging
effects on an organism, in essence, due to oxidation of essential
cellular components (88). Consequently, animals have evolved
mechanisms to avoid or minimize the production of RS of
oxidative metabolism, as well as of antioxidant defense systems needed to limit the prooxidant activity of RS (89). The
Address for reprint requests and other correspondence: Institute for Biodiversity, Animal Health and Comparative Medicine, College of Medical,
Veterinary and Life Sciences, Univ. of Glasgow, Graham Kerr Bldg., Glasgow
G12 8QQ, UK (e-mail: [email protected]).
http://www.ajpregu.org
result has been a high diversity in molecular and structural
antioxidant defenses, as well as in redox signaling pathways
perfectly integrated in the cellular metabolic machinery. However, the antioxidant machinery is not 100% efficient in mopping RS up, especially when there is a strong imbalance
between production of RS and antioxidant response. The consequence of such an imbalance is the generation of oxidative
damage to biomolecules. This kind of biochemical stress of
cells has been called oxidative stress (88, 195), which may be
defined as the rate at which oxidative damage is generated (48).
Implicit in this definition is that oxidative stress is a continuous
variable that is unlikely to ever be exactly zero since prooxidants are continually produced, and some oxidative damage is
always generated. Although the study of oxidative stress physiology has been classically of interest to the medical sciences
because of its potential contribution in the progression of many
human diseases, recent studies on nonhuman animals suggest
that oxidative stress could also represent 1) a universal constraint of life-history evolution, 2) a modulator of phenotypic
development, and 3) a physiological correlate of behavioral
differences between individuals (3, 17, 41, 44, 47, 50, 64, 132,
136, 174, 178, 217). Therefore, oxidative stress could have
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Pamplona R, Costantini D. Molecular and structural antioxidant defenses against
oxidative stress in animals. Am J Physiol Regul Integr Comp Physiol 301: R843–R863,
2011. First published July 20, 2011; doi:10.1152/ajpregu.00034.2011.—In this review, it
is our aim 1) to describe the high diversity in molecular and structural antioxidant
defenses against oxidative stress in animals, 2) to extend the traditional concept of
antioxidant to other structural and functional factors affecting the “whole” organism, 3) to incorporate, when supportable by evidence, mechanisms into models of
life-history trade-offs and maternal/epigenetic inheritance, 4) to highlight the
importance of studying the biochemical integration of redox systems, and 5) to
discuss the link between maximum life span and antioxidant defenses. The
traditional concept of antioxidant defenses emphasizes the importance of the
chemical nature of molecules with antioxidant properties. Research in the past 20
years shows that animals have also evolved a high diversity in structural defenses
that should be incorporated in research on antioxidant responses to reactive species.
Although there is a high diversity in antioxidant defenses, many of them are
evolutionary conserved across animal taxa. In particular, enzymatic defenses and
heat shock response mediated by proteins show a low degree of variation. Importantly, activation of an antioxidant response may be also energetically and nutrient
demanding. So knowledge of antioxidant mechanisms could allow us to identify
and to quantify any underlying costs, which can help explain life-history trade-offs.
Moreover, the study of inheritance mechanisms of antioxidant mechanisms has
clear potential to evaluate the contribution of epigenetic mechanisms to stress
response phenotype variation.
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ANIMAL ANTIOXIDANT DEFENSES
Antioxidant Defense Mechanisms as Evolutionary
Adaptations to Endogenous Reactive Species Generation
In aerobic organisms, about 85–90% of cellular oxygen is
consumed by the mitochondria to produce energy as ATP
molecule. A main side-effect of ATP production is the formation of RS (Fig. 1). RS include a variety of molecules, mostly
physiologically generated from the metabolic activity (12, 88,
185). RS can induce chemical modifications in other molecules, generating oxidative damage, but also act as second
messengers in signal transduction networks.
RS mostly consist of reactive oxygen species (ROS) and
reactive carbonyl species (RCS). Superoxide anion, the product
of a one-electron reduction of oxygen, is the precursor of most
ROS. Dismutation of superoxide anion [either spontaneously
or through a reaction catalyzed by superoxide dismutase (SOD)
enzymes] produces hydrogen peroxide (H2O2), which, in turn,
may be fully reduced to water or in the presence of ferrous or
cuprous ions, forms the highly reactive hydroxyl radical. In
addition, superoxide anion may react nonenzymatically with
other molecules, including nitric oxide (NO) in a reaction
controlled by the rate of diffusion of both reactants. The
product of the reaction between superoxide anion and NO,
peroxynitrite, is also a very powerful oxidant. The oxidants
derived from NO have been called reactive nitrogen species
(RONS). On the other hand, hydrogen peroxide, although
lacking radical properties, can work as a Trojan horse, diffusing away from sites of ROS production to generate the hydroxyl and other reactive radicals at other cellular locations,
thereby propagating oxidative damage. In addition to ROS/
RONS, the oxidation of both carbohydrates and lipids [partic-
Fig. 1. Inside mitochondria, electrons from reduced substrates move from complexes I and II (CI and CII) of the electron transport chain through complexes III
and IV (CIII and CIV) to oxygen, forming water and causing protons to be pumped across the mitochondrial inner membrane. When glucose is metabolized
through the tricarboxylic acid (TCA) cycle, it generates electron donors. The main electron donor is nicotinamide adenine dinucleotide (NADH), which gives
electrons to complex I. The other electron donor generated by the TCA cycle is flavin adenine dinucleotide (FADH2), formed by succinate dehydrogenase, which
donates electrons to complex II [the same electron donors (NADH and FADH2) obtained by glucose oxidation are generated by both ␤-oxidation of fatty acids
and oxidation of free fatty acid-derived acetyl CoA by the TCA cycle]. The proton-motive force set up by proton pumping drives protons back through the ATP
synthase in the inner membrane, forming ATP from their precursors ADP and phosphate (189). The electron transport system is organized in this way so that the
level of ATP can be precisely regulated. UCPs, uncoupling proteins; Ant, adenine nucleotide translocase; PC, pyruvate carboxylase. For more details, see text.
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worked as a relevant selective pressure and that is why animal
taxa have evolved a myriad of mechanisms to regulate and
adjust the redox balance. This high diversity in antioxidant
mechanisms also suggests that organisms have a great deal of
flexibility in how they deal with RS challenges across time,
conditions, and tissue types, as well as that different organisms
may have evolved different strategies for dealing with similar
oxidative challenges.
In the following paragraphs, we review the high diversity of
molecular and structural defenses against RS evolved by animals. In particular, it is our goal to describe physiological
mechanisms underlying the production of RS and the biochemical regulation of defense mechanisms, to extend the traditional
concept of antioxidant to other structural and functional factors
affecting the whole organism, and to link antioxidant defenses
to life-history models and maternal/epigenetic inheritance. Finally, we review the state of the art about the link between life
span and antioxidant defenses.
We have used the term adaptation in a general way throughout the manuscript, including both real adaptations and exaptations. For a character to be regarded as an adaptation, it must
be a derived character that evolved in response to a specific
selective agent (p. 13 in Ref. 93). The emergence of an
antioxidant mechanism could be maintained by natural selection because it confers current selective advantage, but this is
not evidence of why it has been evolved. So it could be
considered as an exaptation sensu (81) rather than as an
adaptation. This means that the protection against oxidative
stress is not the primary reason for why all antioxidant mechanisms have been evolved, but it could have played a role
secondarily to provide selective advantage.
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stable reacting carbohydrate (30) and, consequently, poorly
susceptible to oxidation. The lower cellular concentration of
highly reactive glycolytic intermediates was, in fact, interpreted as an adaptation to suffer less oxidative stress (137).
This intracellular condition is particularly critical for birds,
given their high blood glucose levels (101). Available evidence
demonstrate that birds possess lower glucose cellular permeability (16) and sensitivity to insulin signaling (67), mechanisms
that probably lead to lower intracellular concentration of glycolytic intermediates. So, it is proposed that the high blood
glucose level showed by birds in comparison with mammals
should be interpreted as a nonreactive and stable functional
compartment ready to use exclusively in physiological conditions that require a high energetic demand, thus maintaining
cellular integrity against molecular damage.
Highly unsaturated fatty acids (more than 2 double bonds)
are on average the least abundant fatty acids in cell membranes
and are less abundant in species that live longer (104, 152).
Unsaturated fatty acids are the macromolecules most susceptible to oxidative damage in cells, and this sensitivity increases
as a function of the number of double bonds they contain (100,
104, 152–153). This means that saturated and monounsaturated
fatty acyl chains are essentially resistant to oxidation, whereas
polyunsaturates are easily damaged (104, 152).
Another antioxidant adaptive structural system evolved to
prevent the oxidation of polyunsaturated fatty acids is linked to
the plasmalogens (24, 70, 145). Substantial evidence accumulated in the last decade indicates that plasmalogens, a class of
ethanolamine (and choline) phospholipids, could represent a
major lipid-soluble antioxidant component based on the ability
of the plasmalogens to scavenge several RS, their relatively
high concentrations in many animal species (which markedly
exceeds the alpha-tocopherol concentrations; see Cellular Protection by Enzymatic and Nonenzymatic Antioxidants) and their
subcellular and extracellular locations in close vicinity to
oxidizable substrates. The structural peculiarity of plasmalogens lies in the enol ether double bond present within the
hydrocarbon chain linked to the C-1 atom of the glycerol
backbone of phospholipids.
Guanine is the least abundant nucleotide in mitochondrial
DNA (183). Guanine is the nucleobase with the lowest oxidation potential and is thus generally most easily oxidized (18).
Methionine is the amino acid that on average has the
smallest percentage presence in cellular proteins (2, 157, 171,
176). Methionine residues from proteins are among the amino
acids most susceptible to oxidation by free radicals (204).
Consequently, the lower the protein Met content, the higher
should be the protein resistance to oxidative damage. In accordance with this, the amino acid compositional analysis of
cellular proteins from different tissues (e.g., skeletal muscle,
heart, and brain) in birds and mammals reveal that Met is
present in the smallest percentage in cellular proteins (2, 157,
171, 176). Selection has, therefore, possibly disfavored the
usage of Met in animal tissues, given its higher proneness to be
oxidized compared with other amino acids.
Although available evidence suggests that aerobic life has
evolved by reducing the relative abundance of those structural
components that are highly susceptible to oxidative damage,
there still exists great variation among species in body composition (e.g., degree of unsaturation of cell membranes; Refs.
34, 103, 105), whose causes and meaning are still not well
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ularly, polyunsaturated fatty acids (PUFAs)] originates a new
generation of RS named reactive carbonyl species, or RCS
(152). Compared with both ROS and RONS, RCS have a much
longer half-life (i.e., minutes to hours instead of microseconds
to nanoseconds for most ROS/RONS). Further, the noncharged
structure of carbonyls allows them to migrate with relative ease
through hydrophobic membranes and hydrophilic cytosolic
media, thereby extending the migration distance far from the
production site. On the basis of these features, RCS could be
more destructive than ROS/RONS and may have far-reaching
damaging effects on target sites within or outside membranes.
Diversity in antioxidant mechanisms is thought to be an
expression of this high variety in RS and in their molecular
consequences. Although not clearly and consistently defined
yet, here, we define antioxidant as “any mechanism, structure
and/or substance that prevents, delays, removes or protects
against oxidative nonenzymatic chemical modification (damage) to a target molecule.”
Protection against oxidative damage is pivotal for organism
function. A major consequence of oxidative damage is the loss
of function and structural integrity of modified biomolecules,
which have a wide range of downstream functional consequences, such as induction of cellular dysfunctions and tissue
damage. Alterations of biochemical and physiological pathways can have a number of detrimental consequences for an
individual’s health, potentially decreasing its Darwinian fitness
perspectives. Given that environmental conditions (e.g.,
weather, food quantity and quality, predation risk, and competition) across a range of habitat types, as well as phases of the
life-cycle (e.g., reproduction and hibernation) may be associated with oxidative stress threats, individuals are continually
under pressure. In this scenario, animals have evolved several
antioxidant defenses to control and mitigate the action of RS.
Individuals may, therefore, be exposed to relevant physiological costs, mainly expressed in terms of oxidative damage and
consumption of energy needed to keep the antioxidant defenses
upregulated and to activate repair systems. It is possible that
the need to regulate the redox system exposes individuals to a
number of trade-offs and, therefore, oxidative stress could have
represented a relevant modulator of life-history strategies.
Animals show, however, large variation in antioxidant defenses, and it is, therefore, pivotal to review them to figure out
the association among oxidative stress, life-history strategies,
and response of natural populations to environmental stressors.
Resistance of cellular structural components to oxidative
damage. Cellular protection against oxidative damage includes
RS elimination and repair/turnover systems. Recent evidence,
however, support the notion of another line of defense based on
the inherent susceptibility of macromolecules to oxidative
damage (155). This susceptibility, defined as the ease with
which macromolecules suffer an oxidative injury, is intrinsically linked to the specific structure or chemical composition of
carbohydrates, lipids, nucleic acids, and proteins. Available
evidence on the differential susceptibility to oxidative damage
of biomolecules shows that in living systems:
Glycolytic intermediates (the most unstable and reactive
monosaccharides) occur at concentrations in the micromolar
range, in contrast to the millimolar range for glucose (the most
abundant and stable monosaccharide). It was proposed that
glucose emerged as the most important carrier of energy from
cell to cell in animal species, because it is the slowest and
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conferring to the macromolecules a lower susceptibility to
oxidative stress and, consequently, a higher structural stability.
Oxidative balance regulatory components. From an evolutionary perspective, it could be expected that natural selection
does work to optimize the interaction among mechanisms that
regulate production of RS rather than antioxidant molecules
only. Hence, an obvious question coming out from this expectation would be: Are there any physiological mechanisms that
regulate the rate of mitochondrial free radical generation?
Available evidence suggests that this is the case. These mechanisms can be grouped in “internal” and “external”.
INTERNAL MECHANISMS. In animal cells, the major sites of
physiological ROS generation are the complex I and III of the
mitochondrial electron transport chain, which contain several
redox centers (flavins, iron-sulfur clusters, and ubisemiquinone) capable of transferring one electron to oxygen to form
superoxide anion (21, 104, 155, 185). It is, therefore, proposed
that important internal mechanisms are those inherently linked
with the structure and function of the mitochondrial free radical
generators: 1) modulation of the absolute content of the mitochondrial electron transport chain, and, very specially, of the
complexes I and III; 2) reduction state of complex I;
3) modulation of the free radical production by uncoupling
proteins; and 4) potential posttranslational modifications.
A first internal mechanism operates through the decrease in
the concentration of the respiratory complex/es responsible for
ROS generation. For example, birds have a lower content of
complex I than mammals, as well as a lower degree of ROS
production (117, 155, 157, 203). Therefore, regulation of the
expression of the mitochondrial complexes (and especially
complex I) could be part of an adaptive mechanism to adjust
ROS production.
A second internal mechanism that controls free radical
generation is the regulation of the degree of electronic reduction of these generators: the higher their degree of reduction,
the higher will be their rate of ROS production (154 –155). It is
known that the rate of mitochondrial ROS generation strongly
increases with a sigmoidal kinetics when the ratio between the
reduced (NADH), an oxidized (NAD⫹) form of nicotinamide
adenine nucleotide, is increased, because this dramatically
increases the degree of reduction of the complex I ROS
generator (12, 116).
A third internal mechanism is characterized by regulation of
uncoupling proteins (UCPs). During oxidation of substrates,
the complexes of the mitochondrial electron transport chain
reduce oxygen to water and pump protons into the intermembrane space, forming a proton-motive force (⌬p). However,
some electrons in the reduced complexes also react with
oxygen to produce superoxide. Superoxide can peroxidize
membrane phospholipids, forming hydroxynonenal, which induces proton transport through the UCPs and the adenine
nucleotide translocase. The mild uncoupling caused by proton
transport lowers ⌬p and slightly stimulates electron transport,
causing the complexes to become more oxidized and lowering
the local concentration of oxygen; both of these effects decrease superoxide production. Thus, the induction of proton
leak by hydroxynonenal limits mitochondrial ROS production
as a feedback response to overproduction of superoxide by the
respiratory chain (21, 68, 186). So, a possible antioxidant
physiological function for UCPs has been proposed (68). In
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understood, despite that it seems to be a relevant determinant
of life span (see Antioxidants and Animal Maximum Life Span).
The link between body composition and susceptibility to oxidative stress can, therefore, be ecologically and evolutionary
relevant. For example, an environmental factor that can contribute to explain some body composition variation is the diet
quality. Diet not only is a source of molecular antioxidants, but
it is also a source of nutrients, such as fatty acids and amino
acids, which can expose the organism to different oxidative
stress threats because of their different molecular susceptibility
to be oxidized. Animals can, however, actively make their cell
composition through an active regulation of metabolism of
nutrients. For example, the lower proportion of n-3 to n-6
polyunsaturated fatty acids in marine bird cardiac membranes
compared with their diet suggests a highly selective nature of
fatty acid incorporation into membrane lipids (34). Such discrimination of dietary fats is especially important because n-3
polyunsaturated fatty acids are less resistant to oxidative damage than their n-6 counterparts.
Such regulation abilities of cell membrane composition may
also be important when the female is depositing nutrients into
the egg or milk. Females could influence the future susceptibility to oxidative stress of their offspring not only through the
investment of antioxidants, but also through other nutrients that
do not have any antioxidant properties. For example, the n-6
polyunsaturate, arachidonic acid, forms between 8% and 19%
(wt/wt) of the phospholipid fatty acids of egg yolk of the
northern gannet (Morus bassanus), the great skua (Catharacta
skua), the American white pelican (Pelecanus erythrorhynchos), and the double-crested cormorant (Phalacrocorax auritus) (207). Although for the pelican and cormorant, the fatty
acid profile of yolk is consistent with the consumption of
freshwater fish, in which arachidonic acid may be a significant
acyl constituent, this finding is more difficult to explain for the
gannet and skua, which largely consume marine fish with a low
arachidonic acid content. These results suggest that females
may have an active control on fatty acid deposition in the egg
and that pelicans and cormorants could have increased the
investment of arachidonic acid in the egg compared with its
bioavailability because the incorporation of this fatty acid in
the embryo tissues may render them less susceptible to oxidative stress (206).
Further to these points, recent studies of reptiles and birds
indicate that the blood oxidative balance may have an inherited
component (41, 111, 148). However, the data were collected in
such a manner that these heritability estimates are based on a
combination of additive genetic effects and maternal/paternal
effects (i.e., indirect genetic or environmental effects on the
phenotype of the offspring, caused by the mother or father). It
is, therefore, difficult to say how much variation in oxidative
balance of offspring was due to female allocation strategies of
nutrients or genetics. This appears to be a fruitful area of
investigation for our understanding of transgenerational effects, long-lasting effects of early life conditions on adult
phenotype, and interaction between passive and active passage
of nutrients from female to offspring.
From the available evidence, we can, therefore, infer that
natural selection operated, possibly through directional selection, reducing the relative abundance of those structural components that are highly susceptible to oxidative damage, thus
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diving hypoxia by storing oxygen in blood and skeletal muscles (both very rich in hemoglobin and myoglobin, respectively) and by limiting the distribution of blood-borne oxygen
to all but the most hypoxia-vulnerable tissues (brain, heart),
through cardiovascular adjustments (172).
A second mechanism is dependent on the cardiolipin content
of mitochondria. Cardiolipin, a phospholipid located almost
exclusively within the inner mitochondrial membrane, is particularly rich in unsaturated fatty acids, but with a low degree
of unsaturation (98, 170). This phospholipid plays an important
role in mitochondrial bioenergetics by influencing the activity
of key mitochondrial inner membrane proteins, including several anion carriers and electron transport complexes I, III, and
IV (98). Mitochondrial cardiolipin molecules are potential
targets of ROS attack because of their content of unsaturated
fatty acids and because of their location in the inner mitochondrial membrane near to the site of ROS production. In this
regard, it has been recently demonstrated that mitochondriamediated ROS generation affects the activity of complex I
(161), as well as complexes III and IV (160, 162), via peroxidation of cardiolipin following ROS-mediated damage to its
fatty acid constituents (161). In this context, it is plausible to
postulate that cardiolipin was positively selected likely because
of its low unsaturation degree that ensures a structural resistance against oxidative damage, as well as a correct functionality for the mitochondrial electron transport chain complexes
(152).
Antioxidant diversity is an expression of the variety of
reactive species and their molecular consequences. Although
defense mechanisms have evolved that regulate the generation
of RS, cells continuously produce them, and so their oxidative
action can be controlled only if endogenous cellular antioxidants are present. In the following paragraphs, we review
briefly the major molecular antioxidant defenses, both enzymatic and nonenzymatic, which have been selected and conserved during animal evolution.
CELLULAR PROTECTION BY ENZYMATIC AND NONENZYMATIC
ANTIOXIDANTS. Direct RS scavenging antioxidant enzymes are
superoxide dismutase (SOD), glutathione peroxidase (Gpx)
and catalase. SOD eliminates superoxide radical by converting
it to oxygen and H2O2. There are different forms of this
enzyme: a Cu,Zn form in the cytosol and in the intermembrane
mitochondrial compartment (147), a Mn form in the mitochondrial matrix (MnSOD), and another form in the extracellular
compartment (e.g., blood). The dismutation of superoxide by
SOD generates the less reactive H2O2. Thus, other enzymes
(catalase and glutathione peroxidases) work coordinately to
eliminate the hydrogen peroxide produced by SOD and other
potential sources (Fig. 1). Catalase decomposes H2O2 at high
rates but shows low affinity for the peroxide and should be
most useful during peaks of H2O2 production or accumulation.
These peaks should occur in vivo, because acatalasemia (i.e.,
disorder caused by lack of catalase) increases oxidative stress
and induces pathologies in humans (80). Gpxs, present in
selenium- and nonselenium-dependent forms, are complementary to catalase, since they decompose H2O2 slowly, but with
higher affinity. Thus, they are most useful to decompose the
small amounts of peroxide continuously and physiologically
produced inside cells. At least five selenium-containing glutathione peroxidases have been identified (13), and their activity
can be manipulated by changing dietary selenium levels (7). In
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oxide by catalyzing mild uncoupling, which lowers protonmotive force and would decrease superoxide production by the
electron transport chain (Fig. 1). This negative feedback loop
protects cells from RS-induced damage and might represent the
ancestral function of all UCPs (21, 68).
Finally, posttranslational modifications (i.e., chemical modifications of a protein after its translation), such as acetylation,
S-nitrosation, and glutathionylation, are another important
mechanism regulating RS production. For example, glutathionylation of complex I increases superoxide production by the
complex itself, and when the mixed disulfides are reduced,
superoxide production returns to basal levels (91, 210). Snitrosation results from the reaction of NO with protein thiols,
generating nitrosative stress (28, 58, 76). The implications of
nitrosative stress for animal function are, therefore, clear and
call for attention by physiologists about its potential contribution to explain individual condition and physiological tradeoffs, as stressed in a recent study on effects of immune
response on nitrosative stress in birds (197). Finally, recent
studies seem to indicate that acetylation can be used as posttranslational modification able to regulate the activity of the
mitochondrial electron transport chain complexes by modifying specific subunits (107). In the light of these mechanisms,
mitochondrial activity and structure could be under strong
selection. For example, mitochondria, being the energy makers
of organisms, could expose the individual to energetic constraints because any damage could reduce their efficiency in
ATP synthesis. The daily metabolizable energy intake of an
animal is potentially limited by either the available feeding
time, food availability, or by its capacity to process energy.
Oxidative mitochondrial damage could, therefore, increase
energy demands of the individual, particularly during demanding phases of the year, such as reproduction or migration.
Moreover, organisms can actively respond to environmental
stressors, not only through upregulation or downregulation of
specific mitochondrial proteins, but also through changes in
mitochondria density, as shown in compensatory responses to
changes in physical activity in birds (20) or to cold acclimation
in marine invertebrates or fish (87).
EXTERNAL MECHANISMS. The characteristics of the environment (e.g., local oxygen concentration and membrane phospholipid composition, where complexes are embedded) surrounding the mitochondrial electron transport chain complexes
can be mentioned as external factors able to modify the free
radical generation and, consequently, they can be considered as
potential antioxidant mechanisms.
A first mechanism operates through the regulation of the
mitochondrial partial pressure of O2. Normally, animal cells
are exposed to quite low oxygen concentrations, likely to
minimize oxygen toxicity, which is interpreted as an antioxidant defense (88). When the level of oxygen decreases dramatically and the animal enters a prolonged state of hypoxia,
hypoxia-inducible transcription factors (HIFs), especially
HIF-1, promote expression of genes encoding proteins that
help cells to respond to a hypoxic or anoxic status (72). There
exists, however, high variation in how species can tolerate
hypoxia. For example, low oxygen flux fauna (e.g., marine
organisms) can tolerate prolonged hypoxia without any detrimental consequence for the organism. Also, in high oxygen
flux fauna, we can observe special adaptations to hypoxia, such
as those described in seals. Seals cope with regular exposure to
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GSH and ascorbate can also interact cooperatively in vivo to
cope with RS (130).
Within the lipid compartments, antioxidant protection is
apparently less efficient than in the aqueous environments. In
fact, there is a dramatic difference in the concentrations and
efficiency of antioxidant defense systems between the aqueous
and lipidic compartments, pointing to an evolutionary priority
for the water-soluble antioxidants. The antioxidants functioning optimally in the lipophilic membrane environment are
nonenzymatic components, such as tocopherols and carotenoids, which are both derived from food (60, 208).
At least eight different substances with vitamin E activity
have been described: d-␣, d-␤, d-␥, and d-␦ tocopherol and the
corresponding tocotrienols (23, 208, 220). Vitamin E reduces
lipid peroxyl groups to hydroperoxides, thus inhibiting the
propagation of lipid peroxidation in a chain reaction (71).
Vitamin E can also reduce lipid alkoxyl radicals to lipid
alcohols. Membranes do not contain large amounts of tocopherols: a ratio of one tocopherol per thousand polyunsaturated
fatty acid side chains is common (220). The antioxidant activity of vitamin E is due to the reducing capacity of the hydroxyl
group of its chromanol ring.
Recent studies on wild birds show that plasma vitamin E is
not associated with 1) variation in life histories across species,
2) reproductive success, age, growth and body size within
species, and 3) with diet within and across species (36 –37,
120). The authors proposed that this could be due to recycling
of vitamin E and short-term variation not easily detectable in
large-scale analyses. It is also possible, however, that circulating levels of vitamin E are not very informative, and so we
should also look at it in different body compartments.
Together with vitamin E, carotenoids are main lipophilic
exogenous antioxidants in animal cells. Hundreds of different
carotenoids have been described, although only some of them,
like ␣- and ␤-carotene, lutein, lycopene, zeaxanthine, or criptoxanthine, are present at relevant concentrations in animal
tissues and plasma (113, 208). Despite the antioxidant properties, empirical evidence for carotenoids being important antioxidants in vivo is weak. A recent meta-analysis on the effects
of carotenoids on biomarkers of oxidative damage (e.g., lipid
peroxidation) and of antioxidant capacity in birds shows that
carotenoids have a contribution to protection against oxidative
stress less than 0.002% (45). Further studies published shortly
after reinforced this view, showing that the contribution of
carotenoids to protection against oxidative stress in birds is
also low under stressful conditions (46, 102, 106, 120). In a
similar way, many supplementation trials of carotenoids, as
well as of other dietary antioxidants (vitamins C and E),
administered singly or together in humans (e.g., 86, 122, 143,
211–212) failed to stop disease and aging, and contributed to
increased mortality among participants of the trial. These
results cast serious doubt on the generalization of the paradigm
that availability of antioxidants in the diet is always limiting
for the capacity of the organism to cope with oxidative stress
and that it can represent an important modulator of life histories. The picture is further complicated by the fact that dietary
antioxidants, such as carotenoids or polyphenols, can also exert
prooxidant activities, as well as other physiological functions,
which are independent from their antioxidant properties (43,
90). For example, the life span extension caused by vitamin E
supplementation in mice appeared to be independent of any
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animals, these enzymes use the reduced form of GSH and of
other thiols (e.g., thioredoxin) to decompose the peroxide.
A phylogenetic analysis of the Gpx family, including plant,
fungi, bacteria, invertebrate, and vertebrate taxa, shows complex relationships in this molecular family, suggesting that
basal Gpx classes have originated from independent evolutionary events, such as gene duplication, gene losses, and lateral
gene transfer (128). In particular, an evolutionary pattern can
be recognized in animals leading to the origin of most Gpx
isozymes and a second pattern leading to the Gpx4 isozyme,
which is found in animals, plants, and fungi. This second
cluster would suggest evolutionary convergence mediated by
functional pressures, but it is unclear why this pattern was not
observed for other Gpx isozymes, as well.
The prooxidant activity of hydrogen peroxide is the result of
its one-electron reduction to the hydroxyl radical. This reaction
is among the most damaging faced by living systems: formation and reactions of hydroxyl radical are associated with
chemical, nonenzymatic reactions, which are beyond any cellular control or antioxidant defense mechanism. Reduction of
H2O2, as well as of hydroperoxides, occurs, for instance, in the
presence of metal ions, such as Fe2⫹ or Cu⫹ or superoxide
radical, which reduce them to highly reactive free radicals,
such as hydroxyl, alkoxyl and alkylperoxyl radicals. These
processes are known as the Haber-Weiss reaction and Fenton
reaction, respectively (88) (Fig. 1). This basic chemical process
justifies, in evolutionary terms, the need for the sequestration
of metal ions, mediated by specific proteins (e.g., ferritin)
extensively used by cells, as antioxidant defenses.
In addition to antioxidant enzymes, various types of endogenous nonenzymatic antioxidants are present in animal tissues.
Although they are depleted when they react with ROS, their
oxidized forms are usually recycled back to the antioxidant
form due to reduction by other molecules (e.g., 114). Their low
molecular weight can also be an advantage to eliminate ROS at
sites not accessible to the much larger enzymes. Furthermore,
there are nonenzymatic antioxidants designed to function in
either the hydrophilic or lipophilic cellular compartments. The
main low-molecular-weight hydrophilic nonenzymatic endogenous antioxidants are glutathione, thioredoxin, and ascorbate.
The tripeptide glutathione (188, 196) is particularly abundant
in many tissues, where it can reach levels as high as 10 mM in
its reduced protective form (GSH). Its antioxidant activity is
due to the reduced thiol group of its cysteine residue. Another
thiol-related redox-active substance is the protein thioredoxin
(133). Thioredoxin has a redox-active disulfide/dithiol at the
active site, which regulates transcription factors like NF-␬B
and activated protein-1. Thioredoxin is cytoprotective against
oxidative stress by scavenging ROS in cooperation with peroxiredoxin/thioredoxin-dependent peroxidase (133).
Ascorbate is usually the other most abundant reduced nonenzymatic antioxidant in cells. Although for humans, other
primates, guinea pigs, fruit-eating bats, and many bird species,
vitamin C is acquired by food, in most vertebrates, it is
endogenously synthesized and maintained at high levels in
tissues (e.g., 1 mM in rats) (11). Ascorbate reacts with ROS
and is thus converted to oxidized forms, which can be reduced
back again by NADPH-dependent (175) or GSH-dependent
(127, 221) dehydroascorbate reductases or by a NADH-dependent plasma membrane ascorbate free radical reductase (146).
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antioxidant effect but was likely related to the regulatory role
of vitamin E of signal transduction and gene expression (9).
However, we cannot rule out that the protective role of carotenoids or vitamin E is specific in terms of tissue or phase of
the life cycle rather than general. For example, supplementation of carotenoids may increase parental care of male threespined sticklebacks (Gasterosteus aculeatus) during hypoxic
(but not normoxic) conditions (168) or sperm quality in wild
male great tits (Parus major), especially in those most deficient
in carotenoids (95).
CELLULAR PROTECTION BY REPAIR/TURNOVER AND DETOXIFYING
ANTIOXIDANT SYSTEMS. Despite this plethora of defense sys-
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tems, some oxidative damage still occurs in vivo. Animals
have so evolved other molecular mechanisms to cope with
chemical modifications derived from the interaction between
RS and macromolecules. Among them the following ones may
be mentioned: enzymes that repair oxidized amino acids (e.g.,
methionine sulfoxide reductase), systems that degrade oxidatively damaged proteins (e.g., the proteasome), DNA repair
enzymes, detoxifying systems against carbonyl compounds
derived from carbohydrate and unsaturated fatty acid oxidation, and phospholipid fatty acyl chain removal systems,
among others.
All amino acid residues of proteins are susceptible to oxidative modification by one or more forms of RS. Although
most amino acids in proteins can be modified by diverse
endogenous oxidants, commonly modified residues are lysine
(Lys), arginine (Arg), cysteine (Cys), methionine (Met), and
tyrosine (59). Oxidation of Met and Cys residues generates
methionine sulfoxide and disulfides in proteins, respectively,
which may lead to loss of their biological activity, and deprives
Met of its function as a methyl donor. Most of the protein
oxidative modifications are irreversible, i.e., the only known
way to eliminate the protein adduct is to catabolize the protein
itself. However, particular oxidative modifications, such as
methionine sulfoxide and disulfides, can be repaired by the
methionine sulfoxide reductase (Msr) enzyme in a thioredoxindependent reaction (142).
The degradation of nonfunctional, oxidized proteins is an
essential part of the antioxidant defenses of cells. Eukaryotic
cells have several major pathways for general protein degradation: the lysosomal proteases, calcium-dependent proteases
and the proteasomal system. Additionally, many proteins are
degraded by specific proteases (e.g., caspases). In healthy cells,
the accumulation of oxidatively damaged proteins is prevented
by rapid elimination of such proteins before they can begin to
aggregate, because oxidative modification of proteins makes
them susceptible to proteolysis (83– 84). By selectively recognizing and rapidly degrading oxidized proteins, the proteasome
constitutes an important part of cellular antioxidant defenses
that prevents the build-up of damaged proteins, and their
subsequent aggregation. Thus, intracellular proteases responsible for the selective degradation of oxidized proteins function
as efficient damage removal and repair systems (85). The
removal of physiologically oxidized proteins is an essential
function for maintaining cellular homeostasis and to prevent
the accumulation of highly oxidized and cross-linked proteins,
which are no longer degradable.
Protection of membranes is also achieved by an additional
complex system that involves several mechanisms: lipid repair,
lipid replacement, scavenging of reactive carbonyl species (as
end-products derived from lipid peroxidation), and degradation-removal of modified molecules (104, 152). An important
mechanism for removing peroxidized lipids is membrane lipid
remodeling. Although the enzyme PHGPX is important in
removing peroxidized acyl chains from phospholipids, other
enzymes are also involved in the continual deacylation/reacylation of phospholipids, and this turnover of membrane acyl
chains is very rapid. Phospholipase A2 is a key enzyme for
removing acyl chains from phospholipids, while acyltransferase and transacylase enzymes are responsible for reacylation
of phospholipids (73). Thus, for example, when isolated rat
liver cells are subjected to oxidative stress, lipid peroxidation is
increased; however, there is no change in either the fatty acid
composition of, or in the rate of acyl turnover of membrane
phospholipids. There is, however, a decrease in the unsaturated
fatty acid content of cellular triacylglycerols. It is suggested
that rapid constitutive recycling of membrane phospholipids
rather than selective in situ repair is responsible for eliminating
peroxidized phospholipids, with triacylglycerols providing a
dynamic pool of undamaged unsaturated fatty acids for phospholipid resynthesis (78). This mechanism could have evolved
because it is less energetically demanding than activation of
repair systems, hence possibly less constraining for life-history
decisions.
As mentioned above, RCS are compounds produced under
oxidative stress conditions. These compounds are detoxified in
multiple ways, including conjugation to GSH, oxidation by
aldehyde dehydrogenases, or reduction by aldoketoreductases
(1, 71, 88, 104, 152, 222). Strongly electrophilic carbonyl
compounds induce rapid and significant depletion of intracellular GSH content, suggesting that this is a relevant detoxification mechanism. Once GSH is depleted, cells exhibit an
intracellular change in redox status and propagate an oxidative
stress response following their production. Reaction of RCS
with GSH can proceed in one of two ways: by nonenzymatic
conjugation or through GSH transferase-mediated conjugation
to form Michael adducts. Glutathione-S-transferases (GSTs)
belong to a supergene family of multifunctional enzymes
(192), which are particularly involved in the detoxification of
highly reactive intermediate aldehydes. Interestingly, protection against oxidative stress is the major driver of positive
selection in mammalian GSTs, explaining the overall expansion pattern of this enzyme’s family (75).
Finally, detoxifying mechanisms to protect tissues from
RCS damage also include the cytosolic GSH-dependent glyoxalases, thiol- and histidine-containing dipeptides—presumably
acting as trapping agents—and ascorbic acid.
Cellular antioxidant regulatory factors. Redox regulation is
a signaling system used by cells to convey oxidative stress
information and coordinate cellular functions, which has remained conserved throughout evolution due to its incredible
versatility. Cellular adaptation mechanisms that deserve a special mention are the heat shock response signaling and the
antioxidant-response element and Nrf2.
HEAT SHOCK RESPONSE SIGNALING. The heat-shock response,
through activation of heat-shock transcription factors (HSFs)
and the elevated expression of heat-shock proteins and molecular chaperones, protects the cell against the accumulation of
nonnative proteins (see e.g., 52, 74, 141, 164, 205). Activation
of heat shock factor-1 (HSF1) during this process subsequently
induces the expression of a variety of heat shock proteins
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response by a chemically diverse range of RS indicates that
common sensing mechanisms act as generic stress sensors.
This signaling cascade culminates in the nuclear translocation
of and transactivation by the transcription factor Nrf2 (39, 77,
110) (Fig. 2). A first response acts to protect the cell from
foreign toxins. It includes the initiation of metabolic phase I
and II enzymes, which are responsible for oxidizing xenobiotics and conjugating them to glucoronyl, sulfate, and glycyl
groups to facilitate their cellular export. A second subset of
genes protects the cell from oxidative damage, increasing the
cell’s defensive capacity by upregulating antioxidant genes and
metabolic enzymes involved in glutathione biosynthesis (39).
Cellular metabolism as an integrated antioxidant system.
Inside mitochondria, electrons from reduced substrates move
from complexes I and II of the electron transport chain through
complexes III and IV to oxygen, forming water and causing
protons to be pumped across the mitochondrial inner membrane. When glucose is metabolized through the tricarboxylic
acid (TCA) cycle (or fatty acids through ␤-oxidation), it
generates electron donors. The main electron donor is NADH,
which gives electrons to complex I. The other electron donor
generated by the TCA cycle is FADH2, formed by succinate
dehydrogenase, which donates electrons to complex II. The
proton-motive force set up by proton pumping drives protons
back through the ATP synthase in the inner membrane, forming ATP from their precursors ADP and phosphate (189). The
electron transport system is organized in this way so that the
level of ATP can be precisely regulated. There are, in this
context, two significant main side reactions: electrons leak
from the respiratory chain and react with oxygen to form ROS,
and pumped protons leak back across the inner membrane,
diverting the conserved energy away from ATP biosynthesis
into heat. As mentioned above, the major sites of physiological
ROS generation are the complex I and III of the mitochondrial
electron transport chain.
The rate of mitochondrial ROS generation strongly increases
with a sigmoidal kinetics when the NADH/NAD⫹ ratio is
increased, because this dramatically increases the degree of
reduction of the complex I ROS generator (12, 116). This
effect is also observed when intracellular concentrations of
glucose or fatty acids are increased, e.g., in hyperglycemic
state like diabetes (27) and insulin resistance (99) and can be
reversed upon exposure to agents that act as mitochondrial
uncouplers or electron transport chain inhibitors.
The physiological or pathological overproduction of ROS by
the mitochondrial electron transport chain decreases the activity of the key glycolytic enzyme GAPDH. The inhibition of
GAPDH activity by hyperglycemia does not occur when mitochondrial overproduction of superoxide is prevented by either UCP1 or MnSOD (66). In a step ahead, recent studies have
demonstrated that higher intracellular glucose concentrationinduced superoxide inhibits GAPDH activity in vivo by modifying the enzyme by ADP-ribosylation (65). By inhibiting
mitochondrial superoxide production with either UCP-1 or
MnSOD, both modification of GAPDH by ADP-ribose and
reduction of its activity are again prevented. Most importantly,
both modifications of GAPDH were also prevented by a
specific inhibitor of poly(ADP-ribose) polymerase (PARP), the
enzyme that makes these polymers of ADP-ribose, establishing
a cause-and-effect relationship between PARP activation and
the changes in GAPDH (27).
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(Hsps). Hsps are evolutionary conserved and show many
homologies within and among all prokaryote and eukaryote
organisms, indicating strong stabilizing selection (115, 125,
190). Quantification of stress response mediated by Hsps
appears to be a promising biomarker in ecological research to
evaluate how the organism can maintain cellular functioning
under environmental challenges and protein denaturing conditions. Studies on wild birds, for example, have found associations between the physiological condition, stress response, and
immune or infection status and resistance (pied flycatcher,
Ficedula hypoleuca, in Ref. 140; blue tit, Parus caeruleus, in
Ref. 6). Also, a study on pied flycatchers found that individuals
breeding in closer proximity to predators’ nests (sparrowhawk
Accipiter nisus) had higher levels of Hsps 60 and 70 (213).
This study suggested that there might be an indirect link
between predation risk and oxidative stress, highlighting the
importance of habitat selection decisions for the individual
physiological status.
The study of Hsps also has the potential to provide important
insight into epigenetic effects. In 1998, Rutherford and
Lindquist (177), proposed that the heat shock response could
be a molecular mechanism underlying the emergence of epigenetic effects in natural animal populations. In a later study
(184), further evidence for this mechanism was provided in
plants (184), suggesting that the stress response mediated by
heat shock proteins is a general mechanism. Some methodological issues on Hsp quantification, however, remain to be
addressed before we can properly interpret changes in Hsps
levels and translate results from laboratory to field conditions
(201).
ANTIOXIDANT-RESPONSE ELEMENT AND Nrf2. Redox state is regulated through the action of RS, which act as second messengers in signal transduction networks (77). RS require a receptor
site on the target protein, which effectively allows different
proteins to function as redox sensors for diverse types of RS.
Given that RS can react with any molecule, it is a major
challenge to interpret which of these reactions can be considered as redox signaling or as random chemical modifications.
In addition, redox signaling reactions vary significantly, depending upon cell type and cellular antioxidant levels, as well
as among animal species (77). Nonetheless, it is becoming
clear that RS can affect the expression of a vast number of
genes and cellular functions, including cell-cell communication, metabolism, structure, motility, proliferation, differentiation, and apoptosis. It is possible to establish some general
principles, governing redox regulation (77): 1) oxidant administration to different cell types regulates the expression of a
large number of genes, typically between 1% and 5% of the
genome; 2) antioxidant levels modulate the effect of RS and
also act independently to regulate signaling; 3) changes in the
cellular redox balance result in both the upregulation and
downregulation of subsets of genes; 4) the expression of
individual genes is related to the chemical identity of the
regulatory oxidant or antioxidant; and 5) the extent of redox
regulation is governed by both the magnitude and duration of
the applied stimulus (i.e., the kind of RS).
The response to oxidative stress is characterized by a concerted upregulation of over 100 genes, which encode for
detoxification and antioxidant proteins. Thus, RS can activate
the “antioxidant response” likely to prevent their accumulation
to toxic levels (124, 219). The activation of this antioxidant
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Poly(ADP-ribosyl)ation occurs in almost all nucleated cells
of mammals, plants, and lower eukaryotes, but is absent in
yeast. It represents an immediate cellular response to DNA
damage, as induced by ionizing radiation, alkylating agents,
and oxidants (33, 38, 62). In the absence of DNA single- and
double-strand breaks, poly(ADP-ribosyl)ation is a very rare
event, but it can increase over 100-fold upon DNA damage.
Under these conditions, about 90% of poly(ADP-ribose) is
synthesized by poly(ADP-ribose) polymerase 1 (PARP-1).
PARP-1 is constitutively expressed, but enzymatically activated by DNA strand breaks. It catalyzes the formation of
ADP-ribose from the oxidized form of NAD⫹ by cleavage of
the glycosidic bond between nicotinamide and ribose. Glutamate, aspartate, and carboxy-terminal lysine residues of target
(“acceptor”) proteins are then covalently modified by the
addition of an ADP-ribose subunit, via formation of an ester
bond between the protein and the ADP-ribose residue. Poly(ADP-ribosyl)ation is a posttranslational protein modification
linked with genome protection and mammalian longevity
(15, 33).
In this scenario, it is plausible to suggest that the inhibitory
effect of ADP-ribosylation on GAPDH probably represents a
regulatory pathway to reduce levels of glycolysis and subsequent flux of metabolites to mitochondria, allowing a decrease
in the levels of reducing equivalents and the subsequent mitochondrial ROS production. In addition, this mechanism seems
to indicate that the stress-induced block of glycolysis is not the
result of a passive oxidative damage, but rather an active cell
AJP-Regul Integr Comp Physiol • VOL
adaptation programmed via ADP-ribosylation for cell selfdefense.
Another class of enzymes that utilize NAD⫹ as a substrate
is that of sirtuins. Sirtuins regulate a wide range of cellular and
physiological activities (223). The sirtuin deacetylase activity
closely links NAD⫹ with a variety of cellular functions that
are important for regulating development and longevity, including stress resistance, mitochondrial biogenesis, adipogenesis, proliferation, DNA repair, metabolism, and autophagy
that collectively are thought to be involved in sustaining
healthy cell types and delaying age-related disease states.
Therefore, NAD⫹ may be considered a key signal molecule
able to integrate oxidative stress and metabolism to maintain
organism health and drive a program of robustness that counteracts or delays aging in a broad multifaceted way.
Biochemical Integration of the Redox System
Our overview on antioxidant mechanisms shows that the
machinery offering protection against peroxidation is highly
complex, involving a large number of gene networks, molecular pathways, and families of molecules. This implicates that
the effectiveness of the contribution of one antioxidant component to protection is not totally independent of each other,
but a variable degree of biochemical integration can be observed among different antioxidants. Integration (e.g., morphological, metabolic, biochemical, genetic) is a property of biological systems that refers to the degree and extent to which its
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Fig. 2. Nrf2 signaling in antioxidant response element (ARE)-mediated coordinated activation of antioxidant genes in animal species (vertebrates and
invertebrates). Nrf2 activity is repressed by an inhibitory binding protein, Keap1. Keap1 retains Nrf2 in the cytosol, closely associated with the actin cytoskeleton
and promotes proteasomal degradation of Nrf2 through Cullin3-dependent polyubiquitination (Ub). Following exposure to reactive species, Keap1 can be directly
modified on several cysteine residues, and this modification can promote release of Nrf2. Intracellular modification of Keap1 similarly allows for increased
stabilization and nuclear localization of Nrf2, potentially through direct modification of Keap1 coupled with Keap1 ubiquitination and degradation. Nrf2 contains
a COOH-terminal basic leucine zipper structure that facilitates dimerization and DNA binding, specifically to the ARE, which is located in the promoter region
of the stress-induced genes. The binding of Nrf2 to the ARE, which requires heterodimerization with small Maf proteins, stimulates transcription of downstream
genes, in part, by recruiting transcriptional coactivators, particularly CREB-binding protein (CBP) through the Neh4 and Neh5 domains of the transcriptional
factor. Nrf2 activity is also enhanced through phosphorylation by several kinases, including protein kinase C isoforms and the endoplasmic reticulum
stress-responsive kinase PERK. In essence, reactive species generation induces the nuclear localizaton of Nrf2, and the corresponding activation of the ARE
defends the cell from oxidative stress. One of these responses acts to protect the cell from foreign toxins and includes the initiation of metabolic phase I and II
enzymes, which are responsible for oxidizing xenobiotics and conjugating them to glucoronyl, sulfate, and glycyl groups to facilitate their cellular export; and
a second subset of genes protects the cell from oxidative damage, increasing the cell’s defensive capacity by upregulating antioxidants genes and metabolic
enzymes involved in glutathione biosynthesis. The Nrf2-regulated cytoprotective genes are aldo-keto reductases (AKR), glutamate cysteine ligase [catalytic
subunit (GCLc), and regulatory subunit (GCLm)], glutathione-S-transferase (GST), glutathione synthetase (GS), heme-oxygenase 1 (HO-1), metallothionein,
microsomal epoxide hydrolase (mEH), NAD(P)H:quinine oxidoreductases (NQO), peroxiredoxin 1 (Prx1), superoxide dismutases (SOD), thioredoxin reductases
(TrxR), thioredoxins (Trx), and UDP-glucuronosyltransferases (UGT) (39). SR, thiol group; SH, sulfhydryl.
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Fig. 3. Schematic representation of three hypothetical scenarios of temporal
variation in degree of redox network integration in a tissue. The network has
originally 10 nodes (e.g., 10 molecular components of a tissue redox state) and
12 of a total of 45 potential links among nodes, hence resulting in a k of 1.2.
In scenario A, the network integration remains stable (k ⫽ 1.2) over time (e.g.,
under nonstressful environmental conditions); in B, the network loses integration (k ⫽ 0.9); hence, there is a reduction in the capacity of the tissue to
maintain homeostasis (e.g., senescence); and in C, the network becomes more
integrated (k ⫽ 1.7), resulting in a more efficient response to environmental
perturbations (e.g., maturation of the antioxidant defenses, priming effect).
under stressful conditions (63), suggesting an increase in integration. The comparison of correlation matrices between different states can, therefore, provide a helpful analytical and
conceptual tool to make inferences on the organismal stress
response as a whole and to identify factors that modulate its
behavior.
The analysis of network may also allow to identify which
antioxidant components can constrain the antioxidant response
itself. Some antioxidant mechanisms rely on a delicate equilibrium among molecules involved. Hence, a failure of one of
them could compromise the effectiveness of antioxidant response. However, it cannot be entirely excluded that maintaining a variety of components of the antioxidant machinery could
provide a “fail-safe” to avoid the case that the failure of one
antioxidant compromises the whole system. To answer these
questions would mean to identify any biochemical constraints
present in the network and how these can limit the plasticity of
the stress response. This could be done, for example, by
experimental removal or downregulation of an arbitrary or
most-connected node (i.e., hub) of the network.
A further important outcome of such analytical approach is
the description of hierarchical organization of modularity in
redox networks (173). A module is a discrete unit of an x
number of elements interacting in a tightly integrated way that
performs a specific task, separable from the functions of other
modules (92, 94). Cluster analysis and PLS regression could be
helpful for the identification of these modules, that we could
call “redox modules” when they refer to antioxidant-prooxidant units (see Fig. 4 for a schematic illustration). Only
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components are correlated through functional, structural, developmental, or evolutionary interdependency (29, 51, 79, 112,
134, 173, 191). More specifically, biochemical integration
refers to the functional interdependency among biomolecules
through a direct or indirect reaction or regulation (e.g., concentration of molecule A depends on concentration of molecule
B and vice versa; molecule A affects molecule C through
molecule B) (see also Ref. 159). To make rigorous inferences
on integration, it is, therefore, important that a basic knowledge
of the biochemistry of molecules under study does exist.
Traditionally, redox mechanisms are studied by means of in
vitro or ex vivo systems, which focus on the description of
specific mechanisms, often in isolation from others. We think
that the integration of traditional approaches with the analyses
of correlation/covariance matrices (e.g., multivariate analysis,
interaction networks) among components of the redox state in
in vivo systems could provide a wealth of information on how
the system works and why it works the way it does. For
example, the application of factor analysis and of cluster
analysis on correlation matrix of redox variables can represent
a first analytical step to quantify the degree and sign of association among variables and to identify clusters of variables
strictly connected to each other (Bruner E, personal communication; see Ref. 51 for an exploratory application on blood
redox state in birds). A bootstrap analysis would then be
important to quantify the statistical robustness of each node of
the generated tree. In case the system under study is governed
by nonlinear dynamics, the application of nonlinear dimensionality reduction algorithms (e.g., isometric mapping ISOMAP)
would be more appropriate (193). To determine the degree of
covariance (i.e., integration) between redox statuses of two
tissue compartments, a two-block partial least squares (PLS)
regression could prove valuable. The correlation matrix can
finally be used to visualize a network (Fig. 3), i.e., a set of
mutually interacting elements whose collective behavior gives
rise to emergent properties of the system under study (149,
215). A network is visualized as a set of nodes and links (or
edges), where a node represents a molecular component, and a
link between two nodes indicates that those two molecules are
significantly correlated. The degree of connectedness of the
network can be quantified by the coefficient k, which is the
ratio between the number of links present and the maximum
number of possible links in that specific network. As shown in
Fig. 3A, the value of k is constant when the redox network
remains stable across time. In Fig. 3, B and C, we can observe
a decrease and an increase of k, respectively, which indicate
that the network became less or more integrated, respectively.
Gene and metabolic networks can, for example, become less
integrated as the individual ages, hence, becoming noisier and
less stable because of a decrease in the effectiveness of communication among functional units (53, 200, 202, 224). Therefore, we can predict that a decrease in k with time may mirror
a signal of senescence. On the other hand, we can see an
increase in k when the organism is mounting a stress response.
In general terms, phenotypic integration can increase with
environmental stress, possibly because of the convergence of
the various response mechanisms when there is an increased
need to promote self-maintenance and survival (e.g., 8, 218). If
we look more specifically at the collective behavior of molecules involved in a redox reaction, we can see that the magnitude of correlations among variables can strongly increase
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Fig. 4. Two examples of network topologies. A: schematic illustration of a
highly integrated network, where a modular organization cannot be recognized. B: schematic illustration of a modular network, where three modules
(white, gray, and black circles, respectively) can be recognized by the fact that
nodes within each module are highly connected, while modules are connected
to each other by one link only.
Antioxidants and Animal Maximum Life Span
In this general scenario, what are the fundamental mechanisms of maintenance in long-lived animal species? Available
evidence points to two general mechanisms: 1) decrease in the
rate of generation of RS and 2) possession of macromolecules
less sensitive to oxidative damage.
Concerning the possession of macromolecules less sensitive
to oxidative damage, available evidence clearly shows a negative correlation between the maximum life span of the animal
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relatively few or weak interactions will then connect different
redox modules, possibly depending on their functions and
environmental fluctuations. Ravasz et al. (173) recognize, for
example, a hierarchical organization of modularity in metabolic network of Escherichia coli. The authors show that there
are many highly integrated small modules (submodules),
which group into a few larger modules. The authors also
suggest that the accumulation of changes in the submodules
could slowly impact the properties of the larger ones, hence
affecting the evolution of the whole system. Further modular
organizations have been recently characterized in metabolic
networks and stress response pathways of bacteria. Parter et al.
(165) show that metabolic networks of bacteria living in
variable environments are more modular than networks of
bacteria living in more stable conditions. The authors suggest
that unstable environmental conditions promote modular organization because each module deals with a specific task. Given
that in an unstable environment an organism can be faced with
several environmental pressures, it could be more functionally
efficient to separate the processing of the stimulus and the
response to it among different modules, according to the nature
of the stimulus itself.
In agreement with Parter et al. (165), Singh et al. (198)
report that stress response modules vary in both number and
degree of cohesiveness in bacteria. Moreover, they show that
most of the modular variation is in environmental sensing and
signal transduction genes at the onset and completion of the
response, while structural proteins show the least variation.
These findings also suggest that the modular architecture may
reflect a real functional organization of the metabolic system in
relation to environmental pressures.
We, therefore, suggest that the physiological response to a
stressor could be better appreciated through the analyses of
correlation/covariation matrices of redox variables rather than
through the analysis of the response of one single variable at a
time.
species and the content of guanine, methionine, and unsaturated fatty acids in their cellular structural components. In
other words, the longer the animal life span, the lower the
content-abundance of the structural component susceptible to
oxidative stress (2, 102, 104, 108 –109, 152–153, 156 –157,
171, 176, 183) (Table 1).
The susceptibility of mtDNA to damage could be related to
the simplest property of a DNA sequence, i.e., the proportion
of adenine (A), cytosine (C), guanine (G), and thymine (T) in
the mtDNA molecule. In a recent study (183), analyzing the
“light strand” mtDNA sequence in 94 animal species (including invertebrates, birds, and mammals), it was found that
short-lived species have higher A and T abundances and lower
C abundances than long-lived species, while G was almost
uniformly low in all species. The light strand was given this
name because it is unusually deficient in G, and data from this
study show that this characteristic low G abundance looks to be
very common across animal species. This result could suggest
that the G abundance has been under strong directional selection. On a more mechanistic ground, it is thought that the
asymmetric mode of replication of the mtDNA molecule
causes an increase in the G to A transition mutations on the
light strand, depleting it of G nucleotides. In addition to this
finding, it was shown that this pattern of nucleotides affects an
important DNA sequence property: the free energy (a physical
property of the double-stranded DNA molecule related to the
binding energy between the two DNA strands). The more
negative the free energy is, the less likely is the spontaneous
separation of the two strands through thermal fluctuations,
conferring to the mtDNA a greater structural stability and
lesser susceptibility to damage (183). In support of these
mechanisms, comparative data show a strong relationship between the mtDNA free energy and maximum life span. Thus,
the longer the maximum life span of a species, the lower is the
free energy of mtDNA. Considering that among the four
nucleobases, G has the lowest oxidation potential and is thus
generally most easily oxidized (18), it has been proposed that
these mtDNA base sequence patterns and derived properties
represent an evolutionary adaptation of long-lived species to
increase mtDNA resistance to endogenous damage by chemical side-reactions and so to decrease the susceptibility of
mtDNA to damage and mutation. Accordingly, in a recent
study (144), analyzing the lineage-specific mitochondrial mutation rate across 1,696 mammalian species and comparing it
with the nuclear rate, a selected decrease of nuclear base
substitution rate was reported in long-lived species, reinforcing
the proposal for a causal role of mtDNA mutations in aging.
This suggests that natural selection may have decreased the
mitochondrial mutation rate in long-lived species. Finally, in
accordance with this interpretation, a recent study (109) with a
phylogenomic approach to identify the genetic targets of natural selection for increased life span in mammals shows, by
comparing the nonsynonymous and synonymous evolution of
5.7 million codon sites across 25 species, genes involved in
DNA replication/repair or antioxidation have played no detectable role in the evolution of longevity in mammals, reinforcing
the relevance of the association between mitochondrial free
radical production and mtDNA oxidation-mtDNA mutations.
From an evolutionary comparative approach, a recent study
Aledo et al. (2) address the association between species’ life
span and methionine usage in mitochondrial proteins using a
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ANIMAL ANTIOXIDANT DEFENSES
Table 1. Comparative studies of endogenous levels of tissue antioxidants in animal species differing in their maximum life span
Compared Species (Common Name)
Antioxidant
Source
Correlation With
Maximum Life Span
Reference
Resistance of cellular structural components to oxidative damage
3 Hummingbird species, mammalian species
Chicken, mammalian species
Mammalian species, bird species, reptile species,
insect species
C. elegans (long- vs. short-lived mutants)
25 mammalian species
Red blood cells
Negative
16
Liver, skeletal muscle
Negative
67
Heart, liver, skeletal muscle
Negative
104, 152
Membrane unsaturation
Membrane unsaturation
Whole
Genomic DNA (genes for
fatty acid synthesis)
mtDNA
Negative
Negative
194
109
Negative
183
Negative
144
Negative
139
76 mammalian species, 14 bird species, 4
invertebrate species
1,696 mammalian species
mtDNA guanine content and
free energy per base pair
mtDNA mutation rate
95 mammalian species, 14 bird species, 10
reptile species, 12 amphibian species, 39 fish
species, 24 insect species, 15 crustacean
species, 9 arachnid species
168 mammalian species
Rat, pigeon
Mouse, rat, guinea pig, rabbit, sheep, pig, cow,
horse
Budgerigar, canary, mouse
mtProtein cysteine content
Complete cytochrome b
sequence
mtDNA
mtProtein methionine content
Proteome methionine content
Proteome methionine content
mtDNA
Skeletal muscle
Heart
Negative
Negative
Negative
2
171
176
Proteome methionine content
Brain
Negative
157
Regulatory mechanisms of free radical generation as antioxidant systems
Rat, pigeon, human
Mouse, rat, guinea pig, sheep, dog, pig, cow,
horse
Rat, pigeon
Budgerigar, canary, mouse
Rat, pigeon
Budgerigar, canary, mouse
Cardiolipin unsaturation
Cardiolipin unsaturation
Liver mitochondria
Liver mitochondria
Negative
Negative
158
170
Complex I content
Complex I content
Degree of reduction Complex I
Degree of reduction Complex I
Heart
Brain
Heart
Heart
Negative
Negative
Negative
Negative
118, 203
157
96
97
mitochondria
mitochondria
mitochondria
mitochondria
Antioxidant diversity is an expression of the variety of reactive species and their molecular consequences: Cellular protection by enzymatic and
nonenzymatic antioxidants
Mouse, rat, guinea pig, rabbit, cow, human
Rat, guinea pig, rabbit, pig, cow, rhesus
monkey, human
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Ames dwarf mouse (df/df) vs. normal or
transgenic GH mouse
Pig-tailed macauqe, rhesus monkey, baboon,
chimpanzee, human
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
D. melanogaster (long- vs. short-lived lines)
Mouse, naked mole-rat
Honey bees (queen vs. worker)
Mouse, rat, vampire bat, Mexican free-tailed bat,
naked mole-rat, little brown bat
Mouse, rat, squirrel, rabbit, guinea pig, big
brown bat, sheep, white-tailed deer, dog, pig,
cow, human, Japanese quail, zebra finch
D. melanogaster (wild-type strains)
Marine bivalve species
Field mouse, deer mouse, pig-tailed macaque,
baboon, human
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Ames dwarf mouse (df/df) vs. normal or
transgenic GH mouse
D. melanogaster (long- vs. short-lived lines)
Ascorbate
Ascorbate
Brain
Liver
Negative
Negative
54
57
Ascorbate
Liver
Negative
126
Ascorbate
Brain, lung
Negative
10, 167
Ascorbate
Liver
Negative
25
Catalase
Liver, kidney
Negative
56
Catalase
Brain, liver, lung
Negative
10, 126, 167
Catalase
Catalase
Catalase
Catalase
Whole
Liver
Brain, Thorax, Abdomen
Liver
Negative
NS or Negative
Negative
Negative
135
4–5
40
31
Catalase
Liver, heart, brain
Negative
151
Catalase (gene expression)
Catalase
Glutathione
Whole
Whole
Brain, liver
Negative
Negative
Negative
187
216
57
Glutathione
Liver, lung
Negative
126, 167
Glutathione
Brain
NS
10
Glutathione
Liver
Negative
25
Glutathione
Whole
Negative
135
Continued
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Intracellular glycolytic
intermediates
Intracellular glycolytic
intermediates
Membrane unsaturation
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ANIMAL ANTIOXIDANT DEFENSES
Table 1.—Continued
Compared Species (Common Name)
Antioxidant
Marine bivalve species
Honey bees (queen vs. worker)
Mouse, rat, squirrel, rabbit, guinea pig, big
brown bat, sheep, white-tailed deer, dog, pig,
cow, human, Japanese quail, zebra finch
D. melanogaster (wild-type strains)
Correlation With
Maximum Life Span
Reference
Glutathione
Glutathione reductase
Liver
Liver
NS or Negative
NS
4–5
126
Glutathione reductase
Brain, liver
Negative
10, 167
Glutathione reductase
Liver, heart, brain
Negative
151
Cu,Zn-superoxide dismutase
Liver, brain, heart
NS
214
Cu,Zn-superoxide dismutase
Brain
Negative
150
Cu,Zn-superoxide dismutase
Liver
NS
126
Cu,Zn-superoxide dismutase
Brain, lung
Negative
10, 167
Cu,Zn-superoxide
Cu,Zn-superoxide
Cu,Zn-superoxide
Cu,Zn-superoxide
Whole
Liver
Brain, thorax, abdomen
Liver, heart, brain
Negative
Negative
Negative
Negative
163
4–5
40
151
Cu,Zn-superoxide dismutase
(gene expression)
Cu,Zn-superoxide dismutase
Mn-superoxide dismutase
Mn-superoxide dismutase
Whole
Negative
187
Whole
Brain, thorax, abdomen
Liver, heart, brain
Negative
Negative
Negative
216
40
151
Mn-superoxide dismutase
(gene expression)
Whole
Negative
187
dismutase
dismutase
dismutase
dismutase
Antioxidant diversity as an expression of the variety of reactive species and their molecular consequences: Cellular protection by repair/turnover and
detoxifying antioxidant systems
Rat, rabbit, sheep, dog, cow, monkey, horse,
human
D. melanogaster (long- vs. short-lived lines)
Snell mouse, C57BL/6Ncr1BR mouse, Syrian
hamster, Norway rat, Mongolian gerbil, 13lined ground squirrel, rabbit, guinea pig, big
brown bat, Sussex sheep, white-tailed deer,
domestic dog, Yorkshire/Hampshire pig,
Black angus/Charlet cow, Japanese quail and
zebra finch
Mouse, rabbit, guinea pig, dog, calf, goldfish
Hamster, rat, sheep, pig, chicken, human
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Mouse, naked mole-rat
Honey bees (queen vs. worker)
Mouse, rat, squirrel, rabbit, guinea pig, big
brown bat, sheep, white-tailed deer, dog, pig,
cow, human, Japanese quail, zebra finch
D. melanogaster (wild-type strains)
Marine bivalve species
Honey bees (queen vs. worker)
Honey bees (queen vs. worker)
Mouse, naked mole-rat
2 Bat species
Base excision repair activities
(polymerase ␤)
Disulfide reductase
Glutaredoxin
Dermal fibroblasts
Negative
26
Whole
Liver, heart, brain
Negative
Negative
135
182
Glutathione peroxidase
Glutathione peroxidase
Glutathione peroxidase
Brain, liver
Liver
Brain, liver, lung
Negative
Negative
Negative
61
121
10, 126, 167
Glutathione peroxidase
Glutathione peroxidase
Glutathione peroxidase
Liver
Brain, Thorax, Abdomen
Liver, heart, brain
Negative
Negative
Negative
4–5
40
151
Glutathione peroxidase (gene
expression)
Glutathione peroxidase
Glutathione S-transferase
Methionine sulfoxide reductase
A
Proteasome (level and specific
activities)
Proteasome (20S activity)
Whole
Negative
187
Whole
Brain, thorax, abdomen
Brain, thorax, abdomen
Negative
Negative
Negative
216
40
40
Liver
N.S.
Liver
Negative
166
180
Continued
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Mouse, naked mole-rat
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Mouse, rat, squirrel, rabbit, guinea pig, big
brown bat, sheep, white-tailed deer, dog, pig,
cow, human, Japanese quail, zebra finch
House mouse, deer mouse, tree shrew, squirrel
monkey, Rhesus monkey, chimpanzee,
orangutan, lemur, tamarin, baboon, African
green monkey, gorilla, human
Mouse, rat, hamster, guinea pig, rabbit, dog,
sheep, cat, pig, cow, horse
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Mouse, rat, guinea pig, frog, trout, toad, canary,
pigeon
Ant Lasius Niger (Queen vs. workers and males)
Mouse, naked mole-rat
Honey bees (queen vs. worker)
Mouse, rat, squirrel, rabbit, guinea pig, big
brown bat, sheep, white-tailed deer, dog, pig,
cow, human, Japanese quail, zebra finch
D. melanogaster (wild-type strains)
Source
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ANIMAL ANTIOXIDANT DEFENSES
Table 1.—Continued
Compared Species (Common Name)
Antioxidant
Snell mouse, C57BL/6Ncr1BR mouse, Syrian
hamster, Norway rat, Mongolian gerbil, 13lined ground squirrel, rabbit, guinea pig, big
brown bat, Sussex sheep, white-tailed deer,
domestic dog, Yorkshire/Hampshire pig,
Black angus/Charlet cow, Japanese quail and
zebra finch
Honey bees (queen vs. worker)
Correlation With
Maximum Life Span
Reference
Proteasome (20S and 26S
activities)
Liver, heart, brain
Negative
181
Thioredoxin peroxidase
(mitochondrial)
Thioredoxin reductase
Thioredoxin reductase
Brain, thorax, abdomen
Negative
40
Brain, thorax, abdomen
Liver, heart, brain
Negative
Negative
40
181
Cellular antioxidant regulatory factors
Mouse, hamster, rat, gerbil, squirrel, rabbit,
guinea pig, finch, sheep, deer, pig, cow
D. melanogaster (long-lived for keap1/Nrf2
mutants versus wild type)
Mouse (long-lived Snell dwarf mutant vs. wild
type)
Mouse (long-lived mGsta4 mutant vs. wild type)
Heat shock protein expression
(HSP60, HSP70, GRP78,
GRP94)
Nrf2 (activity)
Brain, heart, liver
Positive
182
Whole
Positive
209
Nrf2 (level and activity)
Skin-derived fibroblasts
Positive
123
Nrf2 (activity)
Liver, skeletal muscle
Positive
199
Positive
82
Cellular metabolism as an integrated antioxidant system
Elephant, pigmy chimpanzee, gorilla, donkey,
rabbit, pig, horse, cattle, rat, guinea pig,
marmoset, sheep, man
Poly(ADP-ribose)polymerase
Mononuclear leukocytes
NS, not significant correlation. Negative correlation means that long-lived species constitutively have lower tissue levels of antioxidants enzymes and
low-molecular-weight endogenous antioxidants than short-lived ones (Positive correlation means the contrary).
meta-examination of mitochondrial genomes from 168 species
of mammals, encompassing 24 different orders. Results
showed that methionine usage and life span exhibit a significant negative correlation. However, species are part of a
hierarchically structured phylogeny, raising the possibility that
data from different species may not necessarily be statistically
independent from one another. Therefore, to correct for nonindependence of the individual-species data, phylogenetically
independent contrasts were calculated. Interestingly, the association between methionine content and life span became more
significant after phylogenetic correction. Extending these findings, Aledo et al. (2) studied the relationship between life span
and methionine content within those eutherian orders with 10
or more species present in their compilation. All of the analyzed orders, Carnivora (n ⫽ 41), Primates (n ⫽ 23), Artiodactyla (n ⫽ 20), Cetacea (n ⫽ 18) and Rodentia (n ⫽ 10),
showed a negative covariance between life span and methionine content. However, after correcting for the phylogenetic
inertia only Carnivora, Primates, and Rodentia exhibited statistically significant negative correlations between the variables
of interest. So, mtDNA-encoded proteins from short-lived
mammals are enriched in methionyl residues compared with
those from long-lived mammals. Interestingly, the results suggest that methionine addition to proteins in short-lived species,
rather than methionine loss from proteins in long-lived species
is responsible for the observed negative correlation between
methionine content and life span. Furthermore, methionine
AJP-Regul Integr Comp Physiol • VOL
abundance in the general group of nDNA-encoded mitochondrial proteins did not significantly correlate with life span,
suggesting that the protein methionine content is circumscribed
to a small subset of mitochondrial proteins localized in the
inner mitochondrial membrane where free radicals are generated. By contrast, when using this data set, cysteine fails to
show a link with life span within the Mammalia class after
correction for the effects of phylogeny. However, when the
metaexamination of mitochondrial genome sequences is extended to 248 animal species with known longevities, including mammals, birds, reptiles, amphibians, fish, insects, crustaceans, arachnids, and helminths, a slightly different picture
seems to appear (14, 138 –139). The analysis reveals that the
frequency with which cysteine is encoded by mitochondrial
DNA is a specific and phylogenetically ubiquitous molecular
indicator of aerobic life span: long-lived species synthesize
respiratory chain complexes, which are depleted of cysteine.
Cysteine depletion was also found on a proteome-wide scale in
aerobic vs. anaerobic bacteria, archaea, and unicellular eukaryotes; in mitochondrial vs. hydrogenosomal sequences; and
in the mitochondria of free-living, aerobic vs. anaerobic-parasitic worms. The association of life span with mitochondrial
cysteine depletion persisted after correction for phylogenetic
interdependence, but it was uncoupled in helminthic species
with predominantly anaerobic lifestyle (139). In contrast, methionine usage in mitochondrially encoded proteins is strikingly higher than in nuclear encoded proteins in the majority of
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Honey bees (queen vs. worker)
Snell mouse, C57BL/6Ncr1BR mouse, Syrian
hamster, Norway rat, Mongolian gerbil, 13lined ground squirrel, rabbit, guinea pig, big
brown bat, Sussex sheep, white-tailed deer,
domestic dog, Yorkshire/Hampshire pig,
Black angus/Charlet cow, Japanese quail and
zebra finch
Source
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ANIMAL ANTIOXIDANT DEFENSES
AJP-Regul Integr Comp Physiol • VOL
the sensitivity to lipid peroxidation is strongly decreased in
long-lived animals, whereas the fluidity of the membrane
would be essentially maintained. This hypothesis, reminiscent
of membrane acclimation to different environments at PUFA
level in poikilotherms and bacteria, has been denominated
homeoviscous longevity adaptation (156). In accordance with
this interpretation, a recent study (109) with a phylogenomic
approach to identify the genetic targets of natural selection for
elongated longevity in mammals has been published. In this
work, comparing the nonsynonymous and synonymous evolution of 5.7 million codon sites across 25 species, shows that
genes involved in lipid composition (and particularly desaturation system) have collectively undergone increased selective
pressure in long-lived species. So, cellular membrane has
apparently been the optimized feature.
With reference to mitochondrial free radical generation and
maximum life span, all the published investigations about this
subject have found that the rate of mitochondrial RS production is lower in the tissues of long-lived than in those of
short-lived animal species independently of their mass-adjusted rates of O2 consumption (12, 117, 154, 156, 185, 186).
In accordance with this low mitochondrial free radical production of long-lived species, there is an adaptive response concerning endogenous cellular antioxidant systems (154 –155).
Long-lived animal species constitutively have lower (instead of
higher) tissue levels of antioxidant enzymes and low-molecular-weight endogenous antioxidants than short-lived ones (Table 1). That characteristic can thus explain why endogenous
tissue antioxidants correlate negatively with maximum life
span across species: long-lived animals have constitutively low
levels of antioxidants because they produce RS at a low rate. If
long-lived animals had high rates of RS production together
with their very low levels of endogenous antioxidants, their
tissue cells would not be able to maintain oxidative stress
homeostasis. Decreasing mitochondrial ROS production instead of increasing antioxidants or repair systems makes sense
when considered from the point of view of evolution of life
span among species and upon the premise that metabolism is
adapted and optimized to achieve balance and economy (131).
It would be very inefficient to generate large amounts of ROS
and, afterward, try to intercept them before they reach cellular
components, or even worse, try to repair after heavily damaging it. This makes even more sense taking into account the high
energetic cost of continuously maintaining high levels of antioxidant and repair molecules in cells, considering that availability of energy is limited by both environment (e.g., food
availability) and physiological constraints. In agreement with
the adaptive response that represents antioxidant defenses, it is
relevant to highlight that long-lived species show high (not
low) levels/activities of regulatory factors (Table 1), confirming the ability of animals (short- or long-lived) to transitorily
induce or repress these protective molecules when needed.
The outcomes of two experimental paradigms deserve also
special mention (reviewed in Refs. 154 and 185): 1) experimentally increasing tissue antioxidants through dietary supplementation, pharmacological induction, or transgenic techniques sometimes moderately increases mean life span (life
expectancy) but does not change maximum life span; and
2) animals in which genes coding for particular antioxidant
enzymes are knocked out can show different pathologies but
their rates of aging do not seem to be affected. These findings
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animals (which is the inverse behavior compared with cysteine), and there is no significant correlation between mitochondrial methionine usage and life span (138). The dichotomy
methionine accumulation vs. cysteine depletion in mitochondrial respiratory chain complexes probably should be considered in the context of antioxidant protection (for methionine)
vs. avoidance of oxidation-derived effects (for cysteine). So,
the topological selectivity of longevity-dependent cysteine depletion to the inner mitochondrial membrane might find its
most probable explanation in the concurrence of three circumstances that are not to be found elsewhere in the cell: exceptionally high concentrations of multispan transmembrane proteins of outstanding hydrophobicity, their particularly high
exposure to free radicals generated in situ, and the unique
chemical reactivity of thiols in nonpolar environments. Under
these premises, cysteine is avoided to prevent cysteine-catalyzed chain-transfer reactions, and thus, the formation of extended networks of internally cross-linked hydrophobic proteins that might eventually lead to a mitochondrial dysfunction
(138).
The mechanisms responsible for the life span-related differences in fatty acid profile can be related, in principle, to the
fatty acid desaturation pathway, and the deacylation-reacylation cycle. The available estimates of delta-5 and delta-6
desaturase activities indicate that they are several folds lower
in long-lived species than in short-lived ones (152–153). This
can explain why 22:6n-3 and 20:4n-6 decrease and 18:2n-6 and
18:3n-3 increase, respectively, from short- to long-lived animals, since desaturases are the rate-limiting enzymes of the n-3
and n-6 pathways synthesizing the highly unsaturated PUFAs
20:4n-6 and 22:6n-3 from their dietary precursors, 18:2n-6 and
18:3n-3, respectively. Thus, desaturation pathways would
make available in situ the n-6 and n-3 fatty acids to phospholipid acyltransferases to remodel the phospholipid acyl groups.
The fact that acyltransferase/n-6 desaturase activity ratio is
about 10:1 in tissues reinforces the idea that regulation of
desaturases can be the main limiting factor responsible for the
observed membrane unsaturation-longevity relationship. Animals with a high life span have a low degree of membrane fatty
acid desaturation based in the redistribution between types of
PUFAs without any alteration in the total (%) PUFA content,
average chain length, and phospholipid distribution. This may
be viewed as an elegant evolutionary strategy, because it
decreases the sensitivity to lipid peroxidation and lipoxidationderived damage to cellular macromolecules without strongly
altering fluidity/microviscosity, a fundamental property of cellular membranes for the proper function of receptors, ion
pumps, and transport of metabolites. This would occur because
membrane fluidity increases acutely with the introduction of
the first and less with the second double bond (due to their
introduction of “kinks” in the fatty acid molecule), whereas
additional (the third and following) double bonds cause a few
further variations in fluidity (22). This is so because the kink
has a larger impact on fluidity when the double bond is situated
near the centre of the fatty acid chain (first double bond) than
when it is situated progressively nearer to its extremes (next
double bond additions). In the case of the sensitivity to lipid
peroxidation, however, double bonds increase it irrespective of
their location at the center or laterally on the fatty acids (100).
Thus, by substituting fatty acids with four or six double bonds
by those having only two (or sometimes three) double bonds,
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ANIMAL ANTIOXIDANT DEFENSES
in particular, when taxa are phylogenetically unrelated. This
last point urgently calls for larger comparative studies than
those carried out so far, as soon as additional data on oxidative
stress physiology in natural populations is accumulated. Finally, we highlight the importance of defining the biological
relevance of measures of oxidative damage, antioxidant status,
and gene expression, which is often overlooked in favor of
more mechanistic explanations. Consequently, more studies
are needed to understand to which extent variation in redox
state traits is functionally significant and, if heritable, to which
extent this variation may be a target of natural selection.
ACKNOWLEDGMENTS
The authors are grateful to the anonymous reviewers for criticisms and
suggestions, which improved the manuscript.
GRANTS
During manuscript preparation, Reinald Pamplona was supported, in part,
by I⫹D grants from the Spanish Ministry of Science and Innovation
(BFU2009-11879/BFI), and the Generalitat of Catalonia (2009SGR735); David Costantini was supported by a postdoctoral NERC research fellowship
(NE/G013888/1).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
Perspectives and Significance
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are probably due to the activation of negative feedback mechanisms to maintain the oxidative stress homeostasis, which is
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