2290 Current Pharmaceutical Design, 2011, 17, 2290-2307
1381-6128/11 $58.00+.00 © 2011 Bentham Science Publishers
Walking the Oxidative Stress Tightrope: A Perspective from the Naked Mole-Rat, the
Karl A. Rodriguez1,2, Ewa Wywial3, Viviana I. Perez1,4, Adrian J. Lambert5, Yael H. Edrey1,2, Kaitlyn N.
Lewis1,4, Kelly Grimes1,4, Merry L. Lindsey1,6, Martin D. Brand7 and Rochelle Buffenstein1,2,4,*
1Sam and Ann Barshop Institute for Aging and Longevity Studies, University of Texas Health Science Center at San Antonio, 15355
Lambda Dr. San Antonio, TX 78245, 2Department of Physiology, University of Texas Health Science Center at San Antonio 15355
Lambda Dr. San Antonio, TX 78245, 3The City College of The City University of New York, Convent Avenue, NY 10031, 4Department
of Cell and Structural Biology, University of Texas Health Science Center at San Antonio 15355 Lambda Dr. San Antonio, TX 78245,
5Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 2XY, UK, 6 Department of Medicine, University
of Texas Health Science Center at San Antonio 15355 Lambda Dr. San Antonio, TX 78245, 7Buck Institute for Research on Aging,
8001 Redwood Blvd., Novato, CA 94945, USA
Abstract: Reactive oxygen species (ROS), by-products of aerobic metabolism, cause oxidative damage to cells and tissue and not sur-
prisingly many theories have arisen to link ROS-induced oxidative stress to aging and health. While studies clearly link ROS to a pleth-
ora of divergent diseases, their role in aging is still debatable. Genetic knock-down manipulations of antioxidants alter the levels of ac-
crued oxidative damage, however, the resultant effect of increased oxidative stress on lifespan are equivocal. Similarly the impact of ele-
vating antioxidant levels through transgenic manipulations yield inconsistent effects on longevity. Furthermore, comparative data from a
wide range of endotherms with disparate longevity remain inconclusive. Many long-living species such as birds, bats and mole-rats ex-
hibit high-levels of oxidative damage, evident already at young ages. Clearly, neither the amount of ROS per se nor the sensitivity in neu-
tralizing ROS are as important as whether or not the accrued oxidative stress leads to oxidative-damage-linked age-associated diseases. In
this review we examine the literature on ROS, its relation to disease and the lessons gleaned from a comparative approach based upon
species with widely divergent responses. We specifically focus on the longest lived rodent, the naked mole-rat, which maintains good
health and provides novel insights into the paradox of maintaining both an extended healthspan and lifespan despite high oxidative stress
from a young age.
Keywords: Comparative biology of aging, mitochondria, naked mole-rat, oxidative stress, proteasome, autophagy, reactive oxygen species.
The oxidative stress theory of aging asserts that the decline in
functionality that characterizes the aging process is due to progres-
sively accumulating levels of oxidative damage. This intuitively
logical theory has been around for more than a century and despite
an equivocal array of data, this invincible theory has held steadfast
as the key proximate mechanism that determines species maximum
lifespan potential (MLSP) . More than a century ago, Max Rub-
ner first noted the inverse correlation between the mass specific rate
of oxygen consumption and longevity in mammals. He further cou-
pled MLSP with body size and metabolism and determined that the
lifetime energy expenditure (LEE) was relatively constant [2, 3].
Twenty years later Raymond Pearl expanded upon this idea and
proposed that differences in metabolic rate explained the lifespan
extension of Drosophila melanogaster maintained at different tem-
peratures and through this observation defined the “rate of living
theory of aging” .
The toxic nature of oxygen was already a well-known phe-
nomenon since the seminal work of Lavoisier in 1781 . How-
ever, free radicals were first regarded as the cause of oxygen toxic-
ity in 1954  and soon afterward in 1956 Denham Harman pro-
posed that physiological metals would cause reactive oxygen spe-
cies (ROS) to form in cells potentially damaging nearby molecules,
including DNA. These would cause mutations, and based on the
belief at the time, such induction of mutations could cause both
cancer and aging. Harman also proposed that administering com-
pounds that could oxidize easily and absorb the ROS in the cell
could slow down this mutation-induced aging . Since that time,
*Address correspondence to this author at the Barshop Institute, STCBM
2.2; 15355 Lambda Drive, San Antonio Texas, 78245; Tel: 210 562 5062;
Fax: 210 562 5028; E-mail: Buffenstein@uthscsa.edu
the free radical theory of aging has been repeatedly modified and
renamed to the “oxidative stress theory of aging [8-11]. As such,
more than 50 years later, it remains a key focal area for aging re-
search. Research has focused on two broad categories to validate
and expand upon the notion that oxidative stress is an integral com-
ponent of aging: testing the levels of oxidatively damaged bio-
molecules in aging tissue and manipulating--either biochemically,
genetically or behaviorally—various stressors to determine their
effects on lifespan (Fig. 1; reviewed in  and ). The boldest
version of the oxidative stress theory of aging makes the all-
encompassing prediction that lifespan is determined by oxidative
damage and, thus, that an increase in oxidative damage will con-
tribute to a shorter lifespan. While a shortened lifespan may not be
the product of accelerated aging, determining the lifespan of animal
or plant species with compromised antioxidant pathways can be
used further to explore this theory: it follows that if an organism has
increased oxidative damage but exhibits no change in lifespan, the
result falsifies the hypothesis.
Here, we offer new insights into oxidative stress, longevity, and
the role of oxidative stress in species longevity. We base our in-
sights on research primarily using mammals and birds, and in par-
ticular, highlight research on the longest-lived rodent known, the
naked mole-rat. This strictly subterranean, eusocial rodent, found in
the northeast horn of Africa lives more than 30 years in captivity
while maintaining cancer-free, good health well into its third dec-
ade of life . The lack of spontaneous neoplasia is most unusual
among captive wild-caught rodents (such as Mus musculus and
Peromyscus species) as well as domesticated laboratory strains of
mice and rats. Approximately 70% of domesticated laboratory ro-
dent deaths are attributed to various types of cancers . Under-
standing the mechanisms that facilitate cancer resistance in captive
naked mole-rats may reveal important insights into cancer preven-
tion. We hypothesized that this extraordinary longevity relative to
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2291
its shorter-lived rodent cousins may be explained by the production
of less ROS and/or extremely efficient mechanisms to protect this
species against oxidative damage.
2. OXIDATIVE STRESS, DISEASE AND LONGEVITY
ROS are formed during oxidative phosphorylation in the mito-
chondria. During this process, electrons from NADH or FADH2 are
transferred through the electron transport chain (ETC) to oxygen
and the energy released in the process is used to power proton
transport across the mitochondrial inner membrane at complexes I,
III, and IV. The proton motive force generated is used to drive the
ATP-synthesis reactions. During this process, however, electron
leakage may occur predominantly in complex I and III, leading to
the generation of superoxide [16, 17] and it is estimated that up to
3%, of oxygen consumed during respiration ends up as oxygen
radicals . This is likely to be an overestimate as other studies
report less than 0.15% of the products of maximum oxygen con-
sumption become ROS . The rate of superoxide production is
primarily dependent upon the local oxygen concentration and the
presence of reducing equivalents, although the exact mechanisms
that lead to superoxide formation are still not fully understood. At
physiological concentrations, ROS may have important intracellular
signaling functions affecting cell function, growth and development
[19-21]. However, excessive mitochondrial ROS production is det-
rimental. Oxygen radicals may react with numerous other com-
pounds to form a variety of other reactive species all of which are
capable of indiscriminately damaging nearby macromolecules in-
cluding phospholipids, proteins and DNA. Oxidized lipids are
themselves potent ROS, autocatalyzing and self-propagating this
unregulated process. Genotoxic intermediates of lipid peroxidation
may also play a role in eliciting age associated DNA mutation .
ROS cause oxidative damage to proteins by showing that oxidative
damage to a histidine in glutamate synthetase resulted in enzyme
inactivation . The damage also modified the residue to a car-
bonyl group. Additionally, carbonyl groups reportedly increase with
age and are resistant to normal proteolytic degradation, making
them the preferred marker of protein damage as a result of ROS
. The harmful effects of ROS have led to the evolution of nu-
merous antioxidant and cytoprotective mechanisms that neutralize
and detoxify the free radicals before they can induce substantial
A complex suite of mechanisms has evolved to eliminate ROS
and thereby offset their potential for accruing oxidative damage.
The primary lines of defense include a) enzymatic antioxidants
including superoxide dismutase (SOD), catalase (CAT) and glu-
tathione peroxidase (Gpx) b) hydrophilic scavengers such as re-
duced glutathione (GSH) c) lipophilic scavengers such as toco-
pherols, flavonoids and carotenoids as well as d) the proteins in-
volved in reducing oxidized antioxidants and thereby recycling
them for further use (e.g., GSH reductase, peroxiredoxins). As a
second line of defense against free radicals, cells possess a number
of detection and repair mechanisms for the different types of DNA
damage (nucleotide excision repair (NER), base excision repair
(BER), mismatch repair (MMR), double-strand break repair
(DSBR)) and also rely on proteasome degradation and autophagy to
remove damaged proteins and organelles . Likewise lipid per-
oxidation can trigger autophagic response leading to a degradation
of oxidized products by lysosomes [26, 27]. Despite the plethora of
antioxidants mechanisms, some ROS always evade detection and
subsequent neutralization, inducing damage that is seldom com-
pletely repaired. As a consequence, according to the oxidative stress
theory, damage accrues and accumulates with age. Furthermore,
ROS have been implicated in the causation of several age-related
diseases from cancer to diabetes. Better processing of these free
Fig. (1). Schematic diagram outlining the components of the oxidative-stress theory of aging. The theory predicts that, as an inevitable byproduct of metabolic
activity, reactive oxygen species (ROS) are produced. If these are not neutralized, oxidative damage to proteins, DNA and lipids may occur. However, as some
damage may occur, repair mechanisms are in place to mitigate the damage. Without repair, accelerated aging occurs. With repair, we may have a slow-down in
the aging process.
2292 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
radicals resulting in less damage could ameliorate these diseases
(reviewed in ).
Research has persistently tried to understand how antioxidant
defenses work, either by overexpressing key genes or knocking
them out in various species. Manipulation of antioxidant genes
shows a myriad of phenotypes in different animal models from
yeast to mice. The invertebrate phenotypes have been described
extensively (reviewed in [12, 13]). Here we focus specifically on
the investigations conducted in vertebrate (mainly mouse) models.
Glutathione peroxidase 1 (Gpx1) is ubiquitous in almost all
mammalian tissues and is thought to be the main cellular scavenger
of hydrogen peroxide (H2O2) . The most significant pathologi-
cal finding to date for Gpx1 knockouts is that the null mice develop
cataracts, potentially resulting from increased ROS damage in the
eye, at a considerably younger age than wild-type mice [30, 31].
Also, Gpx1?/? mice are very sensitive to paraquat and diquat, both
potentiators of ROS which cause fatal oxidative damage to tissues
in these animals . Unfortunately, glutathione peroxidase 4 (Gpx
4) knockout mice are embryonically lethal indicating a vital devel-
opmental function , but recent studies using a spermatocyte-
specific knockout show that the lack of Gpx4 leads to infertility
possibly due to an increase of ROS in the germ cells . Survival
curves of Gpx1 -/- and Gpx4 +/- mice have not shown a difference in
lifespan when compared to wild-type mice [13, 35].
Cu/Zn superoxide dismutase (SOD1) is a highly conserved
enzyme responsible for the scavenging of the superoxide radical
Formerly called erythrocuprein, the enzymatic function was first
described by McCord and Fridovich (1969). They reported that
CuZnSOD1 catalyzes the reaction where the first superoxide mole-
cule is oxidized and the second molecule is reduced, turning two
molecules of superoxide into O2 and H2O2 . In 1993, Rosen et
al., determined that mutations in the SOD1 gene correlated with
familial amyotrophic lateral sclerosis (ALS) . Reportedly,
SOD1 is ubiquitously expressed in all tissues with highest concen-
trations found in neural tissue . Mutations in this gene are the
most common known cause of ALS, comprising close to 10% of all
cases . Copper is rapidly incorporated into both newly trans-
lated and pre-formed SOD1. Mouse fibroblasts labeled with radio-
active copper (64Cu) show that the concentration of apo-SOD1 has
an inverse relationship with copper levels . It has been shown
that SOD1 activity is induced under conditions of oxidative stress
Female mice without CuZnSOD are prone to infertility due to a
dysfunction in ovarian development, in addition to signs of early,
age-related hearing-loss and early onset of cataracts [43-47]. Fur-
thermore, mice lacking SOD1 have an increased risk of developing
hepatocellular carcinoma  and this predisposition to cancer may
be the main reason for the 30% reduction in maximum lifespan of
these animals . Congenic SOD1 knockout animals have high
levels of oxidative damage to all macromolecules . Sod1-/- mice
also exhibit a pronounced loss of muscle mass as they age, such that
by 20 months of age they have ~50% of the muscle mass of that of
their wild-type counterparts and accrue a significant amount of
oxidative damage in skeletal muscle compared to wild-type mice
. More recently, Sod1-/- mice were found to exhibit an increase
in blood glucose and blocked glucose-stimulated insulin production
. These collective differences in Sod1-/- mice phenotype relative
to wild type suggest that the lack of an accelerated enzymatic
method to remove superoxide anions results in biological toxicity
with a long-term pathogenesis of oxidative stress and damage to
Mn superoxide dismutase (SOD2) is the main scavenger of
superoxide in the mitochondrial matrix. Mutations in this gene may
cause idiopathic cardiomyopathy and diabetes and increase the risk
?) in the cytosol and in the mitochondrial inter-membrane space.
of breast cancer [51-53]. Depending on the genetic background,
removal of the SOD2 gene in mice results in neonatal lethality pre-
dominantly via neurodegeneration within the first 24 days after
birth [54-56]. These mice also have a significant increase in DNA
oxidative damage and are very sensitive to hypoxia [57, 58]. Het-
erozygous transgenic mice show that a non-lethal decrease in Sod2
nevertheless increases oxidative damage and the sensitivity to oxi-
dative stressors. However, while the animals have a higher inci-
dence of cancer, their lifespans were indistinguishable from those
of wild-type mice [59-61].
The third, though least abundant SOD enzyme is extracellular
SOD (ECSOD) which is encoded by the Sod3 gene. ECSOD is
located in the extracellular fluids including plasma, lymph and
synovial fluid. It also has a substantial presence in the lung and a
strong affinity to heparin [62, 63]. Polymorphisms in the SOD3
gene in human populations lead to increased susceptibility to type II
diabetes . Sod3 knockout mice are viable, can reproduce nor-
mally and do not show a decrease in lifespan. However, they do
show sensitivity to hyperoxia .
Genetic manipulations of methionine sulfoxide reductase
(MsrA; a reducing enzyme found in the cytoplasm and mitochon-
dria that selectively reduces the sulfoxide of methionine) reveal a
possible impact on longevity . One group found that deletion of
MsrA decreased longevity by 40% and increased sensitivity to oxi-
dative stressors such as paraquat . These knockout animals have
also shown compromised cardiomyocyte activity following oxida-
tive stress . A more recent study also observed sensitivity to
oxidative stress, however, no decrease in either median or maxi-
mum lifespan was observed .
Knockout studies of five of the six known peroxiredoxins--
antioxidants that scavenge H2O2, organic peroxides and peroxyni-
trite--in mice all show some signs of oxidative damage related pa-
thology and increased cancer risk [70, 71]. For instance peroxire-
doxin 1 (Prx1) knockout mice have increased oxidative damage to
both DNA and proteins [72, 73] and suffer from hemolytic anemia,
an increase in cancer incidence and a 15% reduction in lifespan
, but aside from Prx1, no data on lifespan are yet available re-
garding other peroxiredoxin knockout animals.
Although many of these animal models are more susceptible to
oxidative stress and may even show similar dysfunction to that
observed in human disease (See Table 1) clearly, there is no clear
link between genetically reduced antioxidant expression and a de-
crease in lifespan.
Overexpression of Antioxidant Enzymes
An alternative approach to studying the impact of antioxidants
on longevity is to genetically overexpress antioxidant genes with
the intention of extending lifespan. In mice, only a handful of stud-
ies examining lifespan in response to overexpression of antioxidant
enzymes have been published. A key question in these studies is
whether or not an increase of activity in one antioxidant enzyme
will in fact reduce endogenous oxidative stress, cause down-
regulations of other antioxidants or even upset the balance of oxi-
dant removal leading ultimately to a counterintuitive increase in
oxidative damage. A large cohort study showed that overexpression
of CuZnSOD did not increase lifespan . High overexpression of
both CuZnSOD and MnSOD leads to harmful phenotypes such as
muscular dystrophy, neuronal degeneration and infertility, with at
least the muscular dystrophy correlated ironically with higher levels
of oxidative damage such as lipid peroxidation, [75-77]. Further-
more, although overexpression of CAT showed protection in a cell
culture model, at the whole organism level it actually led to an in-
crease in gamma irradiation sensitivity [78, 79]. Mice overexpress-
ing CAT (2- to 4-fold) in the peroxisome did not show a significant
extension of maximal lifespan, although median lifespan showed a
modest 10% increase . Transgenic mice that overexpress human
CAT in mitochondria (MCAT) showed a statistically significant
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2293
Table 1. Diseases and Disorders Associated with Antioxidant Defense Genes and the Effect of Genetic Manipulations of These
Antioxidant Enyzme Antioxidant Function Genetic Manipulation Phenotype(s) Disorder/Disease Association Ref(s).
Glutathione Peroxidase 1 Scavenges H202 Gpx1-/-
Gpx TG in PD model
after viral infection. Protec-
tion against insulin resis-
tance under high fat diet
Lifespan not reported
Reduced neural death,
Cardiovascular Disease, Diabe-
Glutathione Peroxidase 4 Scavenges LOOH Gpx4-/- Embryonic Lethal.
Infertile in spermatocyte-
Scavenger of O2
Multiple pathologies, 30%
Mn Superoxide Dismu-
Scavenger of O2
Protection from insulin
resistance under high fat
Cardiovascular Disease, Cancer,
Scavenger of O2
Protection against LPS-
induced inflammation in
Inflammation, Diabetes 
Catalase Scavenges H202 Cat-/-
Increase lifespan, protec-
tion against insulin resis-
Cardiovascular Disease, Diabe-
Peroxiredoxin 1 Scavenges peroxidea Prdx1-/- Hemolytic anemia Cancer 
Peroxiredoxin 3 Scavenges peroxidea Prx3 TG
protects the heart against
post-MI remodeling and
failure in mice
Cardiovascular disease 
Reduction of proteins
by cysteine thiol-
20% increased lifespan,
increased resistance to
Inflammation, Stroke 
Repairs oxidized me-
MrsA-/- 40% shortened lifespan Cardiovascular disease 
Metal and OH. scaven-
Sensitivity to ROS
15% increase in lifespan
Cardiovascular disease 
a. Peroxiredoxins scavenge H2O2, short chain organic peroxides, fatty acid alkyl hydroperoxides, and in the case of Prdx6, also lipid hydroperoxides
extension of both median and maximum lifespan of approximately
20% and equivocal changes in lifespan when expressed in other
organelles . No increases in lifespan was evident with overex-
pression of MnSOD . Age-related changes in oxidative damage
(8-Oxo-2’-deoxyguanosine and mitochondrial DNA deletions in
skeletal muscle), H2O2 production (in heart-specific mitochondria)
and H2O2 -induced aconitase inactivation (in heart tissue) were
attenuated in MCAT animals . In a more recent study, mice
cardiomyocyte-targeted overexpression of CAT or endothelial nitric
oxide synthase (eNOS) showed a reduction in vascular H2O2 and an
increased expression of EC-SOD potentially leading to improved
cardiac aging [82, 83]. However, whether this finding can lead to
life extension is not yet known. Thioredoxin (Trx) facilitates the
reduction of other proteins by cysteine thiol-disulfide exchange
. Overexpression of human thioredoxin (Hs Trx1) in mice re-
portedly leads to a 20% increase in lifespan, as well as increased
resistance to cerebral ischemia and to UV-induced oxidative stress
[85, 86]. In these animals HsTrx1 was three to six times more
prevalent in tissues than endogenous Trx1 . Metallothionein
(MT), a scavenger of metal ions OH- is also highly induced by oxi-
dative stress . Transgenic mice overexpressing metallothionein
(MT-TG) showed a 15% increase in mean lifespan relative to con-
trol mice . Furthermore, their cardiomyocytes have attenuated
age-related increases in superoxide generation, cytochrome c re-
2294 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
lease, p47phox (Neutrophil cytosol factor-1) expression and higher
aconitase activity  In contrast, knockout mouse models of MT
show enhanced toxicity from ROS especially in cardiomyocytes
[90, 91]. However, a lifespan study of MT null mice has not been
conducted. The Mastushima group reported that overexpression
of Prx3 protects the heart against post-mycardial infarction remod-
eling and failure in mice. Prx3 overexpression reduced left ven-
tricular dilatation and dysfunction and attenuated myocyte hyper-
trophy, interstitial fibrosis and apoptosis of the non-infarcted myo-
cardium. These beneficial effects of the Prx3 gene overexpression
were also associated with attenuation in oxidative stress and
mtDNA decline and dysfunction .
Clearly, overexpression of the antioxidants discussed above
enhance protective mechanisms that could contribute to extended
healthspan and lifespan. While lifespan studies have not been con-
ducted on every antioxidant gene that is overexpressed, despite the
many signs of enhanced protection, the available data show that the
impact on lifespan is modest.
Studies in Humans
In humans, natural variation, measurable by genetic polymor-
phisms is a useful modality to investigate the effects of antioxidant
enzyme alterations on lifespan. An abundant amount of literature
exists on whether or not certain polymorphisms may present risk
factors for disease where a subset of those risk factors includes
oxidative stress. Studies in aged human cells containing mutated
mtDNA or defective mitochondria show that a compromised meta-
bolic function creates a higher rate of ROS production [93-95]. In
contrast, a study on Japanese centenarians reported that 20% of this
population possessed a mitochondrial gene variant in a coding a
subunit of NADH dehydrogenase, Mt5178A  associated with
low ROS production. Not only did the people in this study survive
to be over 100 years of age, but they were 50% less likely to be
hospitalized for age-related disease [96, 97].
A polymorphism in the mitochondrial localization sequence of
MnSOD (the Val/Ala polymorphism at position -9 from the mature
protein or 16 from the full-length precursor; V16A) is a known risk
factor in many oxidative-stress-related pathologies such as diabetic
nephropathy, hypertension and certain, but not all cancers [98-101].
However while the Ala allele has been found by some studies to
increase the risk of breast cancer [102, 103] others cannot find a
correlation [104, 105].
The cumulative indices of oxidative markers and oxidative
stress in healthy humans related to aging and gender have been
measured in multiple biological fluids and in many instances con-
founding results have been reported [106-115]. In several of these
investigations some of the parameters tested showed significant
correlations between age and an increase of pro-oxidative capacity
together with a decrease of antioxidants [109, 110, 112, 113]. This
was demonstrated by the observations of increases of plasma
malondialdehyde (MDA) and 4-hydroxynonenal (HNE), erythro-
cytic glutathione disulfide (GSSG), cysteinylcysteine (CysCys) and
by a slight decrease of erythrocytic GSH with age [108-113]. How-
ever, not all markers behaved predictably and the conclusions var-
ied among the studies. For example the plasma protein carbonyls
reached the lowest value in aged individuals in the Gil et al., (2006)
study. Indeed that study reported that metabolic and nutritional
influences play a greater role than does the balance between anti-
oxidants and oxidants. Age-related changes in antioxidants (SOD,
GPx and CAT, carotenoids and tocopherols) in humans do not show
a consistent pattern; generally erythrocytic activity of SOD and
GPx increased while plasma values were unchanged [108, 110-112,
114, 115]. Similarly oxidative damage indices are also equivocal.
Some studies report positive and linear correlations with increasing
age were significant with regards to MDA, HNE, protein carbonyls
and GSSG [108, 110, 113, 114]; levels of urinary isoprostanes re-
portedly are unchanged with age; and assessment of DNA damage
by comet assay and the FLARE (Fragment Length Analysis using
Repair Enzymes) method are contradictory with the former increas-
ing while the latter remains unchanged . Lastly, there does not
appear to be any sexual dimorphism with regards to the selected
measures of oxidative stress in age-matched men or women [112-
114]. One of the limitations of these studies is the cross-sectional
nature. The subjects cannot be followed to track whether more fa-
vorable markers of oxidative stress (low oxidative damage; high
antioxidant capacity) lead to increased longevity and no study has
examined ROS production. Direct measures of antioxidants, the
results of their manipulations or correlations with disease and oxi-
dant polymorphisms have not shown unequivocal support for the
theory. More than likely, the successful aging seen in these popula-
tions in not only linked to their antioxidant/oxidative damage pro-
file, but also how the body responds to the damage to maintain a
“survivable” set point for homeostasis and thereby avoiding the
common debilitating age-associated diseases that ultimately in-
crease the chances of dying.
Oxidative Damage and Alzheimer’s Disease
Oxidative damage has also been implicated in Alzheimer’s
disease (AD) (reviewed in ), the most common age-related
neurodegenerative disease currently affecting ~30 million people
(www.alz.org). Accumulation of neurotoxic amyloid beta (A?) is
considered one of the pathological hallmarks of AD. The greatest
risk factor for AD is age and to date there is no therapy for this
devastating disease. Lipid peroxidation has been extensively stud-
ied because of the abundance of fatty acyl groups which account for
~10% of neural tissue. Moreover, the brain is an energetically ex-
pensive organ, with high oxygen consumption and having a high
iron content along with ascorbates, which may further damage pro-
teins by binding to them under pro-oxidant conditions. Finally, the
limited antioxidant defenses yield the brain prone to oxidative stress
and damage (reviewed in ).
Clinical assessments and postmortem studies have shown an
inverse relationship between docosahexaenoic acid (DHA) and AD
(reviewed in ) linking oxidative damaged lipids and AD. Fur-
thermore, a clear association has been made between overproduc-
tion of free radicals arising from dysfunctional mitochondria and
from A? with oxidative damage in brain regions with AD pathol-
ogy (hippocampus and cortex). However, the cerebellum which
remains clear of AD pathology, shows low oxidative stress and no
neuronal loss . An increase in antioxidant levels has also been
reported in AD brains and dietary manipulations increasing antioxi-
dant activity have been investigated as a therapeutic targets for AD
(reviewed in ). A promising clinical trial recently showed that
attenuating metal proteins was effective in improving cognition in
patients with AD  further highlighting the role of oxidative
damage in the pathologies observed in this disease.
3. THE COMPARATIVE BIOLOGY OF OXIDATIVE
STRESS; A POTENTIAL DETERMINANT OF SPECIES
As reiterated above, the oxidative stress theory of ageing asserts
that as an inevitable by-product of aerobic metabolism, the electron
transport chain in the mitochondria produce ROS and that those
species capable of living long lives will accrue less cumulative
damage as a result of more complete neutralization by antioxidants.
Furthermore, one would predict that long-lived species may also
have structural macromolecule characteristics and/or better repair
processes that make them more resistant to the cumulative effects
of oxidative damage.
Although there is considerable support for this proximate
mechanism of aging in invertebrates, [120-122], there are several
contradictory studies even when using the same species (e.g. Dro-
sophila, [123, 124]. Furthermore, the high phenotypic diversity and
reproductive modality among invertebrates, coupled with the meta-
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2295
bolic dependence upon temperature and terrestrial or aquatic life-
style differentially influence the interactions between ROS produc-
tion, antioxidant defense and maximum species lifespan potential.
Endothermic vertebrate classes (birds and mammals) exhibit mark-
edly higher oxygen flux than aquatic and terrestrial invertebrates
and the ectothermic vertebrates (fish, amphibians and reptiles).
Given their high metabolic flux, endotherms ought to be more sus-
ceptible to the harmful effects of potentially higher levels of un-
checked ROS. However, birds exhibit a 1.5-fold higher mass spe-
cific basal metabolic rate (BMR) than do similar-sized mammals
and in contrast to the predicted detrimental effects on longevity
caused by the inevitable ROS-induced damage due to oxygen con-
sumption, some live approximately twice as long as do similar-
sized mammals .
Most biologists are aware of the importance of scaling biologi-
cal traits with body size  and the famous “mouse-to-elephant
curve” produced by Benedict  which profoundly demonstrated
mammalian metabolic intensity to be inversely related to body
mass. A very similar allometric relationship exists for birds, from
humming birds to ostriches . The allometric relationships for
metabolic rate between birds and mammals are indistinguishable,
despite the fact that endothermy arose independently in these two
evolutionarily distinct vertebrate classes. In both birds and mam-
mals, doubling of body mass, leads to a 15-20% decline in mass
specific metabolic rate [129, 130]. Lindstedt and Calder 
showed that longevity in birds and mammals increased similarly
with increasing body size, although birds generally live twice as
long as similar-sized mammals . BMR is affected by several
variables including body mass and phylogeny. When these effects
of size and phylogeny on BMR are statistically corrected, the rela-
tionship between BMR and MLSP no longer holds in either birds or
mammals . The combination of a relatively higher metabolic
rate and a longer lifespan also means that birds, on average, will
have three-fold greater lifetime energy expenditure (LEE) than
mammals. This raises critical questions about how aerobic metabo-
lism and susceptibility to oxidative damage might differ between
birds and mammals and if exceptionally long-lived mammals ex-
hibit a profile more in keeping with that of long-lived birds. Both
these outcomes refute the explanatory power of the rate of living
theory for MLSP. Furthermore, when a more comprehensive as-
sessment of >240 mammals is used to determine LEE, calculated
values are not constant, but rather MLSP is negatively correlated
with LEE. Furthermore BMR, the integral component upon which
LEE is determined, is specifically measured at rest in post absorp-
tive, non-growing, non-breeding, healthy adults housed in their
thermoneutral zone and is not an accurate indicator of total daily
energy expenditure; it does not take into account the energy costs
associated with daily activity, foraging, digestion, growth or repro-
duction. If average daily metabolic rates (ADMR) or field meta-
bolic rates (FMR) are used instead of BMR, LEE among mammals
declines with increasing body mass [129, 130]. There are also mul-
tiple exceptions to the presumed constant relationship between
metabolic rate and lifespan posited by the rate of living theory. For
example voluntary exercise and its associated increase in metabolic
rate does not shorten lifespan of rats  or humans  and is
generally thought to extend healthy lifespan. Dietary caloric restric-
tion (CR) is well known to extend lifespan in a wide range of spe-
cies , yet this process is not accompanied by attenuations in
mass specific metabolic rate resulting in elevated LEE . Also,
there is no inverse relationship between lifespan and mass-specific
metabolic rates of individual mice, dogs or flies [137-139]. Intra-
specific data also provide compelling exceptions to this theory
showing that within a species those individuals with the highest
metabolic rates live longest and this is attributed to a decline in
mitochondrial efficiency and metabolic uncoupling processes .
Similarly significant interspecific differences in MLSP within both
groups of endotherms cannot be explained by metabolic rate differ-
ences. Rather, interspecifies differences in metabolic rate generally
reflect climatic zones and geographical regions such as desert or
mesic habitats [140-142], the season during the year when the
measurements were taken  and/or whether animals live above
or below ground [144, 145] Generally all subterranean rodents have
low basal metabolic rates relative to that expected allometrically
[144, 146]. However the low metabolic rates of African mole-rats
species (Bathyergidae) with known MLSP data do not correlate
with species longevity or other life history traits (Table 2) such as
time to sexual maturity, gestation length or litter size, ). Fur-
thermore, while BMR of naked mole-rats is 75% that of age-
matched mice their greater longevity would result in the highest
mass specific LEE of any known mammal .
Membrane composition, and in particular mitochondrial mem-
brane lipid composition, also varies in a systematic manner with
body mass in both endothermic vertebrate classes (see  for
review). Phospholipids in membranes of larger species contain
proportionately less easily oxidized polyunsaturated fatty acids
(PUFA), but rather have more monounsaturated fatty acids
(MUFA) that are more resistant to oxidation. The products of lipid
peroxidation are powerful ROS, and initiate a self-propagating
autocatalytic process of oxidative damage to adjacent macromole-
cules. Interclass variability in membrane composition could explain
the seven-fold difference in metabolism between reptiles and
mammals as well as differences in metabolic rate attributed to intra-
class variability in body size . Differences in membrane com-
position in both mammals and birds also are correlated with their
maximum lifespans such that shorter-lived smaller mammals have
membranes rich in n-3 PUFAs (such as DHA) that are more suscep-
tible to lipid peroxidation [149, 150]. These findings have given
rise to the “membrane pacemaker theory of aging” (see  for
review). Comparative studies assessing membrane composition of
species with disparate longevity provide support for this theory. For
instance long-lived naked mole-rats , echidnas  and hu-
mans  all have membranes with a lower peroxidation index
than predicted by body size. Similarly, longer-lived strains within a
species have membranes with less DHA  and while CR does
not influence BMR, membrane fatty acid composition varies with
the degree of restriction . Membrane leakiness and concomi-
tant mitochondrial membrane potential influences the proton gradi-
ent and thus the rate of ROS production. Membrane leakiness, like
BMR is inversely related to body mass in both mammals and birds
[155, 156]. The extent of membrane leakiness however is report-
edly not directly due directly to membrane fatty acid composition
Both superoxide and H2O2 production per mg of mitochondrial
protein scale inversely with body mass in a number of animals,
from mice to horses [158, 159] further revealing that the potential
for oxidative stress is inherently greater in smaller than in larger
endotherms. Unless countered by other processes, oxidative stress
could account for the relationship between body size and longevity.
The rate of ROS production per unit of oxygen consumed was
lower in pigeons than in rats, thus reconciling their ability to have a
higher metabolic rate than rats and yet live far longer . A simi-
lar inverse correlation between longevity and mitochondrial H2O2
production was found in a comparative study among three similar-
sized rodents with widely divergent longevities  and in a
larger comparative study using mitochondria from heart tissue of
ten mammalian and two bird species that differed substantially in
both body size and MLSP . Significantly, even after statisti-
cally correcting for the influence of body size on longevity and
ROS production, this relationship was upheld. Furthermore recent
studies in reptiles also exhibit a similar relationship between MLSP
and rate of mitochondrial H2O2 production . Despite this gen-
eralized strong support, several inter-specific data sets do not back
2296 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
the oxidative stress theory of aging and these discrepancies lead us
to be wary of fully supporting this august theory. For example, in a
comparative study among long-lived little-brown bats (Myotis luci-
fugis, MLSP 34y); short-tailed shrews (Blarina brevicauda, MLSP
2 years) and white-footed mice (Peromyscus leucopus, MLSP 8
years), while ROS production in heart tissue was lowest in the long-
lived species, ROS production in brain tissue was considerably
higher than that of shorter-lived, white-footed mice and the species
differences in ROS production did not correlate with the 17-fold
and 4-fold difference in MLSP. Furthermore, in the Lambert study
described above, in which rates of H2O2 production were measured
under identical conditions, statistically indistinguishable levels of
ROS production were evident among the two short- and long-lived
rodent pairs, namely white-footed mice and wild-derived mice de-
spite a two-fold difference in longevity; and among the naked mole-
rats and the C57Bl/6 mice despite an eight-fold difference in
MLSP. Similarly, Brazilian free-tailed bats (Tadarida brasiliensis,
MLSP 12y) are approximately three times shorter-lived than the
little brown bat (Myotis lucifugus, MLSP 34 y), yet have only a 10%
lower ROS production rate. While ROS production in heart tissue
was statistically similar among the long-lived naked mole-rats and
C57Bl/6 mice ROS production in naked mole-rat skeletal muscle
and kidneys showed significantly lower levels (Fig. 2) in the
longer-lived species (unpublished data Lambert, Brand and Buffen-
stein). This tissue difference among the heart and other tissues may
reflect the fact that the naked mole-rat heart is a smaller and slower
pump than that of the mouse [139mg±0.6 for the naked mole-rat
heart mass, or 0.36% of average body weight (n=15) and
143mg±0.3 or 0.56% of the average body weight ( n = 10) for that
of the mouse]; also, the naked mole-rat anesthetized heart operates
between 150-220 beats per minute whereas that of mice under simi-
lar conditions beats at 450-500 beats per minute (Grimes, Lindsey,
Lewis, and Buffenstein, unpublished data). Given the large differ-
ence in heart function, it is likely that the heart may not be the most
appropriate organ for comparing mitochondrial function among
species. This could be interpreted to mean that very small differ-
ences in rate of ROS formation have substantial effects on MLSP,
but it also suggests that mitochondrial ROS production rates are
tissue specific and are only one of many factors that may influence
species longevity and that these are not the main determinants of
It is worth mentioning that the detected inverse relationship
between mitochondrial ROS and MLSP we observed is only evi-
dent when one substrate (succinate) is supplied in abundance .
This study suggests the reverse electron transfer through complex I
may be an indicator of longevity. The substrate succinate is known
to induce significant amounts of reverse electron transfer from the
Q pool to complex I, which, in turn, is associated with high rates of
ROS production when the membrane potential is also high .
There is considerable debate regarding whether or not this stalled
electron transport occurs naturally and it is also not known why the
correlation is not evident when multiple other substrates representa-
tive of forward electron transport through the various components
of the ETC were employed. However, fatty acids and ? ?-glycero-
phosphate also enter the electron transport chain through the Q pool
and may reduce it to levels provoking reverse electron flow .
Complex I is considered the dominant site of in vivo ROS formation
 and also the site responsible for the reductions in rates of
mitochondrial ROS production following CR in mammals . In
this regard, it is significant that birds examined thus far (pigeons,
budgerigars, and canaries) have proportionately less complex I in
their mitochondria than mammals (rats, mice) [16, 168, 169]. Fur-
thermore, the maximal rates of complex I ROS production per mg
of heart mitochondrial protein in pigeons was about half that of rat
mitochondria, reflecting exactly the proportionate difference in
complex I content of their mitochondria . If this were a general
characteristic of avian mitochondria, birds ought to be predisposed
to a reduced mitochondrial ROS production when compared to
In addition, it is important to determine whether this phenome-
non would be observed in mitochondria from other vital tissues
such as brain, liver, kidney or skeletal muscle. Mitochondria from
different tissues of the same species have different phenotypes in
physiological and pathological situations [170, 171], (Lambert,
Brand and Buffenstein unpublished data). In addition, interspecies
differences in mitochondrial organization may influence the effi-
ciency of mitochondrial substrate utilization as well as proton leak.
Furthermore, whether mitochondrial ROS would cause similar ex-
Table 2. Bathyergid Key Life History and Metabolic Traits.
Mole Rat 75 11
34 450 (f) 81 3.5
152 15.5 147 0.57 60 35 511(f) 93 2.5
272 20 174 0.60 75 36 unknown 112 2.6 
197 11.2 100 0.60 68 36
44 5 
35 31 381 1.00 66 33 228 (f) 77 16
Information not specifically cited taken from the AnAge Database (http: //genomics.senescence.info/species/)
# % predicted MLSP based upon the allometric equation of JP de Magalhaes 
*% BMR expected based upon the allometric equations of BK McNab 
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2297
tent of damage in intact cells containing endogenous antioxidant
systems is still an open question and probably more meaningful.
Interestingly, targeted disruption of the mitochondrial gene SURF1,
causes an increase in longevity in laboratory mice . However,
the cause of the phenotype may neither be dependent on membrane
potential nor be related to ROS control, but instead depend on the
handling of Ca2+ uptake .
A complex suite of mechanisms have evolved to eliminate ROS
and thereby offset their potential for accruing oxidative damage.
Antioxidant activity as a determinant of MLS remains controversial
with reports of a positive, negative and no correlation between anti-
oxidant activity and MLSP [173-178]. CAT and GPx activities
reportedly negatively correlated with MLSP  whereas Sohal et
al.,  found that SOD and CAT activities were positively corre-
lated and indeed in the case of GPx, levels correlated positively
with MLSP in brain samples but negatively in the liver and heart
. When birds, fish and amphibians were included for compari-
son for antioxidants activities and MLSP, strong negative correla-
tions for some but not all antioxidants were obtained and results
were tissue specific [160, 180, 181]. An often-touted explanation
for this negative correlation is that antioxidant defenses are not the
reason for extended longevity of long-living animals, but rather the
lower antioxidant levels in long-living species may be indicative of
lower levels of oxidative stress [173, 182-184] Similarly, we too
have found that total antioxidant power varies among tissues and is
responsive to dietary manipulation, as seen by the increase in total
antioxidants following supplementation with peroxidation prone
omega n-3 fatty acids (Fig. 3; Wywial and Buffenstein, unpublished
One difficulty in studying the role of antioxidants in longevity
is that there is no consistent stoichiometric relation between pro-
oxidation or antioxidant measures, making inferences between spe-
cies from limited measures of only a few antioxidants essentially
meaningless. Many members of the antioxidant defense system
share redundant roles and are rapidly induced in response to ele-
vated oxidative stress rather than maintained at high basal levels. In
order to estimate the overall ability to defend against ROS, it is
necessary to evaluate the activities of all antioxidants present in
tissues and their interactions and not simply focus on a few key
No consistent differences in known processes that remove radi-
cals and repair the damage have been found to correlate with MLSP
[162, 175, 185, 186]. Many long-lived species, such as the humans,
and naked mole-rat, show unremarkable antioxidant defenses [186,
187]. Clearly levels of antioxidant defense cannot account for
lifespan difference across the animal kingdom and as such the anti-
oxidant arsenal are not integral determinants of species longevity.
These findings concur with studies showing that the effects of anti-
oxidants on human health are equivocal  as are the efficacy of
pharmacological mimetics of antioxidants . Genetic overex-
pression of various antioxidants and their effects on lifespan in
various animal models similarly yield conflicting results and knock-
ing down antioxidant protection, while leading to more oxidative
damage, does not impact upon longevity . This observation
correlates with the negative data from various dietary treatments
with exogenous antioxidants that sometimes lead to extended me-
dian ages, but almost never extends MLSP [176, 183, 184]. If the
oxidative stress theory holds true it would suggest that variation in
rates of ROS importantly influences MLSP and that consequent
oxidative damage is imperfectly countered by antioxidants.
The oxidative stress theory of aging asserts that the decline in
functionality that characterizes the aging process is due to progres-
sively increasing oxidative damage. Most comparative studies
compare data from young healthy adults providing a snap shot of
oxidative damage at a given age of each species. The rationale be-
hind this approach is that traits influencing rates of aging ought to
be present throughout life and if these traits facilitate long life, even
at an early age their impact should be evident. Accrued oxidative
damage may not only influence aging but also may impact consid-
erably on many life history characteristics dependent upon the ap-
portioning of energy metabolism into fecundity, development, so-
matic maintenance and maximum species lifespan.
Several comparative studies show an inverse relationship be-
tween oxidative damage and longevity, already evident in young
healthy adults [173, 190, 191]. However, many exceptions to this
premise have been reported. For example within flies, the longer-
lived Drosophila species has more protein carbonyls than the
shorter-lived blow flies (Calliphora vicina ) . Similarly
longer-lived birds have higher levels of oxidative damage to DNA
than do shorter-lived mammals , and within mammals both
long-lived vampire bats and naked mole-rats have higher levels of
protein carbonyls than short-lived rats and mice measured under
identical conditions [1, 175, 182, 194]. The level of oxidative dam-
age, such as MDA differs substantially among tissues. Despite
lower levels of ROS production in muscle than in heart tissues (Fig.
2), muscle tissue exhibits twice as much lipid peroxidation as ob-
served in heart tissue (Fig. 4) and exhibits a similar level of antioxi-
dant protection (Fig. 3). The degree of oxidative damage is de-
pended upon dietary fatty acid content, as is evidenced by the sig-
nificantly greater MDA levels following chronic dietary supple-
mentation with n-3 rich flaxseed oil. High levels of oxidative dam-
age in mole-rats and bats has been attributed to their high intracellu-
lar iron content wreaking havoc with redox [182, 195]. Such inter-
specific and ontogenetic differences in iron homeostasis are likely
to have a huge impact on rates of aging and sudden increases in
ROS-related damage leading to death.
Oxidative damage measurements made in tissue samples are the
net result of several interacting processes associated with primary
rates of ROS generation in mitochondria including autocatalytic
secondary ROS production as a result of phospholipid peroxidation,
neutralization of ROS by antioxidants as well as damage repair and
the removal of damaged products by nucleases, proteases and auto-
phagy that precede the sampling point. These net levels of oxidative
damage as such may represent a “steady-state” level of damage
tolerance rather than indicative of rates of oxidative stress. Steady-
state levels of oxidative damage, based upon these premises reveal
a lack of consistent patterns, with higher and lower levels of net
damage reported in longer-lived birds, bats, and rodents [178, 186,
196, 197]. While it is possible that all the exceptions to the oxida-
tive stress theory reflect unusual species that have been subjected to
a different collage of evolutionary pressures and thus present with
their own suite of “private mechanisms” to deal with aging, given
the broad range of exceptions both within species, among species
and among vertebrate classes, this seems unlikely . Rather the
lack of a consistent pattern in these measures of oxidative damage
and lifespan challenge the validity of this theory.
This mode of comparative approach, however, is not without
limitations and ignores the rates of age-related changes in oxidative
stress and thereby fails to address the dynamics of aging and how
this differs among species of disparate longevity, even though there
is convincing evidence from numerous studies that aging is associ-
ated with increased oxidative stress and oxidative macromolecular
damage in various tissues studied [193, 198]. Indeed age-dependent
accumulation of oxidative damage is considered an intrinsic factor
determining the rate of aging, with both damage to protein carbon-
yls, lipids and DNA increasing more markedly in the latter third of
life [199-201]. For example oxidative damage to DNA gradually
increases in a non-linear modality with age in rats, however levels
appear to be tissue-specific such that DNA damage in the brain is
greater than in liver and kidney . The few studies that utilized
2298 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
both approaches are obviously more powerful for testing the oxida-
tive stress theory of aging [186, 202-204]. These studies often yield
contrary results to those simply using young adults. For example
although naked mole-rats show high levels of oxidative damage,
evident already at a young age, both cysteine oxidation and lipid
peroxidation are maintained at those high steady state levels for at
least two thirds of the extraordinary lifespan of naked mole-rats,
providing some support for the oxidative stress theory of aging
. Clearly, this species has a higher threshold of damage toler-
ance and has mechanisms in place to facilitate structural resistance
to the harmful effects of oxidative damage and still maintain cellu-
lar integrity and function.
Although antioxidant status and accrual of oxidative damage
show mixed correlations with MLSP, stress and toxin resistance
Fig. (2). Hydrogen peroxide (H2O2) production by heart, skeletal muscle, and kidney mitochondria in mice and naked mole-rats. To assess ROS production by
complex I, succinate was added to the assay and H2O2 generation rate was determined by monitoring the oxidation of either p-hydroxyphenylacetic acid or
amplex red with the reaction being coupled to the enzymatic reduction of H2O2 by horseradish peroxidase as described . While there was no significant
difference in H2O2 production in heart tissue, naked mole-rats showed significantly less H2O2 in both skeletal muscle and kidney. (Lambert, Buffenstein and
Brand, unpublished data).
Fig. (3). Total antioxidant power in naked mole-rats varied substantially among tissues. Furthermore, dietary supplementation for 6 weeks with flaxseed oil, an
omega-3 fatty acid (7g PUFA per 100g ProNutro)  resulted in augmented total antioxidant activity (Wywial and Buffenstein, unpublished data). Total
antioxidant power was measured quantitatively using a commercial colorimetric microplate assay for total antioxidants (Oxford Biomedical Research, Oxford,
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2299
appears to play a significant role in the determination of lifespan in
naturally long-lived species, as well as genetically modified models
of longevity [206-209]. In worms and flies, manipulation of genes
resulting in longer lifespan often result in increased resistance to
exogenous stressors or toxins including heat shock, H2O2 and
paraquat [210-212]. This toxin resistance is observed in rodent
models of longevity as well. Dwarf mice, which can be drastically
long-lived compared to wild-type littermates, show increased resis-
tance in vitro and in vivo to an array of stressors [207, 213].
Most notably, fibroblasts cultured from naked mole-rats prove
to be incredibly resistant to a variety of toxins, including oxidative
stressors, when compared to fibroblasts from standard laboratory
mice [205, 209]. However, while naked mole-rat fibroblasts showed
resistance to toxicity from paraquat, consistent with the model of
oxidative stress and longevity, the cells were acutely sensitive to
H202 . Interestingly, the toxicity of H202 differs from that of
paraquat. Paraquat causes cellular toxicity through the production
of O2 ? in the mitochodria disrupting NAPDH-dependent biochem-
istry and eventually leading to tissue damage and compromised
organismal function . H202, on the other hand, causes oxidative
stress through the production of hydroxyl radicals that cause dam-
age to membranes, lipids and nucleic acids  . Aside from the
different mechanisms of radical formation, the standard culture
conditions of 20% oxygen, or the use of serum-free conditions (af-
fecting membranes and lipids as well) may have contributed to this
seemingly paradoxical result pushing the naked mole-rat fibro-
blasts, already containing high levels of oxidative damage com-
pared to age-typed laboratory mice beyond their ability to mitigate
the damage [205, 209, 216, 217]. In any case, because the naked
mole-rat appears to be resistant to a diverse range of toxins that
have different modes of action, a pathway that may serve as a key
regulator of cytoprotection may serve as the main mechanism for
the profound resistance to toxins observed not only in the naked
mole-rat, but other naturally and genetically-modified long-lived
species. One such pathway is the nuclear factor erythroid-2 related
factor-2 (Nrf2) signaling pathway, which regulates the transcription
of 230 genes . These genes include those involved in GSH
synthesis and conjugation , free radical scavengers  as
well as proteasomal subunits . Nrf2 is a transcription factor
expressed in all tissues of an organism and is conserved from
worms to humans . Under constitutive (non-stressful) condi-
tions, Nrf2 is basally expressed at low levels and has a relatively
short half-life (~15 minutes). Nrf2 is largely regulated by kelch-like
ECH-associated protein 1 (Keap1), which targets Nrf2 for ubiquiti-
nation and degradation via the proteasome [222, 223]. Under stress-
ful conditions (i.e. an increase in ROS or xenobiotic stressor), cys-
teines within Keap1 are modified, impeding the interaction with
Nrf2. Levels of free Nrf2 increase resulting in an increase in half-
life (~60 minutes) and increased nuclear levels of Nrf2. Nrf2 binds
to the antioxidant response element to promote the transcription of
cytoprotective molecules [224, 225].
In C. elegans, the Nrf2 homolog, Skn-1, was shown to be a
mediator for the life-extending effects of CR . Similar results
were observed in Drosophila, in addition to a lifespan extension
observed in flies that had decreased Keap1 expression . In
both of these invertebrate models, long-lived organisms show an
increase in the signaling of Nrf2/Skn-1-regulated molecules, possi-
bly contributing to their longevity. These trends are also observed
in long-lived rodent models including the naked mole-rat .
Caloric and methionine-restricted mice, as well as dwarf mice,
show signs of increased Nrf2-signalling activity further supported
by increased toxin resistance [227-229].
Repair processes play a vital role in maintaining cellular ho-
meostasis and not surprisingly there are numerous cellular path-
ways to ensure quality control and the rapid removal of damaged
molecules before these can impair functionality . A gradual
decline in some of these pathways occur during aging and the mal-
function of these quality control mechanism form the basis of many
Fig. (4). Lipid peroxidation, as indicated by levels of malondehyde (MDA) in various tissues of naked mole rats. Note that MDA varies significantly within
tissues (see Control profile), with skeletal muscle showing the highest levels. The degree of lipid peroxidation is influenced by dietary manipulations. After
only six weeks of dietary supplementation with the omega-3 rich fatty acids present in ground flaxseeds, all tissues showed a significant increase in MDA.
Wywial and Buffenstein, unpublished data). LPO was measured quantitatively using a commercial colorimetric assay (Oxford Biomedical Research, Oxford,
2300 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
age-associated diseases such as neurodegeneration, metabolic dis-
orders and cancers . Cells rely on various molecular chaper-
ones to maintain the three dimensional structure of their proteins.
Chaperones selectively binding to non-native protein conformations
to form stable complexes and prevent the irreversible aggregation
of non-native protein conformations . Not surprisingly many
of these chaperones are induced under stressful conditions to assist
in the maintenance of protein quality. Expression of chaperones
declines with age and manipulations that retard aging enhance
chaperone expression, including the heat-shock response [230, 232,
233]. Previously we have shown that the proteome of naked mole-
rats and bats is more resistant to both urea-induced and heat-
induced unfolding than that of shorter-lived mice [205, 234]. This
implies that long-lived species might have evolved enhanced chap-
erone-like activities to preserve protein structure and prevent mis-
folding/aggregation. If the damaged proteins cannot be repaired by
these chaperones, the proteins are directed towards proteolytic deg-
radation pathways either via the ubiquitin-mediated proteasomal
system or via lysosomal mediated authophagy. Both of these sys-
tems are subject to age-related declines in functionality [235, 236].
Amongst the many biological substrates of proteasome are oxi-
datively damaged proteins, the accumulation of which is one of the
hallmarks of aging [237, 238]. Such proteins are suspected to in-
hibit the proteasome machinery, either by overwhelming the protea-
some or by a noncompetitive inhibition . In parallel, free radi-
cals causing oxidative damage of substrates may modify the com-
ponents of the proteasome system, effectively changing (most likely
inhibiting) its actions . Aging is also accompanied by the in-
crease of protein carbonyl groups and aggregated cross-linked ma-
terial which are resistant to proteolytic degradation and may act as
inhibitors of the proteasome [24, 239-241]. Consequently, a model
with reduced oxidative damage as observed in caloric restricted
animals might reduce these potential inhibitors of the proteasome
system [242, 243].
Data based upon the proteasome in fission yeast (Schizosac-
charomyces pombe) reveal three basic principals of proteasome
function . 1) G0 cells require proteasome function during the
maintenance of G0 quiescence. Temperature sensitive mutants of
26S proteasome subunits perturb this maintenance. 2) proteasome
functions differ between G0 and vegetative proliferation phase
(VEG). This conclusion comes from the examination of defective
phenotypes of proteasome mutants in G0 phase, leading to a huge
increase of oxygen stress responsive compounds and the massive
decrease of mitochondria that did not occur in VEG. 3) proteasome
dysfunction in G0 elicits defensive responses both by triggering the
production of antioxidant components and inducing the degradation
of mitochondria by autophagy. These defensive responses were not
found in VEG cells, suggesting that proteasome functions in G0 are
directly or indirectly involved in minimizing ROS . Oxidative
stress biomarkers, of which 20S proteasome was included, showed
different responses depending on age in brown trout (Salmo trutta)
exposed to paraquat. Fish aged five months showed no changes in
carbonylation in liver tissue and 20S proteasome when exposed to
the drug while the 12-month- old fish had both an increase in pro-
tein carbonylation in liver tissue and a decrease in 20S proteasome
activity in brain tissue .
In mammalian studies, the influence of oxidative stress on the
proteasome is strongly evident. Following injury to the brain or
retina, the immunoproteasome activity is upregulated . Fur-
thermore, following immunoproteasome deficiency, the proteasome
in the mouse retina could not respond to stress and as such became
more susceptible to “oxidation- induced cell death” . In rat
brain and liver tissue preparations, exposure to oxidative damage-
inducing agents, H2O2 and HNE, inhibited peptidase activity from
the proteasome in a dose-dependent manner . The effect was
more pronounced in the brain. Interestingly, neither age of the ani-
mal or dietary restriction produced a significant effect on the oxida-
tive-stress induced decline of proteasome activity observed in that
study . When proteasome activity is inhibited in neuronal cell
crude lysates, an increase in both ubiquitinated and oxidized pro-
teins is observed, as well as a selective increase in newly synthe-
sized but insoluble proteins , again implicating the proteasome
in oxidative-stress related protein homeostasis. An exception to the
age-related decline of the proteasome is seen in the case of the na-
ked mole-rat . Perez et al., postulated that the robust main-
tenace of the proteasome in older mole rats (20yr) when compared
to old mice (2yr), may contribute to a better preservation of the
proteome and thus contribute to the increased longevity seen in the
naked mole-rat . Also, old naked mole-rats have significantly
lower levels of ubiquitinated proteins than do old and young mice
suggesting less accrual of oxidized or misfolded proteins. Chymo-
trypsin-like activity from the proteasome is higher in both young
and old and naked mole-rats than that observed in their similarly
aged mouse counterparts, supporting their hypothesis . Naked
mole-rat cytosolic and nuclear liver preparations also showed a
more than two-fold increase in trypsin-like activity when compared
to the activity from liver preparations of the same subcellular frac-
tions obtained from livers of age-matched mice (Rodriguez KA and
Buffenstein R, unpublished data) (Fig. 5).
Oxidative stress also links the proteasome to human disease. In
a mouse model of ALS (SOD1G93A), a decrease in both the
mRNA and protein expression in catalytic subunits ?1 and ?5 is
observed during the progression of the disease in spinal cord tissue.
A simultaneous increase is also noted in Lmp7 (?5i), an immuno-
proteasome subunit . When these mice were crossed with
ALS-mice possessing a fluorescently-tagged UPP substrate, re-
vealed both and accumulation of the substrate, and ubiquitin sug-
gesting an impairment of motor neuron UPP in the mutant SOD1-
linked ALS mouse . Furthermore, Sod1-/- samples show a
relative decline of proteasome activity with both age and oxidative
stress especially in the cytosolic and microsomal fractions of mouse
liver tissue and a significant reduction in the contribution of activity
from 26S in favor of 20S activity in tissue from young oxidatively
stressed animals and from old animals (Rodriguez KA, Osmulski
PA, and Gaczynska M, unpublished data). If indeed naked mole-
rats have a higher steady-state level of oxidative stress as suggested
by the studies referenced above [1, 175, 182, 194, 205], they might
also have a higher contribution of activity from their 20S protea-
somes and better maintenance of that activity with age.
Autophagy is an evolutionarily conserved catabolic pathway,
present in all eukaryotic cells. This lysosomal system involves deg-
radation of intact proteins and protein aggregates . There are
three pathways by which proteins and cellular organelles are deliv-
ered to the lysosome: microautophagy, macroautophagy, and chap-
erone-mediated autophagy. Surprisingly little research compares
species differences regarding autophagy. Studies conducted on
senescent avian and mammalian fibroblasts reveal that cells from
longer-lived birds better maintain autophagasomal and lysosomal
enzymes whereas these activities are down regulated with age in
mammalian cell lines [252, 253]. Furthermore, our unpublished
data reveal that this may indeed play an important role in the main-
tenance of protein quality in the naked mole-rat (Perez VI and Buf-
fenstein R, unpublished data) as macroautophagy (degradation sen-
sitive to 3-methyladenine, 3-MA) is substantially higher in fibro-
blasts maintained under serum starvation from a naked mole-rat
compared to those from shorter-lived mice. Similarly when auto-
phagy is assessed by monitoring markers of vacuole development
(i.e., the conversion of LC3-I to LC3-II), the LC3-II/LC3-I ratio
induced by serum deprivation was ~ 2-fold greater in naked mole-
rat cells (Perez VI and Buffenstein R, unpublished data) (Fig. 5).
This suggests perhaps that this exceptionally long-lived rodent,
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2301
under basal conditions maintains higher levels of autophagy,
thereby removing potentially toxic proteins and possibly its high
ferritin load, before these can negatively impact upon organ func-
tionality. Nevertheless exceptionally old (>29 yr) naked mole-rats
accrue the age-associated yellowish-brown pigment lipofuscin
which accumulates in various organs including skin, heart and liver,
as a result of incomplete lysosomal digestion of cell products .
The plethora of contradictory results together with the lack of a
general consensus among multiple species across the animal king-
dom as well as among genetically manipulated mice in which oxi-
dative stress is altered strongly suggest that species MLSP is unre-
lated to any of the oxidative stress parameters currently being
measured. Certainly current data from genetically modified mice
that exhibit high levels of oxidative damage in response to experi-
mentally impaired antioxidant defenses show no concomitant
changes in age-related mortality. Similarly high levels of oxidative
damage to lipids, proteins and DNA in the naturally long-lived
naked mole-rat coupled with low levels of Gpx that are not com-
pensated for by other enzymatic antioxidants, do not negatively
impact on their exceptional longevity. Instead they seem to have a
higher set point of equilibrium for oxidative damage. In fact there
may be great differences in tolerance and structural resistance to
oxidative damage that may underlie the insignificant differences in
ROS production and antioxidant protection, despite the large differ-
ences in species longevity. This may limit the role of oxidative
stress in leading to diseases including cancer, myopathy, vascular
disease and neurodegeneration known to result from unchecked
ROS. Through the mitigation of damage naked mole-rats may have
arrived at an adaptive state of flux. Finally, we hope that the studies
reviewed here can act as a springboard to gain insights into the
mechanisms on how certain species, like the naked mole-rat, are
able to tolerate high levels of oxidative stress without an endpoint
of cancer and neurodegenerative disease. Such information in the
future can translate to a better understanding of oxidative stress and
its relation to a healthy lifespan free of age-related disease in our
This work was supported by an Ellison Senior Scholar award, a
Breakthroughs in Gerontology award from the American Federation
for Aging research and by the NIA/NIH to Rochelle Buffenstein
[AG022891-01 and NIGMS S06-GM08168] and by grants from
National Institutes of Health [grant numbers P01 AG025901, PL1
AG032118, P30 AG025708 and R01 AG033542], the W.M. Keck
Foundation and the Ellison Medical Foundation [grant number AG-
SS-2288-09] (to M.D.B.). Both Karl Rodriguez and Kaitlyn Lewis
were supported by NIH Aging training grant [T32 AG021890-08].
 Buffenstein R, Edrey YH, Yang TT, Mele J. The oxidative stress
theory of aging: embattled or invincible? Insights from non-
traditional model organisms. AGE 2008; 30: 99-109.
 Rubner M. Uber den Einfluss der Korpergrosse auf stoff- und kraft-
wechsel. Z Biol. 1883; 19: 535-62.
 Rubner M. Das Problem der Lebensdauer. Munich: Oldenburg;
 Pearl R. The Rate of Living. New York: Knopf; 1928.
 Lavosier AL. Considérations générales sur la nature des acides, et
sur les principes dont ils sont composés Mémoires de l'Académie
Royale des Sciences de Paris. 1781.
 Gershman R, Gilbert DL, Nye SW, Dwyer P, Fenn WO. Oxygen
poisoning and X-irradiation: a mechanism in common. Science
1954; 119: 623-6.
 Harman D. Aging: a theory based on free radical and radiation
chemistry. J Gerontol 1956; 11: 298-300.
 Beckman KB, Ames BN. The free radical theory of aging matures.
Physiol Rev 1998; 78: 547-81.
 Harman D. The biological clock: the mitochondria? J Am Geriatr
Soc 1972; 20: 145-7.
 Hulbert AJ. On the importance of fatty acid composition of mem-
branes for aging. J Theor Biol 2005; 234: 277-288.
 Ishii N. Role of oxidative stress from mitochondria on aging and
cancer. Cornea 2007; 9: S3-9.
 Bokov A, Chaudhuri A, Richardson A. The role of oxidative dam-
age and stress in aging. Mech Ageing Dev 2004; 125: 811-26.
Fig. (5). Both autophagy and proteasome activity show an increase in repair capacity in naked mole-rats when compared to mice. A) In serum-free media, the
ratio of LC3II/LC3I increases significantly in naked mole-rats suggesting greater sensitivity in activating autophagy processes (Perez and Buffenstein, unpub-
lished data). Data were collected using a Western blot technique and the ratios were calculated using Storm Image Quant software package (Molecular Dy-
namics, Sunnyvale, CA). B) Species differences in chymotrypsin-like (ChT-L) activity in liver cytosol lysates of young mice (4 m) and naked mole-rats (2y) in
response to varying doses of proteasome inhibitor (MG132, N-(benzyl-oxycarbonyl) leucinyl-leucinal) (Rodriguez and Buffenstein, unpublished data). Data
(presented as mean + standard deviation) are based on three different experiments, run in triplicate, using a modification of the methods described by Rogers
and Dean using the release of AMC from 100μM of the fluorogenic peptide Succ-LLVY-AMC (Boston Biochem, Boston MA) to measure proteasome ChT-L
activity [268, 269]. Naked mole-rats not only show greater levels of proteasome activity compared to mice but also appear to be markedly resistant to a known
inhibitor of proteasome activity.
0 50 100150 200 250300
Concentration Inhibitor (nM)
pmol AMC/min/ug lysate
NMR - MG132
Mouse - MG132
2302 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
 Muller FL, Lustgarten MS, Jang Y, Richardson A, VanRemmen H.
Trends in oxidative aging theories. Free Radic Biol Med 2007; 43:
Buffenstein R. Negligible senescence in the longest living rodent,
the naked mole-rat: insights from a successfully aging species. J
Comp Physiol B 2008; 178: 439-45.
Ikeno Y, Hubbard GB, Lee S, et al. Housing density does not influ-
ence the longevity effect of calorie restriction. Journals of Geron-
tology Series a-Biological Sciences and Medical Sciences 2005;
St.-Pierre J, Buckingham JA, Roebuck SJ, Brand MD. Topology of
superoxide production from different sites in the mitochondrial
electron transport chain. J Biol Chem 2002; 277: 44784-90.
Cadenas E, Davies K. Mitochondrial free radical generation, oxida-
tive stress, and aging. Free Radic Biol Med 2000; 29: 222-30.
Speakman JR, Selman C. The free-radical damage theory: Accu-
mulating evidence against a simple link of oxidative stress to age-
ing and lifespan. Bioessays 2011; 33: 255-9.
Sun Y, Oberley LW. Redox regulation of transcriptional activators.
Free Radic Biol Med 1996; 21: 335-48.
Chung HY, Sung B, Jung KJ, Zou Y, Yu BP. The molecular in-
flammatory process in aging. Antioxid Redox Signal 2006; 8: 572-
Linnane AW, Kios M, Vitetta L. Healthy aging: regulation of the
metabolome by cellular redox modulation and prooxidant signaling
systems: the essential roles of superoxide anion and hydrogen per-
oxide. Biogerontology 2007; 8: 445-67.
Golden TR, Melov S. Mitochondrial DNA mutations, oxidative
stress, and aging. Mech Ageing Dev 2001; 122: 1577-89.
Levine RL. Oxidative modification of glutamine synthetase. I.
Inactivation is due to loss of one histidine residue. J Biol Chem
1983; 258: 11823-7.
Szweda PA, Friguet B, Szweda LI. Proteolysis, free radicals, and
aging. Free Radic Biol Med 2002; 33: 29-36.
Boesch P, Weber-Lotfi F, Ibrahim N, et al. DNA repair in organ-
elles: Pathways, organization, regulation, relevance in disease and
aging. Biochim Biophys Acta 2011; 1813: 186-200.
Hill BG, Haberzettl P, Ahmed Y, Srivastava S, Bhatnagar A. Un-
saturated lipid peroxidation-derived aldehydes activate autophagy
in vascular smooth-muscle cells. Biochem J 2008; 410(3): 525-34.
Guéraud F, Atalay M, Bresgen N, et al. Chemistry and biochemis-
try of lipid peroxidation products. Free Radic Biol Med 2010; 44:
Madani A, Nehal M, Haque SS, Khan A. Perspective of oxidative
stress in a biological system and prevention by naturally occurring
antioxidant. Proc Natl Acad Sci USA 2010; 80: 287-95.
Lawrence RA, Burk RF. Species, tissue and subcellular distribution
of non Se-dependent glutathione peroxidase activity. J Nutr 1978;
Reddy VN, Giblin FJ, Lin LR, et al. Glutathione peroxidase-1
deficiency leads to increased nuclear light scattering, membrane
damage, and cataract formation in gene-knockout mice. . Invest
Ophthalmol Vis Sci 2001; 42: 3247-55.
Wolf N, Penn P, Pendergrass W, et al. Age-related cataract pro-
gression in five mouse models for anti-oxidant protection or hor-
monal influence. . Exp Eye Res 2005; 81: 276-85.
Fu Y, Cheng WH, Porres JM, Ross DA, Lei XG. Knockout of
cellular glutathione peroxidase gene renders mice susceptible to
diquatinduced oxidative stress. Free Radic Biol Med 1999; 27: 605-
Yant LJ, Ran Q, Rao L, et al. The selenoprotein GPX4 is essential
for mouse development and protects from radiation and oxidative
damage insults. . Free Radic Biol Med 2003; 34: 496-502.
Imai H, Hakkaku N, Iwamoto R, et al. Depletion of selenoprotein
GPx4 in spermatocytes causes male infertility in mice. J Biol Chem
2009; 284: 32522-32.
Pérez VI, Bokov A, VanRemmen H, et al. Is the oxidative stress
theory of aging dead? Biochem Biophys Acta 2009; 1790: 1005-14.
McCord JM, Fridovich I. Superoxide dismutase. An enzymic func-
tion for erythrocuprein (hemocuprein). J Biol Chem 1969; 244:
Rosen D, Siddique T, Patterson D, et al. Mutations in Cu/Zn super-
oxide dismutase gene are associated with familial amyotrophic lat-
eral sclerosis. Nature 1993; 362: 59-62.
 Wang J, Xu G, Borchelt DR. High molecular weight complexes of
mutant superoxide dismutase 1: age-dependent and tissue-specific
accumulation. Neurobiol Dis 2002; 9: 139-48.
Majoor-Krakauer D, Willems PJ, Hofman A. Genetic epidemiology
of amyotrophic lateral sclerosis. Clin Genet 2003; 63: 83-101.
Bartnikas TB, Gitlin JD. Mechanisms of biosynthesis of mammal-
ian copper/zinc superoxide dismutase. J Biol Chem 2003; 278:
Brown NM, Torres AS, Doan PE, O'Halloran TV. Oxygen and the
copper chaperone CCS regulate posttranslational activation of Cu,
Zn superoxide dismutase. Proc Natl Acad Sci USA 2004; 101:
Cuarano-Yzermens AL, Bartnikas TB, Gitlin JD. Mechanisms of
the copper-dependent turnover of the copper chaperone for super-
oxide dismutase. J Biol Chem 2006; 281: 13581-7.
Matzuk MM, Dionne L, Guo Q, Kumar TR, Lebovitz RM. Ovarian
function in superoxide dismutase 1 and 2 knockout mice. Endocri-
nology 1998; 139: 4008-11.
Ho YS, Gargano M, Cao J, Bronson RT, Heimler I, Hutz RJ. Re-
duced fertility in female mice lacking copper-zinc superoxide dis-
mutase. J Biol Chem 1998; 273: 7765-9.
McFadden SL, Ding D, Burkard RF, et al. Cu/Zn SOD deficiency
potentiates hearing loss and cochlear pathology in aged 129, CD-1
mice. J Comp Neurol 1999a; 413: 101-12.
McFadden SL, Ding D, Reaume AG, Flood DG, Salvi RJ. Age
related cochlear hair cell loss is enhanced in mice lacking cop-
per/zinc superoxide dismutase. Neurobiol Aging 1999b; 20: 1-8.
Reddy VN, Kasahara E, Hiraoka M, Lin LR, Ho YS. Effects of
variation in superoxide dismutases (SOD) on oxidative stress and
apoptosis in lens epithelium. Exp Eye Res 2004; 79: 859-68.
Elchuri S, Oberley TD, Qi W, et al. CuZnSOD deficiency leads to
persistent and widespread oxidative damage and hepatocarcino-
genesis later in life. Oncogene 2005; 24: 367-80.
Muller FL, Song W, Liu Y, et al. Absence of CuZn superoxide
dismutase leads to elevated oxidative stress and acceleration of
age-dependent skeletal muscle atrophy. Free Radic Biol Med 2006;
Wang X, Vatamaniuk MZ, Roneker CA, et al. Knockouts of SOD1
and GPX1 exert different impacts on murine islet function and pan-
creatic integrity. Antioxid Redox Signal 2011; 14: 391-401.
Hiroi S, Harada H, Nishi H, Satoh M, Nagai R, Kimura A. Poly-
morphisms in the SOD2 and HLA-DRB1 genes are associated with
nonfamilial idiopathic dilated cardiomyopathy in Japanese. Bio-
chem Biophys Res Commun 1999; 261: 332-9.
Nakanishi S, Yamane K, Ohishi W, et al. Manganese superoxide
dismutase Ala16Val polymorphism is associated with the devel-
opment of type 2 diabetes in Japanese-Americans. Diabetes Res
Clin Pract 2008; 81: 381-5.
Cai Q, Shu XO, Wen W, Cheng JR, Dai Q, Gao YT, Zheng W.
Genetic polymorphism in the manganese superoxide dismutase
gene, antioxidant intake, and breast cancer risk: results from the
Shanghai Breast Cancer Study. Breast Cancer Res 2004; 6: R647-
Li Y, Huang TT, Carlson EJ, et al. Dilated cardiomyopathy and
neonatal lethality in mutant mice lacking manganese superoxide
dismutase. Nat Genet 1995; 11: 376-81.
Lebovitz RM, Zhang H, Vogel H, et al. Neurodegeneration, myo-
cardial injury, and perinatal death in mitochondrial superoxide
dismutase-deficient mice. Proc Natl Acad Sci USA 1996; 93: 9782-
Huang T, Carlson E, Kozy H, Mantha S, Goodman S, Ursell P,
Epstein C. Genetic modification of prenatal lethality and dilated
cardiomyopathy in Mn superoxide dismutase mutant mice. Free
Radic Biol Med 2001; 31: 1101-10.
Melov S, Coskun P, Patel M, et al. Mitochondrial disease in super-
oxide dismutase 2 mutant mice. Proc Natl Acad Sci USA 1999; 96:
Asikainen TM, Huang TT, Taskinen E, et al. Increased sensitivity
of homozygous Sod2 mutant mice to oxygen toxicity. Free Radic
Biol Med 2002: 175-86.
VanRemmen H, Salvador C, Yang H, Huang TT, Epstein CJ,
Richardson A. Characterization of the antioxidant status of the het-
erozygous manganese superoxide dismutase knockout mouse. Arch
Biochem Biophys 1999; 363: 91-7.
VanRemmen H, Williams MD, Guo Z, et al. Knockout mice het-
erozygous for Sod2 show alterations in cardiac mitochondrial func-
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2303
tion and apoptosis. . Am J Physiol Heart Circ Physiol 2001; 281:
VanRemmen H, Ikeno Y, Hamilton M, et al. Life-long reduction in
MnSOD activity results in increased DNA damage and higher inci-
dence of cancer but does not accelerate aging. Physiol Genomics
2003; 16: 29-37.
Marklund SL. Extracellular superoxide dismutase. Methods Enzy-
mol 2002; 349: 74-80.
Marklund SL. Human copper-containing superoxide dismutase of
high molecular weight. Proc Natl Acad Sci USA 1982; 79: 7634-8.
Tamai M, Furuta H, Kawashima H, et al. Extracellular superoxide
dismutase gene polymorphism is associated with insulin resistance
and the susceptibility to type 2 diabetes. Diabetes Res Clin Pract
2006; 71: 140-5.
Carlsson LM, Jonsson J, Edlund T, Marklund SL. Mice lacking
extracellular superoxide dismutase are more sensitive to hyperoxia.
Proc Natl Acad Sci USA 1995; 92: 6264-8.
Hansel A, Kuschel L, Hehl S, et al. Mitochondrial targeting of the
human peptide methionine sulfoxide reductase (MSRA), an en-
zyme involved in the repair of oxidized proteins. . FASEB J 2002;
Moskovitz J, Bar-Noy S, Williams WM, Requena J, Berlett BS,
Stadtman ER. Methionine sulfoxide reductase (MsrA) is a regulator
of antioxidant defense and lifespan in mammals. Proc Natl Acad
Sci USA 2001; 98: 12920-5.
Nan C, Li Y, Jean-Charles PY, et al. Deficiency of methionine
sulfoxide reductase A causes cellular dysfunction and mitochon-
drial damage in cardiac myocytes under physical and oxidative
stresses. Biochem Biophys Res Commun 2011; 402: 608-13.
Salmon AB, Pérez VI, Bokov A, et al. Lack of methionine sulfox-
ide reductase A in mice increases sensitivity to oxidative stress but
does not diminish life span. FASEB J 2009b; 23: 3601-8.
Wood ZA, Schroder E, Harris JR, Poole LB. Structure, mechanism,
and regulation of peroxiredoxins. Trends Biochem Sci 2003; 28:
Dubuisson M, Stricht DV, Clippe A, Etienne F, Nauser T, Kissner
R, Koppenol WH, Rees JF, Knoops B. Human peroxiredoxin 5 is a
peroxynitrite reductase. FEBS Lett 2004; 571: 161-5.
Egler RA, Fernandes E, Rothermund K, et al. Regulation of reac-
tive oxygen species, DNA damage, and c-Myc function by perox-
iredoxin 1. Oncogene 2005; 24: 8038-50.
Neumann CA, Krause DS, Carman CV, et al. Essential role for the
peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour
suppression. Nature 2003; 424: 561-5.
Huang TT, Carlson EJ, Gillespie AM, Shi Y, Epstein CJ. Ubiqui-
tous overexpression of Cu,Zn superoxide dismutase does not ex-
tend life span in mice. J Gerontol A Biol Sci Med Sci 2000; 55:
Rando TA, Crowley RS, Carlson EJ, Epstein CJ, Mohapatra PK.
Overexpression of copper/zinc superoxide dismutase: a novel cause
of murine muscular dystrophy. Ann Neurol 1998; 44: 381-6.
Jaarsma D, Haasdijk ED, Grashorn JA, et al. Human Cu/Zn super-
oxide dismutase (SOD1) overexpression in mice causes mitochon-
drial vacuolization, axonal degeneration, and premature motoneu-
ron death and accelerates motoneuron disease in mice expressing a
familial amyotrophic lateral sclerosis mutant SOD1. Neurobiol Dis
2000; 7: 623-43.
Raineri I, Carlson EJ, Gacayan R, Carra S, Oberley TD, Huang TT,
Epstein CJ. Strain-dependent high-level expression of a transgene
for manganese superoxide dismutase is associated with growth re-
tardation and decreased fertility. Free Radic Biol Med 2001; 31:
Mele J, VanRemmen H, Vijg J, Richardson A. Characterization of
transgenic mice that overexpress both copper zinc superoxide dis-
mutase and catalase. Antioxid Redox Signal 2006; 8: 628-38.
Chen X, Liang H, VanRemmen H, Vijg J, Richardson A. Catalase
transgenic mice: characterization and sensitivity to oxidative stress.
Arch Biochem Biophys 2004; 422: 197-210.
Schriner SE, Linford NJ, Martin GM, et al. Extension of murine
life span by overexpression of catalase targeted to mitochondria.
Science 2005; 308: 1909-11.
Pérez VI, VanRemmen H, Bokov A, Epstein CJ, Vijg J,
Richardson A. The overexpression of major antioxidant enzymes
does not extend lifespan of mice. Aging Cell 2008; 8: 73-75.
 Oppermann M, Balz V, VAdams, et al. Pharmacological induction
of vascular extracellular superoxide dismutase expression in vivo. J
Cell Mol Med 2009; 13: 1271-8.
Suvorava T, Kojda G. Reactive oxygen species as cardiovascular
mediators: lessons from endothelial-specific protein overexpression
mouse models. Biochem Biophys Acta 2009; 1787: 802-10.
Nordberg J, Arnér E. Reactive oxygen species, antioxidants, and
the mammalian thioredoxin system. Free Rad Bio Med 2001; 31:
Takagi Y, Mitsui A, Nishiyama A, et al. Overexpression of thiore-
doxin in transgenic mice attenuates focal ischemic brain damage.
Proc Natl Acad Sci USA 1999; 96: 4131-6.
Mitsui A, Hamuro J, Nakamura H, et al. Overexpression of human
thioredoxin in transgenic mice controls oxidative stress and life
span. Antioxid Redox Signal 2002; 4: 693-6.
Bauman JW, Liu J, Liu YP, Klaassen CD. Increase in metal-
lothionein produced by chemicals that induce oxidative stress.
Toxicol Appl Pharmacol 1991; 110: 347-54.
Yang X, Doser TA, Fang CX, et al. Metallothionein prolongs sur-
vival and antagonizes senescence-associated cardiomyocyte dia-
stolic dysfunction: role of oxidative stress. . FASEB J 2006; 20:
Michalska AE, Choo KH. Targeting and germ-line transmission of
a null mutation at the metallothionein I and II loci in mouse. Proc
Natl Acad Sci USA 1993; 90: 8088-92.
Fu Z, Guo J, Jing L, Li R, Zhang T, Peng S. Enhanced toxicity and
ROS generation by doxorubicin in primary cultures of cardiomyo-
cytes from neonatal metallothionein-I/II null mice. Toxicol in Vitro
2010; 24: 1584-91.
Yang H-Y, Wang Y-M, Peng S-Q. Metallothionein-I/II null car-
diomyocytes are sensitive to Fusarium mycotoxin butenolide-
induced cytotoxicity and oxidative DNA damage. Toxicon 2010;
Matsushima S, Ide T, Yamato M, et al. Remodeling and failure
after myocardial infarction in mice overexpression of mitochon-
drial peroxiredoxin-3 prevents left ventricular remodeling and fail-
ure after myocardial infarction in mice. Circulation 2006; 113:
Xu JX. Radical metabolism is partner to energy metabolism in
mitochondria. Ann NY Acad Sci 2004; 1011: 57-60.
Genova ML, Pich MM, Bernacchia A, Bianchi C, Biondi A, Bo-
vina C. The mitochondrial production of reactive oxygen species in
relation to aging and pathology. Ann NY Acad Sci 2004; 1011: 86-
Block G, Dietrich M, Norkus EP, Packer L. Oxidative stress in
human’s populations. Culter R, Rodriguez H, editors. Singapore:
World Scientific Publishing; 2003.
Tanaka M, Gong JS, Zhang J, Yaneda M, Yagi K. Mitochondrial
genotype associated with longevity. Lancet 1998; 351: 185-6.
Camougrand N, Rigoulet M. Aging and oxidative stress: studies of
some genes involved both in aging and in response to oxidative
stress. Respir Physiol 2001; 128: 393-401.
Nomiyama T, Tanaka Y, Piao L, et al. The polymorphism of man-
ganese superoxide dismutase is associated with diabetic nephropa-
thy in Japanese type 2 diabetic patients. J Hum Genet 2003; 48:
Lee SJ, Choi MG, Kim DS, Kim TW. Manganese superoxide dis-
mutase gene polymorphism (V16A) is associated with stages of al-
buminuria in Korean type 2 diabetic patients. Metabolism 2006; 55:
Mollsten A, Marklund SL, Wessman M, et al. A functional poly-
morphism in the manganese superoxide dismutase gene and dia-
betic nephropathy. Diabetes 2007; 56: 265-9.
Crawford A, Fassett RG, Coombes JS, et al. Glutathione peroxi-
dase, superoxide dismutase and catalase genotypes and activities
and the progression of chronic kidney disease. Nephrol Dial Trans-
plant 2011; 16(epub ahead of print).
Cai Q, Shu X, Wen W, et al. Genetic polymorphism in the manga-
nese superoxide dismutase gene, antioxidant intake, and breast can-
cer risk: results from the Shanghai Breast Cancer Study. Breast
Cancer Res 2004; 6: R647-R55.
Mitrunen K, Sillanpaa P, Kataja V, et al. Association between
manganese superoxide dismutase (MnSOD) gene polymorphism
and breast cancer risk. Carcinogenesis 2001; 22: 827-9.
Tamimi RM, Hankinson SE, Spiegelman D, Colditz GA, Hunter
DJ. Manganese superoxide dismutase polymorphism, plasma anti-
2304 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
oxidants, cigarette smoking, and risk of breast cancer. Cancer Epi-
demiol Biomarkers Prev 2004; 13: 989-96.
Millikan RC, Player J, Cotret ARd, et al. Manganese superoxide
dismutase Ala-9Val polymorphism and risk of breast cancer in a
population-based case-control study of African Americans and
whites. Breast Cancer Res 2004; 6: R264-R74.
Aejemelaeus RT, Holm P, Kaukinen U, et al. Age-related changes
in the peroxyl radical scavenging capacityof human plasma. Free
Radic Biol Med 1997; 23: 69-75.
Kim JW, No JK, Yu BP, Chung HY, editors. Analysis of redox
status in serum during aging. New York: New York Academy of
Meccoci P, Polidori CM, Troiano L, Cherubini A, Cechetti R, Pini
G. Plasma antioxidants and longevity: a study on healthy centenari-
ans. Free Radic Biol Med 2000; 28: 1243-8.
Jones DP, Mody VC, Carlson JL, Lynn MJ, Sternberg P. Redox
analysis of human plasma allows separation of pro-oxidant events
of aging from decline in antioxidant defenses. Free Radic Biol Med
2002; 33: 1290-300.
Erden-Inal M, Sunal E, Kanbak G. Age-related changes in the
glutathione redox system. Cell Biochem Funct 2002; 20: 61-6.
Ozbay B, Dulger H. Lipid peroxidation and anti-oxidant enzymes
in Turkish population: relation to age, gender, exercise, and smok-
ing. Tohoku J Exp Med 2002; 197: 119-24.
Junqueira VB, Barros SB, Chan SS, Rodriguez L, Giavarotti L,
Abud RL. Aging and oxidative stress. Mol Aspects Med 2004; 25:
Gil L, Siems W, Mazurek B, Gross J, Schroeder P, Voss P. Age-
associated analysis of oxidative stress parameters in human plasma
and erythrocytes. Free Radic Res 2006; 40: 495-505.
Frisard MI, Broussand A, Davies SS, Roberts LJ, Rood J, Jonge
LE. Aging, resting metabolic rate,and oxidative stress damage: re-
sults from Louisiana healthy aging study. J Ger Med Sc 2007; 62:
Mendoza VM, Ruiz M, Sanchez MA, Retana R, Mun˜oz JL. Ag-
ing-related oxidative stress in healthy humans. Tohoku J Exp Med
2007; 213: 261-268.
Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med
2010; 362: 329-44.
Candore G, Bulati M, Caruso C, et al. Inflammation, cytokines,
immune response, apolipoprotein E, cholesterol, and oxidative
stress in Alzheimer disease: therapeutic implications. Rejuvenation
Res 2010; 13: 301-13.
Calon F, Cole G. Neuroprotective action of omega-3 polyunsatu-
rated fatty acids against neurodegenerative diseases: evidence from
animal studies. Prostaglandins Leukot Essent Fatty Acids 2007; 77:
Faux NG, Ritchie CW, Gunn A, et al. PBT2 rapidly improves
cognition in Alzheimer's Disease: additional phase II analyses. J
Alzheimers Dis 2010; 20: 509-16.
Abele D, Strahl J, Brey T, Philipp EE. Imperceptible senescence:
ageing in the ocean quahog Arctica islandica. Free Radic Biol Med
2008; 42: 474-80.
Abele D, Brey T, Philipp E. Bivalve models of aging and the de-
termination of molluscan lifespans. Exp Gerontol 2009; 44: 307-15.
Philipp E, Brey T, Pörtner HO, Abele D. Chronological and
physiological ageing in a polar and a temperate mud clam. Mech
Ageing Dev 2005; 126: 598-609.
Miwa S, Riyahi K, Partridge L, Brand MD. Lack of correlation
between mitochondrial reactive oxygen species production and life
span in Drosophila, strategies for engineered negligible senescence:
why genuine control of aging may be foreseeable. Ann NY Acad
Sci 2004; 1019: 388-91.
Ross RE. Age-specific decrease in aerobic efficiency associated
with increase in oxygen free radical production in Drosophila
melanogaster. J Insect Physiol 2000; 46: 1477-80.
Buttemer WA, Battam H, Hulbert AJ. Fowl play and the price of
petrel: long-living Procellariiformes have peroxidation resistant
membrane composition compared with short-living Galliformes.
Biol Lett 2008; 4: 351-4.
Schmidt-Nielsen K. Scaling in biology: the consequences of size. J
Exp Zool 1975; 194: 287-307.
Benedict FG. Vital Energetics: a study in comparative basal me-
tabolism. Washington D.C.: Carnegie Institution of Washington;
 McKechnie AE, Wolf BO. The allometry of avian basal metabolic
rate: good predictions need good data. Physiol Biochem Zool 2004;
Speakman JR. Body size, energy metabolism and lifespan. J Exp
Biol 2005; 208: 1717-30.
Hulbert AJ, Pamplona R, Buffenstein R, Buttemer WA. Life and
death: Metabolic rate, membrane composition, and life span of
animals. Physiol Rev 2007; 87: 175-213.
Lindstedt SL, Calder WA. Body size and longevity in birds. Con-
dor 1976; 78: 91-4.
deMagalhães JP, Costa J, Church GM. An analysis of the relation-
ship between metabolism, developmental schedules, and longevity
using phylogenetic independent contrasts. J Gerontol A Biol Sci
Med Sci 2007; 62: 149-60.
Holloszy JO, Smith EK, Vining M, Adams S. Effect of voluntary
exercise on longevity of rats. J Appl Physiol 1985; 59: 826-31.
Lee IM, Hsieh CC, Jr RSP. Exercise intensity and longevity in
men. The Harvard Alumni Health Study. J Am Med Assoc 1995;
Masoro EJ. Caloric restriction and aging: controversial issues. J
Gerontol 2006; 61: 14-9.
McCarter RJ, Palmer J. Energy metabolism and aging: a lifelong
study of Fischer 344 rats. Am J Physiol 1992; 263: E448-E52.
Hulbert AJ, Usher MJ, Wallman JF. Food consumption and indi-
vidual lifespan of adults of the blowfly, Calliphora stygia: a test of
the “rate of living” theory of aging. . Exp Gerontol 2004; 39: 1485-
Speakman JR, vanAcker A, Harper EJ. Age-related changes in
metabolism and body composition of three dog breeds and their re-
lationship to life expectancy. Aging Cell 2003; 2: 265-75.
Speakman JR, Talbot DA, Selman C, et al. Uncoupled and surviv-
ing: individual mice with high metabolism have greater mitochon-
drial uncoupling and live longer. Aging Cell 2004; 3: 87-95.
Lovegrove BG. The metabolism of social subterranean rodents --
adaptation to aridity oecologia 1986; 69: 551-5.
Lovegrove BG. Thermoregulation in the subterranean rodent
Georychus capensis (Rodentia, Bathyergidae). Physiol Zool 1987;
Lovegrove BG. The zoogeography of mammalian basal metabolic
rate American Naturalist 2000; 156: 201-19.
Li QF, RYSun, Huang CX, et al. Cold adaptive thermogenesis in
small mammals from different geographical zones of China Comp
Biochem Physiol A Mol Int Physiol 2001; 129: 949-61.
McNab BK. Climatic adaptation in the energetics of heteromyid
rodents. Comp Bioche Physiol A Physiol 1979; 62: 813-20.
Bennett NC, Aguilar GH, Jarvis JUM, Faulkes CG. Thermoregula-
tion in 3 species of Afrotropical subterranean mole-rats (Rodentia,
Batherygidae) from Zambia and Angola and scaling with the genus
Crytpomys. Oecologia 1994; 97: 222-7.
Buffenstein R. Ecophysiological responses to a subterranean habi-
tat; A Bathyergid perspective. Mammalia 1996; 60: 591-605.
deMagalhães JP, Costa J. A database of vertebrate longevity re-
cords and their relation to other life-history traits. J Evol Bio 2009;
O'Connor TP, Lee A, Jarvis JU, Buffenstein R. Prolonged longev-
ity in naked mole-rats: age-related changes in metabolism, body
composition and gastrointestinal function. Comp Biochem Physiol
A Mol Integr Physiol 2002; 133: 835-42.
Pamplona R, Portero-Otin M, Riba D, et al. Low fatty acid unsatu-
ration: a mechanism for lowered lipoperoxidative modification of
tissue proteins in mammalian species with long life spans. J Geron-
tol 2000; 55: B286-B91.
Pamplona R, Portero-Otin M, Riba D, Ruiz C, Prat J, Bellmunt MJ,
Barja G. Mitochondrial membrane peroxidizability index is in-
versely related to maximum lifespan in mammals. J Lipid Res
1998; 39: 1989-94.
Hulbert AJ, Faulks SC, Buffenstein R. Peroxidation-resisant mem-
branes can explain longevity of longest-living rodent and similarly-
sized mice. J Gerontol A Biol Sci Med Sci 2006; 61: 1009-18.
Hulbert AJ, Beard LA, Grigg GC. The exceptional longevity of an
egg-laying mammal, the short-beaked echidna (Tachyglossus acu-
leatus) is associated with peroxidation-resistant membrane compo-
sition. Exp Gerontol 2008; 43: 729-33.
Rabini RA, Moretti N, Staffolani R, et al. Reduced susceptibility to
peroxidation of erythrocyte plasma membranes from centenarians.
Exp Gerontol 2002; 37: 657-63.
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2305
 Faulks SC, Turner N, Else PL, Hulbert AJ. Calorie restriction in
mice: effects on body composition, daily activity, metabolic rate,
mitochondrial ROS production and membrane fatty acid composi-
tion. J Gerontol 2006; 61: 781-94.
Porter RK, Hulbert AJ, Brand MD. Allometry of mitochondrial
proton leak: influence of membrane surface area and fatty acid
composition. Am J Physiol Regul Integr Comp Physiol 1996; 271:
Brand MD, Turner N, Ocloo A, Else PL, Hulbert AJ. Proton con-
ductance and fatty acyl composition of liver mitochondria corre-
lates with body mass in birds. Biochem J 2003; 376: 741-8.
Brookes PS, Hulbert AJ, Brand MD. The proton permeability of
liposomes made from mitochondrial inner membrane phospholip-
ids: no effect of fatty acid composition. Biochem Biophys Acta
1997; 1330: 157-64.
Sohal RS, Svensson I, Brunk UT. Hydrogen peroxide production
by liver mitochondria in different species. Mech Ageing Dev
1990a; 53: 209-15.
Sohal RS, Svensson I, Sohal BH, Brunk UT. Superoxide anion
radical production in different animal species. Mech Ageing Dev
1989; 49: 139-45.
Barja G, Cadenas S, Rojas C, Lopez-Torres M, Perez-Campo R. A
decrease of free-radical production near-critical targets as a cause
of maximum longevity in animals. Comp Biochem Physiol Bio-
chem Mol Biol 1994; 108: 501-12.
Shi Y, Buffenstein R, Pulliam DA, VanRemmen H. Comparative
studies of oxidative stress and mitochondrial function in aging. In-
teg Comp Biol 2010; 50: 869-79.
Lambert AJ, Boysen HM, Buckingham JA, et al. Low rates of
hydrogen peroxide production by isolated heart mitochondria asso-
ciate with long maximum lifespan in vertebrate homeotherms. .
Aging Cell 2007; 6: 607-18.
Robert KA, Brunet-Rossinni A, Bronikowski AM. Testing the 'free
radical theory of aging' hypothesis: physiological differences in
long-lived and short-lived colubrid snakes. Aging Cell 2007; 6:
Hansford RG, Hogue BA, Mildaziene V. Dependence of H2O2
formation by rat heart mitochondria on substrate availability and
donor age. J Bioenerg Biomembr 1997; 29: 89-95.
Schönfeld P, Wojtczak L. Fatty acids as modulators of the cellular
production of reactive oxygen species. Free Radic Biol Med 2008;
Miwa S, Brand MD. Mitochondrial matrix reactive oxygen species
production is very sensitive to mild uncoupling. Biochem Soc
Trans 2003; 31: 1300-1.
Lopez-Torres M, Barja G. Lowered methionine ingestion as re-
sponsible for the decrease in rodent mitochondrial oxidative stress
in protein and dietary restriction Possible implications for humans.
Biochem Biophys Acta 2008; 1780: 1337-47.
Pamplona R, Portero-Otin M, Sanz A, Ayala V, Vasileva E, Barja
G. Protein and lipid oxidative damage and complex I content are
lower in the brain of budgerigar and canaries than in mice. Relation
to aging rate. AGE 2005; 27: 267-80.
Lambert AJ, Buckingham JA, Boysen HM, Brand MD. Low com-
plex I content explains the low hydrogen peroxide production rate
of heart mitochondria from the long-lived pigeon, Columba livia.
Aging Cell 2010; 9: 78-91.
Forman HJ, Kennedy J. Dihydroorotate-dependent superoxide
production in rat brain and liver. A function of the primary dehy-
drogenase. Arch Biochem Biophys 1976; 173: 214-24.
Panov A, Dikalov S, Hemendinger NSR, Rosenfeld JTGJ. Species-
and tissue-specific relationships between mitochondrial permeabil-
ity transition and generation of ROS in brain and liver mitochon-
dria of rats and mice. Am J Physiol Cell Physiol 2007; 292: C708-
Dell’Agnello C, Leo S, Agostino A, et al. Increased longevity and
refractoriness to Ca21-dependent neurodegeneration in Surf1
knockout mice. Hum Mol Genet 2007; 16: 431-44.
Barja G. Rate of generation of oxidative stress-related damage and
animal longevity. Free Radic Biol Med 2002; 33: 1167-1172.
Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and
aging. Science 1996; 273: 59-63.
Andziak B, O’Connor TP, Buffenstein R. Antioxidants do not
explain the disparate longevity between mice and the longest-living
rodent, the naked mole-rat. Mech Ageing Dev 2005; 126: 1206-12.
 Perez-Campo R, Lopez-Torres M, Cadenas S, Rojas C, Barja G.
The rate of free radical production as a determinant of the rate of
aging: evidence from the comparative approach. J Comp Physiol B
1998; 168: 149-58.
Sohal RS, Agarwal S, Dubey A, Orr WC. Protein oxidative damage
is associated with life expectancy of houseflies. Proc Natl Acad Sci
USA 1993; 90: 7255-9.
Wilhelm-Filho D, Althoff SL, Dafre AL, Boveris A. Antioxidant
defenses, longevity and ecophysiology of South American bats.
Comp Biochem Physiol C Toxicol Pharmacol 2007; 146: 214-20.
Cutler RG. Peroxide-producing potential of tissues: inverse correla-
tion with longevity of mammalian species. Proc Natl Acad Sci
USA 1985; 82: 4798-802.
Lopez-Torres M, Perez-Campo R, Rojas C, Cadenas S, Barja G.
Maximum life span in vertebrates: relationship with liver antioxi-
dant enzymes, glutathione system, ascorbate, urate, sensitivity to
peroxidation, true malondialdehyde, in vivo H2O2, basal and
maximum aerobic capacity. Mech Ageing Dev 1993; 70: 177-99.
Perez-Campo R, Lopez-Torres M, Rojas C, Cadenas S, Barja G.
Longevity and antioxidant enzymes, non-enzymatic antioxidants
and oxidative stress in the vertebrate lung: a comparative study. J
Comp Physiol B Biochem Syst Environ Physiol 1994; 163: 682-9.
Ferreira-Cravo M, Welker AF, Andrade RG, Drew K, Hermes-
Lima M. Physiological oxidative stress in the animal world. Comp
Biochem Physiol A Mol Integr Physiol 2007; 148: S63-S4.
Sanz A, Pamplona R, Barja G. Is the mitochondrial free radical
theory of aging intact? . Antioxid Redox Signal 2006; 8: 582-99.
Pamplona R, Barja G. Highly resistant macromolecular compo-
nents and low rate of generation of endogenous damage: two key
traits of longevity. Ageing Res Rev 2007; 6: 189-210.
Brunet-Rossinni AK. Reduced free-radical production and extreme
longevity in the little brown bat (Myotis lucifugus) versus two non-
flying mammals. Mech Ageing Dev 2004; 125: 11-20.
Andziak B, O’Connor TP, Qi WB, et al. High oxidative damage
levels in the longest-living rodent, the naked mole-rat. Aging Cell
2006; 5: 463-71.
Voituron Y, deFraipont M, Issartel J, Guillaume O, Clobert J. Ex-
treme lifespan of the human fish (Proteus anguinus): a challenge
for ageing mechanisms. Biol Lett 2011; 7: 105-7.
Meydani M, Lipman RD, Han SN, et al. The effect of long-term
dietary supplementation with antioxidants. Ann NY Acad Sci 1998;
Melov S, Ravenscroft J, Malik S, et al. Extension of life-span with
superoxide dismutase/catalase mimetics. Science 2000; 289: 1567-
Pamplona R, Portero-Otin M, Riba D, Ledo F, Gredilla R, Herrero
A, Barja G. Heart fatty acid unsaturation and lipid peroxidation,
and aging rate, are lower in the canary and the parakeet than in the
mouse. Aging Clin Exp Res 1999; 11: 44-9.
Pamplona R. Mitochondrial DNA damage and animal longevity:
insights from comparative studies. J Aging Res 2011; 2011:
Sohal RS, Sohal BH, Orr WC. Mitochondrial superoxide and hy-
drogen-peroxide generation, protein oxidative damage, and longev-
ity in different species of flies. Free Radic Biol Med 1995; 19: 499-
Hamilton ML, VanRemmen H, Drake J, et al. Does oxidative dam-
age to DNA increase with age?. Proc Natl Acad Sci USA 2001; 98:
Hermes-Lima M, Welker AF, Ferreira-Cravo M, Campos EG.
Physiological oxidative stress and low oxygen. Comp Bioche
Physiol B Mol Integr Physiol 2007; 148: S50-S60.
Buffenstein R. The naked mole-rat; a new long-living model for
human aging research. J Gerontol Biol Sci 2005; 60: 1369-77.
Portero-Otin M, Requena JR, Bellmunt MJ, Ayala V, Pamplona R.
Protein nonenzymatic modifications and proteasome activity in
skeletal muscle from the short-lived rat and the long-lived pigeon.
Exp Gerontol 2004; 39: 1527-35.
Agarwal S, Sohal RS. Relationship between suceptibility to protein
oxidation, aging, and maximum lifespan potential of different spe-
cies. Exp Gerontol 1996; 31: 365-72.
VanRemmen H, Richardson A. Oxidative damage to mitochondria
and aging. Exp Gerontol 2001; 36: 957-68.
Kaneko T, Tahara S, Matsuo M. Non-linear accumulation of 8-
hydroxy-2'-deoxyguanosine, a marker of oxidized DNA damage,
2306 Current Pharmaceutical Design, 2011, Vol. 17, No. 22 Rodriguez et al.
during aging. Mut Res DNA Aging Gene Instab Aging 1996;
Levine RL. Carbonyl modified proteins in cellular regulation, ag-
ing, and disease. Free Radic Biol Med 2002; 32: 790-6
Gil P, Farinas F, Casado A, Lopez-Fernandez E. Malondialdehyde:
A possible marker of ageing. Gerontology 2002; 48: 209-14.
Andziak B, Buffenstein R. Disparate patterns of agerelated changes
in lipid peroxidation in long-lived naked mole-rats and shorter-
lived mice. Aging Cell 2006; 5: 525-32.
Pérez VI, Lewa CM, Cortez LA, et al. Thioredoxin 2 haploinsuffi-
ciency in mice results in impaired mitochondrial function and in-
creased oxidative stress. Free Radic Biol Med 2008; 44: 882-92.
Sasaki M, Ikeda H, Sato Y, Nakanuma Y. Proinflammatory cyto-
kine-induced cellular senescence of biliary epithelial cells is medi-
ated via oxidative stress and activation of ATM pathway: A culture
study. Free Radic Res 2008; 42: 625-32.
Pérez VI, Buffenstein R, Masamsetti V, et al. Protein stability and
resistance to oxidative stress are determinants of longevity in the
longest-living rodent, the naked mole-rat. Proc Natl Acad Sci USA
2009; 106: 3059-64.
Kapahi P, Boulton ME, Kirkwood TBL. Positive correlation be-
tween mammalian life span and cellular resistance to stress. Free
Radical Bio Med 1999; 26: 495-500.
Salmon AB, Murakami S, Bartke A, Kopchick J, Yasumura K,
Miller RA. Fibroblast cell lines from young adult mice of long-
lived mutant strains are resistant to multiple forms of stress. Am J
Physiol-Endoc M 2005; 289: E23-E9.
Harper JM, Salmon AB, Leiser SF, Galecki AT, Miller RA. Skin-
derived fibroblasts from long-lived species are resistant to some,
but not all, lethal stresses and to the mitochondrial inhibitor rote-
none. Aging Cell 2007; 6: 1-13.
Salmon AB, Akha AAS, Buffenstein R, Miller RA. Fibroblasts
from naked mole-rats are resistant to multiple forms of cell injury,
but sensitive to peroxide, ultraviolet light, and endoplasmic reticu-
lum stress. J Ger Biol Sci Med Sci 2008; 63: 232-41.
Jasper H. SKNy worms and long life. Cell 2008; 132: 915-6.
McElwee JJ, Schuster E, Blanc E, Thomas JH, Gems D. Shared
transcriptional signature in Caenorhabditis elegans dauer larvae and
long-lived daf-2 mutants implicates detoxification system in lon-
gevity assurance. J Biol Chem 2004; 279: 44533-43.
Sykiotis GP, Bohmann D. Keapl/Nrf2 signaling regulates oxidative
stress tolerance and lifespan in Drosophila. Dev Cell 2008; 14: 76-
Sun LY, Bokov AF, Richardson A, Miller RA. Hepatic response to
oxidative injury in long-lived Ames dwarf mice. FASEB J 2011;
Bus JS, Cagen SZ, Olgaard M, Gibson JE. Mechanism of paraquat
toxicity in mice and rats. Toxicology and Applied Pharmacology
1976; 35: 501-13.
Puppo A, Halliwell B. Formation of hydroxyl radicals in biologi-
cal-systems - does myoglobin stimulate hydroxyl radical formation
from hydrogen-peroxide. Free Radical Research Communications
1988; 4: 415-22.
Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi
J. Oxygen sensitivity severely limits the replicative lifespan of mur-
ine fibroblasts. Nature Cell Biology 2003; 5: 741-7.
Busuttil RA, Rubio M, Dolle MET, Campisi J, Vijg J. Oxygen
accelerates the accumulation of mutations during the senescence
and immortalization of murine cells in culture. Aging Cell 2003; 2:
Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically
important stress response mechanism. Trends Mol Med 2004; 10:
Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu
Rev Pharmacol 2005; 45: 51-88.
Nioi P, Hayes JD. Contribution of NAD(P)H : quinone oxidoreduc-
tase 1 to protection against carcinogenesis, and regulation of its
gene by the Nrf2 basic-region leucine zipper and the arylhydrocar-
bon receptor basic helix-loop-helix transcription factors. Mutat
Res-Fund Mol M 2004; 555: 149-71.
Kwak MK, Wakabayashi N, Greenlaw JL, Yamamoto M, Kensler
TW. Antioxidants enhance mammalian proteasome expression
through the Keap1-Nrf2 signaling pathway. Mol Cell Biol 2003;
 Tong KI, Kobayashi A, Katsuoka F, Yamamoto M. Two-site sub-
strate recognition model for the Keap1-Nrf2 system: a hinge and
latch mechanism. Biol Chem 2006; 387: 1311-20.
McMahon M, Itoh K, Yamamoto M, et al. The cap 'n' collar basic
leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor
2) controls both constitutive and inducible expression of intestinal
detoxification and glutathione biosynthetic enzymes. Cancer Res
2001; 61: 3299-307.
Thimmulappa RK, Mai KH, Srisuma S, Kensler TW, Yamamato
M, Biswal S. Identification of Nrf2-regulated genes induced by the
chemopreventive agent sulforaphane by oligonucleotide microar-
ray. Cancer Res 2002; 62: 5196-203.
Kwak MK, Wakabayashi N, Itoh K, Motohashi H, Yamamoto M,
Kensler TW. Modulation of gene expression by cancer chemopre-
ventive dithiolethiones through the Keap1-Nrf2 pathway - Identifi-
cation of novel gene clusters for cell survival. J Biol Chem 2003;
Lewis KN, Mele J, Hayes JD, Buffenstein R. Nrf2, a guardian of
healthspan and gatekeeper of species longevity. Integr Comp Biol
2010; 50: 829-43.
Brown-Borg HM, Rakoczy SG. Calorie restriction affects brain
glutathione metabolism in normal and long-living mice. Exp Ger-
ontol 2007; 42: 140-.
Brown-Borg HM, Rakoczy SG, Uthus EO. Growth hormone alters
methionine and glutathione metabolism in Ames dwarf mice. Mech
Ageing Dev 2005; 126: 389-98.
Amador-Noguez D, Dean A, Huang WD, Setchell K, Moore D,
Darlington G. Alterations in xenobiotic metabolism in the long-
lived Little mice. Aging Cell 2007; 6: 453-70.
Morimoto RI, Cuervo AM. Protein homeostasis and aging: taking
care of proteins from the cradle to the grave. J Ger Biol Sci Med
Sci 2009; 64: 167-70.
Fink AL. Chaperone-mediated protein folding. Physiol Rev 1999;
Swanlund JM, Kregel KC, Oberly TD. Autophagy following heat
stress - The role of aging and protein nitration. Autophagy 2008; 4:
Swindell WR, Masternak MM, Kopchick JJ, Conover CA, Bartke
A, Miller RA. Endocrine regulation of heat shock protein mRNA
levels in long-lived dwarf mice. Mech Ageing Dev 2009; 130: 393-
Salmon AB, Leonard S, Masamsetti V. The long lifespan of two
bat species is correlated with resistance to protein oxidation and
enhanced protein homeostasis. FASEB J 2009a; 23: 2317-26.
Ward WF. The relentless effects of the aging process on protein
turnover. Biogerontology 2000; 1: 195-9
Martinez-Vicente M, Sovak G, Cuervo AM. Protein degradation
and aging. Exp Gerontol 2005; 40: 622-33.
Zwickl P, Seemuller E, Kapelari B, Baumeister W. The protea-
some: a supramolecular assembly designed for controlled proteoly-
sis. Adv Protein Chem 2001; 59: 187-222.
Bochtler M, Ditzel L, Groll M, Hartmann C, Huber R. The protea-
some. Annu Rev Biophys Biomol Struct 1999; 28: 295-317.
Friguet B, Stadtman ER, Szweda LI. Modification of glucose-6-
phosphate dehydrogenase by 4-hydroxy-2-noneal. Formation of
cross-linked protein that inhibits the multicatalytic protease. J Biol
Chem 1994; 269: 21639-43.
Bulteau AL, Petropoulos I, Friguet B. Age-related alterations of
proteasome structure and function in aging epidermis. Exp Geron-
tol 2000; 35: 767-77.
Reinheckel T, Ulrich O, Sitte N, Grune T. Differential impairment
of 20S and 26S proteasome activities in human hematopoietic
K562 cells during oxidative stress. Arch Biochem Biophys 2000;
Altun M, Besche HC, Overkleeft HS, et al. Muscle wasting in
aged, sarcopenic rats, is associated with enhanced activity in the
ubiquitin proteasome pathway. J Biol Chem 2010; 285: 39597-608.
Zainal TA, Oberley TD, Allison DB, Sweda LI, Weindruch R.
Caloric restriction of rhesus monkey lowers oxidative damage in
skeletal muscle. FASEB J 2000; 14: 1825-36.
Takeda K, Yoshida T, Kikuchi S, et al. Synergistic roles of the
proteasome and autophagy for mitochondrial maintenance and
chronological lifespan in fission yeast. Proc Natl Acad Sci USA
2010; 107: 3540-5.
Walking the Oxidative Stress Tightrope Current Pharmaceutical Design, 2011, Vol. 17, No. 22 2307 Download full-text
 Carney-Almroth B, Johansson AA, Förlin L, Sturve J. Early-age
changes in oxidative stress in brown trout, Salmo trutta. Comp
Biochem Phys Part B 2010; 155: 442-8.
Ferrington DA, Hussong SA, Roehrich H, et al. Immunoprotea-
some responds to injury in the retina and brain. J Neurochem 2008;
Hussong SA, Kapphahn RJ, Phillips SL, Maldonado M, Ferrington
DA. Immunoproteasome deficiency alters retinal proteasome’s re-
sponse to stress. J Neurochem 2010; 113: 1481-90.
Dasuri K, Nguyen A, Zhang L, et al. Comparison of liver and brain
proteasomes for oxidative stress induced inactivation: influence of
aging and dietary restriction. Free Rad Res 2009; 43: 28-36.
Dasuri K, Ebenezer PJ, Zhang Z, et al. Selective vulnerability of
neurons to acute toxicity after proteasome inhibitor treatment: Im-
plications for oxidative stress and insolubility of newly synthesized
proteins. Free Rad Bio Med 2010; 49: 1290-7.
Cheroni C, Marino M, Tortarolo M, et al. Functional alterations of
the ubiquitin-proteasome system in motor neurons of a mouse
model of familial amyotrophic lateral sclerosis. Hum Mol Genet
2009; 18: 82-96.
Ventruti A, Cuervo AM. Autophagy and neurodegeneration. Cur-
rent Neurology Neurosci 2008; 7: 443-51.
Massey AC, Kiffin R, Cuervo AM. Autophagic defects in aging -
Looking for an "emergency exit"? Cell Cycle 2006; 5: 1292-6
Strecker V, Mai S, Muster B, Beneke S, Burkle A, Bereiter-Hahn J,
Jendrach M. Aging of different avian cultured cells: Lack of ROS-
induced damage and quality control mechanisms. Mech Ageing
Dev 2010; 131: 48-59.
Buffenstein R, Edrey YH, Hanes M, Pinto M, Mele J. Successful
aging and sustained good health in the naked mole rat: a long-lived
Mammalian model for biogerontology and biomedical research.
ILAR J 2011; 52: 41-53.
Ho YS, Magnenat JL, Bronson RT, Cao J, Gargano M, Sugawara
M, Funk CD. Mice deficient in cellular glutathione peroxidase de-
velop normally and show no increased sensitivity to hyperoxia. J
Bio Chem 1997; 272: 16644-51.
Loh K, Deng H, Fukushima A, et al. Reactive oxygen species
enhance insulin sensitivity. Cell Metab 2009; 10: 260-72.
Thiruchelvam M, Prokopenko O, Cory-Slechta DA, Richfield EK,
Buckley B, Mirochnitchenko O. Overexpression of superoxide
dismutase or glutathione peroxidase protects against the paraquat+
maneb-induced Parkinson disease phenotype. J Biol Chem 2005;
Hoehn KL, Salmon AB, Hohnen-Behrens C, et al. Insulin resis-
tance is a cellular antioxidant defense mechanism. Proc Natl Acad
Sci USA 2009; 106: 17787-92.
Bowler RP, Nicks M, Tran K, et al. Extracellular superoxide dis-
mutase attenuates lipopolysaccharide-induced neutrophilic inflam-
mation. Am J Respir Cell Mol Biol 2004; 31: 431-9.
Ho YS, Xiong Y, Ma W, Spector A, Ho DS. Mice lacking catalase
develop normally but show differential sensitivity to oxidant tissue
injury. J Biol Chem 2004; 279: 32804-12.
Anderson EJ, Lustig ME, Boyle KE, et al. Mitochondrial H2O2
emission and cellular redox state link excess fat intake to insulin
resistance in both rodents and humans. J Clin Invest 2009; 119:
Matsui M, Oshima M, Oshima H, Takaku K, Maruyama T, J Yo-
doi, Taketo MM. Early embryonic lethality caused by targeted dis-
ruption of the mouse thioredoxin gene. Dev Biol 1996; 178: 179-
Bennett NC, Cotterill FPD, Spinks AC. Thermoregulation in two
populations of Matabeleland mole-rat (Cryptomys hottentotus nim-
rodi) and remarks on the general thermoregulatory trends within
the genus Cyrptomys (Rodentia: Bathyergidae). Jounal of Zoology
1996; 239: 17-27.
Holmes MM, Goldman BD, Goldman SL, Seney ML, Forger NG.
Neuroendocrinology and sexual differentiation in eusocial mam-
mals. Front Neuroendocrinol 2009; 30: 519-33.
Scharff A, Locker-Grütjena O, Kawalikab M, Burda H. Natural
History of the giant mole-rat, Crytomys mechowi (Rodentia: Bathy-
ergidae) from Zambia. J Mammalogy 2001; 82: 1003-15.
Buffenstein R, Yahav S. Is the naked mole-rat Heterocephalus
glaber an endothermic yet poikilothermic mammal? J Thermal Biol
1991; 16: 227-32.
Abayasekara DRE, Wathes DC. Effects of altering dietary fatty
acid composition on prostaglandin synthesis and fertility. Prosta-
glandins Leukotrienes and Essential Fatty Acids 1999; 61: 275-87.
Rodgers KJ, Dean RT. Assessment of proteasome activity in cell
lysates and tissue homogenates using peptide substrates. Int J Bio-
chem Cell Biol 2003; 35: 716-27.
Rodriguez KA, Gaczynska M, Osmulski PA. Molecular mecha-
nisms of proteasome plasticity in aging. Mech Ageing Dev 2010;
Received: May 12, 2011
Accepted: July 7, 2011