Free Radical Research, December 2006; 40(12): 1359–1365

Role of mitochondrial oxidative stress to explain the different longevity

between genders. Protective effect of estrogens

J. VIÑA1, J. SASTRE1, F. V. PALLARDÓ1, J. GAMBINI1, & C. BORRÁS2

1
Departamento de Fisiologı́a, Universidad de Valencia, 46010 Valencia, Spain, and 2Universidad Católica de Valencia, 46003
Valencia, Spain
Free Radic Res Downloaded from informahealthcare.com by Deakin University on 07/08/13

Accepted by Dr T. Grune

(Received 7 June 2006; in revised form 26 July 2006)

Abstract
Females live longer than males. Work from our laboratory has shown that this may be due to the up-regulation of longevity-
associated genes by estrogens. Estrogens bind to the estrogen receptors and subsequently activate the mitogen activated
protein kinase and nuclear factor kappa B signalling pathways, resulting in an up-regulation of antioxidant enzymes.
For personal use only.

Estrogen administration, however, has serious undesirable effects and of course, cannot be administered to males because of
its powerful feminizing effects. Thus, we tested the effect of genistein, a phytoestrogen of high nutritional importance whose
structure is similar to estradiol, on the regulation of the expression of antioxidant, longevity-related genes and consequently on
oxidant levels in mammary gland tumour cells in culture. Phytoestrogens mimic the protective effect of oestradiol using the
same signalling pathway.
The critical importance of up-regulating antioxidant genes, by hormonal and dietary manipulations, to increase longevity is
discussed.

Keywords: Gender, aging, estrogens, phytoestrogens, antioxidant enzymes, free radicals

Abbreviations: MAP, mitogen activated protein; NFKB, nuclear factor kappa B; 8-oxo-dG, 8-oxo-deoxyguanosine;
Mn-SOD, manganese-superoxide dismutase; GPx, glutathione peroxidase; 16S rRNA, 16S ribosomal RNA; PDTC, pyrrolidine
dithiocarbamate

Introduction: The mitochondrial theory of aging
Denham Harman proposed in 1956 the free radical
theory of aging [1,2]. It postulates that free radicals are
involved in the aging process. Miquel pointed to the
mitochondria as sources and targets of free radical
damage to aging cells [3]. A series of authors produced
evidence in favour of the mitochondrial hypothesis of
aging [4 – 7] This theory is supported by experimental
evidences such as the extension of lifespan obtained
by increasing the antioxidant defense as well as the
inverse relationship between the rate of reactive oxygen
species (ROS) production and the maximum lifespan
of species [8,9]. Administration of antioxidants can
increase the average lifespan of flies [10]. Orr and
Sohal reported that simultaneous overexpression of
copper– zinc superoxide dismutase and catalase genes
in transgenic Drosophila extend their mean and
maximum lifespan [11].
The fact that mitochondria are damaged inside
intact cells was almost simultaneously reported by us
[12] and by Hagen et al. [13].
Mitochondrial components are targets of free
radical damage associated with aging [3,12,14].
More than 90% of the oxygen used by aerobic cells
is consumed in mitochondria and about 1– 2% of
oxygen used by mammalian mitochondria in state four
does not form water but superoxide anion [15,16],

Correspondence: J. Viña, Departamento de Fisiologı́a, Facultad de Medicina, Avenida Blasco Ibáñez 17, 46010 Valencia, Spain.
E-mail: jose.vina@uv.es

ISSN 1071-5762 print/ISSN 1029-2470 online q 2006 Informa UK Ltd.

DOI: 10.1080/10715760600952851
 1360 J. Viña et al.
which is converted to hydrogen peroxide within
mitochondria either spontaneously or by Mn-super-
oxide dismutase. Hydrogen peroxide is released to the
extramitochondrial space [17]. Studying different
mammalian species, Sohal et al. found that mitochon-
dria from shorter-lived species produce higher
amounts of hydroperoxide than those from the
longer-lived species [18,19]. Thus, oxygen free
radicals and hydroperoxides are generated continu-
ously in the mitochondrial respiratory chain [15,16]
and they, particularly hydroxyl radical, cause oxidative
damage to proteins, lipids and DNA [12].
The continuous generation of ROS by mitochon-
dria throughout cell life produces an age-related
“chronic” oxidative stress, which plays a key role in
cellular aging. It is now well established that oxidative
damage to mitochondrial DNA, proteins and lipids
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occurs upon aging [12,14,20,22].
We found that late onset administration of some
sulfur-containing antioxidants protects against the
age-associated glutathione depletion in mice. It also
prevented the age-related decline in neuromuscular
coordination [23]. These antioxidants also increased
the mean life span of Drosophila [23].
More recently, we have investigated the protective
effect of a standardized extract from dried leaves
For personal use only.
of Ginkgo biloba (EGb 761) on the age-associated
oxidative damage to mitochondrial DNA [21]. Oral
administration of EGb 761 to rats for three months is
able to prevent the oxidative damage to mitochondrial
DNA that occurs in liver and brain upon aging [21].
Mitochondrial glutathione oxidation is also prevented.
In fact, age associated mitochondrial glutathione
oxidation and mitochondrial DNA damage are
directly correlated [14] (Figure 1) Hence, EGb 761
or other antioxidants partially prevent the chronic
oxidative stress associated with mitochondrial aging.
In conclusion, administration of certain antioxidants:
such as GSH, thiazolidine carboxylate derivatives,
Figure 1. Relationship between DNA damage and glutathione
oxidation. Age associated mitochondrial glutathione oxidation and
mitochondrial DNA damage (estimated measuring the levels of 8-
oxo-20 -deoxyguanosine) are directly correlated. (see Ref. [14])
Antioxidants given were vitamin C (50 g/Kg body wt) and vitamin E
(30 mg/Kg body wt).
vitamins C and E or the Ginkgo biloba extract EGb
761-, may prevent or delay the mitochondrial
oxidative stress and some of the physiological
impairment associated with aging.
Different life expectancy between males and
females of various species
Life expectancy over time by gender in Spain during
the period 1900 – 1992 was analyzed by Fernandez
Ballesteros et al. in 1999 [24]. Two important features
emerge: On one hand, life expectancy has increased
from around 34 years to approximately 80 years of age
during the 20th century. This is the highest increase
in life expectancy in the human species in recorded
history. Another increase of about 45 years is highly
unlikely to occur again. Indeed, if we accept a maximal
life span for humans of around 110 years, then, even if
all diseases were cured and all humans reached the
maximal life span, one would expect an increase of
up to 30 years. Therefore, the dramatic increase in life
span, which has taken place in the 20th century, is very
unlikely to occur again. Another important feature is
that in all cases females have lived longer than males.
Moreover, when, earlier in the 20th century, both men
and women died of diseases unrelated to aging, the
average life span of women was only 3.8% higher than
in men. However, when at the end of the century,
women died of diseases related to aging, their average
life span was almost 10% higher than that of men.
The higher longevity of females vs. males might be
attributed to social reasons. However, this is not a
phenomenon specific to humans. A survival curve of
Wistar rats in our laboratory is shown in Figure 2.
In this case, also, females lived considerably longer
than males. Average life span for female rats was
15% higher than for males. Thus, the increase in
average life span in women over men is unlikely to
be attributable to social reasons and basic biological
phenomena may be underlying this fact [25].
Biological markers of aging indicate that females
are biologically younger than males of the same
chronological age
A major challenge of gerontology is to find reliable
markers of biological, as opposed to chronological,
aging. In many cases the search for reliable parameters
of biological aging has been sterile.
The level of 16S rRNA is an interesting marker of
aging. Work by the group of Marco, in Madrid [39]
showed an important decrease in mitochondrial
transcripts of 16S rRNA with aging. Moreover, they
reported that these changes correlate with the shape
of the life span curve. Work by the group of Davies
showed that 16S rRNA degradation is associated with
oxidative stress [40]. Thus, we checked the hypothesis
that mitochondria from cells of higher biological age

Figure 2. Females live longer than males in human and rat species. (A) Life expectancy in Spain throughout 1900–2020. Data are taken
from [24]. (B) Female rats live longer than male rats. The longevity curve was made using around 70 animals/sex. Rats were kept under
standard conditions in the Faculty of Medicine, University of Valencia.
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should have lower expression of 16S rRNA. If this is the
case, mitochondria from female animals should have a
higher expression of 16S rRNA than those from males.
Indeed, this is the case and 16S rRNA expression was
over 700% higher in females than in males.
Glutathione is another biomarker of aging. Its
concentration is similar to that of glucose [41].
Mitochondrial GSH levels are double in females than
in males. The intracellular level of the oxidized form
For personal use only.
of glutathione (GSSG) is considered a biological
marker of aging [42], and we have found that mito-
chondrial GSSG is related to the damage associated
with aging [43].
Thus, we conclude that biological markers of aging
indicate that females behave as if they were younger
than males of the same chronological age.
Females produce less mitochondrial oxidants
than males
The rate of oxidant production by mitochondria from
females is significantly lower than that from males. We
measured the rate of H2O2 production and found that
mitochondria from female rats produce approximately
half the amount of H2O2 generated by “male”
mitochondria, as tested in liver and brain mitochon-
dria from mice and rats [25].
Moreover, neuronal mitochondria produce much
greater quantities of oxidants than do glial mitochon-
dria, a finding that agrees with the idea that post-mitotic
and non-dividing cells suffer much more age-associated
damage than do cells that divide normally [26,27].
To determine whether ovarian hormones, such as
estrogens, are involved in the marked differences
in oxidant production between mitochondria from
male and female rats, we tested the effects of
ovariectomy and of estrogen replacement therapy
on mitochondrial H2O2 production. Ovariectomy
caused an increase in H2O2 production by liver and
brain mitochondria, yielding values similar to those
Oxidative stress differences between genders 1361
observed with mitochondria from males, i.e. an
increase of more than 60% from the values found
normally with mitochondria from females. Estrogen
replacement therapy completely abolished the observed
increase in H2O2 generation that was produced by
ovariectomy [25,28].
Mitochondria from females suffer considerably less
oxidative damage to critical molecules such as
mitochondrial DNA (mtDNA) or glutathione than
those from males [25,29]. Mitochondrial DNA is a
key component of the mitochondrion [30], and its
degree of oxidation is directly related to aging
[31,14,32]. The level of 8-oxodeoxyguanosine, an
excellent indicator of oxidative damage to DNA, is
four times as great in mitochondria from males than
in those from females [25,33]. This is the most
pronounced oxidative change we have observed in
mtDNA in any physiological situation, and it indicates
that the higher chronic and continuous H2O2
production in males results in marked oxidative
damage to mtDNA and in mutagenic lesions to
DNA [34].
Estradiol increases the expression of
mitochondrial antioxidant, longevity, genes
The beneficial effects of estradiol have been widely
documented. In particular, estradiol has been shown
to have a cardioprotective role [35]. The possible
importance of estradiol in the prevention of Alzhei-
mer’s disease has been proposed but is not widely
accepted [36]. Moreover, estrogens have a powerful
in vitro antioxidant effect. However, the possible
effects of estradiol on longevity in vivo are unlikely to
be due to its chemical antioxidant properties. In fact,
the recommended dose of estradiol as part of estrogen
replacement therapy is of 50 mg/day. A widely
recommended dose of vitamin E as an antioxidant
supplement is 400 mg/day, i.e. 8000 times more than
that of estradiol. Therefore, to have significant
 1362 J. Viña et al.
antioxidant properties due to its chemical structure,
estradiol should be 8000 times more potent an
antioxidant than vitamin E. This is clearly not the
case. Thus, the beneficial effects of estrogens might be
due to the induction of the expression of antioxidant
genes. To check this hypothesis we tested whether the
effect of estradiol on hydrogen peroxide levels in cells
is mediated by receptors. We used tamoxifen (the
estrogen receptor antagonist) and checked its effects
on hydrogen peroxide levels in a mammary tumour
gland cell line (MCF-7 cells). Estradiol causes a
significant decrease in the levels of H2O2 in cells (in
agreement with our findings in vivo). Tamoxifen
significantly blocked this effect.
We also checked whether estradiol increased the
expression of the antioxidant enzymes superoxide
dismutase and glutathione peroxidase.
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Pinto and Bartley in the late 1960s reported the fact
that glutathione peroxidase activity was higher in
females than in males [37]. However, these authors
did not relate this fact to the different longevity
between males and females. Glutathione peroxidase
activity in males is approximately 50% of the values
found in females. Furthermore, we checked whether
this was due to a different gene expression in females
and males. Molecular expression of the glutathione
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peroxidase gene is significantly lower in males than in
females.
We tested the effect of gender on enzyme activity
and on gene expression of mitochondrial superoxide
dismutase. Mn-superoxide dismutase activity in
hepatic mitochondria from males is approximately
40% of the value found in females. This is due to a
higher expression of the Mn-SOD gene in females
than in males. Thus, the expression of both superoxide
dismutase and of glutathione peroxidase is signifi-
cantly lower in males than in females. This is
important in the context of previous mentioned work
by Orr and Sohal [11] that showed, in Drosophila, that
overexpression of superoxide dismutase or catalase
alone did not increase average life span. However,
Drosophila overexpressing both superoxide dismutase
and catalase did indeed have a higher average life span
than the controls. In this sense, female rats behave as
double transgenics overexpressing both superoxide
dismutase and glutathione peroxidase when compared
with males. This in itself may explain why the average
life span of female rats is higher than that of males.
Signalling pathways involved in the antioxidant
effect of estrogens
Estrogens were long ago recognized as in vitro
antioxidants [38]. In vivo experiments show that
estrogens have a powerful antioxidant effect: mito-
chondrial H2O2 production is significantly increased
(by more than 50%) after ovariectomy and this is
completely prevented when ovariectomised rats are
treated with estradiol at doses similar to those used in
estrogen replacement therapy (for details see [25]).
Then, it is clear that estrogens do not act as chemical
antioxidants in vivo. Instead they exert their antioxi-
dant effect by up-regulating of the expression of
antioxidant genes and antioxidant enzymes (see
above).
We used MFC-7 cells, a human mammary gland
cell line, to test the intracellular mechanism by which
estradiol acts to increase the expression of mitochon-
drial antioxidant enzymes. A direct genomic effect of
estradiol was unlikely because neither Mn-SOD nor
GPx have estrogen-responsive elements in their
promoter region. Thus, it was likely that the action
of estradiol is mediated via intracellular signalling
cascades. We tested the effect of mitogen activated
protein (MAP) kinases by using UO126, an inhibitor
of the phosphorylation of these kinases. Our experi-
ments showed that UO126 completely inhibited the
lowering effect of estradiol on the level of H2O2 in
cells. MAP kinases are known to activate the nuclear
factor kappa B (NF-kappaB). Thus, we tested
whether estradiol acts by activating NF-kappaB in
its up-regulation of the expression of both Mn-SOD
and GPx genes, whose promoters contain putative
NF-kappaB -binding motifs. This was indeed the case:
when cells were incubated with pyrrolidine dithio-
carbamate (PDTC), an inhibitor of the I-kappaB
degradation, and therefore an inhibitor of the
NF-kappaB translocation to the nucleus, the effect
of estradiol on the up-regulation of antioxidant
enzyme expression was prevented. Using these
pharmacological inhibitors of the signalling pathways,
we concluded that estradiol upregulates the expression
of Mn-SOD and GPx-mediated by the following
pathway: interaction with membrane estrogen
receptor, activation of MAP kinases, activation of
NF-kappaB, and up-regulation of gene expression. [28].
Phytoestrogens mimic the beneficial action of
estrogens in promoting mitochondrial
antioxidant defences
The effect of estradiol as an up-regulator of
antioxidant and longevity-related genes indicates that
its administration might be beneficial to increase
life span, particularly in males that should reach in
that way a life span similar to females. However,
considerable evidence has shown that estrogen
replacement therapy after menopause may have set
backs [44]. Phytoestrogens constitute an interesting
alternative. Their beneficial effects have been reported
repeatedly [45 – 48] and, to our knowledge very few, if
any, serious reports have shown detrimental effects.
Genistein, one of the major phytoestrogens in soya
[49], has a structure similar to that of estradiol. Its
antioxidant properties may be due to a direct chemical
effect caused mainly by its phenolic structure.

However, the concentrations of genistein usually
found in plasma, range from 50 to 800 ng/ml, making
it highly unlikely that its antioxidant properties are the
result of direct chemical effects due to its phenolic ring
structure.
Genistein binds preferentially to estrogen receptor b
(ERb). We reported that genistein decreases the basal
level of peroxide production in MCF-7 cells. In this
context, our results are consistent with the view that
diets rich in soy protein do not necessarily increase the
antioxidant capacity of plasma. Indeed genistein (and
very likely other isoflavones) are poorly absorbed
across the gastrointestinal tract, with only low levels
detected in plasma. However, our experiments
establish that the effect of the isoflavone genistein is
catalytic, i.e. it increases the expression of genes,
which in turn increase the translation of antioxidant
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enzymes. As in the case of other hormones, genistein
appears to act through an interaction with classical
estrogen receptors.
The beneficial actions of isoflavone phytoestrogens
have been attributed to their preferential interaction
with ERb and subsequent activation of critical cell
signaling pathways, leading to an upregulation of
antioxidant gene expression. We have shown that
genistein activates phosphorylation of ERK1/2
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and nuclear translocation of the p50 subunit of the
NFkB complex. Both these actions of genistein were
prevented when cells were co-incubated with genistein
and an inhibitor of the MAP kinase pathway [50].
In a similar fashion, genistein increased the
expression of MnSOD and this effect was abolished
when cells were incubated with genistein and an
inhibitor of the MAP kinase pathway [50]. Thus, our
results indicate that the pathway through which
genistein acts to increase antioxidant capacity in cells
is via interaction with estrogen receptor(s)—activation
Figure 3. Estrogens protect against free radical damage associated
to aging by up regulating antioxidant genes.
Oxidative stress differences between genders 1363
of MAP kinase—activation and nuclear translocation
of NFkB—over-expression of MnSOD—and lowering
of the intracellular levels of oxidants.
Concluding remarks
Finding good models of aging is a major aim of
gerontology. The different longevity between genders,
i.e. that females live around 10% more than males
in many species, including humans, offers a unique
opportunity to study fundamental aspects of aging.
In the context of the mitochondrial theory of aging,
we have found that mitochondrial oxidant production
is approximately double in females than in males
(Figure 3). We have traced this advantage of females
to the presence of estrogens, which act via a pathway
that comprises membrane estrogen receptors, MAP
kinase, NF-kappaB signalling and the up-regulation of
the expression of the antioxidant enzymes Mn-SOD
and GPx. Estrogen administration after menopause
has clinical problems and, obviously, cannot be given
to males. Phytoestrogens offer an interesting alterna-
tive. We have found that at concentrations found in
plasma, they increase the expression of antioxidant
enzymes without the feminizing effects of estradiol.
The possibility of using these compounds to increase
the longevity of males to reach the longevity of females
is a fact to be seriously considered.
Acknowledgements
Work reported from this laboratory was supported by
CICYT (BFI-2001-2849 and SAF2004-03755 to
J.V.), from CICYT (SAF2002/00885 to F.V.P.), from
Instituto de Salud Carlos III, RCMN (C03/08),
Madrid, Spain and from European Community
(COST-B35 action). We are grateful to Dolores
Royo for her skillful technical assistance.
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