N 19

October 2015
 Journal of Spanish Society of Anti-Aging Medicine and Longevity and Latin-American
Federation of Anti-Aging Medicine Societies
Editor in Chief
- Prof. Antonio Ayala (aayala@us.es)
- Dr. Jos Serres

Editorial Board

Prof. Antonio Ayala. University of Sevilla. Spain

Prof. Daro Acua Castroviejo. University of Granada. Spain

Prof. Joaqun Calap. University of Cdiz. Spain

Prof. Manuel Castillo. University of Granada. Spain

Prof. Santiago Durn. University of Sevilla. Spain

Prof. Juliana Faria. Complutense University of Madrid. Spain

Prof. Jess Fernndez Tresguerres. Complutense University of Madrid. Spain

Prof. Mnica de la Fuente. Complutense University of Madrid. Spain

Prof. Enrique Lerma. University of Barcelona. Spain.

Prof. Alberto Machado. University of Sevilla. Spain

Prof. M Teresa Mitjavila Cors. University of Barcelona. Spain

Prof. Plcido Navas. University Pablo de Olavide. Spain

Prof. Pedro Puig Parellada. University of Barcelona. Spain

Prof. Enrique Rojas. Complutense University of Madrid. Spain

Jos M Serra Renom. International University of Catalonia, Spain.

Prof. Jos Via. University of Valencia. Spain

International Committee

Dr. Richar G. Cutler (USA) Dr. Mario Kyriazis (United Kingdom)

Dr. Jorge Hidalgo (Peru) Prof. Francesco Marotta (Italy)
Dr. Hasan Insel (Turkey) Prof. Russel J. Reiter (USA)
Dr. Michael Klenze (Germany) Prof. Alfred S. Wolf (Germany)

Information and Subscription Publisher

Secretara Tcnica SEMAL Evento XXI
Depsito Legal: SE-3645-05
Colegio Oficial de Mdicos de Sevilla
ISSN: 1885-4028
Avda. de la Borbolla, 47 - 41013 Sevilla
Tel.: 954 08 47 00
Web: www.semal.org E-mail: info@semal.org
 SUMARY
N 19 - OCTOBER 2015

- Editorial................................................................................................................................................... . 6

- Articles..................................................................................................................................................... . 7

- Loneliness and social isolation worsen immunity in the elderly. .......................................... 7

Julia Cruces, Sergio Portal-Nez, Jess A. Fernndez-Tresguerres, Mnica De la Fuente

- The menopause disease............................................................................................................ 17

Georges Debled.MD

- Toxic Metals, Cell Death and Ageing.......................................................................... 25

Eleonore Blaurock-Busch PhD

- Endogenous opioid. A review .......................................................................................................... 31

Liuyi He, Mario F. Muoz and Antonio Ayala

- Treatment with mesenchymal stem cells in an animal model of parkinsons disease .......... 37
Jos Angel Naranjo, Antonio Ayala, Mercedes Cano*, Sandro Argelles,Victoria Ruiz, Mario Muoz

- Can electromagnetic fields modify the natural processes of aging? ........................................... 45

C. Maestu

- Coordinated cycling of calorie restriction, exercise, and pharmaceuticals

to enhance molecular turnover and removal of age-related neurotoxins ................................... 63

Roy G. Cutler

5
 www.approachestoagingcontrol.org

Editorial
Aging is the result, on the one hand, the passage of time and, secondly, the way that this passing of time affects us. Thus it
depends on the passage of time and also on our ability to resist the inexorable passage of time.The first is not preventable or
easily influenced. The latter itself is influenced. And it can be for the worse (being particularly susceptible or sensitive over
the years) or positive (being resistant, or giving oneself the means to become resistant to the effects of aging).
In spanish, the term aging refers to people and the consequences (mostly negatives) of the passage of time, long time. In
english the term aging -or better said ageing- its more precise and refers only to the pass of time and its consequences.
This term may refer either to a young person or an old person because both are affected from the pass of time and its
negative consequences but also positive because we acquire attributes that didnt have before.
The passing of years affects everyone and everything. Sometimes the effects are positive, but usually negative. In living
beings (and also in objects most susceptible to changes, the most fragile, and also those who are subject to more use) the
impact is particularly noticeable.
In humans it is interesting to insist on prior semantic disquisition and differentiate the consequences of aging in the early
stages of life, with the morphological and functional gain that this implies, what would be the morphological and functional
decline that occurs later in the same subject and the same time in quantitative terms. Focusing on the person now fully
developed, ie the end of youth or early adulthood, if we consider only the passing of years, aging occurs in a specific
way and continues but more subtly. It almost is not a concern, almost not a problem. It would be a purely chronological
aging. The chronological age advances and the biological age is stable. Moreover, in terms of functional capacity and in
terms of appearance noticeable improvements may even occur. This process can take years, decades. It is not uncommon
to find people who are better, and feel better, later on in adult life, at full maturity, then when they were younger. This
phenomenon is clearer in men but can occur similarly in women.
Extending those years, those decades, without the negative effects of aging, and even other positive effects, until an
advanced age is a target of Anti-Aging Medicine. A goal that is perfectly attainable from a morpho-physiological and
strictly scientific perspective: over the years with few adverse effects or even positive effects.
Therefore we would then talk about aging as corresponding to a chronological age that measures only the time and
a biological age that measures how the passage of time has been affecting the various body structures and functions. It
should be noted here that that effect (that biological age) varies greatly from one person to another and within the same
person, the structures and functions to another. So, some people age very fast and some very slow. And within the same
person, there are structures and functions that become affected very fast and early and others which are hardly affected or
just to a very small extent. The affects are visually (structural changes) and functionally noticeable. And both are related.
Consequently, under a biological standpoint, aging would be the structural deterioration and loss of functionality that
occurs over the years. Since the functional capacity of most organs depends on the number of cells and / or relationships
that establish interconnections two phases can be considered: First they are gaining cellularity (and interconnection), and
therefore functionality; and a second step where cellularity and connections are lost. The first stage corresponds to the
process of growth and development. Once we have ceased to grow we do not stay in a stable plateau of cellularity and
functionality begins a slow but inexorable decline and loss of cellularity and functionality that would be aging itself. This
loss of cellularity is caused by cell death, either by apoptosis or necrosis. The first is comparable to the natural death of cells,
the second would be comparable to the traumatic cell death. It would be the death that occurs after a physical, chemical or
biological assault that is the death by necrosis. Death by apoptosis would be the consequence of the accumulation of minor
damage not severe enough to cause necrosis; that happens as a result of the passage of time or an exposure to an overuse.
Alternatively death by apoptosis occurs due to the lack of stimulation or similarly to a lack of use. Overall it is estimated
that the decline in functional capacity, for each function or set of functions, occurs at a rate of 10% per decade of life after
the age of 20. Since the clinical manifestations of lack of decisive cellular functional failure occurs when there is only 20%
cellularity left, therefore 20% of functional capacity, the optimum time horizon of life free from symptoms associated with
aging would be 100 years and the vital horizon would be 120 years. In other words our body structure, our physiology is
designed to live to be 120 years old and live reasonably well, with an active life, free from degenerative disease, to 100 years.
Keeping this 10% loss would represent normal aging. An increase in the level of loss would be aging at an accelerated
rate. Reduce that level of loss would amount to slow the aging process and this can be done at any age but the sooner you
start, the better. Achieving the goal of 100 and 120 years could be extended to even more years and even decades. That
is precisely the current objective, perfectly feasible, of Anti-Aging Medicine not to rejuvenate or prevent aging, but only
alleviate the consequences of the passage of time. Which in itself is a lot.
Prof. Manuel J. Castillo. UGR

6
Approaches to Aging Control. Vol 19. october 2015

Loneliness and social isolation worsen immunity in the

elderly
Julia Cruces1,2, Sergio Portal-Nez3, Jess A. Fernndez-Tresguerres4, Mnica De la Fuente1,2*
1Department of Physiology (Animal Physiology II), Faculty of Biology, Complutense University of Madrid. 28040

Madrid, Spain.
*Corresponding author: Mnica de la Fuente. Departamento de Fisiologa Animal II, Facultad de Biologa, Universidad

Complutense de Madrid. Calle Jos Antonio Novis, 2. 28040, Madrid, Spain.Tel: 0034913944989. Fax:
0034913944935. e-mail: mondelaf@bio.ucm.es.
2Research Institute of Hospital 12 de Octubre (i+12). 28041 Madrid, Spain
3Laboratory of Mineral and Bone Metabolism, Instituto de Investigacin Sanitaria-Fundacin Jimnez Daz. 28040

Madrid, Spain.
4Department of Physiology, Medical School, Complutense University of Madrid. 28040 Madrid, Spain.

Keywords: loneliness, social isolation, aging,
immunity, anxiety.
Abreviatures
APC Antigen presenting cell
Con A Concanavalin A
CRP C-reactive protein
HPA Hypothalamic-pituitary-adrenal
IL Interleukin
LPS Lipopolysaccharide
MCP-1 Monocyte chemotactic protein 1
NFB Nuclear factor kappa B
NK Natural killer
SAM Sympathetic-adrenal-Medullar
TNF Tumor necrosis factor alpha
Abstract
Loneliness and social isolation, in social species such
as humans and rodents, are potent emotional stressors
which alter the homeostatic systems, the nervous,
endocrine, and immune systems, and the relationship
between them, the neuroendocrine-immune
communication. Consequently, both stressors lead to
the loss of homeostasis and hence of health. Similarly,
aging impairs the homeostatic systems functionality
and their communication, increasing morbidity and
mortality. Since older adults are more vulnerable to
feel lonely or to be socially isolated, both situations
may strikingly aggravate health state in the last stages
of life. Immune function is a well proven marker of
health and predictor of longevity and its age-related
changes, denominated immunosenescence, are
involved in oxi-inflamm-aging and, consequently, in
the rate of aging. In this review, the scarcely studies
on the effects of loneliness and social isolation on
immune system both in humans and rodent models
are commented. In general, loneliness and social
isolation increase oxidative and inflammatory stress
and impair immune cell functions. Nonetheless, the
particular characteristics of each individual, such as
age, gender and emotional response to stress, especially
anxiety, may modify these effects. In conclusion,
the maintenance of an active and rich social life,
particularly in elderly, may improve the homeostatic
systems activity, such as immune system, becoming a
useful strategy to slow down the aging process and
therefore to aim a healthy longevity.
Resumen
La soledad o el aislamiento social, en especies sociales
(humano, roedores), se consideran un potente estrs
emocional que altera el funcionamiento de los sistemas
homeostticos (nervioso, endocrino e inmunitario)

7

as como la relacin entre ellos o comunicacin
neuroinmunoendocrina. Este deterioro conlleva la
prdida de la homeostasis y, por tanto, de la salud.
As mismo, al envejecer, los sistemas homeostticos
y su comunicacin se ven deteriorados, aumentando
la morbilidad y la mortalidad. En la vejez aumentara
la vulnerabilidad a sufrir soledad o aislamiento social,
agravndose el estado de salud en las ltimas etapas de
la vida. El sistema inmunitario es un buen marcador
de salud y predictor de longevidad y los cambios que
experimenta al avanzar la edad (inmunosenescencia)
estn especialmente involucrados en el estrs oxidativo-
inflamatorio crnico asociado al envejecimiento
(oxi-inflamm-aging) y, consecuentemente, con la
velocidad de envejecimiento. Esta revisin recoge
los pocos estudios que abordan los efectos de la
soledad y del aislamiento social sobre la inmunidad,
en humanos y en modelos en roedores. En general,
la soledad y el aislamiento social aumentan el estrs
oxidativo-inflamatorio y deterioran diversas funciones
inmunitarias. No obstante, las caractersticas propias
del individuo (edad, sexo y respuesta emocional al
estrs, especialmente la ansiedad), pueden modificar
dichos efectos. Por tanto, mantener una vida social
activa y rica, particularmente en la vejez, podra
mejorar el funcionamiento del sistema inmunitario,
y de los sistemas homeostticos en general,
postulndose como una estrategia clave para frenar el
proceso de envejecimiento y, por tanto, para alcanzar
una longevidad saludable.
Introduction
Mental and physical health depends on the appropriate
functional state of homeostatic systems, namely the
nervous, endocrine and immune system, as well as
of their interrelationship, which is denominated the
neuroendocrine-immune communication [1-3].
Given the bidirectional nature of this communication
any stimuli affecting one of the homeostatic systems
may reflect on the others. Thus, negative situations
affecting the neuroendocrine system such as
emotional stress or anxiety also alters immune system,
disrupting the homeostatic balance and favoring the
appearance of pathologies [4,5]. With aging the three
8
www.approachestoagingcontrol.org
regulatory systems undergo several changes, which
producing a deterioration of their functions and that
finally leads to the loss of the correct neuroendocrine-
immune communication and of homeostatic balance,
increasing morbidity and mortality [6]. According to
the oxidation-inflammation theory of aging, chronic
oxidative stress and inflammatory stress situations
(with higher levels of oxidant and inflammatory
compounds and lower antioxidant and anti-
inflammatory defenses) are the basis of the age-related
impairment of organism functions, including those
of the regulatory systems. Moreover, the immune
system, which has been proposed as a relevant marker
of health [7] due to its production of oxidant and
inflammatory compounds for carrying out its
defensive function, seems to be involved in the levels
of oxi-inflamm-aging and consequently in the rate of
aging. Thus, the age-related changes of the immune
cells, which are denominated immunosenescence,
can be a source of high amounts of oxidant and
inflammatory compounds. These through the over-
activation of transcription nuclear factors such as the
nuclear factor kappa B (NFB), may increase the
oxidative and inflammatory stress of the organism
[8]. In the context of the neuroendocrine-immune
communication, it has been described that an
oxidative-inflammatory situation also occurs in
subjects with anxiety and emotional stress, and this
contributes to their premature immunosenescence
and, consequently, their premature aging and shorter
life span [9].
Social species are characterized by living in organized
social groups, which ensure the survival by providing
protection from environmental threats and contribute
to their reproductive success. In fact strong social bonds
between partners in these species have been related
to enhance neuroendocrine and immunological
functionality and longer life expectancy [10-14].
Conversely the loss of social relationships may have
negative effects on the regulatory systems, impairing
the cross-talk between each of them and leading to
the loss of homeostatic balance and the development
of a wide range of pathologies [15]. Accordingly,
Approaches to Aging Control. Vol 19. october 2015
loneliness and social isolation are considered as major
risk factors of morbidity and mortality [10] (Fig. 1)
In this Review we have briefly addressed how a
potent psychological stress, loneliness, affects the
immune system function, especially in the elderly. In
addition we discussed how the results obtained from
social isolation animal models, particularly in rodents,
may be translatable to humans, highlighting its
importance as useful tools to understand how social
isolation affects immune function and hence health
and longevity. In this regard, some results obtained
from a social isolation protocol carried out in aged
female mice, will be summarized. The fact that the
effects of social isolation on immunologic outcomes
may exhibit strong individual differences, especially at
old ages, will be also considered. Thus, these negative
effects of loneliness or social isolation may vary
depending of the emotional state on the individual,
especially the absence or presence of anxiety.
Loneliness, psychological stress, aging and
immunity
As mentioned above, for social species such as humans
and rodents the maintenance of social interactions
may play a key role in the health state of the
individuals. Conversely a disruption of social network
may negatively impact on the homeostasis and
hence on mental and physical health. Loneliness was
conceptualized by Weiss in 1973 as a perceived social
isolation and defined as a gnawing, chronic disease
without redeeming features [16]. Since loneliness
may be considered as a subjective term, an individual
may feel lonely among a crowd or fulfillment in
isolation. Nevertheless, as a potent psychological
stressor, non-chosen loneliness has been associated to
a wide range of pathologies including cardiovascular
diseases [17], hypothalamic-pituitary-adrenal (HPA)
axis and glucocorticoids regulation alterations [18],
sleep impairments [19], and cognitive deficits [20].
In fact, exposure to a stress results in nervous and
endocrine responses with coordinated activation
of diverse neurotransmitters and hormones [21].
The HPA and the sympathetic-adrenal-Medullar
(SAM) axes as well as their final products, namely
glucocorticoids and catecholamines, respectively, are
the two major pathways involved in stress response. In
addition, immune cells express specific receptors for
both stress hormones [22,23] and therefore, changes
in the levels of glucocorticoids and catecholamines,
induce alterations in immunity [24]. Thus, loneliness
and social isolation exert significant actions on
immune system; especially through the HPA axis
regulation that ultimate impair immune function
[18].
In addition, vulnerability to loneliness and social
isolation may increase in elderly. In todays world, aged
individuals frequently suffer a lack of social support
due to the loss of close relatives, i.e. widowhood, or
the difficulty in establishing or maintaining quality
social relationships. In fact data from the Spanish
National Statistics Institute (INE) reported in 2011
that the 22% from the total older adult population live
alone in Spain (more than 1.5 million of individuals)
of which a 59% feel lonely or isolated. Thus, if aging
leads to alteration of the neuroendocrine-immune
communication and hence to a loss of homeostasis
and health [6], as loneliness and isolation can also
produce this deterioration, feeling lonely or suffering
social isolation in the last stages of life may aggravate
the age-related neuroendocrine and immunologic
decline. Accordingly loneliness is considered a major
risk factor of morbidity and mortality in elderly [25].
The effects of loneliness on the immune system, from
the levels of gene expression to function, which may
extend beyond the immunosuppressive action of
the products of the neuroendocrine system, will be
commented.
Loneliness induces differential gene expression in
immune cells
Peripheral blood leukocytes from chronically-lonely
human middle-aged subjects exhibit a differential
gene expression profile than non-lonely subjects of
the same age [26]. In fact, loneliness was found to
up-regulate genes encoding inflammatory mediators,
activating the pro-inflammatory NFB pathway,
among others. Additionally, anti-inflammatory
9

markers were down-regulated and several genes
involved in the immunological response were also
under-expressed in lonely individuals, including genes
implicated in antiviral resistance as well as in antibody
production and B lymphocytes maturation. These
data seem to suggest that loneliness predominately
affects NFB activity, and consequently increase
many inflammatory factors [27]. Moreover, loneliness
attenuates the anti-inflammatory activity of the
glucocorticoid receptor [18]. Further studies have
identified that this immune system transcriptional
response to loneliness appears to be particularly
mediated by myeloid antigen presenting cells
(APCs), as dendritic cells and monocytes, and to a
lesser extent by B lymphocytes [28]. Together, these
results suggest that loneliness induces changes in gene
expression of leukocytes, some of which may be
related to a pro-inflammatory gene expression profile
and an attenuated APC response. These findings may
account for the proposed effects of loneliness and
social isolation on immunological function.
Loneliness impairs immune function
Few studies have focused on how loneliness
impairs immune function. Since APCs show a key
role in innate immune responses, the first line of
defense against pathogens and induce the specific
immunity, the fact that feeling lonely results in an
overexpression of pro-inflammatory genes as well as
an underexpression of anti-inflammatory genes in
leukocytes, particularly in APCs [26,28], suggest that
loneliness is linked with a dysregulation of immune
function. Although T lymphocytes and NK cells were
seen to be less transcriptionally sensitive to loneliness
than other leukocytes [28], functional impairment
of these cells has been observed in response to
perceived isolation in adult subjects [29,30]. NK
activity constitutes a significant defense against viral
and tumor processes and a functional impairment
in these cells may contribute to the increased
vulnerability of lonely individuals to cancer and viral
infections [31-34]. These results are consistent with
the under-expression of genes involved in innate
antiviral resistance (e.g., type I interferon genes and
10
www.approachestoagingcontrol.org
genes associated with antibody production and B
lymphocytes maturation) observed in leukocytes of
lonely individuals [26,28]. Regarding T lymphocytes,
it has been reported that loneliness may reduce the
proliferation response of these cells to mitogens [29]
and this fact may be also mediate through the effects
of loneliness on APCs and B lymphocytes, which in
turn play a role in T lymphocyte activation. Although
loneliness is clearly linked to immune dysregulation,
and in elderly the risk of suffering loneliness is
increased, scarcely studies on the effects of isolation
on immune activity in elderly have been carried out.
Loneliness and inflammation
Human epidemiological studies have associated
loneliness and social isolation to inflammatory
diseases, which may be triggered by the increased
expression of pro-inflammatory cytokines at both
gene and protein level, and increased activation of
NFB in leukocytes [26], a factor over-stimulated in
these cells with aging [35]. Indeed, higher levels of
serum fibrinogen in isolated men and, particularly
an elevated overall inflammation burden index in
older men have been observed [36]. Moreover,
augmented circulating levels of C-reactive protein
(CRP) again in aged men [37], increased plasma
levels of the pro-inflammatory cytokines IL6 and
TNFwhich have been related to a large number of
inflammatory diseases, in lonely middle-aged subjects
after an acute stress [38,39] as well as higher levels of
the monocyte chemotactic protein 1 (MCP-1) [36], a
cytokine implicated in inflammatory diseases such as
atherosclerosis [40] have been reported.
Animal models of social isolation
Although loneliness and social isolation may be
different psychological stressors since the former is
considered a subjective feeling and the latter is an
objective term which refers a lack of social contacts
(spouse, family, friends, colleagues, etc.) in humans,
both constructs seem to exert similar downstream
effects and susceptibility to disease. Loneliness may
increase health problems, as it was considered in
the present Review; nevertheless the mechanisms
Approaches to Aging Control. Vol 19. october 2015
involved are not well understood. Given the long
life expectancy of human species and the difficulty
to determine when an individual is feeling lonely,
animal models in social species such as rodents may
constitute an important tool to study the effects of
isolation on the neuroendocrine and the immune
systems as well as on the neuroendocrine-immune
communication. Moreover, social isolation animal
models may also allow us to study its possible effects
on longevity. Even though it seems impossible
to detect when a rodent is feeling lonely, socially
isolated mice and rats (i.e. maintaining the animal
alone apart from their counterparts) exhibit similar
outcomes than lonely humans, which finally leads
to the loss of health status [15]. Similarly to what
occurs in humans, the NFB pathway may also have
a key role in the detrimental effects of social isolation
in animals [41], in which immune dysregulation
was also observed. Thus, regarding the effects of
social isolation on the immune function in rodent
models, these socially isolated animals exhibited a
reduced NK activity [42], a decreased proliferative
of both B and T lymphocytes [42,43], as well as
increase levels of pro-inflammatory cytokines such
as TNF [44]. These adverse immunological outcomes
observed in socially isolated rodents are similar to
those found in lonely human individuals and in
fact social isolation in animal models has been also
associated to inflammatory diseases as cardiovascular
disease and atherosclerosis, tumorigenic processes and
Alzheimers disease [45-47].
Given the adverse effects of social isolation on the
immunity and the increased vulnerability of suffer
isolation in elderly, our research group carried out
an experiment with female mice submitted to a
social isolation of 6 months when they are old.
The results showed deteriorate behavioral, with
learning and memory deficit, as well as a decrease
in the NK activity of thymus cells [48]. Recently,
a protocol of social isolation for 1 month has been
carried out in old female mice, and several immune
function parameters have been assessed in peritoneal
leukocytes, which were obtained without sacrificing
of animals. Thus, the longevity of each mouse was
recorded.The most representatives results obtained are
summarized in Table 1 (unpublished data). As we can
see, this social isolation protocol strikingly reduced
important immunological functions. Thus, peritoneal
macrophages and lymphocytes exhibited a decreased
chemotaxis, e.g. an impaired capacity to aim the
inflammatory focus. In addition, the proliferation of
lymphocytes is also altered since these cells show an
augmented basal proliferation in absence of stimulus as
well as a reduced lymphoproliferative response to the
mitogens LPS, a mitogen which particularly activate
B lymphocytes, and Concanavalin A (Con A), more
specific for T lymphocytes. Nevertheless, the 4-weeks
social isolation protocol seems not affect NK activity
and phagocytosis. Apart from these immunological
impairments, those female mice maintained socially
isolated exhibited a slightly reduction of their life
span.Thus, these data may confirm that the disruption
of social interactions in elderly induces an immune
dysregulation, corroborating loneliness and social
isolation as risks factors of morbidity and mortality,
especially in the last stages of life.
The effects of loneliness and social isolation
may depend on the individual emotional
response
Although in this Review has been shown how
loneliness and social isolation affects the immune
system, these effects may vary according to diverse
factors such as the age of the individual or the presence
of stress-related disorders as anxiety [15], which could
be defined as a psychological, physiological, and
behavioral state induced in animals and humans by
a threat to well-being or survival, either actual or
potential [49]. In fact, anxiety state may interfere in
the ability to cope with stressful events. Accordingly,
differences in the anxiety levels may result in different
responses to a certain stressor as loneliness or social
isolation and in turn in inter-individual differences
among groups of study not only in humans but also
in animals. Based on the existence of these inter-
individual differences our research group has recently
reported, in a model of social isolation in old male
rats, that those isolated animals responding with high
11

anxiety levels to this psychological stress showed a
greater impairment of immunity as well as a greater
oxidative and inflammatory stress than those with low
levels of anxiety [50]. Together, these findings suggest
that the negative effects of social isolation on the
neuroimmunoendocrine system depend significantly
on the emotional state of the individual. Similar
results have been recently obtained in mice (work in
the process of publication).
Conclusions and future research
Loneliness and social isolation are considered
psychological stressors for social species, negatively
impacting on the neuroendocrine-immune
network with a consequent increase in morbidity
and mortality. Since vulnerability to feel lonely or
socially isolated is increased in later life due to the
loss of close relatives and friends or the difficulty to
create and maintain quality social relationships, and
with aging the ability to cope with stressful events
is decreased, loneliness and social isolation in elderly
subjects become more potent stressors with more
health negative impact. Nevertheless, few studies
have focused in how these stressors affect immunity
especially in elderly, stage of life showing higher
morbidity and mortality. Therefore, future research
is necessary to fully understand how feeling lonely
or being socially isolated affects to the immune
function and consequently to health, particularly in
the last stages of life. In this regard, animal models
of social isolation, carried out in social species such
as rodents, may become as useful tools to further
comprehend the effects of perceived and objective
isolation in humans. Although it is well known, that
loneliness and social isolation are different constructs,
since loneliness refers to a subjective feeling and
social isolation corresponds with an objective term,
both situations show similar adverse health outcomes.
Indeed, research in animal models of social isolation
has demonstrated that animals socially isolated
exhibited neuroendocrine and immune impairments
as well as vulnerability to diseases similarly to what
occurs in lonely humans. Additionally, given the
long life expectancy of human little is known about
12
www.approachestoagingcontrol.org
the effects of isolation on longevity. Thus, protocols
of social isolation performed in laboratory animals
as mice or rats may allow us to advance in the
understanding of the effects of loneliness on the
longevity of individuals. For all of these reasons,
animal models of social isolation may allow for
mechanistic investigations that are extremely difficult
to perform in humans.
In addition, the response to a certain stressor may
vary according to a wide range of factors such as
the age or the emotional state of each individual.
Thus, a subject may feel lonely among a crowd or
fulfillment in isolation. This different emotional
response to isolation may stem the inter-individual
differences observed among groups of study, even
in animal models. One of these factors, which may
contribute to the differential response to isolation,
could be the anxiety state. In fact, we have recently
demonstrated that responding anxiously to social
isolation induced a severe immunosuppression. In this
situation, it is possible to wonder if, as consequence
of the neuroendocrine-immune communication,
the individual changes in the immune system in
response to the social isolation can affect to the other
homeostatic systems and therefore to the health state.
Since aging, anxiety and emotional stress are related
with an increased oxidative and inflammatory stress
[9], which is especially due to the deregulation of
immune cells in the generation of oxidant and
inflammatory compounds [8], it is possible that to
know the redox and functional state of the immune
system in each individual could allow understand
his/her particular response to loneliness and social
isolation, regarding to the maintenance of health.
In addition, further studies are required to establish
strategies that can alleviate those negative effects
of loneliness in the elderly susceptible individuals.
Thus, to promote and active social life seems to be
very useful for reaching a healthy longevity. This
is appropriated in general, but it is necessary to
discriminate between the positive and the possible
negative effects of this strategy depending on the
particular situation of each individual. Accordingly
Approaches to Aging Control. Vol 19. october 2015
in a recent experiment our group have observed
that old male mice living with chronic isolation,
shown a deterioration of their immune functions
after 2 months of grouping (work in the process of
publication). In this case, other kinds of strategies
should be proved. Nevertheless, although considering
the peculiarity of each individual (age, sex, emotional
state, etc.), in general, it is possible to state that the
maintenance of an active social network and of
quality social relationships during aging seems to be
necessary to improve neural, endocrine and immune
functions, ensuring psychological and physiological
wellbeing, slowing down the aging process and,
consequently, reaching a healthy longevity.
Acknowledgments
This work was supported by grants from the
MINECO (BFU2011-30336), the Research
Group of UCM (910379ENEROINN) and
RETICEF (RD12/0043/0018; RD12/0043/0008;
RD12/0043/0032) from the ISCIII-FEDER
(European Union). Julia Cruces is the recipient of
a PhD fellowship from the Complutense University
of Madrid (UCM). Sergio Portal-Nez is a
recipient of a postdoctoral contract from RETICEF
(RD12/0043/0008).
References
[1]. Blalock, J.E. (1989). A molecular basis for
bidirectional communication between the immune
and neuroendocrine systems. Physiol. Rev. 69: 1-32.
[2]. Besedovsky, H.O., Del Rey, A. (1996).
Immune-neuro-endocrine interactions: facts and
hypotheses. Endocrine Rev. 17: 64-102.
[3]. Besedovsky, H.O., Del Rey, A. (2007).
Physiology of psychoneuroimmunology: a personal
view. Brain Behav. Immun. 21: 34-44.
[4]. Costa-Pinto, F.A., Palermo-Neto, J.
(2010). Neuroimmune interactions in stress.
Neuroimmunomodulation 17: 196-199.
[5]. Arranz, L., Guayerbas, N., De la Fuente, M.
(2007). Impairment of several immune functions in
anxious women. J. Psychosom. Res. 62: 1-8.
[6]. De la Fuente, M. (2008). Role of
the neuroimmunomodulation in ag ing.
Neuroinmunomodulation 15: 213-223.
[7]. Wayne, S.J., Rhyne, R.L., Garry, P.J.,
Goodwin, J.S. (1990). Cell-mediated immunity as a
predictor of morbidity and mortality in subjects over
60. J. Gerontol. 45: 45-48.
[8]. De la Fuente, M., Miquel, J. (2009).An update
of the oxidation-inflammation theory of aging: the
involvement of the immune system in oxi-inflamm-
aging. Curr. Pharm. Des. 15: 3003-3026.
[9]. Vida, C., Gonzlez, E.M., De la Fuente, M.
(2014). Increase of oxidation and inflammation in
nervous and immune systems with aging and anxiety.
Curr. Pharm. Des. 20: 4656-4678.
[10]. House, J.S., Landis, K.R., Umberson, D.
(1988). Social relationships and health. Science 241:
540-545.
[11]. Lillard, L.A., Waite, L.J. (1995). Til death
do us apart - marital disruption and mortality. A. J.
Sociol. 100: 1131-1156.
[12]. Lee, R.M., Robbins, S.B. (1998). The
relationship between social connectedness and
anxiety, self-esteem and social identity. J. Couns.
Psychol. 45: 338-345.
[13]. Kiecolt-Glaser, J.K., Newton, T.L. (2001).
Marriage and health: his and hers. Psychol. Bull. 127:
472-503.
[14]. Townsend, K.C., McWhirther, B.T. (2005).
Connectedness: a review of the literature with
implications for counseling, assessment and research.
J. Couns. Dev. 83: 191-201.
[15]. Cruces, J., Venero, C., Pereda-Prez, I., De la
Fuente, M. (2014). The effect of psychological stress
and social isolation on the neuroimmunoendocrine
communication. Curr. Pharm. Des. 20: 4608-4628.
13

[16]. Weiss, R.S. (1973). Loneliness: the experience
of emotional social isolation. Cambridge, MA: MIT
Press.
[17]. Hawkley, L.C., Masi, C.M., Berry, J.D.,
Cacioppo, J.T. (2006). Loneliness is a unique predictor
of age-related differences in systolic blood pressure.
Psychol. Aging 21: 152-164.
[18]. Hawkley, L.C., Cole, S.W., Capitanio, J.P.,
Norman, G.J., Cacioppo, J.T. (2012). Effects of social
isolation on glucocorticoid regulation in social
mammals. Horm. Behav. 62: 314-323.
[19]. Cacioppo, J.T., Hawkley, L.C., Berntson,
G.G., Ernst, J.M., Gibbs, A.C., Stickgold, R., Hobson,
J.A. (2002). Do lonely days invade nights? Potential
social modulation of sleep efficiency. Psychol. Sci. 13:
384-387.
[20]. Cacioppo, J.T., Hawkley, L.C. (2009).
Perceived social isolation and cognition.Trends Cogn.
Sci. 13: 447-454.
[21]. Jols, M., Baram, T.Z. (2009). The neuro-
symphony of stress. Nat. Rev. Neurosci. 10: 459-466.
[22]. Marchetti, B., Morale, M.C., Testa, N., Tirolo,
C., Caniglia, S., Amor, S., Dijkstra, C.D., Barden, N.
(2001). Stress, the immune system and vulnerability
to degenerative disorders of the central nervous
system in transgenic mice expressing glucocorticoid
receptor antisense RNA. Brain Res. Brain Res. Rev.
37: 259-272.
[23]. Madden, K.S. (2003). Catecholamines,
sympathetic innervation and immunity. Brain. Behav.
Immun. 17: 5-10.
[24]. Costa-Pinto, F.A., Palermo-Neto, J.
(2010). Neuroimmune interactions in stress.
Neuroimmunomodulation 17: 196-199.
[25]. Luo, Y., Hawkley, L.C., Waite, L.J., Cacioppo,
J.T. (2012). Loneliness, health, and mortality in old
age: a national longitudinal study. Soc. Sci. Med. 74:
907-914.
14
www.approachestoagingcontrol.org
[26]. Cole, S.W., Hawkely, L.C., Arevalo, J.M.,
Sung, C.Y., Rose, R.M., Cacioppo, J.T. (2007). Social
regulation of gene expression in human leukocytes.
Genome Biol. 8: R189.
[27]. Cole, S.W., Arevalo, J.M.,Takahashi, R., Sloan,
E.K., Lutgendorf, S.K., Sood, A.K., Sheridan, J.F.,
Seeman, T.E. (2010). Computational identification of
gene-social environment interaction at the human
IL6 locus. Proc. Natl. Acad. Sci. U. S. A.: 107: 5681-
5686.
[28]. Cole, S.W., Hawkley, L.C., Arevalo, J.M.,
Cacioppo, J.T. (2011). Transcript origin analysis
identifies antigen-presenting cells as primary targets
of socially regulated gene expression in leukocytes.
Proc. Natl. Acad. Sci. U. S. A. 108: 3080-3085.
[29]. Kiecolt-Glaser, J.K., Garner, W., Speicher,
C., Penn, G.M., Holliday, J., Glaser, R. (1984).
Psychosocial modifiers of immunocompetence in
medical students. Psychosom. Med. 46: 7-14.
[30]. Kiecolt-Glaser, J.K., Ricker, D., George,
J., Messick, G., Speicher, C.E., Garner, W.,
Glaser, R. (1984). Urinary cortisol levels, cellular
immunocompetency, and loneliness in psychiatric
inpatients. Psychosom. Med. 46: 15-23.
[31]. Glaser, R., Kiecolt-Glaser, J.K., Speicher,
C.E., Holliday, J.E. (1985). Stress, loneliness, and
changes in herpesvirus latency. J. Behav. Med. 8: 249-
260.
[32]. Reynolds, P., Kaplan, G.A. (1990). Social
connections and risk for cancer: prospective evidence
from the Alameda County Study. Behav. Med. 16:
101-110.
[33]. Pressman, S.D., Cohen, S., Miller, G.E.,
Barkin, A., Rabin, B.S.,Treanor, J.J. (2005). Loneliness,
social network size, and immune response to influenza
vaccination in college freshmen. Health Psychol. 24:
297-306.
[34]. Dixon, D., Cruess, S., Kilbourn, K., Klimas, N.,
Fletcher, M.A., Ironson, G., Baum, A., Schneiderman,
N., Antoni, M.H. (2006). Social support mediates
Approaches to Aging Control. Vol 19. october 2015
loneliness and human herpesvirus type 6 (HHV-6)
antibody titers. J. Appl. Soc. Psychol. 31: 1111-1132.
[35]. Arranz, L., Caamao, J.H., Lord, J.M., De
la Fuente, M. (2010). Preserved immune functions
and controlled leukocyte oxidative stress in naturally
long-lived mice: possible role of nuclear factor kappa
B. J. Gerontol. A. Biol. Sci. Med. Sci. 65: 941-950.
[36]. Yang, Y.C., McClintock, M.K., Kozloski, M.,
Li, T. (2013). Social isolation and adult mortality: the
role of chronic inflammation and sex differences. J.
Health. Soc. Behav. 54: 183203.
[37]. Loucks, E.B., Berkman, L.F., Gruenewald,
T.L., Seeman, T.E. (2006). Relation of social
integration to inflammatory marker concentrations
in men and women 7079 years. Am. J. Cardiol. 97:
1010-1016.
[38]. Hackett, R.A., Hamer, M., Endrighi, R.,
Brydon, L., Steptoe, A. (2012). Loneliness and stress-
related inflammatory and neuroendocrine responses
in older men and women. Psychoneuroendocrinology
37: 1801-1809.
[39]. Jaremka, L.M., Fagundes, C.P., Peng, J.,
Bennett, J.M., Glaser, R., Malarkey, W.B., Kiecolt-
Glaser, J.K. (2013). Loneliness promotes inflammation
during acute stress. Psychol. Sci. 24: 1089-1097.
[40]. Lin, J., Kakkar, V., Lu, X. (2014). Impact of
MCP-1 in atherosclerosis. Curr. Pharm. Des. 20:
4580-4588.
[41]. Venna, V.R., Weston, G., Benashski, S.E.,
Tarabishy, S., Liu, F., Li, J., Conti, L.H., McCullough,
L.D. (2012). NF-kappaB contributes to the detrimental
effects of social isolation after experimental stroke.
Acta Neuropathol. 124: 425-438.
[42]. Wu, W., Yamaura, T., Murakami, K., Murata,
J., Matsumoto, K., Watanabe, H. (2000). Social
isolation stress enhanced liver metastasis of murine
colon 26-L5 carcinoma cells by supressing immune
responses in mice. Life Sci. 66: 1827-1838.
[43]. Lu, Z.W., Hayley, S., Ravindran, A.V., Merali,
Z., Anisman, H. (1999). Influence of psychosocial
psychogenic and neurogenic stressors on several
aspects of immune functioning in mice. Stress 3:
55-70.
[44]. Wu, W., Yamaura, T., Murakami, K.,
Ogasawara, M., Hayashi, K., Murata, J., Saiki, I.
(1999). Involvement of TNF-alpha in enhancement
of invasion and metastasis of colon 26-L5 carcinoma
cells in mice by social isolation stress. Oncol. Res. 11:
461-469.
[45]. Bernberg, E., Andersson, I.J., Gan, L.M.,
Naylor, A.S., Johansson, M.E., Bergstrom, G. (2008).
Effects of social isolation and environmental
enrichment on atherosclerosis in ApoE-/- mice.
Stress 11: 381-389.
[46]. Liu, H., Wang, Z. (2005). Effects of social
isolation stress on immune response and survival time
of mouse with liver cancer.World J. Gastroenterol. 11:
5902-5904.
[47]. Dong, H., Goico, B., Martin, M., Csernansky,
C.A., Bertchume, A., Csernansky, J.G. (2004).
Modulation of hippocampal cell proliferation,
memory, and amyloid plaque deposition in
APPsw (Tg2576) mutant mice by isolation stress.
Neuroscience 127: 601-609.
[48]. Arranz, L., Gimnez-Llort, L., De Castro,
N.M., Baeza, I., De la Fuente, M. (2009). Social
isolation during old age worsens cognitive, behavioral
and immune impairment. Rev. Esp. Geriatr. Gerontol.
44: 137-142.
[49]. Steimer, T. (2002). The biology of fear- and
anxiety-related behaviors. Dialogues Clin. Neurosci.
4: 231-249.
[50]. Cruces, J., Venero, C., Pereda-Prez, I., De la
Fuente, M. (2014). A higher anxiety state in old rats
after social isolation is associated to an impairment of
the immune response. J,. Neuroimmunol. 277: 18-25.
15
 www.approachestoagingcontrol.org

Legends for tables to the loss of homeostasis, increasing morbidity and

Table 1. Effects of social isolation (1 month) on
immune functions of peritoneal leukocytes from
aged (684-weeks-old) female ICR-CD1 mice.
Parameters
Macrophages chemotaxis
Socially isolated aged
female mice
(versus grouped mice)
mortality. Thus, suffering loneliness or social isolation
in elderly and responding anxiously to any of these
stressors may worsen the neuroendocrine and
immunological age-related decline, increasing the
vulnerability to pathologies and finally shortening life
span. (Ox: oxidant compounds, PI: pro-inflammatory
compounds, Ax: antioxidant defenses, AI: anti-
inflammatory defenses)

Macrophages phagocytic capacity =

NK activity =

Lymphocytes chemotaxis

Basal

Lymphocytes LPS-induced
proliferative response
ConA-

induced

Mean longevity T
, , T represent lower levels of the respective parameter in socially isolated aged
female mice with respect to grouped female mice (p<0.001, p<0.01, statistical trend,
respectively). represents higher levels of basal lymphoproliferation in socially isolated
aged female mice with respect to grouped female mice (p<0.01). = represents no statistical
differences between socially isolated female mice and grouped female mice.

Figure legends
Figure 1. As emotional stressors, loneliness and social
isolation in social species (i.e. humans and rodents)
impair the function of homeostatic systems, namely
the nervous, endocrine and immune systems and
the cross-talk between them, the neuroendocrine-
immune communication. In fact, it has been suggested
that the immune impairment observed in lonely and
isolated subjects may stem in the establishment of an
oxidative and inflammatory chronic stress (with higher
levels of oxidant and pro-inflammatory compounds
and lower antioxidant and anti-inflammatory
defenses) affecting the organism and, particularly the
homeostatic systems and the communication between
them. Moreover, this oxidative-inflammatory stress
situation has also been described in aging as well
in subjects suffering stress-related disorders such as
anxiety. In any of these situations, the disruption of
the neuroendocrine-immune communication leads

16
Approaches to Aging Control. Vol 19. october 2015

The menopause disease

Georges Debled.MD
Department of Urology, CHU Saint Pierre and
CHU Brugmann, Brussels, Belgium, UE

Abstract dont suffer from androgens deficiencies at the

same age.
Menopause is not a disease as it is only the
definitive stop of menstruations. The new concept Daily production of androgens during the
of menopause disease results from a lack of normal menstrual cycle
androgens production in woman after the definitive
Androgens Productions in g/day
cessation of menstruations. This condition leads during the normal menstrual cycle
to functional symptoms, local consequences,
and general ageing. Mesterolone is indicated for Estra-
DHEA DHEAS
Andro- Testos-
diol stenedione terone
androgens deficiencies in woman.
Proliferative
Introduction 40 5.000 15.000 4.000 200
phase
Secretory
Androgens in woman. The technical term phase
200 5.000 15.000 4.000 200
androgen includes true androgens such as
testosterone and dihydrotestosterone (DHT) active Before menopause normal woman produces each day 5
on their receptors, but also precursors of androgens fold more testosterone than estradiol in the proliferative
such as the dehydroepiandrosterone (DHEA), phase and the same quantity in the secretory phase.
dehydroepiandrosterone sulfate (SDHEA) and Mesterolone Te s t o s t e r o n e and
A4-androstenedione, and androgens metabolites dihydrotestosterone
among which androstanediol, androsterone and
their glucuronides derivatives (which are the
principal metabolites representatives).
Origin of androgens production in woman.
Androgens are produced daily in the ovaries, in Testosterone
the adrenals and in peripheral tissues constituting Mesterolone Dihydrotestosterone
the total androgens production. With ageing
the daily androgens production varies in those
three pathways. When the ovaries production of Depending from its molecular structure mesterolone
androgens decreases for one or another reason has properties similar to those of testosterone and
(ageing, oophorectomy, menopause) the total pool dihydrotestosterone.
of androgens is diminished. When the total daily
production is sufficient they are no symptoms of
androgens deficiencies. This explains why women

17

The clasical medical concept of menopause
The word menopause goes back to 1823.
According to the dictionary this term means the
end of the ovarian function characterized by the
stop of ovulation and the menstrual hemorrhages
(which are physiological changes). A second meaning
means the time when the menopause occurs
(or more usually the female climacteric. These
vague definitions do not make possible to identify
a disease and consequently its treatment.
The menopause presents obvious signs: the
stopping of ovulation and the cessation of menses:
those conditions are physiological and are not a disease.
The random administration of estrogens associated
or not with progesterone or progestogens HRT
(hormonal replacement therapy) is dedicated to
failure and have even noxious effects.
The Womens health initiative is a15-year
project involves over 161,000 women ages 50-79,
and is one of the most definitive, far reaching
programs of research on womens health ever
undertaken in the U.S. (www.whi.org). Results
of the first HRT study on Risks and Benefits of
Estrogen plus Progestin in Healthy Postmenopausal
Women were published in 2002 (JAMA. 2002;
288:321-333) (5). The conclusions about this
randomized clinical trial of great scale relating to
16,608 old women from 50 to 79 years treated by
Progestin (2.5 mg of medroxyprogesterone acetate)
and estrogens (0,625 Mg of combined estrogens
equine) were:
Women taking estrogen plus progestin relative to placebo
JAMA. 2002;288:321-333 (5)
Stroke rates increased by 41%
CHD (coronary heart disease) events was increased by
29%
Venous thromboembolism (VTE) 2-fold greater
Total cardiovascular disease increased by 22%
Breast cancer increased by 26%
18
www.approachestoagingcontrol.org
The study was to proceed until 2005. It was stopped
after a 5.2 years average of follow-up on July 9th,
2002. It was decided to continue the treatment
containing estrogens alone among women with
prior hysterectomy.
The Womens Health Initiative (WHI) Estrogen
Alone (E-Alone) Trial was designed to assess the
health benefits and risks of estrogen use in healthy
postmenopausal women. In the WHI E-Alone
Trial, 10,739 women with prior hysterectomy,
aged 50-79 years, were assigned to take either
estrogen alone (conjugated estrogens [Premarin]
or inactive (placebo) study pills. The National
Institutes of Health stopped the E-Alone Trial
ahead of schedule in February 2004 primarily
because of an increased stroke risk for women
taking study pills with estrogen alone.
Even after the Womens Health Initiative (WHI)
found that the risks of menopausal hormone
therapy (hormone therapy) outweighed benefit for
asymptomatic women, about half of gynecologists
in the United States continued to believe that
hormones benefited womens health. The
pharmaceutical industry has supported publication
of articles in medical journals for marketing
purposes (6).
In June 2011 the U.S. Supreme Court refused to
hear a Pfizer Inc. units appeal of a $58 million
award in a case against Premarin (estrogens) and
Prempro (estrogens plus medroxyprogesterone
acetate) menopause drugs.Three Nevada women who
contracted breast cancer after taking the companys
menopause drugs were awarded the amount in a
2007 case.
The rebuff leaves the amount as the largest to
be upheld on appeal in thousands of hormone-
replacement drug suits. Over six million women took
Prempro and other menopause drugs before a 2002
study pointed out their links to cancer. At one point
Pfizer and his units faced more than 10,000 claims,
according to lawyers for former user.
Androgens deficiencies before menopause
Testosterone secretion decreases regularly from the
age of 20 in woman: The expected Testosterone
Approaches to Aging Control. Vol 19. october 2015
concentration in the blood of a woman of 40 would
be about half that of a woman of 21 (2).
Oral contraceptive troubles
Consequences of oral contraceptive troubles with
Decrease of androgen ovarian synthesis
Harrisons Principles of internal medicine (endocrinol-
ogy and metabolism), 1988.
Hypertension (14/9) after 5
Thrombosis of the
years of continuous use ( 5% of
Deep Veins
the cases)
Pulmonary embolism Disturbances of the plasmatic
(risks x 2 to 12) lipoproteins.
Cerebral thromboem-
bolism (risks x 3 Resistance to insulin.
to 9)
Cerebral hemorrhage
(risks x 2)
Women using OCs (OC=Oral contraceptive) had
significantly lower serum androgen levels compared
to naturally cycling women and free testosterone
levels displayed an inverse relation to breast epithelial
proliferation (3).
OC use, however, has been associated with womens
sexual health complaints and androgen insufficiency.
OC use is associated with a decrease of androgen
ovarian synthesis and an increase in the production of
sex hormone-binding globulin (SHBG) even months
after the stop of OC. Troubles may persist after stopping
the OC(4).
Bilateral oophorectomy: In this condition balance
between estradiol and progesterone could be
impaired leading to fibromyoma of the uterus,
spotting and bleeding. Androgens deficiency will be
also considered in those cases.
Mesterolone therapy will benefit to all those cases
with persisting troubles due to the decreased
testosterone and dihydrotestosterone secretions.
Androgens deficiencies after menopause
The menopause disease
To understand this disease of ageing it is advisable
to establish a precise definition of it. I propound
the following definition:The menopause disease
is the whole of the physiopathological and
psychopathological modifications brought out by the
stop of the ovarian androgens production.
Cause
The ovaries secrete estradiol, progesterone and
testosterone. The cessation of estradiol and
progesterone are linked with the stop of ovulation and
of menstrual hemorrhages which are physiological
changes. The drastic reduction in the secretion of
androgens hormones by the ovaries (at an age where
the production of androgens by the suprarenal glands
is already decreased) is generally not taken into
account and brings about the menopause disease.
Blood rates of ovarian hormones in woman
before menopause. Estradiol: its blood rate varies
from 0.06 nanograms per milliliter in the proliferative
part of the menstrual cycle towards 0.2 nanograms
per milliliter in the secretory part of the menstrual
cycle. Testosterone: its rate is on average of 0.57 + 0, 19
nanograms per milliliter throughout all ovarian cycle
and dihydrotestosterone levels are commonly the
half of the testosterone rates in healthy women (1-
p.432). Progesterone: its blood rate varies from 0.3 to
1.5 nanograms per milliliter (in the proliferative part
of the menstrual cycle towards 3 to 20 nanograms per
milliliter in the secretary part of the menstrual cycle.
Woman before menopause :
Plasmatic hormonal Concentrations in ng /ml (1)
Dihy-
drotes-
Estradiol Progesterone Testosterone
tosterone
(DHT)
Proliferative
0.06 0,3-1,5 0.57 + 0,19
0,27+
phase 0.06
24 hours
before ovu- 0.6
lation
Secretory 0,27+
0.2 3-20 0.57 + 0,19
phase 0.06
In woman concentration of testosterone in plasma
is always greater than estradiol concentration (1).
The plasma concentration of testosterone is + or
10 fold the level of estradiol during the proliferative
19

phase and + or 3 fold the level of estradiol during
the secretory phase. Serum levels of testosterone
increases during ovulation (Direct RIA) in normal
menstrual cycle (7).
Daily production of ovarian hormones. Estradiol:
its daily production varies from 40 micrograms per
day in the proliferative part of the menstrual cycle
around 200 micrograms per day in the secretary
part of the menstrual cycle. Testosterone: its daily
production is of 200 micrograms throughout all
ovarian cycle. The progesterone: its daily production
is of 4,200 micrograms during the proliferative phase
of the ovarian cycle and of 42.000 micrograms in the
secretary part of the menstrual cycle.
Woman before menopause : Normal daily hormonal production
(g/day) (1)
Estradiol Progesterone
Proliferative phase 40 4.200
Secretory phase 200 42.000
The secretion of these three hormones stops
in the ovaries at the time of the menopause.
The secretory and proliferative cycle controlled by
estradiol and progesterone intended to fertilize ovules
does not exist any more after the stop of the menses.
One can logically wonder which reason would justify
a systematic replacement of these hormones except
the fact of wanting to prolong in time an ovarian
cycle become useless in the absence of ovulation.The
total production of testosterone during a menstrual
cycle is more important in quantity compared with
the production of estradiol. Consequently one is
in right to ask for why estradiol substitution was
proposed in the past by neglecting the production
of testosterone? The sharp fall of front testosterone
secretion at the time and after the stop of menses is
responsible for most of the disorders caused by the
menopause disease.
CONSEQUENCES AND SYMPTOMS
The reduction in androgens production causes in
woman to different degree:
Functional symptoms: hot flashes, irritability,
intestinal distension, swollen legs.
20
www.approachestoagingcontrol.org
Local consequences:
The masculine genitalia of woman are: The
clitoris and its foreskin, the labia majora, the bladder
neck (= Inner part of urethra = Homology with
the prostate musculature in man)
Local symptoms resulting from of masculine
genitalia involutions are Chronic cystitis,
incontinence, urgencies, (sclerosis of bladder neck);
Painful or difficult copulation (sclerosis of vulva)
as consequences of a lack of dihydrotestosterone
production (lack of testosterone leads to a decreased
production of dihydrotestosterone).
Balanced treatment with male hor mones
(mesterolone) is indicated in menopause disease as
in andropause disease. This fact is generally ignored
so that the administration of estradiol (or estrogens)
Testosterone associated or not with progesterone or progestogens
200 doesnt constitute the treatment for the menopause
200
disease whose I gave the definition (androgens
deficiencies). One can even wonder whether the
traditional treatments of hormonal replacement
therapy (HRT) do not worsen the state of good
health. See: www.whi.org
General consequences: lipids disorders, vascular
disorders, weakness, hyper coagulation, venous
thromboses, rheumatic problems, nervous breakdown,
cerebral involution, and Alzheimers disease. These
consequences are wrongfully allotted to the lack
of estradiol and progesterone (a polluted concept)
whereas in fact they are the consequences of a lack of
male hormones (testosterone for general consequences
and dihydrotestosterone for local genital involution).
General Symptoms are the same in man suffering
from andropause disease described for the first time
in 1988. See: http://www. man.uk.georgesdebled.
org/history andropause.htm
Treatment of in androgens deficiencies and of
menopause disease with mesterolone
Orally administration of mesterolone in amounts
between 5 milligrams and 25 milligrams per day
constitutes the specific treatment. Each treatment
is unique for each woman after complete biological
check up. If there is no symptom, there is no disease.
Approaches to Aging Control. Vol 19. october 2015

If there is no disease no curative treatment is necessary. Loco motor weakness Women with higher
The treatment covers all indications for androgens with system dis- muscular atro- free T levels have greater
abilities phy lean body mass consis-
women. tent with the anabolic
rheumatic prob- effect of T. Bone density
lems is associated with T lev-
Mesterolone is a hormone prescribed for man to els (13).
Connective Tissue
compensate a lack of production of androgens. Used
Changes of Aging in
for man since 1967 it is henceforth in the public postmenopausal woman
(14).
domain. Its manufacturing technique is known. Cerebral irritability Compar ing women
with and without AD
No harmfulness was described to date. Before conse- nervous break-
(Alzheimer disease),
quences down Female brains levels of
menopause woman secretes each day of the cycle cerebral involu- estrogens and androgens
0.2 milligrams of testosterone = 200 micrograms tion are lower in AD cases
aged 80 years and older.
Alzheimers dis- Age-related depletion of
or 200,000 nanograms or 200,000,000 picograms.
ease androgens and estrogens
Mesterolone makes it possible to replace androgens in women may be rel-
evant to development of
whose production is strongly decreased in woman AD (15)

with menopause disease. It requires a traditional Why is mesterolone the treatment for androgens
deficiencies in woman? Mesterolone cannot be
compounding.
aromatized in estradiol (contrary to testosterone).
Its methyl radical inserted on Carbon 1 of the
Testosterone and Dihydrotestosterone (DHT)
testosterone confers this property. At physiological
deficiencies produce
doses Mesterolone does not influence the secretion
Symptoms Pathology of the pituitary gland so that the secretion of LH is
Functional hot flashes weakness of all smooth not modified (contrary to testosterone). Mesterolone
symptoms intestinal disten- musculatures of arte- prescribed in small amounts adds its effects to
sion ries, veins and bowel
(6,5 meters of smooth those of testosterone secreted by the organism.
swollen legs
musculature for the With the prescribed physiological pharmacological
small intestine;1,5 amounts doping is impossible and the overdose
meters of smooth mus-
culature for the large
too. Mesterolone is prescribed orally in amounts
intestine) varying between 5 milligrams and ten milligrams
Local con- chronic cystitis, sclerosis and inflamma- per day approximately. A substitution amount of 25
sequences i n c o n t i n e n c e, tion of bladder neck milligrams per day can be considered. The secretion
urgencies incon- sclerosis of vulva of LH by the pituitary gland is not inhibited (contrary
tinence)
dyspareunia to testosterone). Mesterolone molecular structure has
(painful or diffi- characteristics of dihydrotestosterone (DHT) which
cult copulation) is directly effective on the masculine sex organs of
Cardiovas- lipids disorders Low testosterone levels women (clitoris, labia majora and bladder neck) and
cular risk vascular disor- predict all-cause mor- on brain tissue (preventing Alzheimers disease) (15).
ders tality and cardiovascular Mesterolone can be prescribed alone.
hyper coagula- events in women: a pro-
tion spective cohort study in No risks of virilism with mesterolone treatment.
German primary care
venous throm- The treatment of androgens deficiency simply
patients (12).
boses consists in replacing missing secretions of testosterone
diabetes 2
(and dihydrotestosterone) thanks to mesterolone

21

properties. In this case the woman finds simply her
former physiological state and prevents the disastrous
consequences described above. Mesterolone used in
small amounts allows that. Virilism secondary to an
excessive administration of mesterolone should be the
consequence of a doping which must be avoided in
all cases.Virilism doesnt exist at physiological doses.
Androgens scientific biological deficit diagnosis
background. Concerning the determination of
testosterone and dihydrotestosterone in serum, The
GC-MS (Gas Chromatography- Mass Spectrometry)
is the most precise method (8). Direct RIA studies
are significant (9). Androgen glucuronides, instead of
testosterone, are interesting markers of androgenic
activity in women (10).
In our study the scientific biological diagnosis of
androgens deficiencies is made on the total pool of
androgens (RIA) in men since 1974 and in women
since 1998 concluding that the level of androgens
daily production is a key diagnosis. This method led
us to the concept of andropause disease (which is
different from hypogonadism) which treatment
is made with mesterolone. The 10 years study on
androgens deficiencies in women is founded on
RIA analyses made by a reference laboratory (Liege
University, CHU, and Liege, Belgium).
The androgens pool study reflects the daily production
of androgens. Androgens in serum (RIA) are: Total
testosterone, Dihydrotestosterone, Androstanediol
glucuronide, Androsterone glucuronide, DHEA,
DHEA Sulfate, 4-Androstenedione. Metabolites
in urine over 24 hours are: Total 17 ketosteroids,
Complete Chromatography of 17 ketosteroids,
Androstanediol glucuronides.
Clinical and biological diagnosis of androgens
deficit. Our study concludes that symptoms of
androgens deficiencies in women are correlated with
a low daily production of androgens reflected by low
serum androgens levels and low levels of metabolites
in serum and urine over 24 hours (the androgens
pool study) leading to treat those women successfully
with mesterolone since 1998 with spectacular results
and without any side effect.
22
www.approachestoagingcontrol.org
The general practice for androgens deficit
in woman. If there is no symptom there is no
disease. And if there is no disease no treatment is
necessary. More than 50 women are followed since
10 years. Our 10 years experience with mesterolone
prescription for androgens deficit in general practice
will continue as an increased number of women
ask to follow this treatment. Hormonal substitution
is founded on the clinical story and on a simplified
hormonal and biological check up (which is not
expensive) for each case individually (11).
Each general practitioner, each gynecologist, each
doctor will be aware of: The clinical diagnosis of
androgens deficiencies in women. The simplified
and non expensive biological diagnosis of androgens
deficiencies in women. The successful and cheap
treatment of androgens deficiencies in women with
mesterolone.
The global androgens pool was evaluated for research
purpose. A simplified biological diagnosis is often
enough in general practice. The masculine genitalia
of woman depend of the strong male hormone
(dihydrotestosterone) like male genitalia. Difficult
copulation, poor sensitivity of the clitoris and
consequently lack of libido are the consequences of a
lack of the strong male hormone (dihydrotestosterone)
production which lack doesnt induces effects on the
masculine genitalia of woman. Those sexual problems
are improved with mesterolone which molecular
structure is similar to the dihydrotestosterone
structure.
Incontinence, urgencies, chronic cystitis and
devastating disorder of interstitial cystitis can be
the consequences of a bladder neck sclerosis and
inflammation. Numerous bladder neck scleroses
in women are the consequences of androgens
deficiencies (mainly dihydrotestosterone) before
menopause (oral contraceptive) or after menopause
(menopause disease: see above). Those bladder neck
consequences (Incontinence, urgencies, chronic
cystitis and devastating disorder of interstitial cystitis)
due to androgens deficiencies may improve with
mesterolone. Results can be spectacular. Bladder neck
fibrosis provokes hypertrophy of bladder musculature
or weak bladder musculature. Bad opening of bladder
Approaches to Aging Control. Vol 19. october 2015
neck leads to secondary narrowing of terminal
ureter, hypertension in upper urinary tract and bad
function of the kidneys. In summary: destruction of
the urinary tract (16).
Mesterolone treatment has cured spectacularly a
woman with proved androgens deficiency (The
Georges Debleds study) whose bladder neck was
congestive with abnormal sensitivity. Urologist of this
woman had proposed an endoscopic surgery which
was postponed thanks the mesterolone treatment.
This woman is free of symptoms since 8 years. There
exists no description of mesterolone effect on bladder
neck in the world medical literature. Those findings
are disclosed here for the first time.
We have an experience about bladder neck sclerosis
(and its endoscopic surgery) in woman since 1974.
The role of dihydrotestosterone on the womans
bladder neck and the benefit effect of mesterolone on
this pathology need future investigations.
About mesterolone in woman. It was not
possible to publish those conclusions before because
consequences of menopause were wrongfully allotted
to the lack of estradiol and progesterone.This polluted
concept is still considered as a dogma by doctors
in general. Even more import and prescription of
mesterolone are not permitted by the US government.
Manufacturers reserved mesterolone only for men.
Our study was conducted during the last ten years
after the first conclusions of the WHI study in 2002.
The study was possible in Europe where mesterolone
is available under traditional compounding. After
the conclusions of the WHI study to date and the
recent judgment (June 2011) of the U.S. Supreme
Court (17) we disclose here for the first time in
a medical review the conclusions of our study on
mesterolone treatment for androgens deficiencies in
woman which is the real solution in the world for
women with androgens deficiencies before and after
menopause.
Conclusion
The menopause disease is a clinical entity with
a specific cause, consequences and treatment.
It is the consequence of a failure in androgens
productions which should be replaced. With the
help of governments and of manufacturers
exhaustive studies will be made to specify contours of
mesterolone as treatment for androgens deficiencies
in women. These future studies on large scale will be
conducted within the general framework of biology
of ageing.
Email: dr. georgesdebled@georgesdebled.org
www.woman.uk.georgesdebled.org/menopause
disease.htm
Bibliography
1. Hormones. E-E Baulieu and Paul A.Kelly. Hermann
publishers1990)
2. Twenty-four hours mean plasma testosterone
concentration decline in non premenopausal women.
Zumoff B, Strain GW,Miller LK, Rosner W. J Clin
Endocrinol Metab, 1995, 80:1429-1430.
3. Effects of oral contraceptives on breast epithelial
proliferation. Isaksson E, von Schoultz E, Odlind V,
Soderqvist G, Csemiczky G, Carlstrom K, Skoog L, von
Schoultz B. Department of Oncology, Radiumhemmet,
Karolinska Hospital, Stockholm, Sweden Breast Cancer
Res Treat2001 Jan; 65(2):163-169.
4. Impact of Oral Contraceptives on Sex
Hormone-Binding Globulin and Androgen Levels:
A Retrospective Study in Women with Sexual
Dysfunction. Panzer et al. The Journal of Sexual
Medicine, January 2006; 3:p.104-113.
5. Risks and benefits of estrogen plus progestin in
healthy postmenopausal women: principal results
From the Womens Health Initiative randomized
controlled trial. Rossouw JE, Anderson GL, Prentice
RL, LaCroix AZ, Kooperberg C, Stefanick ML,
Jackson RD, Beresford SA, Howard BV, Johnson
KC, Kotchen JM, Ockene J; Writing Group for the
Womens Health Initiative Investigators. JAMA 2002
Jul 17; 288(3):321-33.
6. Promotional tone in reviews of menopausal
hormone therapy after the Womens Health Initiative:
an analysis of published articles. Fugh-Berman
A, McDonald CP, Bell AM, Bethards EC, Scialli
AR.Department of Physiology and Biophysics,
Georgetown University Medical Center, Washington,
DC, USA.PLoS Med. 2011 Mar;8(3):e1000425.
Epub 2011 Mar 15.
7. Androgens and osteocalcin during the menstrual
cycle. Massafra C, De Felice C, Agnusdei DP, Gioia D,
23

Bagnoli F. Department of Obstetrics and Gynecology,
University of Siena, Italy. J Clin Endocrinol Metab.
1999 Mar;84(3):971-4.
8. Testosterone measured by 10 immunoassays
and by isotope-dilution gas chromatography-mass
spectrometry in sera from 116 men,women, and
children. Taieb J, Mathian B, Millot F, Patricot MC,
Mathieu E, Queyrel N, Lacroix I, Somma-Delpero
C, Boudou P. Hormonology Laboratory, A. Bclre
Hospital, 92141 Clamart, France. Clinical Chemistry
49, 1381-1395, 2003.
9. Androgen levels in adult females: changes with age,
menopause, and oophorectorny. Davison SL, Bell R,
Donath S, Montalto JG, Davis SR. J Clin Endocrinol
Metab, 2005, 90:3847-53.
10. Androgen glucuronides, instead of testosterone, as
the new markers of androgenic activity in women.
Labrie F, Blanger A, Blanger R Brub R, Martel pdf
C, Cusan L, Gomez J, Candas B, Castiel I, Chaussade
V, Deloche C, Leclaire J.. J Steroid Biochem Mol
Biol, 2006, 99:182-188.
11. Wide distr ibution of the ser um
dehydroepiandrosterone and sex steroid levels in
postmenopausal women: role of the ovary? Labrie
F, Martel C, Balser J. Endoceutics Inc, Quebec City,
Quebec, Canada. Menopause.2011 Jan: 18 (1):30-43.
12. Low testosterone levels predict all-cause mortality
and cardiovascular events in women: a prospective
cohort study in German primary care patients.
Sievers C, Klotsche J, Pieper L, Schneider HJ, Mrz W,
Wittchen HU, Stalla GK, Mantzoros C.Department
of Endocrinology, Max Planck Institute of Psychiatry,
Kraepelinstrasse 2-10, Munich, Germany. Eur J
Endocrinol. 2010 Oct;163(4):699-708. Epub 2010
Aug 4.
13. Higher serum free testosterone concentration
in older women is associated with greater bone
mineral density, lean body mass, and total fat mass:
the cardiovascular health study. Rariy CM, Ratcliffe
SJ, Weinstein R, Bhasin S, Blackman MR, Cauley
JA, Robbins J, Zmuda JM, Harris TB, Cappola
AR. Division of Endocrinology, University of
Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104, USA. J Clin Endocrinol Metab.
2011 Apr;96(4):989-96. Epub 2011 Feb 2.
14. Hormonal Influences Upon Connective Tissue
Changes of Aging, SOBEL H. and MARMORSTON
24
www.approachestoagingcontrol.org
J. Institute for Medical Research, Cedars of Lebanon
Hospital, and the Department of Biochemistry
and Nutrition and the Department of Medicine,
University of Southern California, Los Angeles,
California,in: PINCUS G (ed.) Recent Progress in
Hormone Research, vol. 14. Academic New York
1958.
15. Brain levels of sex steroid hormones in men and
women during normal aging and in Alzheimers
disease. Rosario ER, Chang L, Head EH, Stanczyk
FZ, Pike CJ. Davis School of Gerontology, University
of Southern California, Los Angeles, CA 90089, USA.
Neurobiol Aging. 2011 Apr;32(4):604-13. Epub 2009
May 9.
16. La pathologie obstructive congnitale de luretre
terminal. G. Debled: p. 446- 452; Acta Urologica
Belgica, 1971, 39, 371-465) see: pathology in: http://
www.georgesdebled.org/Pathologie uretre terminal.
17. High Court Rejects Pfizer Hormone Therapy
Lawsuit Appeal Date Published: Wednesday, June
22nd, 2011. The U.S. Supreme Court refused to hear
a Pfizer Inc. units appeal of a $58 million award in
a case against Premarin and Prempro menopause
drugs. Three Nevada women who contracted breast
cancer after taking the companys menopause drugs
were awarded the amount in a 2007 case. The rebuff
leaves the amount as the largest to be upheld on
appeal in thousands of hormone replacement drug
suits. Over six million women took Prempro and
other menopause drugs before a 2002 study pointed
out their links to cancer. At one point Pfizer and
his units faced more than 10,000 claims, according
to lawyers for former users. The Nevada Supreme
Court concluded jurors properly held Pfizers Wyeth
unit responsible for hiding the breast cancer risks
of Premarin and Prempro. The original 2007 case
resulted in an award totaling $134.1 million to Arlene
Rowatt, Jeraldine Scofield and Pamela Forrester. The
trial judge later reduced the verdict to $57.6 million.
Yearly sales of Wyeths hormone replacement drugs
exceeded $2 billion before a 2002 study, sponsored by
the U.S. National Institutes of Health, suggesting that
women using the medicines had a 24 percent higher
risk of breast cancer. Pfizer, the worlds largest drug
maker, acquired Wyeth in 2009 settled a third of the
pending cases over its Prempro menopause drug. The
company said last month that it set aside $772 million
to resolve claims over the medicine.
Approaches to Aging Control. Vol 19. october 2015

Toxic Metals, Cell Death and Ageing

Eleonore Blaurock-Busch PhD
Research director, Micro Trace Minerals nd Trace Minerals International Laboratories. D-91217
Hersbruck. Germany.

Abstract which engulf and degrade the affected cells. The

It is impossible to overstate the relevance of cell death,
and its influence on the ageing process. Toxic metal
exposure affects cell death, but research indicates that
simple nutritional measures can prevent premature
cell death and early ageing. In this paper, we evaluate
cell reactions to foreign metal substances and how to
intercept or slow the cascade of events leading to cell
destruction through antioxidant and detoxification
treatment.
Keywords: Ageing, Cell death, Environmental toxins,
antioxidants, chelation.
INTRODUCTION
Erypthosis
Neurodegeneration and ageing is attributed to the
damage inflicted on cells. Eryptosis, the suicidal death
of erythrocytes, is a condition that normally affects
defective cells. Lang et al state that eryptosis may
be a mechanism of defective erythrocytes to escape
hemolysis. Conversely, excessive eryptosis favors the
development of anemia. Conditions with excessive
eryptosis include iron deficiency, lead or mercury
intoxication, sickle cell anemia, thalassemia, glucose
6- phosphate dehydrogenase deficiency, malaria, and
infection with hemolysin-forming pathogens. [1]
Fller et al state that eryptosis is characterised by cell
shrinkage, membrane blebbing and cell membrane
phospholipid scrambling with phosphatidylserine
exposure at the cell surface. Phosphatidylserine-
exposing erythrocytes are recognised by macrophages,
researchers also report that triggers of eryptosis
include oxidative stress, aluminum, mercury, lead and
copper exposure. Diseases associated with accelerated
eryptosis include sepsis, malaria, sickle-cell anemia,
beta-thalassemia, glucose-6-phosphate dehydrogenase
(G6PD)-deficiency, phosphate depletion, iron
deficiency, hemolytic uremic syndrome and Wilsons
disease.[2]
When it comes to copper, an essential element
that can be potentially toxic, studies suggest that
increased copper and/or homocysteine levels in the
elderly could promote significant oxidant damage
to neurons and may represent an additional risk
factor in the development of Alzheimer s Disease
(AD) or related neurodegenerative diseases. [3]
Homocysteine potentiates copper and amyloid beta
peptide-mediated toxicity, a possible risk factor in
Alzheimers-type neurodegenerative pathways. [4]
Aluminum is largely considered nontoxic, however
Niemller et al demonstrated that aluminum ions
trigger eryptosis. The researchers state that eryptosis
was paralleled by release of hemoglobin, pointing to
loss of cell membrane integrity. In conclusion, Al3+
ions decrease cytosolic ATP leading to activation
of Ca2+-permeable cation channels, Ca2+ entry,
stimulation of cell membrane scrambling and cell
shrinkage. Moreover, Al3+ ions lead to loss of cellular
hemoglobin, a feature of hemolysis. Both effects
are expected to decrease the life span of circulating
erythrocytes and presumably contribute to the
development of anemia during Al3+ intoxication.[5]

25

Thus, eryptosis allows defective erythrocytes to
escape hemolysis, while excessive eryptosis favours
the development of anemia.
Many studies have shown a correlation between iron
deficiency and high blood lead concentration. Lead
intoxication induces eryptosis possibly through a
molecular pathway that includes oxidation, depletion
of reduced glutathione (GSH), increment of [Ca2+]
and -calpain activation. Erythrocytes incubated with
lead have exhibited major eryptosis. [6] Excessive
gold and cadmium exposure significantly induce
eryptosis, Sopjani states. The suicidal death of
erythrocytes is characterized by cell shrinkage and
this erythrocyte shrinkage results, because Cd2+
ions stimulate Ca2+ entry into erythrocytes, which
activates Ca2+ sensitive K+ channels leading to
erythrocyte shrinkage and which triggers Ca2+-
sensitive erythrocyte membrane scrambling leading
to phosphatidylserine exposure. [7]
Apoptosis
Nelson Fausto MD defines cell death as a complex
phenomenon that forms the basis for most disease
processes. He states that It is now known that there
are at least 2 distinct types of cell death: apoptosis
(also known as programmed cell death) and necrosis.
The major importance of this distinction between
types of cell death is that while necrosis is always a
pathological process, apoptosis may take place as a
physiological phenomenon that is essential for life.
Moreover, necrosis generally elicits an inflammatory
reaction while apoptosis is not accompanied by
inflammation. [8]
Ageing is due to cell destruction. Rupert Sheldrake,
biologist states as early as 1973 that dying cells also
have a chemical effect on neighbouring cells and,also
a physical effect as cell to cell contacts are broken. Cell
deaths within a tissue may affect the functioning of
the tissue as a whole: for example, the death of nerve
cells within the brain seems likely to affect pathways
or patterns of nervous conduction, perhaps leading
to the formation of new pathways or patterns. Such
cell deaths could act as a source of random change
within the nervous system that might not always
be deleterious. [9] Neurodegenerative diseases such
as Parkinsons Disease (PD), Huntingtons Disease
26
www.approachestoagingcontrol.org
(HD) and Alzheimers Disease (AD) to name a few
are the most common form of diseases caused by
an increase in cell death. Elemental mercury is able
to penetrate into the CNS, where it can be ionized
and trapped, contributing to its significant neurotoxic
effects. [10] Mercury easily inactivates sulfhydryl
groups in enzymes, which limits the bodys natural
detoxification ability.
Mitochondrial impairment is an attractive candidate
to explain neurodegeneration, states Levy et al.
Neurons are highly metabolically active and
dependent upon aerobic metabolism for energy.
Mitochondria accumulate mutations with aging.
Mitochondrial dysfunction can lead to insufficient
ATP production and the generation of ROS.
Furthermore, mitochondria are critical for the
regulation of apoptosis. [11]
Apoptosis, or the programmed cell death, is the
process of cell self-destruction that is marked by
the fragmentation of nuclear DNA. What leads
to apoptosis is a process that is activated either by
the presence of a stimulus or by the removal of a
stimulus or suppressing agent. Apoptosis is a normal
physiological process eliminating DNA-damaged,
superfluous, or unwanted cells, and when halted (as
by genetic mutation) may result in uncontrolled cell
growth and tumour formation.
Thus, apoptosis is an innate response of the cell to
protect the rest of the organism from a potentially
harmful agent such as chemical damage. Metal
overload, for example, can inactivate enzyme systems
and the initial insult that started with a foreign metal
substance initiates cell reactions. These lead to a
cascade of events and the destruction of the cell.
Certain hexavalent chromium compounds are
human carcinogens and animal research demonstrates
that exposure to soluble sodium chromate
results in apoptosis. Blankenship et al reports:
Morphological changes indicative of apoptosis, as
well as internucleosomal DNA fragmentation, were
detected 24hr after treatment with lead chromate or
soluble sodium chromate. All of the cells killed by
treatments with lead chromate particles underwent
apoptosis as the mode of cell death, and this was
accurately modelled in cell culture by continuous
Approaches to Aging Control. Vol 19. october 2015
treatments with low-dose soluble sodium chromate.
[12] More interestingly, the authors demonstrated
that pre-treatment of cells with antioxidant vitamins
prior to chromium exposure markedly inhibited the
chromosomal aberrations induced by both particulate
and soluble chromate compounds, even though
chromium adduct levels were not decreased by either
vitamin pre-treatment. Cell survival assays showed
that ascorbate, but not alpha-tocopherol, protected
cells from apoptosis induced by sodium chromate.
Ya Wen Chen et al demonstrated that inorganic
mercury causes pancreatic -cell death via the
oxidative stress-induced apoptotic and necrotic
pathways. [13] Intracellular mercury levels were
markedly elevated in HgCl2-treated HIT-T15 cells
and results suggest that HgCl2-induced oxidative stress
causes pancreatic -cell dysfunction and cytotoxicity
leading to apoptotic and necrotic cell death. The
authors also noted that HgCl2 significantly increased
ROS formation in HIT-T15 cells, and the antioxidant
-acetyl cysteine effectively reversed HgCl2-induced
insulin secretion dysfunction in HIT-T15 cells and
isolated mouse pancreatic islets. -acetyl cysteine
significantly reversed HgCl2-induced cell death-
related signals.The neurotoxicity of inorganic lead
(Pb) and mercury (Hg) in animals and humans is well
established, and exposure to these neurotoxic metals
generally results in serious adverse effects, ranging
from cancer to IQ deficit to peripheral neuropathy.
[14, 15] Woo-Sung Choi et al noted that exposure
to inorganic mercury and lead induces neuronal cell
death. [16] In epidemiologic studies of human adults,
cumulative lifetime lead (Pb) exposure has been
associated with accelerated declines in cognition. [17]
Recent data in cell culture has shown that brain
neurons are particularly vulnerable to degeneration
by apoptosis. Cotman and Su state, It appears as if
neurons are in a struggle between degeneration and
repair. As research advances it is critical to reduce the
stimuli that cause the neuronal damage and discover
the key intervention points to assist neurons in the
repair processes. [18]
Medical interest in Alzheimer Disease (AD) has led
to environmental exposure studies, which associated
chronic metal exposure, especially in nanoparticle
form, with late-onset Alzheimers disease. Of the
potentially toxic metals, mercury and lead show
causative effects, but aluminum, copper, zinc and iron
are also associated with neurodegeneration, leading
to AD. Also mentioned are pesticides, solvents and
particulate matter in air pollution. [19, 20]
In Alzheimers disease (AD), the hippocampus
is one of the first regions of the brain to suffer
damage; memory loss and disorientation are included
among the early symptoms. [21] In 2-4 week old
rats, lead-induced cell death in the hippocampus
was demonstrated in vivo [22], and Jung-Mi Han
et al stated that ascorbic acid has protective effects
against lead-induced apoptotic neurodegeneration.
[23] Rivire S et al found that Plasma vitamin C is
lower in AD in proportion to the degree of cognitive
impairment. Studies indicate that this cannot
explained by a lower vitamin C intake, possibly an
indication that toxic metal exposure leads to an
increase in reactive oxygen species (ROS), which
requires more vitamin C as a protective factor for the
prevention of ROS-induced cell injury.
Novel Approaches
The widespread occurrence of potentially toxic
metals in the environment increasingly poses a threat
to human health. Research indicates that toxic metal
exposure significantly influences cell ageing, which
can lead to premature cell death. However, research
also demonstrates that it is possible to slow cell
destruction. By reducing a persons toxic burden, we
intercept cell death and slow down the disease and
ageing process.
Diagnosing acute or chronic metal exposure
Diagnostic measures that enable us to evaluate the
severity of a persons toxic metal load involve blood,
urine, hair testing etc. We must, however recognize
that each of those tests provides information about
one particular aspect. No test tells all and the medical
diagnosis must be made based on a number of
medical and biochemical information. Monitoring
the complexity of toxic exposures, resulting in a
confident diagnosis involves knowledge of the patient
history combined with examination findings. It may
involve more than one test.
27

Blood metal testing, generally favoured in
conventional medicine, allows the detection of
immediate metal exposure and thus is of value in
the diagnosis of acute intoxication. The test is of
lesser value for the detection of a subclinical, chronic
exposure, however randomised population studies
as initiated by environmental agencies demonstrate
that due to environmental factors elevated blood
concentration of specific toxic metals such as lead,
mercury or cadmium are more common than
expected in people of all ages.
The most useful specimen for metal testing is whole
blood, which requires little handling, and because
whole blood does not involve cell packing or tube
transfers, the potential for external contamination is
greatly reduced. False high blood metal concentration
are less likely to occur as they might with erythrocyte,
serum or plasma testing.
Similar principles apply to urine testing. A spot or
random urine or the 24h urine collection, often used
in occupational medicine allows the determination
of an acute exposure. It also allows the detection of a
subclinical, low exposure due to lifestyle (i.e. smoking)
and environment (occupation, water, air pollution,
dietary or pharmaceutical intake). People living in
industrial or agricultural areas where toxic exposure
is greater than expected, urinary metal concentrations
more often rise above baseline urine reference ranges,
whereas the toxic metal concentration in the urine
of people living in cleaner environments is generally
lower, reflecting a reduced daily exposure.
The provocation or challenge urine, a rather new
means of diagnosing chronic metal exposure,
involves the administration of an antidote, also called
chelation agent, either as an injectable or given orally.
These tests are useful for the detection of chronic
exposures. To state it simply, the use of chelating
agents allows the detoxification of organ systems that
have accumulated metals over a given time. Among
those chemical agents are DMSA (Dimercapto
Succinic Acid), DMPS (Dimercaptopropansulfonic
acid or the EDTAs (Ethylenediaminetetraacetic acid).
Each of those agents is capable of chelating a broad
range of metals, but nevertheless has a specific affinity
and binding ability with specific metals. Thus, the use
28
www.approachestoagingcontrol.org
of chelating agents requires knowledge. To use them
safely, involves following protocols.
Treatment Possibilities
After diagnostic results identified the degree of
intoxication and allowed us to identify the main
culprit, we can select the appropriate therapeutic
approach. Diagnostic and treatment protocols for
the use in acute and chronic intoxications have been
established. [24]
DMSA (Dimercapto Succinic Acid)
In 1984, the US Federal and Drug Agency (FDA)
approved oral DMSA as an Orphan drug and In 1991,
the oral use of DMSA was officially approved for lead
intoxication in children. Recommended treatment
doses range from 10-30mg/kg body weight, however
a number of protocols provide varying treatment
approaches. DMSA side effects are rare, usually
limited to digestive discomfort or headaches.
DMPS (Dimercaptopropansulfonic acid)
DMPS has been registered in Germany since 1997
under the name Dimaval (Heyl, Berlin) and was
registered under the same name in Taiwan in 2002.
DMPS is available in capsule form for oral treatment
(1 capsule contains 100mg), and as 5ml ampules
(250mg) for intravenous application (IV or IM).
DMPS is routinely used in clinical practice for its
diagnostic and detoxification abilitiy. The use of
DMPS, administered either intravenously or orally,
is highly effective for the binding and elimination of
arsenic, mercury and lead, and has a strong copper
binding ability. Side effects include allergy reaction,
after repeated use only.
Successful clinical trials have been conducted in
Germany, the USA (University of Rochester), the
Iraq, Bangladesh and the Philippines.
EDTA (Ethylenediaminetetraacetic acid)
With the addition of magnesium, this chelating agent
NaMgEDTA is most effective in the treatment of
vascular problems. The TACT study (Trial to Assess
Chelation Therapy), a randomized, double-blind
trial involving intravenous NaMgEDTA Chelation
Approaches to Aging Control. Vol 19. october 2015
provided surprising benefits for patients who had
suffered at least one Myocardial Infarct (MI) and
was particularly beneficial to increase circulation in
diabetics.
Na2EDTA easily binds with calcium. The drug
is administered intravenously at a rate of 16.6mg/
minute or 1gr/h, never faster. It is recommended
in the treatment of hypercalcemia, atherosclerotic
disease and metal detoxification. It is an effective
chelator for lead, cadmium and many other toxic
metals, including radioactive elements. Side effects are
rare, usually due to added substances such as Vitamin
B1. Na2EDTA is contra-indicated in children or
patients suffering from parathyroid disease.
Antioxidants
For preventive measures, antioxidant therapy provides
an easy and safe intervention method. As has been
outlined in this paper, specific antioxidants such as
vitamin C and N-acetyl cysteine have the ability to
intercept cell destruction, provided sufficient amounts
are administered at the time of, or shortly after
exposure. Treatment protocols are sparse. In general
antioxidant treatment is based on type and extent of
exposure, and patient tolerance.
Summary
Toxic metal exposure affects cell life. If not
intercepted, an acute or chronic intoxication leads
to cell shrinkage and cell death, causing premature
ageing and a wide range of diseases. Early detection
through proper diagnosis and appropriate treatment
with antioxidants and/o chelation therapy allow
early intervention. This combined effort supports and
promotes cell heath and cell life.
References
1 Lang F1, Lang KS, Lang PA, Huber SM, Wieder T.
Mechanisms and significance of eryptosis. Antioxid
Redox Signal. 2006 Jul-Aug;8(7-8):1183-92.
2 Fller M, Huber SM, Lang F. Erythrocyte
programmed cell death.UBMB Life. 2008
Oct;60(10):661-8
3 Donnelly PS, Xiao Z, Wedd AG. Copper and
Alzheimers Disease. Current Opinion in Chemical
Biology. 2007;11(2):128-133
4 White AR, Huang X, Jobling MF, Barrow CJ,
Beyreuther K, Masters CL, Bush AI, Cappay R.
Homocysteine potentiates copper- and amyloid
beta peptide-mediated toxicity in primary neuronal
cultures: Possible risk factors in the Alzheimers-type
neurodegenerative pathways. J Neurochem. 2001;76:
1509-1520
5 Niemoeller OM, Kiedaisch V, Dreischer P, Wieder
T, Lang F. Stimulation of eryptosis by aluminium ions.
Toxicology and Applied Pharmacology; 2006; 217(2):
168175
6 Aguilar-Dorado IC, G, Hernndez, Quintanar-
Escorza MA, Maldonado-Vega M, Rosas-Flores
M, Caldern-Salina:s JV. Eryptosis in lead-exposed
workers. Toxicology and Applied Pharmacology,
2014; 281(2): 195202
7 Sopjani M. Eryptosis induced by gold and cadmium
ions. Thesis, E.Karls Universitt Tbingen. Nov.18,
2014
8 Fausto N. Cell Injury, Cell Death. 2006 Study
Guide. nfausto@u.washington.edu
9 Sheldrake R. The Ageing, Growth and Cell Death.
Nature. 1974; 250(5465): 381-385
10 Davidson PW, Myers GJ, Weiss B. Mercury
exposure and child development outcomes. Pediatrics.
2004; 113:10231029.
11 Levy OA, Malagelada C, Greene LA. Cell Death
Pathways in Parkinsons disease: proximal triggers,
distal effectors, and final steps. Apoptosis. 2009;
14(4):478-500
12 Blankenship LJ, Carlisle DL, Wise JP, Orenstein
JM, Dye LE 3rd, Petierno SR. Induction of apoptotic
cell death by particulate lead chromate: differential
effects of vitamins C and E on genotoxicity and
survival. Toxicol Appl Pharmacol. 1997;146(2):270-
80.
13 Ya Wen Chen, Chun Fa Huang, Ching Yao Yangc,,
Cheng Chieh Yen, Keh Sung Tsai , Shing Hwa Liu.
29
 www.approachestoagingcontrol.org

Inorganic mercury causes pancreatic -cell death via and Cerebellum. J Biochem Mol Toxicol. 2007;21(5):
the oxidative stress-induced apoptotic and necrotic 265272.
pathways. Toxicology and Applied Pharmacology.
22 Sharifi AM, Baniasadi S, Jorjani M, Rahimi
2010; 243(3): 323331
F, Bakhshayesh M. Investigation of acute lead
14 Fitsanakis VA, Aschner M. The importance of poisoning on apoptosis in rat hippocampus in vivo.
glutamate, glycine, and gamma-aminobutyric acid Neuroscience Letters. 2002; 329: 4548.
transport and regulation in manganese, mercury and
23 Jung-Mi Han, Byung-Joon Chang, Tian-Zhu Li,
lead neurotoxicity. Toxicol Appl Pharmacol. 2005;
Nong-Hoon Choe, Fu-Shi Quan, Bong-Jun Jang,
204:343354
Ik-Hyun Cho, Hea-Nam Hong, Jong-Hwan Lee.
15 Taber KH, Hurley RA. Mercury exposure: effects Protective effects of ascorbic acid against lead-induced
across the lifespan. J Neuropsychiatry Clin Neurosci. apoptotic neurodegeneration in the developing rat
2008;20:389. hippocampus in vivo. Brain Research. 2007; 1185:
68-74
16 Woo-Sung Choi, Su-Jin Kim, Jin Suk
Kim. Inorganic lead (Pb)- and mercury (Hg)- 24 Blaurock-Busch E. Toxic Metals and Antidotes.
induced neuronal cell death involves cytoskeletal MTM Publ. 2010. E-book http://www.
reorganization. Lab Anim Res. 2011;27(3): 219225. microtraceminerals.com/en/books-by-eblaurock-
busch/e-book-chelation/
17 Bakulski KM, Rozek LS, Dolinoy DC, Paulson
HL, Hu H. Alzheimers Disease and Environmental
Exposure to Lead: The Epidemiologic Evidence and
Potential Role of Epigenetics. Curr Alzheimer Res.
2012;9(5):563573.
18 Cotman CW1, Su JH. Mechanisms of neuronal
death in Alzheimers disease. Brain Pathol.
1996;6(4):493-506.
19 Caldern-Garcidueas L, Vojdani A, Blaurock-
Busch E, Busch Y, Friedle A, Franco-Lira M, Sarathi-
Mukherjee P, Martnez-Aguirre X, Park SB, Torres-
Jardn R, DAngiulli A. Air pollution and children:
neural and tight junction antibodies and combustion
metals, the role of barrier breakdown and brain
immunity in neurodegeneration. J Alzheimers Dis.
2015; 43(3):1039-58
20 Caldern-Garcidueas L, Reed W, Maronpot RR,
Henrquez-Roldn C, Delgado-Chavez R, Caldern-
Garcidueas A, Dragustinovis I, Franco-Lira M,
Aragn-Flores M, Solt AC, Altenburg M, Torres-
Jardn R, Swenberg JA. Brain inflammation and
Alzheimers-like pathology in individuals exposed to
severe air pollution.Toxicol Pathol. 2004;32(6):650-8.
21 Chao SL, Moss JM, G. Harry JG. Lead-induced
Alterations of Apoptosis and Neurotrophic Factor
mRNA in the Developing Rat Cortex, Hippocampus,

30
Approaches to Aging Control. Vol 19. october 2015
Endogenous opioid. A review
Liuyi He, Mario F. Muoz and Antonio Ayala
Dept. of Biochemistry and Molecular Biology. University of Seville. 41012-Sevilla. Spain.
Introduction
Endogenous opioid is the name given to a set of
substances which are produced endogenously and
have similar effect to morphin. In fact, they are
mainly produced by special neurons localized in the
central and peripheral nerve system. They play a key
role in the nociceptive system reducing the sensation
of pain by increasing pain tolerance and decreasing
perception and reaction to pain. At present, due to
their clinical importance, the relationship between
endogenous opioids and exogenous opioids is the
main area where investigations about endorphins
are focused on. Nonetheless, it must be stated that
endogenous opioids also interact with many other
areas of the body, taking part in other physiological
processes and regulating them. Stimulation of
the immune system and control of the behavior
(wellbeing, reward system) are two examples of
these. Recently, these other secondary functions
of endorphins are drawing the attention of more
and more researches, specially, those ones related
to stress, brain reward system, aging and other
pathophysiologies.
Classification
Endogenous opioids are a big family which includes
many types of widely different chemical substances,
but among them, three types can be stood out:
- The group of endorphins. This is a group
of endogenous opioid polypeptides
which are mainly synthesized by the brain
(hypothalamus and the nucleus of solitary
tract) and peripherally (pituitary gland,
adrenal gland and immune cells) especially
during periods of intense level of exercises.
(1)(2) The division of this group is made
into 3 main types of endorphins: alpha-
endorphin, beta-endorphin and gamma-
endorphin. All of them come from the same
precursor, proopiomelanocortin (POMC)
which produces beta-lipotropin and, in
turn, give rise to the 3 different types of
endorphins (Figure 1). Therefore, these 3
types of endorphins share a similar structure
(Figure 2). Nevertheless, since the discovery
of endorphins, only little investigations have
been made about alpha and gamma type
endorphins. This is because the potency of
beta-endorphins as a ligand is much higher.
Notwithstanding, it does not mean that
the other two can be ignored, in fact, there
are some roles (for example, in dopamine
metabolism) which only are performed by
alpha and gamma-endorphins, thereupon,
more investigations about these two types are
also needed. (3)
Figure 1. Cleavage of POMC giving rise the different
types of endorphin.
31

Structure
Alpha-endorphin Tyr-Gly-Gly-Phe-Met-Thr-
Ser-Glu-Lys-Ser-Gln-Thr-
(16 aa)
Pro-Leu-Val-Thr
Beta-endorphin Tyr-Gly-Gly-Phe-Met-Thr-
Ser-Glu-Lys-Ser-Gln-Thr-
(31 aa)
Pro-Leu-Val-Thr-Leu-Phe-
Lys-Asn-Ala-Ile-Ile-Lys-
Asn-Ala-Tyr-Lys-Lys-Gly-
Glu
Gamma-endorphin Tyr-Gly-Gly-Phe-Met-Thr-
Ser-Glu-Lys-Ser-Gln-Thr-
(17 aa)
Pro-Leu-Val-Thr-Leu
Figure 2. Structure of alpha, beta and gamma-endorphins
- The group of enkephalins. This is a group of
two endogenous opioids pentapeptides: met-
enkephalin and leu-enkephalin. Both differ
only in one amino acid (Figure 3) but they
share a common precursor, proenkephalin
A, which produce a higher number of
met-enkephalin than leu-enkephalin. For
this reason, met-enkephalin (Also known
as opioid growth factor) perform most of
the functions related to enkephalins in the
body (pain modulation, food and liquid
consumption, stimulation of the immune
system) which are suggested to be many.
However, recent researches about met-
enkephalin have been focused only on the
relationship between it and pancreatic cancer,
this is due to the possibility of using OGF as
a treatment for this type of cancer.(4) On
the other hand, leu-enkephalin has been
less studied, nevertheless, it is known it plays
some important roles in gonadal hormone
regulation.(5) Finally, the site of production
of enkephalins is widely distributed along
the central nerve system. For example, those
enkephalins which mediate pain perception
are produced by neurons of the spinal cord
and periaqueductal gray and those affecting
emotional responses act in limbic areas. But,
enkephalins are also found peripherally. This
fact supports the idea that encephalin is
involved in many physiological functions.
32
www.approachestoagingcontrol.org
Structure
Met-enkephalin Tyr-Gly-Gly-Phe-Met
Leu-enkephalin Tyr-Gly-Gly-Phe-Leu
Figure 3. Structure of alpha, beta and gamma-endorphins
- Dynorhpin is the last main group of
endogenous opioids. They are polypeptides
very rich in basic amino acids, specially, lysine
and arginine which grant them part of their
properties. The site of production is usually
distributed along the CNS, nevertheless,
the highest concentrations are found in the
brainstem, hypothalamus and spinal cord.
The reason of this wide distribution is that
depending on where they are produced,
dynorphins exert different physiological
functions, for example, dynorphins produced
by the parvocelular neurons of the supraoptic
nucleus take part in the pattering of electrical
activity and the ones produced in the arcuate
nucleus is part of the appetite regulation
pathway. (6) Dynorphins are cleavage
products of the protein prodynorphin.
This reaction is performed by the enzyme
proprotein convertase 2. Prodynorphins give
rise to different types of dynorphins where
two of them can be stood out: Dynorphin
A and dynorphin B (Figure 4). In general,
dynorphin A is more potent than dynorphin
B.(7)
Figure 4. Prodynorphin cleavage products.
Receptors
All of the endogenous opioids must bind to a receptor
in order to perform their function. Nonetheless,
depending on which is the receptor they bind to,
Approaches to Aging Control. Vol 19. october 2015
the function performed by the ligand can vary. These
receptors are G protein coupled receptors and there
are mainly big three types which are localized mainly
on prejunctional neurons:
- Mu-receptor (MOR). This type of receptor
is characterized by a high affinity for
morphins, therefore, it is the main mediator
in processes where morphin and morphin-
like compounds take part. There are 2 basic
types of MOR: 1 and 2. The first one has
a higher affinity for morphine and act on
supraspinal sites to produce analgesia. The
second one has a lower affinity for morphine
and produce analgesia by acting on the spinal
cord. Notwithstanding, both of them can
bind to other endogenous opioids such as
beta-endorphin, enkephalin and dynorphin,
but with a lower affinity. One last type of
MOR was first described in 2003, 1, but
this variant bind only to opiate alkaloid and
not peptides. (8)
- The second type of opioid receptor is
kappa opioid receptor (KOR). It binds
to dynorphins with high affinity, but it
can bind to other opioid agonists such as
ketocyclazocine. There are 3 different types:
1, 2, 3. Nonetheless, only 1 and 3 disorders. These are performed by the activation of
are functionally important. 1 is implied in
analgesic effects which start from spinal cord.
On the other hand, the analgesic effect of
3 agonist is supraspinal. (6) Nowadays, the
investigations related to KOR are focused on
the possibility of using this type of receptor
in the treatment against drug addiction. This
is possible due to KOR has been shown to
take part in the mechanism which lead to
stress-induced drug-seeker behavior. (9)(10)
(11)
- the neurobiological pathway which lead
The last big group is called delta opioid
receptor (DOR). It has a high affinity for
enkephalins but also for other compounds
such as deltorphin. The mediation of pain
(analgesic function) by this receptor is
performed through spinal cord, specifically,
DOR are mainly presented in dorsal horns.
Unlike KOR and MOR, DOR is not further
divided, since only one variation has been
identified so far but like MOR, traditional
investigations about DOR have been
focused on mechanism of opioid analgesia.
Nonetheless, investigations about the role of
DOR and its ligands as antidepressants have
increased recently. (12)
Figure 5. Opioid receptors location and responses mediated
by them.
Figure 6. Opioid Receptors-their agonists and antagonists
and endogenous ligands.
Why is endogenous opioid system important?
As it was said before, endogenous opioid system
takes part in not only many physiological pathway
of our body, but also many mechanism of different
the different opioid receptors which can be done by
either endogenous ligands (enkephalin, dynorphin)
or opioid receptor agonists/antagonists (Figure 6).
Therefore, in order to stand out the importance of
endogenous opioid system, a brief explanation of
its different roles in physiological and pathological
functions will be given:
- Alcohol consumption, it has been suggested
that the activation of endogenous opioid
system by alcohol consumption is part of
to alcohol dependence and abuse (initial
suggestion). This suggestion is supported
by mainly three lines of evidence: The
first line is the fact that opioid receptor
antagonists (nonselective antagonist and
antagonist selective for mu and delta
receptors) can significantly decrease alcohol
self-administration in rodents and monkeys
33

under different experimental conditions;
The second line is related to the increase of
endorphin and enkephalin gene expression
in isolated cells, tissues exposed to alcohol
or rodents after an event of acute alcohol
consumption; Finally, the last line is related
to some studies which state that alcohol
intake trigger a bigger response in the opioid
system of rodents and humans which have
a predisposition to alcohol consumption.
Namely, the increase of endorphins and
enkephalins is higher in subjects genetically
predispose to alcohol consumption. Apart
from these three lines of evidence, the fact
that a high concentration of endorphins
and enkephalin lead to feeling of well-being
or euphoria, give us the last point which is
needed to make the initial suggestion. (1)
- Stress is a natural response elicited by our
body to help us overcome life-threatening
situations. Nonetheless, our body not only
releases pro-stress compounds, but also
releases anti-stress compounds to prevent
chronic stress state which is detrimental.
Endogenous opioids are mediators which
are involved in both functions: On one side,
anti-stress effects of endogenous opioids
are mainly associated with MOR which
binds to enkephalin, beta-endorphin and
endomorphin with relatively high affinity.
This is the main function of endogenous
opioids related to stress. In general, deficiency
of these compounds (especially beta-
endorphin) lead to several effects related to
an enhanced stress response, for example,
mouse with beta-endorphin deficiency tend
to be more aggressive under stress condition.
(13) On the other side, pro-stress effects are
related to dynorphins which bind to KOR.
Nevertheless, this function is less understood.
- Immune system. As said before, endogenous
opioids are also produced by nonneuronal
cells being immune cells the best example for
this. Endogenous opioids produced by these
cells play various key roles in the immune
system. The first one is immune-derived
opioid antinociception, namely, they can
exert their usual function of morphin-like
34
www.approachestoagingcontrol.org
substance in order to prevent hyperalgesia
which is common during infections and
inflammations. Nonetheless, the second role
is the one we are interesting in. Endogenous
opioids are able to mimic the roles of many
cytokines and take part in the regulation
process of the immune system, this includes
regulation of natural killer activity, antibody
production, mitogen-induced proliferation...
All of these processes are crucial for the
correct homeostasis of the immune system,
namely, endogenous opioids are one of the
key for successful aging, because having a
homeostatic immune system is a necessary
condition for successful senescence.
Notwithstanding, this second role of
endogenous opioids is only beginning to
be discovered, therefore, more studies need
to be performed to know more about the
relationship between endogenous opioids
and immune system. (14)(15) Even so, we
already know some methods which are able
to increase the amount endogenous opioids
in a non-pathological way and therefore, they
are helpful to keep us healthy. Among them,
sunbathing, exercise (known as runners high
phenomenon) or even consuming sugar are
the most effective ways. (16)(17)(18)
Conclusion
There are many types of endogenous opioids, but
endorphin, enkephalin and dynorphin are the most
important. Matching with these three types of opioids,
there are three types of receptor, MOR, KOR, DOR.
The stimulation of these receptors can activate
many different pathways which take part in different
functions. The most investigated and used one is the
analgesic function. Nonetheless, other functions such
as immune system regulation or stress mediation are
important as well, therefore, more studies in these
areas need to be performed.
Acknowledge
This work was supported by Consejeria de
Economa, Innovacion y Ciencia de la Junta de
Andalucia (Spain), Ref. P10-CTS-6494.
Approaches to Aging Control. Vol 19. october 2015

References and receptors. Global Journal of Pharmacology,

3(3), 149-153.
(1) Janice C. Froehlich, P. D. (1997). Opioid peptides.

Alcohol Health & Research World, 21(4), 132-136. (7) James, I. F., Fischli, W., & Goldstein, A. (1984).

Opioid receptor selectivity of dynorphin

(2) Jessop, D. S. (1998). Beta-endorphin in the
gene products. The Journal of Pharmacology and
immune system--mediator of pain and stress?
Experimental Therapeutics, 228(1), 88-93.
Lancet (London, England), 351(9119), 1828-1829.

doi:S0140-6736(05)78799-7 [pii] (8) Stefano, G. B. (2004). Endogenous morphine: A

role in wellness medicine. Medical Science Monitor

(3) Kameyama, T., Ukai, M., Noma, S., & Hiramatsu,
: International Medical Journal of Experimental and
M. (1982). Differential effects of alpha-, beta- and
Clinical Research, 10(6), ED5. doi:5613 [pii]
gamma-endorphins on dopamine metabolism

in the mouse brain. Brain Research, 244(2), 305- (9) Redila,V. A., & Chavkin, C. (2008). Stress-induced

309. doi:0006-8993(82)90089-0 [pii] reinstatement of cocaine seeking is mediated

by the kappa opioid system. Psychopharmacology,

(4) Zagon, I. S., & McLaughlin, P. J. (2014). Opioid 200(1), 59-70. doi:10.1007/s00213-008-1122-y
growth factor and the treatment of human [doi]
pancreatic cancer: A review. World Journal
(10) Xuei, X., Dick, D., Flury-Wetherill, L., Tian, H.
of Gastroenterology : WJG, 20(9), 2218-2223.
J., Agrawal, A., Bierut, L., . . . Edenberg, H. J.
doi:10.3748/wjg.v20.i9.2218 [doi]
(2006). Association of the kappa-opioid system
(5) Dudas, B., & Merchenthaler, I. (2002). Close with alcohol dependence. Molecular Psychiatry,
juxtapositions between LHRH immunoreactive 11(11), 1016-1024. doi:4001882 [pii]
neurons and substance P immunoreactive
(11) Xi, Z. X., Fuller, S. A., & Stein, E. A. (1998).
axons in the human diencephalon. The Journal
Dopamine release in the nucleus accumbens
of Clinical Endocrinology and Metabolism, 87(6),
during heroin self-administration is modulated
2946-2953. doi:10.1210/jcem.87.6.8558 [doi]
by kappa opioid receptors: An in vivo fast-cyclic
(6) Anupama, K., Sreemantula, S., & Shaik, R. (2009). voltammetry study. The Journal of Pharmacology

Endogenous opioids: Their physiological role and Experimental Therapeutics, 284(1), 151-161.

35
 www.approachestoagingcontrol.org

(12) Al-Hasani, R., & Bruchas, M. R. (2011). Immunoreactive beta-endorphin increases after

Molecular mechanisms of opioid receptor- an aspartame chocolate drink in healthy human

dependent signaling and behavior.
Anesthesiology, 115(6), 1363-1381. doi:10.1097/
ALN.0b013e318238bba6 [doi]
(18) Farrell, P. A., Gates, W. K., Maksud, M. G., &
(13) Vaanholt, L. M., Turek, F. W., & Meerlo, P.
(2003). Beta-endorphin modulates the acute
response to a social conflict in male mice but
does not play a role in stress-induced changes
in sleep. Brain Research, 978(1-2), 169-176.
doi:S0006899303028051 [pii]
subjects. Physiology & Behavior, 50(5), 941-944.
doi:0031-9384(91)90418-N [pii]
Morgan, W. P. (1982). Increases in plasma beta-
endorphin/beta-lipotropin immunoreactivity
after treadmill running in humans. Journal of
Applied Physiology: Respiratory, Environmental and
Exercise Physiology, 52(5), 1245-1249.

(14) Bidlack, J. M. (2000). Detection and function

of opioid receptors on cells from the immune

system. Clinical and Diagnostic Laboratory

Immunology, 7(5), 719-723. doi:0098 [pii]

(15) Kapitzke, D., Vetter, I., & Cabot, P. J. (2005).

Endogenous opioid analgesia in peripheral tissues

and the clinical implications for pain control.

Therapeutics and Clinical Risk Management, 1(4),

279-297.

(16) Tejeda, H. A., & Bonci, A. (2014). Shedding

UV light on endogenous opioid dependence.

Cell, 157(7), 1500-1501. doi:10.1016/j.

cell.2014.06.009 [doi]

(17) Melchior, J. C., Rigaud, D., Colas-Linhart, N.,

Petiet, A., Girard, A., & Apfelbaum, M. (1991).

36
Approaches to Aging Control. Vol 19. october 2015

Treatment with mesenchymal stem cells in an animal

model of parkinsons disease
Jos Angel Naranjo, Antonio Ayala, Mercedes Cano*, Sandro Argelles,Victoria Ruiz, Mario Muoz.
Departament of Biochemistry and Molecular Biology. *Department of Physiology. University of
Sevilla.41012-Sevilla..

Introduction differentiation of endogenous stem cells according to

Biological aging is characterized by a progressive
loss of organisms functions from their physiological
optimal values along the time as result of intrinsic
and extrinsic factors (1; 2; 3). Several hypotheses
may explain the causes of aging but only few are
thoroughly accepted. One of them is related to the
the loss of capacity of cell replacement in damaged
tissues (4;5).
Most of the tissues have a specific quantity of adult Stem
Cells (SC) responsible for their periodic renovation
and regeneration (6; 7). SC have the ability to self-
renew by symmetric divisions and the potentiality
to differentiate into other cell types depending on
their multipotency (9; 10). The understanding of
SC viability and regenerative capacity is one of the
pillars of Regenerative Medicine (8). One type of
adult SC are the mesenchymal SC derived from
adipose tissue (ADSC), which have been used in our
study. There are several procedures by which ADSC
can be used to repair and regenerate tissues. For
instance, ADSC transplantation into damaged tissues
provokes a cytokine- and growth factor-induced
response which stimulate their recovery ought to a
SC microenviroment (niches) modulation caused
by the ADSC presence. This could stimulate the
their lineage pathway.
In this paper, the therapeutic potential of ADSC in
Parkinson Disease (PD) has been studied in animal
models by an intracranial injection of bacterial
Lipopolysaccharide (LPS) in the substantia nigra (SN).
This brain area, which is rich in dopaminergic neurons,
is degenerated in PD. LPS-mediated dopaminergic
neurotoxicity depends on the microglial activation,
which releases pro-inflammatory factors (NO,
TNF-, IL-1) (11; 12). Moreover, it was found
that the neurotoxicity was mainly mediated by the
activation of microglial NADPH-oxidase, generating
ROS and neurotoxic factors (12). The aim of this
study is to show how stem cells engraftments in an
animal model of PD can produce a histological and
functional improvement.
Material and methods
Adipose stromal vascular fraction isolation
The stromal vascular fraction (SVF) was obtained from
adipose tissue removed from lumbar or abdominal
area of Wistar rat anesthetized with chloral hydrate
6%. Adipose tissue was washed with phosphate buffer
saline (PBS) to eliminate as much blood as possible.
The tissue was enzymatically processed by a

37
 www.approachestoagingcontrol.org

collagenase solution incubated at 37 C for 150
minutes on an orbital shaker. Then, an equal volume
of stop medium (Dulbeccos Modified Eagle
Medium-DMEM) with 10% fetal bovine serum (FBS)
was added and samples were centrifuged at 600xg for
10 min at 4C. The pellet (SVF) was resuspended in
DMEM + FBS + antibiotic and the cell suspension
was filtered through a 40 m pore-size filter.
Magnetic separation of the ADSC using the
CD90.1 MicroBeads.
In order to perform the extraction of the ADSC from
a cellular suspension of SVF, a magnetic separation
system based on MACS technology (Magnetic
Cell Sorting) were used. This procedure is based on
the use of the so-called microbeads which are a
magnetic micro-particles associated with antibodies
that specifically recognize antigens on surface of
stem cells. In our case, the CD90.1 (THYmocyte
differentiation antigen-1.1) Microbeads antibody
was chosen. After magnetic separation, cells were
centrifuged, resuspended and cultivated with
DMEM+FBS+antibiotic and finally incubated at 37
C.
The Countess Automated Cell Counter was used
to determine the number of cells using the Trypan
Blue assay. To verify if obtained cells are ADSC, the
presence of CD90.1 and CD29 surface markers and
the absence of CD11b, CD34, CD45 was checked by
flow cytometry, using monoclonal antibodies labeled
with fluorophores (mouse antiCD34-PE, CD45-PE,
CD11b-PE, CD29-APC and CD90.1-FITC).
luminescent proteins have enabled the visualization
of stem cells over in vivo and ex vivo periods of time.
ADSCs were infected with pSIN-DUA-Luciferasa-
GFP2 lentivirus with 8 g/ml polybrene for
24 h. The percentage of GFP-positive cells was
determined by flow cytometry. Bioluminiscence
activity was measured in vitro and in vivo using
Xenogen IVIS Lumina II imaging system incubation
with D-Luciferin (150 g/ml). For the in vivo study,
D-Luciferin was injected intraperitoneally at a dose
of 150 mg/kg body weight.
Surgical procedure and injecion of LPS and
stem cells.
Rats were anesthetized with chloral hydrate (400
mg/kg) and positioned in a stereotaxic apparatus
(Kopf Instruments, Tujunga, CA, USA) in accordance
with the brain atlas of Paxinos and Watson (1986).
Intracerebral surgery was performed by trepanation
of the skull and injections into SN were made 5.4
mm posterior, 1.8 mm lateral, and 8.3 mm ventral to
bregma (Fig. 1.). Animals received 2 l of vehicle (1%
Monastral Blue) inert tracer in saline or 2 l of LPS (8
g) (n = 4) or 2 l of ADSC solution + LPS (3 x 105
cells + 8 g) in the SN. Experiments were carried out
in accordance with the Guide-lines of the European
Union Council following the Spanish regulations for
the use of laboratory animals and approved by the
Scientific Committee of the University of Seville.

In vivo tracking of ADSC.

In vivo tracking systems using gene reporters is a useful

tool for cell tracking before, during and after cells
engraftment. Optical imaging technologies combined
with the use of genetically encoded fluorescent and Figure 1. Intracerebral surgery

38
Approaches to Aging Control. Vol 19. october 2015
Functional study of learning in operant
conditioning chamber (Skinner box).
The experiment conducted in a Skinner box was as
follows: the animal was starved for 24 hours. Later,
once the rat pressed the lever, a mechanical device
drops a pellet of food to the feeder installed inside the
same box. After a period of time, the rat activates the
lever and the food falls into the feeder. Rat eats pellets
and returns to the lever. The process is repeated with
insistence and rat runs incessantly from the feeder to
the lever. The scheme of learning is the following:
S (conditioned Stimulus) R (conditioned
Response) C (Consequence or Enhancer)
Goal-oriented learning
SRC
Habit learning
SR
S: lever inside the box. R: pressure on the lever.
C: pellet of food
The rats were trained with programs of fixed and
variable series which result in a learning goal through
enhancers. As a consequence of overtraining, the rats
learnt a habit (i.e., some it is done unconsciously).
On other hand, we could check if the animal kept
pressing the lever even when the enhancer was
devalued.
Immunohistochemical analysis
After perfusing the animal with intracardiac
injection of paraformaldehyde, frontal lobes were
removed. Thaw-mounted 20 m coronal sections
were cut with a cryostat at 20 C and mounted
on gelatin-coated slides. The primary antibodies
used were rabbit-derived anti-tyrosine hydroxylase
(anti- TH, Sigma; 1:300) and mouse-derived OX-6
(Serotec, Oxford, U.K.; 1:200). OX-6 is directed
against a monomorphic determinant of the rat
major histocompatibility complex (MHC) class II
antigens, expressed by activated microglia but not
by the resting cells. Incubations and washes for all
the antibodies were in Tris-buffered saline (TBS), pH
7.4. All work was performed at room temperature.
Sections were washed and then treated with 0.3%
hydrogen peroxide in methanol for 20 min, washed
again, and incubated for 60 min in a humid chamber
in a solution containing TBS and 1% horse serum
(Vector Laboratories, Burlingame, CA, USA) for
OX-6 immunostaining or goat serum (Vector)
for TH immunostaining. Slides were drained and
further incubated with the primary antibody in
TBS containing 1% horse/goat serum and 0.25%
Triton-X-100 for 24 h. Sections were then incubated
for 2 h with biotinylated horse anti-mouse IgG
(Vector, 1:200) for OX-6 immunostaining or
biotinylated goat anti-rabbit IgG (Vector, 1:200) for
TH immunostaining. The secondary antibodies was
diluted in TBS containing 0.25% Triton-X-100,
and prior to its addition three 10-min rinses in TBS
were performed. Sections were then incubated with
ExtrAvidin1-Peroxidase solution (Sigma, St Louis,
MO, USA; 1:100).The peroxidase was visualized with
a standard diaminobenzidine/hydrogen peroxide
reaction for 5 min.
For the measurement of the areas expressing OX-6
and TH, the AnalySIS1 image software (Soft Imaging
System GmbH, Mu nster, Germany) coupled to a
Polaroid DMC camera (Polaroid, Cambridge, MA,
USA) attached to a Leika light microscope (Leika
Mikroskopie, Wetzlar, Germany) was used. For each
animal, five sections (sampling fraction 1:3) were
39
 www.approachestoagingcontrol.org

systematically sampled along the anterior-posterior
axis from a random starting point, following
stereological criteria. The number of TH-positive
neurons in the SN was estimated using a fractionator
sampling design. Counts were made at regular
predetermined intervals (x = 150 m and y = 200
m) within each section. An unbiased counting frame
of known area (40 m x 25 m = 1000 m2) was
superimposed on the tissue section image under a
100x oil immersion objective. Therefore, the area
sampling fraction was 1000/(150 x 200) = 0.033.
The entire z-dimension of each section was sampled;
hence, the section thickness sampling fraction was
1. In all animals, 20 m sections, each 100 m apart,
were analyzed; thus, the fraction of sections sampled
was 20/100 = 0.20. The number of neurons in
the SN was estimated by multiplying the number
of neurons counted by the reciprocals of the area
sampling fraction and the fraction of section sampled.
Statistical analysis
Results are expressed as mean SD. Means were
compared by One-Way ANOVA followed by the
LSD test for post hoc multiple range comparisons. An
alpha level of 0.05 was used. The IBM SSPS Statistics
21 package was used for the analyses.
drawn on a plot displaying flow cytometry data. On
the histogram plot (Fig. 2B) cells are now plotted as
Counts on the y-axis versus GFP fluorescence on the
x-axis. The percentage of GFP+ cells were 69,2%.
Figure 2. Density plot diagram (A) and Histogram plot of
GFP expression (B). Measurement by flow cytometry in
channel FL1.
In vivo tracking of the ADSC.
Bioluminiscence activity in vitro and vivo was
determinated using Xenogen IVIS Lumina II
imaging system and 3 x 105 cells in SN (5.5 mm
posterior, 1.5 mm lateral, and 8.3 mm ventral). The
injected cells emited 1.72 x 108 5.83 x 107 p/s /
cm2/sr at 5 min of exposure time (Fig. 3). This result
shows the possibility of in vivo tracking in central
nervous system.

Results

Transfection of ADSC with lentivirus

Transfected cells reached the confluence in 2 days.

Then, the percentage of infected cells was determined
by flow cytometry.

On the density plot diagram (Fig. 2A), each dot or

point represents an individual cell that has passed
through the instrument. Cells have been gated Figure 3. Photographs of optical bioluminescence 1 day after
as region 1 (R1). Region simply refers to an area transplantation (1,721108 p/s/cm/sr).

40
Approaches to Aging Control. Vol 19. october 2015
Effect of LPS on the microglial population.
In response to certain brain damage, either immune
or inflammatory stimulus, microglia suffers significant
morphological and metabolic changes. The activated
microglia increases the expression of certain proteins,
forming the so-called activated microglia (13, 14).
Thus, this study aims to study the degree of activation
of microglia in the SN in response to LPS injection
by using the OX-6 antibody, which only marks the
activated microglia.
Injection of vehicle into the SN induced only a
slight OX-6 immunoreactivity around the injection
track (Fig. 4). Besides, injecting LPS induced a
clear microglial activation. LPS+ADSC injections
prevented by 50 % this activation.
Figure 4. Quantification of the area expressing OX-6
immunoreactivity in the SN. Results are mean SD.
aStatistical signification compared with control and
bstatistical signification compared with LPS group p< 0.01.
Effect of LPS on the number of TH positive
neurons.
The injection of vehicle did not affect the normal
pattern of TH immunostaining in these structures
(Fig. 5). The number of TH-positive neurons in
the SN diminished significantly after the injection
of LPS. However, in this case the decrease was
partially limited thanks to the ADSC, because the
results obtained have a higher standar deviation ( no
entiendo muy bien en sentido de esta frase). This
result is consistent with previous results (15).
Figure 5. Quantification of the area expressing TH
immunoreactivity in the SN. Results are mean SD.
aStatistical signification compared with control and
bstatistical signification compared with LPS group p<0.05.
Functional study of learning.
Operant conditioning (also known as instrumental
conditioning) is a process by which organisms learn
to behave in such a way as to obtainrewardsand avoid
punishments. Habit learning is the process by which
new behaviors become automatic. If an.acquired
behavior is regularly followed, it becomes almost
involuntary.
To know if the injuries done in SN affect the
dopaminergic activity by the neostriatum (striate
dorsomedial and dorsolateral in rodents) we must
take into account that the dorsomedial part is related
to learning goal behaviours while the dorsolateral
part is related to habits (16). Using the Skinner box,
our results show that control animals acquired the
skill in less time than both LPS- and LPS + ADSC-
treated animals (Fig. 6).
41

Figure 6.Learning acquisition time.
Figure 7. Decline of consumption of the devalued pellet.
To break the RC connection in the operant
conditioning and habit learning we used food pellet
of LiCl that produce gastric discomfort to the animal
so that they show an aversive behavior to eat it. Using
these pellets we can assess whether the learning
behavior was predominantly aimed at a goal or
maintained by habit. As we can be seen in Fig. 7, LPS-
treated animals quickly lower their rate of response,
which indicate that their learning is primarily aimed
at a goal. It is worthy to note that LPS + ADSC
animals showed a higher rate of response than control
subjects, which suggests a greater ability to acquire a
habit.
Discussion
Cell therapy has been studied in a model of
Parkinsons disease (PD). The working hypothesis is
that the ADSC, implanted locally, can recognize the
42
www.approachestoagingcontrol.org
damage and stay in these tissues during a period of
time and, later, contribute to their reparation. The
idea is that autologous treatments with ADSC would
prevent or treat the loss of optimal neurophysiological
functions occurring with age.
An LPS experimental model of PD was used. LPS
induces a strong reaction in the microglia as indicated
by a 67-fold increase in the OX-6 compared to
control data. The SN has special sensitivity to
the inflammation stimulus due to the fact that it
contains the highest density of microglial cells (13).
In addition, LPS led to a loss of more than 70% of
TH positive neurons. It has been described that the
increase in the synthesis of inflammatory cytokines
by glia produces the selective death of dopaminergic
neurons due to oxidative stress (18, 19, 20).
Considering the inflammatory process that occurs
in the SN after injection of LPS (15), it has been
suggested that inflammation could be involved in
certain neurodegenerative processes such as PD.
The results also show that ADSC partially prevented
both the microglial activation caused by LPS and the
loss in the number of TH-positive neurons. Likewise,
ADSC treatment led a slight tendency to observe a
greater ability to acquire a habit. In conclusion, stem
cells are capable of improving inflammation, neuronal
death and possible improvements at the functional
level in PD.
Acknowledgement
This work was supported by Consejeria de Economa,
Innovacion y Ciencia de la Junta de Andalucia (Spain),
Ref. P10-CTS-6494.
Approaches to Aging Control. Vol 19. october 2015
References
1 YU, B. P.; YANG, R. 1996. Critical evaluation of
the free radical theory of aging.A proposal for the
oxidative stress hypothesis. Ann. N.Y. Acad.Sci. 786,1-
11.
2 KASAPOGLU, M.. OZBEN, T. 2001. Alterations
of antioxidant enzymes and oxidative stress markers
in aging. Exp. Gerontol. 36, 209-220.
3 KIRKWOOD, T. B. 2005. Understanding the odd
science of aging. Cell 120,437-447.
4 HO AD, WAGNER W, MAHLKNECHT U. 2005.
Stem cells and ageing. The potential of stem cells to
overcome age-related deteriorations of the body in
regenerative medicine.EMBO Rep.6,3538.
5 ROSENTHAL N. 2005 .Youthful prospects for
human stem-cell therapy. In another few decades,
revised attitudes toward stem cells could lead to
disease prevention and life extension.EMBO Rep. 6
Spec No, S30S34.
6 HIPP J, ATALA A . 2008. Sources of stem cells for
regenerative medicine .Stem Cell Reviews. 4, 311.
7 TEO AK,VALLIER L. 2010.Emerging use of stem
cells in regenerative medicine.Biochem J. 428, 1123.
8 RANDO, T. A. 2006. Stem cells, ageing and the
quest for immortality. Nature 441, 1080-1086.
9 TEO AK,VALLIER L. 2010.Emerging use of stem
cells in regenerative medicine.Biochem J. 428, 1123.
10 TROUNSON, A.; THAKAR, R.G.; LOMAX, G.
& GIBBONS, D. 2011. Clinical trials for stem cell
therapies. BMC Medicine. 9, 52-58.
11 Xing,b.;t. xin;r. l. hunter,and g. bing,
2008. pioglitazone inhibition of lipopolysaccharide-
induced nitric oxide synthase is associated with altered
activity of p38 map kinase and pi3k/ akt,journal of
neuroinflammation, vol.5,art. 4.
12 Liu, M., & Bing, G. 2011. Lipopolysaccharide
animal models for Parkinsons disease.Journal of
Pharmacology and Experimental Therapeutics,
vol.293,no2,607617.
13 LAWSON LJ, PERRY VH, DRI P, GORDON
S. 1990. Heterogeneity in the distribution and
morphology of microglia in the normal adult mouse
brain.Neuroscience. 39:151-170.
14 NIMMERJAHN A, KIRCHHOFF F,
HELMCHEN F. 2005. Resting microglial cells are
highly dynamic surveillants of brain parenchyma in
vivo, Science 308:1314-1318.
15 HERRERA AJ, CASTANO A, VENERO JL,
CANO J, MACHADO A. 2000.The single intranigral
injection of LPS as a new model for studying
the selective effects of inflammatory reactions on
dopaminrgicasystem.Neurobiol Dis 7:429-447
16 DAZ E, VARGAS JP, QUINTERO E, DE LA
CASA LG, ODONNELL P, LOPEZ JC. 2014.
Differential implication of dorsolateral striatum in
encoding and recovery processes of latent inhibition.
Neurobiology of Learning and Memory111:19-25
17 QUINTERO E, DAZ E, VARGAS JP,
SCHMAJUK N, LPEZ JC, DE LA CASA LG.
2010. Effects of context novelty vs familiarity on
latent inhibition with a conditioned taste aversion
procedure.Behavioural Processes.86:242-249
43
 www.approachestoagingcontrol.org

18 DE PABLOS MR, HERRERA AJ, VILLARN

RF, CANO J, MACHADO A. 2005. Dopamine-
dependent neurotoxicity of lipopolysaccharide in
substantianigra.FASEB J 19:407-409

19 Floor e, wetzel mg. 1998. Increase protein

oxidation in human substantianigra pars compacta in
comparison with basal ganglia and prefrontal cortex
measured with an improved dinitrophenylhydrazine
assay. J Neurochem 70:268-75

20 KIM WG, MOHNEY RP, WILSON B Y

COLS. 2000. Regional difference in susceptibility
to lipopolysaccharide-induced neurotoxicity in the
brain: role of microglia.JNeurosci. 20:6309-6316.

44
Approaches to Aging Control. Vol 19. october 2015

Can electromagnetic fields modify the natural processes

of aging?
C. Maestu
Unidad de Bioelectromagnetismo, Centro de Tecnologa Biomdica, Universidad Politcnica de Madrid
Campus de Montegancedo 28332 Pozuelo de Alarcn Madrid. ceferino.maestu@ctb.upm.es

Keywords: Age, bioelectromagnetism, oxidative on the effect on oxidative stress and its relationship to
stress, aging the aging process.

Abstract. Aging process represents one of the most Introduction

important concerns of advanced societies due to that
increased longevity rises the number of old people and
age-related diseases. Electromagnetic fields (EMFs)
originating both from both natural and manmade
sources permeate our environment. As people are
continuously exposed to EMFs in everyday life, it is a
matter of great debate whether they can be harmful
to human health. Old organisms are less responsive to
changing conditions outside in natural evolutionary
scale, so the possibility that the aging process being
influenced most acutely by exposure to a non-
adaptive component such as EMF looks real. On the
basis of two decades of epidemiological studies, an
increased risk has been consistently assessed, inducing
the International Agency for Research on Cancer to
insert them in the 2B section of carcinogens in 2001.
EMFs interaction with biological systems may cause
oxidative stress under certain circumstances. Since
free radicals seems to be involved in both causing and
accelerating the aging process, research focusing on
the possible influence of the EMFs-driven oxidative
stress, especially to the etiology of neurodegenerative
disorders, is still in progress. This short review shows
the emerging evidences about this topic, and focus
Aging is a series of morphological, psychological,
functional and biochemical changes that occur over
the life time. Aging is a slow and gradual process
of deterioration of the functional capacity of the
body, after maturity, and eventually leads to death of
it [1,2,3]. There are certain environmental toxicity
factors that can accelerate these processes. Decreased
longevity by harmful environmental factors changes
in multiple systems from the cellular level to the
homeostatic balance. Stress can affect many aspects
of the physiology, and has an important influence on
health and disease.
Humans have been subjected during the evolutionary
process to natural EMF radiation, to which we are
phylogenetically adapted, coming from the sun, the
earths magnetic field and electric depolarizacin
phenomena (Ray etc.) that occur in the atmospheric
range. New types of EMF radiation have occurred
since the discovery of electricity that are flooding
our environment of artificial radiation for which
there is adapted and for which our bodies do not
have detection systems and defense. Over the past
several decades people have been constantly exposed

45
 www.approachestoagingcontrol.org

to electric (E) and magnetic (H) fields from both the electromagnetic (EM) radiation. Therefore, for
industrial and domestic uses. The EMFs are produced RF and MW exposure, the characteristic interaction
not only for technological applications (e.g., power quantity is the Specific Absorption Rate (SAR)
lines, communications systems, mobile phones [20], defined as the power (W) deposited by an EM
wifi, etc), but they are now widely used also in radiation in a unitary mass (g) of the biological target,
medicine for diagnostic (e.g., magnetic resonance in a fixed time period(s), and it is measured in W/kg.
imaging (MRI) scanner and microwave imaging)
and therapeutic purposes (e.g., radiofrequency and
microwave ablation and hyperthermia) [7,8]. The
EMFs coupling with biological systems depends on
the frequency range of the employed signals, as well
as on their characteristics as amplitude, modulation,
waveform and polarization [16,17]. Mainly three
categories of EMFs signals can be identified. They
are classified as static, electric and magnetic fields (as
direct current, DC, 0Hz), Extremely Low Frequency
fields (ELF, between 1Hz up to 100kHz) and high
frequency (HF) fields, in the band of the Radio
Frequency fields (RF, 100kHz3GHz), and of the
microwaves (MW, above 3GHz) [13, 14]. These
radiations (with frequencies below 300GHz) are all
nonionizing ones (Figure 1).

The established regulations against health hazards [19,

20] are based on two key mechanisms of interaction
with biological systems, one elicited by DC and ELF
sources, and the other by RF and MW exposures. For
DC and ELF exposures, the induced E-field (V/m)
and current density (J, A/m2) are the main physical
quantities to describe the EMF interaction. They
can be generated by both external applied E-fields
and variable H-fields, and their amplitudes have to
be limited in order to avoid hazardous health effects
[20]. When RF and MW exposures are taken into
account, the main mechanism to be considered is
the rise in temperature, as no charges movements
are triggered at these frequencies. The heat effect
is strictly dependent on both the water content of
the biological target, the frequency, and intensity of
FIG. 1: The whole electromagnetic spectrum, with partition
between nonionizing and ionizing radiations, is reported.
Main filed sources at the different frequencies are also
sketched.
The increased social and public interest in this
subject, based the risks that these technologies and
their emissions produced over the whole population
increase in the case of the performance of certain
pathologies such as degenerative processes or simply
the aging process with reduced defense capabilities.
[18,19], prompted the World Health Organization
(WHO) Report (2007) and WHO Environmental

46
Approaches to Aging Control. Vol 19. october 2015
Health Criteria (EHC) Report (2007) to issue
precautions against the ELF-EMFs [19,20].
A growing body of epidemiological evidence suggests
an association between occupational exposure to
extremely low frequency magnetic fields (ELF-MF)
and dementia/Alzheimers disease [15]. ELF-MF
are generated by electric-powered equipment, be
among other sources. They are part of a spectrum of
electromagnetic waves that run from gamma rays at
the highest frequency end, through x-rays, ultraviolet
rays, visible light, infrared radiation, microwaves,
radio waves, very low frequency, and extremely low
frequency waves at the lowest end (FIG1).
BioInitiative Working Group found seven
epidemiologic studies with an association between
ELF-MF and Alzheimers disease, [21]. One of the
mechanisms proposed to mediate the increased
risk of Alzheimers disease with ELF-MF exposure
is reduced melatonin production. Numerous
epidemiologic studies (11 of 13 reviewed in the
BioInitiative Working Group study) found that high
ELF-MF exposure was associated with reduced
melatonin production in occupational and residential
settings. Melatonin is a potent inhibitor of amyloid
beta neurotoxicity, oxidative stress in transgenic mouse
models of Alzheimers disease, and proinflammatory
cytokine production induced by amyloid-beta in
rat brains. In humans, the levels normally decline
with age. The levels are more sharply reduced in
people with Alzheimers disease. [15,82,83]. Another
study in people with Alzheimers disease in group
homes showed that, although melatonin improved
sleep patterns, its effects on cognitive function were
beneficial only when combined with bright lights
during the day [80]. The evidence is suggestive
that ELF-MF could potentially increase risk for
Alzheimers disease by reducing brain levels of
neuroprotective melatonin. [84,85,86,87.88].
The oxidative stress
Aging and the degenerative diseases associated with
it are attributed basically to the deleterious side
attacks of free radicals on cell constituents and on
the connective tissues. The free radicals probably arise
largely through reactions involving molecular oxygen
catalyzed in the cell by the oxidative enzymes and in
the connective tissues [15,31,43,47,82,83].
The mechanisms proposed to explain potential ELF-
MF effects on the brain and biological systems in
general include oxidative stress, calcium ion release in
immune cells and neurons, apoptosis and necrosis in
brain cells, and effects on biomagnetic particles in the
brain [45,62,63].
The cellular stress response is a protective reaction of
individual cells to potentially harmful stimuli in the
environment [65]. It is characterized by the synthesis
of a class of proteins referred to as stress proteins,
which can be used as a markers of cellular damage.
The cellular stress response was first described as a
reaction to elevated temperature, which accounts
for these proteins initially being called heat shock
proteins (HSP)
FIG 2- Oxygen radicals can cause peroxidation of nearby
lipidsThese LPO products can then adduct to proteins,
ie Complex IVStudies have shown that as adduction of
4-HNE (a LPO product) increases, Complex IV activity
decreases Source: Suh, J. H., Heath, S. H., Hagen, T. M.
(2003). Two subpopulations of mitochondria in the aging
rat heart display heterogeneous levels of oxidative stress. Free
Radical Biology & Medicine 35 (9), 1064-1072
47

Several physical and chemical environmental,
including EMF, have been found to induce HSP
[141]. In fact, the cells were far more sensitive to
EMF than to thermal stimuli, the threshold energy of
the EMF stimulus being more than one billion times
weaker than an effective thermal stimulus [141].
EMF can also interact with DNA. It can cause errors
during replication, as well as during protein synthesis,
and higher energy EMF could be expected to cause
DNA strand breaks [139].The correlation between
DNA strand breaks and field strength indicates a
dose-response support the evidence that EMF is a the
causing agent [3,4,108,126].
EMF induce abnormal influx of calcium into cells,
which can lead to and trigger allergic reactions.
.[34.35]. Also, EMF cause mitochondria dysfunction.
Considering that mitochondria are the powerhouse
of the cells creating energy, this could explain the
common link between fatigue and RF radiation
exposure [91].
EMF alter the production of Melatonin and,
consequently, cause sleep difficulties. This clearly
demonstrates that the electromagnetic environmental
monitoring during the period of sleep significantly
improves cellular repair. It also supports that the
electromagnetic fields that surround us, progressively
decrease the antioxidant defenses and, by extension,
causing pathologies [24].
FIG 3- possible consequences of exposure to different types
of EMF http:// www.emf.com/emrsolution/emfissues/
emfissuessummary.html
48
www.approachestoagingcontrol.org
Aging and Interaction of the EMFs with the
Biological Systems
Ageing is a multi-factorial process, which leads to
irreversible damage to macromolecules (i.e. DNA,
proteins, lipids), cells and organs [65,74]. The rate of
this process is attributable to individual genetic as well
as to environmental factors. Several studies have
addressed the relationships between DNA damage
and aging, and have suggested an age-dependent
accumulation of DNA damage .[4,5]. Mutation rate
appears to increase with age, indicating a decline of
DNA repair efficiency with age [138].
EMF penetrate cells unattenuated so that can interact
directly with the DNA in the cell nucleus [5,72]. In
contrast to EMF, most biological agents are impeded
by membranes and require special mechanisms to
gain access to the cell interior. Friedman et al [137]
have demonstrated that, in those situations, the
initial step in transmitting extracellular information
from the plasma membrane to the nucleus of the
cell occurs when NADH oxidase rapidly generates
reactive oxygen species (ROS).
FIG 4- Processes of oxidative phosphorylation in
mitochondria.
These ROS stimulate matrix metalloproteinases that
allow them to cleave and release heparin binding
epidermal growth factor. [47].
Metabolic processes which generate oxidants and
antioxidants can be influenced by environmental
factors, such as EMFs [19]. As EMFs are nonionizing,
Approaches to Aging Control. Vol 19. october 2015
the searches for conventional genotoxic [139]
mechanisms, as potentially responsible events
underlying the interaction with the biological
systems, have shown contradictory results. A
molecular mechanism, disclosing the link between
human diseases and exposure to electromagnetic
fields, is still in revision, and mainly oxidative stress
have been proposed [75,77,116,118,136].
Scaiano et al. [29] first proposed that ELF-EMFs
exposure can stabilize free radicals in such a way
as to increase their lifetime and permit a wider
dispersion rather than their return to the basal
level. This might contribute to an increase in the
activity and concentration of the free radicals, as
also reported in the immune system, mainly mouse
macrophages, human monocytes, and rat neutrophils
[31,47]. Besides, the inhibitory potential of chronic
ELF-EMFs exposure on the availability of the pineal
gland hormone melatonin has been suggested as
an additional pathway in the oxidative stress-driven
interaction of ELF with the biological systems [24].
Concerning the procedures for the protection of
health against EMF, it is considered two possible
effects resulting from the interaction of biological
environment and EMF. One is the thermal effect by
which the temperature rise in tissue and biological
systems. This is the only effect that international law
allows [16,19,20], that rely on the ability of RF fields
to transfer their energy to biological matter, leading
to an increase in average temperature through the
vibration of atoms and molecules) [21].
The non-thermal effects is based on the induction
microcurrent, by resonant action or dynamic changes
in the ionic product of spin reorientation. The latter
only have been correlated to the generation of
oxidative stress. [33]. This non-thermal effects range
from alterations in the permeability of the blood-
brain barrier, to changes in encephalogram and
blood pressure, although this issue is still controversial
[37, 38]. Oxidative stress has been proposed as the
underlying mechanism responsible for this kind of
RF effects.
EMFs and Oxidative Stress in Brain
Brain volume declines with age but at highly different
rates in different brain structures (Fig. 5).
FIG 5. Fjell and Walhovd 2010: Reviews in the
Neurosciences 21:187-221
At the cellular level, with aging there is cumulative,
unrepaired or poorly repaired natural or unnatural
cell injury [140]. Cell injury results when cells
can no longer adapt to stress, have unrecoverable
exposure to damaging agents or suffer from intrinsic
abnormalities, whether genetic or nutrient-based.
Cell injury results when the limits of adaptive
responses of cells are exceeded or if cells are exposed
to injurious agents or stress, are deprived of essential
nutrients, or become compromised by mutations that
affect essential cellular constituents. [25,126,127].
The hallmarks of ireversible injury are reduced
oxidative phosphorylation with depletion of
ATP, cellular swelling caused by changes in ion
concentrations and water influx, mitochondria and
cell skeleton alterations and DNA damage. Free
radicals are essential for physiological processes,
especially in brain metabolism [25,29,31]. The brain
consumes the highest amount of oxygen in the human
49

body and, although most oxygen is converted into
CO2 and water, a small amount of O2 forms ROS
[50]. The high metabolic rate and the composition
rich in polyunsaturated fatty acids which are ROS
targets in brain, make this organ more sensitive to
oxidative damage [46]. The interaction between the
ELF-EMFs and the biological systems directly implies
the involvement of the oxidative stress, in particular
by the radical pair mechanism, as the equilibrium of
the elementary reaction producing a pair of radicals
may be altered by the magnetic field [33]. Thus, ELF-
EMFs may prolong the lifetime of free radicals and
increase their concentration in living cells [31,].
FIG-6 Effects of oxidation and aging
50
www.approachestoagingcontrol.org
A decline of DNA repair efficiency with age raise
the question, if senescence per se leads to a higher
susceptibility to DNA damage upon environmental
exposures, [72], which could point to an age-related
decrease of DNA repair efficiency of ELF-EMF
induced DNA strand breaks [140].
ELF-EMFs exposure (50Hz, 0.11.0mT) is reported
to elicit redox and trophic response in rat cortical
neurons [30], and to induce oxidative stress in mouse
cerebellum [58].
Oxidative stress produced a disfunction between
the production of free radicals and the scavenging
capacity driven by various antioxidant compounds
and enzymes. [62]. As an overall oxidative stress-based
decline in physiologic functions and in resistance
to stressors is an unavoidable consequence of aging
[59], it has been also investigated whether the aging
process per se might reduce resistance towards
EMFs prooxidant attack. In this context, ELF-
EMFs exposure (50Hz, 0.11.0mT) was shown to
significantly affect antioxidant enzymatic capacity in
aged rat brains [60].
Certain types of electromagnetic radiation (EMF)
have been identified in several studies as neurological
effects generators, including headaches, changes in
sleep pattern, modification in the neuronal electrical
activity, and disturbance in the neurotransmitter
release [62]. Although controversial results, increasing
evidence indicates that oxidative stress may be
involved in the adverse effects elicited by RF-EMFs
in the nervous system [118]. They first proved
that RF-EMFs exposure of the brain in rats cause
histopathological changes typical of brain injury,
accompanied by oxidative stress, as biochemically
revealed by increased levels of nitric oxide (NO),
malondialdehyde (MDA), as well as xantine oxidase
(XO), and adenosine deaminase (ADA) activities.
Approaches to Aging Control. Vol 19. october 2015
In animal model with guinea pigs, Meral et al. [70]
evaluated the effects of GSM signal (890915MHz
EMF, SAR 0.95Wkg1, for 12h/day for 30 days) on
the oxidative stress pathway, by assessing MDA, GSH,
CAT and vitamin A, D3, and E (considered part of
antioxidant defense systems of tissues) levels in both
brain and blood. They found an increase of MDA,
and a decrease of both GSH and CAT levels in brains,
thus suggesting that RF exposure could decreases he
capacity of the antioxidant systems, due to increased
lipid peroxidation and formation of free radicals. Xu
et al. [72] also reported a significant increase of ROS
production, and demonstrated, a reduction in the
mitochondrial DNA copy numbers.
EMFs and Neurodegenerative Diseases
It is well established that free radicals can interact
with DNA, leading to mutation, and interfere with
gene regulation to eventually promote carcinogenesis
[74]. But an additional aspect of free radicals is their
potentiality to affect neuropathological conditions
such as Parkinsons disease (PD) and Alzheimers
disease (AD), the oxidative stress being a molecular
hallmark of neurodegenerative diseases [76,78]. The
rapid increase of such diseases would be associated also
to the rapid development of wireless communication
technologies that invade our lives.
Alzheimer disease
Alzheimer disease (AD) is the most prevalent
neurodegenerative disease, and is characterized by
progressive loss of neurons, particularly in the cortex
and hippocampus [80]. One hypothesis of developing
and progression of this disease is increasing the deposit
of the protein beta-amyloid. Oxidative damage has
been implicated as a key mediator and can promote
the synthesis of amyloid beta (A) precursor protein
in an oxidative stress-mediated pathway [ 84,87, 90].
Among the potential environmental factors, exposures
to heavy metalls, solvents, pesticides, and lead and
also EMFs (mainly ELF-EMFs) have been the most
widely studied [80]. Epidemiological studies suggest a
potential association between occupational exposure
to ELF-EMFs (typical of electric power installers and
repairers, power plant operators, electricians, electric
and electronical equipments repairer, telephone line
technicians, welders, carpenters, and machinists)
and AD onset [80], although it not well established
the mechanisms of action. Several hypothesis have
been proposed, such as melatonin depletion, a
neuroprotective factor and biosynthetic enzymes in
the pineal gland, Ca2+ efflux in immune system cells
and neurons, interference with the amyloidogenic
process, and clearly oxidative stress. Sobel and
Davanipour [81] hypothesized that ELF-EMFs
exposure might increase A peripheral and brain
production by modulating the Ca2+ channels. EMFs
may increase the intracellular ion concentration levels,
a molecular factor that positively correlates with the
cleavage of the amyloid precursor protein to give the
soluble A. However Arendash et al. [88] have found
the opposite result. Reported that long-term (79
months) RF-EMFs exposure, directly associated with
cell phone use (918MHz; 0.25WKg1), provide
cognitive benefits, disclosing a potential noninvasive,
nonpharmacological therapeutic strategy against
AD. cognitive-protective and cognitive-enhancing
effects, associated to reduced brain A deposition and
increased cerebral blood flow, were demonstrated in
transgenic mice destined to develop AD over a long
term exposure period, without increasing indices of
oxidative stress in the brain. (88)
Parkinson disease (PD)
PD is the second most common prevalent
neurodegenerative disease. It occurs with loss of
activity of dopaminergic neurons in the substantia
51

nigra and the appearance inclusions (Lewy bodies)
of -synuclein [100]. Oxidative stress, generated
by dopamine redox chemistry and by -synuclein
mutation, is considered one of the pathogenic factors
in PD [113]. The oxidative damage to lipids, protein,
DNA, and elevated RNA oxidation have been
observed in both postmortem substantia nigra tissue
and cerebrospinal fluid from living PD patients [33].
Noonan et al. [104], described that welders, who
are exposed to high levels of magnetic fields as
well as to other potentially neurotoxic agents
such as metals, significantly increase the chance of
developing of PD, Therefore, up to date, convincing
epidemiological data support a correlation between
PD and environmental/occupational factors. These
effects are still under investigation and it is not clear
the underlying mechanisms.
Amyotrophic Lateral Sclerosis
ALS is a neurodegenerative disorder characterized
by progressive degeneration of motor neurons in the
spinal cord, motor cortex, and brainstem.At molecular
level, a mutation in the gene encoding the antioxidant
Cu2+/Zn2+ SOD (SOD1) has been reported in
about 20% of ALS patients, probably by the oxidative
stress [101], Mitochondrial dysfunction may play a
more significant role in the etiopathogenesis of this
disorder, which confer an intrinsic susceptibility to
long-lived, postmitotic motor neurons to energy
deficit, calcium mishandling, and oxidative stress
[109]. Haynal and Regli were the first to raise the
hypothesis that exposure to ELF-EMFs was linked to
ALS in 1964 [12].
Discussion
Although the precise mechanisms of interaction of
EMF with the biological systems by which they can
be harmful to humans are still unknown, we know
some consequences that have led the WHO IARC
52
www.approachestoagingcontrol.org
to consider EMF possibly carcinogenic [7, 8, 114].
The International Agency for Research on Cancer
(IARC) inserted ELF in the 2B section of the table
of carcinogens (possible) in 2001, and recently
classified also the Radio Frequency (RF) fields as 2B
[4, 115]. In addition recent studies indicate that EMF
can be co-promoters of some neurodegenerative
pathologies mainly AD [6, 9, 74]. The study of
neurodegenerative processes and their pathological
associations, with the EMF, have their origin in yet
unknown processes, where certain alterations are
involved in vital cellular process. Dysfunctions in
repair processes and characteristic energy supply of
cellular oxidation, may be typical underlying aging
process. The rhythm of these natural processes of
aging, can be affected by these subtle changes at
the cellular level. Only we know the consequences
when are manifested in the acute phase. But the day
to day process of cellular oxidation and failures in
the mechanisms of repair, will be known when they
encounter some system failure and probably too late.
Therefore we must assume that the cellular aging
process is accelerated to chronic exposure to EMF.
The lack of research in this area difficult to test these
possible associations, although the results of laboratory
and animal models support this hypothesis.
However, this area has quickly acquired attention
because of implications in human health, occupational
exposure, and aging, although, for a number of
methodological reasons, the epidemiology of
neurodegenerative diseases is more difficult to study
than cancer.
Epidemiological studies has not currently contemplate
the aging process due to the lack of precise markers.
Also, it is difficult to accurately establish the real
rate of population exposure. In this case, the direct
measure or numerical evaluation of the emitted
Approaches to Aging Control. Vol 19. october 2015
EM field could be particularly hard and expensive,
due to the elevated number of involved people and
residential places. Estimation of the dose has been
possible based on people interview about the most
common exposure sources present in their daily-life
environment. Therefore, a more careful approach
seems to be necessary such as the use of personal
dosimeters to record in real time the effective EMFs
levels, together with the time and the exact position
of the exposure. Also, it would be necessary to focus
on different cellular systems, along with alterations
of blood parameters, changes in cytokine profiles,
and effects on the immune system, although no clear
understanding of the underlying mechanisms has
been uniformly documented [1519].
Conclusion
Biological systems have adapted in an evolutionary
manner to the presence of natural radiation sources
(sun light geomagnetic field etc.). Thousands of years
have been necessary to adapt to these conditions.
However, the discovery of electricity has flooded
our planet of artificial radiation intensities with
unknown frequencies, which we are not adapted to.
The emergence of this new sources of environmental
disruptors may affect life expectancy and generate
new pathologies associated with these new radiations.
Further studies are needed to know the amount
of exposure that our body can handle. However,
precautionary measures must be taken to limit
the damage so that advances in technology can be
compatible with the delicate balances of biological
systems and their adaptive parameters.
References
1-S.Bondy K.Maiese aging and related disorders.
Oxidative stress in applied basi research and clinical
practice.Human press, Springer NY. 2010.
2-Denham Harman Free radical theory of aging:
Origin of life, evolution, and aging AGE October
1980,Volume 3, Issue 4, pp 100-102
3-Raz, Naftali Aging of the brain and its impact on
cognitive performance: Integration of structural and
functional findings.Craik, Fergus I. M. (Ed); Salthouse,
Timothy A. (Ed), (2000). The handbook of aging and
cognition (2nd ed.). , (pp. 1-90). Mahwah, NJ, US:
Lawrence Erlbaum Associates Publishers, ix, 755 pp.
4-Ivancsits, S., Pilger, A., Diem, E., Schaffer, A., Ru
diger, H.W., 2002b. Vanadate induces DNA strand
breaks in cultured human fibroblasts at doses relevant
to occupational exposure. Mutat. Res. 519,25/35.
Ivancsits, S., Diem, E., Pilger, A., Ru diger, H.W.,
Jahn, O., 2002a.Induction of DNA strand breaks by
exposure to extremely-lowfrequency electromagnetic
fields in human diploid fibroblasts. Mutat. Res. 519, 1
/13.
5-Ivancsits, S., Diem, E., Jahn, O., Ru diger,
H.W. Intermittent extremely low frequency
electromagnetic fields cause DNA damage in a dose
dependent way. Int. Arch. Occup. Env. Health, in press
6-Osman Fikret Sonme, Ersan Odaci, Orhan Bas,
Sleyman Kaplan (2010) Purkinje cell number
decreases in the adult female rat cerebellum following
exposure to 900 MHz electromagnetic field B r a i n
Research1356 95101
7-W. R. Adey, Tissue interactions with nonionizing
electromagnetic fields, Physiological Reviews, vol.
61, no. 2, pp. 435514, 1981.
8-Lacy-Hulbert, J. C. Metcalfe, and R. Hesketh,
Biological responses to electromagnetic fields, The
FASEB Journal, vol. 12, no. 6, pp. 395420, 1998.
9-J. Juutilainen, P. Matilainen, S. Saarikoski, E. Lr,
and S. Suonio, Early pregnancy loss and exposure to
53

50-Hz magnetic fields, Bioelectromagnetics, vol. 14,
no. 3, pp. 229,236, 1993.
10-International Agency for Research on Cancer-
(IARC), Non-ionizing radiation Part I: static and
extremely low frequency (ELF) electric and magnetic
fields, Monographs, vol. 80, 429 pages, 2002.
11-C. Y. Li and F. C. Sung, Association between
occupational exposure to power frequency
electromagnetic fields and amyotrophic lateral
sclerosis: a review, American Journal of Industrial
Medicine, vol. 43, no. 2, pp. 212220, 2003. View at
12- HAYNAL and F. REGLI, Amyotrophic
lateral sclerosis associated with accumulated electric
injury, Confinia Neurologica, vol. 24, pp. 189198,
1964.
13-N. Wertheimer and E. Leeper, Original
contributions. Electrical wiring configurations and
childhood cancer,American Journal of Epidemiology,
vol. 109, no. 3, pp. 273284, 1979.
14-D. P. Loomis and D. A. Savitz, Mortality from
brain cancer and leukaemia among electrical
workers, British Journal of Industrial Medicine, vol.
47, no. 9, pp. 633638, 1990.
15-Z. Davanipour, C. C. Tseng, P. J. Lee, and E. Sobel,
A case-control study of occupational magnetic
field exposure and Alzheimers disease: results from
the California Alzheimers Disease Diagnosis and
Treatment Centers, BMC Neurology, vol. 7, article
13, 2007.
16-WHO, Electromagnetic fields and public health.
Exposure to extremely low frequency fields, Fact
Sheet no. 322, 2007. 17- WHO (Environmental
Health Criteria), Extremely Low Frequency Fields,
vol. 35, WHO, Geneva, Switzerland, 1984.
54
www.approachestoagingcontrol.org
18-C. Polk and E. Postov, CRC Handbook of
Biological Effects of Electromagnetic Fields, CRC
Press, Boca Raton, Fla, USA, 1996.
19-Ahlbom, U. Bergqvist, J. H. Bernhardt et al.,
Guidelines for limiting exposure to time-varying
electric, magnetic, and electromagnetic fields, Health
Physics, vol. 74, no. 4, pp. 494521, 1998.
20-ICNIRP (International Commission on Non
Ionizing Radiation Protection), Guidelines for
limiting exposure to time-varying electric and
magnetic fields (1Hz TO 100kHz), Health Physics,
vol. 99, no. 6, pp. 818836, 2010.
21-BioInitiative Report:A rationale for a biologically-
based public exposure standard for electromagnetic
fields (ELF and RF)C Sage, D Carpenter, BioInitiative
Working Group - 2007 - BioInitiative Working
GroupJ.
22-M. Caraglia, M. Marra, F. Mancinelli et al.,
Electromagnetic fields at mobile phone frequency
induce apoptosis and inactivation of the multi-
chaperone complex in human epidermoid cancer
cells, Journal of Cellular Physiology, vol. 204, no. 2,
pp. 539548, 2005.
23-H. W. Li, K.Yao, H.Y. Jin, L. X. Sun, D. Q. Lu, and
Y. B.Yu, Proteomic analysis of human lens epithelial
cells exposed to microwaves, Japanese Journal of
Ophthalmology, vol. 51, no. 6, pp. 412416, 2007.
View at Publisher
24-F. Oktem, F. Ozguner, H. Mollaoglu, A. Koyu,
and E. Uz, Oxidative damage in the kidney induced
by 900-MHz-emitted mobile phone: protection by
melatonin, Archives of Medical Research, vol. 36, no.
4, pp. 350355, 2005.
25-P. Kovacic and R. Somanathan, Electromagnetic
fields: mechanism, cell signaling, other bioprocesses,
toxicity, radicals, antioxidants and beneficial effects,
Approaches to Aging Control. Vol 19. october 2015
Journal of Receptors and Signal Transduction, vol. 30,
no. 4, pp. 214226, 2010.
27-J. Jajte, J. Grzegorczyk, M. Zmysacute, and E.
Rajkowska, Effect of 7mT static magnetic field and
iron ions on rat lymphocytes: apoptosis, necrosis and
free radical processes, Bioelectrochemistry, vol. 57,
no. 2, pp. 107111, 2002.
28-M. Z.Akdag, M. H. Bilgin, S. Dasdag, and C.Tumer,
Alteration of nitric oxide production in rats exposed
to a prolonged, extremely low-frequency magnetic
field, Electromagnetic Biology and Medicine, vol.
26, no. 2, pp. 99106, 2007.
29-J. C. Scaiano, N. Mohtat, F. L. Cozens, J. McLean,
and A. Thansandote, Application of the radical pair
mechanism to free radicals in organized systems: can
the effects of 60Hz be predicted from studies under
static fields? Bioelectromagnetics, vol. 15, no. 6, pp.
549554, 1994.
30-M. Simk, Cell type specific redox status is
responsible for diverse electromagnetic field effects,
Current Medicinal Chemistry, vol. 14, no. 10, pp.
11411152, 2007.
31-M. Valko, D. Leibfritz, J. Moncol, M. T. D. Cronin,
M. Mazur, and J.Telser, Free radicals and antioxidants
in normal physiological functions and human
disease, International Journal of Biochemistry and
Cell Biology, vol. 39, no. 1, pp. 4484, 2007.
32-S. Harakawa, N. Inoue, T. Hori et al., Effects of a
50Hz electric field on plasma lipid peroxide level and
antioxidant activity in rats, Bioelectromagnetics, vol.
26, no. 7, pp. 589594, 2005.
33-Q. Kong and C. L. G. Lin, Oxidative damage
to RNA: mechanisms, consequences, and diseases,
Cellular and Molecular Life Sciences, vol. 67, no. 11,
pp. 18171829, 2010.
34-J. Rollwitz, M. Lupke, and M. Simk, Fifty-
hertz magnetic fields induce free radical formation
in mouse bone marrow-derived promonocytes and
macrophages, Biochimica et Biophysica Acta General
Subjects, vol. 1674, no. 3, pp. 231, 238, 2004.
35-M. Simk and M. O. Mattsson, Extremely low
frequency electromagnetic fields as effectors of
cellular responses in vitro: possible immune cell
activation, Journal of Cellular Biochemistry, vol. 93,
no. 1, pp. 8392, 2004.
36-S. Roy, Y. Noda, V. Eckert et al., The phorbol
12-myristate 13-acetate (PMA)-induced oxidative
burst in rat peritoneal neutrophils is increased by a
0.1mT (60Hz) magnetic field, FEBS Letters, vol.
376, no. 3, pp. 164166, 1995. 37- M. Simk,
S. Droste, R. Kriehuber, and D. G. Weiss, Stimulation
of phagocytosis and free radical production in murine
macrophages by 50Hz electromagnetic fields,
European Journal of Cell Biology, vol. 80, no. 8, pp.
562566, 2001.
38-S. Thun-Battersby, M. Mevissen, and W.
Lscher, Exposure of Sprague-Dawley rats to a
50-hertz, 100-Tesla magnetic field for 27 weeks
facilitates mammary tumorigenesis in the 7,12-
dimethylbenz[a]-anthracene model of breast cancer,
Cancer Research, vol. 59, no. 15, pp. 36273633,
1999.
39-L. S. Caplan, E. R. Schoenfeld, E. S. OLeary, and
M. C. Leske, Breast cancer and electromagnetic
fieldsa Review, Annals of Epidemiology, vol. 10,
no. 1, pp. 3144, 2000.View at Publisher
40-G. Katsir and A. H. Parola,Enhanced proliferation
caused by a low frequency weak magnetic field in
chick embryo fibroblasts is suppressed by radical
scavengers, Biochemical and Biophysical Research
Communications, vol. 252, no. 3, pp. 753756, 1998.
55

41-K. R. Foster and R. Glaser, Thermal mechanisms
of interaction of radiofrequency energy with
biological systems with relevance to exposure
guidelines, Health Physics, vol. 92, no. 6, pp. 609
620, 2007.
42-M. Gaestel, Biological monitoring of non-
thermal effects of mobile phone radiation: recent
approaches and challenges, Biological Reviews, vol.
85, no. 3, pp. 489500, 2010.
43-M. K. Irmak, E. Fadillio lu, M. Gle, H. Erdo an,
M.Ya murca, and O.Akyol,Effects of electromagnetic
radiation from a cellular telephone on the oxidant
and antioxidant levels in rabbits, Cell Biochemistry
and Function, vol. 20, no. 4, pp. 279283, 2002.
44-R. Stam, Electromagnetic fields and the blood-
brain barrier, Brain Research Reviews, vol. 65, no. 1,
pp. 8097, 2010.
45-M. Zmy lony, P. Politanski, E. Rajkowska, W.
Szymczak, and J. Jajte, Acute exposure to 930MHz
CW electromagnetic radiation in vitro affects reactive
oxygen species level in rat lymphocytes treated by
iron ions, Bioelectromagnetics, vol. 25, no. 5, pp.
324328, 2004.
46-E. Ozgur, G. Gler, and N. Seyhan, Mobile phone
radiation-induced free radical damage in the liver is
inhibited by the antioxidants n-acetyl cysteine and
epigallocatechin-gallate, International Journal of
Radiation Biology, vol. 86, no. 11, pp. 935945, 2010.
47-M. Lantow, M. Lupke, J. Frahm, M. O. Mattsson,
N. Kuster, and M. Simko, ROS release and
Hsp70 expression after exposure to 1,800MHz
radiofrequency electromagnetic fields in primary
human monocytes and lymphocytes, Radiation and
Environmental Biophysics, vol. 45, no. 1, pp. 5562,
2006.
56
www.approachestoagingcontrol.org
48-M. Lantow, J. Schuderer, C. Hartwig, and M.
Simk, Free radical release and HSP70 expression in
two human immune-relevant cell lines after exposure
to 1800 MHz radiofrequency radiation, Radiation
Research, vol. 165, no. 1, pp. 8894, 2006.
49-T. Q. Huang, M. S. Lee, E. H. Oh et al.,
Characterization of biological effect of 1763MHz
radiofrequency exposure on auditory hair cells,
International Journal of Radiation Biology, vol. 84,
no. 11, pp. 909915, 2008.
50-B. Halliwell, J. M. C. Gutteridge, A. C. Andorn,
R. S. Britton, and B. R. Bacon, Lipid peroxidation
in brain homogenates: the role of iron and hydroxyl
radicals , Journal of Neurochemistry, vol. 69, no. 3,
pp. 13301331, 1997.
51-M. Naziro lu, New molecular mechanisms on
the activation of TRPM2 channels by oxidative stress
and ADP-ribose, Neurochemical Research, vol. 32,
no. 11, pp. 19902001, 2007.
52-zmen, M. Naziro lu, H. A. Alici, F. ahin, M.
Cengiz, and I. Eren, Spinal morphine administration
reduces the fatty acid contents in spinal cord and
brain by increasing oxidative stress, Neurochemical
Research, vol. 32, no. 1, pp. 1925, 2007.
53-R. K. Adair, Effects of very weak magnetic fields
on radical pair reformation, Bioelectromagnetics,
vol. 20, no. 4, pp. 255263, 1999.
54-R. ODea,A. F. Curtis, N. J. B. Green, C. R.Tinunel,
and P. J. Hore, Influence of dipolar interactions on
radical pair recombination reactions subject to weak
magnetic fields, Journal of Physical Chemistry A, vol.
109, no. 5, pp. 869873, 2005.View at Publisher
55-J. Hoff, Magnetic field effects on photosynthetic
reactions, Quarterly Reviews of Biophysics, vol. 14,
no. 4, pp. 599665, 1981.
Approaches to Aging Control. Vol 19. october 2015
56-T. Rodgers and P. J. Hore, Chemical
magnetoreception in birds: the radical pair
mechanism, Proceedings of the National Academy
of Sciences of the United States of America, vol. 106,
no. 2, pp. 353360, 2009.
57-S. Di Loreto, S. Falone, V. Caracciolo et al., Fifty
hertz extremely low-frequency magnetic field
exposure elicits redox and trophic response in rat-
cortical neurons, Journal of Cellular Physiology, vol.
219, no. 2, pp. 334343, 2009.
58-L. Y. Chu, J. H. Lee, Y. S. Nam et al., Extremely
low frequency magnetic field induces oxidative
stress in mouse cerebellum, General Physiology and
Biophysics, vol. 30, no. 4, pp. 415421, 2011.
59-E. Ciejka, P. Kleniewska, A. Goraca, and B. Skibska,
Effects of extremely low frequency magnetic field
on oxidative balance in brain of rats, Journal of
Physiology and Pharmacology, vol. 62, no. 6, pp. 657
661, 2011.
60-Jelenkovi, B. Jana,V. Pei, D. M. Jovanovi, I.Vasiljevi,
and Z. Proli, Effects of extremely low-frequency
magnetic field in the brain of rats, Brain Research
Bulletin, vol. 68, no. 5, pp. 355360, 2006.
61- Bediz, A. K. Baltaci, R. Mogulkoc, and
E. ztekin, Zinc supplementation ameliorates
electromagnetic field-induced lipid peroxidation
in the rat brain,Tohoku Journal of Experimental
Medicine, vol. 208, no. 2, pp. 13 140, 2006.
62- Lee, H. M. Johng, J. K. Lim et al., Effects
of extremely low frequency magnetic field on
the antioxidant defense system in mouse brain: a
chemiluminescence study, Journal of Photochemistry
and Photobiology B, vol. 73, no. 1-2, pp. 4348, 2004.
63-M. Z. Akdag, S. Dasdag, E. Ulukaya, A. K. Uzunlar,
M. A. Kurt, and A. Ta kIn, Effects of extremely low-
frequency magnetic field on caspase activities and
oxidative stress values in rat brain, Biological Trace
Element Research, vol. 138, no. 13, pp. 238249,
2010.
64-J. Martnez-Smano, P. V. Torres-Durn, M. A.
Jurez-Oropeza, and L. Verdugo-Daz, Effect of
acute extremely low frequency electromagnetic field
exposure on the antioxidant status and lipid levels in
rat brain, Archives of Medical Research, vol. 43, no. 3,
pp. 183-189, 2012.View at Publisher View at Google
Scholar View at Scopus
65-K. C. Kregel and H. J. Zhang, An integrated
view of oxidative stress in aging: basic mechanisms,
functional effects, and pathological considerations,
American Journal of Physiology, vol. 292, no. 1, pp.
R18R36, 2007.
66-S. Falone, A. Mirabilio, M. C. Carbone et al.,
Chronic exposure to 50 Hz magnetic fields causes
a significant weakening of antioxidant defence
systems in aged rat brain, International Journal of
Biochemistry and Cell Biology, vol. 40, no. 12, pp.
27622770, 2008.
67-H. Kabuto, I. Yokoi, N. Ogawa, A. Mori, and
R. P. Liburdy, Effects of magnetic fields on the
accumulation of thiobarbituric acid reactive
substances induced by iron salt and H2O2 in
mouse brain homogenates or phosphotidylcholine,
Pathophysiology, vol. 7, no. 4, pp. 283288, 2001.
68-K. A. Hossmann and D. M. Hermann, Effects of
electromagnetic radiation of mobile phones on the
central nervous system, Bioelectromagnetics, vol. 24,
no. 1, pp. 4962, 2003.
70-Meral, H. Mert, N. Mert et al., Effects of 900-
MHz electromagnetic field emitted from cellular
phone on brain oxidative stress and some vitamin
levels of guinea pigs, Brain Research, vol. 1169, no. 1,
pp. 120124, 2007.
57

71-M. Ammari, A. Lecomte, M. Sakly, H. Abdelmelek,
and R. de-Seze, Exposure to GSM 900MHz
electromagnetic fields affects cerebral cytochrome c
oxidase activity,Toxicology, vol. 250, no. 1, pp. 7074,
2008.
72-S. Xu, Z. Zhou, L. Zhang et al., Exposure to
1800MHz radiofrequency radiation induces oxidative
damage to mitochondrial DNA in primary cultured
neurons, Brain Research, vol. 1311, pp. 189196,
2010.
73-Hyt, J. Luukkonen, J. Juutilainen, and J. Naarala,
Proliferation, oxidative stress and cell death in cells
exposed to 872MHz radiofrequency radiation and
oxidants, Radiation Research, vol. 170, no. 2, pp.
235243, 2008
74-C. Benz and C. Yau, Ageing, oxidative stress
and cancer: paradigms in parallax, Nature Reviews
Cancer, vol. 8, no. 11, pp. 875879, 2008.
75-K. Jomova, D. Vondrakova, M. Lawson, and M.
Valko, Metals, oxidative stress and neurodegenerative
disorders, Molecular and Cellular Biochemistry, vol.
345, no. 1-2, pp. 91104, 2010.
76-G. McKhann, D. Drachman, and M. Folstein,
Clinical diagnosis of Alzheimers disease: report of the
NINCDS-ADRDA work group under the auspices
of Department of Health and Human Services Task
Force on Alzheimers disease, Neurology, vol. 34, no.
7, pp. 939944, 1984.
77-Shi Du Yan, Shi Fang Yan, X. Chen et al., Non-
enzymatically glycated tau in Alzheimers disease
induces neuronal oxidant stress resulting in cytokine
gene expression and release of amyloid -peptide,
Nature Medicine, vol. 1, no. 7, pp. 693699, 1995.
78-Nunomura, G. Perry, M. A. Pappolla et al., RNA
oxidation is a prominent feature of vulnerable neurons
58
www.approachestoagingcontrol.org
in Alzheimers disease, Journal of Neuroscience, vol.
19, no. 6, pp. 19591964, 1999.
79-Ward, S. Crean, C. J. Mercaldi et al., Prevalence
of Apolipoprotein E4 genotype and homozygotes
(APOE e4/4) among patients diagnosed with
alzheimers disease: a systematic review and meta-
analysis, Neuroepidemiology, vol. 38, no. 1, pp. 117,
2012.
80-M. Santibaez, F. Bolumar, and A. M. Garca,
Occupational risk factors in Alzheimers disease:
a review assessing the quality of published
epidemiological studies, Occupational and
Environmental Medicine, vol. 64, no. 11, pp. 723732,
2007.
81-E. Sobel, J. Louhija, R. Sulkava et al., Lack of
association of apolipoprotein E allele 4 with late-onset
Alzheimers disease among Finnish centenarians,
Neurology, vol. 45, no. 5, pp. 903907, 1995.
82- M. Garca, A. Sisternas, and S. P. Hoyos,
Occupational exposure to extremely low frequency
electric and magnetic fields and Alzheimer disease: a
meta-analysis, International Journal of Epidemiology,
vol. 37, no. 2, pp. 329, 340, 2008.
83- M. Rsli, Commentary: epidemiological
research on extremely low frequency magnetic
fields and Alzheimers disease biased or informative?
International Journal of Epidemiology, vol. 37, no. 2,
pp. 341, 343, 2008.
84-L. Masters and K. Beyreuther, Science, medicine,
and the future. Alzheimers disease, BMJ, vol. 316, no.
7129, pp. 446448, 1998.
85-Josefson, Foods rich in antioxidants may reduce
risk of Alzheimers disease, BMJ, vol. 325, article 7,
2002.
Approaches to Aging Control. Vol 19. october 2015
86-Del Giudice, F. Facchinetti,V. Nofrate et al., Fifty
Hertz electromagnetic field exposure stimulates
secretion of -amyloid peptide in cultured human
neuroglioma, Neuroscience Letters, vol. 418, no. 1,
pp. 912, 2007.
87-Sobel and Z. Davanipour, Electromagnetic field
exposure may cause increased production of amyloid
beta and eventually lead to Alzheimers disease,
Neurology, vol. 47, no. 6, pp. 15941600, 1996.
88-W. Arendash, J. Sanchez-Ramos, T. Mori et al.,
Electromagnetic field treatment protects against and
reverses cognitive impairment in Alzheimers disease
mice, Journal of Alzheimers Disease, vol. 19, no. 1,
pp. 191210, 2010. 89- Dubreuil, T. Jay, and J. M.
Edeline, Head-only exposure to GSM 900-MHz
electromagnetic fields does not alter rats memory
in spatial and non-spatial tasks, Behavioural Brain
Research, vol. 145, no. 1-2, pp. 5161, 2003.
90-W. Arendash, T. Mori, M. Dorsey, R. Gonzalez, N.
Tajiri, and C. Borlongan, Electromagnetic treatment
to old Alzheimers mice reverses -amyloid deposition,
modifies cerebral blood flow, and provides selected
cognitive benefit, PLoS One, vol. 7, no. 4, Article ID
e35751, 2012.
91-N. Dragicevic, P. C. Bradshaw, M. Mamcarz et
al., Long-term electromagnetic field treatment
enhances brain mitochondrial function of both
Alzheimers transgenic mice and normal mice:
a mechanism for electromagnetic field-induced
cognitive benefit?Neuroscience, vol. 185, pp. 135,
149, 2011.
92- F. Sderqvist, L. Hardell, M. Carlberg, and K.
H. Mild, Radiofrequency fields, transthyretin, and
alzheimers disease, Journal of Alzheimers Disease,
vol. 20, no. 2, pp. 599606, 2010.
93-F. Terro, A. Magnaudeix, M. Crochetet et
al., GSM-900MHz at low dose temperature-
dependently downregulates -synuclein in cultured
cerebral cells independently of chaperone-mediated-
autophagy,Toxicology, vol. 292, no. 2-3, pp. 136144,
2012.
94-F. P. De Gannes, G. Ruffi, M. Taxile et al.,
Amyotrophic Lateral Sclerosis (ALS) and extremely-
low frequency (ELF) magnetic fields: a study in the
SOD-1 transgenic mouse model, Amyotrophic
Lateral Sclerosis, vol. 10, no. 5-6, pp. 370373, 2009.
View at Publisher
97-Tasset, F. J. Medina, I. Jimena et al.,
Neuroprotective effects of extremely low-frequency
electromagnetic fields on a Huntingtons disease rat
model: effects on neurotrophic factors and neuronal
density, Neuroscience, vol. 209, pp. 5463, 2012.
98-V. G. Khurana, C. Teo, M. Kundi, L. Hardell,
and M. Carlberg, Cell phones and brain tumors: a
review including the long-term epidemiologic data,
Surgical Neurology, vol. 72, no. 3, pp. 205214, 2009.
View at Publisher View at Google Scholar View
at Scopus
99-M. S. Pollanen, D. W. Dickson, and C. Bergeron,
Pathology and biology of the Lewy body, Journal
of Neuropathology and Experimental Neurology, vol.
52, no. 3, pp. 183191, 1993.
100-L. S. Wechsler, H. Checkoway, G. M. Franklin,
and L. G. Costa, A pilot study of occupational and
environmental risk factors for Parkinsons disease,
NeuroToxicology, vol. 12, no. 3, pp. 387392, 1991.
101-A. Savitz, H. Checkoway, and D. P. Loomis,
Magnetic field exposure and neurodegenerative
disease mortality among electric utility workers,
Epidemiology, vol. 9, no. 4, pp. 398404, 1998. View
at Google Scholar View at Scopus
59

102-C. Johansen, Exposure to electromagnetic fields
and risk of central nervous system disease in utility
workers, Epidemiology, vol. 11, no. 5, pp. 539543,
2000.
103-A. Savitz, D. P. Loomis, and C. K. J.Tse,Electrical
occupations and neurodegenerative disease: analysis
of U.S. Mortality data, Archives of Environmental
Health, vol. 53, no. 1, pp. 7174, 1998.
104- C. W. Noonan, J. S. Reif, M. Yost, and J.
Touchstone, Occupational exposure to magnetic
fields in case-referent studies of neurodegenerative
diseases, Scandinavian Journal of Work, Environment
and Health, vol. 28, no. 1, pp. 4248, 2002.
105-Huss, A. Spoerri, M. Egger, and M. Rsli,
Residence near power lines and mortality from
neurodegenerative diseases: longitudinal study of the
Swiss population, American Journal of Epidemiology,
vol. 169, no. 2, pp. 167175, 2009.
106-S. Boille, C.Vande Velde, and D. Cleveland, ALS:
a disease of motor neurons and their non neuronal
neighbors, Neuron, vol. 52, no. 1, pp. 3959, 2006.
View at Publisher
107-P. Julien and J. Kriz, Transgenic mouse models
of amyotrophic lateral sclerosis, Biochimica et
Biophysica Acta, vol. 1762, no. 11-12, pp. 10131024,
2006.View at Publisher
108-Y. Chang, Q. Kong, X. Shan et al., Messenger
RNA oxidation occurs early in disease pathogenesis
and promotes motor neuron degeneration in ALS,
PLoS ONE, vol. 3, no. 8, Article ID e2849, 2008.
109-M. Cozzolino and M. T. Carr, Mitochondrial
dysfunction in ALS, Progress in Neurobiology, vol. 97,
no. 2, pp. 5466, 2012.
110-Kheifets, J. D. Bowman, H. Checkoway et al.,
Future needs of occupational epidemiology of
60
www.approachestoagingcontrol.org
extremely low frequency electric and magnetic fields:
review and recommendations, Occupational and
Environmental Medicine, vol. 66, no. 2, pp. 7280,
2009
111-Kondo and T. Tsubaki, Case-control studies of
motor neuron disease. Association with mechanical
injuries, Archives of Neurology, vol. 38, no. 4, pp.
220226, 1981.
112-Feychting, F. Jonsson, N. L. Pedersen, and A.
Ahlbom, Occupational magnetic field exposure and
neurodegenerative disease, Epidemiology, vol. 14,
no. 4, pp. 413419, 2003.View at Publisher View at
Google Scholar
113-T. Sorahan and L. Kheifets, Mortality from
Alzheimers, motor neuron and Parkinsons disease
in relation to magnetic field exposure: findings
from the study of UK electricity generation and
transmission workers, 19732004, Occupational and
Environmental Medicine, vol. 64, no. 12, pp. 820826,
2007.
114-L. E. Parlett, J. D. Bowman, and E. Van
Wijngaarden, Evaluation of occupational exposure
to magnetic fields and motor neuron disease
mortality in a population-based cohort, Journal of
Occupational and Environmental Medicine, vol. 53,
no. 12, pp. 14471451, 2011.
115-A. Sorolla, G. Reverter-Branchat, J. Tamarit,
I. Ferrer, J. Ros, and E. Cabiscol, Proteomic and
oxidative stress analysis in human brain samples of
Huntington disease, Free Radical Biology and
Medicine, vol. 45, no. 5, pp. 667678, 2008.
116-Klepac, M. Relja, R. Klepac, S. Heimovi, T. Babi,
and V. Trkulja, Oxidative stress parameters in plasma
of Huntingtons disease patients, asymptomatic
Huntingtons disease gene carriers and healthy
Approaches to Aging Control. Vol 19. october 2015
subjects: a cross-sectional study, Journal of Neurology,
vol. 254, no. 12, pp. 16761683, 2007.
117-Tnez, I. Tasset, V. P. D. La Cruz, and A.
Santamara, 3-nitropropionic acid as a tool to study
the mechanisms involved in huntingtons disease:
past, present and future, Molecules, vol. 15, no. 2, pp.
878916, 2010.
118-M. N. Herrera-Mundo, D. Silva-Adaya, P. D.
Maldonado et al., S-Allylcysteine prevents the rat
from 3-nitropropionic acid-induced hyperactivity,
early markers of oxidative stress and mitochondrial
dysfunction, Neuroscience Research, vol. 56,
no. 1, pp. 3944, 2006. 119- S. Ramaswamy, J.
L. McBride, and J. H. Kordower, Animal models of
Huntingtons disease, ILAR Journal, vol. 48, no. 4,
pp. 356373, 2007.
119-S. Ramaswamy, J. L. McBride, and J. H. Kordower,
Animal models of Huntingtons disease, ILAR
Journal, vol. 48, no. 4, pp. 356373, 2007.
120-L. Kheifets, D. Renew, G. Sias, and J. Swanson,
Extremely low frequency electric fields and cancer:
assessing the evidence, Bioelectromagnetics, vol. 31,
no. 2, pp. 89101, 2010.
121-International Agency for Research on
Cancer-(IARC), Non-ionizing radiation, part II,
radiofrequency electromagnetic fields (RF-EMF),
Monograph, vol. 102, 2011.
122-Arias-Carrin, L.Verdugo-Daz, A. Feria-Velasco
et al., Neurogenesis in the subventricular zone
following transcranial magnetic field stimulation
and nigrostriatal lesions, Journal of Neuroscience
Research, vol. 78, no. 1, pp. 1628, 2004.
126-J. Naarala, A. Hyt, and A. Markkanen, Cellular
effects of electromagnetic fields, ATLA Alternatives
to Laboratory Animals, vol. 32, no. 4, pp. 355360,
2004.
127-J. Luukkonen, A. Liimatainen, A. Hyt, J.
Juutilainen, and J. Naarala, Pre-exposure to 50HZ
magnetic fields modifies menadione-induced
genotoxic effects in human SH-SY5Y neuroblastoma
cells, PLoS ONE, vol. 6, no. 3, Article ID e18021,
2011.
128- M. A. Martnez, A. beda, M. A. Cid, and M.
A. Trillo, The proliferative response of NB69 human
neuroblastoma cells to a 50Hz magnetic field is
mediated by ERK1/2 signaling, Cellular Physiology
and Biochemistry, vol. 29, no. 5-6, pp. 675686, 2012.
129-N. Kuster and F. Schnborn, Recommended
minimal requirements and development guidelines
for exposure setups of bio-experiments addressing
the health risk concern of wireless communications,
Bioelectromagnetics, vol. 21, no. 7, pp. 508514, 2000.
130-Costa, F. Ferreira-da-Silva, M. J. Saraiva, and
I. Cardoso, Transthyretin protects against A-beta
peptide toxicity by proteolytic cleavage of the
peptide: a mechanism sensitive to the kunitz protease
inhibitor, PLoS ONE, vol. 3, no. 8, Article ID e2899,
2008.
131-Ebert, S. J. Eom, J. Schuderer et al., Response,
thermal regulatory threshold and thermal breakdown
threshold of restrained RF-exposed mice at
905MHz, Physics in Medicine and Biology, vol. 50,
no. 21, pp. 52035215, 2005.
132-W. Kainz, N. Nikoloski, W. Oesch et al.,
Development of novel whole-body exposure setups
for rats providing high efficiency, National Toxicology
Program (NTP) compatibility and well-characterized
exposure, Physics in Medicine and Biology, vol. 51,
no. 20, article 5211, 2006.View at Publisher
133-Paffi, M. Liberti,V. Lopresto et al., A wire patch
cell exposure system for in vitro experiments at
wi-fi frequencies, IEEE Transactions on Microwave
61
 www.approachestoagingcontrol.org

Theory and Techniques, vol. 58, no. 12, pp. 4086

4093, 2010.

134-Merla, N. Ticaud, D. Arnaud-Cormos, B. Veyret,

and P. Leveque, Real-time RF exposure setup
based on a multiple electrode array (MEA) for
electrophysiological recording of neuronal networks,
IEEE Transactions on Microwave Theory and
Techniques, vol. 59, no. 3, pp. 755762, 2011

135-Y. Zhao, S. P. Zou, and P. E. Knapp, Exposure

to cell phone radiation up-regulates apoptosis genes
in primary cultures of neurons and astrocytes,
Neuroscience Letters, vol. 412, no. 1, pp. 3438, 2007.

136-R. Ferreira, F. Bonatto, M. A. De Bittencourt

Pasquali et al., Oxidative stress effects on the
central nervous system of rats after acute exposure
to ultra high frequency electromagnetic fields,
Bioelectromagnetics, vol. 27, no. 6, pp. 487493, 2006.
Biochem J. 2007 Aug 1;405(3):559-68.

137-Friedman J1, Kraus S, Hauptman Y, Schiff Y, Seger

R.Mechanism of short-term ERK activation by
electromagnetic fields at mobile phone frequencies.
487493, 2006. Biochem J. 2007 Aug 1;405(3):559-
68.

138-R. M Anson,V A. Bohr Mitochondria, oxidative

DNA damage, and agingJ Am Aging Assoc. 2000 Oct;
23(4): 199218.

139-Lai H, Singh NP. Acute low-intensity microwave

exposure increases DNA single-strand breaks in rat
brain cells Bioelectromagnetics 1995; 16 (3): 207
210

140-Capuani B., Pacifici F., Pastore D., Guadagni

F., Donadel G., Palmirotta R., Sbraccia P., Lauro D.,
Della-Morte D Cellular Repair and Reversal of
Aging: the Role of NAD CellR4 2014; 2 (2): e852

62
Approaches to Aging Control. Vol 19. october 2015

Coordinated cycling of calorie restriction, exercise, and

pharmaceuticals to enhance molecular turnover and
removal of age-related neurotoxins
Roy G. Cutler
Laboratory of Neurosciences, National Institute on Aging, Intramural Research Program, Baltimore,
MD, 21224, U.S.A.
Correspondence: Dr. Roy G. Cutler. Laboratory of Neurosciences, 251 Bayview Blvd,
Baltimore, MD, 21224. Email: rcutler@nih.gov Phone: 410 558-8239.

Abstract

Molecular turnover is the balance between

synthesis and degradation. The rate of molecular
turnover has been shown to decrease with old
age and concomitant accumulation of damaged
molecules. The onset and progression of the major
age-related neurodegenerative diseases has been
linked to the accumulation of protein aggregates
(e.g. Alzheimers: beta-amyloid; Parkinsons: alpha-
synuclein; Huntingtons: Huntingtin; Amyotrophic
lateral sclerosis (ALS): mutated SOD1). Researchers
have been exploring the use of increasing the rate
of molecular turnover to prevent and clean up the Graphical abstract: proposed mechanism and protocol to
accumulation of aberrant disease causing molecules by increase molecular turnover and renewal by cycling master
activating catabolic pathways via intermittent fasting, catabolic and anabolic regulators. Caffeine is underlined
exercise, and drugs aimed at activating autophagy (e.g. to emphasize its unique dual potential as an AMPK
rapamycin). However, the often ignored but equally activator, and its metabolized methyluric acid derivatives (i.e.
important requirement for increasing molecular 1,7-dimethyluric acid) as a putative AMPK inhibitor.
renewal is the activation of anabolic pathways to 1. INTRODUCTION
synthesize new proteins, lipids, and carbohydrates. By
exploring the connections between the oxidative/ 1.1. Redox Regulation of Catabolism and
Autophagy
reduction control of AMP activated protein kinases
(AMPK), autophagy and gluconeogenesis, we propose Nutrient deprivation during starvation or prolonged
a protocol using a properly timed cycle approach of physical exercise increases the production and release
diet, exercise, and drugs to enhance the catabolic/ of reactive oxygen and nitrogen species (ROS, RNS)
anabolic phases of molecular turnover. (1). There is growing body of evidence indicating that
ROS/RNS are primary energy sensors that induce
Keywords: AMPK, aging, antioxidant, autophagy, catabolic pathways (i.e. AMP activated protein kinases
caffeine, fructose, urate oxidase, uric acid. (AMPK)) and autophagy (2). AMPK are found

63

in all eukaryotes and ubiquitously expressed in all
organs, where its major function is as an energy and
oxidative stress sensor that deactivates the majority
of anabolic energy using pathways, while activating
catabolic energy producing pathways (3). AMPK
is activated by hormones released during metabolic
stress (i.e. adiponectin) and physical exercise (i.e.
adrenaline (epinephrine))(4, 5). Natural dietary
factors such as lovastatin from oyster mushroom and
red yeast rice, berberine, and French Lilac diguanidine
(i.e. pharmaceutical derivative metformin) has
been shown to activate AMPK via superoxide
release from inhibition of mitochondria complex I
(6). The data from these findings indicate that the
released superoxide is acting on ataxia telangiectasia
mutated (ATM)/LKB1 and c-Src/PI3K dependent
pathways (7, 8). ROS/RNS activation of ATM also
mediates upstream activation of p53 thereby shifting
transcription towards the activation of autophagy.
Recent research has discovered new roles for p53 in
regulating energy metabolism and autophagy that is
dependent on its subcellular localization, where high
cytosolic levels inhibit, and transport into the nucleus
activates autophagy (9). In most organs tested,
AMPK protein and activity levels decrease with
over nutrition and old age (10). However, chronic
and global activation of these catabolic pathways as
seen in under nutrition and type 2-diabetes, acts to
decrease basil metabolic rate and muscle mass, while
increasing percent fat as preparation for long term
conservation of energy expenditure (11).
1.2. Uric Acid Blocks Fructose Mediated
Catabolic Response By Inhibiting AMPK
According to observations of the currently living
hominoids and the fossils of our ancestral apes 15
million years ago, their diets consist/ed of 50 - 75% of
daily calories coming from fruit, with the exception
of modern gorillas (12, 13). The high quantity and
caloric density of fruits in the ancestral rainforest
led to the evolution of many species of frugivore
primates. The most common types of fruits found to
be eaten by currently living apes nutritionally consists
on average of 48% simple sugars by weight, with near
1:1 ratios of fructose and glucose (12).
64
www.approachestoagingcontrol.org
Unlike glucose, dietary fructose is almost exclusively
metabolized in the liver, where ATP is rapidly used
up by fructokinase yielding fructose-1-phoshate and
thereby bypassing regulation by phosphofructokinase.
Fructose-1-phosphate is then catalyzed by aldolase
B yielding glyceraldehyde and dihydroxyacetone-3-
phosphate with further metabolism into glycerol-
3-phospate providing the glycerol moiety for
triglyceride synthesis. While the catabolism of
glucose is controlled by the negative feedback of
TCA cycle citrate on phosphofructokinase, fructose
catabolism proceeds without any negative feedback
regulation. Fructose catabolite glyceraldehyde-3-P
feeds into the glycolysis pathway resulting in increases
of lactate and citrate, the latter of which can act to
shuttle fructose metabolites towards Acetyl-CoA
(fatty acid synthesis) and both fructose and glucose
metabolites towards the PPP pathway and away from
glycogen by inhibiting phosphofructokinase during
excess caloric intake. Radio labeling of fructose
has shown that over feeding activates transketolase
(TKT) 2.5 times higher then glucose, which pulls
catabolites into PPP synthesis of xylulose-5-P, ribose-
5-P and nucleic acids (14). Xylulose-5-P produced
from PPP activates enzyme activity of protein-
phosphatase 2 (PP2A), which turns on enzymes for
glycolysis, protein synthesis, and lipogenesis pathways
(e.g. carbohydrate-responsive element-binding
protein (ChREBP), acetyl CoA carboxylase, and
fructokinase), while shutting off catabolic pathways
mediated by AMP-activated protein kinase (AMPK).
Figure 1 depicts how these PPP metabolites are acting
as a positive self-propagating feed forward pathway
for rapid fructose metabolism (15).
Approaches to Aging Control. Vol 19. october 2015
Figure 1. Hepatic metabolism of glucose and fructose
cascade under caloric excess of a high in fructose meal.
Metabolites = black, enzymes = blue, transcription factors
= green; activation effect = green line, inhibition effect =
red line. Heavier weighted arrow indicates direction of most
favorable reaction. Only most relevant metabolites, enzymes
and reaction for this discussion are shown, with dashed lines
indicating multiple steps in the pathway.
However, due to the sudden decrease of ATP to
ADP (fructokinase) and AMP (PPP pathway ribose-
phosphate-pyrophosphokinase), along with ROS/
RNS produced during purine catabolism (xanthine
oxidase), an acute caloric excess of dietary fructose can
activate AMPK thereby shutting off anabolic energy
storage, while activating energy producing catabolic
pathways (16, 17). This metabolic phenomena is
made worse by fructoses low glycemic index (i.e.
insulin response), which under a high fructose load
culminates to activate a starvation response, even
thought excess calories are being consumed (18).
Fortunately, the purine catabolite uric acid (UA) that
is produced by fructose via the PPP pathway and
from the breakdown of AMP via AMP deaminase
has been shown to inhibit AMPK activation and has
been hypothesized as a biochemical counter measure
against activation of AMPK (18, 19). Further evidence
that UA is likely playing a key role towards increasing
energy storage efficiency during a high fructose (e.g.
fruit) meal is that it is produced at the time and sites
(liver peroxisome and mitochondria) needed to block
activation of AMPK.
Even though fructose does not elicit an insulin
response, caloric excess amounts of fructose has
been shown to be significantly more efficient than
glucose in being synthesized into fat energy reserves,
but this effect is blunted with xanthine oxidase
inhibitor allopurinol (19). Even more compelling
is that a high fructose diet significantly increases
protein levels of nuclear transcription factor sterol-
regulatory element-binding proteins (SREBP) 2.2
times, and DNA binding activity of ChREBP 3.9
times, with no such change from the high glucose
diet (20). Numerous human epidemiological studies
have confirmed lab results showing a strong positive
association between dietary fructose and increased
fasting levels of blood triglycerides and glucose are
dependent on levels of UA (21, 22). The metabolic
advantage of fructoses efficiency in the conversion
into fat energy may have also been a driving force
for the 2.33 times relative sweetness response over
glucose (as measured in humans), which predicts a
likely coevolution between fruit bearing plants and
frugivores (23).
1.3. Uric Acid Inhibition of AMPK Preserves
Gluconeogenesis During Metabolic
Stress
In hominoids, maintaining glucose levels for the brain
is critical to ensure functionality for survival during
environmental stress that includes starvation. Under
normal conditions, the human brain consumes about
60% of available blood glucose. Acute and non-
diabetic rises in blood pressure and glucose levels
have been linked with higher cognitive performance.
Whereas, a 20 to 25% drop from a normal fasting level
of 72 mg/dl (4.0 mmol/L) is a medical emergency
that causes neuroglycopenia (24). During a fight or
flight situation, the release of adrenaline (epinephrine)
and norepinephrine acts to acutely raise both blood
pressure and glucose levels. However, this effect can
only lasts about 40 minutes, therefore, during periods
of prolonged physical and cognitive metabolic stress,
multiple insurance mechanisms are desirable to insure
optimal levels of blood glucose (25). The calories
consumed after a meal are quickly distributed
for storage either in subcutaneous/visceral fat
triglycerides, muscle proteins, or liver glycogen. Post-
65

absorptive blood glucose levels are then maintained
approximately 50% by liver glycogenolysis, and 50%
by liver (30%) and kidney (20%) gluconeogenesis
from glycerol, alanine, glutamine, and lactate (26).
The relatively high levels of UA in humans is due to
permanent transcriptional loss of urate oxidase (UOX)
that was mainly expressed in liver peroxisomes and
mitochondria, and from the roughly 90% reabsorption
by URAT1 and GLUT9 in the kidney (27). The loss
of UOX in hominoids happened in an ancestor of the
great apes: orangutan, chimpanzee, bonobo, gorilla,
and human, 15 million years ago, and independently
in the gibbons (lesser apes) about 10 million years
ago. Sequence analysis of the UOX pseudogenes
from 5 different species of gibbons revealed that each
species had evolved independent mutations for the
loss of function in UOX (28). Johnson and colleagues
(29) hypothesize that silencing of UOX provided
a survival advantage by increasing the efficiency of
energy storage, particularly from a large high fructose
(fruit) meal. The conserved sequence of the silencing
mutation in UOX within each species of hominoids
indicates that it was fixed within a small founding
group. Fossil studies indicate that the changes in
the physical and social environment during the time
that the UOX mutations were fixed were under
conditions of dwindling fruit availability during the
cooling Miocene epoch climate, and under a socially
dominant hierarchical mating groups competing for
resources (12, 13).
During high intensity anaerobic exercise, contracting
muscle first utilizes energy from available ATP,
creatine, then from glucose and glycogen which
yields lactate that is released into the blood stream.
Blood lactate enters the liver and kidneys where it
is catalyzed by the Cori cycle to pyruvate, which
relies on the gluconeogenesis pathway to produce
glucose that is released back into the blood stream.
Exercise increases levels of AMPK activating
hormones adrenaline, noradrenaline and adiponectin,
while suppressing insulin an AMPK deactivating
hormone (30, 31). As previously discussed, AMPK
is a metabolic energy switch that acts to shut down
energy using anabolic (mTOR, ChREBP, SREBP1c,
and gluconeogenesis) pathways, while activating
energy producing catabolic (glycolysis, protein and
66
www.approachestoagingcontrol.org
lipid oxidative phosphorylation) pathways (11). The
shut down of gluconeogenesis in the liver and kidneys
by AMPK activation would act to limit the source of
blood glucose during metabolic stress to be coming
solely from liver glycogen stores. Lactate released
into the blood from anaerobic muscle contractions
that would normally be metabolized into glucose via
the gluconeogenesis pathway in the liver and kidneys
would also rise and limit physical performance
(Figure 2)(32). However, anaerobic exercise also
results in an acute rise of UA in the liver, kidneys
and blood, as purine (e.g. ATP) are catabolized. The
acute rise in UA acts to maintain clearance of lactate
via gluconeogenesis in the liver and kidney during
metabolic stress by blocking AMPK activation, so
much so that these organs actually have increases
in protein and activity levels of regulatory enzymes
PEPCK and G6Pase (21, 22). Gluconeogenesis in the
liver and kidney is important in maintaining blood
glucose homeostasis as it accounts for approximately
50% of blood glucose during exhaustive exercise
(26). One hypothesis for the evolutionary selection
for the permanent loss of UOX and increases in
reabsorption pathways aimed to increase UA levels
is for maintaining levels of glucose needed for high
brain cognition during times of extended metabolic
stress (33). Human exercise studies have reported
that during exhausting trials, there is a strong
correlation between blood UA levels and homeostasis
of glucose (34). More importantly, maintenance of
blood glucose levels during prolonged exhaustive
exercise challenges are key in preventing declines
in cognitive performance (36, 37). Increases in
cognitive performance, particularly with perceptual
and memory tasks, can be achieved by consumption
of 25 grams of glucose before testing (38, 39).
Further evidence that UA is likely playing a key role
in maintaining gluconeogenesis during metabolic
stress is that it is produced at the time and sites (liver
peroxisome and mitochondria; kidney Urat1 and
Glut9 reabsorbsion) needed to block activation of
AMPK, which are also the two organs that perform
gluconeogenesis.
Approaches to Aging Control. Vol 19. october 2015
Figure 2. Role of uric acid in preventing ROS/RNS
activation of AMPK towards shutting off liver and kidney
gluconeogenesis during fasting and exercise.
1.4. Increasing Molecular Renewal Rates by
Redox Cycling of Catabolic and Anabolic
Pathways
The currently prescribed protocol of drug
administration for type-2 diabetes and cardiovascular
disease (e.g. metformin, statins) is aimed at chronically
activating catabolic pathways. We are proposing a
potentially better outcome in molecular turnover
and reduced risk of age-related disease by using a
coordinated and properly timed regimen of diet,
exercise and low dose drugs to amplify between cycles
of catabolism and anabolism. Understanding the
mechanism and evolutionary tradeoffs that went into
the transcriptional deactivation of UOX to raise UA
levels so that large quantities of dietary fructose could
be consumed and stored into energy can be used to
develop drugs targeting the AMPK pathway. One
candidate drug is caffeine (1,3,7-dimethyxanthine).
While caffeine is a natural plant made pesticide that
is classified as hazardous by OSHA, it is the most
commonly consumed psychoactive drug in the world
(40). Epidemiological studies show that regular use of
caffeine products lowers the risk of many age-related
diseases including type-2 diabetes, cardiovascular,
Parkinsons, and Alzheimers (41). Caffeine has been
shown to activate AMPK and autophagy in a calcium
dependent manner and passes the blood brain barrier
(42). It is metabolized in the liver into paraxanthine
(84%), theobromine (12%) and theophylline (4%),
followed by further metabolism by xanthine oxidase
into mono- and di-methyl-uric acid derivatives.
Compared to UA, caffeine derived methyl-uric
acid derivatives (e.g. 1,7-dimethylxanthine) are
approximately 100-times more water-soluble and
have been shown to cross the blood brain barrier
(43). Caffeine may therefore have the dual property
of activating catabolic and autophagy mechanisms,
while its metabolic end product (e.g. 1,7-dimethyl-
uric acid) helps activate anabolic pathways by
inhibiting AMPK.
Figure 3. Timeline example of a proposed 24-hour
catabolic/anabolic enhanced cycle.
Data from Argelles et al. (44) and colleagues shows
that there is a natural 24-hour circadian rhythm
with peak periods of oxidative stress and antioxidant
protection (45, 46). Since mitochondria generated
superoxide as induced by complex I inhibitors (e.g.
metformin) stimulates catabolic and antioxidants
(e.g. uric acid, NADPH via PPP pathway) promote
anabolic pathways, property timed oral intake of
oxidative stress inducers and antioxidants can be used
to either enhance or blunt the amplitude in there
natural circadian cycle.
Dorsomorphin (i.e. compound C) is the only
commercially available pharmaceutical AMPK
inhibitor. Although Dorsomorphin has been
reported to inhibit activation of AMPK mediated by
AICAR and metformin, it also induces autophagy
by an AMPK independent mechanism and therefore
would not likely be a good candidate in activating
anabolic pathways (47). However, recent reports by
67

Mairet-Coello et al. (48) and Ma et al. (49) show
that CAMKK2-AMPK mediates the synaptotoxic
effects of -amyloid through Tau phosphorylation,
where inhibition of AMPK by dorsomorphin
alleviates these impairments in the hippocampus.
Furthermore, aside from the recognized association
of apolipoprotein E (APOE) locus with Alzheimers
disease, a genome-wide association study identified
SNPs upstream of UA transporter SLC2A9 (GLUT9)
as a strong risk factor (50). Cell culture studies on
the neuroprotective effects of UA in preventing
glutamate and cytosine arabinoside toxicity in
neurons and dopaminergic neurons respectfully,
requires its accumulation in astrocytes; the mediator
of gluconeogenesis and lactate shuttle (51 - 53).
As previously discussed, when caloric excess of a
meal high in fructose is consumed, the liver is flooded
with all the metabolites needed for protein and
triglyceride synthesis. Compared to glucose, fructose
bypasses the glycolytic pathway in such a way that
the metabolites feed into the PPP cycle that produces
the glycerol and reducing agent NADPH needed
for the biosynthesis of free fatty acids and steroids.
As with proteins, de novo synthesis of new lipids is
as important as catabolism for increasing turnover
aimed at lowering the accumulation of reactive lipid
peroxides as seen with a ketosis diet (54). Therefore
the refeeding and anabolic half of our proposed cycle
(figure 3) should ideally include a balance of insulin
inducing carbohydrates, PPP pathway inducing
fructose, and a complete amino acid protein source,
rich in mTORC1 inducing branch chain amino
acids (Leu, Ile, Val)(55). Low dose of both phase
enhancing drugs and natural food factors are needed
for separating the phase and therefore maximizing
the effect during a 24-hour cycle. This concept is
emphasized by the accumulation of data showing that
taking high doses of antioxidants blunts the positive
effects of exercise training on muscle growth and
blood pressure (56, 57). Alternatively, our proposed
cycle could include longer periods of time (i.e. days
or weeks) to allow higher drug doses resulting in
more pronounced peaks of catabolism.
The activation of catabolic processes to remove
damaged tissues after intense exercise is required to
trigger the subsequent repair and renewal anabolic
68
www.approachestoagingcontrol.org
process. In conjunction with the blunting effect of
antioxidants on repair and renewal discussed above,
taking high doses of anti-inflammatory (i.e. NSAID,
alpha omega fatty acids (EPA & DHA) and/or
antioxidant agents have also been shown to inhibit
both the catabolic and anabolic response following an
intense exercise session with the additional blunting
of the beneficial blood pressure lowering effects
(56, 57). These findings indicate the importance of
immune signaling factors in the molecular repair
and renewal pathway, and along with syncing with
the natural circadian peaks of other hormones (e.g.
thyroid, growth hormone, testosterone, etc.)
should be explored to further enhance our proposed
protocol.
Conflict of interest: The author declares no
competing financial interests.
Acknowledgments: This work was supported by
the Intramural Research Program of the National
Institute on Aging, U.S.A.
References
(1) Filomeni G, Desideri E, Cardaci S, Rotilio G,
Ciriolo MR. Under the ROSthiol network is
the principal suspect for autophagy commitment.
Autophagy. 2010, Oct; 6(7): 999-1005.
(2) Chen Y, Azad MB, Gibson SB. Superoxide is the
major reactive oxygen species regulating autophagy.
Cell Death Differ. 2009 Jul; 16(7): 1040-52.
(3) Shirwany NA, Zou MH. AMPK: a cellular
metabolic and redox sensor. Aminireview. Front
Biosci (Landmark Ed). 2014 Jan 1; 19:447-74.
(4) Zhou L, Deepa SS, Etzler JC, Ryu J, Mao X, Fang
Q, Liu DD,Torres JM, Jia W, Lechleiter JD, Liu F, Dong
LQ. Adiponectin activates AMP-activated protein
kinase in muscle cells via APPL1/LKB1-dependent
and phospholipase C/Ca2+/Ca2+/calmodulin-
dependent protein kinase kinase-dependent pathways.
J Biol Chem. 2009 Aug 14;284(33):22426-35.
(5) Koh HJ, Hirshman MF, He H, Li Y, Manabe
Y, Balschi JA, Goodyear LJ. Adrenaline is a critical
Approaches to Aging Control. Vol 19. october 2015
mediator of acute exercise-induced AMP-activated
protein kinase activation in adipocytes. Biochem J.
2007 May 1;403(3):473-81.
(6) Han Y, Wang Q, Song P, Zhu Y, Zou MH. Redox
regulation of the AMP-activated protein kinase. PLoS
One. 2010 Nov 5; 5(11)
(7) Zou MH, Kirkpatrick SS, Davis BJ, Nelson JS,
Wiles WG 4th, Schlattner U, Neumann D, Brownlee
M, Freeman MB, Goldman MH. Activation of the
AMP-activated protein kinase by the anti-diabetic
drug metformin in vivo. Role of mitochondrial
reactive nitrogen species. J Biol Chem. 2004 Oct 15;
279(42): 43940-51.
(8) Turner N, Li JY, Gosby A, To SW, Cheng Z,
Miyoshi H, Taketo MM, Cooney GJ, Kraegen EW,
James DE, Hu LH, Li J,Ye JM. Berberine and its more
biologically available derivative, dihydroberberine,
inhibit mitochondrial respiratory complex I: a
mechanism for the action of berberine to activate
AMP-activated protein kinase and improve insulin
action. Diabetes. 2008 May; 57(5): 1414-8.
(9) Maiuri MC, Galluzzi L, Morselli E, Kepp O, Malik
SA, Kroemer G. Autophagy regulation by p53. Curr
Opin Cell Biol. 2010 Apr; 22(2): 181-5.
(10) Hofmann JW, Zhao X, De Cecco M, Peterson
AL, Pagliaroli L, Manivannan J,Hubbard GB, Ikeno Y,
Zhang Y, Feng B, Li X, Serre T, Qi W, Van Remmen
H, Miller RA, Bath KG, de Cabo R, Xu H, Neretti N,
Sedivy JM. Reduced Expression of MYC Increases
Longevity and Enhances Healthspan. Cell. 2015 Jan
29; 160(3): 477-88.
(11) Park S, Kim da S, Kang S, Shin BK. Chronic
activation of central AMPK attenuates glucose-
stimulated insulin secretion and exacerbates hepatic
insulin resistance in diabetic rats. Brain Res Bull.
2014 Sep; 108:18-26.
(12) Andrews P, Kelley J. Middle Miocene dispersals
of apes. Folia Primatol (Basel). 2007; 78(5-6): 328-43.
(13) Begun DR. Middle Miocene hominoid origins.
Science. 2000 Mar 31; 287(5462): 2375.
(14) Liu H, Huang D, McArthur DL, Boros LG,
Nissen N, Heaney AP. Fructose induces transketolase
flux to promote pancreatic cancer growth. Cancer
Res. 2010 Aug 1; 70(15): 6368-76.
(15) Uyeda K, Yamashita H, Kawaguchi T.
Carbohydrate responsive element-binding protein
(ChREBP): a key regulator of glucose metabolism
and fat storage. Biochem Pharmacol. 2002 Jun 15;
63(12): 2075-80.
(16) Jaeschke H, Mitchell JR. Mitochondria and
xanthine oxidase both generate reactive oxygen
species in isolated perfused rat liver after hypoxic
injury. Biochem Biophys Res Commun. 1989 Apr
14; 160(1): 140-7.
(17) Xie Z, Dong Y, Zhang M, Cui MZ, Cohen
RA, Riek U, Neumann D, Schlattner U, Zou MH.
Activation of protein kinase C zeta by peroxynitrite
regulates LKB1-dependent AMP-activated protein
kinase in cultured endothelial cells. J Biol Chem.
2006 Mar 10; 281(10): 6366-75.
(18) Schwarzmeier JD, Marktl W, Moser K, Lujf A.
Fructose induced hyperuricemia. Effects of fructose
on the de novo synthesis of adenine nucleotides in
the liver and skeletal muscles of rats. Res Exp Med
(Berl). 1974; 162(4): 341-46.
(19) Lanaspa MA, Sanchez-Lozada LG, Choi
YJ, Cicerchi C, Kanbay M, Roncal-Jimenez CA,
Ishimoto T, Li N, Marek G, Duranay M, Schreiner G,
Rodriguez-Iturbe B, Nakagawa T, Kang DH, Sautin
YY, Johnson RJ. Uric acid induces hepatic steatosis by
generation of mitochondrial oxidative stress: potential
role in fructose-dependent and -independent fatty
liver. J Biol Chem. 2012 Nov 23; 287(48): 40732-44.
(20) Koo HY, Miyashita M, Cho BH, Nakamura MT.
Replacing dietary glucose with fructose increases
ChREBP activity and SREBP-1 protein in rat liver
69

nucleus. Biochem Biophys Res Commun. 2009 Dec
11; 390(2): 285-9.
(21) Lin, J.; Yang, R.; Tarr, P.T.; Wu, P.H.; Handschin,
C.; Li, S.; Yang, W.; Pei, L.; Uldry, M.; Tontonoz, P.;
Newgard, C.B.; Spiegelman, B.M. Hyperlipidemic
effects of dietary saturated fats mediated through
PGC-1beta coactivation of SREBP. Cell, 2005,
120(2), 261-273.
(22) Rock, K.L.; Kataoka, H.; Lai, J.J. Uric acid as a
danger signal in gout and its comorbidities. Nat Rev
Rheumatol., 2013, 9(1), 13-23.
(23) Remis MJ. The role of taste in food selection
by African apes: implications for niche separation
and overlap in tropical forests. Primates. 2006
Jan;47(1):56-64.
(24) Cryer PE, Axelrod L, Grossman AB, Heller SR,
Montori VM, Seaquist ER, Service FJ; Endocrine
Society. Evaluation and management of adult
hypoglycemic disorders: an Endocrine Society
Clinical Practice Guideline. J Clin Endocrinol Metab.
2009 Mar; 94(3): 709-28.
(25) Blackman JD, Towle VL, Lewis GF, Spire JP,
Polonsky KS. Hypoglycemic thresholds for cognitive
dysfunction in humans. Diabetes. 1990 Jul;39(7):828-
35.
(26) Cano N. Bench-to-bedside review: glucose
production from the kidney. Crit Care. 2002
Aug;6(4):317-21.
(27) Bobulescu IA, Moe OW. Renal transport of
uric acid: evolving concepts and uncertainties. Adv.
Chronic Kidney Dis. 2012 Nov; 19(6): 358-71.
(28) Oda M, Satta Y, Takenaka O, Takahata N.
Loss of urate oxidase activity in hominoids and its
evolutionary implications. Mol Biol Evol. 2002
May;19(5):640-53.
(29) Johnson RJ, Stenvinkel P, Martin SL, Jani A,
Snchez-Lozada LG, Hill JO, Lanaspa MA. Redefining
70
www.approachestoagingcontrol.org
metabolic syndrome as a fat storage condition based
on studies of comparative physiology. Obesity (Silver
Spring). 2013 Apr; 21(4): 659-64.
(30) Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki
H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K,
Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P,
Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T.
Adiponectin stimulates glucose utilization and fatty-
acid oxidation by activating AMP-activated protein
kinase. Nat Med. 2002 Nov; 8(11): 1288-95.
(31) Miller RA, Chu Q, Le Lay J, Scherer PE, Ahima
RS, Kaestner KH, Foretz M, Viollet B, Birnbaum
MJ. Adiponectin suppresses gluconeogenic gene
expression in mouse hepatocytes independent of
LKB1-AMPK signaling. J Clin Invest. 2011 Jun;
121(6): 2518-28.
(32) Billat VL, Sirvent P, Py G, Koralsztein JP, Mercier
J.The concept of maximal lactate steady state: a bridge
between biochemistry, physiology and sport science.
Sports Med. 2003;33(6):407-26.
(33) Cicerchi C, Li N, Kratzer J, Garcia G, Roncal-
Jimenez CA, Tanabe K, Hunter B, Rivard CJ, Sautin
YY, Gaucher EA, Johnson RJ, Lanaspa MA. Uric
acid-dependent inhibition of AMP kinase induces
hepatic glucose production in diabetes and starvation:
evolutionary implications of the uricase loss in
hominids. FASEB J.2014 Aug;28(8):3339-50.
(34) Hellsten-Westing Y, Sollevi A, Sjdin B. Plasma
accumulation of hypoxanthine, uric acid and creatine
kinase following exhausting runs of differing
durations in man. Eur J Appl Physiol Occup Physiol.
1991;62(5):380-4.
(35) Hellsten-Westing Y, Sollevi A, Sjdin B. Plasma
accumulation of hypoxanthine, uric acid and creatine
kinase following exhausting runs of differing
durations in man. Eur J Appl Physiol Occup Physiol.
1991;62(5):380-4.
Approaches to Aging Control. Vol 19. october 2015
(36) Brisswalter J, Collardeau M, Ren A. Effects of
acute physical exercise characteristics on cognitive
performance. Sports Med. 2002;32(9):555-66.
(37) Moore RD, Romine MW, Oconnor PJ,
Tomporowski PD. The influence of exercise-
induced fatigue on cognitive function. J Sports Sci.
2012May;30(9):841-50.
(38) Foster JK, Lidder PG, Snram SI. Glucose and
memory: fractionation of enhancement effects?
Psychopharmacology (Berl). 1998 Jun;137(3):259-70.
(39) Snram-Lea SI, Foster JK, Durlach P, Perez C.
The effect of retrograde and anterograde glucose
administration on memory performance in healthy
young adults. Behav Brain Res. 2002 Aug 21;134(1-
2):505-16.
(40) Nathanson JA. Caffeine and related
methylxanthines: possible naturally occurring
pesticides. Science. 1984 Oct 12; 226(4671): 184-7.
(41) Carman AJ, Dacks PA, Lane RF, Shineman DW,
Fillit HM. Current evidence for theuse of coffee and
caffeine to prevent age-related cognitive decline and
Alzheimers disease. J Nutr Health Aging. 2014 Apr;
18(4): 383-92.
(42) Mathew TS, Ferris RK, Downs RM, Kinsey
ST, Baumgarner BL. Caffeine promotes autophagy
in skeletal muscle cells by increasing the calcium-
dependent activation of AMP-activated protein
kinase. Biochem Biophys Res Commun. 2014 Oct
24; 453(3): 411-8. doi: 10.1016/j.bbrc.2014.09.094.
(43) Haberman F,Tang SC, Arumugam TV, Hyun DH,
Yu QS, Cutler RG, Guo Z, Holloway HW, Greig NH,
Mattson MP. Soluble neuroprotective antioxidant
uric acid analogs ameliorate ischemic brain injury in
mice. Neuromolecular Med. 2007;9(4):315-23.
(44) Argelles S, Gmez A, Machado A, Ayala A. A
preliminary analysis of within-subject variation in
human serum oxidative stress parameters as a function
of time. Rejuvenation Res. 2007 Dec;10(4):621-36.
(45) Kanabrocki EL, Murray D, Hermida RC, Scott
GS, Bremner WF, Ryan MD, Ayala DE, Third JL,
Shirazi P, Nemchausky BA, Hooper DC. Circadian
variation in oxidative stress markers in healthy
and type II diabetic men. Chronobiol Int. 2002
Mar;19(2):423-39.
(46) Kanabrocki EL,Third JL, Ryan MD, Nemchausky
BA, Shirazi P, Scheving LE, McCormick JB, Hermida
RC, Bremner WF, Hoppensteadt DA, Fareed J, Olwin
JH. Circadian relationship of serum uric acid and
nitric oxide. JAMA. 2000 May 3;283(17):2240-1.
(47) Vucicevic L, Misirkic M, Janjetovic K,
Vilimanovich U, Sudar E, Isenovic E, Prica M,
Harhaji-Trajkovic L, Kravic-Stevovic T, Bumbasirevic
V, Trajkovic V. Compound C induces protective
autophagy in cancer cells through AMPK inhibition-
independent blockade of Akt/mTOR pathway.
Autophagy. 2011 Jan; 7(1): 40-50.
(48) Mairet-Coello G, Courchet J, Pieraut S, Courchet
V, Maximov A, Polleux F. The CAMKK2-AMPK
kinase pathway mediates the synaptotoxic effects of
A oligomers through Tau phosphorylation. Neuron.
2013 Apr 10;78(1):94-108.
(49) Ma T, Chen Y, Vingtdeux V, Zhao H, Viollet B,
Marambaud P, Klann E. Inhibition of AMP-activated
protein kinase signaling alleviates impairments in
hippocampalsynaptic plasticity induced by amyloid .
J Neurosci. 2014 Sep 3;34(36):12230-8.
(50) Hollingworth P, Sweet R, Sims R, Harold D,
Russo G, Abraham R, Stretton A, Jones N, Gerrish
A, Chapman J, Ivanov D, Moskvina V, Lovestone S,
Priotsi P, Lupton M, Brayne C, Gill M, Lawlor B,
Lynch A, Craig D, McGuinness B, Johnston J, Holmes
C, Livingston G, Bass NJ, Gurling H, McQuillin A;
GERAD Consortium; National Institute on Aging
Late-Onset Alzheimers Disease Family Study Group,
71
 www.approachestoagingcontrol.org

Holmans P, Jones L, Devlin B, Klei L, Barmada MM, humans: a double-blind, randomised, controlled trial.
Demirci FY, DeKosky ST, Lopez OL, Passmore P, J Physiol. 2014 Apr 15;592(Pt 8):1887-901.
Owen MJ, ODonovan MC, Mayeux R, Kamboh
MI, Williams J. Genome-wide association study of
Alzheimers disease with psychotic symptoms. Mol
Psychiatry. 2012 Dec;17(12):1316-27.

(51) Du Y, Chen CP, Tseng CY, Eisenberg Y, Firestein

BL. Astroglia-mediated effectsof uric acid to protect
spinal cord neurons from glutamate toxicity. Glia.
2007 Apr 1;55(5):463-72.

(52) Cipriani S, Desjardins CA, Burdett TC, Xu Y, Xu

K, Schwarzschild MA. Protection of dopaminergic
cells by urate requires its accumulation in astrocytes. J
Neurochem. 2012 Oct;123(1):172-81.

(53) Tang F, Lane S, Korsak A, Paton JF, Gourine AV,

Kasparov S, Teschemacher AG. Lactate-mediated glia-
neuronal signalling in the mammalian brain. Nat
Commun. 2014;5:3284.

(54) Milder J, Patel M. Modulation of oxidative stress

and mitochondrial functionby the ketogenic diet.
Epilepsy Res. 2012 Jul; 100(3): 295-303.

(55) Tang JE, Manolakos JJ, Kujbida GW, Lysecki

PJ, Moore DR, Phillips SM. Minimal whey protein
with carbohydrate stimulates muscle protein synthesis
following resistance exercise in trained young men.
Appl Physiol Nutr Metab. 2007 Dec; 32(6): 1132-8.

(56) Ristow M, Zarse K, Oberbach A, Klting N,

Birringer M, Kiehntopf M, Stumvoll M, Kahn CR,
Blher M. Antioxidants prevent health-promoting
effects of physical exercise in humans. Proc Natl Acad
Sci U S A. 2009 May 26;106(21):8665-70.

(57) Paulsen G, Cumming KT, Holden G, Halln J,

Rnnestad BR, Sveen O, Skaug A, Paur I, Bastani
NE, stgaard HN, Buer C, Midttun M, Freuchen F,
Wiig H, Ulseth ET, Garthe I, Blomhoff R, Benestad
HB, Raastad T. Vitamin C and E supplementation
hampers cellular adaptation to endurance training in

72
 Lesiones vasculares
Epilacin
Terpia fotodinmica (PDT)

Fotorrejuvenecimiento
Nd: YAG
Lesiones pigmentarias

93.886.63.40 / Descubre Nordlys en www.ellipse.com

Sociedad Espaola de Medicina Antienvejecimiento y Longevidad
El objetivo y finalidad de la Sociedad es fomentar y llevar a cabo, en inters pblico y sin nimo de lucro, la
Medicina Antienvejecimiento (Anti-Aging) como procedimiento teraputico, procurando la cooperacin
y la unin de especialidades mdicas y de todos los profesionales de la salud, farmacuticos, psiclogos,
biolgos, odontlogos, etc..., que por su actividades y dedicaciones, manifiestan expresamente su inters en
la Medicina Antienvejecimiento, que bsicamente es un sistema integral preventivo y curativo, que a partir
del estudio del envejecimiento natural, descarta los factores perjudiciales que producen un envejecimiento
prematuro, proponindose un sistema de vida de promocin de la salud, aplicando tcnicas correctoras de
los signos estticos y orgnicos de decaimiento corporal.
Para cumplir estos objetivos, vamos a proporcionar a los miembros, la informacin necesaria sobre la
prctica y avances de las tcnicas que nos ocupan, mediante congresos, symposium, cursos y actos (siempre
en unas condiciones especiales para sus miembros), adems recibiras la Approaches to Aging Control,
rgano oficial de la S.E.M.A.L.
Para mayor informacin puedes conectarte a nuestra pgina web: www.semal.org

Nombre ..................................Apellidos ..............................................................................Edad..........

Direccin ...............................................................................................................................................
Localidad ...............................................................................................................................................
C.P. ........................ Provincia......................................................................... Pas ..............................
Telfono ...........................Fax ............................. E-mail.....................................................................
Especialidad.............................................................................................................................................
N Colegiado ........................Colegio de: .............................................................................................
Hospital/Clnica ....................................................................................................................................
Consulta privada ....................................................................................................................................

Miembros Fundadores, de la Junta Directiva y Nume- en Medicina y Ciruga o de Especialista, a la siguiente

rarios: 200 euros/ao
direccin:
Miembros espaoles y miembros correspondientes
extranjeros: 100 euros/ao Sociedad Espaola de Medicina Antienvejecimiento y
La inscripcin a la sociedad incluye la suscripcin a la Longevidad
revista de la misma.
Entidad Bancaria: Real e Ilustre Colegio de Mdicos de Sevilla
La Caixa. N de cta: 2100 2112 11 0200500924 Avda. de la Borbolla, 47 - 41013 Sevilla
Titular: Sociedad Espaola de Medicina Antienveje-
cimiento y Longevidad (S.E.M.A.L.) Tlf.: 954 084 700 - Fax: 954 084 700
Para formar parte de la sociedad, enve este boletn de e-mail: info@semal.org
inscripcin y el justificante de ingreso o transferencia,
junto con una copia del ttulo de Licenciado o Doctor web: www.semal.org
^>^dd/ 6%$'2GH2FWXEUHGH
sE^E>dZdD/EdK>^>KW/^
0RGHUDGRU3URI-RDTXtQ&DODS'U)DELDQR0DJDFKR
 0LH[SHULHQFLDHQDORSHFLDVFRQ69)\353
'UD0DULDQD'tD]
 5HFXSHUDFLyQFDSLODUFRQWpFQLFD)8(0LH[SHULHQFLD
'U(]HTXLHO3DQQR
 1XHYDWpFQLFDGHWUDVSODQWHFDSLODUDXWRPDWL]DGRSDUDHOWUDWDPLHQWRGHODDORSHFLD
'U6HUJLR9Dxy
 $ORSHFLDHQKRPEUHV\PXMHUHV
'UD$XURUD*XHUUD7DSLDV
 7HUDSLDUHJHQHUDWLYDHQDORSHFLDV
'U$OEHUWR*RUURFKDWHJXL
 $ORSHFLDQXWUDFpXWLFRVHLQWUDGHUPRWHUDSLD
'UD&DPLOD7DJOLDUL
 &LUXJtDV&DSLODUHV$FWXDOL]DFLRQHVHQ)866\)8(
'U5DPyQ9LODURYLUD
 $YDQFHVHQWUDWDPLHQWRVGHODVDORSHFLDV
'U-XOLiQ&RQHMR0LU
 3$86$&$)e<9,6,7$$/$(;326,&,1&20(5&,$/
ZDdK>K'1^dd/
0RGHUDGRU3URI-XOLiQ&RQHMR0LU'UD(OLD5RR
 1XWULFRVPpWLFRVHQODSUHYHQFLyQ
'UD$XURUD*XHUUD
 'HUPRFRVPpWLFRVFRPRREWHQHUUHVXOWDGRVGHLPSDFWR
'UD$GULDQD6FKPLGW
 8VRGHDQWLR[LGDQWHVHQODSURWHFFLyQSDUDHOHQYHMHFLPLHQWR
'UD1DWDOLD0D]HU
 7HVWJHQpWLFRVHQSDWRORJtDVGHOD3LHOGHUPDWLWLVDWySLFD
'U-RVH/XLV/RSH](VWHEDUDQ]
 1RYHGDGHVWHFQROyJLFDVDQWLHQYHMHFLPLHQWR
'UD(OLD5RR
 '(%$7(
 &21)(5(1&,$0$*,675$/6$/$$17,$*,1*
'U&ROLQ7&DPSEHOO
 $6$0%/($*(1(5$/'(0,(0%526'(6(0$/&RQYRFDWRULDFRQYRFDWRULD
dZW/^Z'EZd/s^ED//Ez/Zh'1^dd/
0RGHUDGRU'U-RUJH3ODQDV'U5LFKDUG/XQJ
 7HUDSLDDYDQ]DGDFHOXODUGHSURGXFFLyQQRLQGXVWULDOFpOXODVPDGUHPHVHQTXLPDOHVSDUDXQIXWXURXVRDXWyORJRHYHQWXDO
'U-RVH0DQXHO&HUYHUD*UDX
 0HGLFLQD5HJHQHUDWLYD\WHUDSLDWUDVODFLRQDOODPHGLFLQDGHOVLJOR;;,
'U-XDQ3HGUR/DSXHQWH
 &pOXODVPDGUHHQPHGLFLQDHVWpWLFD4XpSRGHPRVRIUHFHU"
'U0DULR&DEUDO
 5HYROXPLQD]DFLyQIDFLDOFRQFpOXODVPDGUHHQSDFLHQWHVKRUPRQDOPHQWHRSWLPL]DGRV
'U+XPEHUWR)HUUHLUD
 OWLPDVQRYHGDGHV35*)HQSLHO
'U(GXDUGR$QLWXD
 1DQRIDWSDUDUHMXYHQHFLPLHQWRIDFLDO
'U-RUJH3ODQDV
 3$86$&$)e<9,6,7$$/$(;326,&,1&20(5&,$/
/Zh'1W>^d/^dd/
0RGHUDGRU'U5DPyQ9LOD5RYLUD'U3HGUR$UTXHUR
 'LVWUHVVHVRIWKHVXUJHRQEHJLQQHU
'U$QWRQLR3DXOR3LWDQJX\
 %OHIDURSODVWLD4XpKDJRFRQODJUDVD"
'U,YDQ0DxHUR
 5HMXYHQHFLPLHQWRIDFLDOLPSRUWDQFLDGHODQDUL]
'U3HGUR$UTXHUR
 $EGRPLQRSODVWLDHQUHMXYHQHFLPLHQWRFRUSRUDO3UHIHUHQFLDV\WpFQLFDSHUVRQDO
'U5LFKDUG/XQJ
 5HMXYHQHFLPLHQWRIDFLDO3RVLELLGDGHVTXLU~UJLFDV\QRTXLU~UJLFDVSDUDSDFLHQWHVMyYHQHV\GHHGDGDYDQ]DGD
'U5DPyQ9LODURYLUD
 7HDUWKURXJK1HZFODVVLILFDWLRQIRUSUDFWLFLRQHUV
'U3URNXGLQ6HUJH\
 3DVDGRSUHVHQWH\IXWXURHQHOUHMXYHQHFLPLHQWRIDFLDOFRQOLSRLQMHUWR
'U)UDQFLVFR1DYDUUR
 $FWXDOL]DFLyQHQOLSRVXFFLyQFRQDQHVWHVLDORFDO
'U6DPXHO+LURRND
 '(%$7( &,5&8167$1&,$6,035(9,67$638('(1352'8&,5$/*1&$0%,2(1(/352*5$0$
^>^dd/ s/ZE^K
^dd/Dh>d//^/W>/EZ
0RGHUDGRU'U*LXVHSSH,]]R'UD0HUFHGHV(JXLOX]
 &RPSOLFDFLRQHVHQHVWpWLFDJHQLWDO0DVFXOLQD
'U-RUJH*LRVFLD)LOKR
 7UDWDPLHQWRFRQPLFURDJXMDVGHUPDUROOHU\iFLGRUHWLQRLFR
'U$UWKXU3LPHQWHO
 0LQLWKUHDGVDQGILOOHUVPHGLFDOIDFHOLIW
'UD3DROD7DUDQWLQR
 &LUXJtDJHQLWDOPDVFXOLQDHQHO6LJOR;;,
'U0DULDQR5RVVHOOy
 &KHPLFDOSHHOVDQGELRVWLPXODWLRQZLWKFRVPHFHXWLFDOVDQGQXWUDFHXWLFDOVLQWKHWUHDWPHQWRIIRWRDJLQJ
'U*LXVHSSH,]]R
 $SOLFDFLyQGHO$FLGR3ROLOiWLFRSDUDHOWUDWDPLHQWRGHODIODFLGH]FRUSRUDO
'UD*LDQQD=)UH\
 3$86$&$)e<9,6,7$$/$(;326,&,1&20(5&,$/
>^ZzEhs^dEK>K'1^
0RGHUDGRU'U0DULR7UHOOHV'UD3DROD7DUDQWLQR
 3HUVSHFWLYDFOtQLFDSUiFWLFDVREUHORVIHQyPHQRVGHODLQWHUDFFLyQOiVHUWHMLGR
'U0DULR7UHOOHV
 9DJLQRSODVWLDOiVHUSDUDHOUHMXYHQHFLPLHQWRtQWLPR
'U&HVDU$UUR\R
 /X]SXOVDGD1G<DJ\&2HQHOWUDWDPLHQWRGHOIRWRUHMXYHQHFLPHQWRFXWiQHR
'UD$ULDQQH0RWWD
 3RUTXpHVFRQVLGHUDGRHOOiVHUHQGRYHQRVRXQWUDWDPLHQWRGHSULPHUDHOHFFLyQSDUDHOWUDWDPLHQWRGHYDULFHV"
'U-RVp$]SLD]X
 1XHYRV$YDQFHVHQWUDWDPLHQWRVGH/iVHU\/X]
'U5DIDHO6HUHQD
 /DQXHYDHUDGHORVSXOVRVHQVXEPLOLVHJXQGRVPVHQOX]SXOVDGD
'U-RVp0LJXHO0XxR]
 &DUER[LWHUDSLD1XHYDVLQGLFDFDFLRQHV
'-XDQ/ySH]
 9HOD%DE\5HFXSHUDFLyQGHODVLOXHWDWUDVHOHPEDUD]R
'UD9LUJLQLD%HQWH]
 3HHOLQJVIRWRDFWLYDGRVFRQ/('\/%,
'U*LRYDQ\%HWWHJD
 '(%$7(
 &2&.7$,/%,(19(1,'$
D//E^dd/
0RGHUDGRU'UD3HWUD9HJD'U)HUQDQGR&DUYDOKR
 0RGXODFLyQ+RUPRQDOVXLQWHUpVHQ0HGLFLQD\&LUXJtD(VWpWLFD
'U-RUJH*LRVFLD)LOKR
 2EHVLGDGHLPDJHQHVWpWLFD
'U/RXLV+RUDFLR&DVWUR
 (VWUDVXQUHWRFRPRWUDWDUODV"
'UD$XGUH\:RUWKLQJWRQ
 5HMXYHQHFLPLHQWR3HULRFXODUPLSURWRFROR
'U)HUQDQGR&DUYDOKR
 5HMXYHQHFLPLHQWRGHPDQRV0LWpFQLFD
'U)DELDQR0DJDFKR
 1RQVXUJLFDOQHFNOLIW+RZZHFDQZRUNZLWKGLIIHUHQWWLVVXHOHYHO
'U3URNXGLQ6HUJH\
 3$86$&$)e<9,6,7$$/$(;326,&,1&20(5&,$/
^h^dE/^Z>>EK
0RGHUDGRU'UD3DORPD7HMHUR'U0DULR&DEUDO
 5HOOHQRVIDFLDOHV&XiOHV\SRUTXp"0LH[SHULHQFLDHQDxRV
'U$UWKXU3LPHQWHO
 5HOOHQRVHQODFRUUHFFLyQGHODSXQWD\GRUVRQDVDO
'U)DELDQR0DJDFKR
 5HOOHQRHQ*ODEHODWpFQLFDSHUVRQDO
'UD$XGUH\:RUWKLQJWRQ
 0LH[SHULHQFLDGHDxRVFRQiFLGRSROLOiFWLFRHQUHMXYHQHFLPLHQWRIDFLDO
'UD$GULDQD6FKPLGW
 8VRFRPELQDGRGHUHOOHQRVFRQFDUERGLVHFFLyQ
'U-XDQ/ySH]
 $OWHUQDWLYDVGHWUDWDPLHQWRVHQFRPSOLFDFLRQHVSRULPSODQWHVGH300$
'U)HUQDQGR&DUYDOKR
 &RPSOLFDFLRQHVHQPHGLFLQDHVWpWLFD
'U0DULR&DEUDO
 *OXWHRSODVWLD*UDVDR300$"
'U6DPXHO+LURRND
 '(%$7( &,5&8167$1&,$6,035(9,67$638('(1352'8&,5$/*1&$0%,2(1(/352*5$0$
 &(1$'(*$/$+27(/1+9(17$61(&(6$5,$,16&5,3&,1(16(&5(7$5$3/$=$6/,0,7$'$6
 :hs^KdhZ
d>>Z^^dd/ >h'ZK>'/KD/K^DZ/
 (QWUHJDGHGRFXPHQWDFLyQ
7$//(5(6*5$78,7266/23$5$,16&5,726$/&21*5(62
3/$=$6/,0,7$'$6
1(&(6$5,20$1'$5%2/(71'(,16&5,3&,1$7$//(5(6
 $SOLFDFLRQHV3UiFWLFDVGHODPHGLFLQDSHUVRQDOL]DGD*HQyPLFD
3URI-XDQ6DEDWHU'$QD6DEDWHU
7$//(53$752&,1$'2325(8*(120,&6
 $OLPHQWDFLyQSDUDXQHQYHMHFLPLHQWRVDOXGDEOH
'UD*ORULD6DEDWHU
7$//(53$752&,1$'23256$/(1*(,
 3KRWRIUDFWLRQDO5HMXYHQHFLPLHQWR)DFLDO,QWHJUDOFRQ/iVHU
'UD3DWULFLD2UWL]
7$//(53$752&,1$'23255()(5(1&(0(',&$/
 (VWLPXODFLyQGHORVILEUREODVWRVDWUDYpVGHODXWLOL]DFLyQGHFROiJHQRHTXLQRKHWHUyORJRWLSR,
'UD0DUtD2UWL]
7$//(53$752&,1$'23256.<0(',&
 7UDWDPLHQWR0HWDEyOLFRGHOD&HOXOLWLV
'UD$QD%HOOyQ
7$//(53$752&,1$'232535212.$/
 9/LIW3UR$OWDWHFQRORJtDMDSRQHVDHQKLORVGHSROLGLR[DQRQD
'UD9LFHQWD/ORUFD'U,VLGUR5HEHOR
7$//(53$752&,1$'2325-$3$1%,2352'8&76
 (VWLPXODFLyQGHFROiJHQR\HODVWLQDGXUDQWHHOWUDWDPLHQWRFLUFXQVIHUHQFLDOGHODJUDVDORFDOL]DGDFRQUDGLRIUHFXHQFLD
ELSRODUFRPELQDGD
'UD9LUJLQLD%HQLWH]
7$//(53$752&,1$'23256<1(521&$1'(/$
 $QiOLVLVGHUPDWROyJLFRHQPHGLFLQDHVWpWLFD\DQWLDJLQJ
'U3DEOR1DUDQMR
7$//(53$752&,1$'23250,&52&$<$
 *pQHVLVODWHFQRORJtDPiVDYDQ]DGD\YHUViWLOSDUDUHMXYHQHFLPLHQWR
'U3DEOR1DUDQMR
7$//(53$752&,1$'2325&87(5$
^>Ed/'/E' ^KK
'E^sZ^h^D/EdED//EEd/Es:/D/EdK
0RGHUDGRU3URI0DQXHO-&DVWLOOR
 ,QWHUFRQH[LRQHVJHQpWLFDV\DPELHQWDOHVHQDQWLHQYHMHFLPLHQWR\ORQJHYLGDG
'U-/0HVD
 6HJXULGDG\(YDOXDFLyQGHO5LHVJR
'U1LFROiV2OHD
 /DHSLJHQpWLFDHQHOHQYHMHFLPLHQWR
'U-RVH0LJXHO*DUFtD6DJUHGR
 '(%$7(
 Wh^&zs/^/d>yWK^//MEKDZ/>
KEdZKsZ^/^EhdZ//KE>^ED//EEd/Es:/D/EdK
^^/MEKEdZh/ME^/Dh>dE/E'>^^WHK>
0RGHUDGRU'U*XVWDYR%DUMD
 7KH(DUWKTXDNHRQIRRGUHFRPHQGDWLRQV
'U&ODXGH'DOOH
 7KHGLHWDQGFDQFHUDVVRFLDWLRQWKDWLVSDUWRIDODUJHUWUXWK
'U&ROLQ7&DPSEHOO
 $QWL$JLQJ1XWULWLRQ7KH5ROHRIWKH&DORULH5HVWULFWLRQ2PHJD)DWW\$FLGVDQG3RO\SKHQROVLQ&KDQJLQJ*HQH
([SUHVVLRQ
'U%DUU\6HDUV
 &OLQLFDOSUDFWLFHEDVHGRQLQWHJUDWHGGUXJIUHHWKHUDSLHVIRUZHLJKWORVV
'UD7DPDUD9RURQLQD
 '(%$7(
 &21)(5(1&,$0$*,675$/
$GLHWDQGFDQFHUFRQQHFWLRQWKDWLVPRUHWKDQJHQHUDOO\NQRZQ
'U&ROLQ7&DPSEHOO
 '(%$7(
 ^D>'EZ>D/DZK^^D>^>^dd/
KdZK^dD^/DWKZdE/ED//EEd/Es:/D/EdK
0RGHUDGRU'UD0HUFHGHV(JXLOX]
 3DSHOGHOD*HULDWUtDHQODDWHQFLyQDODSHUVRQDPD\RU
'U5LEHUD&DVDGR
 $FWXDOL]DFLyQHQFRQWUDWDPLHQWRKRUPRQDODQWLHQYHMHFLPLHQWR+*+7HVWRVWHURQD
'U+XPEHUWR)HUUHLUD
 0RGXODFLyQGHOPHWDEROLVPRYtD$03.\HVWLPXODGRUHVGHOFLFORGH.UHEV
'U-RUJH/XLV&XEUtDV0RUDOHV
 '(%$7(
 Wh^&zs/^/d>yWK^//MEKDZ/>
KdZK^dD^/DWKZdE/ED//EEd/Es:/D/EdK
0RGHUDGRU'U-RVp6HUUHV
 8VLQJQHZWHFKQRORJLHVIRUGHWHUPLQDWLRQDQGWUHDWPHQWRIHQYLURQPHQWDOFKHPLFDOV
'U:LOOLDP6KDZ
 3URWRFRORGH7HUDSLD6XVWLWXWLYD+RUPRQDO
'U$OIUHGR%HO]X]DUUL
 7UDWDPLHQWRVLQWUDYHQRVRVHQPHGLFLQDDQWLHQYHMHFLPLHQWRPLH[SHULHQFLD
'UD9LFHQWD/ORUFD
 0LFUR,QPXQRWHUDSLD\0LFURQXWULFLyQVLQHUJLDSDUDODVDOXG
'U$QWRQLR5XL]3HUH]
 ,QIODPDFLyQGHOWHMLGRDGLSRVRFDXVDV\UHVROXFLyQ
'U,JQDFLR6DMRX[
 7UDFWR*DVWURLQWHVWLQDO\(QIHUPHGDGHV1HXURGHJHQHUDWLYDV8Q&RQFHSWR1XHYR&HUHEUR,QVWHVWLQR\0LFURELRWD
'U2FWDYLR9LHUD
 0HQRSDXVLD(VXQDHQIHUPHGDG"
'U*HRUJHV'HEOHG
 '(%$7(
&,5&8167$1&,$6,035(9,67$638('(1352'8&,5$/*1&$0%,2(1(/352*5$0$
^>Ed/'/E' s/ZE^K
 (QWUHJD'RFXPHQWDFLyQ
 $FWR,QDXJXUDO
s/^/ME/E^d/dh/KE>>D//EEd/Es:/D/EdK
0RGHUDGRU'U-XOLiQ%D\yQ
 (QYHMHFLPLHQWR\6DOXG
'-RVp-DYLHU&DVWURGH]D'LU*UDOGH6DOXG3~EOLFDH,QQRYDFLyQ0LQLVWHULRGH6DQLGDG
 9DORUGHOD0HGLFLQD3UHYHQWLYDHQOD0HGLFLQD$QWLHQYHMHFLPLHQWR
'6XVDQD*UDQDGR-HIDGHiUHDGHSURPRFLyQGHODVDOXG\SUHYHQFLyQ&RPXQLGDGGH0DGULG
 '(%$7(
 Wh^&zs/^/d>yWK^//MEKDZ/>
WZK/Md/K^zWZ/Md/K^E>Es:/D/EdK
0RGHUDGRU'U*XLOOHUPROYDUH]&DODWD\XG
 5HSHUFXVLyQGHORVSURELyWLFRVHQOD,QPXQRVHQHVFHQFLD
'UD$VFHQVLyQ0DUFRV
 3UHELyWLFRVHQOD'LHWDSDUDXQ(QYHMHFLPLHQWR6DOXGDEOH
'UD7HUHVD5HTXHQD
 '(%$7(
EhdZ//MEKDZKEKKDZ
0RGHUDGRU3URI$QWRQLR$\DOD
 (ODEDQGRQRGHODGLHWDPHGLWHUUiQHDHQ(VSDxD+HFKRV\SURSXHVWDVGHVROXFLRQHV
3URI-HV~V5RPiQ
 /LSLGRPLFDGHOHQYHMHFLPLHQWRVDOXGDEOH
3URI5HLQDOG3DPSORQD
 '(%$7(
 K<d/>/EsE/
,KZDKE^d/ZK/^
0RGHUDGRU3URI-HV~V)HUQiQGH]7UHVJXHUUHV
 'LVUXSFLyQ(QGRFULQD7LURLGHDDOJRPiVTXH<RGR\%RFLyJHQRV
'U1LFROiV2OHD
 'LVIXQFLyQWLURLGHD\HQYMHFLPLHQWR
'U-XDQ-RVp'tH]
 '(%$7(
 Wh^&zs/^/d>yWK^//MEKDZ/>
Ed/Ky/Ed^>Ky//MEz^hZh/ME
0RGHUDGRU3URI-RVH9LxD
 (QYHMHFLPLHQWRR[LGDFLyQ\DQWLR[LGDQWHV'HODHYLGHQFLDH[SHULPHQWDODODDSOLFDFLyQFOtQLFD
'U*XLOOHUPR6iH]7RUPR
 /RVDQWLR[LGDQWHVSXHGHQWHQHUHIHFWRVEHQHILFLRVRVHQHOHQYHMHFLPLHQWR
3URI0yQLFDGHOD)XHQWH
 '(%$7(
 &21)(5(1&,$0$*,675$/
$OFRKRO6RFLHGDG\(QYHMHFLPLHQWR
3URI$UWXUR)HUQiQGH]&UX]
 E'>,Kd>E,sEd^E^Z//E^Z/W/MEE^ZdZ1W>^>/D/d^
&,5&8167$1&,$6,035(9,67$638('(1352'8&,5$/*1&$0%,2(1(/352*5$0$
 :hs^KdhZ
WdW/
D
t^
+25$5,2&XUVRFRQWUDGXFFLyQVLPXOWiQHD,QJOpV(VSDxRO
/8*$5'(5($/,=$&,1,OXVWUH&ROHJLRGH0pGLFRVGH0DGULG
6HHQWUHJDUiGLSORPDDFUHGLWDWLYR
WKE/E^Z/W/ME>KE'Z^K^/E/E^Z/W/ME>KE'Z^K
WZK'ZD
d,

D
hdddYD
D
Ks
+25$5,2GHD\D
/8*$5'(5($/,=$&,1,OXVWUH&ROHJLRGH0pGLFRVGH0DGULG
6HHQWUHJDUiGLSORPDDFUHGLWDWLYR
WKE/E^Z/W/ME>KE'Z^K^/E/E^Z/W/ME>KE'Z^K
>D


WZK'ZD
,


D

YD

&,5&8167$1&,$6,035(9,67$638('(1352'8&,5$/*1&$0%,2(1(/352*5$0$
 y/sKE'Z^K>^K/^WHK>D//EEd/Es:/D/EdKz>KE's/ ^D>
&20,7e'(+2125 &20,7e25*$1,=$'25 &20,7e&,(17),&2
^ 35(6,'(17( 35(6,'(17(
WD : W:&d
^:^D 9,&(35(6,'(17( 9,&(35(6,'(17(
^D :^ WD:
^D
D Dd W
/^^> DD WD&
WKDD ^Z W:s
Wd Ws
:>
WKEEd^
 '     D 
     E D 
 W    :> D 
      D 
W     W E 
 :    & E 
W '    E K 
     D K 
     W K 
 s   W Z W 
 '     W 
 D     W 
W :    W W 
 d  hh  : W 
 &    / Z W
W D    d Z 
 >,  hh  :D Z 
 ::   W : Z 
 :D     ZZ 
W : D   D Z 
 :>     Z 
   &   ^ 
W D &   ' ^ 
 '   W : ^ 
 D    ' ^ 
 ::    / ^ 
 D    > ^ 
W  &    ^ 
W : &d    ^ hh
 , & W  Z ^ 
 ' &   W ^ Z
 :D '^   : ^ 
 : '&   t ^ hh
  '    d /
 ^ '   W d /
  'd   W d 
 ^ ,   D d 
 ' / /  ^ s 
 s >   W s 
 :W >   K s 
 : >   Z s 
 :> >  W : s 
 Z > hh  K s 
 & D   d s h<
 / D    t 
^,E,sD