Nutrigenômica e Inflamação - Mocchegiani Et Al., 2014

Mechanisms of Ageing and Development 136-137 (2014) 2949
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Mechanisms of Ageing and Development

journal homepage: www.elsevier.com/locate/mechagedev
Micronutrientgene interactions related to inammatory/immune

response and antioxidant activity in ageing and inammation.
A systematic review
Eugenio Mocchegiani a,*, Laura Costarelli a, Robertina Giacconi a, Marco Malavolta a,
Andrea Basso a, Francesco Piacenza a, Rita Ostan b, Elisa Cevenini b, Efstathios S. Gonos c,
Daniela Monti d
a
Translation Center of Research in Nutrition and Ageing, Scientic and Technological Pole, Italian National Research Centres on Ageing (INRCA), Via Birarelli 8,
60121 Ancona, Italy
b
Department of Experimental Diagnostic and Specialty Medicine (DIMES) and Interdepartmental Centre L. Galvani (CIG), University of Bologna, Via San
Giacomo, 12, 40126 Bologna, Italy
c
National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, 48 Vas. Constantinou Ave., Athens 11635, Greece
d
Department of Clinical and Experimental Biomedical Sciences, University of Florence, Viale Morgagni, 50, 50134 Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Article history:
Available online 2 January 2014
Recent longitudinal studies in dietary daily intake in human centenarians have shown that a satisfactory
content of some micronutrients within the cells maintain several immune functions, a low grade of
inammation and preserve antioxidant activity. Micronutrients (zinc, copper, selenium) play a pivotal
role in maintaining and reinforcing the performances of the immune and antioxidant systems as well as
in affecting the complex network of the genes (nutrigenomic) with anti- and pro-inammatory tasks.
Genes of pro- and anti-inammatory cytokines and some key regulators of trace elements homeostasis,
such as Metallothioneins (MT), are involved in the susceptibility to major geriatric disease/disorders.
Moreover, the genetic inter-individual variability may affect the nutrients absorption (nutrigenetic)
with altered effects on inammatory/immune response and antioxidant activity. The interaction
between genetic factors and micronutrients (nutrigenomic and nutrigenetic approaches) may inuence
ageing and longevity because the micronutrients may become also toxic. This review reports the
micronutrientgene interactions in ageing and their impact on the healthy state with a focus on the
method of proteinmetal speciation analysis. The association between micronutrientgene interactions
and the proteinmetal speciation analysis can give a complete picture for a personalized nutrient
supplementation or chelation in order to reach healthy ageing and longevity.
2013 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Zinc/copper/seleniumgene interactions
Immune system
Antioxidant activity
Ageing
Longevity
1. Introduction
Ageing is an inevitable biological process that is accompanied
by gradual and spontaneous biochemical and physiological
changes including increased susceptibility to diseases, adverse
environmental conditions and loss of mobility and agility.
Alterations in the neuroendocrineimmune interactions as well
as in the antioxidant capacity also play a fundamental role in
ageing. The inability of an organism in coping with these changes
may lead to the onset of some degenerative age-related diseases.
* Corresponding author at: Scientic and Technological Pole INRCA-IRCCS,

Translation Center of Research in Nutrition and Ageing, Via Birarelli 8, 60121
Ancona, Italy. Tel.: +39 0718004216; fax: +39 071206791.
E-mail address: e.mocchegiani@inrca.it (E. Mocchegiani).
0047-6374/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mad.2013.12.007
As a result, the remodelling theory of ageing has been proposed

(Paolisso et al., 2000). Various nutritional factors are directly linked
with these phenomena as for instance in restoring neuroendocrine-immune network, the metabolic harmony and the capacity
to respond to oxidative stress (Meydani, 2001). Approximately, 40
micronutrients (vitamins, essential minerals and other compounds
required in small amount for normal metabolism) have been
reported as essential components of the diet (Alvarez Leon et al.,
2006). The dietary intake of essential micronutrients is often
inadequate in the elderly due to several causes (Ames, 2006). First
of all, the poor socio-economic condition present in a large part of
old people may lead to a consumption of inexpensive foods
decient in micronutrients, such as carbohydrates (Kant, 2000).
The gap is worsened by loss of appetite, lack of teeth, intestinal
malabsorption and decreased requirement of energy that lead, as a
nal result, to frailty, disability and mortality (Semba et al., 2006).
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E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949
Some authors have reported that the deciency of micronutrients

in ageing is strictly related to global impairments of the immune
functions, metabolic harmony and antioxidant defence by external
noxae with subsequent onset of age-related diseases (Failla, 2003).
Indeed, many micronutrients contribute directly or indirectly to
the biological activity of some antioxidant enzymes (superoxide
dismutase, SOD; glutathione peroxidase, GPx; catalase), to the
efciency of the immune system, to the metabolic harmony, to
keep under control the inammatory state and, as such, to
maintain the correct functioning of many body homeostatic
mechanisms with subsequent achievement of the longevity
(Mocchegiani et al., 2008c) and, nally, to the maintenance of
metabolic function especially in preventing mitochondrial decay
(Ames, 2006). In this last context, feeding studies in old rats have
shown that mitochondrial metabolites and antioxidants protect
the neuronal cells from neurotoxin- and oxidant-induced toxicity
and oxidative damage but especially they delay the normal
senescence of human diploid broblasts and the oxidant-induced
senescence (Liu et al., 2002). With advancing age, an increased
oxidative damage to proteins and lipid membranes, particularly in
mitochondria, causes a deformation of the structure of enzymes,
with a consequent decrease of enzyme activity as well as substrate
binding afnity for their substrates. An increased level of substrate
by micronutrients restores the speed of the reaction as well as
mitochondrial function, thus delaying mitochondrial decay and
ageing (Liu et al., 2002). In contrast, recent longitudinal studies in
dietary daily intake in human nonagenarians/centenarians (successful ageing) have shown that an adequate consumption of
micronutrients as well as a satisfactory content of some trace
elements within the cells lead to good performances in several
immune functions, to metabolic compensation and preservation of
the antioxidant activity (Chernoff, 2001). In this context, polyunsaturated fatty acids, highly sensitive to reactive oxygen species
(ROS), decrease in liver mitochondria from human centenarians, a
feature that could represent a protective mechanism to favour
longevity (Anantharaju et al., 2002). Therefore, nutritional factors
can play a pivotal role for healthy ageing and longevity. However,
the effects of the nutrients are strongly inuenced by genetic
factors, in particular by genes involved in inammatory/immune
response and antioxidant activity. The genes of IL-1, IL-6, TNF-a
pro-inammatory cytokines, of IL-10 anti-inammatory cytokine,
of HSP70 chaperone and the regulators of trace elements
homeostasis, Metallothioneins (MT), seem particularly relevant
because they are involved in the susceptibility to major geriatric
disorders, such as diabetes, osteoporosis, osteoarthritis, dementia,
cardiovascular diseases (CVD) and infections (Franceschi, 2007;
Mocchegiani et al., 2006a,b). Indeed, up to 25% of the variation in
human lifespan is heritable (Mitchell et al., 2001); the rest is due to
environmental and life style factors, which impact on the ageing
process contributing, as such, to a large inter-individual variability.
Therefore, current problems are to understand how the interaction
between genetic factors and nutrients may inuence the ageing
process and longevity in view of the high impact of gene
expression, protein production and epigenetic mechanisms in
regulating the life span (Mattson, 2008). Some dietary patterns
fave shown very different impact on long-term disease occurrence
and survival. In this context, a strong evidence for a benecial
effect of higher compliance to the Mediterranean dietary pattern
on causes of death, including those ones by CVD and cancer, has
been reported (Mitrou et al., 2007). Anyway, it is also commonly
accepted that the complex interactions of multiple polymorphisms
play a key role in how individuals may respond to dietary
interventions (nutrigenetic approach) or how some nutrients may
affect the gene expressions (nutrigenomic approach) (Darton-Hill
et al., 2004). For each nutrient, there is a window of intake between
the Recommended Dietary Allowance (RDA), (dened as the
dietary intake sufcient to meet the requirement of 97% of healthy

individuals in a particular stage of the life and gender group), and
the tolerable upper limit (UL), which is the highest nutrient intake
that can be achieved without incurring risk of adverse health
effects for most individuals in the general population (Stover,
2006). Although worldwide research on genetic variation that
requires a different RDA or UL is still in progress, several genes and
alleles have been suggested to affect nutrient utilizations
(Mocchegiani et al., 2012a). During this last decade, it has been
recognized the pivotal role played by some micronutrients (zinc,
copper, selenium) in maintaining the correct functioning of many
body homeostatic mechanisms with subsequent achievement of
longevity (Mocchegiani et al., 2008c). Taking into account the
inuence of these nutrients in many genes involved in inammatory/immune response and antioxidant activity (Mocchegiani
et al., 2012a), the micronutrientgene interactions are fundamental to escape many age-related diseases and to achieve healthy
longevity. We report the specic role played by the most relevant
micronutrientgene interactions in ageing with a focus on the
precise determination of their specic transporter proteins, by
means of the method of proteinmetal speciation analysis
(Malavolta et al., 2012). As such, the association between
micronutrientgene interactions and proteinmetal speciation
analysis may give a complete picture of the personalized
micronutrient supplementation or chelation (in the case of
nutrient overload) in order to reach healthy ageing and longevity.
2. Micronutrients, immune efciency, antioxidant response,
ageing
2.1. Zinc, immunity, antioxidant response and ageing
Zinc (Zn) is an essential micronutrient required for many
cellular processes, including the correct function of the immune
system and antioxidant response. Zinc homeostasis and signaling,
acting as a second messenger, are both critical in immune
activation and against oxidative stress (Powell, 2000; Chasapis
et al., 2012). An imbalance in zinc homeostasis is associated with
the development of chronic diseases. Zinc deciency (genetic or
nutritional) causes signicant impairment in both adaptive and
innate immune responses, promotes systemic inammation and
impaired antioxidant defence (Fraker and King, 2004; Chasapis
et al., 2012; Wong and Ho, 2012). A plethora of data indicates that a
signicant portion of the aged population has inadequate zinc
intake with a decline in zinc state with advancing age (Mocchegiani et al., 2013b).
Zinc deciency in the elderly seems to be due to many factors
related to the ageing process, such as inadequate mastication and
psychosocial factors leading to a reduction of zinc-rich foods
(bovine, ovine and pork meat, hard cheese, nuts, cocoa, eggs)
intake, altered intestinal absorption, alteration in zinc transporter
proteins, drug interactions and competition between zinc and
other bivalent minerals (copper, iron, calcium and selenium) or
vitamins (Mocchegiani et al., 2013b). The intracellular zinc
homeostasis is regulated by buffering Metallothioneins (MT), i.e.
the major storage proteins for zinc, and by zinc transporters (ZnT
and ZIP families) for cellular zinc efux and inux, respectively,
that mediate the intracellular zinc signaling. The main reasons why
intracellular zinc levels are compromised during ageing have been
traced to the increased expression of MT (Mocchegiani et al., 2007)
and to the defective zinc transporters, which could result in
increased sequestration of zinc and low intracellular free zinc
content (Mocchegiani et al., 2013b). Specically, MT preferentially
bind zinc but in ageing they are unable to release zinc in order to be
used by other Zn-dependent antioxidant enzymes and proteins
related to the efciency of the immune system. Indeed, zinc, acting
as a second messenger, modulates the signaling cascades that

improve antioxidant and immune defenses, such as the activation
of Nrf-2 transcription factor as well as a zinc-nger protein A20,
which inhibits Nuclear Factor Kappa B (NF-kB) activation via TRAF
pathway (Prasad, 2008). Consequently, the zinc release from
proteins acts both as antioxidant and anti-inammatory agent.
Therefore, the low free zinc ion availability and the high MT levels
result in the failure of the antioxidant and immune response
provoking increased inammation followed by infection relapses
in the elderly (Mocchegiani et al., 2000a,b, 2003). The major
alterations of a zinc deciency are the involution of the thymus,
defective lymphocytes maturation and function, decline in both
cellular and humoral immune response (Haase and Rink, 2009a,b)
and the maintenance of a systemic low grade of chronic
inammation (named inammaging) (Franceschi et al., 2000).
These alterations can lead to an increased risk for the onset of
degenerative age-related diseases (Mocchegiani et al., 2013b). The
thymic involution is due to enhanced apoptosis of lymphocyte
precursors (Fraker and King, 2001) and to a reduction of the
activity of thymulin (Prasad et al., 1988): an hormone secreted by
thymic epithelial cells requiring zinc as a cofactor for thymulin
biological activity and essential for the differentiation, maturation
and function of T cells. As a consequence, the thymic output
(measured using T cell receptor rearrangement excision circles) is
reduced during ageing leading to a substantial loss of pre-T cells
and a reduced number of naive mature T cells in the circulation
with subsequent inability to substitute activated memory T cells
that undergo to apoptosis after exposure to foreign antigens
(Mitchell et al., 2006). Centenarians display satisfactory zinc pool
(Mocchegiani et al., 2003) and a sufcient maintenance of the
thymic output by IL-7 (Nasi et al., 2006). IL-7 and its receptor act
via zinc nger protein Miz-1 and SOCS1 (Saba et al., 2011), which is
in turn regulated by another zinc nger protein TRIM8/GERP
(Toniato et al., 2002).
Moreover, zinc deciency is accompanied by reduced T cell
proliferation upon mitogen stimulation, reduced precursor number and activity of CD8 cytotoxic T cells, and impaired CD4 helper T
cell functions, resulting in an imbalance of Th1/Th2 cytokines
secretion (Beck et al., 1997; Uciechowski et al., 2008), characterized by decreased IFN-g, IL-2 and TNF-a production by Th1 cells
and increased IL-6 production by Th2 cells and macrophages,
similarly to what occurs at old age in CD4T lymphocytes (Alberti
et al., 2006). In vitro, zinc supplementation can modulate T celldependent immune reactions, for example by leading to cytokinesmediated T cell activation, but higher concentrations of zinc can
directly suppress T cell function by reducing IL-1-dependent T cell
stimulation through the inhibition of interleukin-1 receptor
associated kinase-1 (Wellinghausen et al., 1997).
The absolute low number of mature B cells in ageing is not
affected by zinc deciency. However, low zinc levels cause a
reduction in B cell lymphopoiesis resulting in decreased number of
pre-B and immature B cells due to accelerated apoptosis and
impaired antibody response, particularly against T cell-dependent
antigens (Moulder and Steward, 1989).
Zinc deciency also affects the functions of cells involved in
innate immune response. For example, zinc deciency reduces the
lytic activity of NK cells (Haase and Rink, 2009b), impairs NKT cell
cytotoxicity and immune signaling (Mocchegiani et al., 2009),
decreases the chemotaxis and oxidative burst by neutrophils
(Moroni et al., 2005), inhibits mast cell activation (Kabu et al.,
2006) contributing to the higher incidence of viral infections and
cancer typical of the elderly. Indeed, zinc supply plays a relevant
role in restoring NK cell function in elderly (Mocchegiani et al.,
2003). Both zinc and MT affect NKT cell development, maturation
and function (Mocchegiani et al., 2009). In conditions of chronic
stress, including ageing, the zinc release by MT is limited, leading
31
to low intracellular zinc bioavailability and subsequent reduced

innate immunity (Mocchegiani et al., 2006a,b). Additionally, a zinc
nger motif [Kruppel-like factor 2 (K2] plays an important role in
the immune response of gdNKT cells (Odumade et al., 2010). In
zinc deciency, gdNKT cell cytotoxicity and K2 activity are
impaired (Mocchegiani et al., 2009). The mechanism by which zinc
may affect the adaptive and innate immunity is by targeting NF-kB,
a transcriptional regulatory factor of proinammatory responses
(Hayden and Ghosh, 2008). In dietary zinc deciency, in ageing or
in depletion of intracellular zinc with a zinc-specic chelator, NFkB activation increases with subsequent proinammatory cytokine gene expressions and production leading to the appearance of
chronic inammation coupled with some age-related degenerative
diseases (Bao et al., 2010; Golovine et al., 2008; Mocchegiani et al.,
2013b). In humans, zinc supplementation improves the immune
functions and decreases the inammation (Mocchegiani et al.,
2013b). Zinc supplementation in the elderly reduces the incidence
of infections (Prasad et al., 2007), increases delayed-type
hypersensitivity (DTH) reaction (Duchateau et al., 1981) and
enhances the T cell function (Cossack, 1989). Moreover, the agerelated increase in proinammatory markers (C-reactive protein
and inammatory cytokines) are restored by zinc supplementation
(Bao et al., 2010). Zinc is also involved in the wound healing
process occurring concomitantly to the inammatory response by
conferring resistance to epithelial apoptosis through the cytoprotection against reactive oxygen species (ROS) and bacterial toxins,
probably thanks to the antioxidant activity of MT (Lansdown et al.,
2007).
On the whole, since zinc deciency may affect the immune
functions and antioxidant activity, many studies have been
performed to evaluate the effect of zinc supplementation in
elderly with contradictory results (Haase and Rink, 2009a,b;
Mocchegiani et al., 2013b). In elderly and in old infected patients
(Mocchegiani et al., 2003; Kahmann et al., 2008), as well as in an
accelerated ageing condition, such as Downs syndrome (Franceschi et al., 1988), zinc treatment improves the thymic endocrine
activity, the lymphocyte mitogen proliferative response, CD4T cell
number, Th1/Th2 ratio, NK cell cytotoxicity and reduces the
spontaneous release of inammatory cytokines. Moreover, zinc
supplementation improves DNA repair (Chiricolo et al., 1993) and
increases the gene expressions of some zinc transporters (ZIP 1)
(Andree et al., 2004), leading to the correct maintenance of the
intracellular zinc homeostasis and to signicant clinical reductions
of relapsing infections.
It is advised to supplement zinc at physiological doses (RDA:
1012 mg zinc/day) for short periods (1 month) at alternating
cycles. These doses of zinc maintain a correct balance among
minerals (copper and iron), which closely interact (Mocchegiani
et al., 2012a). However, the accumulation of zinc may become toxic
and it may lead to an abnormal activation of some zinc-dependent
enzymes, such as PARP-1 involved in genomic stability, or favour
the entering of excessive calcium into the cells, with subsequent
cell death in both cases (Mocchegiani et al., 2000b; Frazzini et al.,
2006). It should be noted that the genetic background may also
inuence an individuals response to zinc supplementation. Recent
studies showed that polymorphisms in IL-6 174G/C and in MT1A
+647A/C loci impact on the effect of zinc supplementation upon the
immune and antioxidant response in the elderly (Mocchegiani
et al., 2012a, 2013b; Mariani et al., 2008b), as discussed below in
details (Section 3.1.1).
2.2. Copper, immunity, antioxidant response and ageing
Copper (Cu) is a trace element/micromineral bound to proteins,
8090% to ceruloplasmin (Cp). Copper is important for the
development and maintenance of the immune system, in
32
particular of neutrophil and monocyte functions even if its role is

still controversial/ambiguous in immunity (Maggini et al., 2007).
Copper is also essential for an optimal antioxidant defense, as it
functions as a cofactor of Cuzinc superoxidase dismutase (Cu, Zn
SOD): an enzyme important for free radical eradication (Prohaska
and Gybina, 2004). Thus, copper deciency may compromise
cellular antioxidant defenses via decreased capability to produce
SOD, thereby increasing their susceptibility to oxidative DNA
damage and, consequently, also the efciency of the immune cells
(Pan and Loo, 2000).
The actual estimated dietary allowances for adults lie between
1 and 1.5 mg/day. Copper deciency is not very common in human.
However, some studies report that plasma copper levels are
elevated during ageing strictly dependent by the dietary habits
(Madaric et al., 1994), leading to a concomitant augment of serum
Cp and cytochrome-c oxidase activity in platelets (Linder and
Hazegh-Azam, 1996). The increase in copper might heighten the
rate of lipid peroxidation through enhanced formation of free
radicals with the consequent impairment of the immune response
(Mukhopadhyay et al., 1997). Anyway, the role of copper has to be
considered together with others minerals, such as zinc, since
copper displays its biological function by bounding MT even more
tightly than zinc. This bound-copper becomes unavailable for
transfer out of the cell thereby decreasing zinc absorption (Cousins,
1985). Associations between plasma copper and zinc levels and the
health state have been reported in the elderly. In particular, plasma
Cu/Zn ratio, higher in women than men and increased with
advancing age, was associated with inammatory (CRP, IL-6) and
nutritional (serum albumin) markers, and with predicted 3.5 years
mortality over 70 years of age (Mocchegiani et al., 2012b). Higher
plasma Cu/Zn ratio was also reported in subjects with stable
cardiovascular disease (CVD), mainly due to increased plasma Cu
(Malavolta et al., 2010). However, most of the age-related changes
of Cu/Zn seemed to result from a progressive decline of plasma
zinc. Indeed, zinc supplementation ameliorates abnormally high
plasma Cu/Zn ratio in old hemodialysis patients improving the
inammatory (IL-6) and immune state (CD4/CD8 ratio) (Guo and
Wang, 2013). Consequently, Cu/Zn ratio may be considered as an
important clinical inammatory-nutritional biomarker and a
signicant predictor of all-cause mortality in over 70-year-olds
(Mocchegiani et al., 2012b).
Ageing does not modify signicantly copper metabolism and
dietary copper intake of elderly people seems to be adequate.
Therefore supplementation of copper is in general not necessary
and no recent clinical studies are available where copper, as a
single micronutrient, was therapeutically given to elderly people
oz et al., 2007).
especially in relation to the immune functions (Mun
Some studies were carried out in some age-related neurogenerative diseases (Parkinson, Huntingtons disease, Alzheimers dementia) in relation to the immune functions, but whether copper
dysregulation is a cause or a consequence of altered immune
functions in these neurodegenerative diseases is under investigation (Festa and Thiele, 2011).
With regard to antioxidant response few studies exist in ageing.
The effect of copper supplementation on oxidative stress and life
span was assayed in superoxide dismutase (SOD) mutants
Saccharomyces cerevisiae. Copper supplementation increased the
lifespan of the SOD1 and SOD2 mutants, indicating that copper
supplementation increases longevity by reducing or removing the
superoxide production, via increased gene expression of metallothionein Cup1 (Kirchman and Botta, 2007). However, copper,
being a redox-active transition metal, may initiate repetitive
radical formation through redox cycling (Lalioti et al., 2009).
Therefore, excessive copper can cause increased oxidative
damage to lipids, proteins and DNA and contributes to the onset
of neurodegenerative and age-related disorders (Linder and
Hazegh-Azam, 1996). Furthermore, excessive copper directly

participates in peroxidation of membrane lipids (Chow, 1979;
Powell, 2000) impairing the cellular membrane function, which
can be prevented by some ROS scavengers (Mattie and Freedman,
2001)
2.3. Selenium, immunity, antioxidant response and ageing
Selenium (Se) is a trace element in the organism representing
an essential component of selenoproteins that play an important
role in many biological processes, including adaptative and innate
immune responses and antioxidant defense (Behne et al., 1998;
Kryukov et al., 2003). In humans 25 genes encode for selenoproteins, such as glutathione peroxidases (GPx), the plasma Setransport protein selenoprotein P (SePP) and thioredoxin reductases (TrxR) (Kryukov et al., 2003). These selenioproteins, by
participating in the reduction of hydrogen peroxide and lipoperoxide, regulate the oxidative stress (Papp et al., 2007). SNPs (single
nucleotide polymorphisms) in the selenoprotein genes can
modulate the function of selenoproteins, thus affecting the cellular
response to oxidative stress (Meplan, 2011).
Ageing is characterized by selenium inadequacy due to a
reduced intake of protein dense food, such as red meat (Arnaud
et al., 2007). The Epidemiology of Vascular Ageing (EVA) study has
shown that people with low plasma selenium at the baseline are
susceptible to mortality by cancer (Akbaraly et al., 2005), display
cognitive decline (Akbaraly et al., 2007) and dysglycemia (Akbaraly
et al., 2010). Furthermore, transcriptomics and proteomics
approaches have revealed that the variation in selenium state
inuences different cellular pathways, such as mTOR pathway,
which are proposed to be correlated with the ageing process and
age-related diseases (Kipp et al., 2009). The mTOR pathway is
involved in cell senescence where MT play also a crucial role as
antioxidant agent (Mocchegiani et al., 2013a) suggesting a close
interrelationship among mTOR, selenium and MT against oxidative
stress and cellular senescence. Therefore, selenium supplementation may be useful to prevent, other than oxidative stress, also
cellular senescence (Pagmantidis et al., 2008).
With regard to the immune efciency, Se stimulates the
antibody formation, the activity of helper and cytotoxic T cell and
NK cells (Mehdi et al., 2013). In vitro and in vivo animal studies
showed that glutathione peroxidase and other selenoproteins,
such as thioredoxin reductase (TrxR) and methionine sulfoxide
reductase (Msr), play key roles in regulating the concentration of
peroxide and other oxidant compounds in the microenviroment of
the immune cells. In fact, selenium reduces oxidized MT through
glutathione peroxidase with subsequent zinc release by MT, with a
nal result in the activation of some antioxidant zinc-dependent
enzymes and subsequent benets in maintaining the integrity of
the immune cells (Maret, 2003). Indeed during ageing, some
fundamental functions of leukocytes, such as activation, proliferation, differentiation and phagocytosis, may be compromised by the
increased oxidative damage resulting from continuous production
of ROS due to selenium deciency (Rayman, 2012).
A supplementation of selenium (100 mg/day) for 6 months in
institutionalized elderly individuals leads to an increment in
serum selenium levels and to an improvement of the proliferative
response to pokeweed mitogen (Peretz et al., 1991). In addition, a
daily low dose supplementation of zinc and selenium (20 mg
zinc + 100 mg selenium) for two years in institutionalized elderly
people increased the antibody titers after inuenza vaccine and
contributed to a signicant improvement of the immunological
parameters and infection rates (Girodon et al., 1999). Another
study showed that a supplementation with selenium (400 mg/day)
for 6 months in healthy old humans increased of more than 50% the
percentage of CD4+ T cells. Interestingly, also the NK cell-mediated
cytotoxicity was enhanced after 3 months of Se-supplementation

(Wood et al., 2000). Moreover, a satisfactory serum selenium
concentration was positively associated with good peripheral
CD16+ NK cell numbers in healthy free-living nonagenarians
(Ravaglia et al., 2000). Selenium in vitro enhanced the
chemotaxis, phagocytic functions and MCP-1 synthesis by
peripheral blood mononuclear cells (PBMCs) from old subjects
(Ventura et al., 1994). Of interest is the existence of an inverse
correlation between serum selenium and IL-6. Old subjects with
the lowest selenium levels display high IL-6 levels coupled with an
higher risk of mortality over a period of 5 years, with thus a close
relationship between selenium state and inammation mediated
by IL-6 in elderly (Walston et al., 2006). Overall, selenium state is
generally perceived as an important factor for maintaining the
health in the elderly (Gonzalez et al., 2007; Ray et al., 2006), even if
the mechanisms by which Se-supplementation may improve
immune and antioxidant responses in old individuals have not
been fully elucidated yet. The seleniumgene interactions may
clarify this point (see below Section 3.3).
3. Micronutrients and gene interactions
During this last decade the pivotal role played by some
micronutrients (zinc, copper and selenium) in maintaining the
correct functioning of many body homeostatic mechanisms with
subsequent achievement of the longevity emerged (Mocchegiani
and Malavolta, 2008b; Mocchegiani et al., 2008c). Taking into
account the inuence of these nutrients in many genes involved in
inammatory/immune response and antioxidant activity (Mocchegiani et al., 2012a), the micronutrientgene interactions are
fundamental to escape many age-related diseases and to achieve
the longevity in healthy state through a personalized diet
containing these specic nutrients. In this section, we report the
specic role played by the most relevant micronutrientgene
interactions in ageing with an extension on their deciency or
toxicosis in order to choose a personalized nutrient supplementation or chelation, respectively.
3.1. Zinc and gene interactions
Zinc plays a key role for many biological functions because of its
occurrence in over one thousand enzymes as a catalytic ion and in
at least the same number of proteins as a structural metal (Maret,
2006; Andreini and Bertini, 2012). Such a role is strengthen by the
role of zinc in the processes of genetic stability and gene
expression, taking into account that about 25% of the cellular zinc
content is in the nucleus (Cousins, 1998). Mutations in some genes
coding for zinc-related proteins are the basis for inborn errors of
zinc metabolism, as it occurs in acrodermatitis enteropathica, a
rare disorder with a mutation in the Zip4 (SLC39A4) importer gene
and resulting in severe zinc deciency (Kury et al., 2002).
Furthermore, the risk of type II diabetes in ageing has been
associated with a single nucleotide polymorphism (SNP) resulting
in an Trp325Arg substitution in the pancreatic b-cell-specic zinc
transporter protein ZnT-8 (SLC30A8), which provides zinc for
insulin maturation and/or storage (Sladek et al., 2007). Moreover, a
novel Zip2 Gln/Arg/Leu codon 2 polymorphism is associated with
carotid artery disease in ageing (Giacconi et al., 2008b). These
ndings clearly suggest that zinc, through zinc transporters, may
affect the genetic stability with subsequent altered modications
of the zinc homeostasis and metabolism. Such an assumption is
supported by the fact that zinc, via zinc ngers, is involved in DNA
replication of many proteins essential for cell proliferation, cell
differentiation, cell growth arrest, cell division, signal transmission, growth factors production, protoncogenes activation, chemokine production, and for codifying hormone nuclear receptor
33
superfamily and for nuclear transcription factor activation (Klug,

1999; Mackay and Crossley, 1998). Indeed, the zinc nger motifs
regulate the gene expression of a large number of genes encoding
metallo-regulatory proteins and acting as zinc sensors for many
transcriptional factors with also an autoregulatory mechanism
(Lyons et al., 2000). This is the case of some regulatory or inhibitory
transcription factors (Egr-1, Sp1, A20), which, together with other
zinc-regulated transcription factors (NF-kB, STAT, HSF-1, Kruppel
zinc nger family), are involved in the regulation of the gene
transcription of pro-inammatory cytokines (IL-6, TNF-a) and heat
shock protein (Hsp70) in presence of antigenic stimuli (Rink and
Kirchner, 2000). Of interest, it is the Kruppel zinc-nger family
because implicated as transcriptional factors in many biological
processes, including proliferation, apoptosis, differentiation, development and in controlling the inammatory/immune response
and oxidative stress (Pearson et al., 2008). The characteristic of this
family is the presence of three Kruppel-like zinc ngers, which
bind to CACCC elements and GC-rich regions of DNA, to mediate
activation and/or repression of transcription. Within of this family,
a peculiar role is played by Klf2 because involved in T cell
proliferation as well as in controlling the IFN-g and TNF-a
production, via NF-kB (Das et al., 2006), and consequently the
inammatory state. On the other hand, mutant mice lacking K2
(ZFP36) showed signs of severe generalized inammation caused
by overproduction of TNF-a (Pearson et al., 2008). Of interest is
also the K5 because affecting the nuclear enzyme PARP-1
(Swamynathan, 2010), which is involved DNA-repair during
oxidative stress and inammation in ageing and in age-related
diseases (Mocchegiani et al., 2000b). The lack of zinc in these last
conditions provokes a reduced activity of PARP-1 with subsequent
no DNA-repair, as it instead occurs in centenarians showing good
PARP-1 activity, reduced inammation and satisfactory intracellular zinc ion bioavailability (Mocchegiani et al., 2003). Therefore,
zinc nger proteins are indispensable to transduce the signal from
the cytokine receptors into expression of response genes and,
consequently, in maintaining a correct balance of Th1/Th2
paradigm. In the course of zinc deciency, an unbalance of Th1/
Th2 paradigm appears with a shift towards Th2 cells and chronic
inammation (Prasad, 2000). Other than zinc nger proteins, zinc
also affects a wide range of genes involved in metabolism,
oxidative stress and inammatory/immune response and differently expressed in young and elderly people (Mocchegiani et al.,
2012a). In particular, genes of pro-inammatory cytokines (IL-6,
TNF-a), chemokines (CXCL3) are more expressed in zinc deciency
like in ageing, whereas the peroxisome proliferator activated
receptor (PPAR) signaling (in particular PPARa) is less expressed
(Mazzatti et al., 2007). Taking into account that PPARa inhibits NFkB activity, with thus an anti-inammatory properties (Delerive
et al., 2000), it is evident that a low gene expression of PPARa in
zinc deciency and in ageing provokes an high degree of
inammation. Indirect evidences in PPARa knock out mice support
this assumption. In aged PPARa/ mice, zinc supplementation is
ineffective, demonstrating that PPARa is critical in promoting the
anti-inammatory and anti-oxidative properties (Spencer et al.,
1997). Therefore, zinc plays a peculiar role in affecting the gene
expression of some genes related to the inammation and
oxidative stress. Sophisticated mechanisms are involved in the
regulation the activity of the complex intracellular zincgene
network. In this context, MT are essential regulators for
intracellular zinc homeostasis by the sequestration and release
of the metal at the occurrence and thereby controlling available
free zinc ions and affecting the zincgene network. The cysteine
sulfur ligands in the cluster structure of MT can be reduced (zinc
sequestration) or oxidized (zinc release) with concomitant changes
in the relative amount of bound and free zinc (Maret, 2003). MT are
genetically polymorphous protein families with subfamilies,
34
subgroups and various isoforms, among which MT-I and MT-II

isoforms are the best known and studied (West et al., 1990; Vallee,
1995). One of the main functions of MT-I and MT-II is to regulate
zinc homeostasis and to limit oxidative damage within the cells,
via zinc release from MT by NO and nuclear Nrf2 translocation and
subsequent Gclc gene expression (Spahl et al., 2003). In chronic
stress and inammation, as it occurs in ageing, high MT are less
able in zinc release with subsequent low zinc ion bioavailability to
ght stress and to support immune responses (Mocchegiani et al.,
2000b). As a consequence, a large number of genes that act as zinc
sensors and transcriptional activators and/or repressors are altered
in ageing (Fulop et al., 2003). In mammalian cells, the metal
responsive transcription factor 1 (MTF-1) was recognized as an
important zinc sensory transcriptional factor. MTF-1 binds to zincsensing gene promoter elements (MRE), and induces the gene
transcription of key target genes, including MT-I and MT-II
(Andrews, 2001). The disruption of MTF-1 gene in mice is lethal
(Andrews, 2001). This nding suggests that MTF-1 gene is
fundamental for MT gene expression and the subsequent regulation of many zinc target inammatory genes, such as IL-6, TNF-a,
Rantes, IL-8, MCP-1, PPARa, PPARg (Mazzatti et al., 2007). This
nding is of a great interest in ageing and in very old age taking into
account that in centenarians the MT gene expression is low like in
healthy adults and coupled with good zinc ion bioavailability and
satisfactory innate immune response (Mocchegiani et al., 2002).
Therefore, a good zincgene interaction, especially MFT-1/MT/
zincgene interaction, is preserved in very old age playing a pivotal
role to reach successful ageing and, at the same time, to escape
some age-related diseases. Such an assumption is supported by the
recent discovery of novel polymorphisms of MT2A and MT1A
associated with the inammatory state, zinc ion bioavailability and
longevity. Old subjects carrying AA genotype for MT2A polymorphism display low zinc ion bioavailability, chronic inammation
and high risk for atherosclerosis and diabetes type II (Giacconi
et al., 2005). By contrast, polymorphism corresponding to A/C
(Asparagin/Threonin) transition at +647 nt position in the MT1A
coding region is the most involved in the longevity (Cipriano et al.,
2006). In addition, +1245 MT1A polymorphism and +647/+1245
MT1A haplotype are implicated in cardiovascular diseases
(Giacconi et al., 2010). These ndings are a clear clue of the
relevance of the zincMTgene interaction with a pivotal role in
controlling inammation and longevity.
An intriguing point is also the nding showing an interaction
among MT polymorphisms, albumin, innate immune response and
cognitive functions in elderly (Mariani et al., 2008b; Marcellini
et al., 2008), taking into account that albumin binds zinc with the
task in moving zinc to various organs and tissues (Cousins, 1986).
Low albumin levels during ageing is associated with sarcopenia
(Visser et al., 2005), kidney damage with the appearance of
nephropathy (Martin and Sheaff, 2007) and mortality with
albumin values of 40 g/L (albuminemia) (Sahyoun et al., 1996).
Polymorphisms of some genes, such as MYO16 (myosin heavy
chain Myr 8), IRS2 (insulin receptor substrate 2), NEGR-1 (neuronal
growth regulator 1), are associated with increased albuminuria
and nephropathy in old mice and humans (Tsaih et al., 2010). Thus,
it is evident the relevance of the zincgene interactions in ageing
both in direct form (zincMT gene) and indirect form (albumin
gene) with the subsequent suggestion of possible zinc supplementation in elderly in order to compensate the lack of zinc. However
caution is needed because zinc may be also toxic with subsequent
undeliverable and deleterious side effects in various systems,
organs and tissues (Prasad, 2012).
3.1.1. Zinc supplementation and zinc toxicity
During these last two decades, a lot of papers report the
benecial effects of zinc supplementation upon the immune
system and the oxidative stress in old mice and in elderly with also
an effect in prolonging the survival in old mice due to a strong
reduction of deaths by infections (Mocchegiani et al., 2008c).
However, contradictory data exist in the benecial effects of the
zinc supplementation in old people. The discrepancies are largely
due to various factors, among them the different doses of zinc used,
the duration of the zinc supplementation, the dietary habits of the
old subjects with subsequent adverse or toxic effects on the
immune response (Sandstead, 1995) and genomic stability (Sharif
et al., 2012a,b). Zinc supplementation for short periods and in
alternate cycles, as carried out in Downs syndrome subjects, has
instead benecial effects upon the immune functions (Franceschi
et al., 1988). However, one possible cause of the discrepancies may
be the choice of old subjects who effectively need zinc
supplementation in close relationship with dietary habits and
inammatory status (i.e. IL-6 values). This assumption is supported
by the discovery that old subjects carrying GG genotypes (termed
C-carriers) in IL-6 174G/C locus display increased IL-6 production, low intracellular zinc ion availability, impaired innate
immune response coupled with enhanced MT. By contrast, old
subjects carrying GC and CC genotypes (termed C+ carriers) in the
same IL-6 174 locus display satisfactory intracellular zinc, good
innate immune response and are more prone to become
centenarians than C- carriers (Mocchegiani et al., 2008a).
Therefore, old C- carriers may benet more from the zinc
supplementation, which in turn restores NK cell cytotoxicity, zinc
state (Mariani et al., 2008b) and stress response (Mariani et al.,
2008a; Mocchegiani et al., 2007). When the genetic variations of
the IL-6 polymorphism are associated with also the variations of
MT1A +647A/C gene, the plasma zinc deciency and the altered
innate immune response is more evident (Mocchegiani et al.,
2008a), suggesting that the genetic variations of IL-6 and MT1A are
very useful tools for the identication of old people who effectively
need zinc supplementation. These results suggest that the daily
requirement of zinc might be different in elderly harbouring a
different genetic background with thus the possibility of a
personalized diet. An exhaustive panel showing the effect of the
zinc supplementation on immune and antioxidant parameters in
elderly in relation to IL-6 and MT polymorphisms is reported in
Table 1.
Despite of that, an excess of zinc may become toxic because
inducing copper deciency (Maret, 2006) perhaps due to a
reduction of the gene expression of ATP7A protein involved in
copper absorption in enterocytes, as it occurs in Menkes disease
(Llanos and Mercer, 2002). Toxic levels of zinc are also implicated
in carcinogenesis because inhibiting the activity of some DNA
repair proteins, such as N-methylpurineDNA glycosylase and
DNAligase 1 (Yang et al., 1996; Wang et al., 2006). However, an
excess of zinc has the major effect on the brain provoking neuronal
injury with the appearance of neurodegenerative disease (Mocchegiani et al., 2005). Toxic accumulation of zinc may result from
either trans-synaptic zinc movement or mobilisation from
intracellular sites, such as Zn ux through receptor associated
calcium channels, voltage-sensitive calcium channels, or zincsensitive membrane transporters (Weiss et al., 2000). Recent
ndings have suggested that postsynaptic zinc-sequestering
proteins, such as MT-III, may represent the main sources of toxic
zinc ions (Frederickson et al., 2004) because sequestering and
rapidly releasing zinc after oxidative stimuli (Lee et al., 2003) with
a nal role in activating neuronal apoptotic processes (Sensi and
Jeng, 2004). These phenomena occur in Alzheimer type dementia
(AD), in which zinc may also favour the accumulation of b-amyloid
(Ab) in the senile plaques (Bush, 2000). Although this last role of
zinc in Ab accumulation has been instead suggested to be
protective because preventing Ab-copper interaction with the
subsequent formation of hydrogen peroxide and free radicals
35
Table 1
Effect of zinc supplementationa on immune and antioxidant parameters in elderly according to MT and IL-6 polymorphisms (data obtained from Zincage project).
Condition/target
Parameter
Effects
Stress-related proteins
Poly(ADP-ribosyl)ation capacity
ROS production
ApoJ plasma
Genes involved in Nitrosative stress (ATF2, CSF2, FOS, ICAM1, JUN, LTA, CCL2, SELE, VCAM1,
iNOS, TNF, NFKB1)
Total intracellular carbonyl levels
MsR activity and protein expression
Chymotrypsin-like peptidase activity of proteasome and 20S protein expression
Chaperone (Hsp72) protein levels
Chaperone (Hsp72) inducibility
"
#
"
#
Antioxidant plasma enzymes
Plasma SOD
Erythrocyte SOD
Catalase
Glutathione peroxidase
"
"
#
#
Thymic output
T-cell receptor excision circles (TRECs)
#"
Plasma cytokines and chemokines
IL-6, IL-8, MIP-1a

MCP-1, RANTES
"
Immune functions
NK lytic activity
Basal IFN-g, IL-8, IL- 1ra and IL-6 production
Basal IL-10 and TNFa production
Stimulated IFN-g, IL-6, TNF-a, IL- 1ra and IL-10 production
""
#
##
"
T cells subsets
Activated T cells (CD3 + CD25+)

CD4:CD8
#
""
"
"
""
"", strongly increased; ", increased; not modied; " slightly increased; #", intervariability; #, decreased.
a
The dose = 10 mg/day for 45 days of zinc aspartate (Unizink 50, Kohler Pharma Corp., Alsbach-Hahnlein, Germany; Mocchegiani et al., 2012a, 2013b).
(Cuajungco et al., 2000), an excess of zinc is toxic in the brain with

thus the use of chelating agents in order to avoid the excess of zinc
in AD and neurodegeneration. Among chelating agents, TPEN and
Clioquinol were used. TPEN was used for in vitro studies in order
to better understand the zinc release by NO in neurons, via
p38MAPK, and the subsequent its toxic effect on neuronal cell
death (Bossy-Wetzel et al., 2004). Other in vitro studies have
shown that TPEN attenuates the upregulation of TNF-a, IL-13, IL-4,
IL-6 by high doses of zinc, via PKC and NF-kB inhibition (Fukuyama
et al., 2011; Haase et al., 2008). The other chelator is Clioquinol that
acts as a zinc, copper, and iron chelator (Cuajungco et al., 2000).
Metal chelation is a potential therapeutic strategy for AD because it
can dissolve amyloid deposits by preventing metalAb interactions (Bush, 2000), as shown in transgenic mice with high Ab
peptide levels (Cherny et al., 2001). However, Clioquinol can
potently inhibit the 20S proteasome causing intracellular accumulation of misfolded proteins with thus a deleterious role (Mao
and Schimmer, 2008). This dangerous effect is dependent by the
inammatory genetic susceptibility and by the dose used. Low
doses of Clioquinol (2030 mg/kg/day) provoke relatively lower
toxic effects in humans (Bareggi and Cornelli, 2012) (see Table 2).
Anyway also for zinc chelation, the individual inammatory
genetic background may play a key role in order to visualize
subjects or patients to be treated effectively with zinc chelators. In

such a way, we can establish a nutritional personalized therapy
either in zinc supplementation or zinc chelation.
3.2. Copper and gene interactions
As reported above, copper (Cu) is an essential component for
numerous reactions that are necessary for the growth and
development in men and animals (Olivares and Uauy, 1996).
The most bioavailable source of copper is meat and the major
storage of copper is in the liver primarily bound to MT (Hartmann
et al., 1993). A blood protein, ceruloplasmin (Cp), contains several
molecules of copper and it is a marker of body copper status
because involved, together with albumin, to the transport of
copper within the cells, organs and tissues (Harvey et al., 2009). A
recent adult human dietary recommendation for copper (estimated safe and adequate dietary intake) was set at between 1.5 and
3.0 mg Cu/day (Milne, 1998). Equipped with high redox potential,
copper serves as a cofactor for proteins involved in a variety of
biological reactions, such as photosynthesis and respiration
(cytochrome c oxidase), free radical eradication (SOD), connective
tissue formation (lysyl oxidase), neurological development (dopamine b-hydroxylase), and iron homeostasis (Cp) (Prohaska and
Table 2
Effect of zinc chelators as potential therapeutic strategy in presence of possible zinc toxicosis.
Chelator
Target
Model
Treatment
Effect
Reference
TPEN
TNFa, IL-13, IL-4

production
TNFa, IL-6, IL-1b
production
Airway inammation
(animal model)
Human monocytes after
LPS stimulation for 24 h
(in vitro model)
Alzheimer (human)
HeLa cells exposed to toxic zinc
(50 mM) (in vitro model)
One i.p. injection (10 mg/Kg)
Fukuyama et al. (2011)
1050 mM for 30 min
Haase et al. (2008)
375 mg/day for 25 weeks

2 mM
#
" Blocked inhibition of
MPG by zinc favouring
DNA-repair
Ritchie et al. (2003)

Wang et al. (2006)
TPEN
Clioquinol
EDTA
Ab42 accumulation
Learning memory
N-methylpurine-DNA
glycosylase (MPG)
", increase; #, decrease.
36
Gybina, 2004). However, excessive copper is toxic or even lethal

for the cells because participates in ROS generation through
Fenton chemistry and direct oxidations of lipids, proteins and
DNA (Linder and Hazegh-Azam, 1996). Consequently, intracellular copper content is maintained by evolutionarily conserved cell
transport systems that regulate uptake, export, and intracellular
compartmentalization (Prohaska and Gybina, 2004). Therefore,
in order to prevent the consequences of copper deciency or
overload, living organisms have evolved molecular mechanisms
that regulate its uptake, intracellular trafc, storage and efux
(Ridge et al., 2008). High-afnity copper uptake is mediated by
copper transporters Ctr1p and Ctr3p in yeast (Dancis et al., 1994;
Pena et al., 2000) and copper transporter hCTR1 in humans (Puig
and Thiele, 2002). In humans, Cu2+ can be transported directly
into the cells by the divalent metal transporter DMT1 (Arredondo
et al., 2003). Once in the cytoplasm, copper is delivered to various
cell targets with the help of copper chaperones, such as CCS, to
transfer copper to SOD1; COX17 through SCO1/2 to cytochrome C
oxidase and HAH1 (homologous of Atox1) to ATP7A/B located in
the trans-Golgi network. The latter ATP7A/B genes, identied as
P-type ATPases, are very important because their function in the
trans-Golgi network is to pump copper to copper-dependent
enzyme (lysyl oxidase) involved in elastic and collagen crosslinking and to maintain cellular copper within safe limits (Petris
et al., 1996). Mutations in the ATP7A/B genes are responsible of
Menks or Wilson diseases that are related to copper deciency or
toxicosis, respectively (Camakaris et al., 1980; Gojova et al.,
2008). When ATP7B is mutated, as in Wilsons disease, Cp levels
decrease and copper accumulates in the liver, suggesting the key
role played by ATP7B in copper homeostasis (Bull et al., 1993;
Mercer, 2001).
The interaction of ATP7B with specic copper chaperone Atox1
is required in the hepatocytes for proper biliary excretion of excess
copper and for delivery of copper for Cp synthesis (Culotta et al.,
1999). Deletion of the Atox1 gene in mice causes perinatal
mortality (Hamza et al., 2001), provokes reduced levels of lysyl
oxidase (Prohaska and Gybina, 2004) and does not protect the
neurons from oxidative stress (Kelner et al., 2000). Mutants of
Atox1 in the corresponding gene (lys7) are defective for SOD1
activity, and are unable to incorporate copper into SOD1 (Culotta
et al., 1999). With regard to ATP7A, it is an important coppertransport protein in neurons and astrocytes (Prohaska and Gybina,
2004). When the cells are exposed to increased levels of copper,
ATP7A moves from the trans-Golgi network to the plasma
membrane for copper efux (Lutsenko and Petris, 2003). In
concert with Atox1, ATP7A also plays a key role in providing copper
for secretory cuproenzymes such as peptidylglycine-amidating
monoxygenase (PAM) (El Meskini et al., 2003). When ATP7A is
non-functional, as in brindled mutant mice, PAM activity is altered,
and the selected neuropeptide maturation is impaired (Steveson
et al., 2003). Moreover, the cells lacking Atox1 have impaired
movement of ATP7A in response to copper, providing a mechanistic explanation for copper retention (Hamza et al., 2003). ATP7A is
also required for the transfer of copper to other cuproenzymes,
such as hephaestin (Prohaska and Gybina, 2004). Therefore, a
copper deciency in the diet modies the gene expression and
function of these relevant copper chaperons with the subsequent
altered intracellular copper homeostasis that may also affect the
Cp synthesis. As a consequence, defects in copper distribution and
in iron metabolism may occur because Cp acts also as a ferroxidase,
converting Fe2+ to Fe3+ and increases the rate of loading of Fe into
transferrin (Osaki and Johnson, 1969). Furthermore, Cp stimulates
Fe efux, suggesting that it is relevant for the mobilisation of Fe
from the body stores (Osaki and Johnson, 1969). Therefore, the
synthesis of Cp by dietary copper is crucial not only for copper body
distribution but also for iron metabolism. Thus, it is evident that
dietary copper is fundamental for the copperiron cross linking in

which the synthesis of Cp plays a key role, as shown by the
presence of four exon polymorphisms of Cp in iron overload
individuals: two of which resulted in an amino acid change, a C3/T
transition in position +1099 (R367C) in exon 6 and a C3/T
transition in position +1652 (T551I) in exon 9 (Lee et al., 2001).
The Cp synthesis and secretion by the liver is regulated by proinammatory cytokines (IL-1, IL-6, and TNF-a) indicating a role of
Cp in inammation (Linder and Hazegh-Azam, 1996). Increases of
Cp in inammation are thought to be related to the need for
scavenging oxygen radicals released at inammatory sites by the
immune cells (Linder and Hazegh-Azam, 1996). An increase in Cp
levels in ageing, in atherosclerosis or Alzheimer disease (AD), could
represent a good marker of the inammatory status. On the other
hand, Cp oxidizes LDL through an interaction of one of the copper
molecules carried by Cp (Mukhopadhyay et al., 1997; Fox et al.,
2000), and oxidized LDL is part of the atherogenic and ageing
processes (Windler et al., 2007). With regard to AD, contradictory
data exist on the role played by copper in its pathogenesis. Some
authors report that high copper may be involved in AD because
Amyloid precursor proteins have a copper binding domain which
reduces copper (II) to copper (I) producing oxidative damage
(Multhaup et al., 1996). Moreover, b-Amyloid binds copper and
cholesterol, facilitating copper oxidation of cholesterol to 70H
cholesterol, which is extremely toxic for neurons (Nelson and
Alkon, 2005). In animal models of AD, the addition of copper in the
drinking water greatly enhances the accumulation of b-Amyloid in
the brain and increases learning decits (Sparks, 2004). By
contrast, other authors report that copper induces a reduction of
the b-Amyloid formation (Phinney et al., 2003). All the data,
although some of them are contradictory, suggest in general that
high copper may be deleterious because affecting the gene
expression of copper chaperones, Cp and the copperiron
interactions with subsequent enhanced inammation and increased incidence of some inammatory pathologies, such as
atherosclerosis, AD and type I and II diabetes (Shukla et al., 2006).
Patients with metabolic syndrome (obesity, hypertension,
glucose intolerance and dyslipidemia) also have elevated Cp levels
(Kim et al., 2002). With the exclusion of Menkes disease, in which
copper deciency is of genetic origin (Kaler, 1994), nutritional
copper deciency is well documented in two situations: (a) the
premature newborn and (b) patients maintained on total
parenteral nutrition for long periods (Danks, 1988). A paucity of
data exists on the effect of copper deciency in old age. This fact
may be due because copper homeostasis is maintained over a wide
range of intakes, mainly through changes in endogenous excretion,
especially at lower intakes (Harvey et al., 2003). On the other hand,
plasma copper is not different among young, adult and old age with
signicant increments in presence of acute inammation or
inammatory diseases (Malavolta et al., 2010) mainly due to
increased levels of SOD1 and MT cuproenzymes to ght oxidative
stress and inammation (Mocchegiani et al., 2006b). Indeed, MT
are another possible copper chaperone, as shown in MT-null
broblast cell lines (Tapia et al., 2004). Two MT isoforms, MT-1 and
MT-2, play a role in intracellular copper transfer and storage in the
liver. Indeed, other than zinc, copper can induce MT synthesis
(Coyle et al., 2002). When copper is limiting in liver cells, MT plays
a role by acting as a copper reserve (Suzuki et al., 2002). Therefore,
MT synthesis and production as well as MT polymorphisms
become relevant in presence of inammation especially in ageing
in order to act as copper reserve also in presence of lower copper
intake. In this context, some novel +647A/C and +1245A/G MT1A
polymorphisms are related to increased copper levels and
enhanced SOD in CVD (Giacconi et al., 2008a), suggesting a role
of MT in the copper transfer to SOD to ght the inammation and
ROS. However, also a copper deciency might be relevant in ageing
taking into account that a copper deciency affects some aspects of

the inammatory/immune response (Mocchegiani et al., 2012a). A
low intake of copper (0.38 mg/day) in adult humans signicantly
impairs the in vitro responsiveness of T lymphocytes to
mitogenic activation and decreases IL-2 receptor concentrations
(Kelley et al., 1995). In the copper decient Jurkat T-cell line, the IL2 mRNA synthesis was decreased by 50%, indicating that copper
plays an important role in the transcription of the IL-2 gene
(Hopkins and Failla, 1999). Anyway, data showing the effects of
copper on the immune response are limited in elderly, mainly due
to the inability to precisely assess marginal and moderate copper
deciency state because of the lack of sensitive and specic
biomarkers.
3.2.1. Copper supplementation or copper depletion
The necessity of copper for the human health derives from its
involvement in a myriad of biological processes, including iron
metabolism, antioxidant defense, neuropeptide synthesis and
immune functions (see Section 2.2), but it may be also potentially
toxic when the intake levels are too high (Brewer, 2008) due to its
participation, like iron, in the Fenton-type redox reaction provoking cell oxidative damage (Linder and Hazegh-Azam, 1996).
Therefore, it is important to distinguish two levels of interventions:
the rst one as a copper supplementation in the case of copper
deciency; the second one as a copper depletion in the case of
copper excess. Such a subdivision may seem obvious, but the
matter is not so simple because it is very difcult to nd a precise
biomarker for the copper state and, consequently, the deciding for
a copper supplementation or depletion is somewhat problematic.
The difculty especially arises when the copper state has to be
determined taking into account that neither plasma copper nor
plasma cuproenzymes reects copper status, which is instead
estimated by intake and exposure assessment. The latter are
subject to the error inherent to all dietary intake studies and, while
suitable for the assessment of macronutrient intake, may not be
precise enough to estimate intake of a dietary micronutrient,
especially for copper due to its content in the drinking water that
can vary in dependence of local changes in the chemical water
properties (water hardness and pH) (Danzeisen et al., 2007). As a
consequence, a large error of dietary copper intake assessment
may occur. Thus, regulators and public health professionals adopt a
predominantly conservative approach in copper-exposure regulation. However, this approach may not be suitable for an essential
trace metal, since a low intake of copper is as dangerous as a toohigh intake. The situation is then worsened by the lack of precise
copper biochemical biomarker/s, despite plasma copper, Cp and
Cu/Zn SOD1 routinely are assayed in human studies (Danzeisen
et al., 2007; Harvey et al., 2009). Therefore, a marginal copper
deciency is difcult to diagnose with respect to severe copper
deciency, which is instead present in various pathological
conditions (Danks, 1988), which some of them are also related
to the ageing process, such as macular degeneration, osteoporosis,
myocardial disease (Klevay, 2000). Conversely, copper toxicity as a
result of a dietary excess generally is not considered to be a
widespread health concern, probably as a result of the homeostatic
mechanisms controlling copper absorption and excretion (Turnlund et al., 2005). However, a copper overload has been
documented in various pathological conditions usually also related
to the ageing process, such as atherosclerosis, type II diabetes, and
AD, even if in AD the data are strongly controversial reporting also
copper deciency (Brewer, 2007).
From this complex picture, many studies on the effect of copper
on the immune response, oxidative stress and bone metabolism
and the subsequent copper supplementation or copper depletion
were conducted in animals and in adult humans with low or
excessive copper content in the diet. In order to study the effect of
37
copper in ageing, the budding yeast Saccharomyces cerevisiae was

used as a model for cellular ageing (Kirchman and Botta, 2007). The
majority of these studies are conducted testing plasma copper or
cuproenzymes or ceruplasmin for copper state. In copper-decient
rodents, the reduced secretion and activity of IL-2 by activated
splenocytes is restored by copper treatment (24 mM) (Bala and
Failla, 1992). A similar restoration also occurs for other impaired
immunological functions (burst neutrophil activity, blastogenic
response to T cell mitogens and balance of Th1/Th2 paradigm
(Bonham et al., 2002). Recently, taking into account that copper
deciency is associated with cardiomyopathy (Klevay, 2000),
copper supplementation can reverse hypertrophic cardiomyopathy in some patients. One example of this is a rare congenital
condition of Sco2 mutations, which affects copper metabolism and
predisposes to hypertrophic cardiomyopathy (Papadopoulou et al.,
1999). Copper supplement (copperhistidine) therapy caused
reversal of the hypertrophic cardiomyopathy along with signicant improvement in heart functions, normalization of ECG signs
and blood pressure (Freisinger et al., 2004). The mechanism by
which copper induces its benecial effect is through copper
chaperone for SOD-1 that mediates also VEGF expression and
angiogenesis (Jiang et al., 2007) suggesting a role of copper also in
angiogenesis. This fact is relevant taking into account that reduced
angiogenesis is involved in endothelial cell senescence (Erusalimsky and Kurz, 2006) with the appearance of aortic valve
stenosis: a characteristic of the ageing process (Yetkin and
Waltenberger, 2009).
With regard to ageing, few studies exist in animal models and in
humans on the role played by copper. In order to examine the role
played by copper in cellular ageing and the possibility that copper
may reduce oxidative stress in cells, the effect of copper
supplementation on the life span of superoxide dismutase yeast
mutants was assayed. Mutants with a deletion of the SOD1 and
SOD2 genes had extremely short life spans. Copper supplementation increased the life span of the SOD1 and SOD2 mutants,
indicating that copper supplementation increases longevity by
reducing the production of superoxide (Kirchman and Botta, 2007).
In humans, the copper state, tested by plasma copper or
ceruloplsamin or cuproenzymes, is strictly dependent by individual dietary habits and healthy state. In particular, both excessive
zinc or iron intake can induce copper deciency interfering with
copper absorption in enterocytes (Underwood, 1981). However, in
general, the plasma copper does not have any substantial changes
in condition of healthy state during the life of an individual,
including old age, in which a copper deciency is at subclinical
level (Harvey et al., 2009). By contrast, in condition of specic
intestinal malabsorption (such as celiac disease, bowel syndrome,
in long-term parenteral nutrition), or in bone abnormalities or in
well genetically determined disease (Menkes disease) copper
deciency is severe with dysfunctions related to immune response,
antioxidant activity and bone metabolism (Danks, 1988). In this
last case, copper deciency is linked to the reduced activity of
enzyme lysyl oxidase with subsequent appearance of osteoporosis
(Lowe et al., 2002). Copper supplementation in old postmenopausal women induces a better bone mineral density
(Saltman and Strause, 1993) as well as an improved clinical
outcome of elderly patients with femoral neck fracture (Delmi
et al., 1990). Of great interest is the role played by copper
deciency in the retinal pigment epithelium (RPE) (Ugarte and
Osborne, 2001) provoking apoptosis of RPE and retinal cells as well
as altering the activity of RPE antioxidant metalloenzymes with the
appearance of the age-related macular degeneration (AMD)
(Zarbin, 2004). A reduced zinc pool in RPE may be ascribed to
reduced action of MT (Nicolas et al., 1996), whereas for copper,
reduced copperadenosine triphosphates (ATPases) has been
proposed taking into account that they are localized in the RPE
38
Table 3
A,B. Effects of copper supplementation and copper chelation in copper deciency and in copper overload, respectively.
Condition
Type of supplementation
A. Effect of copper supplementation in copper deciency

Neutropenia (animal model) (Bala and Failla, 1992)
6 mg of Cu in the diet for 5 weeks
Target
Effect
Macrophage functions
Neutrophils activity
IL-2 mRNA
NK cytotoxic activity
Th1/Th2 balance
T-cell proliferation
ECG
"
"
"
"
Normalization
"
Normalization
Menkes disease (human) (Kreuder et al., 1993)
S.c. injections of Cu-histidine

(65 mg/kg/day/120 days)
IL-2
NK cytotoxic activity
SOD
"
"
"
Macular degeneration (human) (AREDS Study, 2001)
80 mg zinc oxide + 2 mg of cupric

oxide/day for 411 years
3 mg CuSO4/day for 2 months
Apoptosis of retinal pigment epithelium

Metallothionein
Bone mineral density
#
"
"
Alzheimer (animal model) (Bayer et al., 2003)
25 g/L of CuSO4 in H2O for 3 months
SOD
Ab accumulation
"
#
B. Effect of copper chelation in copper overload

Condition
Type of Chelator
Target
Effect
Type II diabetes (human) (Cooper et al., 2004)

Wilsons disease (human) (Brewer et al., 2009)
Trientine (1.2 g/day/6 months)

Tetrathiomolybdate (120 mg/day/8weeks)
Left ventricular hypertrophy (LVH)

Ceruloplasmin and copper levels
Normalization
#
Alzheimer (human) (Squitti et al., 2002)
Penicillamine (600 mg/day for 24 weeks)
SOD
Ab accumulation
Cognitive status
"
#
"
Alzheimer (animal model) (Cherny et al., 2001)
Clioquinol (oral treatment of

30 mg/kg/day for 9 weeks)
Ab accumulation
Survival
#
"
Osteogenesis in postmenopausal women (human)

(Saltman and Strause, 1993)
", increase; #, decrease.
and facilitate copper transport between RPE and retina (Krajacic

et al., 2006). Combined oral supplementation of copper plus zinc
reduces the risk of progression of AMD in old patients due to better
MT homeostasis and increased SOD (Erie et al., 2009) (Table 3A).
With regard to high copper, the presence of chronic inammation plays a pivotal role taking into account that pro-inammatory
cytokines affects the synthesis and production of Cp (Linder and
Hazegh-Azam, 1996), which in turn is under the control of copper
status, with thus a strict interrelationship among inammation, Cp
and copper plasma levels. Such a link is evident in some
inammatory pathologies, such as atherosclerosis and type II
diabetes, which are also common age-related diseases. Epidemiologic studies have shown a relationship between high serum
copper or elevated Cp levels and atherosclerosis (Ford, 2000;
Klipstein-Grobusch et al., 1999), whereas other epidemiologic
studies failed (Enbergs et al., 1998). On the other hand, elevated
levels of copper have been found in human atherosclerotic plaques
oxidizing LDL (Stadler et al., 2004), which is in turn a part of the
atherogenic process, especially in the early phase of the
atherogenesis (Ferns et al., 1997). Elevated levels of Cp and copper
have been found in diabetes mellitus with vascular complications
(Shukla et al., 2006). All these studies show, on one side, that high
copper is toxic because generating ROS; on the other side, they
suggest that a clinical trial of copper depletion may be of benet in
atherosclerosis. However, no clinical trial of copper depletion in
atherosclerosis or diabetes mellitus have been carried out up to
date. Promising data are obtained in diabetic animal models using
copper chelator trientine (Cooper et al., 2004) with benecial
effects on the cardiomyocyte structure. Other studies in mouse
model of Wilson disease, the copper-lowering agent tetrathiomolybdate (TM) is capable of producing a greater degree of copper
depletion than other drugs (Brewer, 2007) (Table 3B).
Of particular interest is the role played by copper in AD with
contradictory data and suggestions (Quinn et al., 2009). In this
context, Squitti et al. (2002) have found high free copper coupled
with high levels of serum peroxides in the blood of AD patients.
Penicillamine (a copper chelator) therapy reduced the level of the
serum peroxides (Squitti et al., 2002) (Table 3B). In animal studies,
Sparks (2004) has found that trace amounts of copper added to the
drinking water in a rabbit model of AD greatly enhances the e3/3
accumulation of b-Amyloid in the brain and increased learning
decits. The treatment with clioquinol markedly inhibited bAmyloid deposition in the brain (Cherny et al., 2001). However,
there is signicant controversy over whether an excess of copper is
involved in AD pathogenesis, taking into account that copper
supplementation in AD mouse model (APP23 transgenic mice)
lowered b-Amyloid production, restored SOD, and increased the
longevity (Bayer et al., 2003). By contrast, human AD studies show
that the cognitive decline correlated positively with low plasma
levels of copper (Pajonk et al., 2005) suggesting a possible copper
supplementation. Such a controversy can to be resolved by further
experimentations. Anyway, a signicant help to discern this
problem in AD may come by the analysis of Apolipoprotein-E4
(ApoE4) genotype that is a risk factor for AD involved in copper
binding of the cysteine residue provoking a diminished antioxidant
effect of the E-4 allele (Miyata and Smith, 1996). In AD, the
presence of ApoE-e4 allele is associated with increased brain
vulnerability to the effects of the disease, whereas the presence of
the ApoE genotype e3/e3 appears to provide moderate neuroprotection (Alberts et al., 1995). This nding is very suggestive because
the possible association between these genotypes and copper state
may be very useful to discern the role played by copper in AD with
subsequent possible copper supplementation or deletion.
3.3. Selenium geneinteractions
Selenium is an essential dietary factor for the prevention of
some diseases, including cancer and infections (Schwarz, 1976).
39
Table 4
Main encoded proteins affected by seleniumgene interactions.
Encoded Proteins
Gene name
Functions
Target organs
Glutathione peroxidase 1
Gpx1
Gpx2
Mitochondria
Liver, intestinal tract
Selenoprotein W
Selenoprotein H
Gpx3
Gpx4
Sepw1
SelH
Selenoprotein 15
Selenoprotein P
Sep15
Sepp1
Selenoprotein X1
Sepx1
Selenoprotein I
Selenoprotein T
SelI
SelT
Selenoprotein S
Selenoprotein K
Selenoprotein M
Selenoprotein O
Thioredoxin reductase 1
SelS
SelK
SelM
SelO
Txnrd1
Txnrd2
Txnrd3
Reduction of hydrogen peroxide

Antioxidant defence
Detoxication
Reduction of hydrogen peroxide
Protection against lipid hydroperoxides
Redox reaction
Cell viability
Antioxidant defence
Detoxication
Protection from protein folding
Selenium transport
Protection against inammation
Incorporation in MSRB1
Protection from oxidative stress
Protection against lipid hydroperoxides
Redox regulation
Cell adhesion
Cell anchorage
Protection against ROS
Regulation Ca2+ ux against infections
Protection on ROS mediated-apoptosis
Defence against toxic substances and ROS
Antioxidant and redox regulation
Redox regulation of cell signaling
Defence against oxidative stress
Skeletal muscle, kidney, lung, breast

Cell membrane
Skeletal muscle, heart, brain
Neuronal cells
Endoplasmic reticulum
Neurons
Liver, kidney
Cell membrane
Neuroendocrine secretion (pituitary)
Cell membrane
Immune cells
Brain (hippocampus)
Liver
Against cancer cells (target for cancer therapy)
Prostate, ovary, liver, testis, colon, small intestine, brain, heart
Mitochondria
For specic references see the text (Section 3.3) and Brigelius-Flohe and Banning (2006).
Some relevant papers report a greater development of carcinoma

by various carcinogens (1,2-dimethylhydrazine or dimethylbenz(a)anthracene) in various organs (colon and mammary gland) in
rats fed a selenium decient diet in comparison with rats treated
with 5 ppm of selenium (Pence and Buddingh, 1985). With regard
to infection, decreased dietary selenium can change a normally
avirulent B3 coxsackievirus (CBV3/0) into a virulent virus (CBV3/
20) by inducing changes in viral RNA polymerase mutations (Beck
et al., 1994) provoking dilated cardiomyopathy and death (Funseth
et al., 2000). In food, selenium derives from the consumption of
seafood, liver, and cereals. In vegetables and cereals the amount of
selenium varies depending on the soil where they are grown and
harvested from different geographical regions. Indeed, selenium
deciency and some related diseases have been well documented
in geographic regions where the selenium content on the soil is
low, such as the Chinese province of Keshan, from which derives
the term Keshan disease characterized by selenium deciency
and presence of substantial number of virulent viruses, including
coxsackieviruses (Li et al., 2013).
Mammals can use both inorganic and organic selenium as a
nutrient. Most of the biological functions of selenium are
attributed to selenoproteins, which contain selenocysteine (SeCys)
residues responsible for their specic activity. The human
selenoproteome consists of 25 selenoproteins, mostly involved
in antioxidant defence systems and affecting various target organs
(Brigelius-Flohe and Banning, 2006) (Table 4). The incorporation of
SeCys into selenoproteins occurs during translation by a mechanism that uses UGA codon and the formation of a complex
involving a RNA stem-loop structure, the Selenocysteine Insertion
Sequence (SECIS), which is located in the 30 untranslated region
(30 UTR) of selenoprotein mRNASs (Hateld and Gladyshev, 2002).
Thus, single nucleotide polymorphisms (SNPs) in selenoprotein
gene regions corresponding to 30 UTR sequence could affect the
expression of the selenoproteins with subsequent inuence on the
antioxidant systems or the inammatory/immune response
(Bermano et al., 2007). In this context, a variety of studies report
the role played by a family of the selenoproteins named Glutatione
peroxidises (GPxs), which protect the cells against oxidative
damage by catalysing the reduction of the hydrogen peroxide and
other hydroperoxides by glutathione (Hayes and McLellan, 1999).

It has been reported that a SNP within the coding region of the gene
encoding the selenoprotein glutathione peroxidase 1 leads to an
amino acid change (Pro to Leu) that leads to a lower enzymatic
activity (Forsberg et al., 2000) with subsequent disease susceptibility, in particular to the lung, breast and bladder cancer.
However, such a susceptibility is still more evident when this
SNP is associated with another SNP in the gene encoding the
antioxidant defense protein manganese dismutase or with
environmental factors, such as alcohol consumption or smoking
(Raaschou-Nielsen et al., 2007). Several studies have also reported
a Pro-to-Leu change in the 30 UTR region of other isoforms of GPxs,
such as GPx2, GPx3, GPx4. While no data report a functional
signicance of a SNP for GPx2 gene, a recent study has instead
indentied up to eight strongly linked variants in the promoter
region of GPx3 gene that fell into two haplotype groups indicating
a lower activity of the enzyme with the possible appearance of the
arterial ischemic stroke in adults (Voetsch et al., 2007). Taking into
account that GPx4 removes membrane-bound phospholipid
hydroperoxides inhibiting lipoxygenases and affects the leukotriene biosynthesis, cell growth, apoptosis and inammation (Kim
and Milner, 2001), a SPN for GPx4 gene was extensively studied in
relation to diseases and selenium metabolism. In this context, a T/C
SNP located within the GPx4 region corresponding to the 30 UTR
near the SECIS element in position 718 show that the C variant is
related to a higher frequency to colorectal cancer, suggesting a link
between this SNP and cancer (Bermano et al., 2007). Of interest is
the recent discovery showing more responsiveness to GPx4
activity in CC subjects than TT ones after selenium supplementation, suggesting that a SNP in the GPx4 gene a T/C variation at
position 718 close to SECIS, has also functional consequences
(Meplan et al., 2008). The same paper (Meplan et al., 2008) also
reports a hierarchy on the effect of selenium supplementation on
GPxs expressed in the same 30 UTR coding region, in particular
GPx1 e GPx3, showing that CC subjects are also more responsiveness than TT ones to increase both GPx1 and GPx3 activity. Such a
nding is relevant because indicating the potential importance of
the SNP in the GPx4 gene especially when selenium intake is
suboptimal with subsequent defects also in GPx1 and GPx3 gene
40
expression and activity. Data from GPX-KO mice conrm the

relevance of glutathione peroxidases. From experiments in GPx1KO mice, the absence of GPx-1 causes the loss of protection of mice
from viral-induced myocardititis in comparison to wild-type mice
(Beck et al., 1998), suggesting that GPx-1 provides protection
against viral-induced damage.
Of particular interest is the gene encoding the plasma
selenoprotein P (SEPP) since this protein has a crucial role in
selenium transport and delivery of hepatic selenium to other
tissues (Schomburg et al., 2003). SePP is unique in containing
multiple selecysteine per molecule (up to 10 in the human SePP)
and it has two SECIS in its RNA (Hesketh, 2008). The central role of
SePP in the selenium transport makes likely that variants in the
SePP gene could have signicant functional consequences. In
particular, two SNPs in the SePP gene, one in the coding region 234
in position 24731 (SNP Ala234Thr) causing Ala to Thr change, and
the other one in the 30 UTR region in position 25191 (r25191) are
known (Meplan et al., 2007). In both SNPs, G-to-A variations are
present. The frequencies of the allele variations in position 24731
and 25191 are different in dependence of the ethnicity (Caucasian
vs Chinese groups) (Meplan et al., 2007). This fact is relevant
because it illustrates the importance of considering ethnicity in the
studies of nutrientgene interactions. Such a different frequency is
also evident after Se supplementation (SELGEN study) (Meplan
et al., 2007), in which subjects with different SNPs within the same
SePP gene display, in a gender-specic manner, different baseline
SePP levels, differences in GPx3 activity as well as in erythrocyte
thioredoxin reductase 1 concentrations and lymphocyte glutathione peroxidase 1 and 4 activities. These ndings demonstrate that
a number of SNPs in selenoprotein genes may have functional
differences between the allelic variants. Therefore, when assessing
the impact of the variants on selenium metabolism, it is important
to consider the known hierarchy and competition among
selenoproteins because changes in the synthesis of one selenoprotein may alter another selenoprotein (Hesketh, 2008; Hesketh
and Meplan, 2011). Therefore, the genetic variations of one SNP
may be magnied or counterbalanced by variants in other genes
with subsequent different modulation on selenium metabolism.
For example, an SNP in the SBP2 gene might affect selenium
incorporation but without consequences unless combined with an
SNP in the 30 UTR of the GPx4 gene (Hesketh, 2008). The effect of the
SNP in GPx1 might be magnied by an SNP in SePP gene that affect
the selenium delivery to target tissues (Hesketh, 2008). Another
example is the presence of two SNPs in the region of Sep 15 gene
that correspond to the 30 UTR of the mRNA (Hu et al., 2001). The
combination of these two variants, a C/T substitution at position
811 and a G/A at position 1125 inuences read-through at a UGA

codon (Hu et al., 2001). Here, the signicant differences in Sep15
allele frequencies is affected by ethnicity and the identity of the
nucleotides at the polymorphic sites inuences SECIS function in a
selenium-dependent manner, suggesting that the loss of heterozygosity at the Sep15 locus may be involved in cancer development. More recently, two genetic variants of SePP1 gene at position
31174 and 43881 when associated to genetic variants of another
selenoprotein thioredoxin reductase 1 (TXNRD1) at position 181
were strong predictors for advanced colorectal adenoma and
subsequent selenium dismetabolism (Peters et al., 2008).
Up to date, the research on nutrientgene interactions in
relation to selenium metabolism is quite limited to studies to
individual SNP in relation to measurements of physiological
parameters. A G/A SNP at position 105 in the promoter of
Selenoprotein S appears as functionally signicant. It modulates
the response to stressors and affects the markers of inammation,
such as TNF-a and IL-1b (Curran et al., 2005). The promoter region
of SePP1 gene is also cytokine responsive, suggesting a role of
SePP1 on the inammation (Dreher et al., 1997). Several other
lines of work indicate various polymorphisms of selenoproteins,
such as GPx1 and GPx4, with a functional signicance, in
particular in response to inammation and oxidative stress
(Seiler et al., 2008). Considering the selenium intake and the
genetic factors, the picture that is emerging is a complex one of
multiple, possibly interacting, functional SNPs, the inuence of
which may be modulated by the ethnicity, gender, life style and
dietary factors. Therefore, no data report the role played by SNPs
of selenoproteins in ageing up to date. By contrast, a substantial
number of papers report the effect of some SNPs of the
selenoproteins in age-related diseases (cancer and CVD). On this
topic, an exhaustive review of Ryan-Harshman and Aldoori (2005)
reports all the selenoproteins involved in cancer and CVD, in
which GPx1 and SepS1 are the more relevant SNPs for the risk of
cancer and CVD. The Gpx1 Leu 198 allele confers an increased risk
of bladder cancer. In particular, subjects carrying the Pro/Leu
genotype with respect to Pro/Pro genotype have a higher risk
(Ichimura et al., 2004), as also reported for lung cancer by a Finnish
study (Ratnasinghe et al., 2000). Another study reports a higher
Leu/Leu genotype frequency in breast cancer than Pro/Pro
genotype, while a lower heterozygosity index has been observed
in the breast cancer samples suggesting an allelic loss during the
tumor development. By contrast, A Danish study reports an
association with decreased risk for lung cancer in subjects
carrying Leu/Leu genotype with respect to Pro/Leu genotype
(Raaschou-Nielsen et al., 2007) (Table 5).
Table 5
Associations of selenoprotein polymorphisms with cancer and cardiovascular diseases.
Polymorphism
Disease
Findings
Ref.
GPx4T/C 718 genotype

SEPP1 4166C/G
SEPP1 loci (31,174 bp 30 of STP A/G
SEPP1 43,881 bp 30 of STP G/A
SEPP1 44,321 bp 30 of STP C/T
TXNRD1 IVS1-181
Gpx1 Leu 198 Pro
SEPS1 (rs8025174) A/C

SEPS1 (rs7178239) C/G
SEPS1 105G/A
Sep15 1125 G/A
Colorectal cancer
Colorectal adenoma
Colorectal adenoma
Colorectal adenoma
Colorectal adenoma
Colorectal adenoma
Bladder cancer risk
Lung cancer risk
Breast cancer
Cardiovascular disease (CAD)
Coronary heart disease (CHD)
Stroke
Gastric cancer
Lung cancer
Bermano et al. (2007)

Peters et al. (2008)
Ichimura et al. (2004)
Ratnasinghe et al. (2000)
Hu and Diamond (2003)
Tang et al. (2008)
Alanne et al. (2007)
Alanne et al. (2007)
Shibata et al. (2009)
Jablonska et al. (2008)
SEPP1-Ala234Thr
Prostate cancer
CC genotype increased cancer risk

CG genotype increased cancer risk
GG genotype increased adenoma risk
AA genotype increased adenoma risk
CT genotype reduced adenoma risk
CG + GG genotypes decreased cancer risk
Pro/Leu genotype increased bladder cancer risk
Pro/Leu genotype increased lung cancer risk
Leu/Leu genotype increased breast cancer risk
Pro/Pro genotype increased CAD risk
A allele increased CHD risk in females
G allele increased stroke risk in females
A allele increased cancer risk in males
GG or GA genotype increased lung cancer risk
in patients with low Se status
Ala/Ala genotype associated with SOD2-Ala16+
genotype increased prostate cancer risk
Cooper et al. (2008)
With regard to CVD, subjects with 198Pro/leu and 198Leu/leu

genotypes had a signicantly higher risk of CVD compared to the
198Pro/Pro carriers, suggesting that GPX1 198Pro/Leu variant
genotypes are signicantly associated with CVD risk (Tang et al.,
2008) and with thoracic aortic aneurysm in hypertensive patients
(Kato et al., 2008). The selenoprotein SEPS1 is also of particular
interest for CVD because is able to mediate the inammation
through the protection of the endoplasmic reticulum (ER) (Ross,
1999).
SEPS1 is an ER membrane protein that participates in the
processing and removal of misfolded proteins from the ER to the
cytosol, via NF-kB activation, which in turn activates the
transcription of the pro-inammatory genes (Curran et al.,
2005) and SEPS1 (Gao et al., 2006). Increased expression of SEPS1
suppresses the cytokine production by its ability to remove
misfolded proteins from the ER. This system constitutes a SEPS1dependent regulatory loop in the presence of inammation
(Curran et al., 2005). Variation in the SEPS1 gene affects the
circulating levels of the inammatory cytokines IL-1b, IL-6 and
TNF-a (Curran et al., 2005). Given the known association of SEPS1
with the inammation, the effect of the genetic polymorphisms in
SEPS1 on the risk of CVD was investigated in two independent
Finnish cohorts. A signicant association was found with an
increased risk for coronary heart disease (CHD) in females carrying
the minor allele of rs8025174. Another variant, rs7178239,
increased the risk for the ischemic stroke in females (Alanne
et al., 2007). These results implicate that the selenoprotein SEPS1 is
involved in the risk of CVD in female. With regard to cancer, taking
into account that the 105G > A polymorphism in the promoter of
SEPS1 increased the pro-inammatory cytokines (Gao et al., 2006),
SEPS1 is a key factor in the process of carcinogenesis from chronic
gastritis induced by Helicobacter pylori (El-Omar et al., 2001). The
105G > A promoter polymorphism of SEPS1 was associated with
the type of gastric cancer. In particular, old subjects (>65 years)
carrying the A allele display an increased risk of both gastric cancer
and inammatory state when compared with the GG genotype
(Shibata et al., 2009). Also other SNPs for selenoproteins, such as
Sep 15 and SEPP1, are involved in cancer risk. A casecontrol study
in a population of Polish smokers with low selenium state
(53.3 14.0 mg/L), showed an effect of Sep15 G/A1125 genotype
on lung cancer risk. Among individuals of lower selenium state
(below 50 mg/L), the risk was higher in individuals with AA genotype
as compared to those ones with GG genotype, suggesting that the
subjects carrying AA genotype might have a benet by increasing
dietary in selenium (Jablonska et al., 2008). Recently, it has been also
reported that homozygous for SEPP1 ala234 SNP allele in men had a
high risk for prostate cancer with a more aggressive form when this
SNP was associated with SOD2-Ala16+ allele (Cooper et al., 2008)
(Table 5)
Form all these data, it emerges a complex picture on the effect of
selenoproteins and of their relative SNPs on the health especially
because few intervention studies have been carried out to assess
the functionality of SNPs in vivo up to date. There is the need to
assess SNPs in human intervention studies using genotyped
cohorts and appropriate biomarkers in order to show the
relationship between selenium, SNPs and the biomarkers of the
healthy state. Trascriptomic approaches, as reported above, have
provided interesting data on the response of the selenoprotein
genes and on the role played by some SNPs for selenoproteins in
some degenerative diseases, but in general the data are limited by
the major regulation of selenoprotein synthesis at the level of
translation. Proteomic methods, in combination with inductively
coupled plasma spectroscopy to detect selenoprotein proles and
species, need to be developed.
However, taking into account that the selenoproteins are
mainly involved in the protection against oxidative damage and
41
inammation (Ryan-Harshman and Aldoori, 2005), many studies

report the role played by selenium deciency and selenium
supplementation in ageing and in age-related disease without
considering the SNPs of the selenoproteins, but exclusively based
on the selenium content in the plasma. Although this fact is a limit
on the effect of selenium supplementation on functional parameters related to the stress and inammation, it does not exclude
that these studies of the selenium supplementation may be
relevant to better understand the role played by selenium in
maintaining the healthy state.
3.3.1. Selenium supplementation in Ageing
Selenium deciency is a condition mainly attributed to low
selenium content in the soil or to long-term parenteral nutrition.
During ageing, selenium deciency may occur as a result of
intestinal malabsorption. However, few data are available
reporting a marked selenium deciency in old subjects (Seiler,
2001). Recently, a paper has explored the relationships between
plasma selenium and mortality in an elderly population for a long
period of observation (9 years): the EVA study (Arnaud et al.,
2007). The authors have observed that the mortality rates were
signicantly higher in individuals with low selenium [1.01 mmol/
L: a value below the cut-off considered as optimal (1.25
1.50 mmol/L)] (Arnaud et al., 2007). Considering the causes of
death, an association was found with low selenium and cancerrelated mortality. The same authors suggest that plasma selenium
could be an indicator of the longevity in a middle-aged,
independently living population not specically at risk for cancer
and CVD. The survival curves illustrate that the relationship
between plasma selenium and mortality remained constant
during the entire 9-year period of follow up (Akbaraly et al.,
2005). However, the mechanism of this potential relationship is
still under debate and further research is needed on the role
played by selenoproteins, especially at genetic level, on this
phenomenon. Some other authors demonstrate the selenium
deciency in elderly people in relation to hypothyroidism
(Olivieri et al., 1996). Interestingly, human healthy centenarians
display selenium values quite similar to the normal elderly
(Savarino et al., 2001). Since few trials have been carried out up to
date in elderly, it is difcult to report a specic benecial effect of
selenium in human ageing, even if the benecial effects of the
selenium supplementation on lymphocyte mitogen responsiveness have been reported in institutionalized old individuals
(Peretz et al., 1991). Moreover in a Finnish study, the addition of
selenium to fertilizer leads to an improved selenium state in the
general population (youngadult, old) (Aro et al., 1995). However,
the possible benecial on the health was not reported in this study
(Table 6A).
The major evidences on the benecial effects of selenium
supplementation relate to age-associated diseases, such as cancer,
cardiovascular diseases (CVD), diabetes and infections. On this
topic, various papers report the benecial effect of selenium
supplementation, but the benecial effect of selenium in se are
not conclusive. Different studies [SUVIMAX study (Reid et al.,
2006); SELECT study (Lippman et al., 2009)] have reported an effect
in preventing cancer in adult and old people with also contradictory results. No specic effect of selenium supplementation was
observed both in diabetes (Faure et al., 2004; Bleys et al., 2007;
Stranges et al., 2007; Yang et al., 2010) and CVD (Czernichow et al.,
2005; Nawrot et al., 2007). An intriguing point is the association
between selenium deciency, immune response and increased
incidence of viral infections in elderly (Ellis et al., 2003), suggesting
that the selenium deciency may be considered a relevant risk
factor for the appearance of infections by viruses. Such an
assumption is supported by experiments in animals, in which a
normally-benign strain of coxsackievirus B3 becomes virulent
42
Table 6
Selenium supplementation in ageing (A), age-related diseases (B) and selenium toxicosis (C).
Dose
Effect
A. Effect of selenium supplementation in ageing

Improved response of lymphocytes to mitogens
100 mg/day
6 mg kg-1 in soil
Improved selenium state
B. Effect of selenium supplementation in age-related diseases
Cancer prevention
200 mg/day
Cancer prevention
50 mg/day
Cancer prevention
100 mg/day + other nutrients
No prevention of prostate cancer
200 mg/day + vitamin E
No effect on risk of hypertension
100 mg/day + other nutrients
960 mg/day
NF-kappaB activity reduction and GPx activity
increase in erytrocytes from type 2 diabetic patients
200 mg/day
No prevention of type 2 diabetes risk
C. Selenium toxicosis
1 mg/day
5 mg/day
Thickened, fragile, honeycomb-like ngernails

Nail deformation, hair loss, skin lesion tooth decay,
neurological disorders
when inoculated in selenium-decient animals due to viral

genomic changes (Beck, 1999) (Table 6B).
With regard to selenium toxicosis, it is a rare event in humans
and very limited specic data exist in literature (Table 6C) The
toxicity of selenium, in the form of Seleniummethyl-selenocysteine, mainly occurs in animals eating plants containing from 100
to 10.000 ppm of selenium provoking a subacute selenosis called
blind staggers, characterized by anorexia, emaciation, alopecia,
hoof necrosis, neurologic deterioration-blindness, ataxia, disorientation and respiratory distress and nally also death (Combs and
Combs, 1986).
In conclusion, selenium is a relevant trace element for many
body homeostatic mechanisms but is it important to assess how
the individual genetic background for selenoproteins may affect
the interaction of SNPs with each other or with selenium intake,
ethnicity and gender. In such a way, a more complete picture on
the effect of selenium after supplementation or in the diet may be
given with subsequent benecial effects on the healthy state in
ageing.
4. Conclusion and future perspectives
Despite the pivotal role played by zinc, copper and selenium for
the inammatory/immune response and antioxidant activity in
ageing and in some age-related diseases, controversial ndings
exist on the real necessity of micronutrient supplementation in
deciency or micronutrient chelation in toxicosis. The contradictory results are mainly due to a great interindividual genetic
variability, to the interaction between environmental (dietary
habits) and genetic factors and to the unreliability of specic
biomarkers for testing zinc, copper and, selenium status in the
plasma. Moreover, the discrepancies may be more due to the fact
that these micronutrients affect many genes related to the
inammation and oxidative stress, which in turn affect the
intestinal absorption of the micronutrients. Alterations in the
absorption or dietary intake of zinc, copper and selenium may lead
to an incorrect inammatory/immune response, impaired antioxidant activity and unsatisfactory DNA repair to various external
noxae with the subsequent appearance of some age-related
degenerative diseases. Differently, a correct diet as well as
adequate polymorphisms are useful to reach healthy ageing and
longevity (Fig. 1). On the other hand, the relevance of the cytokine
production in maintaining a correct Th1/Th2 paradigm, with
subsequent satisfactory inammatory/immune response, has been
well documented in ageing in relation to the three micronutrients
Period
References
6 months
6 years
Peretz et al., 1991

Aro et al., 1995
7 years
5 years
8 years
712 years
56 years
3 months
Reid et al. (2002)

Blot et al. (1995)
SU.VI.MAX trial. Meyer et al. (2005)
SELECT study. Lippman et al. (2009)
SU.VI.MAX trial. Czernichow et al. (2005)
Faure et al. (2004)
7.7 years
Stranges et al. (2007)
2 years
10 years
Fan and Kizer (1990)
herein reported. Taking into account that pro-inammatory

cytokines also affect the absorption and the distribution of zinc,
copper and selenium within the body (Prasad, 2000), a vicious
circle arises with deleterious or limited benecial effects of the
micronutrients themselves on inammatory/immune response
and antioxidant activity (Mocchegiani et al., 2012a). In this
context, polymorphisms of pro-inammatory cytokines and MT
are very useful tools to screen old subjects who effectively need
micronutrient supplementations with subsequent benecial effect
upon the inammatory/immune response and antioxidant activity
(Fig. 1). Indeed, subjects can be screened for zinc supplementation/
chelation on the basis of IL-6 and MT polymorphisms and for
copper supplementation/chelation on the basis of SOD1 and SOD2
polymorphisms regardless to the nutritional state and inammatory condition. Such a genetic screening is very useful both in
ageing and in age-related diseases taking into account the
existence of a strictly relationship between copper and zinc and
by the recent discovery showing Cu/Zn ratio as a predictor of
disability and mortality in ageing (Malavolta et al., 2010;
Mocchegiani et al., 2012b). With regard to selenium, specic
selenoprotein polymorphisms, such as SEP-15, SEPP1, SEPS1, can
be very useful to screen subjects to prevent cancer. However,
selenium is important in conferring activity to GPx, which is
fundamental for the zinc release from MT via glutathione oxidation
(Khatai et al., 2004), suggesting also the existence of a close link
between zinc and selenium bioavailability. Such a link is pivotal for
the antioxidant activity of many zinc-dependent enzymes and for
the activity of the nuclear enzyme PARP-1. During ageing, PARP-1mediated poly(ADP-ribosyl)ation and GPx decrease. The decreased
PARP-1 activity might be in part the result of a low zinc ion
bioavailability due to a limited zinc release by the S-glutathionylation of MT (Casadei et al., 2008). As a consequence, PARP-1 activity
is altered and may fail in the activation of DNA-repair mechanisms
causing cell death. Therefore, the combination of polymorphisms
of MT and selenoprotenis cited above may be useful tools to
effectively screen old subjects for a correct selenium supplementation in order to prevent some degenerative diseases, including
cancer. However, such a screening may not be sufcient because it
is necessary also to evaluate the zinc, selenium and copper status
of old individuals. In this context, hyphenated techniques based on
coupling chromatographic separation techniques, mainly HPLC,
with ICP-MS detection are now established as the most realistic
and potent analytical tools available for the speciation analysis of
the essential trace elements (Sanz-Medel and Montes-Bayon,
2003). This novel technology has been recently developed in our
[(Fig._1)TD$IG]
43
Fig. 1. Schematic representation of the possible benecial interactions between dietary micronutrients (Cu, Se, Zn) intake and the related genes in inducing a satisfactory
cellular homeostasis, genomic stability and DNA repair in ageing. The inammatory status (inammaging) determined by an inbalance among anti-inammatory, proinammatory cytokines and chemokines plays a key role affecting both genomic stability and the absorption/transport of micronutrients. A correct balance of the
interrelationship micronutrient-gene-inammation leads to healthy status and longevity.
INRCA laboratory and it is very useful to test precisely the quota of

the trace element bound to its specic protein, such as albumin for
zinc, Cp for copper, selenoprotein-P for selenium with an error of
less 10% (Malavolta et al., 2012). Such determinations were useful
at clinical level to better characterize some pathologies (El Balkhi
et al., 2010; Vinceti et al., 2013).
In conclusion, the study of nutrigenetic and nutrigenomic
approaches can be therefore pivotal for the best use of nutrients as
supplements or chelators in clinical practice during ageing and in
age-related diseases. However, further genetic-nutritional studies
are required to clearly dene the impact of these micro-nutrients
in promoting healthy ageing and longevity. Surely, the micronutrientgene interactions and the metal speciation analysis are
fundamental for personalized nutritional interventions aimed at
preventing many age-related inammatory degenerative diseases,
such as cancer, CVD and type II diabetes.
Conict of interest
No conict of interest by the authors, who have agreed to
submit and to eventually publish the manuscript in Mechanisms of
Ageing and Development.
Acknowledgments
Supported by INRCA; European Zincage project (FP6 n. FOOD2004-506850); European Markage project (FP7 n. HEALTH-F42008-200880); Italian Health Ministry (R.F. 154/GR n. 20091584108 My Mind). We thank Dr. Aurelia Santoro, Scientic
Manager of NU-age project, for useful suggestions and criticisms.
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Mechanisms of Ageing and Development 136-137 (2014) 2949

Contents lists available at ScienceDirect

Mechanisms of Ageing and Development

Micronutrientgene interactions related to inammatory/immune

* Corresponding author at: Scientic and Technological Pole INRCA-IRCCS,

As a result, the remodelling theory of ageing has been proposed

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

Some authors have reported that the deciency of micronutrients

dietary intake sufcient to meet the requirement of 97% of healthy

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

as a second messenger, modulates the signaling cascades that

to low intracellular zinc bioavailability and subsequent reduced

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

particular of neutrophil and monocyte functions even if its role is

Hazegh-Azam, 1996). Furthermore, excessive copper directly

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

cytotoxicity was enhanced after 3 months of Se-supplementation

superfamily and for nuclear transcription factor activation (Klug,

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

subgroups and various isoforms, among which MT-I and MT-II

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

Antioxidant plasma enzymes

T-cell receptor excision circles (TRECs)

Plasma cytokines and chemokines

IL-6, IL-8, MIP-1a

Activated T cells (CD3 + CD25+)

(Cuajungco et al., 2000), an excess of zinc is toxic in the brain with

subjects or patients to be treated effectively with zinc chelators. In

TNFa, IL-13, IL-4

One i.p. injection (10 mg/Kg)

Fukuyama et al. (2011)

1050 mM for 30 min

Haase et al. (2008)

375 mg/day for 25 weeks

Ritchie et al. (2003)

", increase; #, decrease.

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

Gybina, 2004). However, excessive copper is toxic or even lethal

dietary copper is fundamental for the copperiron cross linking in

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

taking into account that a copper deciency affects some aspects of

copper in ageing, the budding yeast Saccharomyces cerevisiae was

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

A. Effect of copper supplementation in copper deciency

Menkes disease (human) (Kreuder et al., 1993)

S.c. injections of Cu-histidine

Macular degeneration (human) (AREDS Study, 2001)

80 mg zinc oxide + 2 mg of cupric

Apoptosis of retinal pigment epithelium

Alzheimer (animal model) (Bayer et al., 2003)

25 g/L of CuSO4 in H2O for 3 months

B. Effect of copper chelation in copper overload

Type II diabetes (human) (Cooper et al., 2004)

Trientine (1.2 g/day/6 months)

Left ventricular hypertrophy (LVH)

Alzheimer (human) (Squitti et al., 2002)

Penicillamine (600 mg/day for 24 weeks)

Alzheimer (animal model) (Cherny et al., 2001)

Clioquinol (oral treatment of

Osteogenesis in postmenopausal women (human)

", increase; #, decrease.

and facilitate copper transport between RPE and retina (Krajacic

E. Mocchegiani et al. / Mechanisms of Ageing and Development 136-137 (2014) 2949

Reduction of hydrogen peroxide