C H A P T E R
20
Effects of Metallic Elements on Reproduction
and Development
PIETRO APOSTOLI AND SIMONA CATALANI
ABSTRACT
Many factors can influence the reproductive or
developmental outcomes of human exposure to metal-
lic elements, including metallic element speciation,
dose, timing, route and duration of exposure, dose-
response relationship, bioavailability, and the distribu-
tion and accumulation of metallic elements in various
organs.
Experimental and epidemiological studies have
reported the adverse effects of many metallic elements
on male and female reproductive function, for exam-
ple infertility or subfertility, malformations, abortion,
and developmental alterations.
Different mechanisms of action have been sug-
gested: increased reactive oxygen species or decreased
activity of the cellular antioxidant defense system;
direct damage on the testes or ovaries; interaction with
the embryonic structure; disruption of reproductive
hormones; and, finally, immune suppression and neu-
rotransmitter alterations.
The targets and effects can be different and specific
to each metallic element (e.g. neurotoxicity of Pb and
methylmercury in the developing brain or the action of
Cd on the testicle) or similar for large groups of metal-
lic elements (reduced sperm motility caused by As, Cr,
Hg, Ni, and Pb).
Knowledge of these mechanisms may be useful to
occupational physicians by providing a better under-
standing of the dose-response relationship, as well
as the choice of current indicators of dose and their
Handbook on the Toxicology of Metals 4E
http://dx.doi.org/10.1016/B978-0-444-59453-2.00020-2
effects. Use of the diagnostic approach in occupational
medicine is necessary to achieve adequate evidence
for a causal relationship between exposure and clinical
findings.
1 INTRODUCTION
The reproductive and developmental toxicity asso-
ciated with metallic elements covers a wide range of
issues, including the number and sources of the metal-
lic elements involved; the difficulty in attributing the
origin of reproductive impairment to males, females,
or both; and the different degree and quality of knowl-
edge regarding pathogenic mechanisms.
The range of these effects in males includes infertil-
ity, subfertility, and reduced sperm quantity and qual-
ity. For females, they include various birth defects such
as intrauterine growth retardation, premature aging,
low birth weight, malformation, postnatal death, abor-
tion, developmental deficits of mental or psychomotor
function, and behavioral effects.
Historically, reproductive toxicity has been demon-
strated for metallic elements of widespread occupa-
tional and environmental interest, such as lead (Pb),
mercury (Hg), arsenic (As), and cadmium (Cd). Other
metallic elements, such as chromium (Cr), cobalt (Co),
selenium (Se), copper (Cu), and zinc (Zn) have been
compared to a double-edged sword in that they are
essential on the one hand and toxic on the other. Fur-
thermore, there is now concern about other metallic
399 Copyright © 2015 Elsevier B.V. All rights reserved.
400 Pietro Apostoli and Simona Catalani
elements [antimony (Sb), vanadium (V), platinum (Pt),
lithium (Li), and uranium (U)] due to their environ-
mental dispersal.
In order to make a critical evaluation, it is also
important to point out the limitations of the studies
into the toxic effects of metallic elements on repro-
duction. The majority of published studies consider
only single metallic element exposures, whereas
environmental and occupational conditions are usu-
ally characterized by combined exposures to several
toxic and essential metals, especially through food.
Evaluation of the possible additive, synergistic,
or antagonistic effects appears to be a key consid-
eration not only from a theoretical but also from a
practical point of view; however, instruments and
methods used for their characterization are still
unsatisfactory.
Other critical points may arise from the complex-
ity of the effects under study and from gender dif-
ferences. For example, clinical and epidemiological
findings on female reproductive function may be
influenced by age, ovarian reserves, hormonal imbal-
ance, male cofactors, and sexually transmitted dis-
eases. Thus, environmental or occupational factors
interact with a wide variety of complex, multiple-
phase processes and it may be difficult to distinguish
occupational or environmental risks from other risk
factors. Finally, we must also consider the possibil-
ity that extrapolating the results from animal stud-
ies to humans is not feasible owing to structural
and functional differences between species, and the
mechanisms of adverse effects are seldom known.
For example, it appears to be difficult to transfer
experimental fetal anomalies, embryo or fetal lethal-
ity, or other developmental effects during organogen-
esis after exposure to metallic elements ions during
periods of pregnancy or development to effects on
humans (Windham and Fenster, 2008). Teratogenetic
bioassays in rodents have yielded positive results for
many metallic elements that produce fetal and early
postnatal deaths, as well as malformations such as
anencephaly, eye defects, cleft palate, and skeletal
anomalies (Wells et al., 2005), but these have not been
confirmed in humans. However, an experimental
approach is essential, especially for investigating the
mechanism of action through which each metallic ele-
ments acts.
In this new edition, we modified the presentation
approach of this chapter by moving from the meth-
odology of dose assessment (see Apostoli et al., 2007)
to the mechanisms of action, hypothesized from
epidemiological and experimental studies on the
various phases of reproductive and developmental
toxicity.
2  HOW TO MEASURE DOSE AND EFFECT
Both the analytical methods adopted, as well as
their quality and accessibility, are crucial for improv-
ing our knowledge of assessing the dose and effect
relationships of metallic elements on reproduction and
development in humans.
Assessment of the occupational or environmental
exposure dose to metallic elements is generally car-
ried out using environmental or biological monitoring
procedures (see Chapter 8, “Biological Monitoring and
Biomarkers”).
Using Pb as an example, one can assess the internal
dose by determining the Pb concentration in the blood
(PbB), although this mainly reflects Pb uptake due to
recent exposure. In fact, there are other indicators that
may provide better information regarding Pb expo-
sure: the adjusted PbB (i.e. PbB × years of exposure),
erythrocyte zinc protoporphyrin (which reflects lon-
ger exposure periods), and indicators of body burden
(such as Pb in the bone or urinary Pb after chelation);
however, these methods are not applicable to large-
scale studies.
The metal concentration in the relevant and criti-
cal tissue would be an ideal indicator of dose; in our
case, Pb concentration in seminal fluid, as a whole or
in its fractions, theoretically correlates with testicular
Pb, and therefore with histopathological alterations.
Bonde et al. (2002) demonstrated that Pb in blood, sem-
inal plasma, and spermatozoa were highly correlated,
although no association between the measurement of
long-term exposure and semen characteristics has been
found (Figure 1). The study showed, however, that Pb
tends to accumulate in spermatozoa and it correlates
with semen volume and sperm density.
Hernandez-Ochoa et al. (2005) showed that
decreases in sperm quality; sperm concentration,
motility, morphology, and viability were associated
negatively with Pb in spermatozoa, whereas semen
volume was negatively associated with Pb in seminal
fluid.
Other problems are exposure to multiple metallic
elements and interaction between toxic and essential
elements. Their concomitant measurement and mak-
ing adjustments for their relative amounts might be
important for understanding interactions relevant to
toxicokinetics and toxicodynamics, and the mecha-
nisms of toxic action on reproduction.
Some studies have attempted an overall evalua-
tion of the relationship between several metallic ele-
ments (Pb, Cd, Cu, Se, and Zn) and the morphology,
number, and motility of spermatozoa (Telisman et al.,
2000, 2007). Apostoli et al. (2000) examined subjects
not occupationally exposed to metallic elements and
 20  Effects of Metallic Elements on Reproduction and Development
FIGURE 1  Scatter plots of lead concentrations in seminal plasma
and spermatozoa (A), spermatozoa and blood (B), and seminal plas-
ma and blood (C) in a subset of 165 industrial workers. All correla-
tions are highly significant. (Note 10 μg/dL = 100 μg/L.) Reproduced
with the permission of Occupational & Environmental Medicine (see
Bonde et al., 2002).
workers exposed to Pb, using inductively coupled
plasma mass spectrometry (ICP-MS). Lead and barium
(Ba) were found in higher amounts in the sperm cells
of Pb-exposed workers, whereas Se, Zn, and calcium
(Ca) were present in significantly lower concentra-
tions, compared to the controls. The Pb and Cd levels
401
were significantly higher than in the seminal plasma
of controls than in Pb-exposed workers, whereas Se
was significantly lower. These results highlight the
potential of this approach, since the determination of
multiple elements may improve our knowledge of the
distribution of metallic elements in mixture exposure
situations.
To obtain better information, an evaluation of expo-
sure to metallic elements should also be made as close
as possible to the “target time.” For example, this may
be a few days in the case of an effect via seminal plasma
mediated by the prostate or seminal vesicles, or up to
12 weeks if spermatogenesis is affected, via the testis.
An ideal risk assessment may be reached when
information from the indicators of exposure is com-
bined with information from indicators of effect. In
this specific case, possible dose indicators include the
environmental or biological concentrations of metallic
elements, and effect indicators range from a synthetic
evaluation of the specific effect to measurements of fer-
tility or neurodevelopmental effects (for example, the
neurobehavioral test), and to the determination of hor-
mones in the blood or urine.
In addition, semen parameters can be used to indi-
cate a direct toxic effect on the male reproductive sys-
tem or to predict adverse effects on fertility or offspring
health.
To date, the semen quality outcomes utilized most
often in investigations of the effects of metallic ele-
ments on male reproduction have been sperm count,
motility, and morphology. Used together, these param-
eters provide a good general assessment of male fer-
tility and reproductive health status. Other techniques
for measuring the characteristics of sperm that predict
reproductive health are simultaneous DNA integrity
assays, which detect DNA strand breakage in single
sperm cells, fluorescence in situ hybridization (sperm-
FISH), which detects abnormalities in chromosome
number, and the sperm chromatin structure assay
(SCSA). Examples of the use of sperm DNA chroma-
tin assays to specifically identify associations between
metallic elements and sperm health include correla-
tions between seminal fluid Zn and SCSAs (Björndahl
and Kvist, 2011), and correlations between seminal con-
centrations of Cd, Pb, and malondialdehydes (MDAs)
as a marker of oxidative stress in sperm (Pandya et al.,
2012).
In an international study of semen quality involv-
ing 503 industrial workers (Project Asclepios), the
effects of Pb exposure were evaluated through semen
studies, semen sampling, and initial processing (vol-
ume, motility, and velocity); semen analysis (morphol-
ogy, computer-aided sperm analysis, SCSA, FISH,
and germ cell genotoxicity); and finally through an
402 Pietro Apostoli and Simona Catalani
exposure assessment. The study showed that current
occupational exposure levels in the United Kingdom,
Belgium, and Italy did not represent a high risk to
male fertility. Adverse effects on sperm concentration,
for example, are unlikely at blood Pb (PbB) concentra-
tions below 450 μg/L (Bonde, 2002). On the other hand,
a decrease in the sperm concentration and sperm count
of 149 Pb-exposed workers was found at PbB concen-
trations below 400 μg/L in studies by other authors
(Telisman et al., 2000).
The most commonly applied nonbiological mark-
ers are time to pregnancy (TTP) or the fecundability
odds ratio (fOR), which compares numbers of progeny
among groups and the number of pregnancy losses.
TTP is the number of months or menstrual cycles that
it takes a couple to conceive and covers the entire dis-
tribution of waiting times from 0 (high-level fecundity)
to several years (low-level fecundity) (Joffe, 1989). The
fOR is the odds of conception in a single menstrual
cycle (fOR = 1/TPP). A fOR of less than one indicates
that the exposed group has a lower fOR, or “sub”
fecundability, than the comparison group.
Fertility studies should not be mistaken for simple
survey studies since they represent an approach that is
exceptionally susceptible to bias. It has become clear
that, to address the various types of bias, TTP studies
need to include information on at least three related
conditions: the TTP distribution, unprotected inter-
course not leading to pregnancy, and accidental preg-
nancies (Bonde et al., 2006).
In addition to selective forces, causal inferences in
TTP studies may be confounded by volitional factors
related to sexual behavior. In occupational studies con-
ducted in groups that are homogeneous with respect
to socioeconomic and cultural factors, volitional fac-
tors such as reproductive behavior, use of contracep-
tion, and persistence of trying may not represent a
severe problem. However, these volitional factors are
certainly relevant to comparisons over time and to
comparisons between different regions and cultures.
Finally, spontaneous abortion has been evaluated in
a number of studies involving metallic elements and
reproduction health. Failure to recognize or consider
very early fetal or even embryonic deaths in the analy-
sis may lead to a failure to detect important exposure
effects. In particular, chromosomal abnormalities are
estimated to occur in 30-40% of spontaneous abor-
tions; therefore, this outcome could be informative in
the investigations of metallic elements that interfere
with the cell cycle, mitosis, or meiosis (Gerhard et al.,
1998). There are published studies (Tabacova and Bala-
baeva, 1993; Borja-Aburto et al., 1999; Bellinger, 2005)
indicating that a history of spontaneous abortion is
related to the plasma:whole PbB ratio. This could be
due to Pb having a greater ability to cross the placental
barrier for a given PbB concentration in some women.
Assessing the influence of genetic polymorphisms of
Pb-binding proteins on the probability of suffering
from miscarriage or other reproductive outcomes will
be very important for identifying groups particularly
susceptible to the effects of Pb exposure during preg-
nancy (Lamadrid-Figueroa et al., 2007).
3  GENDER DIFFERENCES
Gender differences in toxicology begin at the gam-
ete and embryo stages. These differences may be
under genetic and hormonal control, and affect xeno-
biotic exposures, metabolism, susceptibility, risk, and
health through development and maturation. The
prominent role of testosterone and dihydrotestos-
terone in male sex differentiation results in a vulner-
ability to chemicals that can influence their synthesis
and action, such as androgen receptor antagonists
and androgen synthesis inhibitors. As for prenatal sex
differentiation, the process of puberty is also suscep-
tible to environmental factors that alter the levels of
important factors or modify their actions on target tis-
sues. Once adulthood is reached, further differences
in susceptibility may occur as a result of differences in
the hypothalamic-pituitary-gonadal control of game-
togenesis. In the male, the relatively tonic secretion of
follicle-stimulating hormone and luteinizing hormone
from the pituitary gland maintains a constant produc-
tion of sperm, whereas in the female intricate feedback
loops between the central nervous system (CNS) and
the ovary create a cyclical pattern of egg development
(Vahter et al., 2007a,b).
Finally, lifestyle factors may affect toxicity differ-
ently in men and women. These may include exposure
situations in the working or general environment; life-
style factors such as smoking, dietary factors, physical
activity, physical constitution (size, fat), cosmetics, and
fashion; and stress factors.
With respect to the mechanisms of action involved,
there are reported to be gender-related differences in
the activity of antioxidant defenses. There is substantial
evidence indicating that sexual hormones can modify
cell proliferation, neuroplasticity, and vulnerability to
neural insults, including oxidative stress.
Testosterone and other androgens may exert bio-
logical effects on neural cells, such as neuroprotective
effects against specific oxidative insults (Hammond
et al., 2001; Nguyen et al., 2005). Additional protection
against oxidative stress is provided by estrogen, which
has antioxidant effects by acting as a scavenger or by
inducing the synthesis of protective molecules via
 20  Effects of Metallic Elements on Reproduction and Development
activation of estrogen receptors (ERs) (Olivieri et al.,
2002; Kluxen et al., 2012). These hormones play a criti-
cal role in the gender differences reported in the patho-
physiology and outcome of acute neurological injuries
where oxidative stress is clearly involved.
There are also some differences between fetus gen-
ders in pregnancy outcome and developmental altera-
tions. Epidemiological studies of human infants and
children, as well as experimental animal studies, have
reported more developmental defects in males than
in females exposed to methylmercury (methyl-Hg)
(Malagutti et al., 2009; Grandjean et al., 2010; Robinson
et al., 2011). Results stratified by sex suggest that pre-
natal Hg exposure may have a greater adverse effect
on psychomotor development in females than in males
(Llop et al., 2012). Kippler and coworkers (Kippler et al.,
2012) found evidence of sex differences in the associa-
tion between maternal Cd exposure and birth size: the
association was apparent only in female offspring. The
authors hypothesized that Cd might affect sex-specific
programming, which begins very early in pregnancy
and may contribute to the vulnerability of the develop-
ing fetus to maternal stressors (Mueller and Bale, 2008).
In a follow-up of the prospective Cincinnati Pb study,
the analysis of childhood Pb exposure and neuropsy-
chological effects in adolescence indicated a height-
ened risk in males (Ris et al., 2004). This finding was
confirmed by Jedrychowski et al. (2009), who assessed
the relationship between the very low levels of prena-
tal Pb exposure measured in cord blood (< 50 μg/L)
and possible gender-specific cognitive deficits during
the first 3 years of life. Gender-based differences in
immunotoxicity induced by Pb have been observed in
both juvenile chickens and adult rats following low-
level exposure during embryonic development (Bunn
et al., 2001). In utero exposure to Pb resulted in more
severe immunotoxicity in females than males.
Metabolism also plays a key role in the detoxifica-
tion and activation of xenobiotics, and a gender dif-
ference in the methylation of inorganic As has been
observed, with a higher rate in females compared to
males (Huda and Vahter, 2011; Pilsner et al., 2012).
4  MECHANISM OF ACTION IN MALE AND
FEMALE GERMINAL CELLS
The male and female reproductive systems have
sensitive windows for toxic exposure during develop-
ment and throughout adult life. Metallic elements can
have detrimental effects on spermatogenesis and ovar-
ian function and at several levels.
Exposure in utero or during childhood and sub-
sequent development may lead to later reproductive
403
failure. Exposure in adults may have a direct effect
on the germinal cells and, subsequently, on hormones
and sexual performance. In addition, exposure of
adult males may affect offspring by exposing the
mother and the fetus to contaminated semen or by the
transmission of abnormal sperm DNA/chromatin to
the progeny.
4.1  Direct Mechanism of Spermatogenesis
Alterations
Spermatogenesis occurs in one of the most rapidly
proliferating tissues in the body. Damage caused by
exposure to some metallic elements early in the sper-
matogenic cycle (i.e. during spermatogenic cell dif-
ferentiation) may cause long-term defects in semen
quality, whereas damage during spermiation or to
sperm present in the epididymis has more transient
effects.
The main effects on spermatic functionality are: (1)
a decrease in semen volume/concentration/count; (2)
a decrease in sperm motility or quality of motility; (3)
an increase in abnormal sperm morphology, particu-
larly in the head of the sperm; and (4) impairment
of the secretory function of the prostate and vesicula
seminalis. The main results of relevant studies are
shown in Table 1. However, some groups have found
no influence of Pb, Cd, or Hg on sperm (Noack-Füller
et al., 1993; Kasperczyk et al., 2002; Bonde et al., 2002;
Mocevic et al., 2013).
Positive effects on the motility, morphology, and
concentration of spermatozoa have been reported
for Zn, Mg, Ca, and Ni. Zn deficiency is also associ-
ated with disturbances in reproductive function. Zinc
is present in both spermatozoa and seminal plasma,
where its concentration is considerably higher than in
the other body fluids. Zinc also contributes to the form
of sperm motility (Kaludin et al., 1983; Wong et al.,
2001). In addition, higher percentages of motile sper-
matozoa have been reported in semen with a higher
concentration of Mg in the seminal plasma (Wong
et al., 2001). Calcium is required for many physiologi-
cal processes that regulate all living cells, including
spermatozoa. Although the influence of Ca on sperm
motility has not been completely elucidated, the addi-
tion of Ca2+ has been observed to stimulate flagellar
beating (Suarez et al., 1993).
Hypothesized direct mechanisms of action through
which metallic elements exert these effects include (1)
an increase in reactive oxygen species (ROS) and a
decrease in activity of the cell antioxidant defense sys-
tem; (2) direct damage to seminiferous tubules or the
testis; (3) alterations in DNA-protamine binding; and
(4) interference with the sperm acrosome reaction.
404 Pietro Apostoli and Simona Catalani

TABLE 1  The Main Effects of the Metallic Elements on Spermatogenesis

In vitro/animal/
Metal Effects on sperm production human Reference(s)

As Low sperm motility Human Wirth and Mijal, 2010

As Decrease in sperm production Human Xu et al., 2012
Cd Immature forms of spermatozoa Human Chia et al., 1992
Cd Decrease in testis size Human Jurasović et al., 2004
Cd Negative effect on spermatozoa quality, especially on sperm morphology Human Massányi et al., 2004
Cd Correlation with pathological sperm alteration (large heads) Human Slivkova et al., 2009
Cr(VI) Decreased sperm count and percentage of motile sperm Human Li et al., 2001
Cr Increased percentage of morphologically abnormal spermatozoa Human Kumar et al., 2005
Cr Reduction of sperm count and sperm forward motility Monkey Subramanian et al., 2006
Cu Toxic effect on the seminiferous epithelium Human Skandhan, 1992
Cu Sperm copper was positively associated with sperm DNA fragmentation Human Schmid et al., 2013
Hg Low sperm motility Human Ernst and Lauritsen, 1991
Hg Decreased sperm motility and sperm count Rat Fossato da Silva et al., 2011
Ni Morphologically abnormal sperm, decreases in sperm count and motility, Human Massányi et al., 2004
destroyed the regular testicular structure
Ni Separated flagellum of spermatozoa Bull, fox Zemanová et al., 2007
Pb Low semen volume, increased sperm chromatin condensate, decreased sperm Human Hernandez-Ochoa et al., 2005
concentration and motility
Pb Negative impact on the motility and viability of spermatozoa and sperm count Human Eibensteiner et al., 2005
Pb Increased incidence of teratozoospermia, diminution of sperm cell production, Human Naha et al., 2006
low sperm vitality
Pb Increased immature sperm concentration and percentage of pathological sperm, Human Telisman et al., 2007
wide sperm and short sperm
Pb Decreased sperm motility, no effect in sperm viability Mice Oliveira et al., 2009
V Decreased sperm counts, damage to the seminiferous epithelium Rabbit Castellini et al., 2009
V Spermatogenic arrest, increased percentage of abnormal sperm Rat Chandra et al., 2010

Testicular apoptosis
↑Increased Leucocytes (extrinsic ROS) DNA fragmentation
FIGURE 2  Metallic elements as a source of
↑Spermatozoa stress response (intrinsic ROS) ROS and toxicity in semen.  Under normal condi-
Defective tions, reactive oxygen species (ROS) are produced
Metals by spermatozoa and leucocytes, and antagonized
Protaminosis
by circulating antioxidants. In the presence of exog-
↓Circulating Antioxidants Low sperm count enous factors, such as metallic elements, ROS pro-
duction is increased and antioxidant capacity lim-
Structural distortions ited, leading to oxidative stress and sperm damage.

4.1.1  Reactive Oxygen Species as Part of Infertility

Assessments
There is much evidence for the presence of ROS in
both normal sperm and sperm samples from infertile
men (Makker et al., 2009), although excessive ROS pro-
duction is consistent with inflammation and, therefore,
with a potential negative impact on sperm function. It
is established that testicular oxidative stress is com-
monly induced under different normal and/or patho-
physiological conditions, leading to male infertility
(Figure 2).
The effect of metallic elements on abnormalities in
sperm morphology and motility, and in altering viabil-
ity may be attributed to an increased semen concentra-
tion of ROS, especially of hydrogen peroxide (H2O2).
Lipid-rich membranes of the testis, together with the
large number of mitochondria and microsomes, are
presumed to render the organ more vulnerable to
oxidative stress (Lavranos et al., 2012). Spermatogo-
nial cells with gross DNA damage may be eliminated
naturally, while cells with more subtle defects may
survive and are finally manifested as abnormal sperm
(Du Plessis et al., 2010). Therefore, during spermato-
genesis, metal-induced ROS could damage specific
genes or processes that influence the sperm structure.
In addition, metal-induced ROS may inhibit the activ-
ity of DNA polymerase, an important enzyme in DNA
repair mechanisms (Angelopoulou et al., 2009).
Excessive ROS levels have been linked to lipid per-
oxidation of the sperm plasma membrane, resulting
in a loss of membrane fluidity, structure, and function
(Mahfouz et al., 2010).
 20  Effects of Metallic Elements on Reproduction and Development
However, cells have devised a variety of mecha-
nisms to protect themselves from the potential dam-
aging effects of ROS accumulation. Defense systems
include enzymes, such as peroxidases, catalases,
and superoxide dismutases, as well as small mole-
cules, such as glutathione (GSH) and some vitamins
(­Lavranos et al., 2012). A reduction in catalase activity
may reflect a decreased capacity of the testicular mito-
chondria and microsomes to eliminate the H2O2 pro-
duced in response to Cr, Cd, Cu, and V (Chandra et al.,
2007; Wu et al., 2008). The balance of these enzyme
systems may be essential to testicular health; hence, a
significant reduction in enzyme activity, accompanied
by a marked increase in H2O2 and lipid peroxidation,
may reflect the adverse effects of metal-induced ROS
on the antioxidant system.
Oxidative stress and the generation of ROS can also
be a consequence of arsenic exposure. ROS generation,
as well as the binding of As to protein thiol groups,
can alter the function of many proteins. In rats, cotreat-
ment with vitamin E provided significant protection
from the harmful effects of sodium arsenite on sperm
number, motility, viability, and morphology (Das et al.,
2009).
Although the testes express several antioxidant
enzymes, such as superoxide dismutase, catalase, and
glutathione peroxidase (GPx), to counteract oxida-
tive stress, their levels are greatly diminished upon
Cd exposure in rats (Pandya et al., 2012). Studies of
substances with antioxidant activity, such as vitamin
C, vitamin E, Zn, Se, and melatonin, have also dem-
onstrated that oxidative stress is associated with Cd-
induced testicular damage, since these substances
reduced and/or prevented both the oxidative stress
and damage caused by Cd (Acharya et al., 2008; Amara
et al., 2008; Burukoğlu and Bayçu, 2008).
Chronic Cr (as potassium dichromate) exposure
induces reversible oxidative stress in the seminal
plasma and sperm of monkeys by creating an imbal-
ance between ROS and the antioxidant systems, lead-
ing to sperm death and reduced motility of live sperm
(Subramanian et al., 2006). The significant changes
seen in exposed groups relative to controls suggests
that CrO3 exposure suppresses antioxidant enzymes
and ascorbic acid levels, with a concomitant increase in
the level of lipid peroxidation and H2O2, to adversely
affect testicular function (Acharya et al., 2006).
Treatment with Hg was also associated with oxi-
dative stress. Evidence of the induction of oxidative
stress was shown by interference with antioxidant
defense and a significant dose-dependent increase in
testicular lipid peroxidation as a consequence of pro-
oxidant exposure. An increase in free radical formation
associated with loss of the antioxidant defense system
405
after HgCl2 exposure may render the testes more sus-
ceptible to oxidative damage, leading to their func-
tional inactivation (Boujbiha et al., 2011). In the case of
Pb, chronic exposure results in increased lipid peroxi-
dation. Structural abnormalities in spermatozoa were
also significantly limited in antioxidant-enriched ani-
mals, thus supporting an oxidation-based mechanism
(Marchlewicz et al., 2004). However, the effects are
dose dependent: acute exposure increases the activ-
ity of antioxidant defense enzymes, unlike chronic
exposure.
Finally, V-induced alterations in the male repro-
ductive systems of humans and animals may involve
oxidative stress by inducing the oxidative deteriora-
tion of testicular functions. In vitro and in vivo stud-
ies demonstrate that V treatment result in a significant
dose- and time-dependent increase in testicular lipid
peroxidation, a marked inhibition in the level of super-
oxide dismutase and catalase activity, and decreased
sperm counts (Aragon et al., 2005; ­Castellini et al.,
2009).
4.1.2  Direct Toxicity to the Seminiferous Tubules or
Testis
The testes are extremely sensitive to external insults,
including the action of some metallic elements; one
possible explanation for this sensitivity is the unique
morphological structure of the blood-testis barrier
(BTB) (Siu et al., 2009).
Ultrastructural analyses of experimental studies in
rabbits have revealed three types of damage to sperm
head membranes, depending on the metallic element
used: acrosome breakage, with the formation of vari-
ous sizes of microvesicles (As, Cd, Hg, and Pt); a large
round hole (As, Cd, and Cr); and numerous folds in
the acrosome membrane (V) (Castellini et al., 2009).
The best-studied metallic element is Cd, and some
of its mechanisms of action have been elucidated.
In initial studies, morphological analysis showed
that Cd induces profound and irreversible injury to
mammalian testes including disruption of endothe-
lial cells of the microvessels, edema, and hemorrhage
using, apparently as the result of primary disruption
of the vascular system. For instance, successive stud-
ies in vitro and in lung tissue have shown that cad-
herin-1/E-cadherin is one of the primary targets of Cd
toxicity in epithelial cells: Cd interacts with the puta-
tive calcium-binding motif in E-cadherin, thus disrupt-
ing cadherin-based cell adhesion (Prozialeck, 2000).
Cadmium may also disrupt Sertoli cell tight junction
barrier function by not only decreasing the synthesis
and/or expression of proteins but also promoting pro-
tein redistribution at the Sertoli-Sertoli cell interface,
406 Pietro Apostoli and Simona Catalani
perhaps via altering protein endocytosis and/or recy-
cling (Wong and Cheng, 2005).
Cadmium-induced BTB alteration and cell junction
disruption in the seminiferous epithelium is mediated
by the activation of specific signal transduction path-
ways. In detail, in the first step, Cd ions probably enter
Sertoli and/or germ cells via different mechanisms.
Once inside the Sertoli and/or germ cell, Cd induces
the synthesis and release of cytokines, which, by inter-
acting with their respective receptors, activate the
stress-activated p38 mitogen-activated protein kinase
(MAPK) signaling pathway. At the same time, possibly
also mediated by p38 MAPK, the production of prote-
ases (e.g. cathepsin L) is also induced. The net result
is that these mechanisms contribute to disruption of
the BTB and Sertoli-germ cell junctions, which leads to
germ cell loss (Siu et al., 2009). However, Cd is known
to be specifically localized to interstitial testicular cap-
illaries (Nordberg, 1972) and it seems unlikely that
changes in Sertoli cells are crucial for BTB disruption.
A possible role for zinc transporter ZIP8 (Wang et al.,
2007) is discussed in Chapter 32, “Cadmium.”
Nickel destroys the normal testicular structure and
induces dose-dependent alterations in almost all mor-
phometric indicators. In Ni-treated animals, the height
of the germinal epithelium and the diameter of the
seminiferous tubules decreased, and the diameter and
relative volume of the lumen increased (Massányi et al.,
2007b). Qualitative analysis revealed dilatation of the
blood vessels in the interstitium, undulation of the basal
membrane, and several empty spaces in the germinal
epithelium. A specific assay detected a higher frequency
of localized apoptosis in the interstitium of Ni-adminis-
tered animals compared to controls (Forgacs et al., 2012).
4.1.3  Alteration to DNA-Protamine Binding
In mammalian spermatozoa nuclei, DNA is tightly
packaged with protamines, Zn-containing proteins that
protect sperm DNA by binding to it during spermatogen-
esis. Altered levels of protamines may result in increased
susceptibility to injury in spermatozoa DNA, causing
infertility or poor outcomes in assisted reproduction.
Due to their molecular properties, protamines not
only have electrostatic interactions with DNA but also
have the potential to bind metallic elements or other
agents, either as part of normal physiology or through
their potential involvement in chromatin alterations.
One of the first observations that stimulated research
into the possible influence of metallic elements on prot-
amines was that Zn is highly abundant in sperm nucleus.
Subsequent studies have confirmed these observa-
tions, leading to a proposal that Zn can stabilize the
chromatin of spermatozoa through binding to thiol
groups that do not participate in the formation of disul-
fide bridges. The influence of Zn on protamine is there-
fore thought to play a role in the normal function of the
spermatozoon (Björndahl and Kvist, 2010, 2011). How-
ever, in addition to the physiological presence of Zn in
the spermatozoon, there is also clear evidence of the
presence of other metallic elements, such as Pb, Cu, or
Ni (­Quintanilla-Vega et al., 2000a; Massányi et al., 2004;
Hernandez-Ochoa et al., 2005). The toxicity in these cases
may be either direct or mediated through their influence
on protamines. Since some metallic elements bind tightly
to sulfhydryl (-SH) groups in proteins, such as enzymes,
cell membrane proteins, and GSH, they might compete
with or replace the Zn atoms that are normally bound to
nuclear protamines. These changes could affect normal
disulfide bond formation, alter DNA-protamine bind-
ing, or impair chromatin decondensation during fertil-
ization (Quintanilla-Vega et al., 2000b; Silbergeld et al.,
2003). The resulting reduced stability of the chromatin
and abnormal chromatin structure is strongly related to
reduced fertility in humans (Zhang et al., 2012).
4.1.4  Sperm Acrosome Reaction
The metallic elements that specifically modulate
the initiation and duration of the acrosome reaction,
are Cd and Pb. Furthermore, As, Cd, Hg, and Pt cause
acrosome breakage, with the formation of various
sized microvesicles. As, Cd, and Cr cause the forma-
tion of a large round hole and V causes numerous folds
to form in the acrosome membrane (Castellini et al.,
2009). Maintenance of the acrosome is essential for the
functional integrity of sperm, required for binding to
the zona pellucida and for responding to the appropri-
ate signals of the oocytes.
The negative impact of Cd on male fertility is medi-
ated by its chemical affinity for Ca channels in the cell
membranes causing a decreased ability of sperma-
tozoa to undergo the acrosome reaction. Arabi and
­Mohammadpour (2006) also discovered that Cd can
alter the integrity of the acrosomal membranes of sper-
matozoa in bulls and thus cause abnormal acrosome
reactions. A role for Pb is suspected in the inexplicable
infertility of men caused by induction of a spontane-
ous premature acrosome reaction, and the Pb content
of seminal plasma may have a negative impact on
in vitro fertilization rates (Benoff et al., 2003).
4.2  Direct Effects on the Ovary and Ovarian
Production
Evidence of the toxicity of metallic elements on the
ovaries and on ovarian production in humans is scarce
compared to that for sperm.
 20  Effects of Metallic Elements on Reproduction and Development
Some experimental studies have shown that metal-
lic elements can have detrimental effects on ovarian
function at several levels. They cause a direct effect
of ovaries or an indirect effect via the hypothalamus-­
pituitary-ovarian axis. With respect to metallic ele-
ments that directly target the ovary, those that
extensively destroy primordial and primary follicles
can cause premature ovarian failure. Alternatively,
metallic elements that selectively damage large grow-
ing or antral follicles can cause reversible disruption of
cyclicity by impairing ovarian steroid production and
ovulation (Table 2).
In contrast to gametogenesis in males, in females,
oocytes are not continually replenished during repro-
ductive life. After folliculogenesis in the latter half of
gestation, the maximal number of oocytes is fixed.
Human female reproductive toxicity, in terms of
reduced fecundity and fertility, has been reported to
be association with high doses and occupational level
exposure to Hg, Cd, and Pb. Associations with high
doses of these metals have been reported among cou-
ples conceiving with assisted reproductive technolo-
gies, as well as with unassisted conception (Mendola
et al., 2008). Some authors have identified associations
between trace amounts of Pb and Cd in follicular flu-
ids from a single follicle and oocyte fertilization. An
inverse association has been detected between Pb in
follicular fluids and fertilization, although the associa-
tion is positive for Cd (Bloom et al., 2012); furthermore,
embryo fragmentation is reduced by higher blood Hg
(Bloom et al., 2011).
Arsenic-treated rats demonstrated reduced ovar-
ian weight and prolonged estrous cyclicity; these
adverse ovarian effects could be prevented by coad-
ministration with l-ascorbate or sodium selenite. The
results of these studies suggest that arsenic-induced
TABLE 2  The Main Effects of the Metallic Elements on the Ovary and Ovarian Production
Metal Effects on the ovary and ovarian production
As Ovarian DNA damage
Reduced ovarian weight
Cd Reduction of maturation of oocytes
Failure to ovulate
Suppressed oocyte maturation
Decreased relative volume of growing follicles and increased stroma
Cr Reduced number of follicles, reduced number of ova, increased estrous
cycle duration
Cr(VI) Damaged ovarian histoarchitecture in various age groups studied,
­delayed sexual maturation
Pb Reduction of maturation of oocytes
Ovarian follicular cysts, fewer corpora lutea, folliculogenesis dysfunction
Correlation with pathological sperm alteration (flagellum ball)
407
reproductive toxicity may be due to the induction of
oxidative stress or free radical generation; however,
arsenic may have direct ovarian effects in the presence
of reduced gonadotropin levels (Chattopadhyay et al.,
2003).
Akram and colleagues (2009) measured DNA dam-
age in the ovarian cells of rats treated with different
doses of sodium arsenite, and found a long comet-like
tail reflecting a high degree of DNA damage in the
ovarian cells with increased doses of sodium arsenite.
The authors assumed that an imbalance in the antioxi-
dant system caused by arsenite treatment could have
led to abnormal oocyte development, as well as caus-
ing damage to oocyte DNA, which ultimately affects
ovulation.
Many hypothesized mechanisms of action have
been proposed on the basis of experimental studies
on Cd toxicity; these include mechanisms involving
specific targets and more general mechanisms involv-
ing the formation of ROS. Gestational exposure to
Cd affects female reproductive health as a result of
changes in ovarian function associated with altered
ovarian histoarchitecture, oxidative stress, anemia, and
delayed puberty with impaired steroid hormone levels
(Thompson and Bannigan, 2008). ROS not only have
effects on the male reproductive system but also have
effects on the female reproductive system in terms of
interfering with the implantation and fertilization of
eggs, female infertility, and the unregulated synthesis
of female sex hormones (Massányi et al., 2007a).
Other studies on antioxidants and free radicals
clearly suggest the onset of oxidative stress in the ova-
ries of Cd-treated rats: there was a significant increase
in the concentration of H2O2 and lipid peroxidation,
and subnormal activity of most of the antioxidant
enzymes tested. Cd-induced production of ROS was
In vitro/animal/
human Reference(s)
Rat Akram et al., 2009; Mondal et al., 2013
Rat Chattopadhyay et al., 2003
Buffalo Nandi et al., 2010
Rat, frog Piasek and Laskey, 1994; Fort et al., 2001
Sheep, human Leoni et al., 2002; Paksy et al., 1997
Rabbit Massányi et al., 2007a
Mouse Murthy et al., 1996
Rat Samuel et al., 2011
Buffalo Nandi et al., 2010
Mouse Tuapeau et al., 2001
Human Slivkova et al., 2009
408 Pietro Apostoli and Simona Catalani
reported, and ROS may propagate the initial attack on
lipid membranes, causing lipid peroxidation (Samuel
et al., 2011; Stohs et al., 2001). Data on H2O2 and lipid
peroxidation show a dose-dependent increase in H2O2
and lipid peroxidation levels.
Yang et al. (2012) showed that Cd exposure results
in oxidative damage of hen ovarian tissue by altering
antioxidant defense enzyme systems, lipid peroxida-
tion, and apoptosis, as well as inducing endocrine
disturbance. After being treated with Cd for 20, 40,
or 60 days, ovary and serum samples were collected
and examined; results showed that the content of Cd,
malon­dialdehyde, and nitric oxide, as well as the activ-
ity of nitric oxide synthase, increased in the ovaries
and serum, while the level of GPx and the activity of
superoxide dismutase decreased in both the low-dose
and high-dose groups. The number of apoptotic cells
in the ovaries increased in the Cd treatment group, and
extensive damage was observed in the ovaries.
Chromium caused a dose-dependent reduction in
all stages of preantral follicles (primordial, primary,
and growing) and in the number of oocytes ovulated,
as well as lengthening of the estrous cycle. There was
also morphological evidence of follicular disruption.
These data support the observation of fewer corpora
lutea in a study of pregnant mice. Furthermore, the
data suggested that high levels of Cr might disrupt
menstrual cyclicity, ovulation, and fertility in Cr-
exposed women, as well as potentially causing early
menopause. Murthy et al. (1996) reported a number of
reproductive effects (reduced number of follicles at dif-
ferent stages of maturation, reduced number of ova per
mouse, increased estrous cycle duration, and histologi-
cal alterations) in the ovaries of female mice exposed to
potassium dichromate in drinking water for 20 days.
The severity of the reproductive effects appeared to be
dose related.
Nampoothiri et al. (2007) studied the effect of com-
bined exposure to Pb and Cd on granulose cells, dem-
onstrating that metallic element exposure caused a
decrease in the reduced GSH content, along with ele-
vated lipid peroxidation, in all groups. Granulose cells
of Cd-treated and combined treatment groups demon-
strated a maximum increase in lipid peroxides and cat-
alase activity, along with a decrease in both GSH status
and superoxide dismutase activity. Animals exposed to
a combination of Pb and Cd exhibited an intermediate
effect on antioxidant status. Pb and Cd in combination
do not show an additive or synergistic effect, indicat-
ing that competition between them is due to their simi-
lar electronic affinities. Arsenic is known to cause free
radical elevation that results in uterine cell degenera-
tion (Wang et al., 2006). Many studies have suggested
that Pb causes direct damage to the ovaries, resulting
in ovarian follicular cysts and fewer corpora lutea at
high Pb concentrations and folliculogenesis dysfunc-
tion, with fewer healthy primordial follicles and an
increase in the number of atretic antral follicles at low
Pb levels (Taupeau et al., 2001). There are histological
changes in the follicular cells and oocytes of chroni-
cally Pb-intoxicated mice, including disruption of the
follicular membrane, increased pyknosis in the granu-
losa cells, and hypertrophy of the theca layer, indicat-
ing follicular atresia. Lead treatment also increased the
number of oocytes that had resumed meiosis, consis-
tent with the fact that follicular atresia is responsible for
the resumption of meiosis and oocyte degeneration in
several mammalian species (Avazeri et al., 2006).
5  THE ROLE OF HORMONES: METALLIC
ELEMENTS AS ENDOCRINE DISRUPTORS
In 2002, the World Health Organization (WHO)
defined endocrine disruptors (EDs) as “exogenous
substances or mixtures which alter the functions of the
endocrine system and, consequently, cause adverse
health effects in an intact organism, its progeny or
in (sub) populations” (Damstra et al., 2002). The ED
hypothesis is that low-level exposure to certain chemi-
cals may contribute to endpoints such as lowering the
age at menarche, impairing semen quantity and qual-
ity, decreasing the male-to-female sex ratio at birth,
and increasing the rates of hypospadias and testicular
cancer, infertility, spontaneous abortions, and structural
and functional congenital malformations (Sharpe, 2001).
The term ED-like to describe a substance that inter-
feres with the endocrine system is considered inappro-
priate by some authors, who have instead introduced
the term endocrine active (EA). In addition, since not
all chemicals with endocrine-disrupting properties
are equally hazardous, an assessment of potency was
also proposed as a second step to differentiate high-
risk chemicals from those of lower risk (Bars et al.,
2012). Numerous chemical substances belong to this
category, including persistent organic pollutants
(POPs), pesticides, and substances used in cosmetics,
in addition to several metallic elements. Some of these
adverse health effects are attributed to several metallic
elements, while other effects are specific. Early studies
demonstrated that metallic elements can bind the ER;
the binding of certain metals to the zinc fingers of the
ER can alter the interaction of the receptor with DNA
(Predki and Sarkar, 1992). Several metallic elements
can displace or compete with estradiol binding to its
receptor in MCF-7 breast cancer cells (Martin et al.,
2003; Nesatyy et al., 2005, 2006). Investigations of four
metallic elements (As, Cd, Pb, and Hg) have provided
 20  Effects of Metallic Elements on Reproduction and Development 409

insight into the mechanism of their impact on mam-
malian reproductive systems. Exposure to both Cd
and Hg stimulates progesterone synthesis. In addi-
tion, negative effects on spermatogenesis have been
observed in experimental studies using As, Hg, or Pb,
while both Mn and Zn stimulated spermatogenesis.
Furthermore, alterations in the onset of puberty corre-
lated with exposure to Pb and Mn (Iavicoli et al., 2009).
Some effects of EDs may be receptor mediated and
may involve direct interaction with cellular targets. The
majority of studies carried out on EDs have revealed
that these substances have a genomic mechanism of
action. However, the biological effects of these com-
pounds cannot be wholly attributed to their interaction
with hormone receptors since some studies have also
clearly indicated the presence of nongenomic mecha-
nisms capable of altering, or at least influencing, the
synthesis, transport, and availability of endogenous
hormones (Waring and Harris, 2011).
Studies into the effects of metallic elements on the
endocrine system have generally involved exposure
to a single metal; however, both environmental and
occupational exposures are much more complex in
that individuals are simultaneously exposed to dif-
ferent classes of xenobiotics, including both metal-
lic elements and organic compounds that may act as
EDs (e.g. POPs, DDT, and pesticides). For this reason,
at present it is not known whether mixtures of differ-
ent EDs may have additive, synergistic, or antagonis-
tic effects. EDs with the same mechanism of action are
generally assumed to behave additively, but there are
few examples in which this hypothesis has been tested
(Iavicoli et al., 2009; Latini et al., 2003).
We report the main mechanisms of action associ-
ated with metallic elements, as extrapolated from
experimental studies in vivo and in vitro; however, we
are cognizant of the fact that the real effect on humans
may be different and may be mediated by a number
of external and internal confounding factors (Table 3).
The precise mechanism by which As alters
­hormone-stimulated gene regulation by ERs remains
to be determined. In an in vitro study on human
kidney proximal tubule epithelial cells, Zhang and
coworkers (2011) showed that arsenic downregulates
ER mRNA and protein levels. Arsenic is clearly not
only an endocrine-disrupting chemical but also acts
via a unique mechanism quite distinct from those of
previously characterized organic EDs, most of which
act as hormone mimetics and as either agonists or
competitive antagonists. Arsenic clearly alters the
ability of hormone-activated, DNA-bound steroid
receptors to regulate gene transcription. The effects of
As on steroid receptor activity appear to be restricted
to DNA-dependent gene regulation (Bodwell et al.,
2004). Recent evidence indicates that inhibition
occurs at the nuclear level and does not depend on a
mechanism in which the metallic elements compete
for the ligand-binding site of the receptors (Watson
and Yager, 2007).
Overexpression of various estrogen-linked genes
reflects the endocrine disruption effects of inorganic
As at an early stage of life. In fact, similar to estradiol,
arsenite decreased the expression of ERα and increased
the expression of the progesterone receptor. Moreover,
the estrogen-like effects of arsenite were inhibited by
an anti-oestrogen, suggesting that these responses
were mediated by ERα. Arsenic also disrupted the cir-
culating levels of gonadotropins and estradiol, led to
the degeneration of luminal epithelial, stromal, and
myometrial cells of the rat uterus, and downregulated

TABLE 3  Mechanism of Action of Metallic Elements as Endocrine Disruptors, as Extrapolated from Experimental Studies
Metal Mechanism of action Reference(s)

Arsenic Stimulation or inhibition of nuclear transcription activity mediated by several hormone Kaltreider et al., 2001
receptors
Bond with ERs Jana et al., 2006
Down regulation of ER mRNA and protein levels Zhang et al., 2011
Cadmium Bond via ERs Garcia-Morales et al., 1994
Inhibition of transcription of the LDL receptor Jolibois et al., 1999
Inhibition of P450 (cholesterol side-chain cleavage) Kawai et al., 2002
Altered expression of aryl hydrocarbon receptor-associated genes Kluxen et al., 2012
Lead Reduction of the expression of the steroidogenic acute regulatory protein Srivastava et al., 2004
Inhibition of luteinizing hormone secretion Ronis et al., 1996; Srivastava et al., 2004
Inhibition of steroidogenic enzymes (3β and 17β-hydroxysteroid dehydrogenase) activities Pandya et al., 2012
Mercury Induction of 3β-hydroxysteroid dehydrogenase Mondal et al., 1997; Heath et al., 2012
Inhibition of the type I iodothyronine deiodinase Barregård et al., 1994
Binding and activating of ERs Zhang et al., 2008

ER, estrogen receptor; LDL, low-density lipoprotein.

410 Pietro Apostoli and Simona Catalani
downstream components of the estrogen signaling
pathway (Chatterjee and Chatterji, 2010).
Various effects of Cd on reproductive endocrinology
have been described, but definitive conclusions regard-
ing its actions on target tissues vary depending on the
experimental model and the dosage employed. Cd was
found to mimic the effect of estrogen on transcription of
a reporter gene, as well of endogenous genes normally
regulated by estrogen, including the ER receptor. Other
studies involving transfected cells, chimeric receptors,
and competitive receptor binding have demonstrated
Cd binding to the hormone-binding domain of ERs. Cd
binding was weakened by the replacement of cysteine,
glutamic acid, aspartic acid, and histidine in the target
hormone-binding region (Henson and Chedrese, 2004).
A detailed analysis of the Cd-­mediated reduction in
progesterone production by cultured human tropho-
blast cells indicated that it is not due to cell death or
apoptosis. Cd also appears to modulate the expression
of aryl hydrocarbon receptor-associated genes by inter-
acting with the ER (Kluxen et al., 2012). A review of the
scientific evidence for the estrogenic effects of Cd indi-
cated that, although the in vitro and in vivo evidence
of the estrogenic properties of Cd was persuasive,
evidence from population-based human studies
remains contradictory (Silva et al., 2012).
Lead-related reproductive toxicity is also linked to the
altered activity of uterine ERs and their affinity for ovar-
ian steroids; exposure to Pb does not seem to pose a risk
with respect to progesterone secretion (Paksy et al., 2001).
The mechanism of action responsible for the inhibi-
tion of estradiol synthesis was probably a reduction in
ovarian expression of the StAR gene (encoding mito-
chondrial steroidogenic acute regulatory protein). The
administration of pregnant mare serum gonadotropin
restored StAr expression, indicating that metallic ele-
ments do not exert a direct effect on the ovary response
to gonadotropins but might instead act on the hypo-
thalamus-hypophysis axis, thus altering the luteiniz-
ing hormone (LH) release required for both StAR and
estradiol synthesis (Srivastava et al., 2004).
Experimental data have demonstrated that, in rats
exposed to Pb, the activities of steroidogenic enzymes
(3β- and 17β-hydroxysteroid dehydrogenase) also
decreased significantly, leading to altered testosterone
production (Pandya et al., 2012).
Nevertheless, some studies have reported direct
spermatoxicity without hormonal disruption, whereas
others report no effect of Pb on reproductive hormones
or spermatogenesis. In short-term Pb-exposed individ-
uals, high LH and follicle-stimulating hormone (FSH)
levels are usually associated with normal testosterone
concentrations; in contrast, in long-term Pb-exposed
individuals, low testosterone levels do not induce high
LH and FSH concentration (Doumouchtsis et al., 2009).
These findings suggested that Pb initially causes some
subclinical testicular damage, followed by hypotha-
lamic or pituitary disturbance following longer periods
of exposure. Leydig cells appear to be a target because
Pb exposure results in suppression of testosterone syn-
thesis in these cells, while Sertoli cell function appears
to be unaffected (Wirth and Mijal, 2010).
A number of studies have reported an association
between Hg exposure in humans and plasma testos-
terone reduction through inhibition of 3β-hydroxy-Δ5-
steroid dehydrogenase activity in Leydig cells (Heath
et al., 2012). Some authors have suggested that the
increased cholesterol is due to the inhibition of its bio-
synthetic conversion to sex steroid hormones, such as
testosterone (Iavicoli et al., 2009). Another possibility
is that mercury mimics the effect of estrogen on the
testes (i.e. to both inhibit androgen production and
cause accumulation of cholesterol), probably through
upregulation of the high-density lipoprotein receptor,
scavenger receptor class B member I (SRB1) (Tong et al.,
2004). Mercury does not affect the binding of estradiol
to ERs. Therefore, HgCl2 exhibits an estrogen-like effect
by binding to and activating ERs rather than competing
with estradiol binding to ERs (Zhang et al., 2008).
After 10 years of experimental and human investi-
gation, and heated debate, the relevance of EDs was
reviewed by Sharpe and Irvine (2004). It can be sum-
marized as follows:
(1) Few definitive data link human reproductive
disorders or cancer to exposure to environ-
mental synthetic chemicals; this may reflect the
difficulty in obtaining such data or a genuine
absence of effects;
(2) Synthetic chemicals are pervasive in the
environment, but our understanding of their
potential to cause harm is limited;
(3) The reproductive effects of environmen-
tal chemicals in (aquatic) wildlife are well
­established; they may act as sentinels for
human effects, especially for the fetus; and
(4) Recent discoveries raise the possibility that
common environmental chemicals have effects
on endogenous hormones.
  
These conclusions raise some questions for toxicolo-
gists working on the effects of toxic metallics on repro-
ductive and developmental systems.
6 CONCEPTION
Metallic elements can influence the phase of con-
ception and implantation, and can have effects on
 20  Effects of Metallic Elements on Reproduction and Development
couple fertility. In experimental studies on fertility,
metallic elements are generally administered orally for
an extended period of time before, during, and after
mating, in most cases not focusing on the specific stage
or mechanism of action involved.
Some studies have shown that Cd inhibits gap junc-
tion intercellular communication and gap junction
protein/connexin phosphorylation, both of which
are essential processes in the progression from the
eight-cell stage through to compaction (Thompson
and Bannigan, 2008). Cadmium-suppressed synthe-
sis of hyaluronic acid in the oocyte-cumulus complex
(OOC) matrix was linked to the inability of the infun-
dibular cilia to successfully introduce the oocyte into
the fallopian tube (Vrsanska et al., 2003). Cell adhesion
is an integral and important part of OCC pick-up and,
although the precise sequence and pattern of expres-
sion of cell adhesion molecules during the process has
yet to be determined, the involvement of gap junctions
in cumulus expansion has already been identified and
the involvement of other junction types is possible.
Cadmium-induced misexpression of cell adhesion
molecules, including various connections, and also
the cellular delocalization of molecules, such as tight
junction protein ZO-1/zonula occludens protein 1
and cadherin-2/N-cadherin has been associated with
abnormalities in cell adhesion in the gap, occludens,
and adherens junctions (Fang et al., 2001). Trophoblast
formation and invasion have also been identified as
targets for Cd toxicity. Modification of the effect of
Cd on the cell cycle by zaldaride, an inhibitor of the
intracellular calcium-binding protein calmodulin,
indicated that calmodulin has a role in mediating Cd-
induced toxicity in the trophoblast (Thompson and
Bannigan, 2008).
Nampoothiri and Gupta (2008) reported altera-
tions in alkaline phosphatase activity and cathepsin
D, two implantation enzymes, after coexposure to Pb
and Cd; however, no change in reproductive perfor-
mance was observed. The decreased activity could be
due to Pb and Cd competition with the magnesium ion
to bind at its active site. In spite of changes in lyso-
somal enzymes, implantation sites did not seem to be
affected.
In addition to the mechanistic studies described
above, many studies have calculated couple fecundity
through the assessment of the fOR or TTP related to
metallic elements concentrations.
A number of groups have reviewed the association
between the exposure to specific chemicals or groups
of chemicals and TTP, and support the notion that
environmental exposure may be hazardous for human
fertility (Sallmen, 2001; Jensen et al., 2006; Jurewicz
et al., 2007). However, these reviews all focused on the
411
effects, mainly in males, of exposure to a specific chem-
ical or a group of chemicals on fertility or reproductive
function rather than focusing on the broader spectrum
of chemical exposures and their effects on TTP in both
men and women.
Snijder and colleagues (2012) carried out a system-
atic review of the scientific literature up to December
2010 regarding occupational exposure to chemical
substances and TTP. The authors included nine stud-
ies reporting TTP in relation to exposure to metallic
elements, of which two studies were carried out in
women (Sallmen et al., 1995; Wulff et al., 1999), and
seven studies were carried out in men (Bonde, 1990;
Hjollund et al., 1998; Apostoli et al., 2000; Sallmen
et al., 2000; Joffe, 2003; Shiau et al., 2004; Greene et al.,
2010). In the two studies on women, the fOR ranged
from 0.80 to 0.93 for Pb exposure and from 0.82 to
0.91 for exposure to a mixture of metallic elements.
In men, four studies reported that high Pb exposure
(measured as PbB) reduced fecundability (Apostoli
et al., 2000; Sallmen et al., 2000; Joffe, 2003; Shiau et al.,
2004). Figure 3 depicts the exposure-response relation-
ship between different levels of PbB values and fORs,
showing a clear trend of increasing PbB levels with
decreasing fORs.
Between 2005 and 2009, another study was carried
out to evaluate the influence of metallic elements on
couple fecundity (Buck Louis et al., 2012). This study
was included in the Longitudinal Investigation of Fer-
tility and the Environment (LIFE) Study, a prospective
cohort study with a preconception enrolment of 501
couples designed specifically to assess persistent envi-
ronmental chemicals and human fecundity. Cadmium,
Pb, and Hg were observed to be significantly associ-
ated with a reduction in couple fecundity: exposed
couples with a higher degree of exposure took a lon-
ger time to conceive a human chorionic gonadotropin
(hCG)-confirmed pregnancy than couples with lower
blood concentrations. When assessing partner expo-
sure separately, female Cd exposure was associated
with an approximate 22% reduction and male Pb expo-
sure with an approximate 15% reduction in the odds
of conception per standard deviation increase in blood
concentration. The authors did not observe any effect
for Hg. Interestingly, their observed association of a
longer TTP for male PbB concentrations below the cur-
rent occupational Pb exposure limit (> 400 μg/L) was
purported to be associated with a decrease in semen
quality (Apostoli et al., 1998).
Finally, another prospective cohort study with a
preconception enrolment of women studied the asso-
ciation between some metallic elements (As, Cd, Pb,
Mg, Ni, Se, and Zn) and TTP (Bloom et al., 2011). In this
study, no adverse influence was attributed to metallic
412 Pietro Apostoli and Simona Catalani

2,5
<20 20–29 30–39 >40
µg/dL µg/dL µg/dL µg/dL

2
Fecundability ratio

1,5

0,5

0
Shiau 2004 - Blood lead level <20 µg/dL

Apostoli 2000 - Blood lead level 20–29 µg/dL

Joffe 2003 - Blood lead level 20–29 µg/dL
Shiau 2004 - Blood lead level 20–29 µg/dL

Apostoli 2000 - Blood lead level 30–39 µg/dL

Joffe 2003 - Blood lead level 30–39 µg/dL
Shiau 2004 - Blood lead level 30–39 µg/dL

Apostoli 2000 - Blood lead level >40 µg/dL

Joffe 2003 - Blood lead level >40 µg/dL
Shiau 2004 - Blood lead level >40 µg/dL

Sallmen 2000 - 0.5–0.9 µmol/l

Sallmen 2000 - 1.0–1.4 µmol/l
Sallmen 2000 - 1.5–1.8 µmol/l
Sallmen 2000 - >1.9 µmol/l

Sallmen 1995a - <0.5 µmol/l

Sallmen 1995a - 0.5–0.9 µmol/l
Sallmen 1995a - >1.0 µmol/l
FIGURE 3  Forest plot summarizing studies into dose-response relationships between blood lead levels and the fecundability odds ratio
(1 μg/dL = 10 μg/L). Reproduced with permission from Human Reproduction Update (see Snijder et al., 2012).

elements, such as Pb, Cd, or As. Only Mg and Zn blood levels of this metallic element are related to maternal
concentrations were suggestive of a possible signifi- but not to cord blood levels (Esteban-Vasallo et al.,
cant beneficial influence on TTP. 2012). In terms of toxicokinetics, a correlation between
Cd placental levels and the expression of placental
metallothionein has been described (Sonawane et al.,
7  OTHER EFFECTS ON PREGNANCY 1975; Kippler and Hoque, 2010).
The role of the placenta as a barrier against Hg
The effects of metallic elements on female repro- is not completely clear. However, cellular uptake of
duction may derive from their action at several stages, this metal appears to be related to its chemical struc-
beginning with the fetus, and may include manifesta- ture. It has been observed that Hg vapor and methyl-
tions, such as intrauterine growth retardation, sponta- Hg easily pass the placenta by passive transport and
neous abortion, malformation, and birth defects. amino acid carriers, respectively (Ask et al., 2002);
Spontaneous abortion may result from a number in contrast, inorganic Hg is more commonly accu-
of causes, such as genetic alterations in the gametes, mulated in the placenta, thus limiting the amount
failure of implantation processes, or hormonal imbal- reaching the fetus (Yoshida et al., 2011). Lead was
ances. It has therefore been difficult to distinguish the found to easily cross the placental barrier by means
occupational and environmental causes of spontane- of passive diffusion (Goyer, 1990); similarly, a posi-
ous abortion or congenital malformations from those tive correlation was observed in the majority of the
of other factors. studies between placental and cord blood levels
Metallic elements can cross the placenta by various (Esteban-Vasallo et al., 2012). Similarly, in the majority
mechanisms. Maternofetal and fetomaternal diffu- of studies, a positive correlation has been observed
sional transfer depend on the thickness of the sepa- between placental and cord blood levels. Concerning
rating layers. Available studies have shown that Cd toxicodynamics, Pb may alter the calcium-mediated
accumulates in the placental tissues; indeed, placental cellular processes in syncytiotrophoblasts, which
 20  Effects of Metallic Elements on Reproduction and Development
form the epithelial covering of the embryonic pla-
cental villi; according to Goyer (1990), Pb seems to
precipitate, along with calcium, in the microvilli
around the trophoblast (Al-Saleh et al., 2011). The Pb
storage observed in syncytiotrophoblast cells seems
to be related to reduced cytochrome oxidase activity
(Reichrtova et al., 1998).
It is still not known by which mechanisms placenta
cells efflux Hg, Pb, and Cd. There is experimental
evidence supporting the idea that the metals prefer-
ably bind to GSH, forming a complex (reviewed by
Gundacker and Hengstschläger, 2012). In addition, a
number of other protein carriers have been identified
in rat placentae (i.e. organic anion-transporting poly-
peptides, organic anion transporters, organic cation
transporters, and zinc transporters), which might also
be involved in metallic element transport in the human
placenta (Thévenod, 2010).
Ahmed and coworkers (2011) assessed the effect
of As on the placenta and reported that As expo-
sure during pregnancy appears to enhance placental
inflammatory responses (in part by increasing oxida-
tive stress), reduce placental T cells, and alter cord
blood cytokines. Lead interferes with calcium uptake
of the syncytiotrophoblast (Lafond et al., 2004). Mer-
cury toxicity in the placenta includes disturbances to
amino acid transfer, placental oxygen consumption,
enzyme activity, hormonal secretion, and membrane
fluidity (Urbach et al., 1992).
With respect to spontaneous abortion, the best-stud-
ied metallic elements are As, Hg, and Pb. The role of Pb in
spontaneous abortion is controversial. Two studies (one
demonstrating mean PbB levels of 71 μg/L in the study
group and 52 μg/L in the controls, and the other find-
ing maternal PbB levels of approximately 100 μg/L) have
shown spontaneous abortion to be associated with mod-
erately elevated PbB levels (Tabacova and ­Balabaeva,
1993; Bellinger, 2005). Similarly, a Mexican study revealed
that each elevation of 50 μg/L in PbB increased the risk
of spontaneous abortions 1.8 times; they reported mean
PbB levels of 120 μg/L for the study group and 101 μg/L
for controls (Borja-Aburto et al., 1999).
Lamadrid-Figueroa and coworkers (Lamadrid-
Figueroa et al., 2007) reported that women with a large
plasma:whole PbB ratio may have a higher incidence of
spontaneous abortion at a mean PbB level of 62 μg/L,
but failed to demonstrate significant correlations
between Pb concentrations and spontaneous abortion,
either in maternal plasma or in whole blood. However,
no significant relationship was discovered between
early pregnancy PbB levels and spontaneous abortion
in 351 apparently healthy pregnant women. Multiple
logistic regression analysis also showed no statistically
significant association of PbB levels with spontaneous
413
abortion after adjusting for covariates. Consistent with
their findings, Faikoğlu and colleagues (2006) showed
that the Pb blood concentration measured at a gesta-
tional age of ≤ 20 weeks was not significantly higher in
aborting pregnancies than in nonaborting pregnancies.
In 2010, Vigeh and coworkers showed that mean
PbB concentrations did not differ significantly between
spontaneous abortion cases and ongoing pregnan-
cies (351 ± 142 and 383 ± 199 μg/L, respectively) (Vigeh
et al., 2010). Although these differences may have
a methodological explanation, PbB levels change
throughout pregnancy and increase significantly dur-
ing the third trimester. Therefore, a comparison should
be carried out at the same gestational age for all par-
ticipants (Hertz-Picciotto, 2000; Gulson et al., 2004). In
addition, it is necessary to consider that in many stud-
ies blood samples were collected at different gesta-
tional ages for both patients and controls, i.e. full-term
delivery versus spontaneous abortion, and this aspect
makes it difficult to standardize the findings of differ-
ent studies.
The mechanism inducing spontaneous abortion is
not clear; in addition to preconceptional chromosome
damage in the egg cell or in testis (see above), or a
direct teratogenic effect on the fetus, interference with
the maternal/fetal hormone environment or develop-
mental toxicity to the embryo/fetus is possible. Vascu-
lar effects on the placenta are also plausible; elevated
blood pressure during pregnancy represents increased
risks to both mother and fetus (reduced birth weight,
abruption placentae, and perinatal mortality).
The majority of epidemiological studies in As-­
contaminated zones have suggested an increase in
spontaneous abortions/stillbirths or preterm births.
Abortion after exposure to As has been reported in the
USA, Hungary, North Chile, and the Indo-Bangladesh
region (Cherry et al., 2008). These studies attributed the
effect of As on pregnancy as being related to (1) ­oxidant/
antioxidant homeostasis in the embryo; (2) early and
late developmental stage, apoptosis, and inflamma-
tion; and (3) estrogen-mediated gene expression.
Speculation about how As exposure complicates
pregnancy is based on data showing that As is trans-
ferred to the developing embryo through the abundant
placental vasculature, which suggests that As toxicity
causes spontaneous mammalian abortion by virtue
of aberrant placental vasculogenesis and placental
insufficiency.
There is evidence for the possibility that placental
“translucency” to As might facilitate abnormal devel-
opment during vascular invasion of the uterus and
thereby “program” the embryo for future loss during
very early gestation (He et al., 2007). Interruption of
hormone signaling pathways, including the steroid
414 Pietro Apostoli and Simona Catalani

hormones estrogen and progesterone, represents an 2006). The association between Cd and reduced birth
alternative mechanism by which As may interfere weights in girls (an estimated decrease of 45 g for every
with gestation in rodents and humans (Sakurai and 1-μg/L increase in maternal urinary Cd) corresponds
Himeno, 2006). to about one-quarter of the estimated effect of smoking
To evaluate prenatal exposure to As in the general during pregnancy on birth weight (∼200 g lower birth
population and its effects on birth size, Guan et al. weight for mothers who smoke than for nonsmoking
(2012) carried out a cross-sectional study in China and mothers) (Rogers, 2009; Kippler et al., 2012).
showed that maternal As concentration was negatively
associated with birth weight, height, and chest circum-
ference, and that fetal As concentration was negatively 8  DEVELOPMENTAL EFFECTS
associated with head circumference.
It was shown that exposure to Cd during gesta- 8.1  Developmental Effects of Prenatal Exposure
tion was associated with reduced birth weight and an
The effects arising from prenatal exposure to metal-
increased number of preterm births. A possible expla-
lic elements have been discussed mainly in terms of
nation for these effects may be the influence of Cd on
development, particularly the effects of the metal-
the synthesis of some hormones, such as hCG, which
lic elements on morphological development, which
plays a vital role in the maintenance and progression of
might result in birth defects as well as in more subtle
pregnancy or placental Cd accumulation (Nishijo et al.,
effects on the developing organism, such as changes in
2002). Another proposed mechanism for the effect of
the growth or maturation of the CNS.
Cd on birth weight is the induction of metallothionein
Some of the effects observed in experimental stud-
in the placenta and the subsequent chelation of essen-
ies are highly specific to the metallic element, and the
tial trace elements, such as Zn and Cu (­Chmielnicka
effect itself may be the only one recorded for the organ-
and Sowa, 1996).
ism. The nature of the exposure, such as an acute single
One study reported a significant inverse associa-
dose, is unlikely to reflect the usual occupational or
tion of the cord blood Cd level with newborn head
environmental exposure of human beings.
circumference and, consistently, with height, weight,
Several studies have confirmed that prenatal expo-
and head circumference up to 3 years of age (Lin et al.,
sure to Pb impairs behavioral and cognitive develop-
2011). However, the effect on fetal growth may be at
ment of children, even at low levels (Lanphear et al.,
least partly related to the accumulation of Cd in the
2005; Plusquellec et al., 2007; Jedrychowski et al., 2009).
placenta because it was associated with the impaired
Mercury is an environmental pollutant that can have
transport of nutrients to the fetus. There are several pos-
neurotoxic effects on the human CNS, particularly
sible mechanisms by which maternal Cd exposure may
during fetal development. Developmental neurotoxic-
directly or indirectly affect size at birth. Some authors
ity was first reported in a Swedish case report in 1952
have shown that pregnant women in rural Bangladesh
and from a serious outbreak in Minamata, Japan, a few
had elevated Cd concentrations in their placentae, and
years later. The exposed patients manifested neurologi-
that these Cd concentrations were inversely associated
cal signs, and some patients exposed in utero were born
with Zn in the cord blood, suggesting that Cd may
with so-called congenital Minamata disease (this is well
disturb Zn transfer to the fetus (Kippler and Hoque,
described with references in Chapter 46, “Mercury”).
2010). Previously, an interaction between Cd and Zn
The passage of metallic elements through the pla-
in the placenta, leading to decreased Zn transfer to
centa (discussed in the previous paragraph) is a crucial
the fetus, had been shown only in vitro and in women
point in the mechanism of its developmental effects.
with increased Cd exposure due to smoking. Since
The modes of action involved mainly include oxida-
insufficient Zn transfer to the fetus is likely to have
tive stress, a direct effect through interaction with
consequences for pregnancy outcomes, especially in
the embryonic structure, and epigenetic effects (DNA
terms of intrauterine growth retardation and preterm
hypomethylation, interaction with enzymes critical
delivery, this has been suggested as one of the main
for fetal development and programming), endocrine
mechanism of Cd-related reduction in birth weight.
effects, immune suppression, and neurotransmitter
Since the effect of Cd exposure on size and weight
alteration.
at birth is greater in females, another possible expla-
nation may be its endocrine-disrupting properties. Cd
8.1.1  Reactive Oxygen Species
seems to affect corticosteroid 11-beta-dehydrogenase
isozyme 2/11-beta-hydroxysteroid dehydrogenase Developmental pathologies may result from the
type 2 (11β-HSD2), a key regulator of maternal glu- endogenous or the xenobiotic-enhanced formation
cocorticoids in the fetoplacental barrier (Yang et al., of ROS, which cause oxidative damage to cellular
 20  Effects of Metallic Elements on Reproduction and Development
macromolecules and/or alter signal transduction.
Development may also be adversely affected by the
reversible reaction of ROS with transduction proteins,
thereby altering embryonic or fetal signal transduction
pathways.
Teratogenicity probably depends, to a large extent,
upon a balance between the pathogenic pathways of
xenobiotic bioactivation, oxidative macromolecular
damage, and signal transduction, on one hand, and, on
the other, the protective pathways of maternal elimi-
nation, embryonic detoxification of xenobiotic reactive
intermediates and ROS, and embryonic pathways for
the detection and repair of oxidative DNA damage.
A potential indirect mechanism by which maternal
and other extraembryonic pathways can modulate
ROS-mediated teratogenesis is via the production of
diffusible factors that can traverse the placental and
cellular membranes into the embryo and alter embry-
onic determinants of oxidative macromolecular dam-
age or signal transduction. This is exemplified by the
peroxynitrite pathway, in which nitric oxide synthases
(NOSs) produce relatively stable and readily diffus-
ible nitric oxide (NO) (Marnett et al., 2003). Once in the
embryo, NO can alter embryonic signal transduction
pathways, or react with superoxide to directly produce
embryopathic hydroxyl radicals and reactive nitrogen
species or peroxynitrite, the latter being capable of ini-
tiating the hydroxylation and nitration of embryonic
proteins and DNA.
As regards to metallic elements in particular, there
are several recent findings. Arsenic is a known neuro-
toxicant and is capable of traversing the placental bar-
rier and reaching the conceptus in cases of maternal
exposure and also of easily crossing the blood-brain
barrier to modify the CNS metabolism and function
at low doses (Xi et al., 2010). An increased level of
lipid peroxidation and consequent damage to the cell
membrane due to As and its compounds have been
reported by numerous investigators. Oxidative stress
has been related to damage to cellular lipids, but there
are also indications that As, due to its interference in
methylation reactions and coupling with thiol groups,
may induce independent effects on proteins and
DNA (Zhong and Mass, 2001). Xi et al. (2010) found
increased oxidative stress in the brain after prenatal
exposure, resulting in lowered GPx.
Human fetal brain tissue explants maintained in
culture in the presence of As have shown the charac-
teristics of cell division, cellular growth, and the devel-
opment of a neuronal network that oppose the signs
of cellular damage, loss of ground matrix, weakening
of the synaptosomes, and cell loss. Arsenic toxicity
appears to act through interference in the tissue homeo-
stasis of cell growth and development, and apoptosis
415
(Ríos et al., 2009; Zhang et al., 2010). The results indi-
cated that As affects the neurobehavioral parameters
of the developed, as well as the developing, brain.
There is increasing evidence that maternal Cd expo-
sure causes relatively specific forelimb ectrodactyly
(the deficiency or absence of one or more central dig-
its) in mouse strains when administered during early
limb development (Robinson et al., 2009, 2011). Cd-
induced fetal malformations might be associated with
impairment of placental development and a reduction
in placental transport capacity. Maternal Cd exposure
during pregnancy induces placental endoplasmic retic-
ulum stress and an unfolded protein response, which
may contribute to Cd-induced impairment in placen-
tal and fetal development. ROS, possibly originating in
trophoblast giant cells, mediate Cd-evoked placental
endoplasmic reticulum stress and an unfolded protein
response (Wang et al., 2012).
Some neurotoxic effects of mercurial compounds
on the different stages of development can also be
attributed to increased ROS. Methylmercury, a potent
neurotoxicant, can easily pass through the blood-
brain barrier and accumulate in brain regions, causing
severe irreversible damage. However, the neurotoxic
effects and action mechanisms of methylmercury are
still unclear, especially in low-dose and long-term
exposures. Furthermore, the alteration of lipid per-
oxidation, Na+/K+-ATPase activities, and nitric oxide
in brain tissue has contributed to observed neurobe-
havioral dysfunction and hearing impairment (Huang
et al., 2008, 2011).
8.1.2  Epigenetic Effects
Since the DNA sequence is static, genetic suscepti-
bility from DNA sequence variations cannot explain
the mechanisms by which prenatal or early child-
hood metallic elements exposure impact cognition and
behavior later in life. One possible mechanistic path-
way for this phenomenon, which has yet to be fully
explored in humans, is epigenetics. Epigenetics is the
study of heritable changes in gene expression that occur
without changes in DNA sequence. Such changes can
have effects as important as those exerted by mutations
but, unlike mutations, they are reversible and respon-
sive to environmental influences. DNA methylation is
the best studied of the epigenetic processes that regu-
late gene silencing. In general, increased methylation
is inversely associated with gene expression (Wright
and Baccarelli, 2007). The growing interest in epigen-
etic markers is a result of their potential to explain the
fetal origins of a disease, or even simply to explain
the latency between exposure to toxic substances and
subsequent disease phenotypes. Several studies have
416 Pietro Apostoli and Simona Catalani

established an association between DNA methylation 8.1.3  Immune Suppression

and environmental metallic elements, including Ni,
Some metallic elements may have effects on the
Cd, Pb, and, particularly, As (Dolinoy et al., 2007). Oxi-
immune system through prenatal exposure and altera-
dative stress may be a unifying process responsible for
tions to thymic function.
these findings across different metallic elements. Oxi-
Prenatal As exposure is associated with increased
dative DNA damage can interfere with the ability of
infant morbidity and reduced thymus size, indicating
methyltransferases to interact with DNA, thus result-
arsenic-related developmental immunotoxicity. The
ing in the generalized hypomethylation of cytosine
child thymic index for thymic function was evaluated
residues (Valinluck et al., 2004).
at birth in a Bangladeshi cohort: prenatal As exposure
In addition, Takiguchi et al. (2003) showed that
was associated with reduced thymic function, possi-
Cd inhibits DNA methyltransferases in a manner
bly via the induction of oxidative stress and apoptosis,
that is noncompetitive with respect to the DNA sub-
suggesting subsequent immunosuppression in child-
strate. This finding is suggestive of interference in the
hood (Raqib et al., 2009; Ahmed et al., 2012).
enzyme-DNA interaction, possibly through an interac-
Cadmium also dysregulated two signaling path-
tion of Cd with the methyltransferase DNA-binding
ways in the thymus, resulting in altered thymocyte
domain.
development (Hanson et al., 2010).
Most of the information regarding the mechanism
Hanson and colleagues (2012) suggested that even
of action was obtained from experimental studies on
very low levels of Cd exposure during gestation can
As exposure. Arsenic mainly causes the induction of
result in long-term detrimental effects on the immune
hypomethylation, possibly through inhibition of DNA
system of the offspring, and that these effects are, to
methyltransferases (Cui et al., 2006), but hypermeth-
some extent, sex specific. The analysis of cytokine pro-
ylation of the TP53 and p16 tumor suppressor gene
duction using stimulated splenocytes demonstrated
promoters in arsenic-exposed people has also been
that prenatal Cd exposure decreased interleukin-2
reported (Chanda et al., 2006). Since DNA methyla-
(IL-2) and IL-4 production by cells in female offspring
tion is an important mechanism of fetal programming,
at 2 weeks of age. Prenatal Hg exposure exerts organ-
As-induced changes in DNA methylation may have
specific effects on cell number, proliferation, and cyto-
severe consequences for the development of health
kine production in preweaned mouse pups. At adult-
defects both before and after birth.
hood, the effects of prenatal Hg exposure could still
Kile and coworkers (2012) carried out the first epide-
be observed, but they differed in females and males.
miological study to note an association between As and
In adult females, an inhibitory effect was observed on
DNA methylation in newborns, and showed that expo-
cytokine production by thymocytes, lymph node cells,
sure to higher levels of As is positively associated with
and splenocytes; in males, a stimulatory effect was
general DNA methylation and, to a lesser extent, with
observed (Silva et al., 2005). Furthermore, gestational
methylation sites within the promoter region of p16.
exposure to Hg leads to persistent changes in the T-cell
Arsenic exerts its toxic effects on the developing
phenotype. A significant reduction in splenic cells
fetus by inhibiting enzymes such as thioredoxin reduc-
in mercury-exposed females, but not in males, was
tase, methyltransferase, and enzymes of the DNA
observed; moreover, the production of IL-4 and inter-
repair mechanism, and disrupting the homeostasis
feron (IFN) from mercury-exposed male and female
of neurotransmitters and hormone secretion.
mice was significantly increased, while IL-2 and IL-10
Moreover, numerous studies have reported altera-
levels were unaffected (Pilones et al., 2009).
tions in the expression pattern of genes involved in
reproductive cell formation. Flora and Mehta (2009)
8.1.4  Effects on Neurotransmitters
reported altered expression of gene markers of the
development and differentiation of all germ layers Metallic elements, such as Al, As, Hg, and Pb may
during embryogenesis. They studied the expression of affect chemical synaptic transmission in the brain and
approximately 47 genes, including markers of embry- the peripheral nervous system, beginning from the
onic cell development, differentiation, apoptosis, gestational period.
and cell cycle in human embryonic stem cell-derived Perinatal As exposure may disrupt the regulatory
embryoid bodies. Arsenic treatment significantly interaction between the hypothalamic-pituitary-adrenal
downregulated the majority of genes studied. These axis and the serotonergic system in dorsal hippocampal
developmental effects were also validated in vivo, formation in a manner that predisposes affected off-
where a significant reduction in litter size along with spring to depressive-like behavior (­Martinez et al., 2008).
an increased incidence of visceral and skeletal defects Arsenic also affected neurotransmitter metabolism by
was observed following As exposure. increasing acetylcholinesterase activity and glutamate
 20  Effects of Metallic Elements on Reproduction and Development
decarboxylase mRNA expression, and decreasing glu-
tamine synthetase activity and ABAT mRNA (encoding
4-aminobutyrate aminotransferase) expression (Xie et al.,
2007). Maternal Pb exposure can have consequences on
the dopaminergic-nitrergic interaction in the nigrostria-
tal pathway (Nowak et al., 2008) and on monoaminer-
gic neuronal function at an adult stage of life, generally
accentuating the behavioral effects of direct and indirect
monoaminergic agonists (Szczerbak et al., 2007).
8.2  Developmental Effects of Neonatal
Exposure
In addition to evidence for the effects of prenatal expo-
sure to metallic elements, child health outcomes (cogni-
tive function, behavior problems, and motor and sensory
function) related to postnatal/neonatal exposure have
also been reported in several epidemiological studies.
The neurotoxic mechanisms of action in children
are comparable to those in adults; however, a child’s
brain is more susceptible and sensitive to neurotoxins.
The neurotoxicity of metallic elements is specifically
addressed in Chapter 15 “Neurotoxicology of Metals.”
Exposure to As through drinking water has been
associated with impaired cognitive function in school-
aged children. Using multivariable-adjusted regres-
sion analyses checking for all potential confounders
and patients lost to follow-up, the authors found the
verbal intelligence quotient (VIQ) and full scale intelli-
gence quotient (FSIQ) to be negatively associated with
the log of the urinary concentration of As (U-As) in
girls. In boys, the U-As showed a consistently low and
nonsignificant association with all IQ measurements.
An effect size calculation indicated that 100 μg/L U-As
was associated with a decrease of 1-3 points in both
VIQ and FSIQ in girls (Hamadani et al., 2011).
Recently, an epidemiological study of developmental
neurotoxicology has increased the focus on coexposure
to As and Mn (Wasserman et al., 2011). To investigate
the possible synergistic impact of simultaneous expo-
sures, a sample of 299 children (8-11 years old) was
recruited and stratified according to As (above and
below 10 μg/L) and Mn (above and below 500 μg/L)
concentrations in household water wells. Both As
and Mn in whole blood were significantly negatively
related to most Wechsler Intelligence Scale for Children
IV subscale scores. With further adjustment for sociode-
mographic features and for ferritin, levels of Mn in the
blood remained significantly associated with reduced
“Perceptual Reasoning” and “Working Memory”
scores; as expected, the association with As was nega-
tive and was significant for “Verbal Comprehension.”
The neurodevelopmental toxicity of As is mediated
by an epigenetic mechanism: As interferes with the
417
epigenetic modulation of the neuronal gene expression
required for learning and memory.
Early life Pb exposure decreases learning, attention,
and IQ, and contributes to hyperactivity, impulsive-
ness, and aggression (Schettler, 2001; Grandjean and
Landrigan, 2006). Preschool Pb exposure has been
associated, using an ecological correlation, with com-
plex outcomes as varied as poor school achievement,
unwed pregnancy, and violent crime (Nevin, 2009;
­Carpenter and Nevin, 2010).
Lanphear and coworkers (2005) collected data from
1333 subjects, where the IQ score was the primary
outcome measure. After adjustment for covariates,
decreases in the estimated IQ point associated with an
increase in PbB from 24-100 μg/L, 100-200 μg/L, and
200-300 μg/L were 3.9, 1.9, and 1.1, respectively. This
data suggests that the dose-response curve is steeper at
lower levels of exposure than at higher levels (Figure 4).
PbB levels of 100 μg/L, as proposed by the U.S. Cen-
ters for Disease Control and Prevention (CDC, 1991),
were considered worrisome and were, for a long time,
accepted as the guideline value. Since then, there has
been a significant worldwide decrease in the PbB lev-
els in children, and the current geometric mean values
in European countries range from 20 to 30 μg/L.
The European Food Safety Authority (EFSA, 2010)
concluded that an increase in the PbB level by 12 μg/L
could decrease the IQ score by one point. It seems that
this value can be used as a “unit risk” for calculating
a possible decrease in IQ and, consequently, influenc-
ing low-level exposure to Pb (< 100 μg/L) on the health
and socioeconomic status of the exposed population.
The Scientific Committee on Neurotoxicology and
Psychophysiology and the Scientific Committee on the
Toxicology of Metals of the International Commission
105
100
95
IQ
90
85
0 10 20 30 40
Concurrent blood lead (µg/dL)
FIGURE 4  Linear models of concurrent blood lead levels and in-
telligence quotient (IQ) adjusted for some covariates (maternal edu-
cation, maternal IQ, and birth weight). Reproduced with permission
from Environmental Health Perspectives (Lanphear et al., 2005).
418 Pietro Apostoli and Simona Catalani
on Occupational Health declared that the action level
for children should be immediately reduced to a PbB
of 50 μg/L (Landrigan et al., 2007).
In 2012, the CDC defined a reference level of 50 μg/L
to identify children with elevated blood lead levels
(Betts, 2012).
In recent years, the mechanism of action of Pb neu-
rotoxicity in the developmental brain has been eluci-
dated. Lead targets the ionotropic glutamate receptor/
N-methyl-d-aspartate receptor (NMDAR) and alters
those physiological processes that are NMDAR depen-
dent, including hippocampus long-term potentiation.
Learning a behavioral task depends greatly on normal
hippocampus function; thus, during its developmental
period, the brain is highly vulnerable to the presence
of Pb. Lead exposure induces alterations in NR1 and
NR2 (encoding NMDAR subunits) mRNA expression,
essentially in the hippocampus region, indicating the
regional selectivity of Pb.
In addition, Pb disrupts the normal development of
the brain, causing a reduction in cellular development
that can be seen at the dendritic, axonal, and synaptic
levels in different brain regions. Lead exposure also
disrupts the aminergic system in the cortex, cerebel-
lum, and hippocampus, thus possibly contributing
to cognitive and behavioral impairment, especially in
rats exposed to Pb during the developmental period
(Neal and Guilarte, 2010; Neal et al., 2010, 2011; Xu
et al., 2009).
Methylmercury is recognized as another important
developmental neurotoxicant, although this insight
developed slowly over many decades. Nonetheless,
exposure limits for this environmental chemical were
based solely on adult toxicity for 50 years after the
first report on developmental neurotoxicity. Meth-
ylmercury poisoning in children shows more widely
distributed damage on the brain; numerous findings
strongly support the notion that early developmental
exposure causes much more serious disease in chil-
dren than in individuals exposed as adults. Current
evidence is affected by uncertainty, most importantly
by the imprecision of exposure assessment in epide-
miological studies. Detailed calculations suggest that
the relative degree of imprecision may be as high as
50% or greater, thereby substantially biasing the results
toward the null. In addition, as methylmercury expo-
sure usually originates from fish and seafood, which
also contain essential nutrients, so-called negative con-
founding may occur (Grandjean and Herz, 2011).
In the most recent regulatory assessments of meth-
ylmercury neurotoxicity, the experts decided to take
the benefits of fish consumption into account, thereby
allowing greater methylmercury exposure in order to
avoid causing a decrease in fish intake or undue panic
and abstention from fish consumption in the local pop-
ulations (JECFA, 2003; Grandjean and Herz, 2011).
In 1997, amid concerns about a possible link between
vaccines containing mercury-based chemical thiomer-
sal and autism, the U.S. Food and Drug Administra-
tion was called upon to review the risks of all food and
drugs that contain mercury. In 1999, despite the lack of
scientific evidence, the CDC issued a recommendation
to manufacturers that “thiomersal-containing vaccines
should be removed as soon as possible” (Siva, 2012).
More than a decade later, despite several independent
studies and consultations that did not find any asso-
ciation between thiomersal vaccines and autism, the
debate continues (Schultz et al., 2010; Siva, 2012).
Several mechanisms have been proposed through
which methylmercury might damage the developing
brain. Among them are methylmercury-induced alter-
ations in microtubules, oxidative damage to neurons,
impairment of neuronal and glial calcium homeostasis,
and potentiation of glutamatergic neurotransmission
(Castoldi et al., 2008). The effect of disrupting microtu-
bules and consequently mitosis, migration, and cortical
organization of neurons is especially serious prenatally,
although these processes continue postnatally (Rodier,
2004; Clarkson, 1987). Chemically, methylmercury has
a strong affinity for the sulfhydryl groups present in
proteins and GSH. When methylmercury forms com-
plexes with these compounds, it can adversely affect
anabolic processes and protein synthesis. By inactivat-
ing sulfhydryl enzymes, methylmercury can interfere
with cellular metabolism and function. Methylmer-
cury is also known to catalyze the formation of excess
ROS, and the regional distribution of this activity par-
allels the sites of known neuropathological changes.
Methylmercury rapidly binds to reduced GSH, which
is present in most cells in millimolar concentrations
(Farina et al., 2011). Such binding may serve to protect
intracellular proteins. There are also adverse effects of
methylmercury on the synthesis of fetal DNA in astro-
cytes and on the growth cones of neurons.
9  CONCLUDING REMARKS AND THE
NEED FOR FUTURE RESEARCH
At present, for only a few metal ions such as Pb,
As, Cd, and Hg extensive evaluations of their poten-
tial effect on reproduction and development have been
done and their relative mode of action is known. How-
ever, in the general and occupational environments,
many other metallic elements (Cr, Ni, Platinum, Pal-
ladium, Rhodium, V, and Sb) are present, and there is
evidence that they can interfere with reproduction or
development.
 20  Effects of Metallic Elements on Reproduction and Development
A better knowledge of the mechanisms of action
involved and of the different target sites is crucial for
understanding this complex and heterogeneous phe-
nomenon; this represents one of the main goals of
future research.
An appropriate use of the diagnostic approach
in occupational medicine is necessary to reach the
­“Bradford Hill Criteria” and obtain adequate evidence
for a causal relationship between an incidence and a
consequence.
An in-depth characterization of doses is another
critical requirement; this requires knowledge of ele-
mental speciation, the identification of more adequate
matrices, nearest to the critical organs or tissues, and
determination of the dose-response relationship and
the threshold for action.
For some metals, another aspect is new routes of expo-
sure. For example, the release of Co and Cr by metallic
prostheses constitutes a new risk. Metal-on-metal bear-
ings have frequently been used by orthopedic surgeons,
especially in young and active patients (Cui et al., 2005),
and hip surgery can be done during child-bearing age
(Stea et al., 2007). However, the implantation of metal-
on-metal hip devices can increase metal ion serum lev-
els (Hartmann et al., 2013). This has led to a concern that
disseminated metal ions, such as Cr and Co (the main
constituents of the alloy used), could affect pregnan-
cies in women owing to transplacental transfer. Current
research into the transplacental passage of metal ions
suggests that 29% of Cr and 60% of Co ions cross the
placenta in mothers with metal-on-metal hip resurfacing
(Ziaee et al., 2007). A few studies and case reports have
explored the question of pregnancy and childbirth after
total hip arthroplasty (THA) (Stea et al., 2007; Boot et al.,
2003; Sierra et al., 2005). These reports primarily found
that pregnancy and vaginal delivery are safe after THA,
although the majority of women with this type of sur-
gery delivered by elective cesarean. These reports did
not address the teratogenic or fetotoxic risk of the materi-
als used. The available data are reassuring because they
show that pregnancy and childbirth are not affected by
the presence of a hip device (Sierra et al., 2005) and that
pregnancy-related complications in women with THA
do not differ from those in healthy women (Meldrum
et al., 2003). Fritzsche and coworkers (2012) described
a 41-year-old patient with bilateral metal-on-metal hip
arthroplasties, a recurrent pseudotumor, and extremely
high metal blood levels (Cr 39 μg/L, Co 138 μg/L) at 12
weeks of gestation. At 38 weeks of gestation, a healthy
male infant was delivered with elevated Cr and Co cord
blood levels; however, the infant’s development appeared
to be uneventful and no signs of toxicity were obvious.
The release of V has also been reported in patients
implanted with a titanium alloy hip prosthesis
419
(­Catalani et al., 2013), and further research is needed to
define potential risks because exposure to V has been
associated with decreased fertility, embryolethality,
fetotoxicity, and teratogenicity (Domingo, 1996, 2002).
Furthermore, some experimental studies have evalu-
ated the effect of rare earth elements such as scandium,
yttrium, lanthanum (La), cerium (Ce), neodymium,
and gadolinium. The offspring of Ce-exposed, but
not of La-exposed, sperm displayed a significant con-
centration-related increase in developmental defects
(Oral et al., 2010). Cerium also caused significant life-
span shortening, accompanied by oxidative damage,
and extended the developmental time of Drosophila
melanogaster (Wu et al., 2012). Many radiology depart-
ments have discouraged the use of gadolinium-based
contrast media in pregnant or lactating women due to
a lack of knowledge regarding the risks for the fetus
and an unwillingness to expose neonates to unnec-
essary drugs. However, documentation and reports
regarding the possible adverse effects of gadolinium-
based contrast media on the fetus are sorely lacking
­(Sundgren and Leander, 2011). After the introduction
of automobile catalytic converters, platinum, palla-
dium, and rhodium have been emitted with exhaust
fumes, and increasing levels have been found in dif-
ferent environmental matrices such as road dusts, soils
along heavily frequented roads, and the sediments
of urban rivers. Compared with other heavy metals,
the biological availability of platinum, palladium, and
rhodium in some experimental studies on road dusts
ranged between those of Cd and Pb. Morphological
and functional effects on rat ovaries and testicular
tissue in rat (Ciftci et al., 2011), embryo lethality, and
teratogenic effects (Sharani et al., 2011) have been dem-
onstrated after exposure to Pt complexes.
We have already stated that current experimental
and human studies that deal with exposure to a single
metallic element contrast with the real environmental
and occupational exposure, which are generally char-
acterized by many metal ions and other organic sub-
stances. Therefore, identifying the possible synergic,
additive, multiplicative, or competitive effects of metal
coexposure remains an intriguing yet difficult objec-
tive for future research.
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