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The hypothalamic–pituitary–testicular axis and

Cite this: Toxicol. Res., 2018, 7, 102
the testicular function are modulated after silver
nanoparticle exposure†
M. D. Cavallin, ‡a R. Wilk,‡a I. M. Oliveira, a N. C. S. Cardoso,a N. M. Khalil,b
c
C. A. Oliveira, M. A. Romano a and R. M. Romano *a
Published on 23 November 2017. Downloaded on 03/05/2018 03:38:16.

Received 31st August 2017,
Accepted 22nd November 2017
DOI: 10.1039/c7tx00236j
rsc.li/toxicology-research
Silver nanoparticles (AgNPs) are widely used in industrial and medical applications and humans may be
exposed through different routes, increasing the risk of toxicity. We investigated the transcript expression
of genes involved in the regulation of the hypothalamic–pituitary–testicular (HPT) axis and the parameters
associated with sperm functionality after prepubertal exposure. AgNPs modulated the transcript
expression of genes involved in the control of the HPT axis and spermatogenesis in the groups treated
with lower doses, while the functional parameters related to sperm and puberty were affected in the
groups administered higher doses. These results suggest that the HPT axis is disrupted by AgNPs during
the prepubertal and pubertal periods, which are highly susceptible windows for the endocrine-disrupting
chemical activity.

Introduction amount of AgNPs leached by infant products (plush toys, baby

Silver nanoparticles (AgNPs) have potent antimicrobial activity
against a wide range of bacteria, including highly pathogenic
and multidrug resistant species such as Staphylococcus aureus,
Salmonella typhi, Staphylococcus epidermidis and Escherichia
coli.1,2 For this reason, AgNPs are included in medical pro-
ducts such as coatings for burn wound dressings, surgical
devices, and bone prostheses,3–6 as well as in other consumer
products such as toothpaste, cosmetics, sprays, disinfectants,
deodorants, diapers, food packaging materials and water
purifiers.7–11 Currently, there are approximately 442 different
products that contain AgNPs in their chemical composition.12
Therefore, humans may be exposed to AgNPs present in
everyday nano-functionalized consumer products.13 Recently,
it was demonstrated that AgNP-impregnated toothbrushes
release silver, which may cause human and environmental
exposure to AgNPs.14 Furthermore, Quadros et al.15 tested the
a
Laboratory of Reproductive Toxicology, Department of Pharmacy, State University of
Centro-Oeste, Rua Simeao Camargo Varela de Sa, 03, 85040-080, Parana, Brazil.
E-mail: romano@unicentro.br
b
Laboratory of Nanotechnology, Department of Pharmacy, State University of Centro-
Oeste, Rua Simeao Camargo Varela de Sa, 03, 85040-080, Parana, Brazil
c
Laboratory of Hormonal Dosages, Department of Animal Reproduction, Faculty of
Veterinary Medicine, University of Sao Paulo, Av. Prof. Dr. Orlando Marques de
Paiva, 87, 05508-270, Sao Paulo, Brazil
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7tx00236j
‡ These authors contributed equally to the work.
blankets, and sippy cups) and found that 43.8% of the AgNPs
were leached from the spout cover of sippy cups, which rep-
resents 0.93 ± 0.02 mg per kg product per day. The toxic effects
of silver in humans (argyria) are associated with high levels of
exposure during life, but the safety limits of exposure to AgNPs
are still under evaluation, because there is very little information
available and more risk assessment studies are necessary.16,17
The toxic effects caused by inhalation,18 ingestion,19–22
dermal contact23,24 or intravenous injection25,26 have been
described after the in vivo administration of AgNPs in animal
models. These toxic effects include the inhibition of mitochon-
drial activity,27,28 cytotoxicity,29 genotoxicity30 and the pro-
duction of reactive oxygen species, which causes oxidative
stress in the central nervous system.31,32
Regarding reproductive toxicity, in vitro exposure of mam-
malian spermatogonial stem cells to AgNPs damages the cell
membrane, induces apoptosis and necrosis,33 and promotes
the decline in cellular proliferation.34 Rabbits exposed intra-
venously to AgNPs have decreased sperm motility and
increased production of reactive oxygen species.35
Furthermore, studies in rats showed that oral exposure to
AgNPs reduces sperm production22 and integrity, compro-
mises the integrity of plasma and acrosome membranes,
reduces the mitochondrial activity in the mid-piece of the
sperm, and alters the reproductive behavior.21
Healthy spermatozoa are the final product of a complex
process controlled by the hypothalamic–pituitary–testicular
(HPT) axis. In this context, this study sought to investigate the

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molecular mechanisms involved in the dose–response effects Results

of four dosages of AgNPs (1.875, 3.75, 7.5 or 15 µg AgNPs per
kg) on the regulation of the HPT axis and the testicular func-
tion, using prepubertal male Wistar rats as an experimental
model. During the entire experimental period (from postnatal
day (PND) 23 through PND60) the animals were weighed daily
to follow their growth, and upon reaching puberty the weight
and age were recorded.
The secretion of gonadotropins is finely regulated by the
hypothalamus, where the pulse frequency of gonadotropin-
releasing hormone (GnRH) controls the differential expression
of LH and FSH subunit genes by the gonadotrophs in the pitu-
itary.36,37 Thus, the hypothalamus was evaluated for the tran-
script expression (mRNA) of gonadotropin releasing hormone 1
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Characterization of AgNPs
Prior to initiating the experimental protocol it was necessary to
determine the AgNP diameter and polydispersity indexes
before and after dilution of the commercial solution. The
dilution did not alter the particle diameter, which was esti-
mated to be 79.5 ± 11 nm and the polydispersity index
measured was 0.575 ± 118. The stability of AgNPs in simulated
gastric fluid is presented in ESI Table 1.†
The effects of prepubertal AgNP exposure on the body growth,
onset of puberty and reproductive organ weights
Following AgNP prepubertal exposure the rats were routinely

(Gnrh1); and the pituitary was evaluated for the transcript weighed to monitor body growth, and had no effect on any of
expression of gonadotropin releasing hormone receptor the treated groups, as compared to controls (ESI Fig. 1†).
(Gnrhr), luteinizing hormone beta polypeptide (Lhb), and fol- However, the onset of puberty was delayed in the groups
licle stimulating hormone beta subunit (Fshb). Additionally, the receiving AgNP dosages 7.5 and 15 µg kg−1, when compared to
serum LH and FSH concentrations were measured in the blood. the controls (Fig. 1). Furthermore, the weights of the testis, epi-
The negative feedback mechanism was investigated in both didymis segments, seminal vesicle and seminal fluid were not
the hypothalamus and pituitary by the transcript expression of affected by any of the treatment dosages (Table 1).
the androgen receptor (Ar), estrogen receptor 1 (Esr1) and
estrogen receptor 2 (Esr2), since these receptors may be Effects of AgNPs on gonadotropin production
involved in controlling the synthesis and release of GnRH and
Towards the goal of understanding how AgNPs modulate gon-
LH.38 The transcript expression of inhibin B (Inhbb) in the
adotropin production, we first examined the transcript
testis was also analyzed, because of its participation in the
expression of Gnrh in the hypothalamus, since this can influ-
negative feedback of FSH.39
ence the expressions of LH and FSH in the pituitary. As shown
Once in the blood, LH stimulates the synthesis of testoster-
in Fig. 2A, only the 3.75 µg AgNP per kg-treated group dis-
one by binding to the luteinizing hormone/choriogonadotro-
played an increase in Gnrh expression, as compared to con-
pin receptor (LHCGR) in the Leydig cells in the testis.40 Thus,
trols; however, the transcript expression of Gnrhr in the pitu-
the testes were evaluated for the transcript expression of Lhcgr
itary was unaffected (Fig. 2B).
and the serum testosterone concentration was measured in the
While the Lhb transcript expression in the pituitary did not
blood. In the Sertoli cells, testosterone binds to the androgen
change in any of the treated groups (Fig. 3A), the serum con-
receptor (AR) or is converted into estradiol by the aromatase
centrations of LH increased in the 3.75 µg AgNP per kg-treated
(CYP19A1) enzyme, both of these events are related to the sper-
group, as compared to the controls (Fig. 3B). There were also
matogenesis process.40 Therefore, the relative transcript
no significant changes in the Fshb transcript expression in the
expressions of Ar and Cyp19a1 were evaluated in the testis and
pituitary (Fig. 3C), yet the serum concentrations of FSH
the serum estradiol concentration was measured in the blood.
increased in the 1.875 µg AgNP per kg-treated group, when
Spermatogenesis events take place in the Sertoli cells, and
compared to the controls (Fig. 3D).
are mainly influenced by the action of FSH, which binds to the
follicle stimulating hormone receptor (FSHR) to perform its
action.40 Estradiol is another important hormone for the sper-
matogenesis process, interacting with estrogen receptors
(ESR1 and ESR2) located in the nucleus and cytoplasm or with
G protein-coupled estrogen receptor 1 (GPER) in the plasma
membrane, which is crucial for the regulation of proteins
involved in the function of the testis.41 In this sense, we evalu-
ated the transcript expression of Fshr, Esr1, Esr2 and Gper in
the testis.
Finally, the sperm production, sperm reserves and sperm
transit time in the epididymis segments were evaluated. The
sperm functionality was assessed by the verification of acro-
Fig. 1 Age at puberty onset for rats exposed to 0 (control), 15, 7.5, 3.75
some integrity, plasma membrane integrity, sperm defects and
or 1.875 µg of AgNPs per kg BW. The values are expressed as median
mitochondrial activity, since these methods more accurately and interquartile ranges (Kruskal–Wallis followed by the post-test of
indicate subfertility and infertility, which reflect the ability of Dunn), *P < 0.05 vs. control, n = 12 per group. AgNPs: silver nano-
the sperm to fertilize the oocyte.42–44 particles; BW: body weight.

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Table 1 The testis, epididymis, seminal vesicle and seminal fluid relative weights in animals exposed to silver nanoparticles (AgNPs)

AgNPs (µg kg−1)

Organ weight
(mg per 100 g BW) 0 1.875 3.75 7.5 15

Testis 487 ± 18 468 ± 7 494 ± 14 488 ± 10 524 ± 10

Caput + corpus epididymis 69 ± 1.5 64 ± 1.4 73 ± 1.5 74 ± 2 67 ± 2
Cauda epididymis 44.5 ± 1.2 47 ± 0.8 49 ± 1.2 42 ± 0.8 44 ± 1.5
Seminal vesicle undrained 232 ± 9 208 ± 14 223 ± 8 214 ± 11 198 ± 6
Seminal fluid 123 ± 8 139 ± 16 134 ± 7 125 ± 9 109 ± 6

Data were subjected to analysis of variance and are expressed as mean ± standard error of the mean; n = 12 animals per group.

per kg-treated groups, when compared to the controls

(Fig. 4C).
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In the pituitary, the Ar transcript expression was decreased

in the 1.875, 3.75 and 7.5 µg AgNP per kg-treated groups
(Fig. 5A), the Esr1 transcript expression was not altered in any
of the treated groups (Fig. 5B) and the Esr2 transcript
expression was decreased in the 1.875, 3.75 and 7.5 µg AgNP
per kg-treated groups, as compared to the controls (Fig. 5C).
In the testis, the Inhbb transcript expression was found to
be increased only in the 7.5 µg AgNP per kg-treated group,
when compared to controls (Fig. 6).

Effects of AgNPs on testicular steroidogenesis

Fig. 2 Transcript expression of Gnrh (A) and Gnrhr (B) for rats exposed
to 0 (control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per kg BW. The Rpl19
was used as an internal control. The values are expressed as the mean ±
S.E.M. (ANOVA followed by the post-test of Dunnett), *P < 0.05 vs.
control, n = 12 per group. Gnrh: Gonadotropin releasing hormone;
Gnrhr: gonadotropin releasing hormone receptor; Rpl19: ribosomal
protein L19; AgNPs: silver nanoparticles; BW: body weight; SEM: stan-
dard error of the mean.
Effects of AgNPs on the expression of genes controlling the
negative feedback mechanisms in the hypothalamic–pituitary–
testicular axis and estradiol receptors
Next we investigated molecular components of the HPT axis
that are involved in the negative feedback mechanism regulat-
ing the synthesis and secretion of GnRH, LH and FSH. In the
hypothalamus, the Ar transcript expression was not altered in
any of the groups treated with AgNPs (Fig. 4A). However, the
Esr1 transcript expression was decreased in the 7.5 µg AgNP
per kg-treated group (Fig. 4B), and the Esr2 transcript
expression was decreased in the 1.875, 3.75 and 7.5 µg AgNP
Having established that the onset of puberty and that the tran-
script expression levels of some of the molecular components
involved in gonadotropin synthesis and secretion were modu-
lated, we then investigated the effects of AgNP exposure on tes-
ticular steroidogenesis. The Lhcgr transcript expression in the
testis was not altered in any of the groups treated with AgNPs
(Fig. 7A). An increase in the serum concentrations of testoster-
one was observed in the 1.875 µg AgNP per kg-treated group,
when compared to the controls (Fig. 7B). Interestingly, the
estradiol serum concentration was increased in the 7.5 AgNP
per kg-treated group (Fig. 7D), despite not being able to detect
a significant increase ( p = 0.08) in the transcript expression of
aromatase (Cyp19a1) for this group, when compared to the
controls (Fig. 7C).
Effects of AgNPs on the expression of genes related to
spermatogenesis
Spermatogenesis relies heavily on the action of FSH and the
estrogen receptors; thus the transcript expression levels were
measured. The Ar transcript expression was reduced in the
1.875 and 3.75 µg AgNP per kg-treated groups compared to the
control (Fig. 8A), whereas the Fshr transcript expression in the
testis was not altered (Fig. 8B). In relation to the estradiol
receptors, the transcript expression of Gper (Fig. 9A) was
reduced in the 1.875 µg AgNP per kg-treated group, and the
transcript expression of Esr1 was reduced in the 1.875 and
3.75 µg AgNP per kg-treated groups, when compared to the
controls (Fig. 9B). On the other hand, the expression of Esr2
was not affected by AgNP treatment (Fig. 9C).

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Fig. 3 Pituitary transcript expression of Lhb (A) and Fshb (C), serum concentrations of LH (B) and FSH (D) for rats exposed to 0 (control), 15, 7.5,
3.75 or 1.875 µg of AgNPs per kg BW. The Rpl19 was used as an internal control. The values are expressed as the mean ± S.E.M. (ANOVA followed by
the post-test of Dunnett), *P < 0.05 vs. control, n = 12 per group. Lhb: luteinizing hormone beta polypeptide; Fshb: follicle stimulating hormone beta
subunit; LH: lutropin; FSH: follitropin; Rpl19: ribosomal protein L19; AgNPs: silver nanoparticles; BW: body weight; SEM: standard error of the mean.

Effects of AgNPs on the sperm production, sperm reserves and
sperm transit time
With regard to sperm parameters, none of the AgNP dosages,
used in this study, had an effect on sperm production
(Table 2). However, the sperm reserves (Fig. 10A) and the
sperm transit time (Fig. 10C) through the caput epididymis
were decreased in the 1.875 µg AgNP per kg-treated group,
when compared to controls. On the other hand, the sperm
reserves (Fig. 10B) and the sperm transit time (Fig. 10D) in the
cauda epididymis were not affected by AgNP treatment.
Effect of AgNPs on the sperm functionality
Finally, we examined sperm functionality parameters, which
may impact the ability of the sperm to fertilize the oocyte. The
acrosome integrity was reduced in the 15 µg AgNP per kg-
treated group, when compared to controls (Fig. 11A); however,
the plasma membrane integrity and the sperm morphology
were unchanged in all of the AgNP dosages utilized in this
study (Fig. 11B and C). The mitochondrial activity was reduced
in the 7.5 and 15 µg AgNP per kg-treated groups, as compared
to controls (Fig. 11D).
Discussion
Reproductive physiology involves complex biological processes
that may be sensitive to environmental chemical contami-
nants, which may increase the incidence of male reproductive
disorders.45 In the present study, we investigated the mole-
cular mechanisms involved in the dose–response effects of
four dosages of AgNPs (1.875, 3.75, 7.5 or 15 µg AgNPs per kg)
on the regulation of the HPT axis and the testicular function
using prepubertal male Wistar rats as an experimental model.
The mean size of silver nanoparticles estimated by dynamic
light scattering was 79.5 nm, possibly due to differences in the
evaluation method (the manufacturer used transmission elec-
tron microscopy7,46). In the simulated gastric fluid, the mean
particle size of AgNPs increased with time in the low pH
environment of the stomach. These data are similar to those
reported by Pinďáková et al. (2017).47 In the simulated intesti-
nal fluid, the AgNPs did not present this behavior, remaining
dispersed in the solution.47 After oral exposure, the liver, the
kidney and the blood present higher levels of AgNPs, which
consistently decline in the subsequent weeks.48 Nevertheless,
in the testis and brain the levels are still elevated.48–50 This
may contribute to the development of the toxic effects
observed in this study.
The central nervous system is the main initiator of puberty
in rats. A network not yet fully understood controls the GnRH
neurons, in which before puberty their activity is predomi-
nantly inhibitory and after puberty predominantly excitatory.51
Prior to puberty, a pulsatile release of GnRH is increased and
stimulatory events lead to the release of LH and FSH from pitu-
itary, which will perform their action in the testicular cells.51–53

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Fig. 4 Hypothalamic transcript expression of Ar (A), Esr1 (B) and Esr2
(C) for rats exposed to 0 (control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per
kg BW. The Rpl19 was used as an internal control. The values are
expressed as the mean ± S.E.M. (ANOVA followed by the post-test of
Dunnett), *P < 0.05 and **P < 0.01 vs.control, n = 12 per group. Ar:
androgen receptor 1; Esr1: estradiol receptor 1; Esr2: estradiol receptor
2; Rpl19: ribosomal protein L19; AgNPs: silver nanoparticles; BW: body
weight; SEM: standard error of the mean.
The action of FSH in the Sertoli cells helps to coordinate the
onset of puberty and progression, but the androgens play a
more relevant role in the process.52 The balanopreputial separ-
ation is a good indicator of the progress of puberty in rats
because there is a positive correlation between the rise in the
serum concentrations of testosterone at the beginning of
puberty and the externalization of the penis from the glans.54
Exposure to chemicals with estrogenic or antiandrogenic effects
can delay the onset of puberty in males.55,56 The delayed
puberty in the rats, treated with 7.5 and 15 µg AgNPs per kg,
observed in this study suggests that hormonal imbalances
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Fig. 5 Pituitary transcript expression of Ar (A), Esr1 (B) and Esr2 (C) for
rats exposed to 0 (control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per kg BW.
The Rpl19 was used as an internal control. The values are expressed as
the mean ± S.E.M. (ANOVA followed by the post-test of Dunnett), *P <
0.05, **P < 0.01 and ***P < 0.001 vs. control, n = 12 per group. Ar:
androgen receptor 1; Esr1: estradiol receptor 1; Esr2: estradiol receptor
2; Rpl19: ribosomal protein L19; AgNPs: silver nanoparticles; BW: body
weight; SEM: standard error of the mean.
occurred during the prepubertal period. In addition to these
events, peripheral insulin growth factor 1 (IGF-1) stimulates
GnRH secretion, which correlates positively with body growth
and the progress of puberty.51 In this study, the body develop-
ment was not affected by AgNP oral exposure during the prepu-
bertal period. In fact, this parameter is affected by oral exposure
to AgNPs only at higher dosages (125 mg kg−1) and after longer
periods of treatment in male rats.57
After puberty, the hypothalamic neurons release GnRH in a
pulse frequency that stimulates the expression of Lhb and Fshb
and the secretion of LH and FSH by the gonadotrophs in the
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and the transcript expression of Esr2 is reduced (1.875, 3.75

Fig. 6 Testicular transcript expression of Inhbb for rats exposed to 0
(control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per kg BW. The Rpl19 was
used as an internal control. The values are expressed as the mean ±
S.E.M. (ANOVA followed by the post-test of Dunnett), *P < 0.05 vs.
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and 7.5 µg AgNPs per kg), which could suggest that there is a
disruption in estradiol signaling in the hypothalamus.
Furthermore, AgNP treatment (7.5 µg AgNPs per kg) also
reduced the transcript expression of Esr1 in the hypothalamus.
The transcript expressions of gonadotropins (Lhb and Fshb)
were not affected by treatment, but the serum concentrations
of LH and FSH were increased (3.75 and 1.875 µg AgNPs per
kg, respectively). This increase may be related to mechanisms
involved in the posttranscriptional regulation, such as the half-
life of mRNA in the cytoplasm, the translational rate of
mRNA,62 or through altered gonadotropin secretion.63
The negative feedback mechanism in the hypothalamus
and pituitary is mediated by testosterone binding to the AR,

control, n = 12 per group. Inhbb: Inhibin beta B subunit; Rpl19:
ribosomal protein L19; AgNPs: silver nanoparticles; BW: body weight;
SEM: standard error of the mean.
pituitary.36,37 The regulation of GnRH-producing neurons is
still under investigation,58 but is suggestive that estradiol
receptors mediate their activity.59 Estradiol binds to ESR2 acti-
vating the transcriptional complex, which promotes the tran-
scription of the GnRH gene,60 while ESR1 activation is the pre-
dominant mechanism involved in the estradiol-mediated sup-
pression of GnRH synthesis/release.61 In this study, the tran-
script expression of Gnrh1 is increased (3.75 µg AgNPs per kg)
which represses the synthesis and release of LH, while the
negative feedback of FSH is mediated, in the pituitary, by the
action of inhibin b, which represses its synthesis and
release.39 The transcript expression of Ar is reduced in the
pituitary of rats (1.875, 3.75 and 7.5 µg AgNPs per kg), weaken-
ing the negative feedback mechanism controlling the synthesis
and secretion of LH. The transcript expression of Inhbb is only
elevated in the 7.5 µg AgNPs per kg group. These molecular
mechanisms are unclear, but the disruption in the LH syn-
thesis and secretion may perturb the fine regulation of FSH,
since it is produced by the same pituitary cells.
In the testis, LH stimulates the synthesis of testosterone by
the Leydig cells.40 The exposure to AgNPs increased the serum

Fig. 7 Testicular transcript expression of Lhcgr (A) and Cyp19a1 (C), serum concentrations of testosterone (B) and estradiol (D), for rats exposed to
0 (control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per kg BW. The Rpl19 was used as an internal control. The values are expressed as the mean ± S.E.M.
(ANOVA followed by the post-test of Dunnett), *P < 0.05 and **P < 0.01 vs. control, n = 12 per group. Lhcgr: luteinizing hormone/choriogonadotro-
pin receptor; Cyp19a1: aromatase; Rpl19: ribosomal protein L19; AgNPs: silver nanoparticles; BW: body weight; SEM: standard error of the mean.

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Fig. 8 Testicular transcript expression of Ar (A) and Fshr (B) for rats
exposed to 0 (control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per kg BW. The
Rpl19 was used as an internal control. The values are expressed as the
mean ± SEM; ANOVA followed by the post-test of Dunnett; *P < 0.05
and ***P < 0.001 vs. control, n = 12 animals per group; Ar: androgen
receptor; Fshr: follicle stimulating hormone receptor; Rpl19: ribosomal
protein L19; AgNPs: silver nanoparticles; BW: body weight; SEM: stan-
dard error of the mean.
concentrations of testosterone (1.875 µg AgNPs per kg). In pre-
vious studies using higher doses (15, 30 or 50 µg AgNPs per
kg) this parameter was not affected.21,22
Testosterone binds to the AR in the Sertoli cells40 and plays
a key role in the maintenance of spermatogenesis, participat-
ing in the maintenance of the blood–testis barrier, as well as
the adhesion and release of sperm from the Sertoli cells.64,65
The transcript expression of Ar was reduced (1.875 and 3.75 µg
AgNPs per kg).
Normal sexual development and reproduction in mammals
depends on the balance between androgens and estrogens. In
the testis, maintaining this balance relies on endocrine and
paracrine factors, but it is also related to the activity of aroma-
tase, an enzyme complex located in the endoplasmic reticulum
that ensures the conversion of androgen into estrogen.66
Testicular estradiol is produced in the testis from the aromatiza-
tion of testosterone by the aromatase cytochrome P450 family
19 subfamily A member 1 enzyme, encoded by the gene
CYP19A1.67 In the present study, serum estradiol concentrations
were increased (7.5 µg AgNPs per kg), whereas Cyp19a1
expression was not altered in any treated group. Interestingly,
the estradiol serum concentrations and aromatase expression
were not affected in the 1.875 µg AgNP per kg-treated group
(increased testosterone), which may indicate a protective mecha-
nism avoiding higher and more deleterious estradiol levels.68
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Fig. 9 Testicular transcript expression of Gper (A), Esr1 (B) and Esr2 (C)
for rats exposed to 0 (control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per kg
BW. The Rpl19 was used as internal control. The values are expressed as
the mean ± S.E.M. (ANOVA followed by the post-test of Dunnett), *P <
0.05 and **P < 0.01 vs. control, n = 12 per group. Gper: estrogen recep-
tor 1 coupled to protein G; Esr1: estradiol receptor 1; Esr2: estradiol
receptor 2; Rpl19: ribosomal protein L19; AgNPs: silver nanoparticles;
BW: body weight; SEM: standard error of the mean.
The estradiol interacts with estrogen receptors located in
the nucleus and cytoplasm (ESR1 and ESR2) or in the plasma
membrane (GPER), which is crucial for the regulation of pro-
teins involved in the function of the testis.41 In fact, ESR1 and
ESR2 knockout mice exhibit compromised spermatogenesis.69
Furthermore, ESR1 and ESR2 were found in the testis of adult
and immature rats and were also expressed in co-culture of
Sertoli cells obtained from mice at 15 days of age.70
Additionally, GPER has been detected in the extranuclear
regions of Sertoli cells and can regulate the expression of
genes involved in cell apoptosis.71 It has also been shown that
ESR1 and ESR2 are involved in the control of cell proliferation
and differentiation by modulating enhancers and inhibitors of
the cell cycle.72 Additionally, ESR1 is necessary for the viability
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Table 2 Sperm production in animals exposed to silver nanoparticles (AgNPs)

AgNPs (µg kg−1)

Sperm production 0 1.875 3.75 7.5 15

Total
×106 per testis 97.1 ± 3.5 98.3 ± 8.5 92.7 ± 8.3 98.1 ± 10.0 99.4 ± 7.2
×106 per g testis 69.0 ± 2.9 70.4 ± 6.0 64.8 ± 5.9 69.8 ± 7.5 65.6 ± 5.4
Daily
×106 per testis 15.9 ± 0.5 16.1 ± 1.4 15.2 ± 1.3 16.1 ± 1.6 16.3 ± 1.1
×106 per g testis 11.3 ± 0.4 11.5 ± 0.9 10.6 ± 0.9 11.4 ± 1.2 10.8 ± 0.8

Data were subjected to analysis of variance and are expressed as the mean ± standard error of the mean; n = 12 animals per group.
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Fig. 10 Sperm reserves (A and B) and sperm transit time (C and D) in epididymis segments for rats exposed to 0 (control), 15, 7.5, 3.75 or 1.875 µg
of AgNPs per kg BW. The values are expressed as the mean ± SEM; ANOVA followed by the post-test of Dunnett; *P < 0.05 vs. control, n = 12 per
group. AgNPs: silver nanoparticles; BW: body weight; SEM: standard error of the mean.

of germ cells and is involved in chromatin condensation and
DNA integrity of spermatozoa.73 In this study, the expression
of the transcripts of Gper was decreased in the group receiving
the lowest dosage (1.875 µg AgNPs per kg), whereas the tran-
scripts of Esr1 were decreased in the groups treated with 1.875
and 3.75 µg AgNPs per kg. As a consequence of these altera-
tions a reduction in the sperm production could occur, but it
was surprisingly not affected. A possible explanation is that
the spermatic cycle could be compromised, because Sleiman
et al.22 only observed a reduction in the sperm production
thirty-seven days after AgNP exposure. In this sense, the com-
promised spermatogenesis observed in these groups exposed
to lower doses of AgNPs should not be disregarded.
Furthermore, the sperm reserves and the sperm transit
time in caput and corpus epididymis were reduced in the
1.875 µg AgNP per kg-treated group. During the transit
through the epididymis segments, the sperm released from
the lumen of seminiferous tubules undergoes maturation and
acquires motility and the ability to fertilize oocytes.74 The
sperm transit time through the epididymis segments is due to
the movement of the fluid secreted by the Sertoli cells, which
results from the contractile activity of the smooth muscle epi-
didymis, which is controlled by androgen action and the auto-
nomic nervous system.75 In this study, the reduced sperm
reserves and the sperm transit time may be due to increased
serum concentrations of testosterone which could stimulate
the contraction of the smooth muscle of the epididymis; as a
result these increased contractions could contribute to the
observed decrease in sperm transit time.
Next, the sperm collected from the cauda epididymis was
subjected to the functional analysis of acrosome integrity,
plasma membrane integrity, mitochondrial activity and sperm

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Fig. 11 Frequency of acrosome integrity (A), plasma membrane integrity (B), defects (C) and mitochondrial activity (D) of the sperm for rats exposed
to 0 (control), 15, 7.5, 3.75 or 1.875 µg of AgNPs per kg BW. The values are expressed as the mean ± SEM; ANOVA followed by the post-test of
Dunnett; *P < 0.05 vs. control, n = 12 per group. AgNPs: silver nanoparticles; BW: body weight; SEM: standard error of the mean.
morphology. In this study, the sperm morphology and the
plasma membrane integrity were not affected. The acrosome
integrity was reduced (15 µg AgNPs per kg) and the mitochon-
drial activity was reduced (7.5 and 15 µg AgNPs per kg). The
acrosome plays a fundamental role in the fertilization process
and the reduction in its integrity may compromise the ability
to fertilize.76 Mitochondria are located in the middle piece of
the sperm, which produces energy to move the flagellum and
propel the spermatozoa through the genital tract. In this way, a
reduction in its function may compromise sperm mobility and
consequently hinder fertilization.77 Alterations in the group
receiving the higher dose (15 µg AgNPs per kg) were previously
demonstrated by our group,21 and this was included in this
study to represent the highest dosage in the dose–response
curve. Thus, it was possible to infer a LOAEL (Lowest Observed
Adverse Effect Level) of 7.5 µg AgNPs per kg and a NOAEL (No
Observed Adverse Effect Level) of 3.75 µg AgNPs per kg for
spermatic function.
Taking all these results together, some differences were
observed among the AgNP-treated groups. The groups receiv-
ing 1.875 and 3.75 µg AgNPs per kg did not present any
alteration in the functional parameters analyzed, but only in
the analysis at the molecular level. The group receiving
1.875 µg AgNPs per kg was the only group to display
increased serum concentrations of FSH and testosterone. The
group receiving 3.75 µg AgNPs per kg was the only group to
present increased transcript expression of Gnrh1 and
increased LH serum concentrations. The group receiving
7.5 µg AgNPs per kg had delayed puberty onset, reduced mito-
110 | Toxicol. Res., 2018, 7, 102–116
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Toxicology Research
chondrial activity, decreased hypothalamic expression of
Esr1, increased expression of Inhbb and elevated serum con-
centrations of estradiol. The group receiving 15 µg AgNPs per
kg presented delayed puberty onset and decreased sperm
functionality, yet all of the other parameters were unaffected.
These differences may be related to the levels of dose–
response in the HPT axis, and help in understanding the
meaning of the modulation of the studied genes and the
functional parameters. At lower doses (1.875 and 3.75 µg
AgNPs per kg), the modulation of the genes in the HPT axis
avoid disturbances in its function. At higher dose (15 µg
AgNPs per kg), the genes are not being modulated but the
function is compromised.
Finally, humans may be exposed to AgNPs by everyday con-
sumer products which are nano-functionalized13 and for
which the safety limits of exposure are still under evaluation.16
It is known that AgNP-impregnated toothbrushes release Ag,
which may cause human and environmental exposure to
AgNps.14 Quadros et al.15 tested the amount of AgNPs leached
by infant products ( plush toys, baby blankets, and sippy cups),
and found that 43.8% of the AgNPs leached from a sippy cup
(spout cover), representing 0.93 ± 0.02 mg kg−1 product per
day.15 The same study was also evaluated by probabilistic
methods to assess the relative uncertainty and potential risks
of exposure of infants to AgNPs and it was concluded that the
leach from these infant products exhibited a minimal risk.78
The relevance of human exposure to low doses of AgNPs is a
controversial issue and in this sense it is necessary to perform
more risk assessment studies.
This journal is © The Royal Society of Chemistry 2018

Toxicology Research
Experimental
Materials and methods
Characterization of AgNPs. A suspension of silver nano-
particles 60 nm in diameter was purchased from Sigma-
Aldrich (catalog number 730815, Sigma-Aldrich Co., Seelze,
Germany). Dilution with water was performed just before the
suspension was administered to the animals according to our
previous study.21 To evaluate whether diluting the AgNP solu-
tion affected the nanoparticle stability, we analyzed the mean
particle size and polydispersity index by dynamic light scatter-
ing (DSL) (BIC 90 plus – Brookhaven Instruments Corp., USA)
at a scattering angle of 90° and a temperature of 25 °C, with
the help of ZetaPlus Particle Sizing Software Version 5.34
Published on 23 November 2017. Downloaded on 03/05/2018 03:38:16.
(Brookhaven Instruments Corp., USA). The particle size of
AgNPs was evaluated in simulated gastric fluid. For this,
AgNPs were diluted in ultra-purified water or acidic conditions
(simulated gastric fluid, SGF, without pepsin, pH 1.2; USP
201279) to a final concentration of 0.004 mg mL−1 and kept in
a platform shaker at 200 rpm and 37 °C. The samples were
taken after 0, 30, 60 and 120 minutes and analyzed by DLS
under the same conditions as those described above.
Experimental design. The experimental design was based on
a protocol suggested by Stoker et al.56 from the Endocrine
Disrupting Screening and Testing Advisory Committee
(EDSTAC) and includes the evaluation of endocrine-disrupting
chemical effects in prepuberty and puberty. Sixty newly
weaned male Wistar rats (Rattus norvegicus var. albinus) were
collected from pregnant female rats that had been monitored
from the 17th day of pregnancy to determine the exact day of
birth. On postnatal day 4 (PND4), 10 litters were culled to 8
pups (4 males/4 females) per female and kept at this size until
weaning (PND21). On PND23, the male offspring were divided
into five groups containing 12 animals each, and received
either 0 (control), 1.875, 3.75, 7.5 or 15 μg of AgNPs per kg of
body weight (BW) from PND23 to PND60. The highest dose of
AgNPs for the dose–response curve used in this study was
based on the experimental toxicological values for the lowest
observable adverse effect level (LOAEL) for sperm parameters
(15 μg kg−1),21 and corresponds to a dose that is 2000-fold
lower than the NOAEL for liver toxicity.17,57 The AgNP water
suspension was administered every other day at a volume of
0.25 ml per 100 g BW by gavage. The control group received
only water. During all experiments, the animals were kept in
groups of four, housed in polypropylene cages (43 × 43 ×
20 cm) with a 5 cm layer of wood shavings, fed commercial
feed (Nuvilab CR-1, Nuvital, PR, Brazil) and were allowed
access to water ad libitum, under a 12 : 12 hour dark/light cycle
in a temperature-controlled room (23 ± 1 °C). All procedures
were performed in accordance with the Conselho Nacional de
Controle de Experimentação Animal (CONCEA) and were
approved by the Universidade Estadual do Centro-Oeste
Ethical Committee for Animal Research ( protocol 013/2015).
Puberty and body growth. An evaluation of the separation of
the preputial membrane and the externalization of the glands
of the penis (balanopreputial separation, PPS) was used to
This journal is © The Royal Society of Chemistry 2018
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Paper
determine the age at the onset of puberty.54 This evaluation
was performed on PND33 and was continued every other day
throughout the balanopreputial separation period by gentle
tissue manipulation. The animals were weighed every other
day from PND23 to PND60 to determine body growth.
Tissue collection. At PND60, animals were euthanized by
decapitation under deep anesthesia with ketamine and xyla-
zine. Blood was collected by cardiac puncture, immediately
centrifuged at 205g (II Excelsa BL 206, Sao Paulo, SP, Brazil)
for 15 min, and the serum was maintained at −80 °C until
further analysis of hormonal dosages of LH, FSH, testosterone
and estradiol. The hypothalamus, the pituitary and testes were
immediately excised, frozen in liquid nitrogen and kept at
−80 °C until further analysis of gene expression by RT-qPCR.
The testes, epididymis (caput, corpus and cauda), seminal
vesicles and seminal fluid were weighed and converted to mg
of tissue per 100 mg of body weight.
Assessment of sperm functionality. The sperm was collected
from the cauda epididymis by making an incision with a
scalpel blade and squeezing out the fluid. Twenty microliters
of the drained fluid containing spermatozoa were suspended
in 200 µl of a seminal extender (328.8 mM Tris, 91.3 mM fruc-
tose, and 115.8 mM citric acid) at 37 °C. The samples were
then immediately subjected to functional analysis of the acro-
some integrity, plasma membrane integrity, mitochondrial
activity and sperm morphology, as previously described.21,80
The acrosome integrity was evaluated by using a single-
stain solution containing 1% (w/v) rose bengal (cat. number
330000, Sigma-Aldrich Co, MO, USA), 1% (w/v) fast green FCF
(cat. number F7252, Sigma-Aldrich Co, MO, USA), and 40%
ethanol in 200 mM disodium phosphate buffer containing
100 mM citric acid at pH 7.2. Five microliters of each sample
were incubated with 5 µl of the single-stain solution for 1 min
at 37 °C. The total volume (10 µl) was then pipetted onto a
slide and smeared with another slide, air-dried at 37 °C and
analyzed by light microscopy (1000×). Two hundred spermato-
zoa were counted per slide and were classified as having intact
or damaged acrosomes.
The plasma membrane integrity was evaluated using eosin–
nigrosin stain [1% eosin Y (Sigma-Aldrich, MO, USA) and 10%
nigrosin (Sigma-Aldrich, MO, USA) in distilled water]. Five
microliters of each sample were mixed with 5 µl of the stain,
and the total volume was pipetted onto a slide, smeared with
another slide, and air-dried at room temperature. Two
hundred spermatozoa were counted per slide by light
microscopy (1000×) and were classified as having intact or
damaged membranes.
The mitochondrial activity was evaluated by measuring the
enzyme activity of cytochrome c oxidase in the intermembrane
space of the spermatozoa using DAB (3,3′-diaminobenzidine,
Sigma-Aldrich, MO, USA). DAB was diluted in PBS (2.7 mM
KCl, 137 mM NaCl, 8 mM NaHPO4, and 1.4 mM KPO4, pH 7.4)
to a final concentration of 1 mg ml−1 and frozen until use. Ten
microliters of each sample were incubated with 300 µl of a
DAB solution pre-heated to 37 °C for 1 h at 37 °C in the dark.
Ten microliters of each sample were pipetted onto a slide,
Toxicol. Res., 2018, 7, 102–116 | 111

Paper
smeared with another slide, and air-dried at 37 °C in the dark.
The slide samples were then incubated for 10 min in a fixative
solution containing 10% formaldehyde (F8775, Sigma-Aldrich
Co, MO, USA) in PBS and allowed to air-dry at 37 °C in the
dark. Two hundred spermatozoa from each sample were ana-
lyzed by phase-contrast microscopy at a magnification of 400×
and then classified based on the degree of staining of the
intermediate piece as follows: DAB I (completely stained), DAB
II (>50%), DAB III (<50%) and DAB IV (unstained).
The sperm morphology was evaluated as a wet preparation
by phase contrast microscopy (400× magnification) in diluted
samples in buffered formalin–saline solution (34.72 mM
Na2HPO4·2H2O, 18.68 mM KH2PO4, 92.4 mM NaCl, and 12.5%
(v/v) formaldehyde).
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Sperm production, sperm reserves and sperm transit time.
The sperm production, reserves and transit time were deter-
mined as previously detailed by Romano et al.,81 and accord-
ing to the method described by Robb et al.82 Briefly, the testes
were weighed, the tunica albuginea was removed and the testi-
cular parenchyma was homogenized in 5 ml of a saline solu-
tion containing 0.5% Triton X-100, followed by sonication at
12 kHz for 30 seconds (Ultrasonic homogenizer DES500;
Unique, Brazil), and the resistant spermatids were counted
using a hemocytometer. Daily sperm production was obtained
by dividing the sperm production by 6.1 [spermatids counted
in this method represent 48% of one cycle of the seminiferous
epithelium which has a duration of 12.75 days for rats (12.75 ×
0.48 = 6.1)]. Sperm reserves were calculated from epididymis
segments (caput, corpus and cauda) that were weighed and
minced, homogenized in 5 mL of 0.9% NaCl containing 0.5%
Triton X-100 followed by sonication at 12 kHz for 30 seconds
(Ultrasonic homogenizer DES500; Unique, Brazil) and the
spermatozoa were counted using a hemocytometer. Sperm
transit time was calculated by dividing the number of sperma-
tozoa in each segment by the daily sperm production of the
corresponding testis.
Reverse transcription followed by real-time quantitative PCR
(RT-qPCR). The hypothalamus, the pituitary and testes were
pulverized in liquid nitrogen and total RNA was extracted by
the guanidine–phenol–chloroform method,83 using Trizol
reagent (Life Technologies, Carlsbad, USA) according to the
manufacturer’s instructions. The hypothalamus was evaluated
for the transcript expression of Gnrh1, Ar, Esr1 and Esr2 rela-
tive to the expression of the ribosomal protein L19 (Rpl19).
The pituitary was evaluated for the transcript expression of
Gnrhr, Lhb, Fshb, Ar, Esr1 and Esr2 relative to the expression of
Rpl19. The testes were evaluated for the transcript expression
of Lhcgr, Fshr, Inhbb, Ar, Esr1, Esr2, Gper and Cyp19a1 relative
to the expression of Rpl19. Two and a half micrograms of total
RNA were reverse transcribed using oligo(dT) primer with the
help of a GoScript Reverse Transcription System kit (Promega,
Madison, USA) according to the manufacturer’s instructions.
Real-time PCR was carried out with an Applied Biosystems
StepOnePlus Real-time PCR System (Applied Biosystems,
Singapore) using a SYBR Green qPCR Platinum SuperMix-UDG
with the ROX (Life Technologies, Carlsbad, USA) kit under the
112 | Toxicol. Res., 2018, 7, 102–116
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Toxicology Research
following cycling conditions: 50 °C (2 min), 95 °C (2 min) and
40 cycles of 95 °C (15 s) and 60 °C (30 s). At the end of the reac-
tion, a melting curve was generated and analyzed to confirm
the specificity of the amplification. The average cycle threshold
(Ct) was automatically determined using StepOne Software
v2.3 (Applied Biosystems), and quantification was performed
using the 2−ΔΔCt method, as previously described.84 The
primer pairs are shown in ESI Table 2.†
Hormone measurements. Estradiol and testosterone serum
concentrations were analyzed by colorimetric competitive
enzyme immunoassay using a commercial Testosterone ELISA
kit and a 17β-estradiol ELISA kit (Enzo Life Sciences Inc.,
Farmingdale, NY, USA). The serum LH and FSH concentrations
were determined by a chemiluminescent immunoassay using
Luminex xMAP technology (Milliplex MAP rat pituitary panel;
Billerica, MA, USA). The intra-assay coefficients of variation
were <10% for testosterone, <8% for estradiol, <6% for LH and
<4.5% for FSH.
Statistical analysis. The variables were first subjected to the
Kolmogorov–Smirnov test for normality and the Bartlett test
for homoscedasticity. The analysis of body growth was per-
formed by multi-way analysis of variance for repeated
measures (MANOVA) using a general linear model (GLM). The
day of PPS was compared among the groups using nonpara-
metric analyses with the Kruskal–Wallis method followed by
the post hoc Dunn test. All other parameters were analyzed by
ANOVA followed by the post-test of Dunnett, comparing treated
groups only with the control group. Serum concentrations of
estradiol and the Cyp19a1 relative expression were compared
by Pearson’s correlation. All analyses were performed with
Statistica 7.0 (StatSoft Inc., Tulsa, OK). Statistical differences
were considered significant when P was less than 0.05.
Conclusions
In this study, it was observed that AgNPs modulate the
expression of genes related to the control of the HPT axis and
spermatogenesis with repercussions in the production of
serum testosterone and estradiol concentrations only in the
group receiving the lowest dosage of AgNPs, while functional
alterations of the sperm were observed in the group receiving
the higher dosages. This is suggestive of a disruption of the
HTP axis by AgNPs during the prepubertal and pubertal
periods, which are the most susceptible windows for the endo-
crine-disrupting chemical activity.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This study was supported by the Coordenação de
Aperfeiçoamento de Pessoal de Nivel Superior, Brazil
This journal is © The Royal Society of Chemistry 2018

Toxicology Research
[23038.009865/2013-32] and Fundacao Araucaria, Brazil [516/
2014].
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