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Toxicol Mech Methods, 2013; 23(3): 161–167
! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2013.764950
RESEARCH ARTICLE
Toxic effects of repeated oral exposure of silver nanoparticles on small
intestine mucosa of mice
Department of Anatomy, Electron Microscope Facility, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
Abstract
Keywords
As the use of silver nanoparticles (AgNPs) is increasing fast in industry, food, medicines, etc.,
exposure to AgNPs is increasing in quantity day by day. So, it is imperative to know the adverse
effects of AgNPs in man. In this study, we selected mice as an animal model and observed the
effect of AgNPs on small intestinal mucosa. AgNPs ranging from 3 to 20 nm were administered
orally at a dose of 5, 10, 15 and 20 mg/kg body weight to the Swiss-albino male mice for 21 d.
There was a significant decrease (p50.05) in the body weight of mice in all the AgNPs-treated
groups. Mice treated at a dose of 10 mg/kg showed the maximum weight loss. Effects were
noted by using light microscopy as well as transmission electron microscopy. It was found
that AgNPs damage the epithelial cell microvilli as well as intestinal glands. It may be
hypothesized that loss of microvilli reduced absorptive capacity of intestinal epithelium and
hence weight loss.
Microvilli, mitotic figure, silver nanoparticles,
transmission electron microscope
Introduction
Since ancient civilizations, man has used silver in medicine,
eating utensils, ornaments, coins, clothes and as a disinfectant
for water and for treating human wounds (Castellano et al.,
2007; Richard et al., 2002). Now a day’s, new forms of silver
having particle size less than 100 nm, i.e. silver nanoparticles
(AgNPs) comes into focus due to its anti-bacterial activity
(Alt et al., 2004; Martinez-Gutierrez et al., 2010; Nersisyan
et al., 2003; Panacek et al., 2006; Shrivastava et al., 2007;
Sondi & Salopek-Sondi, 2004; Xing et al., 2011), anti-viral
and anti-tumor activity (Huang et al., 2011) as well as antiplatelet property (Shrivastava et al., 2009). AgNPs are use in
daily life such as in medicine, toothpastes, paints, washing
machines, food materials, water purification and shampoos.
Due to these widespread uses, AgNPs released into the
environment are bound to spread either in the air, water or
soil. Already, It has been shown that AgNPs can enter the
human body through several ports, and there have been
several excellent reviews regarding intestinal uptake of
particles (Florence & Hussain, 2001; Hussain et al., 2001;
Jeong et al., 2010). There are reasons to be sure that exposure
*Present address: Department of Anatomy, Gajara Raja Medical College,
Gwalior, Madhya Pradesh, India.
Address for correspondence: Prof. Gajendra Singh, Department of
Anatomy, Electron Microscope Facility, Institute of Medical
Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh,
India. Tel: (O) þ91 542 – 2367568; (R) þ91 542 – 2369341. Fax: þ91
542 – 2368174. Mob: þ91 9415223588. E-mail: gajendrasinghims@
gmail.com
History
Received 4 November 2012
Revised 3 January 2013
Accepted 7 January 2013
Published online 19 February 2013
of gastrointestinal tract to the AgNPs must be taking place
consciously or unconsciously. Nanomaterials can be ingested
directly via water, food, cosmetics, drugs, drug delivery
devices, etc. (Oberdörster et al., 2005; Peters et al., 1997).
Beside its widespread use, toxicity study of AgNPs needs
further investigation. Present work was conducted to know the
effect of AgNPs on small intestine using light microscopy and
transmission electron microscope (TEM).
Methods
Animal model for in vivo study
A total of 50, 8–10 weeks-old Swiss albino male mice were
obtained from animal house of the Department of Anatomy,
Institute of Medical Sciences, Banaras Hindu University,
Varanasi, India. After being checked for infections of any kind
for 1 week, all 50 mice were weighed. Animals with an
average weight of 23 5 g were used for the study.
Commercial mice food (Pashu Aahar Kendra, Varanasi,
India) along with water was given ad libitum. Mice were
housed individually in plastic cages with a bed lined with
absorbent material, i.e. rice husk providing nesting material.
The cages were placed in a conventional room and bedding
changed daily. The room was air conditioned at 23 C and
40–60% humidity with a light/dark cycle of 12 h each. All the
work performed on animals was in accordance with and
approved by the Animal Ethics Committee of Institute of
Medical Sciences, Banaras Hindu University (Varanasi,
India). The animals were treated with utmost humane care
and all aseptic precautions were observed.
20
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Brigesh Shahare*, Madhu Yashpal, and Gajendra Singh
162
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Synthesis of AgNPs
AgNPs were synthesized essentially as described by
Shrivastava et al. (2007). Briefly, AgNO3 (0.017 g) was
dissolved in deionized water (100 ml) along with sodium
hydroxide (0.01 M) to form a solution of stable soluble
complex of silver ions. During this process, liquid ammonia
(30%) was added drop wise. Further, a mixture of D-glucose
(0.01 M) and hydrazine (0.01 M) were also added to the
solution of silver ions with continuous stirring. This ensures the
complete reduction of the silver ions at a final concentration of
0.005 M. The pH of the solution was adjusted to 7.4 with citric
acid (1 M). The brown solutions AgNPs were stored in closed
glass vials at 4 C for future experiments. Before each
experiment, the solution that contains nanoparticles was
sonicated (Labsonic 2000, B. Braun, Apeldoorn, The
Netherlands) for about 2 min and passed through filters of
0.2 mm pore size (Sartorius) in order to remove solid insoluble,
larger nanoparticles or particle agglomerates, if any.
Toxicol Mech Methods, 2013; 23(3): 161–167
recorded using a digital camera system attached to
microscope.
TEM of AgNPs treated cells
Different intestinal samples, either with or without nanoparticle pretreatment, were fixed in Karnovsky’s fixative followed by post-fixation in osmium tetraoxide. The tissues were
dehydrated through an ascending series of acetone concentrations, cleared in toluene and embedded in resin (Araldite
CY212) to prepare the blocks for TEM. Ultra-thin sections
(70 nm thick) were cut with an ultra-microtome (Leica EM
UC6, Leica Microsystems, Wetzlar, Germany). Samples were
mounted on Formvar-coated grids followed by staining with
uranyl acetate and lead citrate, and examined under a
Technai-12 (FEI, Eindhoven, The Netherlands) electron
microscope equipped with SIS Mega View III CCD camera
(FEI, Eindhoven, The Netherlands) at 120 KV. Measurements
were done using AnalySIS software (SIS, Muenster,
Germany).
Characterization of AgNPs
The size, morphology and dispersal of AgNPs were
characterized using a TEM model Technai 12 G2, FEI of
the Department of Anatomy, BHU, Varanasi. For TEM, the
sample were prepared by placing a drop of AgNPs on copper
grids (TAAB, England, UK) coated with 2% Collodion in
Amyl acetate (TAAB, England, UK) and subsequently dried
in air, before transferring it to the microscope operated at an
accelerated voltage of 120 kV.
Treatment of animals with AgNPs
Six to eight week old mice were identified by an ‘‘Ear Notch
Code’’ and subsequently divided into five groups (n ¼ 5 in
each group). Each group was treated with or without AgNP
solution as follows: first group (Control) received deionised
water as vehicle; second group (Group A) received 5 mg/kg
body weight (b.w.)/d AgNPs solution; third group (Group B)
received 10 mg/kg b.w./d AgNPs solution; fourth group
(Group C) received 15 mg/kg b.w./d AgNPs solution and
fifth group (Group D) received 20 mg/kg b.w./d AgNPs
solution. All mice were exposed to oral gabbage administration of vehicle and AgNP solution for 21 d. Body weight was
recorded weekly i.e. on day 0, 7, 14 and 21. Each mouse from
the experimental sets of study was carefully observed for any
signs of toxicity or ill health throughout the experiment and
was sacrificed on day 21 of AgNP treatment.
Collection of organ from mice
Body weight of all animals was recorded before killing. All
animals were sacrificed by the cervical dislocation.
Abdominal cavities of mice were cut open by a midline
abdominal incision to collect small intestine.
Histopathology of small intestine
Small intestine was fixed in aqueous Bouin’s fluid (Bouin,
1897) for histological studies. Thereafter, the organs were
embedded in paraffin, stained with hematoxylin and eosin,
and examined under light microscopy. Observations
were made on a binocular microscope. The results were
Statistical analysis
The experiment was terminated at the end of day 21 of
AgNP’s treatment and repeated three times to check the
reproducibility of the results obtained in the first experiment.
The data represented is the mean SEM of one experiment.
All the data were analyzed by one-way analysis of variance
followed by post-hoc test (Student Newman Keul’s test). The
level of significance was tested at p50.05 and confidence
limit 95% using SPSS software (Version 16.0; DST Centre for
Interdisciplinary Mathematical Sciences, Faculty of Science,
Banaras Hindu University, Varanasi, India).
Results
Characterization of AgNPs
TEM imaging of the AgNPs were performed to assess the
range of primary particle size, obtain the size distribution and
observe the general morphology of the particles. The size of
particles ranged from 3 to 20 nm (average diameter of AgNP
in the solution ¼ 10.15 nm) (Figure 1) and the shape of the
particles, in general, was either oval or circular (Figure 2).
Body weight of mice and food consumption
The most important finding observed during the course of
treatment was decrease in the body weight of mice. A
significant decrease in the body weight of mice was observed
in all the treated groups compared to the control on day 14
and 21 during the experiment (Figure 3). Among treated
groups, there was no significant change in the body weight
for the first 7 d, i.e. from day 0. However, after day 7, a
significant decrease in the body weight of mice was observed
on day 14 and 21 of the experiment. It is noteworthy that,
despite decrease in the body weight of mice in all AgNPs
treated groups, no significant difference was observed in the
food consumption (food supplied after due measurement) of
mice between the treated and the control groups.
The maximum decrease in the body weight was observed
in the group B which received AgNPs at the dose of 10 mg/kg
body weight for 21 d. Hence, this dose was considered as an
DOI: 10.3109/15376516.2013.764950
Effects of silver nanoparticles on small intestine mucosa of mice
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Figure 1. Histogram showing average diameter and percentage of different sizes of
AgNPs. The distribution pattern shows most
of the particles in the size range of 5–10 nm.
Figure 2. Transmission electron photomicrograph of AgNPs showing circular and mono-dispersed particles (Scale bar-200 nm).
optimum dose and further description is on mice treated with
AgNPs at a dose of 10 mg/kg of body weight of mice for 21 d.
Histopathology of small intestine
Histological observation under light microscope revealed that
in control mice the villi of small intestine were lined by simple
columnar epithelium containing the tall absorptive cells with
striated border due to microvilli, sometimes called terminal
bar (Figure 4A). A few goblet cells were also observed
between the columnar epithelial cells. Lamina propria was
present in the core of each villus (Figure 4A). Intestinal glands
were observed in the lamina propria with mitotic figures
(Figure 5A). After oral administration of AgNPs, the following
changes were observed in the small intestine.
(a) The striated border (terminal bar) was not demarkable as
the microvilli were lost (damaged) (Figure 4B).
(b) Increased number of inflammatory cells in the lamina
propria of each villus leading to the widening of lamina
propria zone (Figure 4B).
(c) Increased number of mitotic figure in the intestinal
glands (Figure 5B and C). This increased number of
mitotic figure indicates that AgNPs damages the intestinal epithelial cell and to replace these damaged
absorptive cells, stem cells in intestinal glands starts
proliferating and show high mitotic activity.
TEM of small intestine
In control mice, striated border of enterocytes shows layer of
densely packed microvilli (Figure 6A) which are nothing but
the cylindrical protrusion of the apical cytoplasm to amplify
area for absorption covered by plasmalema (cell wall).
However, after oral administration of AgNPs in mice, the
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Toxicol Mech Methods, 2013; 23(3): 161–167
Figure 3. Histogram representing change in the body weight in control and AgNP treated groups of mice. Control group received deionised water
a vehicle; Group A received 5 mg/kg body weight (b.w.)/d AgNPs solution; Group B received 10 mg/kg b.w./d AgNPs solution; Group C received
15 mg/kg b.w./d AgNPs solution and Group D received 20 mg/kg b.w./d AgNP solution. Values are represented as mean SEM. *Denotes the level of
significance (p50.05) from the control group.
microvilli were totally disrupted. A few distorted one were still
present in the area with shrunken lumen, which showed beadlike swelling probably because of AgNPs inside lumen. Small
fragments of microvilli could be seen scattered in the area, i.e.
lumen of intestine. AgNPs were present in various strata of
small intestine including broken microvilli (Figure 6B).
Discussion
In the present work, we have attempted to investigate the
adverse effects of repeated sustained oral exposure of AgNPs
on the mucosa of the small intestine in mice. Intestine is lined
by simple columnar epithelium. On the luminal aspect of each
epithelial cell, microvilli were projected which are nothing
but the cytoplasmic extension of enterocytes. These microvilli
give the border a striated appearance in light microscopy,
which is also known as ‘‘terminal bar’’.
In the present study, we observed that after administration
of AgNPs, this terminal bar got lost which clearly indicated
the damage to the microvilli. TEM also revealed that these
microvilli were completely disrupted and broken, which
indicates that AgNPs can cross the protective barrier of the
small intestine i.e. the intestinal mucins and glycoproteins and
cause impairment to the microvilli.
From the above histological finding, we can explain the
significant decrease in body weight of mice in AgNPs treated
mice. The main function of small intestine is absorption. Due
to the loss of microvilli of enterocytes, the absorptive surface
area of the intestine was markedly reduced which led to
decrease in the absorption of nutrient materials resulting in a
reduction in the body weight of mice.
There are certain evidences that after subcutaneous
injection, AgNPs are translocated to the blood circulation
and distributed throughout the main organs, especially in the
kidney, liver, spleen, brain and lung in the form of particles
(Tang et al., 2009). Tang et al. postulates that AgNPs crossed
the BBB and by transcytosis of capillary endothelial cells
these particles get accumulated in the brain micro-vessels
vascular endothelial cells of rat (Tang et al., 2010). They also
proposed that AgNPs induce degenerative changes in the
some endothelial cells, which lead to the loosening of tight
junction between the endothelial cells and AgNPs cross
through these crevices. In our study, we observed the
structural changes in the enterocytes, i.e. loss of microvilli,
which may give passage to AgNPs for entering into the
intestinal wall and then hence into the portal circulation and
systemic circulation.
Ultra-structural studies have shown that activated platelets,
when exposed to AgNPs in a concentration-dependent
manner, lost their characteristic well-developed hyaloplasmic
processes, pseudopods (Shrivastava et al., 2009). In addition,
AgNPs were also found to be accumulated within platelet
granules. After 28 d of repeated oral administration of AgNPs,
a dose-dependent increased accumulation of AgNPs was also
observed in the lamina propria in both the small and large
intestine of Sprague–Dawley rats (Jeong et al., 2010). Jeong
et al. (2010) also reported frequent cell shedding at the tip of
the villi in intestine. Dose-dependent increase in the pigmentation of the villi observed after 90 days oral exposure of
AgNPs, which was apparent treatment related effects (Kim
et al., 2010). More recently, Loeschner et al. (2011) also
reported presence of silver granules in the intestinal system of
rats after 28 d repeated oral exposure of AgNPs or to silver
acetate to rats (Loeschner et al., 2011). Similarly, in the
present study, AgNPs were present in the various strata of
small intestine including broken microvilli. A few distorted
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DOI: 10.3109/15376516.2013.764950
Effects of silver nanoparticles on small intestine mucosa of mice
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Figure 4. Photomicrographs of cross-sections of small intestine showing
intestinal villi in control (A) and AgNPs administered mice (B). (A) In
the control small intestine, each villous is lined by simple columnar
epithelium containing absorptive cells with uniform and continuous
striated border of microvilli, i.e. the terminal bar (arrow) and lamina
propria (asterisk) in the core of each villous. Inset showing terminal bar
(arrowhead). H&E 400. (B) Showing disrupted border of microvilli
(arrow). Note the widening of lamina propria zone (asterisk). Inset
showing the loss of terminal bar (arrowhead). H&E 400.
microvilli were still present in the area with shrunken lumen,
which showed bead-like swelling probably because of AgNPs
inside lumen. Furthermore, in AgNPs treated mice, the
microvilli on the intestinal absorptive cells were found to be
badly damaged and broken (Figure 4B). It is possible that the
nanoparticles are taken up across the intestinal barrier since
particles with diameters less than 1 mm are particularly
susceptible to absorption by the intestinal lymphatic system
(Florence et al., 1997).
We may speculate that AgNPs somehow interact with the
structural elements of the microvillus of the intestinal
absorptive cells causing structural changes, and finally
destruction of the microvilli. As evident from Figure 2(B),
the terminal bar was absent in AgNPs treated intestinal cells
and could be made out at light microscopy level with 1000X
magnification. This view corroborates with Sondi & SalopekSondi (2004).
Sondi & Salopek-Sondi (2004) reported that AgNPs
treated E. coli show significant changes in bacterial membranes. They also reported the formation of ‘‘pits’’ on the
surfaces of the bacterial cell wall and accumulation of AgNPs
Figure 5. Photomicrographs of cross-sections of small intestine of
control (A) and AgNPs administered mice (B, C). (A) Showing intestinal
gland cells with few numbers of mitotic figures (circles). Note the
narrow lumen of each intestinal gland. Inset showing a mitotic figure.
H&E 400. (B) Showing a large number of mitotic figures (circles)
along with dilated intestinal glands (arrowheads). H&E 400. (C) Same
as B. Inset showing mitotic figure at higher magnification. H&E 400.
in the bacterial membrane. A similar effect was also reported
in E. coli bacteria treated with highly reactive metal oxide
nanoparticles (Stoimenov et al., 2002). More recently,
Shrivastava et al. (2007) also reported AgNPs managing to
enter the gram-negative bacteria by making perforations in
the membrane and thus resulting in cell lysis. A bacterial
membrane with pits on the surfaces shows a considerable
increase in permeability, leaving the bacterial cells incompetent of regulating transport through the plasma membrane
and, finally, resulting in cell death (Sondi & Salopek-Sondi,
2004). Sondi & Salopek-Sondi, (2004) hypothesized that the
change in membrane permeability is probably due to
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Toxicol Mech Methods, 2013; 23(3): 161–167
At this point, we may speculate that AgNPs somehow
interact with this protective barrier and other structural
elements of the microvilli of the intestinal absorptive cells
causing structural changes, resulting in the membrane
permeability and finally destruction of the microvilli.
Subsequently, the epithelial cells of the gastro-intestinal
tract get destroyed. Therefore, it is conceivable that in order to
replace these damaged enterocytes, the neck cells of the crypt
of Lieberkuhn start mitosis at a faster rate. Hence, the AgNPs
administrated mice show larger number of mitotic figures as
compared to the control animals. If the normal cells do not
replace the damaged enterocytes, the site may result in the
formation of ulcer.
Furthermore, it could also be hypothesized that the AgNPs
may possibly interfere with the cell cycle kinetics without
inducing cell death. Because small intestine is an organ,
which affords a generous population of continuous dividing
cells, it may also be speculated that when AgNPs breach the
protective barrier of the intestinal cells, somehow these
nanoparticles trigger the regulators of the cell cycle and the
process of cell division is accelerated and cellular uptake of
AgNPs will be reduced over time due to augmented cell
division. Several studies have shown that the uptake of the
nanoparticles by cells is influenced by different phases of
their cell cycle and cell division can dilute the concentration
of nanoparticles in the cell population (Errington et al., 2010;
Kim et al., 2012; Summers, 2012; Summers et al., 2011).
Moreover, it may also be speculated that AgNPs affect the
cellular response at the molecular level and may increase the
expression of certain genes related to cell cycle pathway and
cause abnormal cell proliferation (Kawata et al., 2009).
Figure 6. Transmission electron photomicrograph of small intestine of
control (A) and AgNPs administered mice (B). (A) The striated border of
an enterocyte showing a layer of densely packed microvilli (arrow)
(Scale bar-500 nm). (B) Showing damaged and broken microvilli (arrow)
due to prolonged exposure of AgNPs. Note the fragments of the
microvilli are scattered in the lumen of intestine. Hardly any microvillus
is in its normal shape and site. The zone just below the microvilli shows
homogenous lamina propria, which appears edematous (asterisk).
Nanoparticles can be seen in the deeper layers and in the lumen of the
intestinal villous. Note the presence of AgNPs in various strata of small
intestine (circles) (Scale bar-1 mm).
progressive release of lipopolysaccharide molecules and
membrane proteins. It is well known that the outer membrane
of gram-negative bacterial cells is primarily made of tightly
packed lipopolysaccharide molecules, which impart an effective permeability barrier (Nikado et al., 1985; Raetz, 1990;
Silva et al., 1973).
The above supposition of Sondi & Salopek-Sondi (2004)
may possibly explain the destruction of hyaloplasmic
processes (pseudopods) of AgNPs-treated platelets
(Shrivastava et al., 2009) and microvilli of the intestinal
absorptive cells in mice in the present study. These AgNPs
also pass through a thick layer of glycocalyx, which is a
polysaccharide, covers the microvilli of the intestinal absorptive cells and platelets too. The thick layer of glycocalyx
imparts protective function to microvilli from any corrosive
material. A similar layer of glycocalyx is also present on the
outer surface of the platelets.
Conclusion
Based on our results, it is postulated that AgNPs somehow
interact with the protective layer of the glycocalyx and other
structural elements of the microvilli of the intestinal absorptive cells causing structural changes, resulting in the alteration
of membrane permeability and finally destruction of the
microvilli. Subsequently, the epithelial cells of gastrointestinal tract get destroyed and are the reason for the
decrease in body weight of mice. We thus conclude that
AgNPs destroy the mucosa of small intestine and impede its
function.
Declaration of interest
Authors have no conflict of interest. Madhu Yashpal is thankful to
University Grants Commission, New Delhi, India for financial support in
the form of Dr D.S. Kothari Postdoctoral Fellowship [F.4-2/2006 (B SR)/
13-455/2011 (BSR)].
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