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Drug Chem Toxicol, Early Online: 1–12

! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.866134

Effects of sub-acute exposure to TiO2, ZnO and Al2O3 nanoparticles on

oxidative stress and histological changes in mouse liver and brain
Rupal Shrivastava*, Saimah Raza*, Abhishek Yadav, Pramod Kushwaha, and Swaran J. S. Flora
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Division of Regulatory Toxicology, Defence Research and Development Establishment, Gwalior, India

Abstract Keywords
Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. Al2O3, morphological changes, metal oxides
However the information regarding toxicity of these nanoparticles on humans and environ- nanoparticles, nanotoxicity, oxidative
ment is still deficient. The present study investigated the toxic effects of three metal oxide stress, TiO2, ZnO
nanoparticles, TiO2, ZnO and Al2O3 on mouse erythrocytes, brain and liver. Male mice were
administered a single oral dose of 500 mg/kg of each nanoparticles for 21 consecutive days. The History
results suggest that exposure to these nano metallic particles produced a significant oxidative
stress in erythrocyte, liver and brain as evident from enhanced levels of Reactive Oxygen Received 11 March 2013
Species (ROS) and altered antioxidant enzymes activities. A significant increase in dopamine Revised 8 October 2013
and norepinephrine levels in brain cerebral cortex and increased brain oxidative stress suggest Accepted 10 November 2013
neurotoxic potential of these nanoparticles. Transmission electron microscopic (TEM) analysis Published online 17 December 2013
indicated the presence of these nanoparticles inside the cytoplasm and nucleus. These changes
were also supported by the inhibition of CuZnSOD and MnSOD, considered as important
biomarkers of oxidative stress. The toxic effects produced by these nanoparticles were more
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pronounced in the case of zinc oxide, followed by aluminum oxide and titanium dioxide,
respectively. The present results further suggest the involvement of oxidative stress as one of
the main mechanisms involved in nanoparticles induced toxic manifestations.

Introduction to oxidative stress. There are toxicological evidences that

ultrafine nanoparticles are capable of producing adverse
Nanoparticles (NPs) are currently having a widespread use in
effects (Englert, 2004; Oberdörster, 2000; Utell & Frampton,
modern technology, however there is insufficient data for
2000). A well defined mechanism of toxicity induced by NPs
their possible health and environmental hazards in human.
is not completely clear, however, several possible mechanisms
The rising concern for nanoparticles emerges because of their
have been proposed based on reported results. The change in
unique chemistry, extremely small size, large reactive surfaces
the physicochemical and structural properties of NPs may be
and non-biodegradability. They rapidly get dispersed through-
responsible for the interactions that could lead to toxico-
out the environment with unknown consequences (Wang
logical effects (Brown et al., 2001). These changes could lead
et al., 2009). Their adverse effects in human have raised
to specific surface groups (e.g. thiol groups, carboxyl groups,
serious concerns. It is therefore critical to understand the
carbonyl groups, SDS, DMSO, surfactants) to be reactive sites
nature and origin of the toxicity of these nanoparticles.
(Hussain et al., 2009). These reactive sites act as electron-
Metallic NPs are closely related to the generation of reactive
donors or electron-acceptors and work with oxygen to induce
oxygen species (ROS) and reactive nitrogen species (RNS)
superoxide radical (O2), which can then generate reactive
due to redox-cycling reactions. Currently, ROS and oxidative
oxygen species (ROS) by dismutation or the Fenton reaction
stress induced by NPs have been one of the best-developed
(Cabiscol et al., 2000).
paradigms for nanoparticle toxicity (Dick et al., 2003; Xia
Various metal oxide nanoparticles, such as, titanium
et al., 2006). Oxidative stress is a normal cellular process
dioxide (TiO2), aluminum oxide (Al2O3), and zinc oxide
involved in many aspects of cellular signaling, though
(ZnO) are receiving increasing attention for a large variety of
excessive oxidative stress can be harmful (Huang et al.,
applications due to their unique physical and chemical
2010). Recent studies have increased our understanding of the
properties (Huang et al., 2010; Nel et al., 2006; Schrand
nanotoxicity of metal oxide particles, particularly with respect
et al., 2010). TiO2 NPs possess inherent advantages of
physical stability, anticorrosion and nanoscale-enhanced
photocatalysis due to which they are widely used in paints,
*These authors contributed equally to the study.
printing ink, rubber, paper, cosmetics, sunscreens, car mater-
Address for correspondence: Swaran J. S. Flora, Division of Regulatory ials, cleaning air products, industrial photocatalytic processes,
Toxicology, Defence Research and Development Establishment, Jhansi
Road, Gwalior 474 002, India. Fax: +91 751 2341148; E-mail: and decomposing organic matters in wastewater (Iavicoli
sjsflora@hotmail.com or sjsflora@drde.drdo.in et al., 2012). Titanium dioxide and zinc oxide NPs are used in
 2 R. Shrivastava et al.
toothpaste, beauty products, sunscreens and textiles. While
Aluminum oxide possess good dielectric and abrasive proper-
ties and are widely used as an abrasive agent or insulator (Lai
et al., 2008; Prabhakar et al., 2012). TiO2 is known to get
accumulated in liver, kidney, and spleen with the related
toxicity (Schrand et al., 2010). There is also concern about
possible neurotoxicity, as it is known that TiO2 NPs may pass
through the blood brain barrier on exposure (Li et al., 2010).
Several studies have shown that TiO2 NPs could be
translocated into the central nervous system (CNS) via the
olfactory pathway in mice and accumulate in the entire brain,
mainly in the hippocampus regions (Wang et al., 2007; Wang
et al., 2008). However, to verify the generalization of this
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phenomenon, further studies are needed in other animal
species. Titanium dioxide (TiO2) NPs injected in the abdom-
inal cavity of mice for 14 days significantly increased LPO,
decreased GSH levels and altered antioxidant enzyme activity
in a dose-dependent manner along with histopathological
changes in (Ma et al., 2010) Previous reports also indicate that
exposure to TiO2 NPs through various routes lead to enhanced
oxidative stress and also alteration of neurotransmitters in brain
(Wang et al., 2008; Shimizu et al., 2009). Adverse effects of
ZnO NPs can be mainly attributed to the generation of ROS
leading to membrane damage, directly or indirectly (Jingxia
et al., 2009). However little is known about the neurotoxic
potential of zinc and the main mechanism involved. Prabhakar
et al. (2012) have shown the possible involvement of oxidative
For personal use only.
stress and altered antioxidant status in eliciting toxicity of
Al2O3 NPs after acute oral treatment. There is evidence that
exposure to aluminum may lead to an increased oxidative
stress, inflammatory events, and/or the breakdown of the
blood-brain barrier (BBB) (Lockman et al., 2004). Due to their
extremely small size, nanoparticles can translocate from these
entry portals into the more sensitive circulatory and lymphatic
systems, and ultimately to body tissues and organs (Borm et al.,
2006). There are also reports which show their accumulation in
different organs leading to systemic toxicity (van der Zande
et al., 2012).
The study was designed to elucidate the sub-acute toxicity
of oral exposure to metal NPs TiO2, Al2O3 and ZnO
nanoparticles in terms of oxidative stress and antioxidant
defense system in erythrocytes and soft tissues of mice. Most
of the toxic chemicals are metabolized in liver due to which
there is a high risk of free radical attack leading to lipid
peroxidation, which in turn leads to hepatotoxicity (Patlolla
et al., 2011). High metabolic rate and poor antioxidant
defense system renders brain highly vulnerable to oxidative
stress. The ability of NPs to cross the blood–brain barrier
further enhances the risk (Ma et al., 2010). Hence, liver and
brain were selected for studying the oxidative stress-inducing
potential of TiO2, ZnO and Al2O3 NPs.
Materials and methods
Chemicals and reagents
Titanium dioxide (TiO2) and Zinc oxide (ZnO) nanoparticles
were procured from Sigma Aldrich (St. Louis, MO). Alumina
(Al2O3) was procured from Alfa Aesar (Ward Hill, MA). All
other laboratory chemicals and reagents were purchased from
Merck (Darmstadt, Germany), Sigma (St. Louis, MO) or
Drug Chem Toxicol, Early Online: 1–12
BDH chemicals (Mumbai, India). Triple distilled water
prepared by Millipore (New Delhi, India) was used through-
out the experiment to avoid contamination, and for the
preparation of reagents and buffers used for various biochem-
ical assays in the study. According to the information
provided by manufacturer the particles size of TiO2, ZnO
and Al2O3 nanoparticles are 575 nm, 5100 nm and 45 nm
respectively. According to the company’s data sheet, TiO2
nanoparticles obtained is a mixture of rutile and anatase
nanoparticles, in the form of suspension. Prior to dosing the
suspension was sonicated for 30 minutes.
Characterization of nanoparticles by SEM
The particle size and distribution were measured using
electron microscopic studies by FEI Quanta-400, SEM.
Samples were coated with a thin layer of gold produced by
ion sputtering in Ion Sputter JFC-1100 before observing these
in the electron microscope at 5 kV.
Animals and treatment
Experiments were performed on healthy 6 weeks old Swiss
albino male mice, weighing approximately 25–30 g. Animals
were obtained from the animal house facility of the Defence
Research and Development Establishment (DRDE), Gwalior.
All animals received humane care in compliance with the
guidelines of the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA). The
Animal Ethical Committee of DRDE, Gwalior, India also
approved the protocols for the experiments. Prior to dosing,
they were acclimatized for 7 days to light from 06:00 to
18:00 h, alternating with 12 h darkness. The animals were
housed in stainless steel cages in an air-conditioned room
with temperature maintained at 25  2  C. Mice were allowed
standard chow diet (Amrut feeds, Pranav Agro, New Delhi,
India; metal content of diet, in ppm dry weight: Cu 10.0, Zn
45.0, Mn 55.0, Co 5.0, Fe 75.0) throughout the experiment
and water ad libitum.
Thirty two mice were randomized into four groups of
8 animals each and were treated as follows for 21 days:
Group I: Control (received normal water)
Group II: TiO2 nanoparticles (500 mg/kg b.w.) oral, once,
daily
Group III: ZnO nanoparticles (500 mg/kg b.w.) oral, once,
daily
Group IV: Al2O3 nanoparticles (500 mg/kg b.w.) oral, once,
daily
We selected oral route of exposure for mouse as these
nanoparticles are generally being used in various products like
food packaging, cosmetics and coating etc and there is
possibility of gaining a direct entry into the body. They may
also enter into the gastrointestinal tract after their accidental
release into the environment. The doses at which humans
may get exposed to these nanoparticles at the above
mentioned scenario are not clear as no guidelines and
standard methodologies for in vivo toxicity assessment of
these nanoparticles. We thus chose to work with the highest
dose suggested by OECD guidelines for a new compound.
This dose does not necessarily reflect the actual concentration
of these nanoparticles in the real environment however, this
 DOI: 10.3109/01480545.2013.866134
can be used to assess the health risks from exposure to these
nanoparticles (Sharma et al., 2012).
After 21 days the exposure was stopped and animals were
sacrificed under light ether anesthesia, 48 h, after last dosing.
Blood was collected in heparinized vials. Liver and brain
were collected and stored in RNA later for gene expression
analysis (RT-PCR). For biochemical estimation, the tissues
were washed with cold normal saline, blotted and all the
extraneous materials were removed. Liver and brain tissues
from two animals were processed for the TEM analysis.
Whole brain of four animals was used for the biochemical
assays while the brain of remaining four animals was excised
quickly and cerebral cortex was used for the estimation of
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neurotransmitter under cold conditions.
Separation of red blood cells
Blood was collected from orbital plexus of mice in
heparinized tubes. RBCs were isolated following the
method of Steck and Kant (1974). Briefly, blood was
centrifuged at 1500  g for 10 min at 4  C. Plasma and
buffy coat were removed by aspiration. The RBCs were
washed three times in phosphate buffer saline (0.1 M). The
packed cell volume (PCV) obtained was divided into two
parts, one part of which was diluted with chilled distilled
water kept for the analysis of reactive oxygen species (ROS),
catalase, thiobarbituric acid reactive substances (TBARS) and
reduced glutathione (GSH). Other part of the packed cell
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volume was used for the estimation of other antioxidant
enzymes i.e. glutathione peroxidase (GPx), superoxide
dismutase (SOD) and glutathione-S-tranferase (GST). For
this, hemoglobin was precipitated in PCV by means of
chloroform and ethanol. After centrifugation at 3000  g for
10 min at 4  C, the supernatant obtained was used for all
above mentioned enzymatic assays.
RNA isolation and quantification
Total RNA was isolated from the liver and brain samples
using kit protocol as provided by the manufacturers (RNeasy
Protect Mini Kit and RNeasy Lipid Tissue Mini Kit respect-
ively, QIAGEN Sciences, Maryland). About 30 mg of liver
tissue and 100 mg brain tissues stored in RNA later were
homogenized using a rotor-stator homogenizer until it was
uniformly homogenous. This was carried out in the presence
of specialized highly denaturing guanidine-thiocyanate con-
taining buffer (1 ml). The lysate was centrifuged for 3 min at
high speed. Supernatant was carefully removed and transfered
to a microcentrifuge tube. 1 volume of 70% ethanol was
added to the cleared lysate, and mixed immediately by
pipetting. 700 ml of the sample was transferred to an RNeasy
spin column placed in a 2 ml collection tube and centrifuged
for 15 s at 10 000 rpm. The flow-through was discarded and
spin column membrane was washed with 700 ml RW1 Buffer
by centrifuging at 10 000 rpm for 15 sec. Washing of spin
column was repeated again using 500 ml RPE Buffer. The
above mentioned steps were repeated twice. Finally, RNA was
eluted using 30–50 ml RNase-free water directly to the spin
column membrane.
Concentration of isolated RNA was spectrophotometrically
determined at 260 nm and absorbance of 1 unit at 260 nm
Metal oxides nanoparticles induced toxicity 3
corresponded to 44 xg RNA per ml. The purity of RNA was
determined by checking A260/A280 ratio, where a value in
the range of 1.9–2.1 indicated the extraction of pure RNA.
Integrity of RNA isolated was checked using formamide gel
electrophoresis. Firstly, 10X MOPS buffer was prepared
(0.4 M MOPS was mixed with 0.1 M NaOAc and 10 mM
EDTA, pH of the buffer was adjusted to 7.0 with conc.
NaOH). To prepare formamide gel (1.2%) agarose was
prepared and 1X MOPS buffer and formaldehyde was further
added to it to cast the gel.
RT-PCR (reverse transcriptase PCR)
Once the purity of RNA had been confirmed, the same was
used for the preparation of cDNA by means of reverse
transcription followed by specific amplification using Qiagen
One Step RT-PCR Kit. For this purpose, template RNA,
specific primer solutions, dNTP mix, 5X Qiagen OneStep RT-
PCR Buffer, and RNase-free water was thawed and placed on
ice. As per the reaction volume, a master mix was made and
its appropriate volumes were dispensed into PCR tubes. To
the master mix the RNA sample was added and preceded to a
thermal cycler leading to both, cDNA preparation by reverse
transcription, as well as amplification of the formed cDNA.
Reverse transcription was carried out by setting thermal
cycler at 45  C for 50 min followed by a 5 min heating step at
95  C during which the DNA polymerase gets activated,
reverse transcriptases get inactivated and the cDNA template
is denatured.
PCR was performed with the initial denaturation cycle at
94  C for 5 min, followed by 25–40 cycles of 94  C for 1 min,
annealing at 50–68  C for 1 min and extension at 72  C for
10 min. Sequence of the primer and the size of the PCR
product generated are described in the Table 1.
Biochemical assays
Reactive Oxygen Species (ROS)
Amount of ROS in blood was measured using 20 , 70 -
dichlrofluorescein diacetate (DCF-DA) that gets converted
into highly fluorescent DCF by cellular peroxides (including
hydrogen peroxide). The assay was performed as described by
Socci et al. (1999). For estimation of ROS in blood, 5% RBC
hemolysate was prepared and diluted to 1.5% with ice–cold
40 mM tris-HCl buffer (pH 7.4). Similarly for estimation of
ROS in tissues, 10% tissue homogenate was prepared. The
tissue was homogenized (10 mg) in 1 ml of ice–cold 40 mM
tris –HCl buffer (pH 7.4), further diluted to 0.25% with the
same buffer and placed on ice. Thenafter, 40 ml of 1.25 mM
DCF-DA in methanol was added for ROS estimation. All
samples were incubated for 15 min in a 37  C water bath.
Fluorescence was determined at 488 nm excitation and
525 nm emission using a fluorescence plate reader (Tecan
Spectra Fluor Plus).
Glutathione (GSH)
Analysis of blood GSH concentration was performed by the
method described by Ellman (1959) and modified by Jollow
et al. (1974). In brief, 0.2 ml of whole blood was added to
1.8 ml of distilled water and incubated for 10 minutes at
 4 R. Shrivastava et al.
Table 1. Sequence of primer and size of the PCR product.
Genes Primer sequence (50 to 30 ) Ta
b-actin F0 -GAGAGGGAAATCGTGCGTGAC 65  C
R0-CATCTGCTGGAAGGTGGACA
Catalase F0 -TGGCCTCCGAGATCTTTTCAATG 63  C
R0-GCGCTGAAGCTGTTGGGGTAGTA
GPx F0 -CGCTCATGACCGACCCCAAGT 65  C
R0-GCCAGCCATCACCAAGCCAATA
CuZnSOD F0 -GCGGCTTCTCTCGTCTCCTTGC 65  C
R0-TTGATGGACATGGAACCCATGCTCG
MnSOD F0 -ACAGCAAGCACCACGCGACC 67  C
R0-AACACCCACCACGGGCCTGA
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b-actin was used as an internal control. The PCR-amplified products
were then subjected to electrophoresis on 1.5% agarose gels.
37  C, for complete hemolysis. After adding 3 ml of 4%
sulfosalicylic acid, tubes were centrifuged at 2500 rpm for 15
minutes. To the supernatant, 0.2 ml of 10 mM solution of 5,
50 -dithiobis-(2-nitrobenzoic acid), DTNB, was added in
presence of phosphate buffer (0.1 M, pH 7.4). Absorbance
recorded at 412 nm was used for calculation of GSH
concentration.
Superoxide dismutase (SOD)
Superoxide dismutase (SOD) activity in whole brain, liver and
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blood was assayed spectrophotometrically as described by
Kakkar et al. (1984). Reaction mixture contained 1.2 ml
(0.052 mM) sodium pyrophosphate buffer, 0.1 ml (186 mM)
phenazine methosulphate, 0.3 ml (300 mM) nitro blue tetra-
zolium. 0.2 ml of the supernatant obtained after centrifugation
(1500  g, 10 min followed by 10 000  g, 15 min) of 5%
hemolysate/homogenate was added to the reaction mixture.
Reaction was initiated by adding 0.2 ml NADH (780 mM) and
stopped by adding 1 ml glacial acetic acid. Color intensity of
the chromogen was measured at 560 nm and the activity was
expressed as units/min/mg of protein.
Catalase
Catalase activity in tissue was assayed following the proced-
ure of Sinha (1972), at room temperature. 0.1 ml of 5% RBC
hemolysate/tissue homogenate was incubated with 0.5 ml of
H2O2 (0.2 M) at 37  C for 90 seconds precisely, in the
presence of 0.01 M phosphate buffer (pH 7.4). Reaction was
stopped by adding 5% dichromate solution. Further samples
were incubated at 100  C for 15 min in boiling water bath.
Amount of H2O2 consumed was determined by recording
absorbance at 570 nm.
Thiobarbituric acid reactive substances (TBARS)
Measurement of lipid peroxidation was done by the method
described by Ohkawa et al. (1979). Tissue lipid peroxidation
was measured in tissue homogenate [5% homogenate (w/v)
in 150 mM KCl for brain, and 10% homogenate (w/v) in
150 mM KCl for liver] for 30 min at 37  C. The incubation
was interrupted by adding 0.1 ml of 10% trichloroacetic acid.
After centrifugation, 1 ml of the supernatant was mixed with
1 ml of 0.65% thiobarbituric acid. The mixture was then kept
Drug Chem Toxicol, Early Online: 1–12
in a boiling water bath for 15 min to get the red color of
thiobarbituric acid-malondialdehyde complex, the absorbance
Product
size
of which was recorded at 535 nm. The amount of TBARS was
calculated using a molar extinction coefficient of 1.56  105/
453 bp M/cm.
In case of blood, 0.1 ml of 5% RBC hemolysate was added
452 bp
to 0.2 ml of 8.1% SDS (w/v) and incubated for 10 min. 1.5 ml
221 bp
of 20% acetic acid (pH 3.5) was added followed by the
addition of 1.5 ml of 0.8% thiobarbituric acid (w/v) and 0.7 ml
201 bp distilled water. The mixture was then incubated for 1 hour in
boiling water bath. 1 ml of distilled water was added to the
560 bp solution after cooling and centrifuged at 6000 rpm for 15
minutes. The absorbance of supernatant was read at 532 nm
and the values were expressed as moles of MDA/ml.
Glutathione peroxidase (GPX)
Glutathione peroxidase activity was measured by the proced-
ure of Flohe and Gunzler (1984). Supernatant obtained after
centrifuging 5% tissue homogenate for 10 min at 1500  g,
followed by 10 000  g for 30 minutes at 4  C, was used for
GPx assay. 1 ml of reaction mixture was prepared which
contained 0.3 ml of phosphate buffer (0.1 M, pH 7.4), 0.2 ml
of GSH (2 mM), 0.1 ml of sodium azide (10 mM), 0.1 ml of
H2O2 (1 mM) and 0.3 ml of tissue supernatant. After incuba-
tion at 37  C for 15 min, reaction was terminated by addition
of 0.5 ml of 5% TCA. Tubes were centrifuged at 1500  g for
5 min and supernatant was collected. 0.2 ml of phosphate
buffer (0.1 M, pH 7.4) and 0.7 ml of DTNB (0.4 mg/ml) were
added to 0.1 ml of the reaction supernatant. After mixing well,
absorbance was recorded at 420 nm.
Glutathione-S-transferase (GST)
GST activity was determined following the procedure of
Habig et al. (1974). Supernatant was obtained after centrifu-
ging 5% tissue homogenate at 1500  g for 10 min followed
by 10 000  g for 30 minutes at 4  C. Reaction mixture
contained 0.02 ml of 1-chloro-2, 4-dinitrobenzene (1 mM),
2.9 ml of GSH (0.3 mg GSH/ml in 0.2 M phosphate buffer, pH
7.4) and 30ml of tissue supernatant. Change in color was
monitored by recording absorbance (340 nm) at 30 sec
intervals for 3 min.
Brain biogenic amines level
The frozen brain tissue samples were weighed and homo-
genized in acidified butanol. Dopamine (DA), norepinephrine
(NE) and 5-hydroxytryptamine (5-HT) were estimated
according to the procedure of Jacobwitz and Richardson,
(1978). The aliquots of butanol extracts were re-extracted
with phosphate buffer (0.1 M, pH 6.5), part of which was
derivatized by adding sodium EDTA and iodine solution. The
reaction was terminated by alkaline sulfite and neutralized
with acetic acid (5 M). Fluorescence was read by excitation at
385 nm and emission at 485 nm for NE, and 320 and 385 nm
respectively, for DA. To determine 5-HT, the butanol layer
was extracted with 0.1 M HCl in the presence of n-heptane
and the acid layer was mixed with o-phthaldehyde. After
boiling and cooling, fluorescence was read by excitation at
360 nm and emission at 470 nm.
 DOI: 10.3109/01480545.2013.866134
Transmission electron microscopy (TEM) of biological
samples
Transmission electron microscopy was performed to assess
the uptake and subcellular localization of TiO2, ZnO and
Al2O3 nanoparticles in the brain and liver tissues of mice.
Slices of brain and liver from the animals post sacrifice (one
animal per group) were randomly removed and fixed with
2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for
4 h. The specimens were postfixed with 2% osmium tetroxide,
dehydrated through graded alcohol solutions and embedded in
Epon. Ultrathin sections mounted on copper grids (300 mesh)
were contrasted with uranyl acetate and lead citrate for TEM
analysis (ZeissbEM 10 A; Carl Zeiss, Oberkochen, Germany)
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at an accelerating voltage of 60 kV. To obtain a global
characterization of the tissues, several grids were prepared (4–
5 grids per animal, each one having 3–4 sectioning cuts) from
the different regions.
Statistical analysis
The results are expressed as the mean  SEM of number of
observations. Comparisons of means were carried out using
one way analysis of variance (ANOVA) followed by Turkey’s
post hoc test to compare means between the different
treatment groups. Differences were considered significant at
p50.05 unless otherwise stated in the text.
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Results
Particle characterization
The SEM was done for the characterization of TiO2, ZnO and
Al2O3 NPs and chemical analysis of these nanoparticles by
EDX method and the data is shown in Figure 1. The spectrum
suggested that the nanoparticle size of TiO2 nanoparticles is
between 50–75 nm, ZnO nanoparticles is 80–100 nm and
Al2O3 nanoparticles is 40–50 nm (Figure 1).
Effects on oxidative stress variables in mouse
erythrocyte
Erythrocytes were more commonly employed in the evalu-
ation of oxidative stress. Effects of TiO2, ZnO and Al2O3
nanoparticles on the generation of reactive oxygen species
(ROS) are depicted in Table 2. The fluorescence intensity of
Figure 1. Particle size distribution of NPs, (A) SEM image of TiO2 NPs, (B) SEM image of ZnO NPs and (C) SEM image of Al2O3 NPs.
Metal oxides nanoparticles induced toxicity 5
DCF, an indicative of oxidative stress increased significantly
in erythrocytes on exposure to TiO2, ZnO and Al2O3
nanoparticles compared to the controls, however increased
free radical generation was observed in case of ZnO NPs,
indicating highest toxicity in blood followed by TiO2 and
Al2O3. Table 2 also depicts effect of TiO2, Al2O3 and ZnO
nanoparticles on TBARS level in mouse erythrocyte, which is
an important marker of lipid peroxidation. Following NP
exposure, TBARS level increased significantly in all the
groups compared to the control, however the comparative
level of TBARS among exposed groups were not found to be
significant.
TiO2 induced alterations in GSH, GPx and GST activities
in mouse erythrocytes as depicted in Table 2. Erythrocyte
GSH increased specifically on TiO2 and Al2O3 nanoparticles
exposure while ZnO nanoparticles had no significant effect on
blood GSH. Significant reduction in the activity of GPx was
observed in mouse erythrocyte on exposure to TiO2 and ZnO
nanoparticles, However Al2O3 nanoparticles had no effect on
GPx activity compared to the control. Activity of GST on the
other hand remained unaffected following exposure to all the
three nanoparticles.
Table 2. Effect of TiO2, Al2O3 and ZnO nanoparticles on oxidative
stress variables/phase II drug metabolizing enzyme in mouse
erythrocyte.
Erythrocyte
Normal TiO2 ZnO Al2O3
ROS 0.73  0.02* 0.86  0.04y 0.83  0.01y 0.86  0.02y
GSH 0.96  0.02* 1.10  0.01y 1.09  0.01* 1.18  0.04y
TBARS 270.8  1.29* 345.0  6.93y 351.8  4.76y 372.5  34.32y
SOD 0.25  0.03* 0.10  0.01y 0.12  0.02y 0.09  0.04y
Catalase 0.79  0.07* 0.54  0.05y 0.63  0.04y 0.56  0.02y
GPx 1.17  0.10* 3.20  0.55y 2.22  0.38y 1.57  0.43*
GST 0.06  0.01* 0.08  0.02* 0.08  0.01* 0.13  0.02y
Abbreviation used and unit: ROS – Reactive Oxygen Species as Zmoles/
min/ml of RBC; GSH – Reduced Glutathione as as mg/ml of RBCs;
TBARS – Thiobarbituric Acid Reactive Substances as nmole MDA
produced/ml RBC; SOD – Superoxide Dismutase as U/mg protein/min;
Catalase as m moles H2O2 degraded/min/mg protein; GPx –
Glutathione Peroxidase as U/mg protein/min; GST – Glutathione-S-
Transferase as U/mg protein/min. Values are mean  SE; n ¼ 5.
*,yp50.05 compared to normal control as evaluated by Student’s
‘‘t’’ test.
 6 R. Shrivastava et al. Drug Chem Toxicol, Early Online: 1–12

Table 3. Effect of TiO2, Al2O3 and ZnO nanoparticles on oxidative (Table 4) on exposure to TiO2, ZnO and Al2O3 nanoparticles
stress variables in mouse liver.
compared to the control group. However in both the tissues,
Liver
levels of ROS were found to be highest in the group exposed to
ZnO NP, indicating it to be most toxic, followed by Al2O3 and
Normal TiO2 ZnO Al2O3 TiO2. In liver, TBARS level was found to be significantly
ROS 230.6  13.2* 270.5  3.1y 327.8  3.8y 313.4  6.4y elevated on exposure to TiO2 and Al2O3 (Table 3) whereas, it
TBARS 2.1  7.5* 3.0  33.5y 2.0  8.4* 2.9  5.2y remained unaltered in the group exposed to ZnO nanoparticle.
SOD 1.33  0.29* 0.45  0.08y 0.52  0.10y 0.61  0.08y
Catalase 1.76  0.31* 0.76  0.02y 0.76  0.13y 0.60  0.14y None of the nanoparticle was found to alter the level of
GPx 2.60  0.51* 1.43  0.07y 1.50  0.17y 1.29  0.04y TBARS in brain (Table 4). Activity of antioxidant enzymes
GST 1.01  0.22* 0.84  0.04* 0.72  0.04* 0.75  0.11* was also evaluated to validate the role of oxidative stress in
elucidating toxic manifestations. Activity of both SOD and
Abbreviations used and units: ROS – Reactive Oxygen Species as
Fluorescent Intensity unit (FIU); TBARS – Thiobarbituric Acid Catalase declined significantly in both liver and brain on
Reactive Substances as mg/gm of tissue weight; SOD – Superoxide exposure to all the nanoparticles compared to control.
Dismutase as units min1 mg protein1; Catalase as nmoles of H2O2
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However highest decrease was observed in case of ZnO NPs,

consumed min1 mg protein1; GPx – glutathione peroxidase as
ngmin1 mg protein1; GST – Glutathione-S-transferase as nmole
suggesting enhanced toxicity compared to Al2O3 and TiO2
conjugate1 mg1 protein. NPs. Activity of GPx was also depleted significantly in the
Values are mean  SE; n ¼ 5. *,yDifferences between values with group exposed to ZnO NPs in both liver and brain compared to
matching symbol notations within each column are not statistically control and other exposed groups. GST activity remained
significant at 5% level of probability
unaltered in all the nanoparticle exposed groups except for a
significant depletion in ZnO exposed group in liver and brain.
Table 4. Effect of TiO2, Al2O3 and ZnO nanoparticles on oxidative
stress variables and neurotransmitter levels in mouse brain. Effects on the level of norepinephrine (NE), dopamine
(DA) 5-hydroxytryptamine (5-HT) in cortex region of
Brain
mouse brain
Normal TiO2 ZnO Al2O3
Table 4 depicts the levels of norepinephrine, dopamine and
Oxidative Stress Biomarkers
ROS 317.7  3.5* 374.7  10.2y 428.6  6.0y 407.7  8.0y
5-hydroxytryptamine in cortex region of mouse brain.
TBARS 3.3  31.4* 3.7  26.0* 2.6  11.3* 2.5  12.7* Administration of all the three nanoparticles TiO2, ZnO and
For personal use only.

SOD 0.29  0.03* 0.24  0.02y 0.26  0.02y 0.42  0.05 Al2O3 led to a significant increase in norepinephrine and
Catalase 1.79  0.11* 0.75  0.29y 1.18  0.19y 0.79  0.19y dopamine levels compared to the control group. Among the
GPx 1.77  0.20* 1.06  0.02y 1.14  0.04y 1.08  0.08y
GST 0.08  0.01* 2.09  0.03y 0.15  0.01* 0.17  0.01* exposed groups, levels of norepinephrine and dopamine were
Neurotransmitters (cerebral cortex) found to be the highest in case of ZnO NP, suggesting
NE 10.37  2.90* 25.84  3.28y 131.4  38.39y 58.72  12.37y enhanced neurotoxic potential of ZnO NP in comparison to
DA 24.90  7.12* 45.49  8.87y 86.53  16.15y 46.39  9.07y other NPs. However, the levels of 5-hydroxytryptamine
5-HT 35.69  2.10* 35.65  1.00* 36.14  1.90* 35.48  1.70*
remained unaltered in all the groups compared to control.
Abbreviations used and units: ROS – Reactive Oxygen Species as
Fluorescent Intensity unit (FIU); TBARS – Thiobarbituric Acid Effects on antioxidant mRNA expression in mouse
Reactive Substances as mg/gm of tissue weight; SOD – Superoxide liver
Dismutase as units min1 mg protein1; Catalase as nmoles of H2O2
consumed min1 mg protein1; GPx – glutathione peroxidase as Total RNA was isolated from all control and exposed samples
ngmin1 mg protein1; GST – Glutathione-S-transferase as nmole by a kit protocol as described earlier and purity and quantity
conjugate1 mg1 protein; NE – Norepinephrine as mg/g tissue weight;
DA – Dopamine as mg/g tissue weight; 5-HT – 5-hydroxytryptamin as of isolated RNA were checked in denaturing formamide
mg/g tissue weight. agarose gel. Figure 2 depicts the expression level of different
Values are mean  SE; n ¼ 4. *,y,Differences between values with antioxidant enzymes in mouse liver following exposure to
matching symbol notations within each column are not statistically
TiO2, ZnO and Al2O3 nanoparticles. Unaltered expression
significant at 5% level of probability
was observed in catalase and GPx transcript on exposure to
NPs, however a marked decrease in the expression of
CuZnSOD and MnSOD transcript was observed. A more
Effects of TiO2, ZnO and Al2O3 nanoparticles on SOD pronounced inhibition in the expression of CuZnSOD and
and catalase activities are shown in Table 2. Activity of SOD MnSOD transcript was noted in case of mice exposed to ZnO
and Catalase reduced significantly following exposure to all and Al2O3 NPs, indicating their enhanced toxicity in
the three NPs in mouse erythrocytes. No difference however comparison to TiO2. The expression of b-actin gene was
was noted in comparative activity of these enzymes among used as control (housekeeping gene) to reveal any variation in
exposed groups. antioxidant expression due to purity of RNA or error in
estimation or loading.
Effects on oxidative stress variables in mouse liver
and brain Effects of TiO2, ZnO and Al2O3 nanoparticles on
antioxidant mRNA expression in mouse brain
Effect of TiO2, ZnO and Al2O3 nanoparticles on the
generation of reactive oxygen species in liver and brain of Figure 2 shows RNA expression level of b-actin and
mice is depicted in Table 3 and Table 4. Levels of ROS, antioxidant enzymes isolated from brain after exposure to
increased significantly in both liver (Table 3) and brain nanoparticles for 21 days. An unaltered expression of catalase
 DOI: 10.3109/01480545.2013.866134 Metal oxides nanoparticles induced toxicity 7
Figure 2. Differential mRNA expression of
antioxidant enzymes of control and TiO2,
ZnO and Al2O3 nanoparticles exposed
mouse. mRNA expression of b-actin (453 bp,
housekeeping gene), catalase (452 bp), GPx
(221 bp), Cu-Zn SOD (201 bp) and MnSOD
(560 bp) in nanoparticles exposed mouse
liver and brain compared with control.
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For personal use only.

transcript was noted following exposure to TiO2, ZnO and
Al2O3 nanoparticles, however a slight upregulation in GPx
expression was observed on exposure to Al2O3 nanoparticles
compared to other nanoparticles. Significant inhibition in the
expression of CuZnSOD and MnSOD transcript was observed
following exposure to ZnO and Al2O3 NPs compared to
control and TiO2 NP. The expression of b-actin gene was used
as control (housekeeping gene) to reveal any variation in
antioxidant expression due to purity of RNA or error in
estimation or loading. No alteration in the level of b-actin was
observed in brain.
Effects on TEM micrographs of mouse liver sections
TEM analyses of the liver sections were performed to study
the biodistribution of nanoparticles. No abnormality was
noted in normal tissues (Figure 3A). TiO2 treated groups
showed the presence particles entrapped within compartments
resembling endosomes and Kupffer cells (Figure 3B).
Numerous cytoplasmic vesicles and Golgi complexes con-
taining nanoparticles trapped inside were observed in liver
sections of ZnO and Al2O3 exposed mice (Figure 3C and D).
Ultrastructure of hepatocytes isolated from Al2O3 treated
group revealed tumescent mitochondria and vacuolization.
Additionally, enhanced deformity and damaged structure near
the nucleus exhibiting signs of necrosis were also observed in
hepatocytes (Figure 3D).
Effects on TEM micrographs of mouse brain
No abnormalities were noted in normal brain tissues
(Figure 4A), whereas the presence of endosomes was observed
near the cell membrane with a large number of nanoparticles
trapped inside in case of ZnO and TiO2 exposed groups
(Figure 4C and D). Clumps of nanoparticles were observed
inside the endosomes resembling nanoaggregates. However,
magnified images show the presence of individual nanoparti-
cles inside the clump (Figure 4B and C). Few nanoparticles
were also noted inside the cylindraxile of neurons (Figure 4C).
The presence of rounded nuclei in neuron was observed in the
control group, however, the ultrastructure of neuron in mice
exposed to Al2O3 NP revealed significant shrinkage of the
nucleus (Figure 4). The phenotypic appearance bears resem-
blance to classical morphological characteristics of apoptosis.
Discussion
The present study investigated the toxicological profile and
general mechanism involved in TiO2, ZnO and Al2O3
nanoparticles-induced sub-acute toxicity in mice. We selected
oral route of administration as the route of exposure for mice
as most of these nanoparticles are being used in food
packaging and thus may gain entry into the body principally
through this route. These nanoparticle are also used in other
consumer products (coating and dermal applications) and thus
there is always a risk of ingestion during use. The doses at
 8 R. Shrivastava et al. Drug Chem Toxicol, Early Online: 1–12
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For personal use only.

Figure 3. TEM micrographs of mouse liver showing no abnormalities (A); presence of particles entrapped within compartments resembling endosomes
and Kupffer cells in TiO2 exposed (B); cytoplasmic vesicles and Golgi complexes containing nanoparticles in ZnO exposed (C); cytoplasmic vesicles
and Golgi complexes containing nanoparticles and also necrosis of nucleus in Al2O3 exposed mouse.

which humans may be exposed to nanoparticles in these
scenarios are yet to be identified as there are no available
guidelines for conducting in vivo toxicity assessment. We thus
selected a dose defined in literature for studying sub-acute oral
toxicity. This dose does not necessarily reflect the actual
concentrations of nanoparticles found in the real environ-
ments, however, they can provide a baseline data to assess the
health risks from exposure to these nanoparticles. Mice were
chosen as the in vivo model of exposure because of their
similarity with human metabolic, biochemical and physio-
logical pathways. Numerous reports are available suggesting
the toxicity of metal oxide NPs at different doses and
durations, without any reported mortality following exposure
to these three NPs (TiO2, ZnO and Al2O3) (Sharma et al.,
2012; Sycheva et al., 2011). Moreover, acute toxicity studies
have been carried out using high doses of these metal oxide
NPs upto 5 g/kg (Klien & Godnic-Cvar, 2012). Thus an
attempt was made in this study to further investigate the toxic
effects of TiO2, ZnO and Al2O3 nanoparticles at an increased
dosage (500 mg/kg) for an increased period of time (21 days)
at both effecter (biochemical) and expression (mRNA) levels.
Oral administration with 500 mg/kg of TiO2, ZnO and Al2O3
nanoparticles for 21 consecutive days induced biochemical
alterations in blood, liver and brain in mice. Cellular oxidative
stress was evident by elevated ROS level, reduced glutathione
level, increased lipid peroxidation and impaired antioxidant
defense status. Despite the fact that there is growing concern in
nanotoxicology studies, little is known about the interaction of
nanoparticles with biological system. TiO2, ZnO and Al2O3
NPs are three metal oxide nanoparticles widely used in
industrial products, such as cosmetics and pharmaceuticals.
Therefore, potential widespread exposure may occur during
both manufacturing and their applications. After entering the
body through different routes, they reach the target organs
through blood. Erythrocytes, being the dominant cell type in
blood are vulnerable to the toxic effect by these nanoparticles
(Li et al., 2008). Due to their characteristics small size and
enhanced absorption properties, nanoparticles interact with
 DOI: 10.3109/01480545.2013.866134 Metal oxides nanoparticles induced toxicity 9
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Figure 4. TEM micrographs of mouse brain showing normal brain tissue with no abnormalities i.e. rounded nuclei in neurons (A); presence of
individual nanoparticles inside clump in TiO2 exposed (B); presence of nanoparticles inside the cylindraxile of neurons in ZnO exposed (C); and
significant shrinkage of nucleus, endosomes near cell membrane with large number of nanoparticles trapped inside in case of Al2O3 nanoparticles (D).

erythrocyte membrane and cause its agglutination by changing
the cell membrane structure and properties (Li et al., 2008).
We studied the effects of TiO2, ZnO and Al2O3
nanoparticles on erythrocyte oxidative stress in the present
study. Our results indicate an increased generation of reactive
oxygen species and elevated TBARS levels in all the
nanoparticles intoxicated groups indicating oxidative stress.
This can be attributed to the phagocytosis of nanoparticles by
erythrocytes which in turn activate a membrane bound
NADPH oxidase leading to the formation of ROS (Afaq
et al., 1998). Elevated level of TBARS in erythrocyte
following exposure to TiO2, ZnO and Al2O3 nanoparticles
revealed enhanced lipid per-oxidative products. Nanoparticles
can get embedded in the lipid protein layer present in the
erythrocyte membrane and cause damage by exerting strong
oxidizing ability (Afaq et al., 1998). Several other studies also
support lipid peroxidation via free radical generation due to
nanoparticles induced toxicity (Li et al., 2008; Park et al.,
2008; Xia et al., 2006). Further, a significant increase in
reduced glutathione level in TiO2, and Al2O3 NPs exposed
group shows the adapting response against the toxic effect of
nanoparticles. GSH can function directly in the destruction of
ROS and an increased level of GSH indicates enhanced
defense mechanism of the body (Cantin & Begin, 1991; Khan
et al., 1990; Martensson & Meister, 1991). Whereas, the level
of GSH in erythrocytes remain unaffected upon exposure ZnO
nanoparticles.
Organisms use a diverse array of antioxidant enzymes
which play crucial role in providing protection against toxic
effects of free radicals. Our studies in erythrocytes have shown
a significant alteration in the activities of most of the
antioxidant enzymes investigated. Decreased activities of
SOD and catalase in TiO2, ZnO and Al2O3 nanoparticles
exposed groups indicate increased generation of free radicals.
However, increased activities of GPx in TiO2 and ZnO exposed
groups again depict an adaptive mechanism against nanopar-
ticles induced oxidative stress. Moreover, increased activity of
GPx can be directly related to the increased level of GSH
observed indicating elevated defense mechanism by these
antioxidants during detoxification mechanism (Nel et al.,
 10 R. Shrivastava et al.
2006). However, GST activity remained unaltered on exposure
to all the nanoparticles studied.
There are few reports which indicated that nanoparticles
can cross the blood-brain barrier and enter the central nervous
system (Kreyling et al., 2002; Lockman et al., 2004;
Oberdörster et al., 2004; Sharma & Sharma, 2007; Wang
et al., 2008). Rapid utilization of nano particles and their
potential risk to central nervous system (CNS) has elicited
much concern recently. Considering this, one of the major
focuses of the present study was to study the potential role of
metal oxide nanoparticles (TiO2, ZnO and Al2O3) in the
induction of brain oxidative stress and alterations in neuro-
transmitters level in mouse. Our data showed increased
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production of reactive oxygen species following exposure to
all the three metal oxide nanoparticles, indicating that mice
treated with these nanoparticles underwent oxidative stress.
Generally direct particles are translocated to various tissues in
case of TiO2 and Al2O3 NPs, whereas ZnO nanoparticles are
mostly dissolved in the stomach to form Zn2þ ions, and are
then taken up in the tissues by interaction between zinc and
sulfur-containing ligands in proteins (Baek et al., 2012;
Huang et al., 2010). The binding of zinc to L-histidine (His)
in cerebrospinal fluids seems to be involved in transferring
zinc to target sites, which regulate its uptake across the brain
barrier systems. Free Zn2þ modulates many membrane
receptors, transporters and channels (Takeda et al., 2002).
The role of oxidative stress in nanoparticles induced CNS
For personal use only.
toxicity has been widely reported. These studies suggest that
nanoparticles could be phagocytized by neurons and micro-
glia, resulting in the release of ROS. Overproduction of ROS
would break down the balance of the oxidative/antioxidative
system in the brain, which is closely related to the reduction
of the antioxidant enzymes (Long et al., 2007; Wang et al.,
2008). In the present study also, the activities of SOD,
Catalase, and GPx, were significantly inhibited in the groups
exposed to the nanoparticles except a significant enhancement
in SOD activity in Al2O3 nanoparticles. Ma et al. (2010) has
also reported decreased activities of antioxidant enzymes in
rat brain exposed to TiO2 nanoparticles. Whereas, GST
remained unaffected, except a significant elevation in TiO2
nanoparticles. Role of GST during TiO2 nanoparticles
administration needs to be further investigated.
Administration of chemicals disturbs the spontaneous
activity of the cells and influences neurotransmitter turnover.
Thus the level of neurotransmitters in cerebral cortex region
of mice has been evaluated. Nanoparticles led to an increased
level of norepinephrine (NE) and dopamine (DA) in cortex
region of mouse brain suggesting weakened ability to
maintain an appropriate state of activation in the central
nervous system due to toxicity. However, the level of 5-HT
remained unaffected following exposure of these nanoparti-
cles. Some researchers have also reported impaired glutamate
and AChE activities in TiO2 nanoparticles exposed animals
indicating disruption in the normal functioning of central
nervous system (Ma et al., 2010). Neurotransmitters are
known to play a key role in memory, awareness, thought, and
consciousness and allow the organism to become alert and
guards against the intensification of reflex reactions and other
behavior. Our results suggest that accumulation of these
nanoparticles in brain resulted in altered synthesis and release
Drug Chem Toxicol, Early Online: 1–12
of certain neurotransmitters and receptors in nerve cells,
leading to neural damage. In addition, ROS could attack the
various kinds of neuronal connections and decrease the
memory in experimental animals (Ma et al., 2010). The
resulting oxidative stress and brain injury is due to a cascade
of reactions triggered by metal oxide nanoparticles, such as
lipid peroxidation, decreased activity of antioxidant enzymes,
release of nitric oxide, reduction of glutamic acid, and
downregulation of acetylcholinesterase activity.
Nanoparticles have been found to be accumulated in liver
which means that nanoparticles could be transported to the
organs after uptake by the gastrointestinal tract (Liu et al.,
2009; Ma et al., 2009). Moreover, some recent studies have
unequivocally showed that exposure to nanoparticles causes
oxidative damage and liver dysfunction in vivo (Cui et al.,
2010; Liu et al., 2009; Ma et al., 2010; Wang et al., 2007).
In the present study also, elevated level of ROS following
exposure to TiO2, ZnO and Al2O3 nanoparticles in liver was
observed. Nanoparticles can generate ROS by different
mechanisms. The uptaken metal oxide nanoparticles can
lose the metallic ions which directly interact with NADPH
oxidases from the plasma membrane or mitochondria thus
disturbing the electron transport chain and generating a
superoxide anion (Klotz & Sies, 2009). Level of TBARS
increased significantly in TiO2 and Al2O3 nanoparticles
exposed groups while ZnO nanoparticles did not produce
any change. Moreover, activities of SOD, CAT and GPx, were
significantly inhibited due to exposure of all the three
nanoparticles suggesting triggered oxidative stress via
increased ROS production and lipid peroxidation with a
subsequent reduction in the level of antioxidant defense
enzymes of the mouse liver (Cui et al., 2010). On the basis of
above findings, it can be concluded that being the primary
metabolizing organ of the body liver might be the most
susceptible organ to nanoparticle exposure and liver damage
is a consequence of direct enzyme inhibition by these
nanoparticles (Liu et al., 2009; Ma et al., 2009). This study
is first in line to report the effect of three metal oxides
nanoparticles (TiO2, ZnO and Al2O3) on gene expression
profile of antioxidant enzymes in mice liver and brain.
However, there are few reports which have investigated the
changes in gene expression of inflammatory markers (Park
et al., 2008) and antioxidant enzymes in lung after inhalation
and intratracheal instillation exposure to nanoparticles
(Janssen et al., 1994). Inhalation of ultra-fine TiO2 nanopar-
ticles led to an increase in gene expression of manganese
superoxide dismutase (MnSOD), catalase and GPx (Janssen
et al., 1994). In contrast, the expression profile of copper-zinc
superoxide dismutase (CuZnSOD) was less striking in these
studies. On the contrary, our results show that following the
exposure to TiO2, ZnO and Al2O3 nanoparticles the expres-
sion level of catalase and GPX remained unaffected in both
mice liver and brain. However, significant inhibition in
mRNA expression profile of CuZnSOD and MnSOD tran-
script was observed which is an important finding of the
present study (Ma et al., 2010). The downregulated expression
of CuZnSOD and MnSOD transcripts suggests decreased
activity of detoxifying excess NPs and ROS scavenging which
supports our biochemical results. Cui et al. (2010) also found
decreased expressions of liver antioxidant enzymes after
 DOI: 10.3109/01480545.2013.866134
intragastric administration of TiO2 nanoparticles to mice.
There was a slight upregulation in GPx mRNA expression
level upon exposure to Al2O3 nanoparticles in brain which
needs to be further investigated.
In order to study the biodistribution of TiO2, ZnO and
Al2O3, Transmission Electron Microscopy (TEM) analyses of
liver and brain tissues were performed. The results obtained
with TEM data are in line with oxidative stress parameters.
The nanoparticles were found to be distributed throughout the
cytoplasm and nucleus. Clumps of nanoparticles are found
inside endosomes and in cytoplasm. Large endosomes with
nanoparticles in the cytoplasm of the cells and near the cell
and nuclear membrane were observed, which suggested that
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Defence Research & Development on 12/17/13
nanoparticles were entering the cells through endocytosis.
The current evidence from electron micrographs sheds light
on the endocytic pathway of nanoparticle uptake. Further, a
detailed study should be conducted to unravel the mechanism
involved in metal oxide nanoparticles uptake. Moreover,
deformity and damaged structure near the nucleus in hepato-
cytes could be observed which exhibited signs of necrosis
whereas, ultrastructure of neurons from the Al2O3 exposed
group presented significant shrinkage of the nucleus with
apoptotic signal.
As indicated at the beginning that the nanoparticles of the
three chosen metals induced toxicity might be attributed due
to high dosing rather than the particle size and distribution. It
is known that metal ions released by three nanoparticles might
For personal use only.
be contributing to the toxicities. Although we didn’t measure
concentration of these metals in blood or soft tissues, this
might be the main lethal mechanism for the more pronounced
toxicity of ZnO NPs that the other two. TiO2 NPs is known to
enter central nervous system and may damage brain. Our
result suggest that among the three nanoparticles the changes
in the biogenic levels were more pronounced in ZnO
nanoparticle exposed mice while TiO2 and Al2O3 exposed
mice elicited similar effects. It has recently been reported that
ZnO NPs are slightly soluble after entering the organism, it
can be ionized to release Zn2þ, which may access cells
through ion channels (Deng et al., 2009; Liu et al., 2013).
These authors further suggested that Zn2þ can subsequently
enter the blood circulation to damage cell metabolism and
stimulate cells to produce large amounts of ROS to cause
oxidative stress and ultimately apoptosis (Deng et al., 2009;
Liu et al., 2013).
Conclusions
In conclusion, the study confirms that interaction of absorbed
nanoparticles with cellular components could generate ROS
and lead to cellular toxicity if the magnitude of ROS
production overwhelms the antioxidant defense status of the
cell. Accumulation of these nanoparticles into brain subse-
quently disturbs the normal metabolism of neurotransmitters,
ultimately leading to brain damage and thereby indicating the
neurotoxic potential of these NPs. Interestingly, inhibition in
gene expression profile of CuZnSOD and MnSOD in liver
and brain, also validates the hypothesis, in turn establishing
oxidative stress as the major mechanism inducing toxic
manifestations. Electron micrographs confirmed significant
number of nanoparticles in vital organs such as cytoplasm,
Metal oxides nanoparticles induced toxicity 11
mitochondria and nucleus. The study revealed ZnO NP as the
most toxic NP, followed by Al2O3 and TiO2, as evident from
the observed results. Nanoparticles thus, possess potential
toxicity and risk assessment is needed prior to their wide
consumption to ensure safe utilization and disposal of
nanoparticles.
Acknowledgements
We thank Dr. M.P. Kaushik, Director, Defence Research and
Development Establishment for offering all facilities and
support required for this study. Ms. Rupal Shrivastava is
recipient of MAPCOST Junior Research Fellowship.
Declarartion of interest
None declared.
This work was supported by the grant from Ministry of
Defence, Government of India.
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