Genotoxicity analysis of cerium oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral administration
Genotoxicity analysis of cerium oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral administration
Monika Kumari 0 1
Srinivas Indu Kumari 1
Paramjit Grover 1
0 Department of Genetics, Osmania University , Osmania University Main Road, Hyderabad 500007, Andhra Pradesh , India
1 Toxicology Unit, Biology Division, Indian Institute of Chemical Technology , Hyderabad 500007, Andhra Pradesh , India
The applications of cerium oxide nanoparticles (CeO2 NPs; nanoceria) extend to polishing agents, diesel fuel additives and as a putative antioxidant in therapeutics. Therefore, understanding the long-term toxic effects of CeO2 NPs is of particular importance. This study investigated the 28 days of repeated toxicity of 30, 300 and 600 mg/kg body weight (bw)/day of nanoceria and CeO2 microparticles (MPs) in Wistar rats after oral exposure. Genotoxicity was analysed using comet, micronucleus (MN) and chromosomal aberration (CA) assays. The results demonstrated a significant increase in DNA damage in peripheral blood leukocytes and liver, MN and CA in bone marrow as well as MN in peripheral blood after exposure to CeO2 NPs at 300 and 600 mg/kg bw/day. Significant alterations were observed in alkaline phosphatase and lactate dehydrogenase activity in serum and reduced glutathione content in the liver, kidneys and brain at 300 and 600 mg/kg bw/day in a dose-dependent manner. Conversely, CeO2 MPs did not induce any significant toxicological changes. A much higher absorptivity and significant tissue distribution of CeO2 NPs was perceived in comparison to CeO2 MPs in a dose-dependent manner. A substantial fraction of CeO2 NPs was cleared by urine and faeces. Histopathological analysis revealed that CeO2 NPs caused alterations in liver, spleen and brain. Further, distinct difference in the data among genders was not obvious. In general, the results suggested that prolonged oral exposure to nanoceria has the potential to cause genetic damage, biochemical alterations and histological changes after retention in vital organs of rats at high concentrations.
Nanoparticles (NPs) differ from larger materials in the number
of atoms at the surface due to their small size (<100 nm). Their
physical properties comprising high surface area per unit
mass, enhanced chemical and surface reactivity, magnified
cell permeability, which are different from bulk materials,
have led to increased use of NPs in various commercial
). However, size-dependent toxicity of various
nanomaterials (NMs) has also been reported (3). Therefore,
toxicological profiling of NMs, which have wide consumer
value, is warranted.
The ability of cerium oxide (CeO2) NPs (nanoceria) to switch
oxidation states between III and IV based on environmental
conditions makes CeO2 NPs comparable to that of biological
). Nanoceria has been included in the list of 14
representative manufactured NMs for testing owing to their
existing commercial use and high production volume (
potential uses of CeO2 NPs incorporate pharmaceutical industry
) and in nanotherapeutics (
). Among the several uses of CeO2
NPs, some are used as a polishing agent for glass mirrors, plate
glass, television tubes, ophthalmic lenses and precision optics
). In cosmetic industry, CeO2 NPs are used as UV-absorbing
compound in sunscreen (
) and as a UV-scattering agent in
non-irritating lipsticks (
). Additionally, CeO2 NPs are used as
a diesel fuel-borne catalyst to reduce particulate matter ejection
in emission control system of automobiles (
hazard of accidental exposure to environment and their entry
into human body through the food chain is inevitable (
Some recent publications have suggested that the exposure
to CeO2 NPs in various organisms and cells can cause adverse
effects on their growth and development (
). The potential
for human health hazards and the environmental effects
following exposure to CeO2 NPs has been examined by in vitro studies
). Further, there are studies that have investigated CeO2
NPs induced toxicity through inhalation, intratracheal
instillation and intravenous (iv) administration routes of exposure in
). On the contrary, there is scarcity of oral exposure
reports. Therefore, the objective of the current study was to
evaluate the toxicity of CeO2 NPs through oral route.
Characterisation is important for the assessment of the
particle behaviour in the biological system because physical
and chemical properties are likely to play an important role
in determining the behaviour, reactivity and potential
toxicity of NPs (
). Hence in the present study, measurement of
size, hydrodynamic diameter and surface charge of CeO2 NPs
was described and compared with CeO2 microparticles (MPs).
Genotoxicity studies are fundamental to assess hazardous
effects of NPs as despair of genetic diseases and cancers is well
known. Therefore, genetic toxicity studies may be used to draw
conclusions about the carcinogenicity potential of chemicals.
The comet assay is useful for the detection of DNA damage in
genotoxicity testing. This assay in animal models can give
information on secondary effects such as inflammation due to
reactive oxygen species (ROS) production (
). Maffei et al. (
suggested micronucleus test (MNT) as a promising biomarker
for the early detection of colorectal cancer and demonstrated
the significant association of micronucleus (MN) frequency
with the clastogenic activity in the plasma. During anaphase,
MN are formed from chromosomal fragments or whole
chromosomes that are left behind when the nucleus divides. The
chromosomal aberration (CA) test can diagnose agents that
cause structural chromosome or chromatid breaks, dicentrics
and other abnormal chromosomes, translocations that are
implicated in the various human genetic diseases and cancers (
Injuries to the liver and oxidative stress may increase alkaline
phosphatase (ALP), lactate dehydrogenase (LDH) and
diminish the reduced glutathione (GSH) content. Therefore,
evaluation of biochemical indices needs to be addressed. Further, NPs
can have different toxicokinetic profiles such as absorption,
distribution, elimination and biopersistence along with tissue
pathology compared with MPs due to stronger interaction of
NPs with biological systems (
). Hence, to gain an insight into
the uptake, retention, kinetics and affects on tissue architecture,
biodistribution and histopathology studies are also mandatory.
Therefore, in the present investigation, a 28-day repeated oral
dose study of CeO2 NPs and CeO2 MPs was carried in albino
Wistar male and female rats. The effect of the particles on the
behavioural symptoms, body weight (bw) and feed intake was
examined. Genotoxicity assays such as the comet assay in
peripheral blood leukocytes (PBL) and liver MNT in bone
marrow and peripheral blood (PB) and CA in bone marrow cells
were performed. Target biochemical enzymes namely ALP for
liver damage and LDH for membrane damage were assessed in
serum. GSH content assay was carried out to study oxidative
stress in various tissues such as the liver, kidneys and brain.
To get insight of morphological alterations, histopathological
examination of the liver, kidneys, spleen, heart and brain was
carried out from treated and control rats. Furthermore,
biodistribution of cerium (Ce) in rat’s whole blood, vital organs, urine
and faeces was analysed using inductively coupled plasma
optical emission spectrometer (ICP-OES). The doses used to
evaluate the toxicity of CeO2 NPs were 30, 300 and 600 mg/kg
bw/day. The lowest treatment dose was to signify the
conceivable human exposure. In order to ascertain the amount of Ce in
animal’s organs as well as toxicity through accidental exposure
to large amounts of CeO2 NPs, two higher doses were selected.
Materials and methods
NPs and chemicals
CeO2 NPs (CeO2 < 25 nm, 99.95%, CAS No. 1306-38-3) and CeO2 MPs (CeO2
< 5µm, 99.9%, CAS No. 1306-38-3) were purchased from Sigma Chemical Co.
Ltd (St Louis, MO, USA). Phosphate buffered saline (Ca2+, Mg2+ free; PBS),
cyclophosphamide (CP), normal melting agarose (NMA), low melting agarose
(LMA), etc. were also purchased from Sigma Chemical Co. Ltd.
Characterisation of CeO2 NPs and CeO2 MPs
The particles were characterised using transmission electron microscopy
(TEM), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV)
to evaluate the material size, size distribution, state of dispersion and zeta
potential of the NMs in the Milli-Q water. Characterisation of CeO2 NPs and
CeO2 MPs was performed to assess the size and morphology using a TEM
(JEM-2100, JEOL, Japan). The images were obtained from TEM with an
accelerating voltage of 120 kV. The TEM was equipped with a plunge freezer and
cryo transfer holder to fix specimens in the frozen state and fitted with a Gatan
2Kx2K CCD camera for acquiring high-resolution images. Particles were
suspended in Milli-Q water at a concentration of 0.01 mg/ml, and one drop of
suspension was placed on a carbon-coated copper TEM grid and evaporated at
room temperature. The software for advanced microscopy techniques was used
for the digital TEM camera. This software was calibrated for nanoscale size
measurements for the accurate examination of NPs. For the size measurement,
100 particles were calculated from random fields of view and images showing
the general morphology of the particles.
The size of the NPs and agglomerates was measured through DLS and
LDV using a Malvern Zetasizer Nano-ZS (Malvern Instruments, UK). This
device uses a 4 mW He–Ne 633 nm laser to analyse the samples and an electric
field generator for the LDV measurements. At the concentration of 40 µg/ml
freshly prepared CeO2 NPs and MPs, suspension in Milli-Q water was
ultrasonicated using a probe sonicator (UP100H, Hielscher Ultrasonics GmbH,
Teltow, Germany) for 10 min at 90% amplitude. The high concentration of the
suspension was further diluted two times and adjusted to a lower
concentration of 20 µg/ml to acquire enough counts per second. The prepared samples
were transferred to a 1.5 ml square cuvette for DLS measurements and 1 ml
of the suspension was transferred to a Malvern Clear Zeta Potential cell for
LDV measurement. The mean NPs diameter was calculated using the same
software program as utilised in the NPs distribution, and the polydispersity
index (PdI) was used to measure the size ranges present in the solution. The
PdI scale ranges from 0 to 1, where 0 indicates monodisperse and 1 indicates
polydisperse state of particles.
Male and female albino Wistar rats, aged 6–8 weeks and weighing 80–120 g,
were obtained from the National Institute of Nutrition, Hyderabad, India. The
animals were acclimatised for 1 week in groups of five in polypropylene cages.
The animals were fed with a standard laboratory pellet diet and reverse
osmosis water was provided ad libitum and maintained under standard conditions
of humidity (55–65%), temperature (22 ± 3°C) and light (12 h light/12 h dark
cycles). The study was approved by the Institutional Animal Ethics Committee.
Treatment of animals
Twenty-eight days of repeated dose oral toxicity study was conducted with
male and female rats with CeO2 NPs and CeO2 MPs following Organization for
Economic Co-operation and Development (OECD) Guideline 407 (
). For the
treatment, rats were weighed daily and concentrations were calculated for the
doses 30, 300 and 600 mg/kg bw for CeO2 NPs and CeO2 MPs and administered
orally. The rats were administered once daily for 28 days using a suitable
intubation cannula with dosing volume 2 ml/100 g bw. Particles were suspended
in Milli-Q water, properly ultrasonicated (UP100H, Germany) and vortexed
before every treatment of the rats. Animals were divided into four groups (10
rats; 5 males and 5 females in each group): control, low dose (30 mg/kg bw/
day), medium dose (300 mg/kg bw/day) and high dose (600 mg/kg bw/day)
of CeO2 NPs and CeO2 MPs. Control group received only Milli-Q water. The
highest dose was selected based on induction of a toxic effect without severe
sufferings and mortality, whereas the lowest dose demonstrated slight adverse
effects. Feed consumption and bw were monitored weekly for 4 weeks. All
treated rats were sacrificed by cervical dislocation after 24h of last
administration of a dose. A known mutagen CP was used as the positive control for
genotoxicity studies at a dose 40 mg/kg bw and the volume injected was 0.01 ml/g
bw. This was given intraperitoneally (ip) 24 h before sacrifice.
The alkaline comet assay was used for the assessment of DNA damage in the
rats after 28 days of repeated oral exposure to the CeO2 NPs and CeO2 MPs.
It was performed according to the method described by Tice et al. (
slight modifications. Liver samples were collected after the sacrificing of rats.
Whole blood withdrawal was done in EDTA-coated tubes from retro-orbital
plexus. The comet assay in liver tissue was performed following the technique
described by Miyamae et al. (
). The tissue was removed from the rats after
sacrificing at various time intervals, minced and suspended at ~100mg/ml in
chilled homogenising buffer (pH 7.5) and homogenised gently at a speed of
500–800 r.p.m. Cell viability was determined by the trypan blue exclusion
). Three slides were prepared for each experimental condition. In
brief, microscope slides were pre-coated with 120 µl of 0.75% NMA in PBS
and allowed to solidify overnight at 37°C after covering with cover slip for
uniform layer. For second layer preparation, 10 µl of whole blood (10 000–
30 000 lymphocytes) or liver homogenate was suspended in 120 µl of 0.37%
LMA. The suspension of cells and LMA was pipetted onto pre-coated slides
and spread uniformly covering with cover slip. The slides were dried at 4°C
for 10 min. A third layer of plain 0.37% LMA (120 µl) was applied and a cover
slip was quickly put to get an even layer and dried at 4°C. After removing the
cover slip, the slides were immersed in chilled lysis buffer (2.5 M NaCl, 0.1 M
Na2 EDTA, 0.2 M NaOH, 1% Triton X-100, 10% dimethyl sulphoxide, pH
10.0) for 10 h at 4°C. The slides were pre-soaked for 20 min in alkaline buffer
(10 M NaOH, 200 mM Na2 EDTA, pH > 13.0) and then electrophoresis was
performed at 25V adjusted at 300 mA for 20 min. The slides were neutralised
twice in 0.4 M Tris buffer, pH 7.5, for 5 min and once in absolute methanol for
5 min. Coded slides were scored after staining with ethidium bromide (20 μg/
ml) using a fluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan)
with a blue (488 nm) excitation filter and yellow (515nm) emission (barrier)
filter at ×400 magnification. A total of 150 randomly selected cells per rat (50
cells per slide) were used to measure the amount of DNA damage and expressed
as percentage of DNA in the comet tail. Quantification of DNA breakage was
realised by using a Comet Image Analysis System, version Komet 5.5 (Single
cell Gel Electrophoresis analysis company, Andor Technology 2005, Kinetic
Imaging Ltd, Nottingham, UK).
The MNT in the rat bone marrow cells was carried out following the method
described by Schmid (
). After 28 days of repeated oral treatment, the bone
marrow was removed from both femur and tibia by aspiration into hypotonic
solution of 1% sodium citrate and centrifuged at ~1000 r.p.m. for 5 min. The
cell pellet was resuspended in a drop of 1% sodium citrate and a smear was
prepared on a microscope slide and allowed to dry in humidified air overnight.
The MNT in PB cells was performed according to the protocol described by
Celik et al. (
) with some modifications and according to the OECD Guideline
). Whole blood was collected from retro-orbital plexus of rat from each
group and smears were made on clean microscope slides. The slides were air
dried, fixed in methanol for 2min and stained with 0.5% Giemsa stain
prepared in PBS for 3 min. The stained slides were used for the assessment of
the MN occurrence. Three slides were made for each animal; the slides were
microscopically analysed at ×1000 magnification. Randomly, 2000
polychromatic erythrocytes (PCEs) per animal were selected from three slides and the
frequency of micronucleated PCEs (MN-PCEs) was determined. In order to
determine the ratio of PCEs to normochromatic erythrocytes (NCEs) in the
bone marrow and PB, ~1000 cells from each animal were examined and the
ratio was expressed as percentages: (PCEs × 100/PCEs + NCEs).
The method described by Adler (
) was used for CA analysis and performed in
bone marrow cells. It is globally recommended to follow the OECD Guideline
) as a test method to identify CAs. For analysis of metaphase cells, cell
division was arrested by a mitotic inhibitor, colchicine (0.020%), 0.01 ml/g bw
ip 2 h prior to sacrifice after 28 days of repeated oral treatment. The bone
marrow was collected from femur and tibia bones by rinsing in hypotonic solution
with 0.9% sodium citrate centrifuged at 2000 r.p.m. for 20 min. Cells were then
fixed through several changes of ice-cold Carnoy’s solution (methanol:acetic
acid, 3:1 v/v) until the pellets were clean. After refrigeration for at least 24 h,
cells were centrifuged and resuspended in fresh fixative, i.e. Carnoy’s solution,
dropped onto slides, dried and stained with Giemsa. Three slides for each
animal were made by the flame-dried technique. Five hundred well-spread
metaphases per dose (100 per animal) were selected to detect the presence of CAs.
The mitotic index (MI) was determined with 1000 or more cells.
ALP activity assay
The blood samples were collected in a tube without having anticoagulant after
28 days of repeated oral treatment with CeO2 NPs and CeO2 MPs. Serum
(30 µl) was pooled by centrifuging blood at 1500 r.p.m. for 10 min for ALP
activity determination. This assay was based on kinetic reaction. The activity of
ALP was determined following p-nitrophenyl phosphate method as described
in kit from M/s. Siemens Healthcare Diagnostics Ltd, Baroda, Gujarat. This
method utilises p-nitrophenyl phosphate that is hydrolyzed by ALP into a
yellow coloured product p-nitrophenol having maximal absorbance at 405 nm.
The rate of reaction is directly proportional to the enzyme activity. The
absorbance of the test was read against blank at 405 nm at intervals of 30 s for 2 min
by using spectrophotometer (Spectra Max Plus, Molecular Devices, Sunnyvale,
CA, USA). The enzyme activity was expressed as U/L using molar extinction
coefficient of 18.75/mM/cm.
The activity of LDH was estimated in serum according to the procedures
described by McQueen (
). In a quartz cuvette, 1000 µl of Sorensen
phosphate buffer, 100 µl reduced nicotinamide adenine dinucleotide and 20 µl of
serum were added and mixed well. Next, 150 µl sodium pyruvate was added
and again mixed well. The absorbance was measured spectrophotometrically at
340 nm at 10-s intervals for 2 min using spectrophotometer. The LDH activity
was expressed as µmol/h/ml using molar extinction coefficient of 6.3/mM/cm.
Reduced GSH content
The reduced GSH content was measured using the method of Jollow et al. (
After 28 days of repeated oral exposure with CeO2 NPs and CeO2 MPs, rats
were sacrificed and liver, kidneys and brain were removed. One gram of tissue
from liver, kidneys and brain of treated and control rats was rinsed in ice-cold
physiological saline, perfused with cold potassium chloride buffer (1.15% KCl
and 0.5 mM EDTA) and homogenised in potassium phosphate buffer (KPB,
0.1 M, pH 7.4) using Miccra D-1 high-speed tissue homogeniser. An aliquot
of 0.5 ml of each tissue homogenate was incubated with 0.5 ml of
sulfosalicylic acid (4% w/v) for 1 h in ice and centrifuged at 10 000 r.p.m. for 10 min.
A 0.4 ml aliquot of the supernatant was mixed with 0.4 ml of Ellman’s reagent,
5,5'-dithiobis-(2-nitrobenzoic acid (4 mg/ml in 5% sodium citrate) and 2.2 ml
KPB (0.1 M, pH 7.4). The yellow colour developed was read at 412 nm. The
amount of GSH present was expressed as µg GSH/g wet weight of tissue.
Histopathology was conducted in the different organs, i.e. heart, liver, spleen,
brain and kidneys of albino Wistar rats after 28 days of repeated oral dose
of CeO2 NPs and CeO2 MPs. After sacrifice, tissues were washed with 1%
ice-cold saline and fixed in 10% neutral buffered formalin. The formalin-fixed
tissues were processed in a Leica TP 1020 tissue processor and then
embedded in paraffin blocks using Leica EG 1160 paraffin embedder. The paraffin
blocks were sliced into ribbons of 3-µm thick sections using a Microm HM 360
microtome and mounted on a glass microscope slide. After that the slides were
stained in haematoxylin and eosin (H&E) using a Microm HMS-70 stainer
and examined with Nikon Eclipse E 800 microscope at ×400 magnification.
A minimum of three random sections per slide and at least three different fields
were assessed for histopathological conditions.
Cerium content analysis in tissues
The biodistribution study of the CeO2 NPs and CeO2 MPs in the female Wistar
rats was carried upon 28 days of repeated oral treatment. The animals were
placed in metabolic cages after treatment to collect the urine and faeces
samples. Rats were sacrificed through cervical dislocation and whole blood, liver,
kidneys, heart, brain and spleen were collected after 28 days. The samples were
processed using the method of Gómez et al. (
). The samples were
predigested in nitric acid overnight, and heated at 80°C for 10 h, followed by
additional heating at 130–150°C for 30 min. Subsequently, a volume of 0.5 ml of
70% perchloric acid was added, and the samples were again heated for 4 h and
evaporated nearly to dryness. Following digestion, the samples were filtered,
and 2% nitric acid was added to a final volume of 5ml for analysis. The
standard solution of Ce was serially diluted to 100, 50, 10 and 1 p.p.m., and
wavelength of 418.66 nm was found to get intensity of samples. The Ce content in
the samples was determined using ICP-OES (JY Ultima, Jobin Vyon, France).
The statistical significant changes between treated and control groups were
analysed by one-way analysis of variance. All results were expressed as mean
± SD. Multiple pair-wise comparisons were done using the Dunnett’s multiple
comparison post-test to verify the significance of positive response. Statistical
analyses were performed using GraphPad Instat Prism 3 Software package for
windows (GraphPad Software, Inc., La Jolla, CA, USA). The statistical
significance for all tests was set at P < 0.05.
Characterisation of CeO2 NPs and CeO2 MPs
The data of physico-chemical characteristics of CeO2 particles
that were determined by TEM, DLS and LDV analysis are
shown in Table I. Over 100 particles were measured in random
field to calculate the mean size of CeO2 NPs and CeO2 MPs
using TEM. The size obtained of CeO2 NPs and CeO2 MPs was
24.2 ± 1.63 nm (Figure 1A) and 3.14 ± 1.29 μm (Figure 1B),
respectively. The morphology of particles was observed as
polyhedron crystals. The hydrodynamic diameter and PdI of
CeO2 NPs in Milli-Q water suspension obtained by DLS was
Zeta potential ζ (mV)
Electrophoretic mobility (μm·cm/s V)
CeO2 NPs and CeO2 MPs at the concentration of 40 µg/ml were dispersed in Milli-Q water and mixing was done via probe sonication for 10 min just before
estimations. ND = not detectable.
191.2 ± 20.25 and 0.304, respectively. The larger hydrodynamic
diameter than the TEM size indicated that CeO2 NPs formed
larger agglomerates in Milli-Q water suspension than in the dry
state. Zeta potential (ζ) and electrophoretic mobility of CeO2
NPs in Milli-Q were quantified by LDV and found to be −17.0
mV and −1.15 µm·cm/s V, respectively, at pH 7.0. In case of
CeO2 MPs, DLS and LDV data were found to be out of the
detection limit (Table I).
Animal observation, food consumption, bw and organ weight
Mortality was not observed in male and female Wistar rats after
28 days of repeated doses of 30, 300 and 600 mg/kg bw/day of
CeO2 NPs and CeO2 MPs in rats. However, rats treated with high
dose of CeO2 NPs showed dullness, irritation and moribund
symptoms. Further, both CeO2 NPs- and CeO2 MPs-treated
animals showed insignificant loss in feed intake (Figure 2A), bw
(Figure 2B) and relative organ weight (Figure 2C). Moreover,
average was calculated combining data obtained from feed
intake, bw and relative organ weight of male and female rats
due to lack of apparent difference in data. The % loss in feed
intake was in the range of 13.0–20.0% in CeO2 NPs-treated
group and 1.7–12.4% in CeO2 MPs-treated group. Likewise,
% bw loss was in the range of 10.9–16.7% and 8.0–10.2% in
CeO2 NPs- and CeO2 MPs-exposed rats, respectively.
After 28 days of repeated oral treatment, a significant (P < 0.01)
increase in % tail DNA was observed in the PBL and liver of
male and female Wistar rats at 300 and 600 mg/kg bw/day dose
of CeO2 NPs (Figure 3A and B). However, significant DNA
damage was not observed at 30 mg/kg bw/day of CeO2 NPs and
all doses of CeO2 MPs in comparison to control. In all samples,
the cell viability by the trypan blue exclusion technique was
>90% (data not shown). The mean % tail DNA of positive
control rats was significantly (P < 0.01) higher compared with
the control. Moreover, gender-dependent variation in % tail
DNA was not apparent.
The MNT in bone marrow and PB was conducted after
28 days of oral treatment with 30, 300 and 600 mg/kg bw/
day of CeO2 NPs and CeO2 MPs in male and female Wistar
rats. The data indicated statistically significant increment
in MN-PCEs frequency in bone marrow (Table II) and PBL
(Table III) in the CeO2 NPs-treated groups of male and female
rats at 600 mg/kg bw/day (P < 0.01) and at 300 mg/kg bw/
day (P < 0.05) in gender-independent manner. However, CeO2
MPs-treated groups did not show any significant increase in
the frequency of MN-PCEs. On the other hand, CP-treated
group induced a substantially significant (P < 0.01) effect on
MN-PCEs frequency. The dose of 600 mg/kg bw/day of CeO2
NPs exhibited a significant decrease in % PCEs in bone marrow
of male and female rats in comparison to the negative control
rats (Table II).
The results of the CA assay in bone marrow cells examined
after the oral administration of CeO2 NPs and CeO2 MPs for
28 days at various doses (30, 300 and 600 mg/kg bw/day) to
male and female Wistar rats are shown in Tables IV and V,
respectively. The structural changes observed were gaps,
breaks, minutes and acentric fragments. The CeO2 NPs induced
significant (P < 0.01) total aberrations including gaps in bone
marrow cells at 300 and 600 mg/kg bw/day dose, whereas total
aberrations excluding gaps incurred significantly (P < 0.01)
only at 600 mg/kg bw/day in the both male and female rats.
Moreover, dose-dependent increments in structural aberrations
were observed in bone marrow cells of CeO2 NPs-treated rats.
However, reciprocal translocations were observed only in
positive control group treated with CP but not in treated groups.
The MI was used to conclude the rate of cell division. There
was no significant decrease in MI after repeated exposure with
CeO2 NPs and CeO2 MPs at any dose level. Moreover, there
was no revelation of alterations in chromosomes by CeO2 MPs
after repeated oral study compared with the control group
(Tables IV and V).
The activity of serum ALP was found to be increased significantly
(P < 0.01) at 300 and 600 mg/kg bw/day of CeO2 NPs in both
male and female rats. The percent increase in ALP activity in
male was 18.31 and 37.48%, respectively, for the exposure with
300 and 600 mg/kg bw/day CeO2 NPs. Likewise, in female rats,
19.18 and 38.00% increase in ALP activity was documented
at 300 and 600 mg/kg bw/day dose of CeO2 NPs, respectively.
However, exposure of CeO2 MPs at all dose level and CeO2 NPs
at 30 mg/kg bw/day dose did not induced significant increase in
ALP activity and percent increase was in the range of 1–6% when
compared to control in both male and female rats (Figure 4A).
The activity of serum LDH significantly increased (P < 0.01) at
300 and 600 mg/kg bw/day dose of CeO2 NPs after 28 days of
repeated oral exposure. In male rats, percent increase in LDH
activity with 300 and 600 mg/kg bw/day dose of CeO2 NPs
with respect to control was observed as 78.56 and 179.53%,
respectively. Similarly in female rats, LDH activity was noted
to be 79.06 and 180.85% higher than the control at 300 and
600 mg/kg bw/day of CeO2 NPs. Conversely, CeO2 MPs and
30 mg/kg bw/day of CeO2 NPs did not induce significant
damage to the cellular systems (Figure 4B).
Dose (mg/kg bw/day)
Data represented as mean ± SD, significantly different from control *P < 0.05 and **P < 0.01, n = 5 animals per group.
aMilli-Q water (negative control).
bCP (positive control).
Data represented as mean ± SD, significantly different from control *P < 0.05 and **P < 0.01, n = 5 animals per group.
aMilli-Q water (negative control).
bCP (positive control).
Significantly different from control at *P < 0.01. One hundred metaphases were analysed per animal; n = 5 animals per group. Data represented as mean ± SD;
TA, total aberrations = structural aberrations.
aNegative control (Milli-Q water).
bCP (40 mg/kg bw).
Reduced GSH content
The 28 days of repeated oral treatment with CeO2 NPs revealed
that GSH content in liver, kidneys and brain was inhibited in a
dose-dependent manner compared with controls in both sexes
of rats in gender-independent manner. There was significant
inhibition in the GSH content in liver, kidneys and brain at
300 and 600 mg/kg bw/day dose of the CeO2 NPs-treated rats
Significantly different from control at *P < 0.05 and **P < 0.01. One hundred metaphases were analysed per animal; n = 5 animals per group. Data represented as
mean ± SD; TA, total aberrations = structural aberrations.
aNegative control (Milli-Q water).
bCP (40 mg/kg bw).
respectively, at 300 and 600 mg/kg bw/day of CeO2 NPs. On
the contrary, CeO2 MPs at all doses and CeO2 NPs at 30 mg/kg
bw did not induce any significant alterations in GSH content
in liver, kidneys and brain and this was in the range of 2–17%
The slides of organs namely brain, heart, liver, spleen and
kidneys stained with H&E were studied. All the organs were
evaluated for any toxic changes as well as presence of any
extraneous material deposits. In liver, significant damage
was observed as dilated portal tract with exposure to CeO2
NPs in high dose groups (Figure 6B). Splenic hyperplasia
(Figure 6F) was found along with inflammation in brain
tissue (Figure 6J) upon exposure with 600 mg/kg bw/day of
CeO2 NPs. However, there were no abnormal pathological
changes in the kidneys and heart of the nanoceria-treated
groups (Figure 6D and H). The histomicrographs of control
liver, kidneys, spleen, heart and brain has been shown
in Figure 6A, C, E, G and I, respectively. Significant
histopathological changes were not found in the liver,
kidneys, spleen, heart and brain of 600 mg/kg bw/day CeO2
MPs-treated rats (data not shown). Further, there were no
evident effects in the low and medium dose groups of
nanoand micro-sized CeO2.
Tissue biodistribution of cerium particles
The exposure of male and female Wistar rats with CeO2 NPs
and CeO2 MPs repeatedly for 28 days with 30, 300 and 600 mg/
kg bw/day doses resulted in a significant increment in Ce
concentration, indicating absorption of Ce (Figure 7). There
was significant accumulation of Ce in all the tissues, i.e. liver,
kidneys, brain, spleen, heart and whole blood (Figure 7A–F) in
the groups of animals treated with CeO2 NPs. The distribution
of Ce was maximum in 600 mg/kg bw/day followed by 300
and 30 mg/kg bw/day in male and female rats treated with
CeO2 NPs. The maximum amount of Ce was distributed in
the liver, kidneys, spleen and blood. However, the intake of
Ce in various tissues was very high in the NPs-treated groups
when compared to MPs-treated groups at all the dose level.
Significant accumulation of Ce was observed in rats treated
with CeO2 MPs at 300 and 600 mg/kg bw/day in the liver and
spleen, whereas in kidneys and blood only at 600 mg/kg bw/
day. Moreover, significant amount of Ce was excreted in urine
at 600 mg/kg bw/day of CeO2 MPs-treated rats. In the CeO2
NPs-treated rats, a significant amount of Ce was removed via
urine in dose-dependent manner (Figure 7G), whereas CeO2
MPs-treated rats showed more excretion in faeces, probably
due to their larger size (Figure 7H).
The present study was executed to provide the first testimony
of the genotoxic, biochemical and biodistribution of the Ce in
different tissues, urine and faeces with CeO2 NPs and CeO2
MPs in albino Wistar male and female rats after 28 days of
repeated oral treatment. The results obtained have revealed
that CeO2 NPs induced toxic effects at medium and high
doses without any severe distress symptoms and mortality. In
contrast, CeO2 MPs did not demonstrate toxicity. Thus, CeO2
NPs can be categorised as substances with toxic effects when
exposed at higher dose for a longer period of time. Moreover,
the physical and chemical properties can influence NPs
behaviour and may have an impact on toxicity. The specific
physico-chemical parameters of NMs may govern unexpected
biological interactions (
). To interpret the likely fate and
behaviour of NPs in the environment, it is essential to establish
their interaction in aqueous medium. Accordingly, in this study,
the mean size of CeO2 NPs and CeO2 MPs was calculated and
found to be 24.2 nm and 3.14 µm, respectively, in dry state,
whereas hydrodynamic diameter in Milli-Q water was noted to
be 191.2 nm. The difference in size in two states can be explained
by possible agglomeration in aqueous medium due to
physicochemical interactions between NPs, which tend to increase
the particle diameter. Zeta potential measurement delves
into surface charge and it was perceived to have an incipient
instability of CeO2 NPs in suspension. The agglomeration of
metal oxides in aqueous medium depends on particle charge
and has a significant effect on their bioavailability. Moreover,
zeta potential is a surface electrical characteristic that has been
used to quantify the influence of NPs in biological cells (
Several studies have revealed that NPs caused more toxicity
than micron-sized particles (
In this study, significant increase in % tail DNA in PBL and
liver cells at a high and medium dose compared to control was
found due to repeated CeO2 NPs exposure through comet assay.
However, CeO2 MPs did not reveal any significant increase.
The results suggest that CeO2 NPs may induce DNA lesion at
alkali-labile sites in leukocytes. There are several in vitro
studies depicting DNA damage with comet assay in divergent
cellular systems with CeO2 NPs (
), which support our findings.
In contrast, there are no in vivo studies with comet assay after
oral exposure of CeO2 NPs. However, an investigation with zinc
oxide (ZnO) NPs is in line with our results. In this study, mice
treated for 14 continuous days resulted in significant increase in
DNA damage with the comet assay in the liver (42).
There was significant increment in MN-PCEs frequency in
both bone marrow and PB cells in CeO2 NPs-treated groups.
It is possible that clastogenic events are involved in the
formation of these MN with CeO2 NPs. There are studies that have
demonstrated that CeO2 NPs exposure induced chromosome
damage and DNA lesion in distinct cellular systems using MNT
and comet assay (
). Moreover, decrease in % PCEs
suggests that cell death had occurred in the treated groups. There
were significant chromosomal damages in bone marrow cells
in CeO2 NPs high dose treated groups but not with CeO2 MPs.
A decrease in MI results compared with the control groups
suggested a slower progression of cells from the S (DNA synthesis)
phase to the M (mitosis) phase of the cell cycle. It is most likely
that this impairment in cell cycle progression is associated with
CeO2 NPs and CeO2 MPs toxicity. During mitosis, NPs could
interact with chromosomes and might introduce breaks into
chromosomes or disturb the process of mitosis, mechanically
or by chemical binding (24). Although there is no literature
with CeO2 NPs utilising CA assay, a study with manganese
oxide (MnO2) NMs has recorded structural aberrations in bone
marrow cells of rats after 28 days of repeated dosing, which is
in accordance with our results (
In the present study, CeO2 NPs significantly increased the
activities of ALP and LDH enzyme in the serum of exposed
rats in dose-dependent manner, suggesting probable injuries to
the tissues. The ALP is the foremost enzyme that is found to
increase in liver disease. Similarly, serum activity of the ALP
has been reported to elevate after 14 days of repeated oral
exposure with ZnO NPs in mice (
). Nanoceria (8 nm) was also
found to induce membrane damage and resultant cytotoxicity
using LDH assay in H4IIE rat hepatoma cells (
). In the
current study, in the liver, kidneys and brain of rats treated with
300 and 600 mg/kg bw/day dose of nanoceria, the GSH content
declined significantly. GSH plays a crucial role in antioxidant
defence, nutrient metabolism and regulation of cellular events.
Its insufficiency contributes to oxidative stress, and this stress
is prime culprit in aging and many diseases (
). The reason for
this is that GSH effectively scavenges free radicals and other
ROS (e.g. hydroxyl radical, lipid peroxyl radical, peroxynitrite
and H2O2) directly and indirectly through enzymatic reactions
). Similarly, cell membrane breakage along with decrease
of GSH levels in different cellular systems due to nanoceria
exposure has been documented (
histopathological damage to rats treated with 600 mg/kg bw/day
dose of the nanoceria for 28 days showed alterations in the
liver, spleen and brain tissues, such as dilation of hepatic
portal tract, splenic hyperplasia and inflammation in brain tissue,
respectively. Similar alterations were documented with other
metal NPs (
The evaluation of consequences of NMs exposure at the
whole body and organ levels is important in their toxicokinetic
study. Hence, another important aspect of this study was the
tissue biodistribution of CeO2 NPs. The significant distribution
of CeO2 NPs at all doses and higher dose of CeO2 MPs broadly
destined to liver, spleen, kidneys, heart and brain through their
translocation into the blood. The distribution was in
dosedependent order. On the other hand, no gender-dependent
tissue distribution was witnessed. A small but significant amount
of Ce in nanoceria-treated rats was excreted in urine, whereas
only higher dose of CeO2 MPs was found to expel Ce in urine.
Because of larger average diameter of glomerular filtration
pore, nanoceria of comparatively small size could leak out. The
decrease in particle size might have enhanced the penetration
of nanoceria into the systemic circulation (
). Further, large
amount of either of the Ce particles were rapidly removed from
the gastrointestinal system into faeces. Similar pattern of
retention in liver and spleen of rats treated with 30 nm CeO2 NPs
with single iv infusion was revealed after 14 days (
suggested mechanism for the tissue distribution was that the CeO2
NPs may have been phagocytised and transported to the liver
or spleen by macrophages to help reduce the filtered amount or
bound to haem in blood cells (
). Furthermore, limited
gastrointestinal absorption of nanoceria was reported and their
timedependent excretion via bile was found in faeces after oral dose
). The biochemical alterations such as increased activity of
serum ALP and LDH and reduction in liver, kidneys and brain
GSH content can be probably explained by the accumulation
of nanoceria. Tissues like liver and spleen are rich in
mononuclear phagocyte cells and hence sensitive to CeO2 NPs exposure
). Brain is sensitive to oxidative stress due to high metabolic
activity. The GSH plays an important role in countering stress.
Subsequently, nanoceria was reported to incite little
oxidative stress in brain components after 20 h of iv infusion in rats
). The body distribution of particles significantly depends
on surface characteristics and the size of the particles
irrespective of the uptake route (
). In support of the present
findings, repeated oral dose studies performed with diverse NMs
revealed that ZnO NPs (300 mg/kg) for 14 days in male mice
and silver NPs and MnO2 NPs (30, 300 and 1000 mg/kg) for
28 days in male and female rats were found to be distributed
into liver, spleen, brain and kidneys leading to several hepatic
alterations after getting translocated into the blood (
Persistence and clearance of NPs in different organs are the
major determinants of the toxicity of NPs. There are no reports
addressing the long-term impact of oral exposure of CeO2 NPs,
whereas a study in rats demonstrated that a single iv infusion
of aqueous dispersion of 85 mg/kg 30 nm nanoceria induced
hepatic apoptosis after 30 days of exposure (
The orally treated CeO2 NPs led to liver damage as
exhibited by the histopathological study that showed dilated portal
tract. The DNA damage, increased ALP level and decreased
GSH content in liver cells were in concurrence with the
probable hepatotoxicity. Moreover, our results suggest
bioaccumulation of CeO2 NPs for the significant genotoxic potential
in rats. However, the mechanisms of NPs genotoxicity are still
not well understood. The possible mechanisms of
genotoxicity may be either by direct interaction of NPs with the genetic
material or by indirect damage from NPs-induced ROS or by
toxic ions released from soluble NPs (
intracellular ROS generated in the metabolising cells could attack
DNA base guanine and form mutagenic 8-OHdG lesions after
unbalancing the redox potential in the cellular environment
(56). Likewise, H2AX-Nox1/Rac1 pathway was reported as
the possible mechanism for the DNA damage and
intracellular ROS signalling for the induction of cell death (
The data obtained with the tested compounds were gender
independent. Further, the NPs showed more bioaccumulation
compared with the MPs at the same dose level in male and
female rats after 28 days of repeated oral dose study. Hence,
particles in the nano-size range can enter into animal system
via intestines. Moreover, high doses were used to provide
detectable quantities after distribution into the animal system
and were not intended to reflect human exposure. Further,
there is always a wide scope for the next level of study in order
to get conclusive information regarding CeO2 NPs toxicity.
This work was supported by Asian Office of Aerospace
Research and Development (AOARD), Japan, under the grant
The authors express sincere thanks to the Director, IICT, Hyderabad, for
providing facility to execute this study. M.K. gratefully acknowledges Mr Shailendra
P. Singh for his insightful comments during research work. Further, M.K.
(senior research fellow) is thankful to University Grant Commission, India, for the
award of fellowship.
Conflict of interest statement: None declared.
1. Hochella , M. F. , Jr , Lower, S. K. , Maurice , P. A. , Penn , R. L. , Sahai , N. , Sparks , D. L. and Twining , B. S. ( 2008 ) Nanominerals, mineral nanoparticles, and Earth systems . Science , 319 , 1631 - 1635 .
2. Oberdörster , G. , Oberdörster , E. and Oberdörster , J. ( 2005 ) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles . Environ. Health Perspect. , 113 , 823 - 839 .
3. Zhu , X. , Zhu , L. , Chen , Y. and Tian , S. ( 2009 ) Acute toxicities of six manufactured nanomaterial suspensions to Daphnia magna . J. Nanopart. Res . 11 , 67 - 75 .
4. Celardo , I. , De Nicola , M. , Mandoli , C. , Pedersen , J. Z. , Traversa , E. and Ghibelli , L. ( 2011 ) Ce³+ ions determine redox-dependent anti-apoptotic effect of cerium oxide nanoparticles . ACS Nano , 5 , 4537 - 4549 .
5. OECD ( 2010 ) List of Manufactured Nanomaterials and List of Endpoints for Phase One of the Sponsorship Programme for the Testing of Manufactured Nanomaterials: Revision. Environment Directorate, Pesticides and Biotechnology, Organisation for Economic Cooperation and Development, Paris, p. 16 , ENV/JM/MONO 46. http://search.oecd.org/officialdocuments/ displaydocumentpdf/?cote=env/jm/mono( 2009 ) 20/rev&doclanguage=en (accessed on April 14, 2014 ).
6. Celardo , I. , Pedersen , J. Z. , Traversa , E. and Ghibelli , L. ( 2011 ) Pharmacological potential of cerium oxide nanoparticles . Nanoscale , 3 , 1411 - 1420 .
7. Hirst , S. M. , Karakoti , A. , Singh , S. , Self , W. , Tyler , R. , Seal , S. and Reilly , C. M. ( 2013 ) Bio-distribution and in vivo antioxidant effects of cerium oxide nanoparticles in mice . Environ. Toxicol. , 28 , 107 - 118 .
8. Kilbourn , B. T. ( 2003 ) Cerium and Cerium Compounds; Kirk-Othmer Encyclopedia of Chemical Technology . John Wiley and Sons, New York, NY, USA.
9. Wu , W. , Li , S. S. , Liao , S. , Xiang , F. and Wu , X. H. ( 2010 ) Preparation of new sunscreen materials Ce1−xZnxO2−x via solid-state reaction at room temperature and study on their properties . Rare Metals 29 , 149 - 153 .
10. Kawamoto , Y. , Tanabe , Y. [assignee: Nippon Shikizai Inc.]. (2005) pat . Lipstick composition having minimized skin irritation (Japan) . Japanese Patent No. 2005220099 A2 . Japan Kokai, Tokyo Koho, 8 pp. Abstract from Toxcenter 232470 .
11. Batley , G. E. , Halliburton , B. , Kirby , J. K. , Doolette , C. L. , Navarro , D. , McLaughlin , M. J. and Veitch , C. ( 2013 ) Characterization and ecological risk assessment of nanoparticulate CeO2 as a diesel fuel catalyst . Environ. Toxicol. Chem ., 32 , 1896 - 1905 .
12. Arnold , M. C. , Badireddy , A. R. , Wiesner , M. R. , Di Giulio , R. T. and Meyer, J. N. ( 2013 ) Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans . Arch. Environ. Contam. Toxicol. , 65 , 224 - 233 .
13. Rodea-Palomares , I. , Boltes , K. , Fernández-Piñas , F. , Leganés , F. , GarcíaCalvo , E., Santiago , J. and Rosal , R. ( 2011 ) Physicochemical characterization and ecotoxicological assessment of CeO2 nanoparticles using two aquatic microorganisms . Toxicol. Sci. , 119 , 135 - 145 .
14. Park , B. , Martin , P. , Harris , C. , Guest , R. , Whittingham , A. , Jenkinson , P. and Handley , J. ( 2007 ) Initial in vitro screening approach to investigate the potential health and environmental hazards of Enviroxtrade mark-a nanoparticulate cerium oxide diesel fuel additive . Part. Fibre Toxicol. , 4 , 12 .
15. Park , E. J. , Choi , J. , Park , Y. K. and Park , K. ( 2008 ) Oxidative stress induced by cerium oxide nanoparticles in cultured BEAS-2B cells . Toxicology , 245 , 90 - 100 .
16. Lin , W. , Huang , Y. W. , Zhou , X. D. and Ma, Y. ( 2006 ) Toxicity of cerium oxide nanoparticles in human lung cancer cells . Int. J. Toxicol ., 25 , 451 - 457 .
17. Geraets , L. , Oomen , A. G. , Schroeter , J. D. , Coleman , V. A. and Cassee , F. R. ( 2012 ) Tissue distribution of inhaled micro- and nano-sized cerium oxide particles in rats: results from a 28-day exposure study . Toxicol. Sci. , 127 , 463 - 473 .
18. Gosens , I. , Mathijssen , L. E. , Bokkers , B. G. , Muijser , H. and Cassee , F. R. ( 2014 ) Comparative hazard identification of nano- and micro-sized cerium oxide particles based on 28-day inhalation studies in rats . Nanotoxicology , 8 , 643 - 653 .
19. Ma , J. Y., Mercer , R. R. , Barger , M. , Schwegler-Berry , D. , Scabilloni , J. , Ma, J. K. and Castranova , V. ( 2012 ) Induction of pulmonary fibrosis by cerium oxide nanoparticles . Toxicol. Appl . Pharmacol., 262 , 255 - 264 .
20. Tseng , M. T. , Lu , X. , Duan , X. et al. ( 2012 ) Alteration of hepatic structure and oxidative stress induced by intravenous nanoceria . Toxicol. Appl . Pharmacol., 260 , 173 - 182 .
21. Baalousha , M. , Ju-Nam , Y. , Cole , P. A. et al. ( 2012 ) Characterization of cerium oxide nanoparticles-part 2: nonsize measurements . Environ. Toxicol. Chem ., 31 , 994 - 1003 .
22. Dusinska , M. , Fjellsbø , L. M. , Magdolenova , Z. , Ravnum , S. , Rinna , A. and Rundén-Pran , E. ( 2011 ) Safety of nanoparticles in medicine . In Hunter, R. J. and Preedy , V. R. (eds), Nanomedicine in Health and Disease . CRC Press, Boca Raton, FL, USA, Chap. 11 , pp. 203 - 226 .
23. Maffei , F. , Zolezzi Moraga , J. M. , Angelini , S. , Zenesini , C. , Musti , M. , Festi , D. , Cantelli-Forti , G. and Hrelia , P. ( 2014 ) Micronucleus frequency in human peripheral blood lymphocytes as a biomarker for the early detection of colorectal cancer risk . Mutagenesis , 29 , 221 - 225 .
24. Magdolenova , Z. , Collins, A. , Kumar , A. , Dhawan , A. , Stone , V. and Dusinska , M. ( 2014 ) Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles . Nanotoxicology , 8 , 233 - 278 .
25. OECD ( 2008 ) Guidelines for the Testing of Chemicals: Repeated Dose 28-Day Oral Toxicity Study in Rodents . Guideline 407. Organization for Economic Cooperation and Development , Paris.
26. Tice , R. R. , Agurell , E. , Anderson , D. et al. ( 2000 ) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing . Environ. Mol. Mutagen ., 35 , 206 - 221 .
27. Miyamae , Y. , Yamamoto , M. , Sasaki , Y. F. , Kobayashi , H. , Igarashi-Soga , M. , Shimoi , K. and Hayashi , M. ( 1998 ) Evaluation of a tissue homogenization technique that isolates nuclei for the in vivo single cell gel electrophoresis (comet) assay: a collaborative study by five laboratories . Mutat. Res. , 418 , 131 - 140 .
28. Pool-Zobel , B. L. , Lotzmann , N. , Knoll , M. , Kuchenmeister , F. , Lambertz , R. , Leucht , U. , Schröder , H. G. and Schmezer , P. ( 1994 ) Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples . Environ. Mol. Mutagen ., 24 , 23 - 45 .
29. Schmid , W. ( 1975 ) The micronucleus test . Mutat. Res. , 31 , 9 - 15 .
30. Celik , A. , Ogenler , O. and Cömelekoglu , U. ( 2005 ) The evaluation of micronucleus frequency by acridine orange fluorescent staining in peripheral blood of rats treated with lead acetate . Mutagenesis , 20 , 411 - 415 .
31. OECD ( 1997 ) Guidelines for Genetic Toxicology: Micronucleus Test . Guideline 474. Organization for Economic Cooperation and Development , Paris.
32. Adler , I. D. ( 1984 ) Cytogenetic tests in mammals . In Venitt, S. and Parry , J . (eds), Mutagenicity Testing: A Practical Approach . IRL Press, Oxford, pp. 273 - 306 .
33. OECD ( 1997 ) Guidelines for Genetic Toxicology: In Vivo Mammalian Bone Marrow Cytogenetic Test-Chromosome Analysis . Guideline 475. Organization for Economic Cooperation and Development , Paris.
34. McQueen , M. J. ( 1972 ) Optimal assay of LDH and α-HBD at 37ºC . Ann. Clin. Biochem . 9 , 21 - 25 .
35. Jollow , D. J., Mitchell, J. R. , Zampaglione , N. and Gillette , J. R. ( 1974 ) Bromobenzene-induced liver necrosis . Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite . Pharmacology , 11 , 151 - 169 .
36. Gómez , M. , Sánchez , D. J. , Llobet , J. M. , Corbella , J. and Domingo , J. L. ( 1997 ) The effect of age on aluminum retention in rats . Toxicology , 116 , 1 - 8 .
37. Doak , S. H. , Griffiths , S. M. , Manshian , B. , Singh , N. , Williams , P. M. , Brown , A. P. and Jenkins , G. J. ( 2009 ) Confounding experimental considerations in nanogenotoxicology . Mutagenesis , 24 , 285 - 293 .
38. Zhang , Y. , Yang , M. , Portney , N. G. , Cui , D. , Budak , G. , Ozbay , E. , Ozkan , M. and Ozkan , C. S. ( 2008 ) Zeta potential: a surface electrical characteristic to probe the interaction of nanoparticles with normal and cancer human breast epithelial cells . Biomed. Microdevices , 10 , 321 - 328 .
39. Karlsson , H. L. , Gustafsson , J. , Cronholm , P. and Möller , L. ( 2009 ) Sizedependent toxicity of metal oxide particles-a comparison between nanoand micrometer size . Toxicol. Lett., 188 , 112 - 118 .
40. De Marzi , L. , Monaco , A. , De Lapuente , J. , Ramos , D. , Borras , M. , Di Gioacchino , M. , Santucci , S. and Poma , A. ( 2013 ) Cytotoxicity and genotoxicity of ceria nanoparticles on different cell lines in vitro . Int. J. Mol. Sci ., 14 , 3065 - 3077 .
41. Auffan , M. , Rose , J. , Orsiere , T. et al. ( 2009 ) CeO2 nanoparticles induce DNA damage towards human dermal fibroblasts in vitro . Nanotoxicology , 3 , 161 - 171 .
42. Sharma , V. , Singh , P. , Pandey , A. K. and Dhawan , A. ( 2012 ) Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles . Mutat. Res. , 745 , 84 - 91 .
43. Kumari , M. , Singh , S. P. , Chinde , S. , Rahman , M. F. , Mahboob , M. and Grover , P. ( 2014 ) Toxicity study of cerium oxide nanoparticles in human neuroblastoma cells . Int. J. Toxicol ., 33 , 86 - 97 .
44. Singh , S. P. , Kumari , M. , Kumari , S. I. , Rahman , M. F. , Mahboob , M. and Grover , P. ( 2013 ) Toxicity assessment of manganese oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral exposure . J. Appl. Toxicol. , 33 , 1165 - 1179 .
45. Rosenkranz , P. , Fernández-Cruz , M. L. , Conde , E. , Ramírez-Fernández , M. B. , Flores , J. C. , Fernández , M. and Navas , J. M. ( 2012 ) Effects of cerium oxide nanoparticles to fish and mammalian cell lines: an assessment of cytotoxicity and methodology . Toxicol. In Vitro, 26 , 888 - 896 .
46. Townsend , D. M. , Tew , K. D. and Tapiero , H. ( 2003 ) The importance of glutathione in human disease . Biomed. Pharmacother. , 57 , 145 - 155 .
47. Fang , Y. Z. , Yang , S. and Wu , G. ( 2002 ) Free radicals, antioxidants, and nutrition . Nutrition , 18 , 872 - 879 .
48. He , X. , Zhang, H., Ma, Y. , Bai , W. , Zhang , Z. , Lu , K. , Ding , Y. , Zhao , Y. and Chai , Z. ( 2010 ) Lung deposition and extrapulmonary translocation of nano-ceria after intratracheal instillation . Nanotechnology , 21 , 285103 .
49. Yokel , R. A. , Au , T. C. , MacPhail , R. et al. ( 2012 ) Distribution, elimination, and biopersistence to 90 days of a systemically introduced 30 nm ceriaengineered nanomaterial in rats . Toxicol. Sci. , 127 , 256 - 268 .
50. Hardas , S. S. , Butterfield , D. A. , Sultana , R. et al. ( 2010 ) Brain distribution and toxicological evaluation of a systemically delivered engineered nanoscale ceria . Toxicol. Sci. , 116 , 562 - 576 .
51. Hoet , P. H. , Brüske-Hohlfeld, I. and Salata , O. V. ( 2004 ) Nanoparticlesknown and unknown health risks . J. Nanobiotechnology , 2 , 12 .
52. Kim , Y. S. , Kim , J. S. , Cho , H. S. et al. ( 2008 ) Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of silver nanoparticles in Sprague-Dawley rats . Inhal. Toxicol., 20 , 575 - 583 .
53. Tseng , M. T. , Fu , Q. , Lor , K. , Fernandez-Botran , G. R. , Deng , Z. B. , Graham , U. , Butterfield , D. A. , Grulke , E. A. and Yokel , R. A. ( 2014 ) Persistent hepatic structural alterations following nanoceria vascular infusion in the rat . Toxicol. Pathol , 42 , 984 - 996 .
54. Kisin , E. R. , Murray , A. R. , Keane , M. J. et al. ( 2007 ) Single-walled carbon nanotubes: geno- and cytotoxic effects in lung fibroblast V79 cells . J. Toxicol. Environ. Health. A , 70 , 2071 - 2079 .
55. Barnes , C. A. , Elsaesser , A. , Arkusz , J. et al. ( 2008 ) Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity . Nano Lett. , 8 , 3069 - 3074 .
56. Singh , N. , Manshian , B. , Jenkins , G. J. , Griffiths , S. M. , Williams , P. M. , Maffeis , T. G. , Wright , C. J. and Doak , S. H. ( 2009 ) NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials . Biomaterials , 30 , 3891 - 3914 .
57. Kang , M. A. , So , E. Y. , Simons , A. L. , Spitz , D. R. and Ouchi, T. ( 2012 ) DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway . Cell Death Dis. , 3 , e249 .