Toxicity Study of Cerium Oxide Nanoparticles in Human

International Journal of Toxicology
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Toxicity Study of Cerium Oxide Nanoparticles in Human Neuroblastoma Cells
Monika Kumari, Shailendra Pratap Singh, Srinivas Chinde, Mohammed Fazlur Rahman, Mohammed Mahboob and
Paramjit Grover
International Journal of Toxicology published online 6 February 2014
DOI: 10.1177/1091581814522305
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Article
Toxicity Study of Cerium Oxide
Nanoparticles in Human
Neuroblastoma Cells
International Journal of Toxicology
1-12
ª The Author(s) 2014
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DOI: 10.1177/1091581814522305
ijt.sagepub.com
Monika Kumari1,2, Shailendra Pratap Singh1, Srinivas Chinde1,2,
Mohammed Fazlur Rahman1, Mohammed Mahboob1, and Paramjit Grover1
Abstract
The present study consisted of cytotoxic, genotoxic, and oxidative stress responses of human neuroblastoma cell line (IMR32)
following exposure to different doses of cerium oxide nanoparticles (CeO2 NPs; nanoceria) and its microparticles (MPs) for 24
hours. Cytotoxicity was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase
assays whereas genotoxicity was assessed using the cytokinesis-block micronucleus and comet assays. A battery of assays
including lipid peroxidation, reactive oxygen species (ROS), hydrogen peroxide, reduced glutathione, nitric oxide, glutathione
reductase, glutathione peroxidase, superoxide dismutase, catalase, and glutathione S-transferase were performed to test the
hypothesis that ROS was responsible for the toxicity of nanoceria. The results showed that nanosized CeO2 was more toxic than
cerium oxide MPs. Hence, further study on safety evaluation of CeO2 NPs on other models is recommended.
Keywords
cerium oxide nanoparticles, human neuroblastoma cells, cytotoxicity, oxidative stress, genotoxicity
Introduction
The application of nanomaterials has had a great impact on
biomedical science and engineering in last few decades
because of its novel characteristics. The unique physical and
chemical properties of nanoparticles (NPs) due to their small
size, chemical composition, structure, and large surface area
have resulted in their incorporation into thousands of commercial products. Increased use of nanotechnology enhances the
risk of exposure to NPs. Hence, the routes of entry, interaction
with cells and tissues, molecular mechanisms of cytotoxicity,
and different effects on biological systems relative to the
composition, size, and shape of emerging nanomaterials need
to be well understood.1,2
Cerium is a lanthanide series rare earth element that can
exist either as a free metal or as a cycle between the cerium
(III) and the cerium (IV) oxidation states.3 Cerium oxide NPs
(CeO2 NPs; nanoceria) consist of a cerium core surrounded by
oxygen lattice. The CeO2 NPs have shown many promising
applications because of their high performance as an oxygen
buffer and active support for noble metals in catalysis, which
relies on an efficient supply of lattice oxygen at reaction sites
governed by oxygen vacancy formation.4 Reactions involving
redox cycles between the Ce3þ and Ce4þ oxidation state allow
nanoceria to react catalytically with free radicals and reactive
oxygen species (ROS) often produced during inflammatory
cascades. The unique properties of CeO2 NPs have resulted
in widespread use in the treatment of medical disorders caused
by oxidative species.5 Moreover, nanoceria is commonly used
as polishing agent,6 ultraviolet-absorbing compound in sunscreen,7 electrolyte in solid oxide fuel cells,8 as a fuel additive to
promote combustion, and as a subcatalyst for automotive
exhaust cleaning.9,10 These applications of engineered CeO2
NPs may increase the risk of exposure to humans and the
environment. Therefore, human health risk assessment of CeO2
NPs is important.
Neuronal cells are of special interest to evaluate the
NP-induced toxicity. The use of NPs has been explored for
neuroimaging strategy due to its optical and electrical functionalities. 11 Hence, this study evaluated the cytotoxicity,
genotoxicity, and oxidative stress caused by CeO2 NPs in
human neuroblastoma (NB) cell line (IMR32). As the physical
and chemical properties of NPs can vary significantly from
those of their bulk counterparts, CeO2 microparticles (MPs)
were used to compare the size effect. The CeO2 NPs were
characterized by transmission electron microscopy (TEM),
1
Toxicology Unit, Biology Division, Indian Institute of Chemical Technology,
Hyderabad, Andhra Pradesh, India
2
Department of Genetics, Osmania University, Osmania University Main
Road, Hyderabad, Andhra Pradesh, India
Corresponding Author:
Paramjit Grover, Toxicology Unit, Biology Division, Indian Institute of
Chemical Technology, Hyderabad, Andhra Pradesh 500 007, India.
Email: [email protected]; [email protected]
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International Journal of Toxicology
dynamic light scattering (DLS), and laser Doppler velocimetry
(LDV) studies to characterize the size, mean hydrodynamic
diameter, and z potential of CeO 2 NPs, respectively.
Cytotoxicity was evaluated by the formazan reduction 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
assay.
There is evidence of both induction and mitigation of
oxidative stress by CeO2 NPs in both in vivo and in vitro in
reports. Owing to their lesser size, CeO2 NPs were found to be
more toxic than equimolar bulk CeO 2 in Caenorhabditis
elegans and showed dose-dependent growth inhibition.12 In a
study with male Sprague Dawley rats, oxidative damage of
protein in liver and spleen was reported and it was suggested
that the ceria NPs toxicity was time dependent with respect to
peripheral organs and this effect may be related to the oxidative
state of the ceria NPs.13 Further, CeO2 NPs were shown to
induce apoptosis and autophagy in human peripheral blood
monocytes at relatively low doses.14 When the human lung
adenocarcinoma (A549) cell line was exposed to various concentrations of 20 nm CeO2 NPs, a dose- and time-dependent
alteration was observed in indicators of oxidative stress and
cytotoxicity. 15 In addition, human lung epithelial cells
(BEAS-2B) exposed to CeO2 NPs showed an increase in the
expression of oxidative stress-related genes, including catalase
(CAT), glutathione S-transferase (GST), heme oxygenase 1,
and thioredoxin reductase.16 In contrast, CeO2 NPs suppressed
ROS production and induced cellular resistance to an exogenous source of oxidative stress in BEAS-2B and RAW 264.7
cells.17 Further, CeO2 NPs were reported to be neuroprotective
to the cells derived from rodent nervous system (HT22 cell
line) through the reduction in endogenous ROS induced by
glutamate.18 Moreover, Niu et al19 suggested that nanoceria
partially prevented heart dysfunction through inhibition of the
myocardial oxidative stress, endoplasmic reticulum stress, and
inflammatory processes in monocyte chemoattractant protein
(MCP) 1 transgenic mice (MCP mice) that normally exhibit
progressive heart damage. Administration of CeO2 NPs to mice
with induced liver toxicity showed therapeutic property of
reducing oxidative stress by decreasing ROS.20 Therefore, in
the present study, lipid peroxidation (LPO), ROS, hydrogen
peroxide (H2O2), reduced glutathione (GSH), lactate dehydrogenase (LDH), nitric oxide (NO), glutathione reductase (GR),
glutathione peroxidase (GPx), superoxide dismutase (SOD),
CAT, and GST estimations were carried out.
The in vitro micronucleus (MN) test has become a standard
cytogenetic test for genotoxicity testing. It is simple to score
and accurate and applicable in different cell types.21 The
cytokinesis-block MN (CBMN) assay is the preferred method
for measuring micronuclei (MNi) in cultured cell lines because
scoring is specifically restricted to binucleated cells that have
undergone 1 cell division.22 At the same time, CBMN assay is a
good approach to evaluate other damage events including
nucleoplasmic bridges, a biomarker of DNA misrepair and/or
telomere end fusions and nuclear buds (NBUDs), a biomarker
of elimination of amplified DNA and/or DNA repair
complexes, and the number of apoptotic and necrotic cells.23
The single-cell gel electrophoresis or comet assay is important
to assess the DNA damaging potential of these particles
because of rapid and sensitive detection of DNA damage in
individual cells.24 Hence, the CBMN and comet assays were
additionally performed in the present study.
Materials and Methods
Particles and Chemicals
Cerium oxide NPs (CeO2 < 25 nm, CAS No. 1306-38-3) and
CeO2 MPs (CeO2, 99.9%, <5 mm, CAS No. 1306-38-3) were
purchased from Sigma Chemical Co Ltd (St Louis, Missouri).
Phosphate-buffered saline (PBS; Ca2þ, Mg2þ free), Dulbecco
modified eagle medium (DMEM), trypsin–EDTA, fetal bovine
serum (FBS), penicillin, streptomycin, dihydrorhodamine 123
(DHR), MTT powder, 2 0 ,7 0 -dichlorofluorescein diacetate
(DCF-DA), cyclophosphamide (CP), normal melting agarose,
low melting agarose, trichloroacetic acid (TCA), thiobarbituric
acid (TBA), 5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB),
diethylene triamine pentaacetic acid (DTPA), 1-chloro-2,4dinitrobenzene (CDNB), and dimethyl sulfoxide (DMSO) were
also purchased from Sigma Chemical Co Ltd. All other
chemicals were obtained locally and were of analytical grade.
Cell culture plastic wares were obtained from Tarsons Products
Pvt Ltd (Kolkata, India).
Characterization of CeO2 NPs and MPs
Transmission electron microscopy (JEM-2100; JEOL, Japan)
was performed to obtain the size and morphology of the CeO2
NPs and MPs at an accelerating voltage of 120 kV. Cerium
oxideNPs and MPs were examined after suspension in MilliQ
water and subsequent deposition onto TEM grids. Information
on mean size and standard error was calculated by measuring
over 100 particles in random fields of view, in addition to
images that show general morphology of the CeO2 NPs and
MPs.
Dynamic light scattering and z potential measurements of
40 mg/mL CeO2 NPs suspension were performed with a Zetasizer Nano ZS (Malvern Instruments, United Kingodm) provided with a He/Ne laser of 633 nm wavelength. The DLS and
LDV were used for the size and charge characterization of
CeO2 NPs in DMEM suspension. To avoid agglomeration of
NPs, a freshly prepared stock solution in DMEM was ultrasonicated using probe sonicator UP100H (Hielscher Ultrasonics
GmbH, Teltow, Germany) for 10 minutes at 90% amplitude.
Samples thus prepared were transferred to a 1.5-mL square
cuvette for DLS measurements, and 1 mL was transferred to
a Malvern Clear Zeta Potential cell for LDV measurement.
Average size was calculated by the software from the intensity,
volume, and number distributions measured.
Cell Culture
Human NB cell line (IMR32) was obtained from American
Type Culture Collection (Manassas, VA) and cultured in
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Kumari et al
3
DMEM supplemented with 10% FBS, 0.2% sodium bicarbonate, and 10 mL/L antibiotic solution at 37" C under a humidified atmosphere of 5% CO2/95% air. After IMR32 cells were
seeded in a 96-well plate, CeO2 NPs and MPs were added to the
culture medium with the final concentration of 10, 20, 50, 100,
and 200 mg/mL and incubation was continued for 24 hours at
37" C. Depending on the number of cells required in the assay,
12-, 24-, and 96-well plates were used and all the assays were
performed in triplicates.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl Tetrazolium
Bromide Assay
Cytotoxicity of CeO2 particles was assessed using MTT assay
to assess the cell viability following the method described by
Hansen et al.25 The assay is dependent on the reduction in the
tetrazolium salt MTT by the mitochondrial dehydrogenase of
viable cells to form a blue formazan product that gets dissolved
in DMSO and read at 570 nm. Briefly, 100 mL of IMR32 cells
were suspended in 96-well plate, and after 50% to 60% confluency, the cells were treated to different concentrations of
CeO2 NPs and MPs suspended in DMEM media with 5% serum
for a time period of 24 hours. Then, medium in each well was
discarded and fresh supplemented medium (100 mL) followed
by 10 mL of MTT solution (5 mg/mL in PBS, filtered sterile) was
added. Medium blank was put up with only medium (100 mL)
and MTT (10 mL). Plates were incubated at 37" C for 2 hours.
The formazan crystals formed by the action of mitochondrial
dehydrogenase on MTT was dissolved in 100 mL of DMSO and
absorbance was measured at 570 nm using Spectra Max Plus 384
UV-Visible plate reader (Molecular Devices, Sunnyvale,
California).
Lactate Dehydrogenase Release
Lactate dehydrogenase release was measured in a 96-well plate
with 60% to 80% confluent cells treated with different concentrations of CeO2 NPs and MPs. This estimation was done
according to the procedures described in Cytoscan LDH cytotoxicity assay kit (Geno Biosciences Pvt Ltd) and LDH release
was measured spectrophotometrically at 340 nm using Spectra
Max Plus 384 UV-visible plate reader (Molecular Devices).
The percentage of LDH activity was calculated by dividing the
amount of activity in the medium by the total activity (medium
and cell lysate).
Measurement of ROS
Intracellular superoxide was estimated fluorometrically using
the oxidation-sensitive fluorescent probe DCF-DA.26 In the 96well plate containing 60% to 80% confluent cells, DCF-DA (20
mmol/L) was added to each well and incubated for 30 minutes.
The cells were washed with PBS to remove extra DCF-DA and
then 5% culture medium was added. Further, cells were inoculated with different concentrations of CeO2 NPs and MPs and
incubated for 24 hours, and finally PBS was added to each well,
and fluorescence intensity was read on a spectrofluorometer
(Dynex Technologies, Virginia) at the excitation and emission
wavelengths of 485 and 528 nm, respectively.
Hydrogen Peroxide Assay
The intracellular production of H2O2 was measured with DHR
as described by Park et al.16 During the cellular production of
ROS, the nonfluorescent DHR was oxidized by H2O2 and
irreversibly converted to the green fluorescent compound
rhodamine 123 (R123). The R123 is membrane impermeable
and accumulates in the cells. An aliquot of DHR (to produce a
concentration of 10 mmol/L in each well) was added to each 96well plate and preincubated for 30 minutes at 37" C. Thereafter,
the medium was removed and IMR32 cells were incubated with
CeO2 NPs and MPs for 24 hours at 37" C. After incubation,
fluorescence intensities of each well were analyzed by spectrofluorometer with excitation filter of 485 nm and emission filter
of 535 nm.
Nitric Oxide Assay
Nitric oxide concentration was determined via the Griess reaction. Briefly, IMR32 cells were plated in 96-well plate up to
60% to 80% confluency and inoculated with different concentrations of CeO2 NPs and MPs for 24 hours. An aliquot of 100
mL from each well was mixed with same amount of Griess
reagent in wells of a separate 96-well plate. After 15-minute
incubation at room temperature (RT), the developed pink color
was read at 540 nm.27
Preparation of Cell Lysate and Protein Estimation
The cells were allowed to grow up to 60% to 80% confluency
before treatment with different concentrations (10, 20, 50, 100,
and 200 mg/mL) of CeO2 NPs and MPs in a 12-well plate for 24
hours. Thereafter, cells were washed with PBS and 100 mL lysis
buffer (146 mmol/L NaCl, 0.62 mmol/L EDTA, 5% Triton
X-100, and 10 mmol/L Tris buffer) was added to each well and
left for 30 minutes on ice. The cell lysate was centrifuged at 13
000 rpm at 4" C for 5 minutes. The supernatant was collected
for further analysis.
The total protein concentration was estimated in cell supernatant following the method of Bradford.28 The Bradford
reagent (200 mL) was added to the 100 mL of cell supernatant
in a 96-well plate and incubated at RT for 30 minutes. The
developed purple color was measured at 595 nm. Bovine serum
albumin was used as standard and experiment was performed in
triplicates.
Lipid Peroxidation
Malondialdehyde (MDA), the end product of LPO, was
estimated according to the method of Wills.29 Cell supernatant
(100 mL) was added to 2 mL of reaction mixture (15% TCA and
0.375% TBA) and then 800 mL of deionized water was added,
mixed thoroughly, heated in a boiling water bath for 20
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International Journal of Toxicology
minutes, and cooled to RT. The color developed was extracted
with 3 mL of butanol and centrifuged at 3000 rpm for 10
minutes. The top butanol layer was collected and read at 532
nm. The MDA level of the sample was calculated using an
extinction coefficient of 1.56 # 105/mol/L/cm. The levels of
MDA were expressed as mmol/mg protein.
Reduced Glutathione Content
The GSH content in cell supernatant was determined according
to the method described by Ellman.30 The cell supernatant of
100 mL was mixed with 2000 mL GSH buffer (0.746 mmol/L,
pH 7.4), 500 mL Ellman reagent (4 mg/mL DTNB in 0.34
mmol/L sodium citrate), and 400 mL milliQ water and mixed
well. The mixture was incubated for 15 minutes at RT and then
absorbance of developed yellow color was read at 412 nm
wavelength. The GSH content in the cell samples was
expressed as mmol of GSH/mg protein and calculated using
extinction coefficient of 13 600/mol/L/cm.
Superoxide Dismutase Activity
The estimation of SOD in cell supernatant was done by the
method of Marklund and Marklund.31 Briefly, 3 mL of assay
mixture contained 50 mmol/L Tris-HCl buffer (pH 8.2) with 1
mmol/L DTPA, 45 mL of 10 mmol/L pyrogallol in 10 mmol/L
HCl, and 50 mL cell supernatant. The rate of inhibition of
pyrogallol autooxidation after the addition of enzyme extract
was noted. The amount of enzyme required to give 50% inhibition of pyrogallol autooxidation was considered as 1 unit of
enzyme activity. The SOD activity was expressed in units/mg
protein.
Catalase Activity
Catalase was estimated spectrophotometrically using the
method of Aebi et al.32 The assay mixture of 3 mL contained
0.063% H2O2 in 0.1 mol/L KPB pH 7.4 and 20 mL cell supernatant. The decrease in absorbance was then observed for 60
seconds at every 5-second interval at 240 nm. Activity was
expressed as mmol of H2O2 decomposed/min/mg protein using
a molar extinction coefficient of 43.6/mol/L/cm. The CAT
activity was expressed as units/mg protein.
Glutathione Reductase Activity
Glutathione reductase activity was measured using the procedure described by Carlberg and Mannervik.33 The GR assay
consisted of potassium phosphate buffer (0.2 mol/L, pH 7), 2
mmol/L NADPH in 0.1% NaHCO3, and oxidized glutathione
(20 mmol/L). Enzyme assay was carried out by pipetting 600
mL of potassium phosphate buffer, 250 mL milliQ water, 50 mL
oxidized glutathione, 50 mL NADPH, and 50 mL sample (cell
supernatant) and the extinction of the sample was recorded at
340 nm based on the molar absorption coefficient of 6.22/
mol/L/cm and the results were expressed in mmol/min/mg
protein.
Glutathione Peroxidase Activity
Glutathione peroxidase activity was measured by the enzymecoupled assay.34 Assay mixture constitution was potassium
phosphate buffer (0.1 mol/L, pH 7), NADPH (2.25 mmol/L
NADPH in 0.1% NaHCO3), GR (7.1 mL/mL), glutathione
(11.52 mg/mL), and H2O2 (1.5 mmol/L). The enzyme assay
was performed by pipetting 750 mL of potassium phosphate
buffer, 60 mL NADPH, 1 U GR, and 25 mL GSH in 1 mL
cuvette. Next, the enzymatic reaction was initiated by the addition of 50 mL of sample (cell supernatant) and 100 mL of H2O2.
Extinction of the sample was registered at 340 nm every minute
for a period of 120 seconds in the spectrophotometer. A unit of
enzyme activity was reported as 1 mmol NADPH oxidized/min
assuming 6.2 # 103/mmol/L/cm to be the molar extinction
coefficient of NADPH at 340 nm. The GPx activity was
expressed as U/mg protein.
Glutathione S Transferase Activity
The GST activity was determined according to the method
of Habig et al.35 The reaction mixture consisting of 2.75 mL
KPB (0.1 mol/L, pH 6.5), 0.1 mL GSH (75 mmol/L), 0.1
mL CDNB (30 mmol/L in 95% ethanol), and 0.05 mL cell
supernatant in a total volume of 3 mL was taken in cuvette.
The change in absorbance was recorded at 340 nm for every
10 seconds for 2 minutes. Activity was expressed as mmol
CDNB conjugate/min/mg protein using a molar extinction
coefficient of 9.6 # 103/mol/L/cm.
Cytokinesis-Block MN Assay
The in vitro MN test was conducted according to the Organisation for Economic Co-operation and Development guidelines
487.36 After cell cultures attained the 60% to 80% confluent
stage in a 24-well plate, the cells were exposed to CeO2 NPs
and MPs at different concentrations (10, 20, 50, 100, and 200
mg/mL) for 24 hours. The CP (0.20 mg/mL) was used as positive control. After treatment, the cells were aspirated and
treated with freshly prepared DMEM medium containing cytochalasin B (3 mg/mL) for 18 hours. For each concentration, 2
wells were prepared. The cells were then harvested with trypsin
and centrifuged at 2000 rpm for 10 minutes. The cell pellets
were dissolved in media and slides were prepared in triplicate
for each culture. Slides were fixed with methanol and air dried
and stained with 10% Geimsa in Sorensen buffer just before the
evaluation with a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany) using a 1000# magnification. The
scoring of slides followed the criteria adopted by Fenech23 for
each end point. We evaluated 2000 binucleated cells per concentration, that is, 1000 binucleated cells per well to calculate
the total numbers of MNi in 1000 binucleated cells. Cell
proliferation was calculated by analyzing cytokinesis-block
proliferation index (CBPI) that indicates the average number
of cell cycles per cell during the period of exposure to cytochalasin B. Moreover, 500 cells were scored to evaluate the
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Kumari et al
5
percentage of mono-, bi-, tri-, and multi-nucleated cells and the
CBPI was calculated as an index of cytotoxicity by comparing
values in the treated and control cultures. Finally, other damage
events were scored in once-divided binucleated cells per 1000
cells. The number of apoptotic and necrotic cells and mitotic
figures per 500 cells were also evaluated.
CBPI ¼ fð1 # Number ofmononucleated cellsÞ
þ ð2 # Number of binucleated cellsÞ
þ ð3 # Number of multinucleated cellsÞg
=Total Number of cells
Comet Assay
The in vitro comet assay was done using the method described
by Tice et al.37 A 24-well plate containing 60% to 70% confluent cells was inoculated with CeO2 NPs and MPs of the same
concentration. The CP (0.20 mg/mL) was used as the positive
control. After 24 hours incubation, cell pellets were collected
and mixed with 0.37% low-melting point agarose (LMPA) in
PBS. Microscope slides were precoated with 120 mL of 0.75%
normal-melting point agarose in PBS and allowed to solidify
overnight at 37" C. The precoated slides were coated with a
second layer of cells suspended in 120 mL of 0.37% LMPA
and dried at 4" C. A third layer of plain 0.37% LMPA (120 mL)
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 mol/L NaCl, 0.1
mol/L Na2 EDTA, 0.2 mol/L NaOH, 1% Triton X-100, and
10% DMSO, pH 10.0) for 1 hour at 4" C. The slides were
presoaked for 20 minutes in alkaline buffer (10 mol/L NaOH
and 200 mmol/L Na2 EDTA, pH > 13.0) and then electrophoresis was performed at 25 V adjusted at 300 mA for 20 minutes.
The slides were neutralized twice in 0.4 mol/L Tris buffer, pH
7.5, for 5 minutes and once in absolute methanol for 5 minutes.
Coded slides were scored after staining with ethidium bromide
(20 mg/mL) using a fluorescence microscope (Olympus,
Shinjuku-ku, Tokyo, Japan) with a blue (488 nm) excitation
filter and yellow (515 nm) emission (barrier) filter at 400#
magnification. A total of 150 randomly selected cells per sample (3 replicates, each with 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 realized using a Comet Image Analysis System, version
Komet 5.5 (single-cell gel electrophoresis analysis company,
Andor Technology 2005; Kinetic Imaging Ltd, Nottingham,
United Kingdom).
except where it is differently indicated. Multiple pairwise comparisons were done using the Dunnett multiple comparison
posttest 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, California). The statistical significance for all tests was
set at P < 0.05.
Results
Characterization
The mean size of CeO2 NPs and CeO2 MPs was calculated
using TEM by measuring over 100 particles in random
fields. The size obtained for CeO2 NPs and CeO2 MPs was
25 + 1.512 nm (Figure 1A) and 3.01 + 1.023 mm (Figure 1B),
respectively. In DMEM, an average hydrodynamic diameter of
CeO2 NPs was 269.7 + 27.398 nm using DLS, revealing the
tendency of agglomeration in DMEM suspension. z potential and
electrophoretic mobility of CeO2 NPs in DMEM were determined
by LDV and found to be '7.74 mV and '1.24 mm cm/s V,
respectively, at pH 7.4. The size and charge of CeO2 NPs in
DMEM using TEM, DLS, and LDV, respectively, are presented
in Table 1. In culture medium, NPs showed a slight increase in the
size with a concomitant decrease in the z potential.
Cytotoxicity Assay
A significant dose-dependent decrease in mitochondrial function was observed after IMR32 cells were exposed to the CeO2
NPs. The cell viability after 24 hours incubation with CeO2
NPs and MPs at concentrations 10, 20, 50, 100, and 200 mg/
mL is shown in Figure 2A. The inhibitory concentration 50
(IC50) calculated for CeO2 NPS was 1.09 mg/mL and for CeO2
MPs was 1.64 mg/mL using probit analysis. The concentrations
lower than that of IC50 of CeO2 NPs and MPs were used in this
study, which was in order to avoid the physical hindrance due
to overaccumulation of the particles in the culture medium. The
cytotoxicity due to loss in cell viability was obvious at 200,
100, and 50 mg/mL of CeO2 NPs and found to be 76.40%,
77.49% (P < 0.01), and 78.68% (P < 0.05), respectively, as
compared to control (100%) while CeO2 MPs did not significantly decrease the cell viability.
Exposure to CeO 2 NPs for 24 hours resulted in a
concentration-dependent increase in LDH leakage and exhibited a significant (P < 0.01) cytotoxicity at 100 and 200 mg/mL
(Figure 2B). The evident difference between CeO2 NPs and
MPs was noted, as CeO2 MPs did not induce a significant
change.
Intracellular Release of ROS and H2O2
Statistical Analysis
The statistically significant changes between treated and control groups were analyzed by 1-way analysis of variance. All
results are expressed as mean and standard deviation (SD;
mean + SD) of the mean for 3 replicates of experiments,
Reactive oxygen specieslevels were quantified to examine the
involvement of oxidative stress in CeO2 NPs (Figure 3A).
It was noted that CeO2 NPs induced a significant 1.61- and
2.44-fold increase at 100 and 200 mg/mL, respectively, in ROS
levels, whereas CeO2 MPs did not show a significant change
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International Journal of Toxicology
Figure 1. Transmission electron microscopy image of cerium oxide nanoparticles (CeO2 NPs; A) and CeO2 microparticles (MPs; B) in MilliQ
water.
Table 1. Characterization of Cerium Oxide Nanoparticles (CeO2 NPs) and CeO2 Microparticles (MPs).a
DLS
Particles
CeO2 NPs
CeO2 MPs
LDV
Size using TEM
Average diameter, nm
PDI
z potential, mV
Electrophoretic mobility, mm cm/s V
pH
25 + 1.512 nm
3.01 + 1.023 mm
269.7 + 27.398
ND
0.436
ND
'7.74
ND
'1.25
ND
7.4
7.4
Abbreviations: PDI, polydispersity index, DLS, dynamic light scattering, LDV, laser Doppler velocimetry; DMEM, Dulbecco modified Eagle medium; ND, not
detectable; TEM, transmission electron microscopy.
a
CeO2 NPs and MPs at the concentration of 40 mg/mL were dispersed in DMEM medium, mixing was done via probe sonication for 10 minutes just before
estimations.
relative to control. Therefore, the production of ROS in IMR32
cells incubated with CeO2 NPs was much higher than the
corresponding MP-exposed cells.
Cerium oxide NPs significantly increased intracellular H2O2
within IMR32 cells at concentrations of 100 (P < 0.05) and
200 mg/mL (P < 0.01) in comparison to control (Figure 3B).
There was no significant induction in the H2O2 levels in cells
treated with CeO2 MPs.
Glutathione Content
Nitric Oxide Production
Activity of Enzymes Associated With Oxidative Stress
Nitric oxide production in IMR32 cells following 24 hours
exposure to CeO2 NPs at 0 to 200 mg/mL is shown in Figure
3C. The NO levels were significantly (P < 0.01) increased at
100 to 200 mg/mL in relation to control. However, in CeO2
MP-exposed cells, there was no significant alteration.
Cerium oxideNP-exposed cells at concentrations of 100 and
200 mg/mL revealed significant reduction in SOD (Figure 3F)
and CAT activity (Figure 3G) when compared to control at
P < 0.05 and P < 0.01, respectively. Likewise, GR activity
in IMR32 cells increased significantly (P < 0.01) in dosedependent manner (Figure 3H) when incubated with CeO2
NPs at the concentrations of 100 and 200 mg/mL. However,
the cells exposed to CeO2 MPs did not exhibit any significant change in the activities of these enzymes.
Further, CeO2 NPs exposure for 24 hours did not cause any
significant changes in the GPx and GST activity in the IMR32
cells when compared to control and same result was found with
CeO2 MPs (data not shown).
Lipid Peroxidation Assay
Lipid peroxidation assay was performed to determine the
MDA levels in the IMR32 cell suspension after 24 hours
treatment with CeO2 NPs and MPs. A significant increase
(P < 0.01) in MDA level was observed at 200 mg/mL of
CeO2 NPs (Figure 3D).
IMR32 cells exposed to CeO 2 NPs showed a dosedependent depletion in GSH levels. The exposure concentrations of 100 and 200 mg/mL exhibited statistically significant (P < 0.01) depletion of 79% and 76%, respectively,
after 24 hours (Figure 3E).
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Kumari et al
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Figure 2. Effects of CeO2 NPs and CeO2 MPs on mitochondrial function (A) and LDH leakage (B) in human neuroblastoma IMR32
cells. Cells were treated with different concentration of CeO2 NPs and MPs for 24 hours. At the end of incubation, mitochondrial
function was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and LDH leakage by LDH assay.
Control cells cultured in NP- and MP-free media were run in parallel to treatment groups. Treatment group included NP- and MPtreated cells. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented as mean + SD. Significantly
different from control at aP < 0.05 and bP < 0.01. CeO2 NP indicates cerium oxide nanoparticle; LDH, lactate dehydrogenase; MP,
microparticle.
Cytokinesis-Block MN Assay
Discussion
Chromosomal damage induced by CeO2 NPs was evaluated
using the CBMN assay, in which cell division is blocked to
allow the counting of once-divided binucleated cells. In the
untreated cells, MN frequency was 2 per 1000 binucleated
cells (Figure 4A). At dose levels of 100 and 200 mg/mL of
CeO2 NPs, the number of MN formed per 1000 binucleated
cells were 10.67 + 2.08 and 16 + 2, which was significant at P < 0.05 and P < 0.01, respectively (Figure 4A).
The cell proliferation was assessed during the CBMN
assay by the calculation of CBPI. Cell proliferation index
was reduced significantly at CeO2 NPs doses of 200 mg/mL
(1.30 + 0.02), 100 mg/mL (1.31 + 0.04), and 50 mg/mL
(1.36 + 0.02) in comparison to control (1.45 + 0.03;
Figure 4B). Therefore, there was a dose-dependent
increase in MN frequency whereas the cell proliferation
index decreased as the dose increased after CeO2 exposure.
Moreover, mono-, bi-, and multinucleated cells (Figure 5A)
along with an insignificant number of necrotic cells (Figure
5B), apoptotic cells (Figure 5C), and NBUDs (Figure 5D)
were observed along with MN in binucleated cells (Figure
5E-H).
Wide application of nanoceria in different sectors of human
welfare and its scanty data on toxicity prompted us to investigate the cellular response of CeO2 NPs and its bulk analog.
The findings of the present study demonstrated significant
ROS generation at 100 and 200 mg/mL doses of CeO2 NPs
in human NB cells. Further, the alterations in ROS production
and various oxidative stress-related indicators were concentration and size dependent. Moreover, the CeO2 NPs showed
DNA damaging potential at these 2 higher doses. The correlation between ROS generation and DNA damage indicated that
CeO2 NPs can lead to oxidative stress and could cause DNA
damage and cell death.
The physicochemical characterization is mandatory for the
toxicity study of NPs. Therefore, the size analysis of CeO2
NPs was done by TEM and DLS. The size obtained from TEM
analysis was 25 nm whereas the mean hydrodynamic size
obtained from DLS was 269.7 nm. This difference in size
could be due to difference in the principles used for the measurement (ie, TEM and DLS). The TEM provides the direct
measurement of particle size, distribution, and morphology by
image analysis in dry state while DLS measures the size distribution in the aqueous state, which is usually larger than the
TEM diameter. The increased diameter may be due to
agglomeration in aqueous medium, which could be due to
physicochemical interactions between NPs. Therefore, in
vitro testing with homogeneous NPs suspension is a challenging task and for that proper sonication is essential. The polydispersity index (PDI) is a measure of the heterogeneity of
molecules in a mixture. The CeO2 NPs were found to be
unstable in DMEM and instantly forming agglomerates
DNA Damage
Comet assay of CeO2 NP- and MP-treated cells showed a
concentration-dependent increase in the percentage of tail
DNA (Figure 6) when compared to control, which indicates
the extent of DNA damage. However, only the highest dose
of 200 mg/mL depicted significant (P < 0.01) increases in the
percentage of tail DNA (17.53 + 2.83).
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International Journal of Toxicology
Figure 3. Effects of CeO2 NPs and CeO2 MPs on (A) intracellular reactive oxygen species (ROS), (B) hydrogen peroxide (H2O2), (C) nitric
oxide (NO), (D) lipid peroxidation (LPO), (E) reduced glutathione (GSH), (F) superoxide dismutase (SOD), (G) catalase, and (H) glutathione
reductase (GR). All assays were performed on the culture medium after 24 hours incubation with CeO2 NPs and MPs at the concentrations of
10, 20, 50, 100, and 200 mg/mL. The control group was treated with media only. Bars indicate the standard deviation (SD) from 3 replicates. The
data are represented as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01. CeO2 NP indicates cerium oxide nanoparticle;
MP, microparticle.
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Kumari et al
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Figure 4. The genotoxicity of cerium oxide nanoparticles (CeO2 NPs) and CeO2 microparticles (MPs) using in vitro micronucleus test. A,
Frequency of micronucleus in binucleated cells and (B) cytokinesis-block proliferation index (CBPI). The control group was treated with media
only, cyclophosphamide (CP) was used as positive control. Bars indicate the standard deviation (SD) from 3 replicates. The data are represented
as mean + SD. Significantly different from control at aP < 0.05 and bP < 0.01.
Figure 5. Photomicrographs of the IMR32 cells scored in the cytokinesis-block micronucleus (CBMN) assay. A, Mononucleated cell, binucleated
cell, and multinucleated cell, (B) necrotic cells, (C) apoptotic cells, (D) binucleated cells containing nuclear buds (indicated by arrow), and (E-H)
binucleated cells containing a micronucleus (indicated by arrow).
(PDI ¼ 0.436) and showed incipient instability due to slightly
negative surface charge (z ¼ '7.74). This negative charge
may be because of adsorption of serum proteins.38
Viability assays are vital steps in toxicology evaluation that
explain the cellular response to a toxicant and they give
information on cell death, survival, and metabolic activities.
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International Journal of Toxicology
Figure 6. Mean percentage of tail DNA in IMR32 cells after 24 hours
exposure of different doses of cerium oxide nanoparticles (CeO2 NPs)
and microparticles (MPs). Bars indicate the standard deviation (SD)
from 3 replicates. The data are represented as mean + SD. Significantly
different from control at aP < 0.05 and bP < 0.01.
Both the MTT and the LDH assays are crucial for cytotoxicity
study. Our data demonstrated dose-dependent cytotoxicity on
exposure to CeO2 NPs with significant decrease in cell viability
and increase in LDH release at higher doses. The significant
loss in cell viability was observed at 100 to 200 mg/mL whereas
weak but significant toxicity was evident at 50 mg/mL. The
LDH release assay indicated that 25 nm CeO2 NPs induced a
loss in cell membrane integrity and cell death of IMR32 cells at
higher doses of 100 to 200 mg/mL but not in lower doses. In
affirmation of our finding, it was shown that 30 nm CeO2 NPs
did not cause significant decrease in the viability of cultured
BEAS-2B cells up to 40 mg/mL after 24 hours exposure.16 In
the same study, no significant cytotoxicity was shown either in
rat cardiomyocytes (H9C2) or in human brain fibroblast cells
(T98G) upon exposure to 5 mg/mL dose of 30 nm CeO2.16
However, even a 10.5 mg/mL dose of 20 nm CeO2 NPs significantly induced the LDH activity in A549 cells after 75 hours
exposure. 15 Hence, these studies suggested differential
sensitivity of the cells toward nanoceria. Further, duration of
exposure also had an important role in induction of toxicity.
The interactions between 2 commercial CeO2 NPs and A549
cells were investigated and weak cytotoxicity was observed
only at the highest concentration of 200 mg/mL. 38
Size-dependent toxicity of CeO2 NPs was quite evident in the
present study. Rosenkranz et al39 also reported the size, concentration, and time-dependent cytotoxicity of CeO2 particles
with H4IIE rat hepatoma cells and rainbow trout-derived RTG2 cells. Further, CeO2 NPs caused membrane damage and
inhibited colony formation in the long term but with different
degrees depending on the cell lines.40 The oxidative stress
induced in IMR32 cells upon 24 hours exposure of CeO2 NPs
was reflected in the ROS, H2O2, and NO production, MDA
levels, GSH content, and SOD, GR, and CAT activity in the
present study. In our study, CeO2 NPs exposure induced a
significant increase in the production of ROS, H2O2, NO,
MDA, and SOD whereas a significant decrease in GSH content, CAT, and GR activity at the higher dose levels was
observed. The ROS are produced in many processes in humans
which include atheroma, asthma, joint disease, aging, and
cancer.41 Zhang et al42 studied the mechanism of toxicity of
nanoceria of 8.5 nm size in C. elegans and suggested that its
ability to catalyze the ROS generation was involved in the
induction of toxicity at environmentally relevant concentrations. The NO and ROS are specialized chemical mediators
produced in an active program during the resolution of inflammation. One of the repercussions of elevated oxidative stress is
the production of MDA, an indicator of oxidative membrane
damage. A study has revealed that CeO2 NPs exposure aggravated LPO and oxidative stress in A549 cells.15 Cheng et al43
concluded from their study that CeO2 NP-induced oxidative
stress can lead to cytotoxicity in human hepatoma SMMC7721 cells after finding significant increase in the production
of ROS and MDA and significant reduction in the activity of
SOD, GPx, and CAT. The GSH is a ubiquitous sulfhydrylcontaining molecule in cells which is responsible for maintaining cellular oxidation–reduction homeostasis.44 Similarly, copper oxide (CuO) NPs induced LPO and loss in cell viability at
higher concentrations (400 mg/mL) after 24 hours of treatment
with mouse NB cell line (Neuro 2A).45 The toxicity study of
different NPs in rat liver-derived cell line (BRL 3A) after 24
hours exposure showed that the alterations in mitochondrial
function, LDH activity, GSH levels, and ROS were the basis
for toxicity evaluation of NPs.46
A combination of CBMN and in vitro comet assays are
excellent for interpreting the results from in vitro genotoxic
hazard assessment of compounds. 47 The MN assay with
cytokinesis facilitates the possibility of measuring important
biomarker of DNA damage, cytostasis, and cytotoxicity, which
can only be measured in once-divided binucleated cells.23 A
comet-like tail implies the presence of a damaged DNA strand
that lags behind when electrophoresis is done with an intact
nucleus. The length of tail increases with the extent of DNA
damage. DNA damage by CeO2 NPs was studied using CBMN
and comet assay. There was common pattern of dosedependent increase in MN frequency and DNA damage.
However, significant genotoxicity was observed only at highest
dose (200 mg/mL) of CeO2 NPs. Results of some investigations
are in accordance with our findings. Perreault et al45 reported
significant increase in MN frequency at 12.5 mg/L CuO NPs
after 24 hours treatment in Neuro 2A cells. The induction of
chromosome damage and DNA lesion related to oxidative
stress was observed in vitro in human dermal fibroblasts using
CBMN and comet assay on exposure to the 7-nm CeO2 NPs at
very low doses indicating the possible nanotoxocity and size
effect of CeO2 NPs.48 Moreover, toxicity was observed within
human hepatic carcinoma cells (HepG2), human colon carcinoma (CaCo2), and A549 cells when exposed to ceria NPs over
a concentration range of 0.5 to 5000 mg/mL for 10 days and
comet assay along with a cytotoxicity test were performed.49 In
the same study, 24 hours exposure of ceria NPs to various cell
lines did not induce DNA damage.49 These differences might
be due to the difference in cell type and culture conditions.50 In
NPs, toxicity ROS generation has been proposed as a possible
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Kumari et al
11
mechanism involved51 and ROS play a major role in the
genotoxicity.52 The data from the present study suggested that
CeO 2 NPs induce ROS and oxidative stress leading to
genotoxicity in IMR32 cell at higher doses.
Conclusion
Overall, the results obtained in this study revealed that CeO2
NPs have size- and dose-dependent toxicity in the tested cell
line whereas CeO 2 MPs did not induced any significant
changes in the exposed cells. Moreover, CeO2 NPs did not
induce toxicity below 100 mg/mL concentration and IMR32
cells were found to be less sensitive to CeO2 NPs. The cytotoxicity observed in the present study was in concurrence with
oxidative stress and genotoxicity induced by CeO2 NPs. This
investigation provides a better understanding of CeO2 NPs
toxicity in IMR32 cells. However, further investigations are
warranted on in vivo system in order to achieve a firm conclusion regarding the toxicity of CeO2 NPs and to get clear picture
of its toxicokinetics and tolerance in the system.
Acknowledgments
Monika Kumari (SRF) is grateful to the University Grant Commission (UGC), India, and Shailendra Pratap Singh (SRF) is grateful
to Indian Council of Medical Research (ICMR), India, for the
award of a fellowship. The authors express their sincere thanks
to the Director, IICT, Hyderabad, India, for providing facilities for
this study.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This research was supported by Asian Office of Aerospace
Research and Development (AOARD), Japan under the Grant
no. FA2386-11-1-4085.
References
1. Medina C, Santos-Martinez MJ, Radomski A, Corrigan OI,
Radomski MW. Nanoparticles: pharmacological and toxicological significance. Br J Pharmacol. 2007;150(5):552-558.
2. Bucchianico SD, Fabbrizi MR, Misra SK, et al. Multiple cytotoxic and genotoxic effects induced in vitro by differently
shaped copper oxide nanomaterials. Mutagenesis. 2013;28(3):
287-299.
3. Hu Z, Haneklaus S, Sparovek G, Schnug E. Rare earth elements in
soils. Commun Soil Sci Plan. 2006;37(9-10):1381-1420.
4. Esch F, Fabris S, Zhou L, et al. Electron localization determines
defect formation on ceria substrates. Science. 2005;309(5735):
752-755.
5. Wason MS, Zhao J. Cerium oxide nanoparticles: potential applications for cancer and other diseases. Am J Transl Res. 2013;5(2):
126-131.
6. Yu T, Park YI, Kang MC, et al. Large-scale synthesis of water
dispersible ceria nano crystals by a simple sol–gel process and
their use as a chemical mechanical planarization slurry. Eur J
Inorg Chem. 2008;2008(6):855-858.
7. Wu W, Li S, Liao S, Xiang F, Wu X. Preparation of new sunscreen
materials Ce1'xZnxO2'x via solid-state reaction at room temperature and study on their properties. Rare Metals. 2010;29(2):149-153.
8. Joo JH, Choi GM. Micro-solid oxide fuel cell using thick-film
ceria. Solid State Ion. 2009;180(11-13):839-842.
9. Park B, Donaldson K, Duffin R, et al. Hazard and risk assessment
of a nanoparticulate cerium oxide-based diesel fuel additive—a
case study. Inhal Toxicol. 2008;20(6):547-566.
10. Masui T, Ozaki T, Machida K, Adachi G. Preparation of ceriazirconia sub-catalysts for automotive exhaust cleaning. J Alloys
Compd. 2000;303-304(1):49-55.
11. Jan E, Byrne SJ, Cuddihy M, et al. High-content screening as a
universal tool for fingerprinting of cytotoxicity of nanoparticles.
ACS Nano. 2008;2(5):928-938.
12. Arnold MC, Badireddy AR, Wiesner MR, Di Giulio RT, Meyer
JN. Cerium oxide nanoparticles are more toxic than equimolar
bulk cerium oxide in Caenorhabditis elegans . Arch Environ Contam Toxicol. 2013;65(2):224-233.
13. Yokel RA, Au TC, MacPhail R, et al. Distribution, elimination, and
biopersistance to 90 days of a systemically introduced 30 nm ceriaengineered nanomaterial in rats. Toxicol Sci. 2012;127(1):256-268.
14. Hussain S, Al-Nsour F, Rice AB, et al. Cerium dioxide nanoparticles induce apoptosis and autophagy in human peripheral blood
monocytes. ACS Nano. 2012;6(7):5820-5829.
15. Lin W, Huang YW, Zhou XD, Ma Y. Toxicity of cerium oxide
nanoparticles in human lung cancer cells. Int J Toxicol. 2006;
25(6):451-457.
16. Park EJ, Choi J, Park YK, Park K. Oxidative stress induced by
cerium oxide nanoparticles in cultured BEAS-2B cells. Toxicology. 2008;245(1-2):90-100.
17. Xia T, Kovochich M, Liong M, et al. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based
on dissolution and oxidative stress properties. ACS Nano. 2008;
2(10):2121-2134.
18. Schubert D, Dargusch R, Raitano J, Chan SW. Cerium and
yttrium oxide nanoparticles are neuroprotective. Biochem
Biophys Res Commun. 2006;342(1):86-91.
19. Niu JL, Azfer A, Rogers LM, Wang X, Kolattukudy PE. Cardioprotective effects of cerium oxide nanoparticles in a transgenic murine
model of cardiomyopathy. Cardiovasc Res. 2007;73(3):549-559.
20. Hirst SM, Karakoti A, Singh S, et al. Bio-distribution and in vivo
antioxidant effects of cerium oxide nanoparticles in mice. Environ
Toxicol. 2013;28(2):107-118.
21. Kirsch-Volders M, Decordier I, Elhajouji A, Plas G, Aardema MJ,
Fenech M. In vitro genotoxicity testing using the micronucleus
assay in cell lines, human lymphocytes and 3D human skin models. Mutagenesis. 2011;26(1):177-184.
22. Fenech M. Cytokinesis-block micronucleus assay evolves into a
‘‘cytome’’ assay of chromosomal instability, mitotic dysfunction
and cell death. Mutat Res. 2006;600(1-2):58-66.
23. Fenech M. Cytokinesis-block micronucleus cytome assay. Nat
Protoc. 2007;2(5):1084-1104.
Downloaded from ijt.sagepub.com at UNIV OF KANSAS MEDICAL CENTER on February 13, 2014
12
International Journal of Toxicology
24. Olive PL, Banath JP. The comet assay: a method to measure DNA
damage in individual cells. Nat Protoc. 2006;1(1):23-29.
25. Hansen MB, Nielsen SE, Berg K. Re-examination and further
development of a precise and rapid dye method for measuring
cell growth/cell kill. J Immunol Methods. 1989;119(2):203-210.
26. Royall JA, Ischiropoulos H. Evaluation of 2,7-dichlorofluorescein
and dihydrorhodamine 123 as fluorescent probes for intracellular
H2O2 in cultured endothelial cells. Arch Biochem Biophys. 1993;
302(2):348-352.
27. Lee JY, Park W, Yi DK. Immunostimulatory effects of gold
nanorod and silica-coated gold nanorod on RAW 264.7 mouse
macrophages. Toxicol Lett. 2012;209(1):51-57.
28. Bradford MM. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem. 1976;72:248-254.
29. Wills ED. Lipid peroxide formation in microsomes. Relation of
hydroxylation to lipid peroxide formation. Biochem J. 1969;
113(2):333-341.
30. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys.
1959;82(1):70-77.
31. Marklund S, Marklund G. Involvement of superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for
superoxide dismutase. Eur J Biochem. 1974;47(3):469-474.
32. Aebi H. Catalase. In: Packer L, ed. Methods in Enzymology. Vol
105. Orlando, FL: Academic pres; 1984:121-126.
33. Carlberg I, Mannervik B. Glutathione reductase. Methods Enzymol.
1985;113:484-490.
34. Floche L, Gunzler WA. Assay of glutathione peroxidase. In:
Packer L, ed. Methods Enzymol. Vol 105. New York: Academic
Press; 1984:114-121.
35. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferase. The
first enzymatic step in mercapturic acid formation. J Biol Chem.
1974;249(22):7130-7139.
36. Organisation for Economic Co-operation and Development.
Guideline for Testing of Chemicals: In Vitro Mammalian Cell
Micronucleus Test (MNvit) (487). Paris: Organization for
Economic Co-operation and Development; 2007.
37. Tice RR, Agurell E, Anderson D, et al. Single cell gel/comet
assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35(3):206-221.
38. Zhou X, Wang B, Chen Y, Mao Z, Gao C. Uptake of cerium oxide
nanoparticles and their influences on functions of A549 cells.
J Nanosci Nanotechnol. 2013;13(1):204-215.
39. Rosenkranz P, Fernandez-Cruz ML, Conde E, et al. Effects of
cerium oxide nanoparticles to fish and mammalian cell lines: an
assessment of cytotoxicity and methodology. Toxicol In Vitro.
2012;26(6):888-896.
40. Kim IS, Baek M, Choi SJ. Comparative cytotoxicity of Al2O3,
CeO2, TiO2 and ZnO nanoparticles to human lung cells. J Nanosci
Nanotechnol. 2010;10(5):3453-3458.
41. Afonso V, Champy R, Mitrovic D, Collin P, Lomri A. Reactive
oxygen species and superoxide dismutases: role in joint diseases.
Joint Bone Spine. 2007;74(4):324-329.
42. Zhang H, He X, Zhang Z, et al. Nano-CeO2 exhibits adverse
effects at environmental relevant concentrations. Environ Sci
Technol. 2011;45(8):3725-3730.
43. Cheng G, Guo W, Han L, et al. Cerium oxide nanoparticles induce
cytotoxicity in human hepatoma SMMC-7721 cells via oxidative
stress and the activation of MAPK signaling pathways. Toxicol In
Vitro. 2013;27(3):1082-1088.
44. Sies H. Glutathione and its role in cellular functions. Free Radic
Biol Med. 1999;27(9-10):916-921.
45. Perreault F, Melegari SP, da Costa CH, Rossetto ALOF, Popovic
R, Matias WG. Genotoxic effects of copper oxide nanoparticles
in Neuro 2A cell cultures. Sci Total Environ. 2012;441:117-124.
46. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In
vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In
Vitro. 2005;19(7):975-983.
47. Kimura A, Miyata A, Honma M. A combination of in vitro comet
assay and micronucleus test using human lymphoblastoid TK6
cells. Mutagenesis. 2013;28(5):583-590.
48. Auffan M, Rose J, Orsiere T, et al. CeO2 nanoparticles induce
DNA damage towards human dermal fibroblasts in vitro . Nanotoxicology. 2009;3(2):161-171.
49. Marzi LD, Monaco A, Lapuente JD, et al. Cytotoxicity and genotoxicity of ceria nanoparticles on different cell lines in vitro . Int
J Mol Sci. 2013;14(2):3065-3077.
50. Lanone S, Rogerieux F, Geys J, et al. Comparative toxicity of 24
manufactured nanoparticles in human alveolar epithelial and
macrophage cell lines. Part Fibre Toxicol. 2009;6:14.
51. Gurr JR, Wang AS, Chen CH, Jan KY. Ultrafine titanium dioxide
particles in the absence of photoactivation can induce oxidative
damage to human bronchial epithelial cells. Toxicology. 2005;
213(1-2):66-73.
52. Schins RPF, Knaapen AdM. Genotoxicity of poorly soluble particles. Inhal Toxicol. 2007;19(s 1):189-198.
Downloaded from ijt.sagepub.com at UNIV OF KANSAS MEDICAL CENTER on February 13, 2014

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