Environmental and Molecular Mutagenesis 00:00^00 (2014)

Research Article

TiO2 Nanoparticles Induce DNA Double Strand Breaks

and Cell Cycle Arrest in Human Alveolar Cells
Krupa Kansara,1 Pal Patel,1 Darshini Shah,1 Ritesh K. Shukla,1
Sanjay Singh,1 Ashutosh Kumar,1* and Alok Dhawan1,2*
1
Institute of Life Sciences, Ahmedabad University, University Road,
Navrangpura, Ahmedabad, Gujarat, India
2
Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research,
Mahatma Gandhi Marg, Lucknow, Uttar Pradesh, India

TiO2 nanoparticles (NPs) have the second high-
est global annual production (3000 tons)
among the metal-containing NPs. These NPs are
used as photocatalysts for bacterial disinfection,
and in various other consumer products includ-
ing sunscreen, food packaging, therapeutics,
biosensors, surface cleaning agents, and others.
Humans are exposed to these NPs during syn-
thesis (laboratory), manufacture (industry), and
use (consumer products, devices, medicines,
etc.), as well as through environmental expo-
sures (disposal). Hence, there is great concern
regarding the health effects caused by exposure
to NPs and, in particular, to TiO2 NPs. In the
present study, the genotoxic potential of TiO2
NPs in A549 cells was examined, focusing on
their potential to induce ROS, different types of
DNA damage, and cell cycle arrest. We show
that TiO2 NPs can induce DNA damage and a
corresponding increase in micronucleus fre-
quency, as evident from the comet and
cytokinesis-block micronucleus assays. We dem-
onstrate that DNA damage may be attributed to
increased oxidative stress and ROS generation.
Furthermore, genomic and proteomic analyses
showed increased expression of ATM, P53, and
CdC-2 and decreased expression of ATR,
H2AX, and Cyclin B1 in A549 cells, suggesting
induction of DNA double strand breaks. The
occurrence of double strand breaks was corre-
lated with cell cycle arrest in G2/M phase.
Overall, the results indicate the potential for
genotoxicity following exposure to these TiO2
NPs, suggesting that use should be carefully
monitored. Environ. Mol. Mutagen. 00:000
000, 2014. V C 2014 Wiley Periodicals, Inc.

Key words: TiO2 NPs; genotoxicity; DNA double strand breaks; oxidative stress; micronucleus; cell cycle
arrest

INTRODUCTION Grant sponsor: UK India Education and Research Initiative (UKIERI)

Metal oxide nanoparticles (NPs) are finding a variety
of applications in catalysis, drug delivery, cosmetics,
clothing, energy conversion, water disinfection, lubrica-
tion, home appliances, and environmental remediation
[Aitken et al., 2004; Hoet et al., 2004a,b; Nohynek et al.,
2008; Akimov and Koh, 2010; Kumar et al., 2011c;
Kumar and Dhawan, 2013b]. TiO2 NPs have the second
highest annual production (estimated 3000 tons), with
major applications as a photocatalyst for bacterial disin-
fection and as a component of sunscreens in the cosmet-
ics industry [Bondarenko et al., 2013]. TiO2 NPs are also
used in products such as food packaging, therapeutics,
biosensors and surface cleaning agents, among others
[Shukla et al., 2013, 2014].
Worldwide use of TiO2 NPs has led to environmental
release with reports of their presence as contaminants in
standard award to Institute of Life Sciences, Ahmedabad University,
India; Grant number: IND/CONT/E/1112/217.
Grant sponsor: European Union Seventh Framework Programme (FP7/
20072013); Grant number: 263147.
Grant sponsor: The Gujarat Institute for Chemical Technology (GICT).
*Correspondence to: Alok Dhawan, Institute of Life Sciences, School of Sci-
ence and Technology, Ahmedabad University, University Road, Navrangpura,
Ahmedabad 380009, Gujarat, India. E-mail: alok.dhawan@ahduni.edu.in (or)
Ashutosh Kumar, Institute of Life Sciences, School of Science and Technol-
ogy, Ahmedabad University, University Road, Navrangpura, Ahmedabad
380009, Gujarat, India. E-mail: ashutosh.kumar@ahduni.edu.in
Conflict of Interest: The authors declare that there is no conflict of
interest.
Received 19 August 2014; provisionally accepted 19 October 2014; and
in final form 29 October 2014
DOI 10.1002/em.21925
Published online 00 Month 2014 in
Wiley Online Library (wileyonlinelibrary.com).

C 2014 Wiley Periodicals, Inc.

V
Environmental and Molecular Mutagenesis. DOI 10.1002/em
2 Kansara et al.
water, air, and soil [Klaine et al., 2008; Kumar et al.
2011b]. Humans are exposed to these NPs at various
stages including: synthesis (laboratory), manufacture
(industry), use (consumer products, devices, medicines,
etc.) and through environmental exposure (through dis-
posal). The main routes exposure are inhalation, ingestion
and dermal [Kumar and Dhawan, 2013a; Magdolenova
et al., 2014]. As the NPs are small in size and have high
diffusion coefficients, they can migrate rapidly in the air
[Aitken et al., 2008]. Therefore, inhalation is considered
to be the primary route of exposure by which humans are
exposed to TiO2 NPs suspended in air. In addition, expo-
sure of biota and humans to NPs is known to induce
adverse health effects including lung cancer, silicosis, and
autoimmune diseases [Donaldson, 2006; Fubini et al.,
2011; Glista-Baker et al., 2014]. Hence, in this study, the
potential toxicological effects of TiO2 NPs in the human
alveolar cell line A549 were assessed.
TiO2 NPs have been shown to induce cytotoxicity in
organ specific cell lines. These effects have been attrib-
uted to reactive oxygen species (ROS) generation, pulmo-
nary inflammation, oxidative stress, and fibrosis [Borm
et al., 2006; Nel, 2006; Singh et al., 2007; Jin et al.,
2008; Shukla et al., 2011b; Pfuhler et al., 2013]. Addi-
tionally, it has been shown that the free radicals (O22
production) produced by TiO2 NPs interact with cellular
components (nucleus, mitochondria, cytoplasm, etc.) and
induce damage by protein oxidation [Gajjar et al., 2009].
The DNA damaging potential of the TiO2 NPs and their
correlation with ROS and oxidative stress is also well
documented [Shukla et al., 2011a, 2013]. Recently, Pra-
sad et al. [2013] demonstrated the genotoxic potential of
TiO2 NPs in human BEAS-2B lung cells. These authors
observed that genotoxicity is independent of the amount
of agglomeration, cellular interaction, and cell-cycle alter-
ations [Prasad et al., 2013]. However, micronucleus for-
mation induced by TiO2 NPs was dependent on serum
concentration and the type of cell culture media used to
grow the BEAS-2B cells. Micronucleus frequency was
highest in keratinocyte growth medium with 10% FBS
concentration. This was attributed to the concentration of
FBS, as the protein present in the FBS induces the forma-
tion of NP protein corona and reduces the formation of
agglomeration. This also leads to higher internalization
and subsequent interaction of NPs with cellular compo-
nents [Prasad et al., 2013, 2014]. In contrast, it has also
been reported that TiO2 NP dispersion with large agglom-
erates (3 min sonication and no serum in stock solution)
induced DNA damage in three cell lines (TK6-human
lymphoblast cells, EUE-human embryonic epithelial cells
and Cos-1-monkey kidney fibroblasts), while the TiO2
NPs dispersed with agglomerates less than 200 nm (foetal
serum in stock solution and sonication 15 min) had no
effect on genotoxicity [Magdolenova et al., 2012]. It is
also known that the genotoxicity induced by TiO2 NPs in
cells relies on a variety of cell cycle checkpoints and
DNA repair pathways [Arenz et al., 2006; Cui et al.,
2012; Prasad et al., 2013]. However, the type of DNA
damage and its correlation with the cell cycle arrest and
apoptosis is not understood.
Therefore, in this study, we investigated the genotoxic
potential of TiO2 NPs in A549 cells, including their
potential to induce ROS, different types of DNA damage
and cell cycle arrest. The correlation between DNA dam-
age and cell cycle arrest was investigated using genomics
and proteomics approaches. A schematic representation of
the study design is depicted in Figure 1.
MATERIALS AND METHODS
Materials
Phosphate buffered saline (PBS; Ca21, Mg21 free), Dulbeccos
Modified Eagles Medium (DMEM F12), fetal bovine serum (FBS), tita-
nium oxide 10% in water (CAS No. PL-TiO-10p-10g), low melting
point agarose, ethidium bromide (EtBr), 2,7-dichlorofluorescein diace-
tate, cell lysis buffer, protease inhibitor, cytochalasin B, ethyl methane-
sulfonate (EMS), sodium dodecyl sulfate, RNase from bovine pancreas,
and bovine serum albumin, sodium chloride, 3-(4, 5-dimethylthiazoyl-
2-yl) 2,5-diphenyltetrazolium bromide (MTT), sodium citrate, normal
melting agarose, triton X-100, ethylenediaminetetraacetic acid diso-
dium salt [(EDTA)Na2], trypsin-EDTA, L glutamine, tris buffer, antibi-
otic/antimycotic solution (10,000 U/ml penicillin, and 10 mg/ml
streptomycin and amphotericin-B) were purchased from Hi-Media,
Mumbai, India. Tissue culture flasks (25 cm2, 75 cm2, and chamber
slide), and black bottom 96 well plates were purchased from Corning,
India. Syringe filters (0.22 mm) and filter holders were purchased from
Millipore, Mumbai, India. Syringes and needles were purchased from
Dispovan, India. End frosted conventional glass slides (75 3 25 mm2,
with 19 mm frosted end) and cover slips (No. 1, 24 3 60 mm) were
purchased from Hi-Media, Mumbai, India. Cytofunnel was purchased
from Thermo Scientific, India.
METHODS
Characterization of TiO2 NPs Using Dynamic
Light Scattering (DLS)
TiO2 NPs at a concentration of 100 mg/ml were suspended in MilliQ
water and complete DMEM F-12 medium; 1 ml of TiO2 NPs suspension
was transferred into a disposable polystyrene cuvette to determine the
size and zeta potential by DLS and phase analysis light scattering,
respectively, using a Zetasizer Nano-ZS equipped with 4.0 mW, 633nm
laser (Model ZEN3600, Malvern instruments, Malvern, UK). The stabil-
ity of NPs in the culture medium was also monitored up to 72 hr.
Cell Culture and Exposure to NPs
The human lung alveolar carcinoma cell line (A549) was obtained
from the National Centre for Cell Sciences, Pune, India, and cultured in
DMEM-F12 supplemented with 10% FBS, 0.2% sodium bicarbonate,
and 10 ml/l antibiotic and antimycotic solution at 37 C under a humidi-
fied atmosphere of 5% CO2.
A stock suspension of TiO2 NPs (1 mg/ml) in DMEM-F12 was
diluted to concentrations ranging from 1 to 200 mg/ml, respectively.
A549 cells were exposed at varying concentrations for each experiment.

Fig. 1. Schematic showing the study design. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Subsequent experiments involving: cytotoxicity assays; genotoxicity
assays (comet assay, micronucleus induction); oxidative stress (ROS
generation); cell cycle; gene expression (polymerase chain reaction);
proteomics (immunoblot assay) and apoptosis markers (Annexin-V-PI)
were conducted and analyzed. For different assays, 96-, 12-, 6-well cell
culture plates and 75 cm2 cell culture flasks were used having a treat-
ment volume of 0.1, 1.2, 3, and 24 ml, respectively. The volume of the
treatments were chosen to ensure that the concentration of NPs per cm2
area was equivalent in 6, 12, 96 well plates and 75 cm2 flask for all
assays. For different plates and flasks, the concentration was equivalent
to 7.89 lg/cm2 for 25 lg/ml; 15.78 lg/cm2 for 50 lg/ml; 23.68 lg/cm2
for 75 lg/ml and 31.57 lg/cm2 for 100 lg/ml.
Cytotoxicity Assays
Cytotoxicity assessment of TiO2 NPs was determined by MTT and
neutral red uptake (NRU) assays. These assays were carried out accord-
ing to the method of Mosmann (1983) and Borenfreund and Puerner
(1985), respectively, with slight modification as described in our earlier
study [Shukla et al., 2011a, 2013]. Briefly, cells (1 3 104 cells/well) in
100 ml of culture medium were seeded in 96-well plates and kept for 24
hr. Cells were then exposed to different concentrations (1 200 mg/ml)
of TiO2 NPs for 6, 24 and 48 hr.
NP interference with the assay reagents was also checked using a cell
free system, where NPs alone were incubated with the assay reagents,
including dye and buffers, and the absorption was monitored by spec-
Environmental and Molecular Mutagenesis. DOI 10.1002/em
DNA Double Strand Breaks and Cell Cycle Arrest 3
troscopy. The results were assessed by measuring the absorbance of end
product at their respective wavelengths using a SYNERGY-HT multi-
well plate reader (Bio-Tek, USA) using Gen5 software.
Genotoxicity Assessment
The genotoxic potential of TiO2 NPs was assessed by comet and
cytokinesis-block micronucleus (CBMN) assays. The treatment scheme
was the same for both assays. Approximately 3 3 105 cells in 3 ml of
DMEM-F12 were seeded in a 6-well cell culture plate. After 24 hr, the
cells were exposed to TiO2 NPs (25, 50, 75, and 100 mg/ml) for 6 h.
EMS at a concentration of 2 and 4 mM was used as a positive control
for the comet and CBMN assay, respectively.
Comet assay for Detection of DNA Damage
Briefly, cells were harvested and slides were prepared by the method
described by Singh et al. (1988) and modified by Bajpayee et al. (2005).
Slides were kept overnight at 4 C in lysis solution (2.5 M NaCl,
100 mM EDTA, 10 mMTris (pH10), 1% Triton X-100 added prior to
lysis step). After lysis, the slides were placed in a horizontal gel electro-
phoresis tank containing fresh and chilled electrophoresis solution
(1 mM EDTA and 300 mM NaOH, pH >13) for 20 min of alkaline
unwinding of DNA, followed by electrophoresis at 0.7 V/cm and 300
mA at 4 C for 30 min. Neutralization was done using Tris buffer
Environmental and Molecular Mutagenesis. DOI 10.1002/em
4 Kansara et al.
(400 mM, pH 7.4) and slides were stained with 20 mg/ml EtBr and
stored at 4 C in a humidified slide box until scoring.
Scoring was done at a final magnification of 4003 on a microscope
with a fluorescent attachment (DM 2500, Leica,Wetzlar, Germany) and
using Komet 6.0 software, which was provided with the image analysis
system (Andor Technology Belfast, U.K.). The comet parameters meas-
ured were tail DNA (%) and Olive Tail Moment (OTM). Analysis of 50
comets (25 from replicate slides) was carried out for each experiment
according to Tice et al. (2000).
CBMN Assay
The CBMN assay was carried out according to the method described
by Fenech (2000). After 6 hr of exposure, the treatment of TiO2 NPs
was aspirated, cells were washed with PBS and grown further for 16 hr
in complete medium containing Cytochalasin-B at final concentration 3
mg/ml in 5% CO2 incubator. The cells were harvested and slides were
prepared by centrifugation using a Cytospin (Thermo Shandon, Hamp-
shire, UK); 300 ml of cell suspension was loaded in each block of cyto-
funnel and centrifuged at 250g for 1 min. Two slides (two dots on one
slide from each replicate culture) were prepared for each concentration.
The slides were fixed in chilled methanol (90%) for 5 min, air-dried and
stored until staining. The slides were stained with 10% Giemsa dissolved
in Sorensons buffer and examined for the presence of micronuclei in
binucleate cells using a bright field microscope (DM 2500, Leica, Wet-
zlar, Germany) at 10003 magnification. Two thousand binucleate cells
from each concentration (1000 binucleate cells from each slide, 500 cells
per dot) were scored.
Micronucleus Assay Using Flow Cytometry
The micronucleus assay using flow cytometry was carried out by the
modified method of Nusse and Kramer [1984]. After 6 hr of exposure,
cells were harvested and centrifuged at 1200 rpm for 10 min. Cells were
washed in 1 3 PBS and transferred to 1 ml of solution I containing
sodium chloride (584 mg/l), sodium citrate (1000 mg/l), RNase (10 mg/
l), EtBr (25 mg/l), Igepal (0.3 ml/l) and incubated for 1 hr at room tem-
perature; 1 ml of solution II containing citric acid (15 mg/L), sucrose
(0.25 M), EtBr (40 mg/L) was then added to same tube and the samples
were analyzed for the presence of micronuclei in the cells using flow
cytometry (FACS Calibur, BD Biosciences, CA).
Intracellular ROS Measurement
Intracellular ROS generation was estimated by the method of Wan
et al. [1993] modified by Shukla et al. (2011b) using 2, 7-
dichlorofluorescein diacetate (DCFDA) dye. Briefly, cells (1 3 105cells/
well) were seeded in a 12-well plate. After 24 hr, the cells were exposed
to TiO2 NPs for 3, 6, 24, and 48 hr. The TiO2 NP-containing media was
aspirated and the cells were washed twice with PBS. Thereafter, culture
medium containing DCFDA dye (20 mM) was added to each well. The
plate was incubated for 30 min at 37 C and the medium containing
DCFDA was discarded; 200 ml of PBS was then added to each well and
fluorescence intensity was measured using flow cytometer at excitation
and emission wavelengths of 485 and 528 nm, respectively. In addition,
qualitative analysis of ROS generation was done using a microscope
with fluorescence attachment (Model DM 2500 Leica, Wetzlar,
Germany).
Cell Cycle Progression Analysis
A549 cells (1 3 105) were seeded on 6-well plate and treated with
different TiO2 NP concentrations (25100 mg/ml) for 24 hr. After the
treatment, medium was aspirated into individual tubes and cells were
harvested and fixed in 70% ethanol (at 220 C for 30 min). After fixa-
tion, cells were lysed in 1ml PBS containing 0.2% triton X 100 at 4 C
for 30 min. Cells were pelleted by centrifugation and re-suspended in
500 ml of PBS containing 20 ml RNAse (10 mg/ml) for 30 min at 37 C.
Finally, cells were stained with 10 ml propidium iodide (1 mg/ml) in
500 ml PBS for 1015 min at 4 C and analyzed on FACS (FACS Cali-
bur, BD Biosciences, CA). A histogram of count vs. intensity was made
to calculate ratio of cells under Go/G1 (2n), S (2n1), G2/M phase (4n),
and under apoptosis (2n-).
Apoptosis Assay Using Annexin V Binding Assay
Cells actively undergoing apoptosis were identified by staining with
fluorescein isothiocyanate-conjugated (FITC)-Annexin V and PI accord-
ing to manufacturers protocol (BD Biosciences, San Jose, CA). Briefly,
2 3 105 cells/well were seeded in a 6-well culture plate for 24 hr prior
to treatment. Cells were treated with TiO2 NPs at different concentra-
tions (0, 25, 50, 75 and 100 mg/ml) for 24 and 48 hr; 1 mM camptothe-
cin was used as a positive control in this assay. After removal of
treatment, cells were harvested and washed twice with PBS, resuspended
in 0.1 ml binding buffer containing 5 ml of FITC-Annexin V and PI and
kept at room temperature in the dark. After 10 min of incubation, 0.4 ml
of binding buffer was added to each sample and analyzed using flow
cytometer.
Genomics Studies
RNA Extraction and cDNA Synthesis
Total RNA was extracted from the control and TiO2 NP-treated (25,
50, and 100 mg/ml) A549 cells using TRIzol reagent (Life Technolo-
gies). cDNA was synthesized using a High-Capacity cDNA Reverse
Transcription Kit (Applied Biosystems). For RT, the 20 ml reaction mix-
ture contained 10 ml 23 master mix [23 RT buffer (2 ml), 0.8 mM
dNTP Mix (0.8 ml), 23 random primers (2 ml), RNase inhibitor (1 ml),
one liter MultiScribe reverse transcriptase (RT) and 3.2 ml nuclease-free
H2O] and an equal volume of diluted RNA sample. The RT reaction
was carried out in a thermal cycler consisting of one cycle at 25 C for
10 min, 50 C for 15 min, 85 C for 5 min, and 4 C on hold.
RT-PCR Analysis
RT-PCR was performed in a 25 ll volume containing 100 ng cDNA,
100 lM dNTPs, 2.5 mM MgCl2, 2.5 ll PCR buffer, 1 unit of DNA
polymerase (Thermo Scientific) and 1 lM gene specific (ATM, ATR,
P53, H2AX, CDC2, and CyclinB1) forward and reverse primers (Table
I). Thirty cycles of PCR amplification were performed and the intensity
of the amplified product was analyzed after electrophoresis using the
ImageQuantLAS 500 software (GE Healthcare Bio-Sciences AB,
Sweden).
Immunoblot Analysis
A549 cells were treated with TiO2 NPs at concentrations of 25, 50,
and 100 mg/ml for 24 hr. After removal of treatment, cells were har-
vested and lysed in lysis buffer (150 mM NaCl, 1% NP-40, 0.5%
sodium deoxycholate, 0.1% SDS, 50 mMTrisHCl, pH 8) containing
protease inhibitor cocktail (Sigma). Protein concentration was esti-
mated using Bradfords method [Bradford, 1976]. Protein (30 mg) from
control and treated groups was separated on Glycine-SDS-
polyacrylamide gel (8%) and transferred to a nitrocellulose membrane
by electroblotting. The membrane was incubated with primary antibod-
ies specific for Hsp70, HMOX1, and b-actin (Abcam). Secondary anti-
body incubation was done and protein bands were detected using
chemiluminescence and densitometry analysis was carried out using
ImageQuantLAS 500 software (GE Healthcare Bio-Sciences AB,
Sweden).
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
DNA Double Strand Breaks and Cell Cycle Arrest 5

TABLE I. Primers for Different Genes

Gene Forward primer Reverse primer
0 0
ATM 5 -TGCCAGACAGCCGTGACTTAC-3 5 -ACCTCCACCTGCTCATACACAAG-30
0

ATR 50 -TGCAGTAATGTCAATGGTTGG-30 50 -CTGGAACTTCAAAGGTTTCTCC-30

p53 50 -TCAGATCCTAGCGTCGAGCCC-30 50 -GGGTGTGGAATCAACCCACAG-30
p21 50 -GTTCCTTGTGGAGCCGGAGC-30 50 -GGTACAAGACAGTGACAGGTC-30
CdC2 50 -TGGGGTCAGCTCGTTACTCA-30 50 -CACTTCTGGCCACACTTCATTTA-30
Cyclin B1 50 -CAGTCAGACCAAAATACCTACTGGGT-30 50 -ACACCAACCAGCTGCAGCATCTTCTT-30
PCNA 50 -GCCCTCAAAGACCTCATCAA-30 50 -TCTGGGATTCCAAGTTGCTC-30
H2AX 50 -AGCAAACTCAACTCGGCAAT-30 50 -ACTCCCCAATGCCTAAGGTT-30
B actin 50 -CGGCATCGTCACCAACTG-30 50 -AACATGATCTGGGTCATCTTCTC-30
GAPDH 50 -CAGGAGGCATTGCTGATGAT-30 50 -GAAGGCTGGGGCTCATTT-30

TABLE II. Characterization of TiO2 NPs in Different to 100% of control) at 150 and 200 mg/ml of TiO2 NPs
Dispersant and Stability in Complete DMEM F12 exposure after 48 hr. However, after 6 and 24 hr expo-
Medium Using DLS sure, no significant cytotoxicity was observed (Fig. 2A).
In the NR uptake assay, a statistically significant
Hydrodynamic Zeta
Dispersant size (d.nm) potential (mV) (P < 0.05) reduction in neutral red uptake was observed
down to 67, 54, and 8% (relative to 100% of control) at
Milli Q 106.7 6 8.0 213.0 6 0.9 the three higher concentrations (100, 150, and 200 mg/ml)
DMEM-F12 (10% FBS) after 0 hr 23.28 6 2.0 210.1 6 1.0
of TiO2 NPs, respectively, after 48 hr exposure (Fig. 2B).
DMEM-F12 (10% FBS) after 24 hr 22.54 6 2.4 212.6 6 1.2
DMEM-F12 (10% FBS) after 72 hr 23.97 6 1.6 211.9 6 1.1 However, a reduction in percent cell viability was also
observed after 6 and 24 hr exposure, but it was not statis-
Values represent mean 6 standard error of three experiments. tically significant.

Statistical Analysis Genotoxic Potential of TiO2 NPs

Results from each experiment were expressed as mean 6 SEM of
three individual experiments and data were analyzed using one way
analysis of variance (ANOVA). The post hoc comparisons of mean of
independent groups were done by Dunnetts test at statistically signifi-
cant values (P < 0.05).
RESULTS
Size and Zeta Potential Analysis of TiO2 NPs Using DLS
The average hydrodynamic size and zeta potential of
TiO2 NPs in culture medium (DMEM F12) supplemented
with 10% FBS were 23.28 6 2.0 nm and 210.1 6 1.0 mV,
respectively. However, in MilliQ water the size and zeta
potential was 106.7 6 8.0 nm and 213 6 0.9 mV, respec-
tively (Table II). The particles were also found to be stable
up to 72 hr as measured by their size and zeta potential
(Table II). Additionally, the primary particle size as meas-
ured by transmission electron microscopy and surface area
of TiO2 NPs by BrunauerEmmettTeller method (as
described in the technical sheet by the supplier) was 4
8 nm 1215 m2/g, respectively. The particles were 99.7%
pure and had an anatase crystal structure.
Cytotoxicity
Cytotoxicity of TiO2 NPs was assessed in human alve-
olar cells (A549) for 6, 24, and 48 hr, respectively. In the
MTT assay, the mitochondrial succinate dehydrogenase
enzymatic activity was reduced to 68 and 64% (relative
DNA Damage
A significant (P < 0.05) concentration-dependent
increase in DNA damage was observed in A549 cells at
different TiO2 NP concentrations (75 and 100 lg/ml) after
6 hr exposure, as evident by the comet assay parameters,
namely, OTM and % Tail DNA respectively (Table III).
Micronucleus Induction
CBMN Assay
Human alveolar cells (A549) showed a statistically sig-
nificant (P < 0.05) increase in micronucleus formation after
6 hr exposure to 75 and 100 lg/ml TiO2 NPs (12.33 and
14.66 MN/1000 BNCs) compared with control (6 MN/
1000 BNCs; Table III). Using flow cytometry, we observed
a significant increase (200 and 500%) in micronucleus for-
mation in A549 cells following exposure to 75 and 100
mg/ml of TiO2 NPs relative to control cells (Fig. 3).
Measurement of Intracellular ROS
A significant qualitative and quantitative increase in %
ROS generation was observed in A549 cells after 6 hr
exposure to TiO2 NPs. The flow cytometric analysis
showed a concentration-dependent significant (P < 0.05)
increase in % ROS generation based on an increase in the
fluorescence intensity of DCFDA dye following treatment
with TiO2 NPs (115 and 128% at 75 and 100 mg/ml,
respectively; Fig. 4).
Environmental and Molecular Mutagenesis. DOI 10.1002/em
6 Kansara et al.

Fig. 2. A significant concentration- and time-dependent increase in cytotoxicity of TiO2 NPs in A549 cells. A: % MTT
reduction. B: % Neutral red uptake. The viability of the control cells was considered as 100%. The data are expressed
as mean 6 SEM from three independent experiments. *P < 0.05, when compared with control. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]

Apoptosis
TABLE III. Genotoxic Potential of TiO2 NPs in A549 Cells as Our data demonstrate a significant (P < 0.05)
Assessed by the Micronucleus and Comet Assay concentration-dependent increase (23, 74, 189, and 249%)
in the apoptotic A549 cells population after 24 hr of TiO2
No. of Micronucleus
in 1000 binucleated NPs exposure (25, 50, 70, and 100 mg/ml; Fig. 5). How-
Groups cells Tail DNA (%) ever, after 48 hr of treatment, cells were necrotic.
Control 6.00 6 2.80 4.48 6 0.11 Cell Cycle Analysis
TiO2 NPs (25 mg/ml) 7.33 6 1.20 5.14 6 0.12
TiO2 NPs (50 mg/ml) 9.66 6 2.84 6.06 6 0.15 Our data demonstrate that TiO2 NPs caused a signifi-
TiO2 NPs (75 mg/ml) 12.33 6 2.96a 8.25 6 0.24a cant increase in the proportion of cells that are arrested in
TiO2 NPs (100 mg/ml) 14.66 6 2.33a 9.49 6 0.25a
Positive Control- EMS(4 mM) 16.66 6 3.66a 48.2 6 1.14a
G2/M phase in a concentration-dependent manner after
24 hr exposure. More cells exposed to TiO2 NPs were
a
P < 0.05 when compared with control using one way ANOVA. arrested in G2/M phase compared to control at the 50,
Values represent mean 6 S.E. of three experiments. 75, and 100 mg/ml concentrations, respectively (Fig. 6).

Fig. 3. A: Flow cytometric analysis of A549 cells after exposure to dif-
ferent concentrations of TiO2 NPs showed a concentration-dependent
increase in micronucleus formation compared to controls. B: Bar graph
representing the percent increase in micronucleus formation. Values repre-
Expression of DNA Damage Responsive Genes
Expression levels of DNA damage responsive genes
were analysed in A549 cells after 24 hr exposure to dif-
ferent concentrations (25, 50, and 100 mg/ml) of TiO2
NPs by RT-PCR. The expression of ATM, P53, and CdC-
Environmental and Molecular Mutagenesis. DOI 10.1002/em
DNA Double Strand Breaks and Cell Cycle Arrest 7
sent mean 6 S.E. of three experiments for each concentration. *P < 0.05,
compared with control. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
2 gene increased by 1.24, 1.42, and 1.25 fold respectively
over control cells at the 100 mg/ml concentration. How-
ever, expression levels of ATR, H2AX and Cyclin B1
decreased by 0.67, 0.21 and 0.24 fold, respectively, com-
pared with controls at 100 mg/ml (Fig. 7).
Environmental and Molecular Mutagenesis. DOI 10.1002/em
8 Kansara et al.

Fig. 4. Significant concentration-dependent increase in intracellular ROS after 6 hr exposure. Values represents mean 6 S.E. of three experiments
using DCFDA dye. A: Control cells. BF: Cells treated with TiO2 NPs (*P < 0.05). [Color figure can be viewed in the online issue, which is
(5100 lg/ml). (Magnification X-400). G: Bar graph representing the available at wileyonlinelibrary.com.]
increase in intracellular ROS generation as detected by flow cytometry

Immunoblot Analysis
cant increase (P < 0.05) in the expression of the stress
Immunoblot analysis of A549 cells exposed to TiO2 proteins HSP70 (20, 40, and 52%) and HMOX-1 (13, 24,
NPs (25, 50, and 100 mg/ml) for 24 hr showed a signifi- and 32%), as shown in Figure 8.

Fig. 5. Bar graph showing percent apoptotic cells. Camptothecin (1 lM)
was used as positive control. Data represent means 6 SEM of three inde-
pendent experiments.*P < 0.05, when compared with control. [Color fig-
ure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
DISCUSSION
This study reveals the genotoxic potential of TiO2 NPs
in human alveolar (A549 cells) cells and provides insight
into the possible mechanism by which TiO2 NPs exert
their genotoxic effects in these cells. Our results demon-
strate that TiO2 NPs induce DNA damage leading to cell
cycle arrest in A549 cells through the ATM/ATR path-
way (Fig. 9).
Characterization of NPs is the most essential part of
nano-toxicological studies. The behavior and activity of
NPs is largely dependent on a number of physical and
chemical properties such as, particle number, mass con-
centration, surface area, charge, chemistry and reactivity,
size distribution, aggregation, elemental composition, as
well as structure and shape [Dhawan and Sharma, 2010].
Therefore, a complete characterization is essential for bet-
ter understanding and interpretation of results. In this
study, characterization of TiO2 NPs was done in MilliQ
and complete medium (DMEM-F12) using DLS. Our data
demonstrate that the TiO2 NPs used in the study were in
nano form. Also, the stability of the NPs was monitored
up to 72 h, as NPs have a tendency to agglomerate in
aqueous medium [Dusinska et al., 2009; Gurbani et al.,
2011]. Our data demonstrate that the TiO2 NPs were sta-
ble in the DMEM-F12; however, the hydrodynamic size
of NPs increased in MilliQ over time. This can be attrib-
uted to the protein coating over the NPs, or the formation
of corona, as the coating reduces the tendency of
agglomeration [Lynch and Dawson, 2008; Elsaesser and
Howard, 2011]. Additionally, the particle-plus-corona
plays an important role in the biological fate of NPs as it
enhances the internalization into the cells.
The interference of the NPs (due to their unique physi-
cal and chemical properties) with dyes that are commonly
being used for cytotoxicity test systems is well docu-
mented [Dhawan and Sharma, 2010; Dhawan et al.,
2011]. Therefore, it has been suggested that findings from
cytotoxicity assays should be validated by two or more
Environmental and Molecular Mutagenesis. DOI 10.1002/em
DNA Double Strand Breaks and Cell Cycle Arrest 9
independent test systems along with a parallel set of cell-
free systems [Sharma et al., 2009, 2012; Shukla et al,.
2011b]. A range of TiO2 NPs concentrations (1200 mg/
ml) for different time points (6, 24 and 48 hr) were used
for the cytotoxicity experiments in this study. MTT and
NR uptake assays are based on two different mechanisms.
MTT represents mitochondrial activity and NR provides
information on the lysosomal activity of the cells. It was
observed that TiO2 NPs cause no significant cytotoxicity
at concentrations ranging from 1 to 200 lg/ml after 6 and
24 hr of exposure in A549 cells. Hence, these non-
cytotoxic concentrations and time points were used for
genotoxicity and mechanistic studies (ROS, cell cycle and
expression of DNA damage responsive genes/proteins).
However, mild cytotoxic responses were observed after
48 hr of exposure in MTT assay (150 and 200 lg/ml) and
NR uptake assay (100, 150, and 200 lg/ml). Therefore,
these concentrations were used for annexin V binding
assay to demonstrate apoptosis. Our data demonstrate that
TiO2 NPs induce apoptosis in A549 cells after 48 hr of
exposure.
Oxidative stress is the most prevalent mechanism pro-
posed in the literature to explain the toxicity of NPs
[Murray et al., 2009; Zhong et al., 2010; Srinivas et al.,
2012; Kumar et al., 2013]. The Hsp family of proteins is
the first tier of cellular defence and induces the protein
folding response to minimize protein degradation and cel-
lular stress [Beere et al., 2000]. Our study indicates that
TiO2 NPs induce stress in A549 cells as evident by an
induction of Hsp 70. Additionally, ROS generation is
hypothesized as the probable mechanism involved in the
toxicity of NPs [Gurr, 2005; Long et al., 2006; Nel, 2006;
Kumar et al., 2011a; Dey et al., 2012]. This has been
attributed to the small size and hence large surface area
of NPs, which is generally thought to produce ROS and
oxidative stress [Borm et al., 2006]. In the present study,
TiO2 NPs were found to generate intracellular ROS when
examined by the cell permeable dye DCFH-DA using
flow cytometer and fluorescence microscope. A significant
induction in % ROS generation in the form of fluores-
cence was observed in A549 cells exposed to TiO2 NPs
(25, 50, and 75 mg/ml) for 6 h using fluorescence micro-
scope (Figs. 4A4F). Also, a significant (P < 0.05) dose-
dependent increase in % ROS generation was observed
by flow cytometry, in the form of increased FITC fluores-
cence intensity following treatment with TiO2 NPs (115
and 128% at 75 and 100 mg/ml; Fig. 4G). This observa-
tion is consistent with earlier studies where TiO2 was
shown to produce ROS even without photoactivation
[Shukla et al., 2011a,b, 2013, 2014]. These free radicals
can oxidize and reduce macromolecules (DNA, lipid, and
protein) in the cellular system, resulting in significant oxi-
dative damage to cells.
Free radicals can also damage DNA, which can lead to
genetic instability and consequently, carcinogenesis or
Environmental and Molecular Mutagenesis. DOI 10.1002/em
10 Kansara et al.

Fig. 6. TiO2 NP-induced alterations in the cell cycle of A549 cells. A: Controls. BE: Cells treated with different con-
centration of TiO2 NPs. F: Cells treated with 4 mM EMS as positive control. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]

cell death [Luo et al., 2011]. To elucidate the genotoxic
potential of TiO2 NPs in A549 cells, the alkaline comet
assay was performed. We found that TiO2 NPs induce
DNA damage in A549 cells using the comet assay. This
supports ROS generation as the mediator of TiO2 NP-
induced DNA damage. Furthermore, a statistically signifi-
cant (P < 0.05) dose-dependent increase in MN frequency
was observed at TiO2 NP concentrations of 25, 50, 75,
and 100 lg/ml, which further confirms the genotoxicity
of the TiO2 NPs. The comet assay measures single and
double strand breaks. However, increase in micronucleus
frequency indicate an abundance of DNA double strand
breaks [Han et al., 2010].
Earlier, Kitchin et al. [2011] demonstrated that ROS
and radical-induced DNA damage is clustered and yields
DNA double strand breaks that can activate the ATM-
Chk2 signalling pathway [Kitchin et al., 2011]. ATM
belongs to a family of kinases that have sequence homol-
ogy to phosphoinositide 3-kinase (PI3K). The phospho-
inositide 3-kinase related kinases (PIKKs) are the key

Fig. 7. Densitometry analysis for the expression of ATM, ATR, P53, cH2AX, CdC2, Cyclin B1, and b-actin in A549
cells, at exposure concentrations of 25, 50, and 100 mg/ml TiO2 NPs by RT-PCR. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
Fig. 8. Immunoblot analysis of proteins (HSP70 and HMOX1) of A549
cells treated with different concentrations (25, 50, and 100 lg/ml) of
TiO2 NPs. [Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
Environmental and Molecular Mutagenesis. DOI 10.1002/em
DNA Double Strand Breaks and Cell Cycle Arrest 11
components of the checkpoint signalling network, repre-
sented by Ataxia-telangiectasia mutated (ATM), ATM
and RAD3-related, and DNA-dependent protein kinase
[Shiloh, 2003]. These protein kinases play an important
role in the cell signaling pathway involved in response to
DNA double strand break in higher eukaryotes [Shiloh,
2003]. In our study, a dose-dependent increase in the
expression of ATM was observed after 24 hr of TiO2 NPs
exposure, which suggests an increase in double strand
breaks. On the contrary, a dose-dependent decrease in the
expression of ATR indicates no single strand breaks in
DNA induced by TiO2 NPs in A549 cells. ATR is a ser-
ine/threonine-specific protein kinase that is involved in
sensing DNA damage and is activated in response to per-
sistent single-stranded DNA, which is a common interme-
diate formed during DNA damage detection and repair
[Horne et al., 1996; Shiloh, 2003]. Moreover, our
Environmental and Molecular Mutagenesis. DOI 10.1002/em
12 Kansara et al.
Fig. 9. A schematic showing possible mechanisms of TiO2 NP-induced double strand DNA breaks and cell cycle arrest.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
observations for double strand DNA breaks were also
confirmed by decreased expression of cH2AX, which is a
sensitive biomarker of DNA double strand breaks [Pod-
horecka et al., 2010]. H2AX is one of several genes cod-
ing for histones; it is a major component of nucleosome-
formation and the structure of DNA. H2AX is phospho-
rylated on serine 139, and then called gamma-H2AX, in
the presence of DNA double strand breaks [Flores-Perez
et al., 2014]. Because of this modification, the DNA
becomes less condensed, potentially allowing space for
the recruitment of proteins necessary during the repair of
double strand breaks. Our data revealed a significant
decrease in the expression of the cH2AX gene, indicating
that the double strand breaks induced by TiO2 NPs were
not repairable at the higher concentration. However, at
lower concentrations the expression of cH2AX was on
higher side (relative to controls), suggesting that this
damage can be repaired by the different repair enzymes.
These findings suggest that exposure to TiO2 NPs acti-
vates the ATM/Chk2 DNA damage signalling pathway in
A549 cells.
Finally, we observed a significant dose-dependent
increase in the expression of CdC2, with concomitant
decreases in the expression of Cyclin B1 [Hinds et al.,
1992]. Cyclin B1 is a regulatory protein involved in mito-
sis. This gene product binds with P34 (Cdk1) and forms
the maturation-promoting factor, which is predominantly
expressed during the G2/M phase of the cell cycle and
promotes cell division [Horne et al., 1996; Zimmermann
et al., 2012]. We observed a dose-dependent decrease in
the expression of Cyclin B1, which indirectly provides
support to our cell cycle finding that TiO2 NP exposure
arrests the cells in the G2/M phase. The correlation
between the DNA damage and cell cycle arrest induced
by TiO2 NPs in A549 cells is shown in Figure 8.
CONCLUSIONS
In the present study we investigated the effects of TiO2
NPs in a human lung alveolar carcinoma cell line (A549)
and explored the different mechanisms underlying their
cytotoxic and genotoxic potential. Comet and CBMN
assays demonstrated that TiO2 NPs can induce DNA
damage and a corresponding increase in micronucleus fre-
quency. The DNA damage induced by TiO2 NPs may be
attributed to increased oxidative stress and ROS genera-
tion. Furthermore, genomic and proteomic changes were
consistent with the induction of DNA double strand
breaks. The occurrence of double strand breaks was cor-
related with cell cycle arrest in G2/M phase. Our results
provide valuable insights into the mechanism of TiO2

NPs induced toxicity in human lung alveolar cells. These
observations once again reiterate concern over the safety
of TiO2 NPs in consumer products.
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