Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells.
ABSTRACT Titanium dioxide nanoparticles (TiO(2)-NPs) induced cytotoxicity and DNA damage have been investigated using human amnion epithelial (WISH) cells, as an in vitro model for nanotoxicity assessment. Crystalline, polyhedral rutile TiO(2)-NPs were synthesized and characterized using X-ray diffraction (XRD), UV-Visible spectroscopy, Fourier transform infra red (FTIR) spectroscopy, and transmission electron microscopic (TEM) analyses. The neutral red uptake (NRU) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assays revealed the concentration dependent cytotoxic effects of TiO(2)-NPs (30.6nm) in concentration range of 0.625-10μg/ml. Cells exposed to TiO(2)-NPs (10μg/ml) exhibited significant reduction (46.3% and 34.6%; p<0.05) in catalase activity and glutathione (GSH) level, respectively. Treated cells showed 1.87-fold increase in intracellular reactive oxygen species (ROS) generation and 7.3% (p<0.01) increase in G(2)/M cell cycle arrest, as compared to the untreated control. TiO(2)-NPs treated cells also demonstrated the formation of DNA double strand breaks with 14.6-fold (p<0.05) increase in Olive tail moment (OTM) value at 20μg/ml concentration, vis-à-vis untreated control, under neutral comet assay conditions. Thus, the reduction in cell viability, morphological alterations, compromised antioxidant system, intracellular ROS production, and significant DNA damage in TiO(2)-NPs exposed cells signify the potential of these NPs to induce cyto- and genotoxicity in cultured WISH cells.
- SourceAvailable from: Vaiyapuri Subbarayan Periasamy[Show abstract] [Hide abstract]
ABSTRACT: The use of engineered nanoparticles (NPs) across multiple fields and applications has rapidly increased over the last decade owing to their unusual properties. However, there is an increased need in understanding their toxicological effect on human health. Particularly, iron oxide (Fe3O4) have been used in various sectors, including biomedical, food, and agriculture, but the current understanding of their impact on human health is inadequate. In this investigation, we assessed the toxic effect of Fe3O4 NPs on human mesenchymal stem cells (hMSCs) adopting cell viability, cellular morphological changes, mitochondrial transmembrane potential, and cell-cycle progression assessment methodologies. Furthermore, the expression of oxidative stress, cell death, and cell-cycle regulatory genes was assessed using quantitative polymerase chain reaction. The Fe3O4 NPs induced cytotoxicity and nuclear morphological changes in hMSCs by dose and time exposure. Cell-cycle analysis indicated that Fe3O4 NPs altered the cell-cycle progression through a decrease in the proportion of cells in the G0–G1 phase. The hMSC mitochondrial membrane potential loss increased with an increase in the concentration of Fe3O4 NPs exposure. The observed expression levels of the CYP1A, TNF3, TNFSF10, E2F1, and CCNC genes were significantly upregulated in hMSCs in response to Fe3O4 NPs exposure. Our findings suggest that Fe3O4 NPs caused metabolic stress through altered cell cycle, oxidative stress, and cell death regulatory gene expression in hMSCs. The results of this investigation revealed that Fe3O4 NPs exhibited moderate toxicity on hMSCs and that Fe3O4 NPs may have biomedical applications at low concentrations. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.Environmental Toxicology 12/2014; · 2.56 Impact Factor
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ABSTRACT: In this work, sunflower oil was utilized for the biomimetic synthesis of silver (Ag) nanoparticles (NPs), leading to highly mono-dispersed hexagonal-shaped silver nanoparticles (NPs) at various concentrations. It was found that the biomolecules of the oil not only have the capability to reduce silver ions, due to its extended phenolic system, but also appear to recognize and affect the Ag nanocrystal growth on the (110) face, leading to hexagonal growth of the NPs of 50nm size. Initially, some spherical AgNPs of less than 10nm diameter were observed; however, over a longer period of time, a majority of hexagonal-shaped nanocrystals were formed. The one step synthesis can be extended for other metals. The as prepared sunflower oil capped AgNPs being completely free of toxic chemicals can be directly utilized for in vitro studies and offer a more rational approach for cellular applications. The NP solution exhibited dose-dependent cytotoxicity in human lung carcinoma cells and physiologically relevant cell model (3T3L1cells).Materials Science and Engineering C 11/2014; 44:209–215. · 2.74 Impact Factor
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ABSTRACT: Abstract Background and aim: Zinc oxide (ZnO) and titanium dioxide (TiO2) nanomaterials (NMs) are used in many consumer products, including foodstuffs. Ingested and inhaled NM can reach the liver. Whilst their effects on inflammation, cytotoxicity, genotoxicity and mitochondrial function have been explored, no work has been reported on their impact on liver intermediary metabolism. Our aim was to assess the effects of sub-lethal doses of these materials on hepatocyte intermediary metabolism. Material and methods: After characterisation, ZnO and TiO2 NM were used to treat C3A cells for 4 hours at concentrations ranging between 0 and 10 μg/cm(2), well below their EC50, before the assessment of (i) glucose production and glycolysis from endogenous glycogen and (ii) gluconeogenesis and glycolysis from lactate and pyruvate (LP). Mitochondrial membrane potential was assessed using JC-10 after 0-40 μg/cm(2) ZnO. qRT-PCR was used to assess phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression. Dihydroethidium (DHE) staining and FACS were used to assess intracellular reactive oxygen species (ROS) concentration. Results: Treatment of cells with ZnO, but not TiO2, depressed mitochondrial membrane potential, leading to a dose-dependent increase in glycogen breakdown by up to 430%, with an increase of both glycolysis and glucose release. Interestingly, gluconeogenesis from LP was also increased, up to 10-fold and correlated with a 420% increase in the PEPCK mRNA expression, the enzyme controlling gluconeogenesis from LP. An intracellular increase of ROS production after ZnO treatment could explain these effects. Conclusion: At sub-lethal concentrations, ZnO nanoparticles dramatically increased both gluconeogenesis and glycogenolysis, which warrants further in vivo studies.Nanotoxicology 04/2014; · 7.34 Impact Factor
Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA
damage in human amnion epithelial (WISH) cells
Quaiser Saquiba, Abdulaziz A. Al-Khedhairya, Maqsood A. Siddiquia, Faisal M. Abou-Tarbousha,
Ameer Azamc, Javed Musarrata,b,⇑
aDepartment of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia
bDepartment of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India
cCentre of Nanotechnology, King Abdulaziz University, Jeddah, Saudi Arabia
a r t i c l e i n f o
Received 20 September 2011
Accepted 12 December 2011
Available online 19 December 2011
a b s t r a c t
Titanium dioxide nanoparticles (TiO2-NPs) induced cytotoxicity and DNA damage have been investigated
using human amnion epithelial (WISH) cells, as an in vitro model for nanotoxicity assessment. Crystalline,
polyhedral rutile TiO2-NPs were synthesized and characterized using X-ray diffraction (XRD), UV–Visible
spectroscopy, Fourier transform infra red (FTIR) spectroscopy, and transmission electron microscopic
(TEM) analyses. The neutral red uptake (NRU) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] (MTT) assays revealed the concentration dependent cytotoxic effects of TiO2-NPs (30.6 nm) in
concentration range of 0.625–10 lg/ml. Cells exposed to TiO2-NPs (10 lg/ml) exhibited significant reduc-
tion (46.3% and 34.6%; p < 0.05) in catalase activity and glutathione (GSH) level, respectively. Treated cells
showed 1.87-fold increase in intracellular reactive oxygen species (ROS) generation and 7.3% (p < 0.01)
increase in G2/M cell cycle arrest, as compared to the untreated control. TiO2-NPs treated cells also dem-
onstrated the formation of DNA double strand breaks with 14.6-fold (p < 0.05) increase in Olive tail
moment (OTM) value at 20 lg/ml concentration, vis-à-vis untreated control, under neutral comet assay
conditions. Thus, the reduction in cell viability, morphological alterations, compromised antioxidant sys-
tem, intracellular ROS production, and significant DNA damage in TiO2-NPs exposed cells signify the
potential of these NPs to induce cyto- and genotoxicity in cultured WISH cells.
? 2011 Elsevier Ltd. All rights reserved.
Titanium dioxide (TiO2) is a naturally occurring mineral used in
domestic and cosmetic products including anti-foulingpaints, coat-
ings, ceramics, additives in pharmaceuticals, food colorants (Jin
et al., 2008; Vamanu et al., 2008), and as a sunscreen additive owing
to its typical characteristics of surface adsorption, photo-catalysis
and UV absorption (Douglas et al., 2000). Titanium either pure or
inalloysis alsoextensivelyused fora widerange ofimplantedmed-
ical devices, such as dental implants, joint replacements, cardio-
vascular stents, and spinal fixation devices. However, under
mechanical stress or altered physiological conditions such as low
pH, titanium-based implants can release large amounts of particle
debris (4.47 mg/g dry tissue weight from titanium-alloy (Ti–6Al–
4V) implants) both in the micrometer and nanometer size range
(Brien et al., 1992; Buly et al., 1992; Arys et al., 1998; Cunningham
et al., 2002). It is reported that the biological responses to nanopar-
ticles (NPs) may exceed those elicited by micron-sized particles
(Borm et al., 2006; Nel et al., 2006) due to their small size, high
number per given mass, large specific surface area, and generation
of free radicals (Lynch et al., 2006). The dimensions of the TiO2-NPs
are critical from the toxicity point of view, as the ultrafine TiO2
causes more pronounced toxicity compared with fine TiO2particles
(Driscoll and Maurer, 1991; Oberdörster et al., 1994; Oberdörster,
2000). UltrafineTiO2particles (620 nm)have beenshown to induce
impairment of macrophage function, persistently high inflamma-
toryreactions, and increased pulmonaryretention compared to fine
TiO2 (particle size > 200 nm) (Baggs et al., 1997). The TiO2-NPs
could be absorbed through inhalation, ingestion and dermal
penetration into the body, and distributed in the important organs
such as lung (Warheit et al., 2007; Wang et al., 2007), lymph nodes
(Bermudez etal., 2004),brain(Thomasetal., 2006),liverandkidney
(Wang et al., 2007).
There are growing concerns about the possible influence of NPs
on human health, particularly with the exposures during prenatal,
pregnancy or early childhood (Lacasana et al., 2005). Nanosized
materials including the carboxylic polystyrene, gold and TiO2-NPs
are reported to cross the placental tissue (Semmler-Behnke et al.,
2007; Tian et al., 2009). In an ex-vivo human placental perfusion
model, Wick et al. (2010) demonstrated the uptake of nanosized
0887-2333/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding author at: Department of Zoology, College of Science, King Saud
University, Riyadh, Saudi Arabia. Tel.: +966 4675768; fax: +966 4675514.
E-mail address: firstname.lastname@example.org (J. Musarrat).
Toxicology in Vitro 26 (2012) 351–361
Contents lists available at SciVerse ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
fluorescently labeled polystyrene beads of 50, 80, 240, and 500 nm
across the placental barrier. Also, in animal models, the transloca-
tion of TiO2-NPs has been reported in brain of prenatally exposed
mice. Since the blood barriers are under developed in the foetus,
the NPs could easily pass into brain during the early stages of foetal
development. The TiO2-NPs in anatase (crystals of eight-faced
tetragonal dipyramids) form, administered subcutaneously to
pregnant ICR mice, were found to be transferred to and affected
the genital and cranial nerve systems of the offspring (Takeda
et al., 2009).
Furthermore, Gurr et al. (2005) demonstrated that anatase-sized
(10 and 20 nm) TiO2-NPs in the absence of photoactivation induce
oxidative DNA damage, lipid peroxidation, and micronuclei forma-
tion, and cause increased hydrogen peroxide and nitric oxide pro-
duction in human bronchial epithelial (BEAS-2B) cell line. Vevers
and Jha (2008) have reported the enhanced level of TiO2-NPs in-
tissue cells. Whereas, in the goldfish skin cells (GFSk-S1), the TiO2-
NPs caused DNA damage in the absence of UV light (Reeves et al.,
2008). Also, in human monoblastoid and bronchial epithelial cells,
the TiO2-NPs induce apoptosis mainly by destabilizing the lyso-
somal membrane and lipid peroxidation (Vamanu et al., 2008; Zhao
apoptosis have also been demonstrated in human lymphocytes,
U937 human monoblastoid cells, A549 alveolar epithelial cells,
NRK-52E normal rat kidney cells, and A431 human epidermal cells
(Vamanu et al., 2008; Gopalan et al., 2009; Park et al., 2007; Barillet
et al., 2010; Shukla et al., 2011). However, to the best of our under-
standing, no systematic study on assessment of the TiO2-NPs cyto-
toxicity and genotoxicity on cells of placental origin are reported
line established as WISH (Wistar Institute, Susan Hayflick) cells
maintains the similar characteristics of growth, cell morphology,
prostaglandin production, and susceptibility to apoptotic agents,
as the primary amnion cells (Lundgren et al., 1997; Moore et al.,
2002). Perhaps, the stability of these cells makes them more useful
(Kumar et al., 2004). Therefore, the amniotic WISH cells have been
chosen as a model in this study, with the aim to assess the effects
of sonomechanically synthesized TiO2-NPs on the (i) cell viability
as cytotoxic end point, (ii) intracellular ROS production and antiox-
idative enzymes, (iii) induction of DNA strand breaks, and (iv) pro-
gression of normal cell cycle, in order to elucidate the plausible
markers for placental toxicity.
2. Materials and methods
Dimethyl sulfoxide (DMSO) cell culture grade, propiodium io-
dide, Na2-EDTA, Tris [hydroxymethyl] aminomethane, RNAse,
20,70-dichlorofluorescin diacetate (DCFH-DA), normal melting aga-
rose (NMA), low melting agarose (LMA), ethyl methanesulphonate
(EMS) and neutral red were purchased from Sigma Chemical Com-
pany, St. Louis, MO, USA. RPMI-1640, L-glutamine, MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, antibi-
otic–antimycotic solution, phosphate buffered saline (PBS, Ca2+,
Mg2+free) were obtained from Hi-Media Pvt. Ltd. (India). Foetal
bovine serum (FBS) was procured from GIBCO-BRL Life Technolo-
gies Inc. (Gaithersburg, MD, USA). Culture wares and other plastic
consumables used in the study were procured commercially from
Nunc, Denmark. Kits for the catalase (Cat # 707002) and glutathi-
one (Cat # 703002) assays were purchased from Cayman Chemi-
cals, USA. Powdered TiO2-NPs (1 mg/ml) were suspended in
Milli-Q water and subjected to sonication using Pro Scientific
Inc., USA for 15 min at 40 W to form a homogeneous suspension
before the treatments.
2.2. Synthesis and characterization of TiO2-NPs
TiO2-NPs were synthesized using sonomechanical method. In
brief, 50 g of bulk TiO2(rutile form) was taken in 250 ml beaker
and 30 ml of methanol was added to form a slurry, which was son-
icated for 30 min. The slurry was dried at 100 ?C for 12 h in an
oven, and the methanol evaporates during drying process. The
slurry was then ground in a planetary ball mill, (Retsch, Germany).
Initial milling was done for 10 h at 350 rpm and then extended up
to 30 h. Finally, the milled slurry was dried at 100 ?C for 12 h in an
oven. Several techniques were employed for characterizing the fi-
nally obtained white powder. The X-ray diffraction (XRD) was used
for crystal phase identification and estimation of the average crys-
tallite size. The particle size and morphology of the powder were
observed by transmission electron microscope (TEM). UV–visible
absorption spectrum was carried out in order to characterize the
optical properties of the TiO2-NPs. Fourier transform infra red
(FTIR) spectrum was used to study the functional groups and
stretching vibrations of the bonds in the TiO2-NP lattice. The NPs
aggregation in suspension and secondary particles sizes were esti-
mated using dynamic light scattering (DLS).
2.3. X-ray diffraction analysis
Powdered sample of TiO2-NPs was analyzed using X’pert PRO
analytical diffractometer (Rigaku X-ray diffractometer, Japan) using
CuKa radiation (k = 1.54056 Å) in the range of 20? 6 2h 6 80? at
40 keV. In order to calculate the particle size (D) of TiO2sample,
the Scherrer’s relationship (D = 0.9k/Bcosh) has been used, where,
k is the wavelength of X-ray, B is the broadening of diffraction line
measured as half of its maximum intensity in radians and h is the
Bragg’s diffraction angle. The particle size of sample has been esti-
mated from the line width of XRD peak.
2.4. UV–visible spectroscopy, FTIR and TEM analyses
The spectra of TiO2-NPs were recorded using a UV–Vis spectro-
photometer, Cintra 10e GBC (Victoria, Australia) at wavelengths
between 200 and 800 nm. The band gap of TiO2-NPs was deter-
mined from the well known Tauc relation, ahm = A(hm ? Eg)n (Tauc
et al., 2006), where, hm is photon energy, a = 2.303A/t is called the
absorption coefficient, A is the absorbance, t is the thickness of the
cuvette, Egis the band gap and n = 2 for indirect band gap semicon-
ductor. The FTIR was employed for examining the functional
groups on TiO2-NPs. For FTIR analysis, the dried powder of TiO2-
NPs was diluted with spectroscopic grade potassium bromide
(KBr) in the ratio of 1:100 and the spectrum was recorded. FTIR
measurements were carried out on Perkin–Elmer FTIR spectropho-
tometer (USA) in the diffuse reflectance mode at a resolution of
4 cm?1in KBr pellets. For TEM analysis, the samples were prepared
by dropping the ultrasonically treated TiO2-NPs suspension onto a
TEM copper grid and dried at room temperature. A total of six TEM
samples were prepared, and at least ten micrographs of each were
analyzed to determine the ensemble average of the sample pri-
mary particle size. TEM was performed on a Field Emission Trans-
mission Electron Microscope (JEM-2100F, JEOL, Japan) at 200 keV.
2.5. Dynamic light scattering and particle dosimetry
TiO2-NPs stock suspension of 2 mg/ml was prepared in deion-
ized Milli-Q water and sonicated for 15 min at 40 W. Stock suspen-
sion was then instantly diluted in the Milli-Q water and RPMI cell
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
culture medium. Dynamic light scattering was performed on a
ZetaSizer-HT, Malvern, UK to determine the hydrodynamic sizes
of the TiO2-NPs in suspensions. The theoretical estimations of
exposure, delivered and cellular doses at the nominal media mass
concentration of 10 lg/ml of an average secondary TiO2particle
size of 152 nm in RPMI medium were performed assuming the par-
ticles to be spherical, following the simplifying assumptions and
Eqs. (1) and (2), as specified by Teeguarden et al. (2007)
Surface area concentration ¼mass concentration
¼ # Concentration ?pd2
# Concentration ¼mass concentration
¼Surface area concentration
where particles are assumed to be spherical, or can be repre-
sented as spheres, d is the particle diameter in cm, surface area
concentration in cm2/ml media, mass concentration is in g/ml, #
indicates particle number and particle density is in g/cm3.
2.6. Cell culture and TiO2-NPs treatment
Human amnion epithelial (WISH) cell line (American Type Cul-
ture Collection, accession No. CCL25, Rockville, MD, USA) was
maintained in our laboratory and have been used for toxicity anal-
ysis of TiO2-NPs. This cell line was chosen as a model due to its
higher stability as compared to the primary cultures of amnion
cells (Kumar et al., 2004). Cells were grown in RPMI 1640, supple-
mented with 10% FBS and antibiotic–antimycotic solution (100?,
1 ml/100 ml of medium) in 5% CO2with 95% atmosphere in humid-
ity at 37 ?C. Each batch of cells was assessed for cell viability by
trypan blue dye exclusion test prior to the experiments and
batches showing more than 95% cell viability and passage number
between 10 and 15 were used in the study. For TiO2-NPs treatment,
the cells (1 ? 104/100 ll/well) in complete RPMI medium were
seeded in 96-well plates, and exposed to varying concentrations
(0.625–10 lg/ml) of TiO2-NPs for 24 h at 37 ?C. The concentration
range and time of exposures were determined based on the NRU
and MTT cytotoxicity assays. Similarly related dose ranges have
also been used in earlier studies on TiO2induced oxidative DNA
damage, lipid peroxidation, micronuclei formation, and the hydro-
gen peroxide and nitric oxide production in human bronchial epi-
thelial (BEAS-2B) cell line and reactive oxygen species production
in immortalized brain microglia (BV2) cells (Gurr et al., 2005; Long
et al., 2007).
2.7. TiO2-NPs uptake in WISH cells
The TEM analysis of TiO2-NPs treated WISH cells for uptake and
internalization have been performed following the method, as de-
scribed by Hussain et al. (2009). Briefly, cells (5 ? 104) were seeded
in 6 well tissue culture plate and allowed to adhere for 24 h in CO2
incubator at 37 ?C. Cells were then exposed to TiO2-NPs (10 lg/ml)
for 24 h. The untreated and TiO2-NPs treated cells were harvested
and fixed with 10% glutaraldehyde in 0.1 M cacodylate buffer (pH
7.4) for 20 min. Cells were then suspended in 1% OsO4in 0.1 M cac-
odylate buffer (pH 7.4) for 1 h at 4 ?C, followed by 1 h incubation in
2% aqueous uranyl acetate at room temperature. The samples were
dehydrated in an ethanol series and embedded in low viscosity
araldite resin. Ultrafine sections (60 nm thick) were visualized un-
der high vacuum at 100 kV using JEOI-1011 Electron Microscope
(JEOL, Tokyo, Japan). The images were captured without any
contrast agent to avoid potential artifacts due the deposition of
contrast agent crystals.
2.8. Tetrazolium bromide salt (MTT) assay
The viability of TiO2-NPs treated WISH cells was assessed by
MTT assay, as described by Siddiqui et al. (2010). In brief, cells
(1 ? 104) were allowed to adhere for 24 h under high humid envi-
ronment in 5% CO2-95% atmospheric air at 37 ?C in 96-well culture
plates. Cells were exposed to TiO2-NPs in the concentration range
of 0.625 to 10 lg/ml for 24 h. Subsequently, MTT (5 mg/ml stock
in PBS) was added in the volume of 10 ll/well in 100 ll of cell sus-
pension, and the plate was incubated for 4 h at 37 ?C. At the end of
incubation period, the aqueous medium was carefully aspirated
and 200 ll of DMSO was added to each well and mixed gently.
The plate was then kept on a rocker shaker for 10 min at room tem-
perature and the purple color developed was read at 550 nm using
microplate reader (Multiskan Ex, Thermo Scientific, Finland). Un-
treated controls were also run under identical conditions. The
probability of TiO2-NPs interference with the cytotoxicity assay
was assessed by measuring the MTT reduction to formazan with
ascorbic acid following the method of Belyanskaya et al. (2007).
Briefly, the TiO2-NPs (0.625 to 100 lg/ml) were incubated with
1 mg/ml ascorbic acid in the presence or absence of 1 mg/ml
MTT for 60 min at 37 ?C. The suspension was centrifuged at
14,000 rpm for 30 min and the supernatant was discarded. The
precipitate containing the purple formazan product was dissolved
in 1 ml of 90% ethanol and sonicated in an ultrasonic bath (Ultra-
sonic Cleaners, Jeiotech, UC-10, Seoul, Korea) at 25 ?C for 5 min.
The suspension was again centrifuged at 3000 rpm for 5 min, and
the absorbance of the dissolved formazan was measured at
550 nm using UV–visible spectrophotometer (Evolution 300 LC,
Thermo Scientific, USA).
2.9. Neutral red uptake (NRU) assay
NRU assay was carried out following the protocol described by
Siddiqui et al. (2008). Briefly, cells were exposed to TiO2-NPs in the
concentration range of 0.625 to 10 lg/ml for 24 h. After the expo-
sure time, medium was aspirated and cells were washed twice
with PBS, and incubated for 3 h in a medium supplemented with
neutral red (50 lg/ml). Medium was washed off rapidly with a
solution containing 0.5% formaldehyde and 1% calcium chloride.
Cells were subjected to further incubation of 20 min at 37 ?C in a
mixture of acetic acid (1%) and ethanol (50%) to extract the dye
and the absorbance was read at 540 nm on microplate reader.
The values were compared with control set run under identical
conditions. The probability of TiO2-NPs interference with the assay
was tested by measuring the reactivity of NR dye with TiO2-NPs in
the absence of cultured cells. Briefly, the TiO2-NPs in the concen-
tration range of 0.625 to 20 lg/ml were mixed with NR dye
(50 lg/ml) in complete RPMI medium, in a total reaction volume
of 100 ll in a microtitre plate. The mixture was incubated at
37 ?C for 3 h. The absorbance was read at 540 nm using a micro-
plate reader (Multiskan Ex, Thermo Scientific, Finland).
2.10. Effect of TiO2-NPs on catalase activity
Catalase activity of the cells treated for 24 h with varying con-
centrations (0.625 to 10 lg/ml) of TiO2-NPs was measured using
commercially available kit (Cayman Chemicals, USA). In brief, the
TiO2-NPs exposed cells were sonicated in cold buffer (50 mM
potassium phosphate, pH 7.0, containing 1 mM EDTA) and centri-
fuged at 10,000 rpm for 15 min at 4 ?C. The supernatant (20 ll)
was collected followed by the addition of 100 ll of assay buffer
and 30 ll of methanol in each well of the 96-well plate. Reaction
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
was initiated by adding 20 ll of hydrogen peroxide as a substrate
and incubated on shaker for 20 min at room temperature. The reac-
tion was stopped by adding 30 ll of potassium hydroxide. To this
mixture, 30 ll of 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole
(purpald) was added as chromogen, and incubated for 10 min fol-
lowed by the addition of 10 ll potassium periodate. The purple col-
or formaldehyde product formed was measured colorimetrically at
540 nm using a microplate reader.
2.11. Effect of TiO2-NPs on glutathione (GSH) level
The changes in GSH level in TiO2-NPs exposed cells were deter-
mined using a commercially available kit (Cayman Chemicals,
USA). In brief, TiO2-NPs (0.625 to 10 lg/ml) treated cells for 24 h
were homogenized in cold MES buffer (50 mM, pH 6.0, containing
1 mM EDTA). The homogenate was centrifuged at 10,000 rpm for
15 min at 4 ?C, and the supernatant was collected and deproteinat-
ed. The samples (50 ll) were transferred to respective wells in 96-
well plate, and 150 ll freshly prepared reagents and enzyme mix-
tures were added and the absorbance was read after 25 min at
405 nm using a microplate reader.
2.12. Reactive oxygen species (ROS) measurement
Intracellular ROS production was determined in WISH cells
using a fluorescent probe DCFH-DA following the method of Wu
et al. (2007). Cells were treated with increasing concentrations of
TiO2-NPs (0.625 to 10 lg/ml) for 24 h and harvested by spinning
at 3000 rpm for 5 min. The pellet was washed twice with cold
PBS and resuspended in 500 ll of PBS. Cells were then incubated
with DCFH-DA (5 lM) for 60 min at 37 ?C in dark. Immediately
after incubation, the cells were washed twice with PBS and finally
suspended with 500 ll PBS. DCFH-DA stained cells without TiO2-
NPs treatment and H2O2(100 lM) were taken as negative and po-
sitive controls, respectively. The fluorescence was recorded upon
excitation at 488 nm. Green fluorescence from 20,70-dichlorofluo-
rescein (DCF) was measured in the FL1 Log channel through
525 nm band-pass filter on Beckman Coulter flow cytometer (Coul-
ter Epics XL/Xl-MCL, USA). The interference of TiO2-NPs with fluo-
rescent DCF was also assessed under acellular conditions following
the method of Messer et al. (2006). In brief, 500 ll of DCFH-DA
stock (500 lM) was mixed with equal volume of 10 mM NaOH.
The mixture was incubated at room temperature for 30 min in dark
for the hydrolysis of diacetate ester. Prior to incubation with the
TiO2-NPs, the solution was neutralized by phosphate buffer (pH
7.4). Subsequently, the TiO2-NPs in increasing concentrations
(0.625 to 20 lg/ml) were mixed with 5 and 10 lM DCFH in a final
reaction volume of 100 ll in a 96 well microtitre plate. The plate
was incubated at 37 ?C for 60 min. The fluorescence of DCF was
measured at 485 nm excitation and 520 nm emission wavelengths
using microplate fluorometer (Thermo Scientific Fluoroskan As-
2.13. Assessment of cell cycle progression of TiO2-NPs treated cells
WISH cells treated with increasing concentrations (0.625 to
10 lg/ml) of TiO2-NPs for 24 h were harvested and centrifuged at
3000 rpm for 5 min, and the pellet was resuspended in 500 ll of
PBS. Cells were fixed with equal volume of chilled 70% ice-cold eth-
anol, and incubated at 4 ?C for 1 h. After two successive washes, the
cell pellet was again suspended in PBS and stained with propiodi-
um iodide (50 lg/ml) solution containing 0.1% Triton X-100 and
0.5 mg/ml RNAase A for 1 h at 37 ?C in dark. The red fluorescence
of 10,000 events of propiodium iodide stained cells were acquired
in FL4 Log channel through a 675 nm band-pass filter using flow
cytometer (Darzynkiewicz et al., 1992). The data were analyzed
excluding the cell debris, characterized by a low FSC/SSC, using
Beckman Coulter flow cytometer (Coulter Epics XL/Xl-MCL, USA
and System II Software, Version 3.0.
2.14. Assessment of DNA damage by neutral comet assay
TiO2-NPs induced strand breaks in DNA were assessed by neu-
tral comet assay, as described by Omidkhoda et al. (2007). Slide
preparation was done following the method of Saquib et al.
(2009). In brief, the cells at a density of 6.0 ? 104cells/well were
exposed to varying concentrations of TiO2-NPs (0.625 to 20 lg/
ml) in a 12 well plate for 6 h at 37 ?C. The cells were washed with
serum free medium and harvested by adding 0.065% trypsin and
incubated at 37 ?C. The cell suspension was centrifuged at
3000 rpm, 5 min and the pellet was resuspended in 100 ll of
PBS. The cells were mixed with 100 ll of 1% LMA and layered on
one-third frosted slides, pre-coated with NMA (1% in PBS) and kept
at 4 ?C for 10 min. After gelling, another layer of 90 ll of LMA (0.5%
in PBS) was added. The cells were lysed in a lysing solution for
overnight. After washing with TBE buffer, the slides were subjected
to neutral electrophoresis in cold TBE (Tris-base, 90 mM; boric
acid, 90 mM; Na2EDTA, 2.5 mM) buffer. Electrophoresis was per-
formed at 1 V/cm for 30 min (16 mA, 32 V) at 4 ?C. All preparative
steps were conducted in dark to prevent secondary DNA damage.
Each slide was stained with 75 ll of 20 lg/ml ethidium bromide
solution for 5 min. The slides were analyzed at 40? magnification
(excitation wavelength of 515–560 nm and emission wavelength
of 590 nm) using fluorescence microscope (Nikon ECLIPSE E600, Ja-
pan) coupled with charge coupled device (CCD) camera. Images
from 50 cells (25 from each replicate slide) were randomly selected
and subjected to image analysis using software Comet Assay IV
(Perceptive Instruments, Suffolk, UK).
2.15. Statistical analysis
Data were expressed as mean ± S.D for the values obtained from
at least three independent experiments. Statistical analysis was
performed by one-way analysis of variance (ANOVA) using Dun-
nett’s multiple comparisons test (Sigma Plot 11.0, USA). The level
of statistical significance chosen was
⁄p < 0.05, unless otherwise
3.1. XRD and TEM analysis of TiO2-NPs
The X-ray diffraction pattern of TiO2-NPs obtained by sonome-
chanical method is shown in Fig. 1. The peaks were indexed using
Powder ? software and were found corresponding with the tetrag-
onal rutile structure of TiO2(ICDD card No. 78–2485). No impurity
phase was observed in the sample. The average crystallite size of
the samples was calculated using Debye Scherrer’s formula. The
estimated size corresponding to the most intense crystallographic
plane (110) was determined to be 30.6 nm. The typical TEM image
shown in Fig. 1 (inset) clearly suggests that most of the TiO2-NPs
are crystallites with polyhedral morphologies.
3.2. UV- Vis absorption characteristics of TiO2-NPs
Fig. 2A shows the optical absorbance spectra of TiO2-NPs. The
absorbance is expected to depend on several factors, such as band
gap, oxygen deficiency and impurity centers. The absorbance
spectrum exhibits an absorption edge around 280–320 nm, which
may be due to the photo-excitation of electrons from valence
band to conduction band. The Tauc plot between (ahm)1/2and hm
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
of TiO2-NPs (rutile) is shown in Fig. 2B. The extrapolation of the lin-
ear region of a plot of (ahm)1/2vs hm provided the value of the opti-
cal band gap Eg. The band gap measured from Tauc plot was
determined to be 4.0 eV, which is higher than the value of Egfor
bulk TiO2(3.2 eV). This increase in the value of band gap confirms
the reduction of the particle size. Moreover, the higher value of
band gap indicates the higher photooxidation as well as photore-
duction ability of TiO2-NPs.
3.3. FTIR analysis of TiO2-NPs
The FTIR spectrum shown in Fig. 3 was recorded in solid phase
using KBr pellets technique in the region of 4000–500 cm?1with
the resolution of 4 cm?1. The peak located at 615 cm?1is the char-
acteristic vibration of the Ti–O bond in the TiO2lattice. The band at
1627 cm?1is assigned to the molecular water bending mode, while
the broad peak at 3439 cm?1shows the presence of –OH stretching
vibration. The broad peak appearing at 3439 cm?1was assigned to
fundamental stretching vibration of O-H hydroxyl group, which
was further confirmed by a weak band at about 1627 cm?1caused
by bending vibration of coordinated H2O. The band observed at
650 cm?1is assigned to the Ti-O stretching vibration in the TiO2
lattice. The above assignments are in good agreement with the re-
sults reported in literature (Yoko et al., 1990; Zhang and Gao, 2002;
Zhang et al., 2002; Gao et al., 2004).
3.4. Hydrodynamic diameter of TiO2-NPs in culture medium
The results of hydrodynamic size of the TiO2-NPs using dynamic
light scattering are shown in Fig. 4. The dispersion of TiO2-NPs of
average size 30.6 nm was heterogeneous due to the presence of
both the primary particles and larger aggregates. The distribution
curves show the TiO2-NPs with a small population of an average
13 nm particle size and larger aggregates of 152 nm in RPMI
medium as compared to the much larger particle aggregates of
380 nm in deionized Milli-Q water.
3.5. TEM analysis for TiO2uptake and accumulation in WISH cells
TEM of WISH cells exposed to TiO2-NPs for 24 h exhibit the
aggregates of NPs, localized either inside the vesicles, as shown
Fig. 1. (A) X-ray powder diffraction (XRD) analysis for phase identification of
sonomechanically synthesized crystalline TiO2-NPs. (B) Inset shows the TEM image
of TiO2-NPs at 200 keV.
Fig. 2. Absorption characteristics of TiO2-NPs. Panel (A) shows the absorption
spectrum of TiO2-NPs; whereas, the panel (B) depicts the Tauc plot for determining
optical gap in amorphous material. The axes represent the quantity hm (the energy
of light) on the abscissa and the quality (ahm)1/2on the ordinate, where a is the
absorption coefficient of the material, as a function of TiO2-NPs.
Fig. 3. FTIR spectrum of TiO2-NPs. The peak located at 615 cm?1represents the
characteristic vibration of Ti–O bond in the TiO2lattice. The band at 1627 cm?1
signifies the molecular water bending mode, while the broad peak at 3439 cm?1
corresponds to –OH stretching vibration in the sample.
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
in the representative Fig. 5 B, B1 or free in the cytoplasm (Fig. 5 B2).
More than 85% of the analyzed cell sections exhibited internalized
TiO2-NPs aggregates. Comparison with the untreated controls con-
firmed the presence of aggregated NPs and nuclear condensation in
3.6. TiO2-NPs induced cytotoxicity in WISH cells
Cells exposed to TiO2-NPs for 24 h exhibited a concentration
dependent decline in the cell survival as compared to untreated
control in MTT assay. The data exhibited about 9.6%, 17.5%,
20.0%, 24.3% and 24.5% reduction in cell viability at varying particle
mass concentrations of 0.625, 1.25, 2.5, 5.0 and 10 lg/ml, respec-
tively vis-à-vis untreated control (Fig. 6). Furthermore, the results
related to the influence of TiO2-NPs on ascorbic acid induced MTT
reduction under acellular conditions, showed no interference in
the development of purple colored MTT-formazan product forma-
tion up to 40 lg/ml. However, addition of TiO2-NPs at higher con-
centrations of80 lg/mland
(p < 0.010) increase in the color intensity, as compared to the con-
trol (Supplementary Fig. S1).
The results of neutral red (3-amino-7-dimethyl-amino-2-meth-
ylphenazine hydrochloride) uptake (NRU) are also shown in Fig. 6.
The data exhibited a concentration dependent decline in the sur-
vival of cells exposed to TiO2-NPs for 24 h. The cellular uptake of
neutral red is proportional to the number of viable cells. A signifi-
cant TiO2-NPs cytotoxicity has been noticed at concentrations
above 1.25 lg/ml. The percent decline in the cell viability at con-
centrations of 2.5, 5 and 10 lg/ml was determined to be 13.1%,
19.7% and 42.5%, respectively as compared to the untreated con-
trol. Under acellular conditions, the TiO2-NPs did not show any
interference with NR dye in concentration range of 0.625 to
10 lg/ml, However, some interference occurred at a higher con-
centration of 20 lg/ml, marked with a significant increase
(p < 0.05) in the absorbance at 540 nm, as compared to the control
(Supplementary Fig. S2).
Fig. 4. Size distribution and secondary size analysis of TiO2-NPs in suspension.
Dynamic light scattering (DLS) analysis of TiO2-NPs (10 lg/ml) after suspending in
RPMI culture medium and Milli-Q water to determine the size distributions and
hydrodynamic diameters of NPs.
Fig. 5. TEM analysis of internalized TiO2-NPs in WISH cells. Cells after 24 h exposure to RPMI medium alone (control) and TiO2-NPs (10 lg/ml) in RPMI medium were
analyzed by TEM. The representative images in the panels are depicted as (A) control, ?6000; (B) TiO2-NPs treated, ?6000; (B1 and B2) TiO2-NPs treated cells, same as in
panel B at higher magnifications of ?25,000. Dark arrows indicate the accumulated TiO2-NPs in vesicles and cytoplasm. Peripheral chromatin condensation is apparent in
panel B as compared to control (panel A).
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
3.7. TiO2-NPs mediated change in catalase and glutathione (GSH)
Catalase and GSH levels of TiO2-NPs treated cells were mea-
sured to assess the extent of oxidative stress in WISH cells. Fig. 7
shows a significant decrease in the catalase activity in WISH cells
treated at higher TiO2-NPs concentrations vis-à-vis untreated con-
trol cells. Considering the enzyme activity in untreated control as
100%, the reductions in catalase activity were determined to be
31.4% and 46.3% (p < 0.05) at 5 and 10 lg/ml, respectively. Further,
the cells exposed to TiO2-NPs for 24 h have also exhibited deple-
tion of GSH level at higher concentrations (Fig. 7). Compared to
the untreated control, the percent decline in the glutathione levels
at 5 and 10 lg/ml were determined to be 23.3 and 34.6% (p < 0.05),
3.8. TiO2-NPs-induced intracellular ROS generation
The TiO2-NPs induced intracellular ROS generation in WISH
cells was analyzed by flow cytometry using a fluorescence probe
DCFH-DA. Fig. 8A shows a significant shift in the DCF fluorescence
peak, exhibiting increased ROS production at 5.0 and 10 lg/ml con-
centrations of TiO2-NPs in treated cells. Almost 1.2- and 1.87-fold
enhancements in the levels of intracellular ROS were determined
after 24 h of TiO2-NPs exposure at 5 and 10 lg/ml concentrations,
respectively as compared with the untreated control cells (Fig. 8B).
However, no significant changes in the fluorescence intensity was
Fig. 6. Cytotoxicity assessment of TiO2-NPs in WISH cells exposed for 24 h.
Histogram shows the percent cell viability using MTT and NRU assays, respectively.
Data are the mean ± SD of three independent experiments.⁄p < 0.05 vs control.
Fig. 7. Effect of TiO2-NPs on antioxidative enzymes, catalase and glutathione (GSH)
levels in WISH cells exposed for 24 h. Each histogram represents the mean ± SD of
three independent experiments.⁄p < 0.05 vs control.
Fig. 8. Flow cytometric analysis of intracellular ROS generation in WISH cells
treated with TiO2-NPs exposed for 24 h. Panel A shows the representative spectra of
fluorescent DCF as a function of TiO2-NPs concentration. Panel B exhibits the
comparative analysis of the fluorescence enhancement of DCF with increasing TiO2-
NPs concentrations. CtI and CtII represent the negative and positive (H2O2, 100 lM)
controls, respectively. Each histogram represent the values of mean ± SD of three
independent experiments.⁄p < 0.05,⁄⁄p < 0.01 vs control.
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
observed after interaction of TiO2-NPs up to 20 lg/ml with 5 and
10 lM DCFH under acellular conditions (Supplementary Fig. S3).
Also, the lower concentrations (0.625 to 2.5 lg/ml) of TiO2-NPs
did not show any significant change in the intracellular ROS levels.
Therefore, the spectra at low concentrations overlaps with the con-
trol and are not shown in Fig. 8A.
3.9. Effect of TiO2-NPs on cell cycle progression
The results shown in Fig. 9 exhibit the effect of TiO2-NPs on cell
cycle progression, as measured in terms of DNA content of the total
cell population. After 24 h of TiO2-NPs exposure, the treated cells at
1.25 lg/ml and higher concentrations showed a significant G2/M
cell cycle arrest. The mean ± SEM values of three independent
experiments exhibited change in DNA contents to the extent of
31.7 ± 1.04, 33.1 ± 0.57, and 34.4 ± 0.76% (p < 0.01) at G2/M phase
at 2.5, 5.0, and 10 lg/ml concentrations, respectively, as compared
to 27.1 ± 0.31% in untreated control cells.
3.10. TiO2-NPs-induced DNA damage
Cells exposed to varying concentrations of TiO2-NPs for 6 h have
exhibited a significant induction (p < 0.05) in DNA damage at a
concentration of 20 lg/ml. The representative images of DNA dam-
age obtained with the neutral comet assay of TiO2-NPs treated
WISH cells are shown in Fig. 10A. Treated cells at this concentra-
tion exhibited 14.6-fold increase in Olive tail moment (OTM) value
as compared to the untreated control (Table 1). The data generated
in term of OTM reveals the product of tail length and the fraction of
total DNA in the tail. It incorporates a measure of both the smallest
detectable size of migrating DNA (reflected in the comet tail
length) and the number of relaxed/broken pieces (represented by
the intensity of DNA in the tail). The frequency distribution analy-
sis of treated cells at the highest concentration of 20 lg/ml exhib-
ited the extent of DNA damage comparable to positive control
(EMS, 1 mM) (Fig. 10B).
The synthesis and applications of metal oxide NPs are consis-
tently expanding due to their distinctive physico-chemical charac-
teristics, and increased industrial and medical applications. This
has evoked serious concerns about their potential impact on the
environment and human health. Owing to their small aerodynamic
diameter, the ultrafine particles (<100 nm) from natural and
anthropogenic sources including viruses, biogenic magnetite, ferri-
tin, metal oxides, fullerenes, carbon, polymers and other fumes
Fig. 9. Effect of TiO2-NPs on cell cycle progression in WISH cells treated for 24 h.
Each histogram represents mean ± SEM values of different phases of cell cycle
obtained from three independent experiments done in triplicate tubes.⁄p < 0.01 as
compared to control. G1, S, G2/M represents the percentage of cells present in
normal phases of cell cycle, SubG1 represents percentage of cells undergone
Fig. 10. TiO2-NPs induced strand breaks in cellular DNA of WISH cells. Panel (A) shows the representative epi-fluorescence images of DNA damage in neutral comet assay, as
(1) untreated control; (2) EMS (1 mM) as positive control; (3) TiO2-NPs (20 lg/ml). Panel (B) shows the percent distribution of DNA damage in WISH cells treated with varying
concentrations of TiO2-NPs for 6 h. Olive tail moment (OTM) values were determined following the algorithm (Olive Tail Moment = (Tail Mean – Head Mean) ? Tail % DNA/
100) using Comet Assay IV software.
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
may contaminate ambient air, penetrate deep into the lungs, and
reach different body organs through the blood circulatory system
(Oberdörster et al., 2005). The tendency of TiO2-NPs to readily dif-
fuse through the protective cellular barriers may also involve risks
to human health, and warrants systematic and in-depth investiga-
tion of their possible toxicological effects. Therefore, in this study,
we have determined the cytotoxicity and genotoxicity of crystal-
line TiO2-NPs using human amniotic (WISH) cells for understand-
ing the trans-placental toxicity under in vitro conditions. WISH
cells have been used as a model in earlier studies to assess the oxi-
dative stress and its impact on gestational prostanoid metabolism
and apoptosis in fetal membrane (Keelan et al., 2001; Biondi et al.,
2002; Kumar et al., 2004). The sonomechanically synthesized and
well characterized polyhedral rutile TiO2-NPs, suspended in RPMI
medium were used for cellular treatment and toxicity assessment
in cultured WISH cells. Since, the primary and secondary sizes of
the NPs are regarded as important parameters for in vitro cytotox-
icity in a cell culture medium, therefore, the behavior of TiO2-NPs
in cell culture medium was evaluated through dynamic light scat-
tering (DLS), to understand the extent of aggregation and second-
ary size of these NPs before cellular exposure. DLS is widely used
to determine the size of brownian NPs in colloidal suspensions in
the nano and submicron ranges (Berne and Pecora, 2000). The
average hydrodynamic particles diameters (secondary TiO2-NPs
particle sizes) in RPMI medium were determined to be much smal-
ler (152 nm) as compared to Milli-Q water (380 nm), which indi-
cates relativelylesser particle
compared to water. The presence of variable sized (13 to
152 nm) TiO2-NPs aggregates in cell culture medium corroborates
well with the earlier reports (Singh et al., 2007; Xia et al., 2006;
Limbach et al., 2005). Thus, the DLS data revealed the formation
of TiO2-NPs aggregates in the RPMI cell culture medium, which
were also found to be internalized in the TEM images of the treated
cells. These aggregated NPs in culture medium, have been reported
to enter into cells mainly through endocytosis, and their localiza-
tion in the vacuoles and cell cytoplasm of the exposed cells corre-
sponds well with the observations of Hussain et al. (2009, 2010).
Assessment of TiO2-NPs cytotoxicity through the MTT and NRU
assays exhibited the dose dependent toxic effects on cell viability
in a concentration range of 0.625 to 10 lg/ml. In context of dose,
it is important to realize the significance of nominal and effective
dosimetry of NPs in cytotoxicity assays. Teeguarden et al. (2007)
suggested that owing to their small size and large surface area,
the insoluble NPs are not affected by the gravitational force and
generally form stable suspensions or sols. Such NPs might cause
problems in the in vitro assay systems, as the cells adhering to
the bottom of a culture plate may not be exposed to the majority
of nanoparticles in suspension. Based on the equations (1) and
(2), specified in the methodology section, the TiO2-NPs (10 lg/
ml) treatment to WISH cells, adhered as monolayer at the bottom
of the 96 well plates (surface area of 0.36 cm2) was theoretically
estimated to be equivalent to the exposure dose of 1.27 ? 108par-
ticles/ml, delivered dose of 3.52 ? 108particles/cm2and cellular
dose of 1.56 ? 104particles/cell in RPMI medium. Significant cyto-
toxicity, intracellular ROS generation, and to some extent G2/M cell
cycle arrest were induced at the above specified treatment dose,
and attributed to TiO2-NPs mediated oxidative stress in the WISH
cells. Thus, the results suggest that the TiO2-NPs in the form of a
heterogeneous suspension of primary (30.6 nm) and secondary
(152 nm) sizes are accessible to the cells in RPMI medium and
exhibited the toxic effects. It is likely due to the convection forces
in sols, as also suggested by Lison et al. (2008), that these particles
may reach to the target cells and exert their potential toxicity.
The morphological analysis of TiO2-NPs treated cells explicitly
demonstrated the toxicity, manifested as detachment of the adher-
ent cells at increasing concentrations of TiO2-NPs (results not
shown). These results are in agreement with the cellular detach-
ment and morphological changes observed in fish cell line (RTG-
2 cells) by Vevers and Jha (2008). The cytotoxic effect at the higher
concentration of 10 lg/ml was more pronounced with the NRU vis-
à-vis MTT assay, which could be attributed to a greater lysosomal
damage as compared to the mitochondria. The results also support
the observations of Hussain et al. (2010), who have showed that
the TiO2-NPs do not cause any significant loss of mitochondrial po-
tential, whereas, the lysosomal membrane destabilization occurs,
which further leads to the release of lysosomal proteases like
cathepsin B, and may directly cause proteolysis or activate caspas-
es, as a pathway for TiO2-NP induced death in bronchial epithelial
Biochemical analysis of the cell extracts, flow cytometry and co-
met data revealed the TiO2-NPs induced changes in the levels of
oxidative markers (GSH and catalase), intracellular ROS generation
and consequent DNA damage. The data revealed significant deple-
tion in GSH level and catalase activity in TiO2-NPs treated cells at 5
and 10 lg/ml, as compared to the untreated control. The results
support the study of Nemmar et al. (2011) on the rutile Fe-doped
TiO2nanorods induced dose dependent decrease in the SOD and
GSH levels in the hepatic and heart tissues of rats. The results also
corroborate the earlier reports on TiO2-NPs induced ROS produc-
tion in bronchial epithelial cell line (BEAS-2B) at 10 lg/ml concen-
tration (Gurr et al., 2005). However, Hussain et al. (2009) reported
ROS production in bronchial epithelial cell line (16HBE14o-) at 50
and 100 lg/ml. Also, significant intracellular ROS production has
been demonstrated in the human epidermal (A431) and brain
microglia (mouse BV2) cells at TiO2-NPs doses of 80 lg/ml and
25 lg/ml, respectively (Long et al., 2007; Shukla et al., 2011). Thus,
the dose comparison of our study with earlier reports suggests the
induction of oxidative stress in WISH cells at relatively lesser con-
centrations of TiO2-NPs.
Surface reactivity has been suggested to plays an important role
in ROS production by NPs (Stone et al., 1998; Oberdörster et al.,
2005). Significant ROS generation at 5.0 lg/ml (p < 0.05) and
10 lg/ml (p < 0.01) concentrations of TiO2-NPs signifies that the
primary mechanism of NPs induced toxicity is due to oxidative
stress, resulting in damage to cellular membranes and biological
macromolecules, as reported earlier (Dalton et al., 2002; Donald-
son and Stone, 2003; Nel et al., 2006; Shukla et al., 2011). Our re-
sults have also demonstrated that the TiO2-NPs induces DNA
damage at a critical concentration of 20 lg/ml. The data suggest
that the TiO2-NPs at lower concentrations up to 10 lg/ml modu-
lates the antioxidant enzymes levels, whereas, at higher concentra-
tions, the cellular DNA repair machinery may be adversely affected.
Consequently, the unrepaired DNA damage was detected upon sin-
gle cell gel electrophoresis under neutral assay conditions, which
TiO2-NPs induced DNA damage in WISH cells, analyzed using different parameters of
GroupsOlive tail moment
EMS (1 mM)
0.66 ± 0.013
11.08 ± 0.10⁄
40.46 ± 0.19
107.50 ± 0.29⁄
4.21 ± 0.29
26.70 ± 0.19⁄
0.62 ± 0.02
0.86 ± 0.05
0.81 ± 0.19
0.90 ± 0.08
1.15 ± 0.71
9.66 ± 0.12⁄
42.13 ± 0.10
40.85 ± 0.22
40.35 ± 0.20
42.57 ± 0.10
41.86 ± 0.19
106.61 ± 0.29⁄
3.43 ± 0.22
5.33 ± 0.29⁄
4.71 ± 0.11
4.21 ± 0.63
5.62 ± 0.43⁄
23.94 ± 0.66⁄
Data represent the mean ± SD of three independent experiments done in duplicate.
⁄p < 0.05; EMS: Ethyl methanesulphonate.
Q. Saquib et al./Toxicology in Vitro 26 (2012) 351–361
substantiates the earlier studies on TiO2-NPs induced DNA damage
(Vamanu et al., 2008; Gopalan et al., 2009; Barillet et al., 2010).
Furthermore, the flow-cytometric analysis of 24 h treated WISH
cells has suggested the activation of DNA repair process with dis-
cernible cell cycle arrest in G2/M phase in damaged cells at higher
TiO2-NPs concentrations. It is known that the cellular DNA repair
mechanisms are highly conserved (Ferreira et al., 2002), and exten-
sive DNA damage may lead to cell-cycle arrest and cell death (Kon-
opa, 1988; Tsao et al., 1992). Most likely, the DNA damage induced
with TiO2-NPs at lower concentrations up to 10 lg/ml could be
adequately repaired during the G2/M phase, as no DNA damage
(comet) was also observed at this concentration. However, the
greater DNA damage occurred at a threshold concentration of
20 lg/ml TiO2-NPs, as evident with the appearance of a prominent
comet tail due to irreparable double strand breaks. We have ob-
served that the WISH cells were sensitive to alkaline conditions,
which resulted in severe DNA damage even in untreated control
cells. Therefore, the neutral comet assay was performed to deter-
mine the extent of induced DNA damage in this cell type at a
threshold concentration of 20 lg/ml TiO2-NPs. However, in other
cell lines such as goldfish skin (GFSk-S1) and rat kidney proximal
(NRK-52E) cells, the DNA damage responses have been observed
at much higher concentrations up to 100 lg/ml (Reeves et al.,
2008; Barillet et al., 2010). It has been reported that the TiO2-NPs
could directly bind to DNA or repair enzymes leading to the gener-
ation of strand breaks (Hartwig, 1998; Reeves et al., 2008). Most
likely, the TiO2-NPs induced _OH radicals are responsible for the
DNA damage in the exposed cells (Reeves et al., 2008). There are
contradicting results in literature on exposure with different NPs
at different stages of embryo development and also with the differ-
ences in experimental models (Challier et al., 1973; Bosman et al.,
2005). Thus, the extent of induced genetic damage, and risk assess-
ment of the nanomaterials and nanoproducts should be assessed
prior to their larger applications in spite of their apparent extraor-
It is concluded that this study for the first time explicitly dem-
onstrated the cyto- and genotoxicity of TiO2-NPs in human amnion
epithelial (WISH) cell line. Significant reduction in marker antiox-
idant levels and intracellular ROS generation suggested their role
in inducing oxidative stress leading to DNA damage in treated cells.
It is contemplated that the differential susceptibility of cell types
could be due to differences in their metabolic rate, antioxidant en-
zyme machinery, and DNA repair capabilities, which may exhibit
variability in TiO2-NPs induced toxic effects on human health. Fur-
thermore, the TiO2-NPs predominantly present in aggregated form
in culture medium, enter into cells mainly through endocytosis.
Therefore, the extrapolation of in vitro dose response data to
in vivo situation, i.e., in placenta or embryo is still a challenge. Fur-
ther studies are warranted to investigate the particokinetics and
rate of transport of TiO2-NPs in amniotic cells to ascertain more
realistic NPs induced toxicity and risk assessment.
6. Conflict of interest
There is no conflict of interest.
Financial support through the National Plan for Sciences and
Technology (NPST Project No. 10-NAN1115-02) and Al-Jeraisy
chair for DNA research, King Saud University, Riyadh, for this study,
is greatly acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tiv.2011.12.011.
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