Mutation Research 628 (2007) 99106

Cyto- and genotoxicity of ultrafine TiO2 particles

in cultured human lymphoblastoid cells
Jing J. Wang a, , Barbara J.S. Sanderson b,1 , He Wang a,2
b

a Department of Public Health, Level 9, 10 Pulteney Street, University of Adelaide, SA 5005, Australia
Department of Medical Biotechnology, School of Medicine, Flinders University, G.P.O. Box 2100, Adelaide, SA 5001, Australia

Received 14 July 2006; received in revised form 30 November 2006; accepted 9 December 2006
Available online 15 December 2006

Abstract
Titanium dioxide is frequently used in the production of paints, paper, plastics, welding rod-coating material, and cosmetics,
because of its low toxicity. However, recent studies have shown that nano-sized or ultrafine TiO2 (UF-TiO2 ) (<100 nm in diameter)
can generate pulmonary fibrosis and lung tumor in rats. Cytotoxicity induced by UF-TiO2 in rat lung alveolar macrophages was
also observed. This generates great concern about the possible adverse effects of UF-TiO2 for humans. The cytotoxicity and
genotoxicity of UF-TiO2 were investigated using the methyl tetrazolium cytotoxicity (MTT) assay, the population growth assay,
the apoptosis assay by flow cytometry, the cytokinesis block micronucleus (CBMN) assay, the comet assay, and the hypoxanthineguanine phosphoribosyltransferase (HPRT) gene mutation assay. WIL2-NS cells were incubated for 6, 24 and 48 h with 0, 26, 65
and 130 g/ml UF-TiO2 . Significant decreases in viability were seen in the MTT assay at higher doses; for example, 61, 7 and
2% relative viability at 130 g/ml for 6, 24 and 48-h exposure (P < 0.01). A dose-dependent relationship was observed, while a
time-dependent relationship was seen only at the highest dose (130 g/ml) after exposure for 24 and 48 h. Treatment with 130 g/ml
UF-TiO2 induced approximately 2.5-fold increases in the frequency of micronucleated binucleated cells (P < 0.01). In addition, a
significant reduction in the cytokinesis block proliferation index was observed by the CBMN assay (P < 0.05). In the comet assay,
treatment with 65 g/ml UF-TiO2 induced approximately 5-fold increases in olive tail moment (P < 0.05). In the HPRT mutation
assay, treatment with 130 g/ml UF-TiO2 induced approximately 2.5-fold increases in the mutation frequency (P < 0.05). The results
of this study indicate that UF-TiO2 can cause genotoxicity and cytotoxicity in cultured human cells.
2006 Elsevier B.V. All rights reserved.
Keywords: Ultrafine particles; Human cells; Cell damage; Chromosomal damage; DNA damage; HPRT mutation

1. Introduction
Titanium dioxide (TiO2 ) is a poorly soluble particulate (PSP) that has been widely used as a white pigment

Corresponding author. Tel.: +618 8303 3562; fax: +618 8223 4075.
E-mail addresses: wang0311@flinders.edu.au (J.J. Wang),
Barbara.sanderson@flinders.edu.au (B.J.S. Sanderson),
he.wang@adelaide.edu.au (H. Wang).
1 Tel.: +618 8204 5788; fax: +618 8204 4101.
2 Tel.: +618 8303 3562; fax: +618 8223 4075.
1383-5718/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2006.12.003

in the production of paints, paper, plastics, welding

rod-coating material and food colorant. Nano-sized or
ultrafine TiO2 (UF-TiO2 ) (<100 nm) is used increasingly in other industrial products, such as cosmetics and
pharmaceuticals [14]. Therefore, potential widespread
exposure may occur during both manufacturing and
use.
Coarse and fine (>100 nm in diameter) particles of
TiO2 were classified as biologically inert in both human
and animals [57]. However, Garabrant et al. [8] reported
that 50% of workers exposed to TiO2 suffered from

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J.J. Wang et al. / Mutation Research 628 (2007) 99106

respiratory symptoms, accompanied by impairment of

pulmonary function. Furthermore, Ahn et al. [9] showed
that goblet cell hyperplasia and Muc 5ac expression were
induced in rats after a single intratracheal instillation of
TiO2 .
On the other hand, the cytotoxicity induced by TiO2
was relevant to the size of particles [10]. There is evidence that UF-TiO2 can cause inflammation, fibrosis,
pulmonary damage and even DNA damage [1113]. UFTiO2 might be able to enter the human stratum corneum
and interact with the immune system [1416], since
UFP can be translocated to the subepithelium space to
a greater extent than the fine particles [17]. An increase
in the level of IL-8 was seen in human endothelial cells
after exposure to UF-TiO2 [18]. A significant decrease
in the level of glutathione was observed in rat alveolar macrophage following exposure to UF-TiO2 [11].
The authors suggested that induction of reactive oxygen
species (ROS) might be responsible for this depletion.
Oxidative DNA damage and increases in the level of
cellular nitric oxide were observed in human bronchial
epithelial cells after exposure to UF-TiO2 [19]. There are
potential multiple effects of UF-TiO2 , and the possible
adverse effects of TiO2 exposure need further clarification.
In the present study, we have investigated the toxicity of UF-TiO2 particles. The cytotoxic and genotoxic
effects of UF-TiO2 were assayed in cultured WIL2-NS
human lymphoblastoid cells.

(Trace Biosciences, Melbourne, Australia), supplemented with

5% fetal bovine serum (FBS; Trace Biosciences) and 50 IU/ml
penicillin (Thermo Trace, Melbourne, Australia). Cells were
seeded in tissue culture flasks at 3 105 cells/ml and incubated
in a fully humidified atmosphere at 37 C with 5% CO2 . The
cells were subcultured every 23 days when they reached a
density of 1 106 cells/ml or greater.
2.3. Particle preparation
The particles used in this study were TiO2 (99% purity),
purchased from SigmaAldrich. The particles were suspended
in culture medium (see above), and sonicated to ensure a
uniform suspension. The mixture was spun at 78 g for 5 min.
The supernatant was sterilized by filtration (0.20 m pore size)
and stored at 20 C. The final dose of the particle extraction
stock was 130 g/ml measured with a spectrophotometer
(Shimadzu, Kyoto, Japan) as absorbance at 400 nm. The
optimal wavelength of 400 nm was chosen on the basis of
peak absorption when a scan from 200 to 800 nm was done
with different doses of particle suspensions. The absorption of
different doses of particles was measured by spectrophotometer at 400 nm, thus generating a standard curve for absorption
versus dose of particles. The absorption of the particle extraction prepared as described above was measured at 400 nm,
which was converted to micrograms per milliliters of particles
by using the standard curve. The particle size distribution
in the final extract was measured by the HPPS: by volume
6.57 nm (100%); by intensity: 8.2 nm (80.4%) and 196.52 nm
(19.4%).
2.4. Cell treatment

2. Materials and methods

2.1. Chemicals and instruments
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl tetrazolium, MTT, 97.5%), cytochalasin B
(Cyt-B), 2-amino-6-mercaptopurine (6-thioguanine, 6-TG)
and sodium dodecyl sulfate (SDS, approximately 99%) were
purchased from SigmaAldrich (St. Louis, MO). DiffQuick
fixative, Stain 1, and Stain 2 were purchased from Lab Aids
(Sydney, Australia). MTT was dissolved in phosphate-buffered
saline (PBS) at 5 mg/ml, and Cyt-B was dissolved in dimethyl
sulfoxide (DMSO) at 1.8 mg/ml. MTT and Cyt-B solutions
were sterilized by passage through a 0.2 m pore size filter
and stored at 20 C. The comet assay kit was purchased
from Trevigen (Bio Scientific, Australia). We used cytospin
(Shandon Scientific, England) and a high-performance
particle sizer (HPPS) (Malvern Instruments, Worcestershire,
UK).
2.2. Cell line and cell culture
WIL2-NS (ATCC, CRL 8155), a human B-cell lymphoblastoid cell line, was maintained in RPMI 1640 culture medium

Cells (1 106 ml1 ) were seeded in flasks and treated for 6,

24 and 48 h with 0, 26, 65 and 130 g/ml UP-TiO2 . Cells were
then centrifuged at 78 g for 5 min. The supernatant was aspirated, and the cell pellet was resuspended at 5 105 cells/ml in
fresh medium. Samples of the cell suspensions were used for
the bioassays described below.
2.5. Bioassays
2.5.1. Methyl tetrazolium cytotoxicity assay
The cytotoxicity of particles was determined using the
MTT assay as described [20,21]. Briefly, 1 104 cells were
seeded in a volume of 100 l into wells of a 96-well (roundbottom) plate. MTT was added to each well at 0.5 mg/ml,
and then plates were incubated at 37 C for 18 h, after which
80 l of 20% SDS in 0.02 M HCl was added to each well and
mixed thoroughly. The plates were kept in the dark at room
temperature for 1.5 h. Optical density (OD) was read on an
ELISA reader at 570 nm, with 630 nm as a reference wavelength. The OD values were converted to cells/well using a
standard curve run with each experiment [21]. The results were
expressed as percentage viability compared to the untreated
control.

J.J. Wang et al. / Mutation Research 628 (2007) 99106

2.5.2. Population growth assay

Previously treated cells (Section 2.4) were maintained at a
concentration of 5 105 cells/ml (see above) in tissue culture
flasks at 37 C. Cell number and viability were determined after
48 and 96 h of growth using trypan blue dye-exclusion staining.
Additional fresh medium was added to the cultures at 48 h.
Relative population growth was estimated by expressing the
viable cell number of the treated cell cultures as a percentage
of the untreated control.
2.5.3. Apoptosis assay by ow cytometry
Double staining for annexin V-fluorescein isothiocyanate
(FITC) and propidium iodide (PI) was carried out with an
annexin V-FITC apoptosis detection kit (BD Biosciences,
USA). After treatment (Section 2.4) cells were washed twice
in cold PBS/sodium azide and centrifuged at 78 g for 5 min.
The pellets were resuspended in binding buffer at a density of
1 106 cells/ml. A sample (100 l) of the solution was transferred to a culture tube and double-stained with 5 l of annexin
V-FITC and 5 l of PI. After incubation in the dark at room
temperature for 15 min, 400 l of binding buffer was added
to the mixture. The intensity of annexin V-FITC and PI was
recorded by FACScan flow cytometry (Becton & Dickson, San
Jose, CA) and analyzed with CellQuest software. A total of
20,000 cells in each sample were analyzed and the percentage
of positive cells was determined for each histogram.
2.5.4. Cytokinesis-block micronucleus (CBMN) assay
The CBMN assay was carried out using the protocol of
Fenech [22], with the minor modifications described below.
Briefly, after treatment (Section 2.4), Cyt-B was added at
4.5 g/ml and the cultures were incubated at 37 C for another
26 h. Cells were collected onto slides by processing at 40 g
for 5 min with a cytospin. Slides were air-dried, fixed for 10 min
in DiffQuick Fixative, and then stained with ten 1-s dips in
DiffQuick Stain 1 and then Stain 2. Slides were scored at a
magnification of 250. Criteria for scoring micronucleated
binucleated cells (MNBNCs), nucleoplasmic bridge in binucleated cells (NPB), and apoptotic cells were as described by
Fenech [23]. The MNBNCs frequency and the NPB frequency
were determined in 1000 BNCs according to established criteria [23]. The cytokinesis block proliferation index (CBPI),
the cytotoxicity induced by treatment, and the percentage of
apoptosis were evaluated in 500 cells and calculated according
to published formulae [2325].
2.5.5. Comet assay
Single-cell gel electrophoresis was performed according to
the guidelines of the comet assay kit. After treatment (Section
2.4), cells were washed twice with prechilled PBS (Mg2+ and
Ca2+ -free), centrifuged at 78 g for 5 min and resuspended
in PBS. Cell viability was over 85% for the tested dose in
this study as assessed using trypan blue dye-exclusion staining. To the slides was added a 75 l sample of a mixture of
50 l of cells at a density of 1 105 cells/ml and 500 l of
low melting-point agarose. The slides were immersed in cold

101

lysis buffer for 30 min, then in an alkaline solution (pH > 13)
for 30 min, and then placed in a horizontal gel electrophoresis tank filled with fresh electrophoresis solution (300 mM
NaOH, 1 mM EDTA). Electrophoresis was conducted at a low
temperature for 30 min at 23 V and 300 mA. All the steps
were conducted under dimmed light. After electrophoresis, the
slides were rinsed with deionized water and immersed in 70%
ethanol for 5 min, then drained and 50 l of SYBR green 1
was added. Slides were scored with a fluorescence microscope
(BX50, Olympus) at a magnification of 200 using a blue
filter (450490 nm), and photographed with a high-resolution
cooled CCD camera (CoolSNAP, Olympus). At least 50 randomly selected images were analyzed from each sample and
the DNA damage was analyzed with the CASP software package. The olive tail moment and the percentage of DNA in the
tail (%Tail DNA) were used as DNA damage indicators in our
study, since they are considered as the most informative and
reliable measurements [26,27].
2.5.6. HPRT gene mutation assay
Mutation frequency was determined by a clonal selection
assay for the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene, as described by Sanderson et al. [28].
After treatment (Section 2.4), cells were grown in flasks for
7 days to allow mutations to be expressed. Cells with 6-TG
(2.5 g/ml) were plated at 104 cells/well and cells without 6TG were plated at 2 cells/well in 96-well (round-bottom) plates.
The plates were incubated at 37 C for 1427 days, and then
clonal growth was scored. The HPRT mutant frequency was
calculated as:
PE =

ln(total wells/empty wells)

cells per well plated

MF =

PE in the presence of 6-TG

PE in the absence of 6-TG

where PE is the plating efficiency and MF is the mutant

frequency.
2.6. Statistical analysis
The data are presented as mean S.E.M. The experiments
were replicated at least three independent times. Statistical
analysis of the data was carried out using ANOVA, followed
by Tukeys HSD post hoc test (equal variances) or Dunnetts
T3 post hoc test (unequal variances); otherwise, the nonparametric KruskalWallis test was used. In the study of DNA
damage by the comet assay, Students t-test for independent
samples was also used. These tests were performed using SPSS
software, version 12.0.1. Differences were considered statistically significant when the P-value was less than 0.05.

3. Results
The sensitivity of the assays was indicated by the use
of styrene oxide, which is a well-known toxicity inducer

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J.J. Wang et al. / Mutation Research 628 (2007) 99106

Table 1
Cell viability (%), population growth (%), and micronucleated binucleate cells (MNBNCs) per 1000 BNCs following treatment of WIL2-NS cells
with and without 0.2 mM styrene oxide for 6, 24 and 48 h
Exposure time (h)

Treatment

Cell viability (%)

(MTT assay)

Population growth (%)

(population growth assay)
48 h

96 h

MNBNCs/1000 BNCs
(CBMN assay)

Untreated control
Styrene oxide (0.2 mM)

100
51 2

100
64 5

100
80 5

7.7 0.7
51 3

24

Untreated control
Styrene oxide (0.2 mM)

100
66 5

100
65 9

100
69 6

3.7 1.2
64 5

48

Untreated control
Styrene oxide (0.2 mM)

100
69 10

100
63 4

100
71 10

5 0.6
72 7

Data represent the means S.E.M. from three separate experiments.

[29]. Treatment with styrene oxide (0.2 mM) decreased

viability and population growth and induced MNBNCs
in WIL2-NS cells as compared with the untreated control
(Table 1).
The values of cytotoxicity induced by UF-TiO2 are
given in Figs. 1 and 2. Treatment with UF-TiO2 produced
a dose-dependent decrease in cell viability. A significant reduction was found at 130 g/ml UF-TiO2 for 6,
24 and 48 h, with a decrease to 62, 7 and 2%, respectively (Fig. 1). Treatment with UF-TiO2 also induced
cytotoxicity in a time-dependent manner, as the percentage of viability decreased significantly following longer
exposure (24 h), at a dose of 130 g/ml (Fig. 1). The
inhibition of population growth by UF-TiO2 was both
dose-dependent and time-dependent (Fig. 2), which is
consistent with the pattern in the MTT assay (Fig. 1).
When cells were treated with lower doses for a short
period of time, i.e. 26 and 65 g/ml for 6 h, however,
the size of the population increased significantly during

Fig. 1. Cell viability monitored by the MTT assay following 6, 24, 48h exposure of WIL2-NS to UF-TiO2 . Data are shown as % viability
compared to the untreated control and are presented as mean S.E.M.
of three separate experiments. Treatments significantly different from
untreated control at P < 0.05 are presented as * and at P < 0.01 as
**.

4 days (96 h) of culture in the flasks (Fig. 2 A). The

size was unchanged at the same doses for 24 and 48 h
exposure (Fig. 2 B and C).
Table 2 summarizes the results of apoptosis induced
by UF-TiO2 detected by the CBMN assay and the flow
cytometry assay. There was a general trend that apoptosis increase was dose-dependent, however, statistically
significant differences (P < 0.01) were observed only
in the CBMN assay for 6 h exposure at 26, 65 and
130 g/ml and for 24 h exposure at 65 g/ml. No significant increase was observed in the apoptosis flow
cytometry assay.
In the micronucleus assay, 6 h exposure to UF-TiO2
resulted in significant (P < 0.01) increases in MNBNCs

Table 2
Apoptosis induction detected by the CBMN assay and the apoptosis
assay by flow cytometry following 6, 24 and 48-h exposure of WIL2NS to UF-TiO2 , respectively
Exposure
time (h)

Dose of particles
(g/ml)

Apoptosis (%)
By CBMN
assay

By flow
cytometry

0
26
65
130

1.7 0.5
4.1 0.3**
4.4 0.3**
3.9 0.2**

3.7
4.7
5.8
4.8

0.65
1.2
1.6
1.5

24

0
26
65
130

2.1 0.1
2.8 0.3
3.7 0.4*
N/A

5.2
5.9
4.8
2.4

0.8
0.5
0.6
0.7

48

0
26
65
130

1.8 0.2
6.2 1.7
5.0 0.7
N/A

4.5
4.7
5.6
2.1

0.2
0.3
1.2
0.3

Data are presented as mean S.E.M. of three separate experiments.

Treatments significantly different from untreated control at P < 0.05
are presented as * and at P < 0.01 as **. N/A: not available.

J.J. Wang et al. / Mutation Research 628 (2007) 99106

103

Fig. 3. Frequency of micronucleated binucleate (MNBNCs) and frequency of nucleoplasmic bridge (NPB) per 1000 BNCs as measured by
the CBMN assay following exposure of WIL2-NS to UF-TiO2 . The
data are the mean S.E.M. from three separate experiments. Treatments significantly different from untreated control at P < 0.05 are
presented as * or # and at P < 0.01 as ** (*, MNBNCs; #, NPB).

Fig. 2. Population growth of WIL2-NS cells following exposure to UFTiO2 . Population size was monitored by the trypan blue dye-exclusion
at 48 and 96 h following the completion of 6-h (A), 24-h (B) and
48-h (C) exposure. Data are shown as percentage population growth
compared to the untreated control and are mean S.E.M. of three
separate experiments. Treatments significantly different from untreated
control at P < 0.05 are presented as * and at P < 0.01 as **.

at a dose of 130 g/ml (17/1000) compared to untreated

control (7.7/1000) (Fig. 3). There was no time-dependent
increase in MNBNCs at 130 g/ml because of the high
toxicity, i.e. greater than 60% for 24 and 48 h exposure.
There were very few cells on the slides and the assay
was not carried out [24,30]. For other doses and exposure times, the cytotoxicity was between 6 and 11%.
Significant increases (P < 0.05) in NPB were found at
130 g/ml for 6 h and 65 g/ml for 24 h (Fig. 3). Fig. 4

shows that there was a significant decline in CBPI following 48 h exposure (P < 0.05), with a decrease to 1.94
for 26 g/ml and 65 g/ml from the value of 2.1 for the
untreated control.
In the comet assay, there was a 3-fold significant
(P < 0.05) increase in %Tail DNA when the cells were
treated with UF-TiO2 at a dose of 65 g/ml for 24 h exposure, i.e. 16 3% for treated cells versus 5 2% for
untreated cells. Furthermore, in the olive tail moment
there was a 5-fold significant (P < 0.05) elevation when
the cells were treated with UF-TiO2 at a dose of 65 g/ml
for 24 h exposure, i.e. 11.4 2.9 for treated cells versus
2 0.9 for untreated cells.
In the HPRT assay, a strong linear relationship
(R2 = 0.99) was observed between the mutation frequency and the doses. After exposure to UF-TiO2 for
24 h, the mutation frequencies (106 ) were 15 2

Fig. 4. The cytokinesis block proliferation index (CBPI) was measured

by the CBMN assay following exposure to UF-TiO2 for 6, 24 and 48 h,
respectively. The graph is shown as mean S.E.M. of three separate
experiments. Treatments significantly different from untreated control
at P < 0.05 are presented as *.

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J.J. Wang et al. / Mutation Research 628 (2007) 99106

for 130 g/ml, 10 3 for 65 g/ml and 6 1 for the

untreated control (P < 0.05).
4. Discussion
The results of this study demonstrate that UFTiO2 particles induce significant cytotoxicity in cultured
human cells. The results from the MTT assay (Fig. 1)
indicated that UF-TiO2 killed the cells in both a dosedependent and a time-dependent manner. In agreement
with our results, Renwick et al. [31] demonstrated a
significant increase in lactate dehydrogenase activity in
bronchoalveolar lavage fluid following exposure of UFTiO2 . Interestingly, it was shown that the cell population
recovered to different extents in response to different
treatment conditions after the particles were removed,
as detected by the population growth assay (Fig. 2). This
was in accordance with the results of the work done by
Baggs et al. [12]. It has been suggested that UFP may
exert pro-inflammatory effects through a ROS-mediated
mechanism [13,32,33]. The recovery might indicate cell
killing is not the main toxicity of UFP, since they may
be absorbed and translocated directly into circulation in
vivo [3436]. The inflammatory reaction, caused by free
radicals, may be transient and therefore insignificant in
the induction of cytotoxicity.
Apoptosis induced by mutagenic carcinogens is a
unique type of programmed cell death. It has been
reported that the reaction of particles with cell membranes results in the generation of ROS, and the
generated oxidative stress may cause a breakdown of
membrane lipids, an imbalance of intracellular calcium
homeostasis, alterations in metabolic pathways, and
results finally in apoptosis [37,38]. In our study, induction of apoptosis was observed following exposure to
UF-TiO2 as measured by the CBMN assay and the apoptosis flow cytometry assay (Table 2). However, a significant difference was found by the CBMN assay only after
exposure for 6 and for 24 h at 65 g/ml. This could be
because apoptotic cells may have disintegrated into cellular debris at later time-points. It has been well reported
that the p53 gene plays a crucial role in the induction of
apoptosis [39]. Therefore, even though WIL2-NS is an
ideal cell line for monitoring MN formation [40], appreciable numbers of apoptotic cells were not detected when
WIL2-NS cells were exposed to UF-TiO2 . This may be
because WIL2-NS cells carry a point mutation in exon
7 of the p53 gene [41]. However, there is a minor pathway of p53-independent apoptosis that would be active
in WIL2-NS [42]. In the current study, the mechanisms
underlying apoptosis are unclear. Further studies are
needed and other cell lines should be introduced.

CBPI, a cytotoxicity index, reflects the average number of cell cycles undergone by a treated cell [25].
According to the results of the CBMN assay, CBPI
declined with an increasing dose of particle, though a
significant difference was found only at the 48-h exposure time-point (Fig. 4). This indicates that cell division
is inhibited after longer exposure to UF-TiO2 , which is
consistent with the results of the study done by Gurr et
al. [19]. However, which stages of the cell cycle were
arrested in the present study is unclear.
The frequency of the induced micronuclei (MN)
indicates the extent of chromosomal changes induced
by particles or chemical regents [43]. In our study, we
found significant increases in the MN frequency as
detected by the CBMN assay (Fig. 3), which showed
a dose-dependence. Consistent with this, significant
induction of sister chromatid exchanges (SCE) and
MN were observed in Chinese hamster ovary K1 cells
after exposure to TiO2 for 24 h [44]. Rahman et al. [13]
reported significant induction of MN and production
of apoptosis in Syrian hamster embryo fibroblasts
after exposure to UF-TiO2 . The authors suggested
that MN arose mainly from clastogenic events. In the
current study, there was a slight but significant increase
in NPB observed by the CBMN assay. The ratio of
NPB/MN was around 0.120.33 (data not shown). This
indicates that MN might originate from a combination of
chromosome break and/or spindle poison mechanisms
[45].
The CBMN assay detects acentric chromosome fragments formed following mis-repair of DNA strand
breaks, and whole chromosomes lagging at cell division caused by defects in the chromosomal segregation
mechanisms, while the comet assay detects un-repaired
DNA strand breaks and alkali-labile sites in viable cells
that are not undergoing necrosis or apoptosis. Hence, the
use of both methods was suggested [46]. In our study,
the sensitivity of the comet assay and the CBMN assay
were similar. Our result is consistent with the study of
Dunford et al. [47], which demonstrated that sunscreen
TiO2 (2050 nm) and ZnO can catalyze oxidative damage to DNA in vitro and in cultured human fibroblasts
measured by the comet assay. However, one limitation of
the comet assay is that the cells undergoing necrosis and
early stages of apoptosis could contain fragmented DNA
and give rise to comets. This means that some damage
indicated by the comet assay may not be induced DNA
damage per se.
In conclusion, the present study shows that UF-TiO2
can induce significant cytotoxicity and genotoxicity in
cultured human cells. However, the precise mechanism
of MN, apoptosis formation and inhibition of cell divi-

J.J. Wang et al. / Mutation Research 628 (2007) 99106

sion by UF-TiO2 is unclear. Additional work needs to be

undertaken to understand the mechanisms of damage.
[14]

Acknowledgements
[15]

We are thankful to Ms. Kylie Lange for valuable suggestions about the statistical analysis. This work was
supported by the Flinders Medical Centre Foundation,
the Workers Compensation Dust Diseases Board, and
the Establishment Grant, Faculty of Health Sciences,
University of Adelaide.

[16]

[17]

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