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
100
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 =
MF =
3. Results
The sensitivity of the assays was indicated by the use
of styrene oxide, which is a well-known toxicity inducer
102
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
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
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
**.
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
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 **.
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
104
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-
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.
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