Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 121-123
http://dx.doi.org/10.4236/jcdsa.2013.31018 Published Online March 2013 (http://www.scirp.org/journal/jcdsa)
121
TiO2 Nanoparticles Induced Genotoxicity in Cultured Cells
Using Atmospheric Scanning Electron Microscopy (ASEM)
Shigeru Sato1, Takashi Takaki2, Hidetoshi Nishiyama2, Tokuya Omi3*
1Division of Morphological and Biomolecular Research, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan; 2R &
D business Support Department Group 1 DATUM Solution Business Operations, JEOL Ltd., Tokyo, Japan; 3Department of Der-
matology, Queen’s Square Medical Center, Yokohama, Japan.
Received November 14th, 2012; revised December 18th, 2012; accepted December 27th, 2013
ABSTRACT
Nano-sized titanium oxide nanoparticles (TiO2 NPs) are widely used as a dye in food and cosmetics. TiO2 NPs are
known to induce DNA damage when incorporated into cells. However, no bioassay is currently available to easily de-
termine the cell incorporation of TiO2 NPs or related DNA damage, and to date, few studies have examined the differ-
ent degrees of incorporation into cells according to the size of the TiO2 NPs particles and the presence or absence of cell
specificity regarding DNA damage. This present study was therefore designed to examine COS7 cells that had incorpo-
rated TiO2 NPs using atmospheric scanning electron microscopy (ASEM). The results indicated that absorption of TiO2
NPs into cells and nuclear abnormalities had occurred. ASEM is a rapid and simple technique that enables the observa-
tion of samples immediately after fixation with glutaraldehyde and staining with phosphotungstic acid, and this method
was suggested to be useful in screening for DNA damage.
Keywords: COS7 Cells; TiO2; Atmospheric Scanning Electron Microscopy (ASEM); Nanoparticle
1. Introduction
Nano-sized titanium oxide (TiO2) powder is widely used
as a dye for food, cosmetic and plastic products. The size
of the nanoparticles (NPs) is important in that NPs with
sizes of up to 50, 25 and 200 nm are taken up by endo-
cytosis into alveolar A549 epithelial cells [1], human
keratinocytes [2], and red blood cells [3], respectively, but
NPs with larger sizes are just adsorbed to the cell surface.
TiO2 NPs incorporated into cells are known to cause
oxidative stress [4,5], and TiO2-induced inflammation [6].
In addition, it has also been reported that TiO2 NPs in-
duce DNA damage and micronuclei formation [6-10].
From these data, it has become clear that cell injuries
including genetic damage are induced by TiO2 NPs. In-
deed, it has also been reported that lung cancer was
caused by inhalation of TiO2 NPs [11].
We are surrounded by a large amount of TiO2 NPs.
The complicated biochemical and morphological meth-
ods should be simply introduced and related literatures
should be cited. Recently, atmospheric scanning electron
microscopy (ASEM) has been developed with which
samples in their natural state, either within a liquid or a
body of gas, may be observed by allowing a gaseous en-
vironment in the specimen chamber. Moreover, the ad-
vantages and disadvantages between these complicated
methods and ASEM method in this paper should be ana-
lyzed, which is important to the necessary and signifi-
cance of the ASEM method in this paper. We report
herein that nuclear changes in cultured cells could be
rapidly and simply detected using ASEM.
2. Materials and Methods
COS7 cells were cultured on the SiN film of the polysty-
rene ASEM dish, in Dulbeco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and 100
μm/ml kanamycine, at 37˚C for one day in the 5% CO2
atmosphere of a CO2 incubator [12]. For experiments in
cell culture, TiO2 NPs powder form (with a diameter of
50 nm) dispersed in culture medium. After then, COS7
cells were cultured during 6 hr.
For ASEM (JASM-6200 Clair ScopeTM) observations,
these cells were fixed with 1% glutaraldehyde in phos-
phate buffer solution for 10 minutes, washed with water,
stained with 2% PTA (phosphor tungsten acid) in water
for 20 minutes, and washed with water, Water was sup-
plemented with 10 mg/ml glucose as a radical scavenger.
3. Results
When COS7 cells were observed with ASEM after cul-
turing in a medium without TiO2 NPs, the nuclei of all

TiO2 Nanoparticles Induced Genotoxicity in Cultured Cells Using Atmospheric Scanning Electron Microscopy (ASEM) 122
cells were stained (Figure 1) and, in particular, nucleoli
were strongly stained (Figure 1). In the cytoplasm, re-
ticular or tubular endoplasmic reticulum was observed
around the nuclei (Figure 1).
When COS7 cells were observed with ASEM after cul-
turing in a medium with TiO2 NPs, cells with stained
nuclei and those with unstained nuclei were observed at a
ratio of approximately 1:1 (Figure 2(a)). In cells with well-
stained nuclei, abundant endoplasmic reticulum vesicles
were observed around the nuclei (Figure 2(b)). In cells
with phosphotungstic acid-unstained nuclei, nuclear mem-
branes were not observed and the endoplasmic reticulum
was also somewhat unclear around the nuclei. In addition,
many granules (diameter, 600 - 1000 nm) were observed
around the nuclei (Figure 2(c)). Such granules were not
found in normal COS7 cells.
4. Discussion
When COS7 cells were cultured in a medium containing
TiO2 NPs with a diameter of 50 nm for 6 hours, granules
stained with phosphotungstic acid were observed in the
cells. These granules were not observed in normal COS7
cells and in TiO2-treated cells with stained nuclei. The
diameter of the granules ranged from 600 to approxi-
mately 1000 nm. It has been reported that TiO2 NPs are
incorporated into cells by endocytosis [1-3]. In addition,
it was observed by transmission electron microscopy that
many incorporated TiO2 NPs aggregated in lysosomes
[13]. This suggests that the intracellular granules ob-
served in this study were lysosomes containing TiO2 NPs.
Usually, phosphotungstic acid is often used for nega-
tive staining. It stains the nuclei, unit membranes and
stromal collagen fibers in section staining. In addition,
phosphotungstic acid is also known to stain basic pro-
Figure 1. An ASEM photomicrograph of normal COS7
cells. (2% phosphotungstic acid staining) The nuclei and
endoplasmic reticulum (Arrow) in the cytoplasm are well
stained.
(a)
(b)
(c)
Figure 2. ASEM photomicrographs of cells cultured in me-
dium containing TiO2 NPs (2% phosphotungstic acid stain-
ing). (a) Cells with stained nuclei (upper insert) and those
with unstained nuclei (lower insert) are observed; (b) The
nucleus is stained and well-stained endoplasmic reticulum
(Arrow) is observed in the cell; (c) The nucleus is not stain-
ed and there are many granules (Arrow) (600 - 1000 nm in
diameter) in the cell.
teins. In this study, phosphotungstic acid stained the cy-
toplasm, but not the nucleus, in some cultured cells, sug-

TiO2 Nanoparticles Induced Genotoxicity in Cultured Cells Using Atmospheric Scanning Electron Microscopy (ASEM) 123
gesting that basic proteins in the nuclei of cultured cells
were denatured by TiO2 NPs and became unstained. The
genotoxicity of TiO2 is a well-known fact [6-10,14]. How-
ever, it is necessary to clarify biochemically in the future
what specific injuries the unstained nuclei have.
There were more granules in cells with unstained nu-
clei than in cells with stained nuclei. As described above,
these granules were considered to be TiO2 NPs. In other
words, it can be said that only cells that incorporated a
large amount of TiO2 NPs had nuclear abnormalities.
There was a variation in the amount of incorporated TiO2
NPs within the same cultured cell population, and this is
an issue to be addressed in the future.
There is an abundance of TiO2 NPs in this world. Sko-
caj et al. described in their review [14] that “Until rele-
vant toxicological and human exposure data that enable
reliable risk assessment are obtained, TiO2 nanoparticles
should be used with great care.” In addition, the toxicity
of TiO2 NPs varies depending on the size of the particles
and the types of cells and tissues exposed to them [14].
This actually makes it difficult to understand various tis-
sue and cell injuries induced by TiO2 NPs.
Complicated techniques of biochemistry and transmis-
sion electron microscopy are needed to assess injuries
induced by TiO2 NPs, and a great deal of labor is re-
quired to assess many samples for analysis of the time
course and dose dependence. However, the ASEM ob-
servation used in this study is a rapid and simple tech-
nique in that samples can be observed immediately after
fixation with glutaraldehyde and staining with phospho-
tungstic acid. It is an excellent technique that can rapidly
and simply detect nuclear abnormalities and intracellular
incorporation of TiO2 NPs. In the future, ASEM will be
used frequently, not only to assess injuries induced by
TiO2 NPs, but also for application to drug screening, in-
cluding screening of drug-induced toxicity.
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