Toxicology Letters 199 (2010) 389–397

Contents lists available at ScienceDirect

Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity
of ZnO nanoparticles
Wenhua Song a,b,∗ , Jinyang Zhang b , Jing Guo a , Jinhua Zhang a , Feng Ding c , Liying Li a , Zengtian Sun a
a
School of Environmental and Chemical Engineering, Tianjin Polytechnic University, Tianjin, 300160, China
b
College of Environmental Science and Engineering, Shanghai Jiaotong University, Shanghai, 200240, China
c
College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China

a r t i c l e i n f o a b s t r a c t

Article history: With large-scale production and wide application of nanoscale ZnO, its health hazard has attracted exten-
Received 21 July 2010 sive worldwide attention. In this study, cytotoxicity of different sized and shaped ZnO nanoparticles in
Received in revised form mouse macrophage Ana-1 was investigated. And contribution of dissolved Zn2+ and ROS in toxicity of
28 September 2010
ZnO particles was analyzed. The results indicated that ZnO particles manifested dose-dependent toxic
Accepted 1 October 2010
effect on Ana-1 cells without size-dependence, and the particles shape may impact cytotoxicity of ZnO
Available online 8 October 2010
particles. When the concentration of dissolved Zn2+ tended to equilibrium in the complete cell medium,
the zinc ion concentration was approximately 10 ␮g/ml, inducing about 50% cell death, which was close
Keywords:
ZnO nanoparticles
to the cytotoxicity of ZnCl2 (IC50 = 13.33 ␮g Zn/ml). The Zn2+ concentration had significant correlations
Ana-1 cell with cell viability and LDH level induced by the supernatant of ZnO particle suspensions (incubation at
Cytotoxicity 37 ◦ C for 24 h). Thus, the dissolved Zn2+ played the main role in toxic effect of ZnO particles. Moreover,
ROS ROS generation assays demonstrated that ZnO particles produced intrinsically a small quantity of ROS,
Zinc ion intracellular ROS was mainly produced after ZnO particles or the dissolved Zn2+ entered into the cells.
Although intracellular ROS had significant correlations with cell viability and LDH induced by ZnO par-
ticles, intracellular ROS may not be a major factor in cytotoxicity of ZnO nanoparticles, but the cytotoxic
response.
© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Nano scale ZnO has become a nano-functional material, widely
concerned following carbon nanotubes. It is one of the materials
with well-established production process and test method, and also
is one of the materials of first commercial application. Since the
Bayer Corporation (Bayer Co., Ltd.) first provided with nano-ZnO
products in the market, various countries started the large-scale
industrial production of nano-ZnO. Nano-ZnO is widely used in
many fields, such as rubber manufacture, cosmetics, pigments, food
additives, medicine, chemical fiber and electronics industries (Ji
and Ye, 2008). However, theoretical study of nano-ZnO lags behind
its production and application. And due to the traditional concept
that zinc oxide is non-toxic, the toxicology study of nano-ZnO is
farther behind the speed of its application development. In recent
years, with the rise and development of nanotoxicology, health
risks of nano-ZnO are gradually concerned. The study on the acute
toxicological impact of ZnO nanoparticles on mice showed that the
liver, spleen, heart, pancreas and bone are the target organs for 20
∗ Corresponding author at: School of Environmental and Chemical Engineering,
Tianjin Polytechnic University, Tianjin, 300160, China.
E-mail address: songwenhua9316@sina.com (W. Song).
and 120 nm ZnO oral exposure (Wang et al., 2008). And exposures
to nanoscale or fine-sized ZnO particles by intratracheal instilla-
tion produced potent but typical “metal fume fever”-like reversible
inflammation (Sayes et al., 2009). Moreover, many in vitro studies
also demonstrated that ZnO nanoparticles are toxic to mammalian
cells. For example, ZnO nanoparticles induced toxicity in RAW264.7
and BEAS-2B cells, leading to the generation of reactive oxygen
species (ROS), oxidant injury, excitation of inflammation, and cell
death, and ZnO dissolution could happen in culture medium and
endosomes (Xia et al., 2008). In addition, some studies have demon-
strated that nano-ZnO is more toxic than other nanometer-scale
structured metallic oxides (Jeng and Swanson, 2006; Lai et al., 2008;
Horie et al., 2009).
However, there are still a lot of controversies on the toxicity
of ZnO nanoparticles. Firstly, there is not a consistent conclusion
on toxicity of ZnO nanoparticles and the impact of particle size on
toxicity of ZnO nanoparticles. Some research showed that differ-
ent sized ZnO nanoparticles had similar cytotoxicity on different
cells, 24 h IC50 was about 10–20 ␮g/ml, and particle size had no
effect on cytotoxicity (Lin et al., 2009; Deng et al., 2009; Yuan et
al., 2010). Nair et al. found 100 ␮M (8.1 ␮g/ml) ZnO nanoparticles
induced osteoblast cancer cells (MG-63) viability decrease to about
40%, and when particle size was under 350 nm, the size did not
impact cytotoxicity, but nanoparticles were more toxic to MG-63

0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2010.10.003
390 W. Song et al. / Toxicology Letters 199 (2010) 389–397
cell than microparticles (Nair et al., 2009). Reddy et al. reported
ZnO nanoparticles (13 nm) still were not cytotoxic at 5 mmol/L
(400 ␮g/ml), only at 10 mmol/L (800 ␮g/ml), ZnO nanoparticles
caused 57% cell death. And cytotoxicity is limited to ZnO in the
nanoscale size range as no significant effect of bulk ZnO powder
was observed (Reddy et al., 2007; Hanley et al., 2008). Secondly,
the effect of particle shape on cytotoxicity of ZnO nanoparticles is
not yet clear. Lee et al. found spherical ZnO particles induced lower
toxicity than rod-shaped ZnO particles (Lee et al., 2008). However in
the study of Lee et al., ZnO exposure concentration was based on the
bottom area of culture plate, if mass-based dosage conversed, the
exposure concentration of rod ZnO particles was much higher than
spherical ZnO particles. Nair et al. showed that rod ZnO nanoparti-
cles were lower toxic than spherical ZnO nanoparticles, but rod ZnO
nanoparticles were coated by starch and spherical ZnO nanopar-
ticles were coated by poly ethylene glycol (PEG), starch and PEG
may impact on toxicity of ZnO nanoparticles (Nair et al., 2009).
In addition, now there are main two explanations on the toxicity
mechanism of ZnO nanoparticles: zinc dissolution and oxidative
damage, but the contribution of two sides is uncertain. Deng et al.
found that ZnO nanoparticles and ZnCl2 had similar toxicity, and
did not observe ZnO particles within cells with the TEM (Deng et
al., 2009). Xia et al. also suggested that zinc ion played an impor-
tant role in cytotoxicity of ZnO nanoparticles, and did not find ZnO
nanoparticles in cells (Xia et al., 2008). However, Jeng and Swan-
son compared cytotoxicity of a variety of metal oxide nanoparticles
on the Neuro-2A cell, and considered oxidative damage had an
important effect on cytotoxicity (Jeng and Swanson, 2006). Lin
et al. showed nanoparticles and microparticles of ZnO decreased
A549 cell viability, and observed particles in cells with TEM. They
still found toxicity of ZnO particles was reduced by antioxidant N-
Acetylcysteine, this demonstrated toxicity of ZnO particles related
with oxidative damage. Moreover, they considered that the low
dissociation of ZnO in the cell culture medium (Ksp = 3.0 × 10−16 )
was not enough to cause cell toxicity. Thus, they inferred that
oxidative damage induced by ZnO particles was due to sequential
oxidation–reduction reactions occurring at ZnO particles surface to
produce reactive species (Lin et al., 2009).
Macrophages were multifunctional immune cells. Their phago-
cytosis was a main pathway to clean up particles in vivo, and
macrophages had a variety of stimulating rapid response capaci-
ties, played an important part in regulating the immune response
and inflammation. ZnO nanoparticles were widely added to the
feed as additives. Ding et al. showed that the oral exposure to low
dosage of ZnO nanoparticles in mice increased the phagocytosis of
mouse peritoneal macrophages (Ding et al., 2007). However, the
oral exposure to high dosage of ZnO nanoparticles induced the
edema and degeneration of hepatocytes, inflammation of pancreas
and damages of stomach and spleen (Wang et al., 2008). Mean-
while, ZnO nanoparticles at high dose still may destroy the immune
system. Especially, peritoneal macrophage had a close connection
with pancreatitis (Ma et al., 2009). At present, the impact of ZnO
nanoparticles on the immune system is not clear. Thus, in this
study, we took mouse peritoneal macrophages Ana-1 cells as sub-
ject, investigated cytotoxicity of different sized and shaped ZnO
particles, and analyzed the effect of the dissolved Zn2+ and ROS in
cytotoxicity.
2. Materials and methods
2.1. ZnO particles
Four different sized ZnO particles were supplied from commercial companies.
Both fine-ZnO particles (<1 ␮m) were purchased from Hangzhou Wanjingxin Mate-
rial Co. Ltd., China. 100 ± 10 nm and 30 ± 10 nm ZnO particles were purchased from
Beijing Nachen Technology Co. Ltd., China. 10–30 nm ZnO particles were purchased
from Shenzhen Nanuo Nanomaterials Corp., China. Visualize particles size and shape
of ZnO particles was measured by transmission electron microscopy (TEM, H7650).
Crystal structure of ZnO particles was characterized using X-ray diffraction (XRD,
Bruker D8 Discover).
2.2. Cell culture and treatment with ZnO particles
Mouse macrophage (Ana-1) cell line was purchased from the Cell Bank of Type
Culture Collection of Chinese Academy of Sciences. Cells were maintained in the
complete cell medium, RPMI1640 supplemented with 10% fetal bovine serum (FBS),
100 U/ml penicillin, 100 ␮g/ml streptomycin, and grown at 37 ◦ C in a humidified 5%
CO2 environment.
A stock solution up to 1 mg ZnO/ml complete cell medium was prepared to
yield a more accurate weight of ZnO powder, dispersed for 20 min by a sonicator to
prevent aggregation, and diluted to the specified concentrations (2.5, 5, 10, 20, 40,
80, 100 ␮g/ml) for treatment of cells.
2.3. Cell viability assay
Cellular viability was determined using the CCK-8 assay (Beyotime). Cells were
seeded with equal density in each well of 96-well plates (104 cells per well), 100 ␮l
of the cell culture medium per well, and incubated for 24 h at 37 ◦ C. Then cells
were treated in 96-well plates with 2.5 ␮g/ml, 5.0 ␮g/ml, 10.0 ␮g/ml, 20.0 ␮g/ml,
40.0 ␮g/ml, 80.0 ␮g/ml or 100.0 ␮g/ml of ZnO particles for 24 h at 37 ◦ C. Untreated
cells served as a control group. At the end of the treatment, CCK-8 dye was added to
each well and the plates were incubated for another 2 h at 37 ◦ C. To prevent particles
from interfering with this assay, the solution in each well of each plate was quanti-
tatively transferred to an empty well in another plate. Subsequently, the absorbance
was measured by dual wavelength spectrophotometry at 450 nm and 630 nm using
a microplate reader. Each treatment was repeated five times.
In order to investigate the effect of zinc ions released from ZnO particles on
cell viability, 2.5 ␮g/ml, 5.0 ␮g/ml, 10.0 ␮g/ml, 20.0 ␮g/ml, 40.0 ␮g/ml, 80.0 ␮g/ml
or 100.0 ␮g/ml of ZnO particle suspensions was incubated at 37 ◦ C in a humidi-
fied 5% CO2 environment for 24 h, and then centrifuged at 20,000 rpm for 30 min.
The supernatant was used to treat cells, and the cell viability was assayed by
CCK-8. Moreover, the viability of Ana-1 exposed to ZnCl2 was also determined.
Because ZnO nanoparticles were similar toxicity to ZnCl2 in some studies (Franklin
et al., 2007; Deng et al., 2009), the concentrations of ZnCl2 were set accord-
ing to the concentration of ZnO particles, with 4.2 ␮g/ml, 8.4 ␮g/ml, 16.8 ␮g/ml,
33.6 ␮g/ml, 67.2 ␮g/ml, 134.4 ␮g/ml or 168.0 ␮g/ml (correspond to 2.5 ␮g ZnO/ml,
5.0 ␮g ZnO/ml, 10.0 ␮g ZnO/ml, 20.0 ␮g ZnO/ml, 40.0 ␮g ZnO/ml, 80.0 ␮g ZnO/ml or
100.0 ␮g ZnO/ml).
2.4. LDH measurement
Release of lactate dehydrogenase (LDH) to the cell culture medium indicates
cell membrane damage. LDH level in the cell culture medium was determined
using a LDH Kit (Jiancheng Bioengineering Co. Ltd., Nanjing, China) according to the
manufacturer’s protocols. LDH catalyzed the oxidation of lactate to pyruvate with
simultaneous reduction of NAD+ to NADH. The rate of NAD+ reduction is directly
proportional to LDH level. The treatment method of cells was the same as the
CCK-8 assay, but a cell culture medium volume of 200 ␮l was used to provide suffi-
cient volume for LDH detection. After ZnO exposure, half the amount of the 200 ␮l
cell culture medium was collected for LDH analysis. Absorption was measured at
340 nm.
2.5. Intracellular reactive oxygen species measurement
The intracellular reactive oxygen species (ROS) was determined using a well-
characterized probe, 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) (Wan et al.,
1993). DCFH-DA passively enters the cell, and is hydrolyzed by esterases to DCFH.
This nonfluorescent molecule is then oxidized to fluorescent compound dichlo-
rofluorescein (DCF) by cellular oxidants. A DCFH-DA stock solution (in methanol)
of 10 mM was diluted 1000-fold in the cell culture medium without serum or other
additives to yield a 10 ␮M working solution. Cells were washed twice with PBS and
then incubated with DCFH-DA working solution for 20 min in the dark environ-
ment (37 ◦ C incubator) followed by treatment with ZnO particles for 24 h. Then the
cells were washed three times with cell culture medium without serum to elimi-
nate DCFH-DA that did not enter the cells. Cells were collected in suspension, the
fluorescence was determined at 488 nm excitation and 525 nm emission using a
fluorospectrophotometer.
2.6. ROS activity determination of ZnO particles under the abiotic condition
ZnO particles suspensions (2.5, 5, 10, 20, 40, 80, 100 ␮g/ml) were prepared with
the complete cell medium, and a part of suspensions was incubated for 24 h at
37 ◦ C. ROS activities of ZnO particles suspensions and supernatants after incuba-
tion for 24 h were determined by HRP–DCF fluorometric method (Venkatachari et
al., 2007; Jiang et al., 2008), using the complete cell medium as control. The mea-
sured fluorescent intensity of controls was subtracted from those of the ZnO particles
suspensions or supernatants samples. Briefly, activation of DCFH-DA to DCFH was
performed immediately prior to analysis with 1 vol of 5 mM DCFH-DA and 9 vol of
 W. Song et al. / Toxicology Letters 199 (2010) 389–397 391

0.01 N sodium hydroxide incubation at room temperature in the dark for 30 min,
which yielded an unstable DCFH solution. Further dilutions were prepared in the
25 mM phosphate buffer (pH 7.2). The solution was kept on ice without exposing it
to light until use. To catalyze the reaction between DCFH and the ROS, Horseradish
peroxidase (HRP) was added to the DCFH solution in a ratio such that the working
reagent contained 2.2 units of HRP/ml of the reagent. In order to correlate the fluo-
rescent intensities and concentrations in terms of equivalent H2 O2 concentrations,
an assay of standard solutions of H2 O2 (2.5, 5, 10, 20, 30, 40 ␮M/L) was performed.
0.1 ml of H2 O2 solutions or the samples were added to 3 ml of the DCFH–HRP reagent
mixture. Blank was prepared with 0.1 ml distilled and deionized water instead of
H2 O2 . And then solutions and blank were incubated at 37 ◦ C for 15 min. The flu-
orescent intensity was measured at 488 nm excitation and 525 nm emission by a
fluorospectrophotometer.

2.7. Dissolved zinc concentrations in the cell culture medium

ZnO particle suspensions (2.5, 5, 10, 20, 40, 80, 100 ␮g/ml) were incubated for
24 h at 37 ◦ C in a humidified 5% CO2 environment. Then centrifugation at 20,000 rpm
for 30 min removed solid phase ZnO from the complete cell medium. 1.5 ml of the
supernatant was added to 1 ml of ultrahigh purity HNO3 , and digested at about
120 ◦ C until the solutions were colorless and clear. At last, the remaining solutions Fig. 1. X-ray diffraction pattern of different sized ZnO particles.
were diluted to 10 ml with 3% nitric acid. The Zn content was analyzed by ICP-AES
(Thermo IRIS Intrepid IIXSP, America).

3. Results
2.8. Statistical analysis
3.1. Characterization of ZnO particles
The data were expressed as mean ± standard deviation. For statistical analysis,
the experimental values were compared to their corresponding control values. A
statistical analysis was done by SPSS 16.0. The significant difference was judged at The X-ray diffraction analysis (Fig. 1) clearly showed that all four
p < 0.05. types of ZnO particles were the hexagonal structure.

Fig. 2. TEM image of ZnO particles.

392 W. Song et al. / Toxicology Letters 199 (2010) 389–397

Fig. 3. Cell viability of Ana-1 cell exposed to different sized ZnO particles. Control cells treated without ZnO particles are considered to have 100% activity. *p < 0.05 vs. control
cells (n = 5).

Table 1
The 24 h IC50 values and 95% confidence limits for Ana-1 cells exposed to different sized ZnO and ZnCl2 . Data were calibrated to Zn concentrations and ZnO concentrations.

ZnO ZnCl2

fine-ZnO 100 nm 30 nm 10–30 nm

Zn concentration(␮g/ml) 35.76 35.26 32.43 24.84 13.33

95% confidence limits 32.32–39.39 31.10–40.05 28.96–37.23 21.97–28.17 11.81–14.99
ZnO concentration(␮g/ml) 44.56 43.94 40.41 30.95
95% confidence limits 40.28–49.09 38.76–49.91 36.09–46.39 27.38–35.10

The TEM micrographs demonstrated the particle shapes and
sizes (Fig. 2). Fine-ZnO, 100 nm ZnO and 30 nm ZnO were rod-
shaped, with lengths of 341.75 ± 173.34 nm, 107.59 ± 38.44 nm
and 70.89 ± 34.18 nm, diameters of 173.48 ± 72.73 nm,
60.21 ± 21.76 nm and 40.44 ± 12.51 nm, respectively. 10–30 nm
ZnO was spherical with diameters of 18.76 ± 4.98 nm. Among
them, the mean size of 30 nm ZnO was very different from the
manufacturer’s information.
3.2. Cytotoxicity and disruption of cell membrane induced by ZnO
particles
Exposure of Ana-1 cells to ZnO particles was characterized by a
steep response pattern. ZnO particles initiated cytotoxicity around
5–10 ␮g/ml with a steep decline in cell viability between 10 and
40 ␮g/ml (Fig. 3). 10–30 nm ZnO particles at the dosage of 5 ␮g/ml
decreased significantly the cell viability (p < 0.05), whereas other
three ZnO particles decreased significantly the cell viability at the
dosage of 10 ␮g/ml. The IC50 values of four ZnO particles were 40.28,
43.94, 40.41 and 30.95 ␮g ZnO/ml for fine-ZnO, 100 nm, 30 nm and
10–30 nm ZnO particles, respectively (Table 1). In order to compare
the toxicity of ZnO particles and ZnCl2 , we studied the cytotoxicity
of ZnCl2 to Ana-1. Cell viability obviously declined at the dose above
5 ␮g/ml. The IC50 value of ZnCl2 was 13.33 ␮g Zn/ml.
Cell membrane damage is reflected in the elevated LDH level in
the cell culture medium. Fig. 4 shows LDH level in the cell culture
medium following 24 h exposure to ZnO particles. LDH levels fol-
lowing exposure to 10–30 nm ZnO particles above 10 ␮g/ml were
increased significantly (p < 0.05). At the dose above 20 ␮g/ml, other
three sized ZnO particles induced the steep elevation of LDH levels
(p < 0.05). However, ZnCl2 caused an obvious elevation of LDH at
5 ␮g/ml (p < 0.05).
3.3. Cytotoxicity and disruption of cell membrane induced by
supernatants of ZnO particles suspensions
As shown in Figs. 5 and 6, at the dose above 10 ␮g/ml, the super-
natant of 10–30 nm ZnO particles suspension had a significant toxic
effect on Ana-1 cell, cell viability obviously decreased (p < 0.05),
and the level of LDH evidently increased (p < 0.05). When the con-
centration of ZnO particles exceeded 20 ␮g/ml, the supernatants
of fine-ZnO, 100 nm and 30 nm ZnO particles suspensions reduced
cell viability markedly (p < 0.05), and definitely elevated the level of
LDH. However, when the concentrations of ZnO particles exceeded

Fig. 4. LDH level in the Ana-1 cell culture medium following a 24 h exposure to ZnO particles and ZnCl2 (n = 5).
 W. Song et al. / Toxicology Letters 199 (2010) 389–397 393

Fig. 5. Cytotoxicity of supernatant of ZnO particles suspensions incubated for 24 h.

*p < 0.05 vs control cells (n = 5).
Fig. 7. Zn contention of ZnO particles dissolution in the cell culture media (n = 3).

Table 2
The correlation analysis of the dissolved Zn concentration with cell viability and
LDH level induced by the supernatant (R2 ).

The dissolved Zn concentration

Fine-ZnO 100 nm 30 nm 10–30 nm

** ** **
Cell viability 0.990 0.982 0.987 0.977**
LDH level 0.991** 0.970** 0.991** 0.951**
**
p < 0.01.

Fig. 6. LDH level in the Ana-1 cell culture medium following a 24 h exposure to
supernatant of ZnO particles suspensions incubated for 24 h (n = 5).
40 ␮g/ml, the supernatants of four sized ZnO particle suspensions
decreased cell viability to about 50%, and cell viability had little
change with the increase of ZnO particles concentrations.
3.4. Dissolution of ZnO particles in cell culture medium
for fine-ZnO, 100 nm, 30 nm and 10–30 nm, respectively, when the
dissolved Zn2+ content tended to stable. The equilibrium concentra-
tion of zinc ion was about 10 ␮g/ml. When the concentration of ZnO
particles was 100 ␮g/ml, the dissolved Zn concentrations of fine-
ZnO, 100 nm, 30 nm and 10–30 nm ZnO particles are 9.89 ± 0.02,
9.71 ± 0.10, 9.58 ± 0.32 and 10.25 ± 0.33 ␮g/ml, respectively.
The correlation analysis revealed there was an inverse correla-
tion between the dissolved Zn concentration of each ZnO particles
and cell viability induced by the supernatant, and a positive correla-
tion between the dissolved Zn concentration of each ZnO particles
and LDH level induced by the supernatant (Fig. 8 and Table 2).

The dissolution profiles from different sized ZnO particles in
cell culture medium are shown in Fig. 7. The results suggested
that the dissolution of ZnO nanoparticles was similar to fine-ZnO
particles under experiment conditions. When the concentration of
ZnO particles exceeded 40 ␮g/ml, the dissolved Zn concentration
tended to equilibrium. The concentration of zinc ion was almost
constant with the increases of the concentrations of ZnO parti-
cles. Through calculation, we estimated that the concentrations
ZnO nanoparticles were 41.33, 41.33, 44.92 and 38.59 ␮g/ml and
their rates of solution were 29.61%, 29.17%, 26.05% and 32.44%
3.5. Intracellular reactive oxygen species
Intracellular ROS levels were significantly increased after expo-
sure to all sized ZnO particles at the dose above 5 ␮g/ml (p < 0.05)
(Fig. 9). But the effects of different sized ZnO particles were dif-
ferent. 10–30 nm ZnO particles appeared more effective, when
the dosage exceeded 10 ␮g/ml. Intracellular ROS levels induced
by 10–30 nm ZnO particles were much higher than other sized
ZnO particles (p < 0.05). 30 nm, 100 nm and fine-ZnO particles
showed the similar characteristics. Compared to ZnO particles,

Fig. 8. The inverse correlation (R2 = 0.990) between the dissolved Zn concentration of fine-ZnO particles and cell viability induced by the supernatant (A), the positive
correlation (R2 = 0.991) between the dissolved Zn concentration of fine-ZnO particles and LDH level induced by the supernatant (B). The figures of other ZnO particles were
not showed, R2 values are shown in Table 2.
394 W. Song et al. / Toxicology Letters 199 (2010) 389–397

Fig. 9. Oxidative stress induced by exposure of Ana-1 cells to ZnO particles and ZnCl2 . *p < 0.05 vs. control cells; ␣ p < 0.05 vs. other sized ZnO particles and ZnCl2 ; ␤ p < 0.05
vs. ZnO particles.

Fig. 10. The inverse correlation (R2 = 0.966) between intracellular ROS levels of fine-ZnO particles and cell viability induced by the supernatant (A), the positive correlation
(R2 = 0.981) between intracellular ROS levels of fine-ZnO particles and LDH level induced by the supernatant (B). The figures of other ZnO particles and ZnCl2 are not showed,
R2 values are shown in Table 3.

ZnCl2 induced relatively less ROS generation at the dosage above
20 ␮g/ml (p < 0.05).
The correlation analysis reveals that there was an inverse cor-
relation between intracellular ROS levels and cell viability induced
by ZnO particles suspensions, and a positive correlation between
intracellular ROS levels and LDH level induced by ZnO particle sus-
pensions (Fig. 10 and Table 3).
and 30 nm. After incubation at 37 ◦ C for 24 h, ROS activities of the
supernatants were distinct lower than ZnO particles suspensions
(Fig. 12). Except that ROS activities in the supernatants of 10–30 nm
ZnO particles were obviously higher than other three ZnO particles,
there was no difference between 100 nm, 30 nm and fine-ZnO par-
ticles. However, ROS activities of all sized ZnO particles under the

3.6. ROS activity determination of ZnO particles under the abiotic

condition

Fig. 11 shows acellular ROS activities of ZnO particles. ROS gen-

erated by all ZnO particles in the cell medium increased with the
increasing of the concentration. The order of ability to generate ROS
of different sized ZnO particles was 10–30 nm, fine-ZnO, 100 nm

Table 3
The correlation analysis of intracellular ROS level with cell viability and LDH level
induced by ZnO particles(R2 ).

intracellular ROS levels

Fine-ZnO 100 nm 30 nm 10–30 nm ZnCl2

Cell viability 0.966** 0.948** 0.929** 0.923** 0.911**

LDH level 0.981** 0.945** 0.922** 0.952** 0.968**
** Fig. 11. Non cellular ROS level of ZnO particles suspensions.
p < 0.01.
 W. Song et al. / Toxicology Letters 199 (2010) 389–397
Fig. 12. Non cellular ROS level of the supernatants after incubation for 24 h.
abiotic condition were lower, not exceeding 4.0 ␮M H2 O2 , either in
the supernatants or in the ZnO particles suspensions.
4. Discussion
4.1. Cytotoxicity of ZnO particles
Much research has showed that ZnO nanoparticles resulted
in cytotoxicity to many types of cell, such as human bronchial
epithelial cells (BEAS-2B) (Huang et al., 2010), human lung epithe-
lial cells (A549) (Lin et al., 2009; Karlsson et al., 2008; Horie et
al., 2009), human keratinocyte HaCaT cells (Horie et al., 2009),
human epidermal cell (A431) (Sharma et al., 2009), L2 rat lung
epithelial cells and rat alveolar macrophages (Sayes et al., 2007,
2009), the mouse macrophage cell RAW 264.7 (Xia et al., 2008),
mesothelioma MSTO-211H and rodent 3T3 fibroblast cells (Brunner
et al., 2006), primary mouse embryo fibroblasts (PMEF) (Yang et al.,
2009), human embryonic lung fibroblasts (HELF) cells (Yuan et al.,
2010), mouse NSC (C17.2) (Deng et al., 2009), human astrocytoma
U87 (astrocytes-like) cells (Lai et al., 2008), human T lympho-
cytes (Reddy et al., 2007; Hanley et al., 2008) and human aortic
endothelial cells (HAECs) (Gojova et al., 2007). And ZnO nanoparti-
cles induced steep dose-cytotoxicity without cell line-dependent.
According to previous research, ZnO nanoparticles had the highest
toxicity in human keratinocyte HaCaT cells and human epidermal
cell (A431), the concentration range of toxic effect was 5–10 ␮g/ml,
cell activity was completely inhibited after exposure of 10 ␮g/ml
ZnO nanoparticles(Horie et al., 2009; Sharma et al., 2009). Secondly,
ZnO nanoparticles had comparative toxicity in most cells, for exam-
ple, lung cells (A549, BEAS-2B, MSTO-211h, RAW 264.7), fibroblast
(3T3, PMEF, HELF) and nerve cells (C17.2, U87), their IC50 value was
about 10–20 ␮g/ml. However, the toxicity of ZnO nanoparticles in
human T lymphocytes was much lower than other cells, exposure
of 400 ␮g/ml ZnO nanoparticles did not yet cause cytotoxicity. T
lymphocytes, as a kind of immune cells, had great tolerance range
to ZnO nanoparticles. And the toxicity of ZnO nanoparticles toward
normal T cells was lower than that to cancerous T cells (Reddy et
al., 2007; Hanley et al., 2008).
In this study, Ana-1 cell also was a kind of important immune
cell. IC50 values of four ZnO particles in Ana-1 cells were 30.95,
40.41, 43.94 and 44.56 ␮g/ml for 10–30 nm, 30 nm, 100 nm and
fine-ZnO particles, respectively. The tolerance range of Ana-1 cell
to ZnO nanoparticles was much lower than that of human T cells.
However, in contrast, toxicity of ZnO nanoparticles was lower
395
than other cells. Compared with similar cells, the toxicity of ZnO
nanoparticles toward normal mouse macrophage Ana-1 was lower
than that to cancerous macrophage RAW 264.7. After exposure
to 25 ␮g/ml ZnO particles (13 nm) for 16 h, all RAW264.7 cells
were almost dead (Xia et al., 2008). This also demonstrated that
the toxicity of ZnO particles to normal cells may be lower than
the corresponding tumor cells. Moreover, LDH leakage reflected
cell membrane damage and the degree of cell necrosis. The steep
elevation of LDH indicated that ZnO particles destroyed cell mem-
brane integrity and could cause cell necrosis. However, at low dose,
5 ␮g/ml of 10–30 nm ZnO and 10 ␮g/ml other three sized ZnO
particles obviously reduced cell viability, but failed to induce the
elevation of LDH level compared with the control, which implied
that low dose of ZnO particles did not result in cell membrane
damage and cell necrosis, but induced apoptosis.
4.2. Impact of ZnO particle size and shape on toxicity
In the present study, the toxicity of three rod-like ZnO particles
were very near, this indicated that particle size had no effect on
toxicity of ZnO particles. There was also the similar conclusion in
previous studies. For example, Lin et al. found that the toxicity of
70 nm ZnO particles was similar to 420 nm ZnO particles (Lin et
al., 2009). Deng et al. showed 10, 30, 60 and 200 nm ZnO particles
had similar toxicity to mouse NSC (C17.2) (Deng et al., 2009). Yuan
et al. also found 20, 30 and 40 nm ZnO particles had comparative
toxicity in human embryonic lung fibroblasts (HELF) cells (Yuan et
al., 2010). However, Nair et al. revealed that the toxicity of 40, 150
and 350 nm ZnO nanoparticles had a little difference in osteoblast
cancer cells (MG-63), but ZnO microparticles had been less toxic
than ZnO nanoparticles (Nair et al., 2009). Reddy et al. also showed
that ZnO nanoparticles had a higher toxicity on human T cells than
ZnO microparticles (Reddy et al., 2007; Hanley et al., 2008). Thus,
we inferred there may be a critical size, when ZnO particle size
was below the critical size, particle size had no effect on toxicity;
when ZnO particle size exceeded the critical size, the toxicity of ZnO
particle was relatively lower. It needs further research to prove this
inference.
At present, there are few reports about the impact of ZnO
particle shape on toxicity. In this study, 10–30 nm ZnO particles
were spherical, 30 nm, 100 nm and fine-ZnO particles were rod-
shaped. 10–30 nm spherical ZnO particles were slightly highly toxic
than three rod-like ZnO particles. Thus, particle shape may affect
its toxicity. Yang et al. inferred that the genotoxicity of different
nanoparticles may primarily be due to particle shape (Yang et al.,
2009). However in the study of Deng et al., the toxicity of 10 nm
spherical ZnO particles was similar to 30 nm, 60 nm and 200 nm
rod-like ZnO particles (Deng et al., 2009). Therefore, the impact
of particle size on the toxicity of ZnO nanoparticles needs further
study.
4.3. The role of dissolved zinc ion in the toxicity of ZnO
nanoparticles
ZnO is slightly soluble, and can release zinc ions in solution.
Some researchers considered that dissolved zinc ions in the toxi-
city of ZnO nanoparticles played an important role. Brunner et al.
inferred that toxic effects of ZnO nanoparticles on MSTO or 3T3
cells may be attributed to the dissolution of zinc ions (Brunner
et al., 2006). Deng et al. found ZnO nanoparticles and ZnCl2 had
the similar toxic effect on mouse NSCs (Deng et al., 2009). How-
ever they did not determine the content of dissolved zinc ions.
Xia et al. determined the content of dissolved zinc ions in the
cell culture medium, and inferred when concentrations of ZnO
nanoparticles were less than ZnO solubility, the cell cultures were
mainly exposed to aqueous zinc ions (Xia et al., 2008), whereas
396 W. Song et al. / Toxicology Letters 199 (2010) 389–397
the content of zinc ions was not determined under the experimen-
tal conditions, and the contribution of zinc ions in toxicity of ZnO
nanoparticles was not clear. In this study, we measured the con-
centration of dissolved zinc ions in the cell culture medium under
the experimental conditions, and investigated the toxic effect of
dissolved zinc ions. Our results showed that the dissolved Zn2+
concentrations tended to equilibrium at 40 ␮g/ml, and the equilib-
rium concentrations of dissolved Zn2+ were about 10 ␮g/ml, which
was close to the result of the work done by Xia et al. By equili-
brating excess ZnO nanopowder with the cell medium for 4 days
at 22 ◦ C, Xia et al. estimated that the maximum total dissolved
zinc concentrations could be 190 ␮M in bronchial epithelial growth
medium (corresponding to 12.35 ␮g/ml) and 225 ␮M in complete
DMEM (corresponding to 14.63 ␮g/ml) (Xia et al., 2008). Never-
theless, Horie et al. found 1 mg/ml ultrafine ZnO particles released
about 60 ␮g/ml Zn2+ into the DMEM-FBS (Horie et al., 2009). More-
over, ZnO nanoparticles in simulated uterine fluid containing HAS
released 7.6 ␮g/ml zinc ions (Yang and Xie, 2006). Rapid dissolu-
tion of the bulk ZnO and nanoparticulate ZnO in Milli-Q water (pH
7.6) was also observed, a dialyzed zinc concentration was approx-
imately 8 mg/l by 6 h, an equilibrium concentration was 16 mg/l
after 72 h (Franklin et al., 2007). And the solubility of ZnO nanopar-
ticles in seawater was 3.7 ␮g/ml (Wong et al., 2010). Thus, the
solubility of ZnO nanoparticles in different medium was differ-
ent. However, zinc ions in different media all had an important
effect on the toxicity of ZnO nanoparticles. Toxicity test results of
supernatants showed that dissolved zinc ions severely inhibited
cell viability. When the concentration of zinc ions reached equilib-
rium (10 ␮g/ml), cell viability reduced by 50%, the toxic effect was
close to ZnCl2 (IC50 = 13.33 ␮g/ml). Furthermore, dissolved zinc ions
still induced LDH leakage. And correlation analysis showed that the
zinc ion content had a significant correlation with cell viability and
LDH level. Thus, zinc ions played a main role in the toxicity of ZnO
particles.
Comparing the toxicity of supernatants with ZnO particle sus-
pensions, we found that the toxic effect of supernatants was similar
to ZnO particles suspensions at dosage under 10 ␮g/ml, which indi-
cated that the toxicity was mainly due to dissolved zinc ions at
the low dosage. And when the concentration of ZnO particles was
10–40 ␮g/ml, the sharp decline of cell viability may be related to
rapid increase of dissolved Zn2+ content. Bozym et al. found free
zinc ions outside a narrow concentration range were toxic to a vari-
ety of cells in vitro (Bozym et al., 2010). When the concentration of
ZnO particles exceeded 40 ␮g/ml, the effect of zinc ions on cytotox-
icity was almost the same, the enhancement of cytotoxicity with
the increase of ZnO particles concentration was mainly caused by
ZnO particles.
4.4. The role of ROS in the toxicity of ZnO nanoparticles
Evidently, ROS plays an important role in toxicity of nanopar-
ticles (Nel et al., 2006). In order to study the effect of ROS in
cytotoxicity of ZnO nanoparticles, we measured ROS generation
under abiotic conditions and intracellular ROS after exposure to
ZnO particles. The determination results of intracellular ROS levels
showed that intracellular ROS levels were significantly increased
after exposure to all ZnO particles at the dose above 5 ␮g/ml. And
there was an inverse correlation between intracellular ROS levels
and cell viability, and a positive correlation between intracellular
ROS levels and LDH level after a 24 h exposure to ZnO particles and
ZnCl2 , which is similar to the result of the work done by Deng et al.
(2009). However, test results of ROS generation under abiotic con-
ditions showed that although ROS generations evidently enhanced
with the increase of ZnO particles concentrate, they were lower
in both ZnO particles suspensions and supernatants, which could
not be enough to cause cytotoxicity (Wiseman et al., 2007). There-
fore, the high level of intracellular ROS may be not a major factor
in cytotoxicity of ZnO nanoparticles, but the cytotoxic response. In
this study, intracellular ROS could produce after zinc ions or ZnO
particles entering into the cells. On the one hand, Ana-1 cell was a
kind of phagocytic cells, the NADPH oxidase was the major source
of ROS production in phagocytic cells (Babior et al., 2002). On the
other hand, the entry of zinc ions and ZnO particles into cells led
to mitochondrial damage, leading to ROS generation (Wiseman et
al., 2007; Xia et al., 2008). It has been demonstrated that nanoma-
terials of various sizes and chemical compositions preferentially
mobilized to mitochondria and destructed mitochondrial function
(Foley et al., 2002; Li et al., 2003; Savic et al., 2003). And the ZnO par-
ticles taken up by cells may cause rapid dissolution, releasing Zn2+
ions (Xia et al., 2008), elevated intracellular Zn2+ induced the dis-
ruption of mitochondrial function (Bishop et al., 2007; Wiseman et
al., 2007) and triggered ROS generation (Xia et al., 2008). Moreover,
metabolisation of particles by P450 cytochrome can also be a source
of ROS, as demonstrated in the case of other particles (Takano et al.,
2002; Lanone and Boczkowski, 2006).
5. Conclusions
In summary, different sized ZnO particles induced slightly dif-
ferent cytotoxicity. The particles shape may impact on cytotoxicity
of ZnO particles. And the dissolved Zn2+ equilibrium concentra-
tions of ZnO particles in the complete cell medium were about
10 ␮g/ml, meanwhile, the supernatants induce 50% cell death. Thus,
the dissolved Zn2+ played a main role in toxic effect of ZnO parti-
cles. Furthermore, ROS generation of ZnO particles under abiotic
conditions is not enough to cause cytotoxicity. Although intracel-
lular ROS has the significant correlation with cell viability and LDH
induced by ZnO particles, ROS was not necessarily the main factor
of cytotoxicity of ZnO particles.
Conflict of interest statement
None.
Acknowledgement
The authors are grateful to the financial support from
the National Natural Science Foundation of China (30771771,
30800934 and 30901221) and the Natural Science Foudation of
Tianjin (10JCZDJC17100).
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