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Environment International
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a r t i c l e i n f o a b s t r a c t
Article history: Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly used nanomaterials in consumer products and
Received 24 December 2012 biomedical applications. As a result, human exposure to these NPs is highly frequent and they have become an
Accepted 28 February 2013 issue of concern to public health. Although toxicity of ZnO NPs has been extensively studied and they have been
Available online xxxx
shown to affect many different cell types and animal systems, there is a signicant lack of toxicological data for
ZnO NPs on the nervous system, especially for human neuronal cells and tissues. In this study, the cytotoxic and
Keywords:
Zinc oxide nanoparticles
genotoxic effects of ZnO NPs on human SHSY5Y neuronal cells were investigated under different exposure condi-
Cytotoxicity tions. Results obtained by ow cytometry showed that ZnO NPs do not enter the neuronal cells, but their presence
Genotoxicity in the medium induced cytotoxicity, including viability decrease, apoptosis and cell cycle alterations, and
Oxidative damage genotoxicity, including micronuclei production, H2AX phosphorylation and DNA damage, both primary and oxida-
SHSY5Y cells tive, on human neuronal cells in a dose- and time-dependent manner. Free Zn2+ ions released from the ZnO NPs
Zn+ 2 ions were not responsible for the viability decrease, but their role on other types of cell damage cannot be ruled out. The
results obtained in this work contribute to increase the knowledge on the genotoxic and cytotoxic potential of ZnO
NPs in general, and specically on human neuronal cells, but further investigations are required to understand the
action mechanism underlying the cytotoxic and genotoxic effects observed.
2013 Elsevier Ltd. All rights reserved.
1. Introduction (Vandebriel and De Jong, 2012), so they can potentially reach any organ
or tissue and involve a risk for human health. Toxicity of ZnO NPs has
Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly been extensively studied and they have been shown to affect many differ-
used nanomaterials in consumer products and biomedical applications ent cell types and animal systems (Chiang et al., 2012; De Berardis et al.,
due to their specic properties, e.g. transparency, high isoelectric 2010; Osman et al., 2010; Sharma et al., 2012a, 2012b; Wahab et al.,
point, biocompatibility, and photocatalytic efciency. They are widely 2011). Commonly, the toxicity of NPs is associated with their small size
employed in a variety of devices including cosmetics, toothpaste, sun- and high specic surface area and therefore, nanoforms are theoretically
screens, llings in medical materials, textiles, wall paints, and other expected to be more toxic than their bulk counterparts (Xiong et al.,
building materials, and they can be also utilized in environmental remedi- 2011).
ation for elimination or degradation of pollutants in water or air (Qiang, In recent years, there have been an increasing number of works
2001). Furthermore, ZnO NPs have promising applications in the medi- reporting that different NPs can reach the brain and cause neurological
cine eld since they have been proposed as a possible treatment for can- injuries, being associated even with neurodegenerative diseases (Block
cer and/or autoimmune diseases after being found to be selectively toxic et al., 2004; Hu and Gao, 2010; Peters et al., 2006). This translocation
towards potential disease-causing cells (Hanley et al., 2008; Premanathan can happen both directly, through axonal transport from olfactory
et al., 2011; Akhtar et al., 2012), and they are being considered to be used epithelium, or indirectly by passing to the bloodstream and crossing
in fabrication of nerve guidance channels for treatment of nerve injury the blood brain barrier (Oberdrster et al., 2004). Similarly to other
(Seil and Webster, 2008). As a result of all these uses, human exposure metal oxide NPs, it has been recently found that ZnO NPs reach the
to these NPs is highly frequent. They can enter the organism through brain of experimental animals after oral (Lee et al., 2012) and inhalatory
different pathways (respiratory tract, digestive system and parenteral (Kao et al., 2012a) administration. Still, there is a signicant lack of toxico-
routes) and have shown a systemic distribution in in vivo studies logical data for ZnO NPs on nervous system, especially for human neuro-
nal cells and tissues.
Corresponding author. Tel.: +351 223401147; fax: +351 223401149.
In vitro data have shown that ZnO NPs induce cytotoxicity in mouse
E-mail address: cstcosta@gmail.com (C. Costa). neuroblastoma Neuro-2A cells and neural stem cells (Deng et al., 2009;
1
These authors contributed equally to this work. Jeng and Swanson, 2006), in rat RSC96 Schwann cells and primary
0160-4120/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.envint.2013.02.013
Author's personal copy
neuronal cells (Chiang et al., 2012; Yin et al., 2012), and in human glioma (1%) were employed as positive controls in the cellular uptake and the
cells (Ostrovsky et al., 2009). Besides, they were found to enhance the lactate dehydrogenase (LDH) assays, respectively.
excitability of rat neurons by altering the ion channels (Zhao et al.,
2009) and to decrease the adhesion of rat astroglial cells (Seil and 2.4. Cellular viability
Webster, 2008), although in this last case the cells were exposed to
composite materials with ZnO NPs. MTT assay (according to Mosmann, 1983) and NRU assay (according
The only in vivo study describing neurological effects after ZnO NP to Borenfreund and Puerner, 1985) were used to test the potential effects
exposure reported attenuation in spatial learning and memory ability of ZnO NPs on viability of neuronal SHSY5Y cells. In each assay, 8 differ-
by alteration of synaptic plasticity in rats after intraperitoneal adminis- ent NP concentrations (080 g/ml) and 4 exposure times (3, 6, 24, and
tration (Han et al., 2011). Besides, morphological and histochemical 48 h) were evaluated. Absorbance was measured at 595 nm (MTT) or
changes in brains of rats fed with bulk ZnO were also described (Kozik 540 nm (NRU) using a Cambrex ELx808 microplate reader (Biotek,
et al., 1980). KC4). A parallel set of experiments conducted without cells was carried
Given the wide and frequent use of ZnO NPs in many elds closely out to exclude the potential interaction of the NPs with the dyes used
related to human beings, their promising benecial applications in in MTT and NRU assays. Data obtained demonstrated no interaction
medicine, and the scarce knowledge on their potential neurotoxic effects, between the ZnO NPs tested and the dyes used for cytotoxicity assess-
the main objective of this work was to investigate the cytotoxic and ment. From the results obtained in the viability experiments, two expo-
genotoxic effects of ZnO NPs on human SHSY5Y neuronal cells and to sure times (3 and 6 h) and three different concentrations of ZnO NPs (20,
explore the underlying mechanisms involved in these effects. 30 and 40 g/ml) were selected to perform the subsequent experiments.
Vuorescein isothiocyanate (FITC) (FL1) and PI (FL2) were analyzed were set according to the results obtained with FAAS. Cell viability was
using Cell Quest Pro software (Becton Dickinson, Madrid, Spain). evaluated using the MTT assay (procedure described above).
2.10. Dissolved zinc concentrations in the cell culture medium Cell percentages in the different phases of the cell cycle (G0/G1, S,
G2/M) were assessed by ow cytometry after ZnO NP treatment. Signif-
To quantify the Zn+2 ion concentrations released from the ZnO NPs, icant alterations of cell cycle were observed after both 3 and 6 h
suspensions (2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55 g/ml) (Table 2). Specically, ZnO NPs induced decreases in the percentage of
were incubated in complete cell medium for 3, 6 and 24 h at 37 C in a cells in G0/G1 and G2/M phases, and an increase in the percentage of
humidied 5% CO2 environment. Then centrifugation at 14,000 rpm for cells in S-phase. Besides, signicant doseresponse relationships were
30 min removed solid phase ZnO from the liquid medium. The Zn
content in the supernatant was analyzed by ame atomic absorption Table 1
spectroscopy (FAAS) (Thermoelemental Solaar S4 v.10.02). As negative Characterization of ZnO NPs.
control, cell culture medium without NPs but subjected to the same con- Particle size Specic surface area Hydrodynamic Zeta potential
ditions was used. (nm)a (m2/g)a diameter (mV)(DLS)
(nm)(DLS)
Fig. 1. Characterization of ZnO NPs in complete medium. Hydrodynamic diameter (a) and zeta potential (b).
found in phase G2/M (r = 0.631, P b 0.01) at 3 h, and in phases S 3.8. Oxidative damage
(r = 0.759, P b 0.01) and G2/M (r = 0.743, P b 0.01) at 6 h.
The NP potential to induce oxidative DNA damage was evaluated by
3.5. Membrane integrity means of comet assay modied by adding an incubation step with the
enzyme OGG1. Increases in DNA damage (%tDNA) in cells incubated
LDH activity in extracellular medium was evaluated as indicator of with OGG1 regarding those incubated only with buffer (indicative of
membrane integrity, since LDH is released when the membrane is oxidative DNA damage) were observed after ZnO NP treatment at all
damaged. Results obtained in this test are collected in Fig. 4. No sig- the concentrations and exposure times tested (Fig. 5).
nicant alteration in the percentage of LDH released was observed
in the neuronal cells after ZnO NP exposure at any treatment time. 3.9. Zn +2 ions: release from NPs and effects
a) Table 2
Results of cell cycle analysis in neuronal cells treated with ZnO NPs (%cells).
120
3H 6H 24H 48H Exposure time Concentration G0/G1 S-phase G2/M
100
3h Control 64.55 0.95 11.79 0.61 23.66 0.39
80 20 g/ml 54.98 1.09* 21.07 0.44* 23.96 1.44
% Viability
80
(Premanathan et al., 2011).
60 The potential of the NPs to be taken up by the cells was evaluated by
40 means of ow cytometry. Surprisingly, no uptake of ZnO NPs was
observed in any case. These negative results in cellular uptake disagree
20 with a few previous studies in other different cell types which observed
0 internalization of these NPs either by scanning electron microscopy or by
0 10 20 30 40 50 60 70 80 90 ow cytometry (Sharma et al., 2011, 2012b). However, it should be
ZnO NP concentration (g/ml) highlighted that a number of studies on in vitro cytotoxic and/or
genotoxic effects of ZnO NPs did not report their uptake by the cells
Fig. 2. Viability of neuronal cells treated with ZnO NPs. Results from MTT assay (a) and NRU (Akhtar et al., 2012; Hsiao and Huang, 2011; Lin et al., 2009). It was
assay (b). Values inside the rectangle are statistically different from the corresponding previously shown that NPs suspended in supplemented medium can
control.
be covered by the proteins of the medium reducing their ability to be
taken up by the cells (Lesniak et al., 2010). Moreover other parameters,
2012), or after oral (Pasupuleti et al., 2012; Sharma et al., 2012b) or including the cell cycle phase or the NP size and shape, may inuence
inhalatory (Wang et al., 2010) administration. However, there is a lack the internalization rate of NPs in cells (Kim et al., 2011). Thus, it seems
of information on the effects of these NPs on neurons and on the nervous that the specic conditions employed in our experiments, especially
system, particularly those from humans. regarding the use of medium supplemented with FBS, are limiting in
In this study, the potential neurotoxic effects of ZnO NPs were someway the entry of ZnO NPs into the neuronal cells.
assessed in vitro in SHSY5Y cells. Several concentrations and exposure The potential cell cycle alterations and apoptosis induced by ZnO NPs
times were tested and a battery of cytotoxicity and genotoxicity method- were assessed by different ow cytometry methodologies. Results
ologies were employed with the aim of characterizing in depth the obtained from the cell cycle analysis show that the ZnO NPs induce
effects of ZnO NPs on human neuronal cells as well as attempting to important cell cycle alterations, including mitotic arrest, in SHSY5Y
understand the underlying action mechanisms. cells after both 3 h and 6 h treatments. Similar to these results, ZnO
Results obtained from MTT and NRU assays showed that ZnO NPs NPs were also recently found to induce cell cycle alterations and mitotic
diminished the viability of the neuronal cells in a dose-dependent man- arrest in neuronal RSC96 Schwann cells treated for 12 h (880 g/ml)
ner at all the treatment times. According to our results, a concentration- (Yin et al., 2012).
dependent decrease in cellular viability after ZnO NP exposure was pre- Likewise, results obtained from the annexin V/PI analysis showed
viously described for several cell types, including astrocytoma U87 (Lai et increases in the percentage of apoptosis induction following both 3 h
al., 2008), hepatocarcinoma HepG2 (Sharma et al., 2012b), lung carcino- and 6 h ZnO NP treatments. Under experimental conditions similar to
ma A549 (Fukui et al., 2012), and epidermal A431 (Sharma et al., 2009) the ones employed in this study, ZnO NPs were previously reported to
cells. There is also evidence that ZnO NPs are particularly cytotoxic to induce cell death in a variety of cell lines (Akhtar et al., 2012; Sharma
rapidly dividing cancer cells relative to quiescent cells of the same et al., 2012b), including cells from the nervous system such as neural
stem cells and glioma cells (Deng et al., 2009; Wahab et al., 2011; Yin
et al., 2012). On the basis of our data, this apoptosis was scarcely associ-
30
3h 6h ated with losses of the mitochondrial membrane potential, as shown by
*
% Cells with NPs
25 the slight effects observed in the MMP assessment. Therefore, our results
20 in apoptosis evaluation suggest that ZnO NPs induce death cell mainly by
a mitochondria-independent pathway. Some previous studies reported
15
mitochondrial alterations after ZnO NP treatments in other different ex-
*
10 perimental systems (De Berardis et al., 2010; Li et al., 2012; Sharma et al.,
5 2012b). Nonetheless, in the study by Sharma et al. (2012b) the NPs were
efciently taken up by the HepG2 cells, penetration of the NPs in the
0
LoVo cells was not evaluated in the work by De Berardis et al. (2010),
Control 20 30 40 PC
and Li et al. (2012) exposed rat mitochondrion directly to the ZnO
ZnO NP concentration (g/ml)
NPs. Thus there are substantial differences between these studies and
Fig. 3. Uptake of ZnO NPs by SHSY5Y cells as analyzed by ow cytometry. PC: positive the current one, so further investigation is required to conrm our
control. *P b 0.01, signicant difference with regard to the corresponding control. observation.
Author's personal copy
100 3h 6h
90
80
70
% LDH activity
60
50
40
30
20
10
0
PC
-10 0 5 10 15 20 25 30 35 40 45
Fig. 4. Results of membrane integrity assessment (LDH assay) in SHSY5Y cells exposed to ZnO NPs.
Different kinds of ZnO NP-induced genotoxic damage were evaluated explain these effects. On one hand the ZnO NPs can potentially interact
by means of several approaches, i.e., MN test, H2AX analysis and comet with the cell membrane to cause the observed toxicity. In this study,
assay. Results obtained from all these biomarkers showed DNA damage the potential alterations in the membrane integrity caused by ZnO NP
(comet assay) induction by the ZnO NPs since the 3 h exposure, but exposure were assessed by LDH assay, but negative results were
only after the 6 h exposure an important dose-dependent MN induction obtained after both 3 and 6 h of treatment. However, we cannot rule
could be observed, which was accompanied by an increase in phosphor- out the possible interaction of NPs with some component of the cellular
ylation of the histone H2AX. It should be taken into account that the membrane without altering its integrity. Zhao et al. (2009) studied the
highest concentration of ZnO NPs used (40 g/ml) induced around 40% effect of ZnO NPs on ion channels and excitability of rat neurons. On
of cytotoxicity at 3 and 6 h, so this fact can be inuencing the non- the basis of their results, they suggested that ZnO NPs can enhance the
signicant results obtained with this concentration for histone phos- excitability of neurons by increasing the opening number of sodium
phorylation (at both treatment times) and comet assay (at 6 h). ZnO channels and delaying rectier potassium channels. These alterations
NPs were previously reported to induce genotoxicity in other different in ion channels may disturb the ionic homeostasis and consequently
cell types including primary keratinocytes (Sharma et al., 2011), bro- the physiological functions of neurons.
blasts (Chiang et al., 2012), epidermal A431 cells (Sharma et al., 2009), On the other hand, several previous studies concluded that ZnO NPs
lung A549 cells (Fukui et al., 2012), cervix HEp-2 carcinoma cells may induce cytotoxicity and genotoxicity by releasing Zn2+ ions. Deng
(Osman et al., 2010), and HepG2 cells (Sharma et al., 2012b). However, et al. (2009), after evaluating the cytotoxicity of ZnO NPs in mouse neural
studies evaluating the effects of these NPs on genetic material of neuro- stem cells at different levels (cell viability, apoptosis and necrosis),
nal cells are highly scarce. Particularly, Chiang et al. (2012) observed reported that ZnO NP toxicity mainly came from the Zn2+ ions dissolved
DNA damage after exposing rat primary neuronal cells to ZnO NPs, and in the culture medium or inside cells. Fukui et al. (2012) found cytotox-
Wahab et al. (2011) found results very parallel to ours in MN induction icity due to the increased levels of Zn+2 ions in vitro, in ZnO NP-treated
on human U87 glioma cells treated with different Zn nanostructures, at human lung carcinoma cells, and in vivo, in rat lung cells after
similar concentrations to those employed in the current study. intratracheal instillation of ZnO NPs. And also cytotoxic effects linked to
The NP potential to induce oxidative DNA damage was also evaluated the presence of high levels of Zn+2 ions in both cytosol and mitochondria
by means of comet assay modied with the enzyme OGG1. Results of broncho-alveolar lavage (BAL) cells and white blood cells were found
obtained show that ZnO NPs induced oxidative damage at both 3 and in rats exposed to ZnO NPs (Kao et al., 2012b). Furthermore, increases in
6 h treatments. These data agree with previous studies reporting in- intracellular levels of Zn+2 ions released from ZnO NPs were correlated
creased values of oxidative stress and ROS production after ZnO NP expo- with high levels of ROS and, consequently, with cell death (Fukui et al.,
sure in a variety of cell types (Akhtar et al., 2012; De Berardis et al., 2010;
Sharma et al., 2009) and in animal studies (Fukui et al., 2012; Hao and
Chen, 2012; Sharma et al., 2012b).
Table 4
As expounded so far, although no cellular uptake of ZnO NPs was Genotoxicity evaluation of ZnO NPs. Results of MN assay (a), comet assay (b) and H2AX
observed, we obtained induction of cytotoxic and genotoxic effects by analysis (c).
these NPs on neuronal cells. Two main reasons may be suggested to
3h 6h
PC: positive control. **P b 0.01, signicant differences regarding the control. PC: positive control. *P b 0.05, **P b 0.01, signicant differences regarding the control.
Author's personal copy
120
a) *
30 100
buffer OGG1
**
% Viability
25 80
20 ** 60
%tDNA
* **
15 * 40
**
10 20
5 0
0 0.05 0.1 0.2 0.4 0.6 0.8 PC
0 Zn+2 concentration (mM)
Control 20 30 40 PC
+2
ZnO NP concentration (g/ml) Fig. 7. Cytotoxicity of Zn ions: MTT assay in SHSY5Y cells treated with ZnSO4.
**P b 0.01, signicant difference with regard to the control.
b)
35
buffer OGG1 *
30
25
* means of FAAS. For all the times assayed (3, 6 and 24 h) linear increases
%tDNA
0,40
3h 6h 24h
0,35
Zn2+ concentration (mM)
0,30
0,25
0,20
0,15
0,10
0,05
0,00
0 2 4 6 8 10 15 20 25 30 35 40 45 50 55
ZnO NP concentration (g/ml)
Fig. 6. Analysis of Zn+2 ions in cell culture medium released from ZnO NPs.
Author's personal copy
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