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Environment International 55 (2013) 92100

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Neuronal cytotoxicity and genotoxicity induced by zinc oxide nanoparticles

Vanessa Valdiglesias a, b, 1, Carla Costa c,, 1, Gzde Kili a, d, Solange Costa c, Eduardo Psaro a,
Blanca Laffon a, Joo Paulo Teixeira c
a
Toxicology Unit, Department of Psychobiology, University of A Corua, Edicio de Servicios Centrales de Investigacin, Campus Elvia s/n, 15071, A Corua, Spain
b
Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166, Roma, Italy
c
Department of Environmental Health, Portuguese National Institute of Health, Rua Alexandre Herculano, 321, 4000-055, Porto, Portugal
d
Department of Cell and Molecular Biology, University of A Corua, Facultad de Ciencias, Campus A Zapateira s/n, 15071, A Corua, Spain

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
Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly
used nanomaterials in consumer products and biomedical applications
due to their specic properties, e.g. transparency, high isoelectric
point, biocompatibility, and photocatalytic efciency. They are widely
employed in a variety of devices including cosmetics, toothpaste, sun-
screens, llings in medical materials, textiles, wall paints, and other
building materials, and they can be also utilized in environmental remedi-
ation for elimination or degradation of pollutants in water or air (Qiang,
2001). Furthermore, ZnO NPs have promising applications in the medi-
cine eld since they have been proposed as a possible treatment for can-
cer and/or autoimmune diseases after being found to be selectively toxic
towards potential disease-causing cells (Hanley et al., 2008; Premanathan
et al., 2011; Akhtar et al., 2012), and they are being considered to be used
in fabrication of nerve guidance channels for treatment of nerve injury
(Seil and Webster, 2008). As a result of all these uses, human exposure
to these NPs is highly frequent. They can enter the organism through
different pathways (respiratory tract, digestive system and parenteral
routes) and have shown a systemic distribution in in vivo studies
Corresponding author. Tel.: +351 223401147; fax: +351 223401149.
E-mail address: cstcosta@gmail.com (C. Costa).
1
These authors contributed equally to this work.
(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
been extensively studied and they have been shown to affect many differ-
ent cell types and animal systems (Chiang et al., 2012; De Berardis et al.,
2010; Osman et al., 2010; Sharma et al., 2012a, 2012b; Wahab et al.,
2011). Commonly, the toxicity of NPs is associated with their small size
and high specic surface area and therefore, nanoforms are theoretically
expected to be more toxic than their bulk counterparts (Xiong et al.,
2011).
In recent years, there have been an increasing number of works
reporting that different NPs can reach the brain and cause neurological
injuries, being associated even with neurodegenerative diseases (Block
et al., 2004; Hu and Gao, 2010; Peters et al., 2006). This translocation
can happen both directly, through axonal transport from olfactory
epithelium, or indirectly by passing to the bloodstream and crossing
the blood brain barrier (Oberdrster et al., 2004). Similarly to other
metal oxide NPs, it has been recently found that ZnO NPs reach the
brain of experimental animals after oral (Lee et al., 2012) and inhalatory
(Kao et al., 2012a) administration. Still, there is a signicant lack of toxico-
logical data for ZnO NPs on nervous system, especially for human neuro-
nal cells and tissues.
In vitro data have shown that ZnO NPs induce cytotoxicity in mouse
neuroblastoma Neuro-2A cells and neural stem cells (Deng et al., 2009;
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
V. Valdiglesias et al. / Environment International 55 (2013) 92100
neuronal cells (Chiang et al., 2012; Yin et al., 2012), and in human glioma
cells (Ostrovsky et al., 2009). Besides, they were found to enhance the
excitability of rat neurons by altering the ion channels (Zhao et al.,
2009) and to decrease the adhesion of rat astroglial cells (Seil and
Webster, 2008), although in this last case the cells were exposed to
composite materials with ZnO NPs.
The only in vivo study describing neurological effects after ZnO NP
exposure reported attenuation in spatial learning and memory ability
by alteration of synaptic plasticity in rats after intraperitoneal adminis-
tration (Han et al., 2011). Besides, morphological and histochemical
changes in brains of rats fed with bulk ZnO were also described (Kozik
et al., 1980).
Given the wide and frequent use of ZnO NPs in many elds closely
related to human beings, their promising benecial applications in
medicine, and the scarce knowledge on their potential neurotoxic effects,
the main objective of this work was to investigate the cytotoxic and
genotoxic effects of ZnO NPs on human SHSY5Y neuronal cells and to
explore the underlying mechanisms involved in these effects.
2. Materials and methods
2.1. Chemicals
ZnO NPs (CAS No. 1314-13-2), mytomycin C (MMC) (CAS No.
50-07-7), bleomycin (BLM) (CAS No. 9041-93-4), camptothecin
(Campt) (CAS No. 7689-03-4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) (CAS No. 298-93-1), 5,5,6,6-tetrachloro-
1,1,3,3-tetraethyl-imidacarbocyanine iodide (JC-1) (CAS No. 3520-43-
2), neutral red dye (CAS No. 553-24-2), and propidium iodide (PI) were
purchased from Sigma-Aldrich Co. (Madrid, Spain). MMC, BLM, and
Campt were dissolved in sterile distilled water prior use.
2.2. Nanoparticle suspension: preparation and characterization
A stock suspension of ZnO NPs (nal concentration 80 g/ml) was
prepared in either deionized water or complete cell culture medium
(with fetal bovine serum, FBS). Prior to each treatment, this suspension
was ultrasonicated (Branson Sonier, USA) at 30 W for 5 min (1.5 min
on and 1 min off twice, and 2 min on), and diluted to prepare the differ-
ent NP concentrations tested. Average hydrodynamic size, size distribu-
tion and zeta potential of particles in suspension were determined by
Dynamic Light Scattering (DLS) using a Zetasizer Nano-ZS equipped
with 4.0 mW, 633 nm laser (Model ZEN 3600, Malvern Instruments
Ltd., Malvern, UK).
2.3. Cell culture and treatments
Human neuroblastoma SHSY5Y cell line was obtained from the
European Collection of Cell Cultures and cultured in nutrient mixture
EMEM/F12 (1:1) medium with 1% non-essential amino acids, 1% antibi-
otic and antimycotic solution, and supplemented with 10% heat-
inactivated fetal bovine serum (FBS), all from Invitrogen (Barcelona,
Spain). The cells were incubated in a humidied atmosphere with 5%
CO2 at 37 C. To carry out the experiments, cells were seeded in
96-well plates (at bottom) and allowed to adhere for 24 h at 37 C.
Cell densities were approximately in the range of 25 105 cells/well
at the beginning of cell culture. For cell treatment, these were incubated
at 37 C for different periods in the presence of a variable number of NP
concentrations, depending on the assay performed, or the control solu-
tions. Complete medium was used as negative control in all experiments.
The following chemicals were used as positive controls: Campt (10 M)
in apoptosis assessment, cell cycle and H2AX analysis; and MMC
(1.5 M), BLM (1 g/ml) and H2O2 (2 M for 3 h treatments, and 1 M
for 6 h treatments) in MN test, comet assay, and oxidative DNA damage
evaluation, respectively. Besides, TiO2 NPs (120 g/ml) and triton X-100
93
(1%) were employed as positive controls in the cellular uptake and the
lactate dehydrogenase (LDH) assays, respectively.
2.4. Cellular viability
MTT assay (according to Mosmann, 1983) and NRU assay (according
to Borenfreund and Puerner, 1985) were used to test the potential effects
of ZnO NPs on viability of neuronal SHSY5Y cells. In each assay, 8 differ-
ent NP concentrations (080 g/ml) and 4 exposure times (3, 6, 24, and
48 h) were evaluated. Absorbance was measured at 595 nm (MTT) or
540 nm (NRU) using a Cambrex ELx808 microplate reader (Biotek,
KC4). A parallel set of experiments conducted without cells was carried
out to exclude the potential interaction of the NPs with the dyes used
in MTT and NRU assays. Data obtained demonstrated no interaction
between the ZnO NPs tested and the dyes used for cytotoxicity assess-
ment. From the results obtained in the viability experiments, two expo-
sure times (3 and 6 h) and three different concentrations of ZnO NPs (20,
30 and 40 g/ml) were selected to perform the subsequent experiments.
2.5. Cellular uptake
A ow cytometry methodology (FACSCalibur Flow Cytometer, Becton
Dickinson, Madrid, Spain) was employed to assess the potential of the
ZnO NPs to enter the cells. The analysis was carried out on the basis of
the size and complexity of the cells by measuring the forward scatter
(FSC) and the side scatter (SSC) following the protocol described by
Suzuki et al. (2007).
2.6. Cell cycle
The cell distribution along the different phases of the cell cycle was
examined in cells treated with ZnO NPs or the controls by evaluating
the relative cellular DNA content with a ow cytometry technique
(FACSCalibur Flow Cytometer, Becton Dickinson, Madrid, Spain) follow-
ing Valdiglesias et al. (2011a). DNA content was assessed from the PI
signal detected by the FL2 detector in a minimum of 104 events. Cell
Quest Pro software (Becton Dickinson, Madrid, Spain) was used to
analyze cell cycle histograms, to calculate the percentage of occupancy
of G0/G1, S and G2/M regions.
2.7. Membrane integrity
Lactate dehydrogenase (LDH) activity in cell culture medium was
measured by using a commercial kit (Roche Diagnostics Corp) according
to the manufacturer's instructions. After exposure, half the amount of the
cell culture medium was collected for LDH measurement. Absorption
was measured at 490 nm with a reference wavelength of 655 nm
using a Cambrex ELx808 microplate reader (Biotek, KC4). Positive con-
trol experiments were performed with 0.1% Triton X-100 and set as
100% cytotoxicity. LDH release was calculated by the following equation:
Asample Amedium
LDH%  100
Apositivecontrol Amedium
where [A]sample, [A]medium, and [A]positive control denote the absorbance of
the sample, medium negative control and Triton X-100 positive control,
respectively.
2.8. Apoptosis
2.8.1. Analysis of apoptosis by PIannexin V staining
Apoptosis rate was determined by means of annexin V/PI double
staining, using the BD Pharmingen annexin VFITC apoptosis detection
kit I (Becton Dickinson, Madrid, Spain), following the manufacturer's rec-
ommendations. At least 104 events were acquired with a FACSCalibur
Flow Cytometry (Becton Dickinson, Madrid, Spain). Data from annexin
 Author's personal copy
94 V. Valdiglesias et al. / Environment International 55 (2013) 92100
Vuorescein isothiocyanate (FITC) (FL1) and PI (FL2) were analyzed
using Cell Quest Pro software (Becton Dickinson, Madrid, Spain).
2.8.2. Mitochondrial membrane potential analysis
Changes in mitochondrial membrane potential (MMP) after ZnO
NP treatments were evaluated by ow cytometry (FACSCalibur,
Becton Dickinson, Madrid, Spain) as previously described (Sharma
et al., 2012b). Data obtained from at least 10 4 events were analyzed
by Cell Quest Pro software (Becton Dickinson, Madrid, Spain).
2.9. Genotoxicity
2.9.1. Micronuclei evaluation by ow cytometry
After incubation of the cells in the presence or absence of ZnO NPs,
they were cultured in fresh medium for an additional period of 24 h.
Then a suspension of nuclei and micronuclei (MN) was prepared
according to the procedure described by Nsse et al. (1994) and
Roman et al. (1998), with some modications (Valdiglesias et al.,
2011b). The nal suspension was analyzed with a FACSCalibur Flow
Cytometer (Becton Dickinson, Madrid, Spain) as previously reported
(Avlasevich et al., 2006).
2.9.2. H2AX assay
The general protocol proposed by Tanaka et al. (2009) with minor
changes (Valdiglesias et al., 2011b) was followed for the evaluation of
H2AX histone phosphorylation after ZnO NP treatments. Data from
Alexa Fluor 488 (FL1) and PI (FL2) intensities from a minimum of
10 4 events were acquired with a FACSCalibur Flow Cytometer (Becton
Dickinson, Madrid, Spain). Data analyses were performed using Cell
Quest Pro software (Becton Dickinson, Madrid, Spain).
2.9.3. Comet assay
After treatments with ZnO NPs, the alkaline comet assay was
performed following Singh et al. (1988), with minor changes (Costa et
al., 2008). Comet IV Software (Perceptive Instruments, Haverhill, UK)
was used for image capture and analysis. In all cases 100 cells were
scored (50 from each replicate slide), and percentage of DNA in the
Comet tail (%tDNA) was used as DNA damage parameter.
2.9.4. Oxidative DNA damage
A modied version of the comet assay incorporating incubation with
8-oxoguanine DNA glycosylase (OGG1) repair enzyme was employed to
evaluate the oxidative DNA damage, specically the presence of
8-oxoguanine, following the protocol described by Smith et al. (2006).
Treatments following the same protocol with just buffer and no OGG1
enzyme served as reference. Similar to the standard comet assay, 100
cells per condition were scored to determine %tDNA.
2.10. Dissolved zinc concentrations in the cell culture medium
To quantify the Zn+2 ion concentrations released from the ZnO NPs,
suspensions (2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55 g/ml)
were incubated in complete cell medium for 3, 6 and 24 h at 37 C in a
humidied 5% CO2 environment. Then centrifugation at 14,000 rpm for
30 min removed solid phase ZnO from the liquid medium. The Zn
content in the supernatant was analyzed by ame atomic absorption
spectroscopy (FAAS) (Thermoelemental Solaar S4 v.10.02). As negative
control, cell culture medium without NPs but subjected to the same con-
ditions was used.
2.11. Cytotoxic effects of Zn +2 ions
In order to test whether cytotoxicity of ZnO nanoparticles was due to
Zn2+ ions released from ZnO NPs, cells were treated for 3 h with
ZnSO4.7H2O at 0.05, 0.1, 0.2, 0.4, 0.6, and 0.8 mM. These concentrations
were set according to the results obtained with FAAS. Cell viability was
evaluated using the MTT assay (procedure described above).
2.12. Statistical analysis
Statistical analyses were performed using SPSS for Windows statisti-
cal package (version 20.0). Distribution of the response variables depart-
ed signicantly from normality (KolmogorovSmirnov goodness of t
test) and therefore non-parametric tests were considered adequate for
the statistical analysis of these data. Differences among groups were
tested with KruskalWallis test and MannWhitney U-test. The associa-
tions between two variables were analyzed by Spearman's correlation. A
minimum of three independent experiments were performed for each
experimental condition tested. Experimental data were expressed
as mean standard error and a P-value of b 0.05 was considered
signicant.
3. Results
3.1. Nanoparticle characterization
Table 1 summarizes the description and characterization data of the
ZnO NPs employed in this study. The average hydrodynamic size, size
distribution and zeta potential of ZnO NPs in water and in complete
cell culture medium suspensions were assessed by Dynamic Light
Scattering (DLS). Slightly higher values of the mean hydrodynamic diam-
eter and the zeta potential (negative) were observed in culture medium
supplemented with FBS (distributions shown in Fig. 1(a) and (b), respec-
tively) than in water.
3.2. Cellular viability
Results obtained from both cytotoxicity tests, MTT and NRU, are
represented in Fig. 2. ZnO NPs induced dose-dependent decreases in
viability percentages of SHSY5Y cells, particularly pronounced at 24
and 48 h treatments in MTT assay. For MTT assay signicance was
obtained from 10 g/ml on treatment periods longer than 6 h, and for
both cytotoxicity tests from 25 g/ml on all the treatment times.
3.3. Uptake
Fig. 3 shows the results obtained from the evaluation of the cellular
uptake by ow cytometry. No uptake of ZnO NPs by the SHSY5Y cells
was observed at any concentration or exposure time tested.
3.4. Cell cycle
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-
icant alterations of cell cycle were observed after both 3 and 6 h
(Table 2). Specically, ZnO NPs induced decreases in the percentage of
cells in G0/G1 and G2/M phases, and an increase in the percentage of
cells in S-phase. Besides, signicant doseresponse relationships were
Table 1
Characterization of ZnO NPs.
Particle size Specic surface area Hydrodynamic Zeta potential
(nm)a (m2/g)a diameter (mV)(DLS)
(nm)(DLS)
Water Medium Water Medium
100(BET) 1525 243.7 273.4 8.23 11.7
BET: BrunauerEmmettTeller.
DLS: Dynamic Light Scattering.
a
Provided by the commercial supplier.
 Author's personal copy
V. Valdiglesias et al. / Environment International 55 (2013) 92100
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
(r = 0.759, P b 0.01) and G2/M (r = 0.743, P b 0.01) at 6 h.
3.5. Membrane integrity
LDH activity in extracellular medium was evaluated as indicator of
membrane integrity, since LDH is released when the membrane is
damaged. Results obtained in this test are collected in Fig. 4. No sig-
nicant alteration in the percentage of LDH released was observed
in the neuronal cells after ZnO NP exposure at any treatment time.
3.6. Apoptosis
Table 3 shows the results obtained from apoptosis assessment by
annexin V/PI analysis and MMP assay. Increases in the percentage of
apoptotic cells were found after both 3 h and 6 h ZnO NP treatments
(annexin V results). However, only slight increases in mitochondrial
membrane potential, with just a statistically signicant result at
20 g/ml after 3 h of exposure, were observed.
3.7. Genotoxicity
Results obtained from the MN test, comet assay, and analysis of
H2AX are collected in Table 4. ZnO NPs did not induce MN production
after the 3 h exposure. However, an important dose-dependent MN
induction was observed after the 6 h treatment (r = 0.635, P b 0.01).
Furthermore, increases in the levels of H2AX phosphorylation and DNA
damage were observed in all cases. These increases were statistically
signicant in all the conditions tested in the comet assay, except for the
highest concentration after the 6 h exposure. However, statistically
signicant values of phosphorylated H2AX were obtained only at the
30 g/ml treatments.
95
3.8. Oxidative damage
The NP potential to induce oxidative DNA damage was evaluated by
means of comet assay modied by adding an incubation step with the
enzyme OGG1. Increases in DNA damage (%tDNA) in cells incubated
with OGG1 regarding those incubated only with buffer (indicative of
oxidative DNA damage) were observed after ZnO NP treatment at all
the concentrations and exposure times tested (Fig. 5).
3.9. Zn +2 ions: release from NPs and effects
After incubation of ZnO NPs (2 to 55 g/ml) in complete cell media
for 3, 6 and 24 h, Zn+2 ions were quantied by FAAS in the liquid super-
natant fraction. Results obtained were very similar at all the treatment
times tested (Fig. 6). A dose-dependent linear increase in Zn+2 ions
was observed in cell culture medium up to approximately 30 g/ml
ZnO NPs; then a plateau is reached.
To investigate the potential cytotoxicity of the released Zn+2 ions, the
cells were treated with a soluble Zn salt (ZnSO4) to obtain Zn+2 ion
concentrations comparable to those existing in the initial ZnO NP suspen-
sions (FAAS results: 0.05 to 0.8 mM). Signicant decreases in the cell
viability were found only at the highest concentrations tested (0.6 and
0.8 mM) (Fig. 7).
4. Discussion
The increasing use of the ZnO NPs in a number of worldwide indus-
tries and, specially, their potential benets in the medicine eld have
brought attentions to their potential toxicity and health risks. It is well
established that, under specic conditions, ZnO NPs may result toxic to
a variety of mammalian and human cells (De Berardis et al., 2010; Kim
et al., 2010; Osman et al., 2010; Sharma et al., 2009, 2012b; Wahab et
al., 2011) and to animals after intratracheal instillation (Fukui et al.,
 Author's personal copy

96 V. Valdiglesias et al. / Environment International 55 (2013) 92100

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

30 g/ml 60.19 1.22* 20.47 0.86* 19.33 2.07*

60 40 g/ml 61.25 0.51* 18.19 0.88* 20.56 1.29*
PC 69.77 0.67* 13.55 0.73 16.68 0.20*
40 6h Control 62.50 0.57 9.75 0.85 27.75 1.12
20 g/ml 50.63 0.61** 20.78 0.77** 28.59 0.77
20 30 g/ml 57.71 0.23** 32.50 3.39** 9.79 3.41**
40 g/ml 61.18 0.44 25.72 2.13** 13.10 2.33**
0 PC 65.10 1.43 15.92 0.27** 18.98 1.19**
0 10 20 30 40 50 60 70 80 90
-20 PC: positive control. *P b 0.05, **P b 0.01, signicant differences regarding the control.
ZnO NP concentration (g/ml)

b) lineage suggesting that ZnO NP toxicity is related to the proliferative po-

120 3H 6H 24H 48h
tential of the cell (Hanley et al., 2008). Our results also support that ZnO
100 NPs present an antiproliferative effect; this capacity led different authors
to indicate ZnO NPs as a promising antibacterial and anticancer agent
% Viability

80
60
40
20
0 internalization of these NPs either by scanning electron microscopy or by
0 10 20 30 40 50 60 70
ZnO NP concentration (g/ml)
Fig. 2. Viability of neuronal cells treated with ZnO NPs. Results from MTT assay (a) and NRU
assay (b). Values inside the rectangle are statistically different from the corresponding
control.
2012), or after oral (Pasupuleti et al., 2012; Sharma et al., 2012b) or
inhalatory (Wang et al., 2010) administration. However, there is a lack
of information on the effects of these NPs on neurons and on the nervous
system, particularly those from humans.
In this study, the potential neurotoxic effects of ZnO NPs were
assessed in vitro in SHSY5Y cells. Several concentrations and exposure
times were tested and a battery of cytotoxicity and genotoxicity method-
ologies were employed with the aim of characterizing in depth the
effects of ZnO NPs on human neuronal cells as well as attempting to
understand the underlying action mechanisms.
Results obtained from MTT and NRU assays showed that ZnO NPs
diminished the viability of the neuronal cells in a dose-dependent man-
ner at all the treatment times. According to our results, a concentration-
dependent decrease in cellular viability after ZnO NP exposure was pre-
viously described for several cell types, including astrocytoma U87 (Lai et
al., 2008), hepatocarcinoma HepG2 (Sharma et al., 2012b), lung carcino-
ma A549 (Fukui et al., 2012), and epidermal A431 (Sharma et al., 2009)
cells. There is also evidence that ZnO NPs are particularly cytotoxic to
rapidly dividing cancer cells relative to quiescent cells of the same
30
3h 6h
% Cells with NPs
(Premanathan et al., 2011).
The potential of the NPs to be taken up by the cells was evaluated by
means of ow cytometry. Surprisingly, no uptake of ZnO NPs was
observed in any case. These negative results in cellular uptake disagree
with a few previous studies in other different cell types which observed
80 90 ow cytometry (Sharma et al., 2011, 2012b). However, it should be
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
(Akhtar et al., 2012; Hsiao and Huang, 2011; Lin et al., 2009). It was
previously shown that NPs suspended in supplemented medium can
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,
including the cell cycle phase or the NP size and shape, may inuence
the internalization rate of NPs in cells (Kim et al., 2011). Thus, it seems
that the specic conditions employed in our experiments, especially
regarding the use of medium supplemented with FBS, are limiting in
someway the entry of ZnO NPs into the neuronal cells.
The potential cell cycle alterations and apoptosis induced by ZnO NPs
were assessed by different ow cytometry methodologies. Results
obtained from the cell cycle analysis show that the ZnO NPs induce
important cell cycle alterations, including mitotic arrest, in SHSY5Y
cells after both 3 h and 6 h treatments. Similar to these results, ZnO
NPs were also recently found to induce cell cycle alterations and mitotic
arrest in neuronal RSC96 Schwann cells treated for 12 h (880 g/ml)
(Yin et al., 2012).
Likewise, results obtained from the annexin V/PI analysis showed
increases in the percentage of apoptosis induction following both 3 h
and 6 h ZnO NP treatments. Under experimental conditions similar to
the ones employed in this study, ZnO NPs were previously reported to
induce cell death in a variety of cell lines (Akhtar et al., 2012; Sharma
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-
ated with losses of the mitochondrial membrane potential, as shown by
*

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

V. Valdiglesias et al. / Environment International 55 (2013) 92100 97

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

ZnO NPs concentration (g/ml)

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
by means of several approaches, i.e., MN test, H2AX analysis and comet
assay. Results obtained from all these biomarkers showed DNA damage
(comet assay) induction by the ZnO NPs since the 3 h exposure, but
only after the 6 h exposure an important dose-dependent MN induction
could be observed, which was accompanied by an increase in phosphor-
ylation of the histone H2AX. It should be taken into account that the
highest concentration of ZnO NPs used (40 g/ml) induced around 40%
of cytotoxicity at 3 and 6 h, so this fact can be inuencing the non-
signicant results obtained with this concentration for histone phos-
phorylation (at both treatment times) and comet assay (at 6 h). ZnO
NPs were previously reported to induce genotoxicity in other different
cell types including primary keratinocytes (Sharma et al., 2011), bro-
blasts (Chiang et al., 2012), epidermal A431 cells (Sharma et al., 2009),
lung A549 cells (Fukui et al., 2012), cervix HEp-2 carcinoma cells
(Osman et al., 2010), and HepG2 cells (Sharma et al., 2012b). However,
studies evaluating the effects of these NPs on genetic material of neuro-
nal cells are highly scarce. Particularly, Chiang et al. (2012) observed
DNA damage after exposing rat primary neuronal cells to ZnO NPs, and
Wahab et al. (2011) found results very parallel to ours in MN induction
on human U87 glioma cells treated with different Zn nanostructures, at
similar concentrations to those employed in the current study.
The NP potential to induce oxidative DNA damage was also evaluated
by means of comet assay modied with the enzyme OGG1. Results
obtained show that ZnO NPs induced oxidative damage at both 3 and
6 h treatments. These data agree with previous studies reporting in-
creased values of oxidative stress and ROS production after ZnO NP expo-
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).
As expounded so far, although no cellular uptake of ZnO NPs was
observed, we obtained induction of cytotoxic and genotoxic effects by
these NPs on neuronal cells. Two main reasons may be suggested to
explain these effects. On one hand the ZnO NPs can potentially interact
with the cell membrane to cause the observed toxicity. In this study,
the potential alterations in the membrane integrity caused by ZnO NP
exposure were assessed by LDH assay, but negative results were
obtained after both 3 and 6 h of treatment. However, we cannot rule
out the possible interaction of NPs with some component of the cellular
membrane without altering its integrity. Zhao et al. (2009) studied the
effect of ZnO NPs on ion channels and excitability of rat neurons. On
the basis of their results, they suggested that ZnO NPs can enhance the
excitability of neurons by increasing the opening number of sodium
channels and delaying rectier potassium channels. These alterations
in ion channels may disturb the ionic homeostasis and consequently
the physiological functions of neurons.
On the other hand, several previous studies concluded that ZnO NPs
may induce cytotoxicity and genotoxicity by releasing Zn2+ ions. Deng
et al. (2009), after evaluating the cytotoxicity of ZnO NPs in mouse neural
stem cells at different levels (cell viability, apoptosis and necrosis),
reported that ZnO NP toxicity mainly came from the Zn2+ ions dissolved
in the culture medium or inside cells. Fukui et al. (2012) found cytotox-
icity due to the increased levels of Zn+2 ions in vitro, in ZnO NP-treated
human lung carcinoma cells, and in vivo, in rat lung cells after
intratracheal instillation of ZnO NPs. And also cytotoxic effects linked to
the presence of high levels of Zn+2 ions in both cytosol and mitochondria
of broncho-alveolar lavage (BAL) cells and white blood cells were found
in rats exposed to ZnO NPs (Kao et al., 2012b). Furthermore, increases in
intracellular levels of Zn+2 ions released from ZnO NPs were correlated
with high levels of ROS and, consequently, with cell death (Fukui et al.,
Table 4
Genotoxicity evaluation of ZnO NPs. Results of MN assay (a), comet assay (b) and H2AX
analysis (c).
3h 6h

(a) Increase in %MN 20 g/ml 0.72 0.78 8.85 2.71**

Table 3 30 g/ml 0.66 0.33 12.55 2.31**
Apoptotic cell rates (%) after ZnO NP exposure. 40 g/ml 0.30 0.59 12.06 1.11**
PC 1.92 0.73* 13.34 1.20**
3h 6h (b) Increase in %tDNA 20 g/ml 4.88 1.93** 3.23 0.79*
30 g/ml 5.37 0.57** 11.34 0.28**
Annexin-V MMP Annexin-V MMP
40 g/ml 4.29 0.53** 5.01 3.70
Control 4.71 0.27 4.93 0.33 6.94 0.34 5.03 0.52 PC 9.37 3.44** 7.71 1.29**
ZnO NP 20 g/ml 6.63 0.15** 7.57 0.24** 9.80 0.98** 3.52 0.48 (c) Increase in %H2AX 20 g/ml 1.81 0.53 1.19 0.58
30 g/ml 6.38 0.31** 6.91 0.72 8.17 0.37 7.68 1.74 30 g/ml 2.20 0.30* 3.87 0.82**
40 g/ml 6.33 0.21** 5.62 0.49 8.98 0.74 5.62 0.69 40 g/ml 0.07 0.42 1.39 1.29
PC 8.28 0.14** 6.69 1.09 10.27 0.41** 9.57 0.70** PC 38.23 4.20** 45.41 1.92**

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

98 V. Valdiglesias et al. / Environment International 55 (2013) 92100

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

20 ** in ion concentrations were observed until 30 g/ml of NPs, and then a

15
10
5 centrations in Dulbecco's modied Eagle's medium (DMEM) (Reed et al.,
0
Control 20 30 40
ZnO NP concentration (g/ml)
Fig. 5. Results of OGG1-modied comet assay in neuronal cells treated with ZnO NPs
for 3 h (a) or 6 h (b). PC: positive control. *P b 0.05, **P b 0.01, signicant difference
with regard to the corresponding buffer.
2012), agreeing with the positive results obtained in our study from
apoptosis and oxidative DNA damage assessments. Contrarily, Lin et al.
(2009) concluded that neither free Zn2+ nor metal impurity in the ZnO
NP samples was the cause of the observed cytotoxicity and oxidative
stress in A549 cells treated with these NPs. Regarding the brain, zinc is
an essential nutrient for normal neurological functions and may play
an important role in the processing of memory formation (Daniels et
al., 2004). To understand the possible effects of this ion in brain cells is
particularly relevant as transition metals (such as zinc) may contribute
to the etiology of neurodegenerative diseases, such as Alzheimer's
disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke or
epilepsy (Pavlica et al., 2009).
In order to check the possibility that Zn+2 ions could be released from
the ZnO NPs to the cell culture medium, and so they could be involved in
the cytotoxic and genotoxic effects observed, we tested the presence of
free Zn2+ ions released from the NPs in our cell culture medium by
plateau was reached at around 0.3 mM Zn2+. Other studies reporting
experiments of Zn2+ ion release from ZnO NP showed similar Zn2+ con-
2012), but lower in seawater (Wong et al., 2010) or in complete minimal
PC essential medium (CMEM) (Sharma et al., 2012b). But Reed et al. (2012)
demonstrated using nanopure water and different cell culture media
that solubility of ZnO NPs may be highly variable and dependent on
the matrix in which they are suspended.
When neuronal cells were treated with Zn2+ ions (from ZnSO4 salt) at
a range of concentrations including the ones obtained as released from
ZnO NPs (0.050.8 mM), decrease in cell viability was only observed at
doses higher than 0.4 mM. Therefore, as the NP concentrations used in
this study released Zn2+ ions at levels up to 0.3 mM, the decrease in
neuronal cell viability observed after ZnO NP treatments cannot be attrib-
uted to the Zn2+ ions released from the NPs, but to the NPs themselves.
Other authors also suggest a size dependent toxicity, distinct from
adverse effects associated with the presence of dissolved ions, also re-
leased from bulk forms (Nair et al., 2009; Wong et al., 2010). Contrarily,
Franklin et al. (2007) found that for ZnO, there are no size related effects;
rather, the most likely impact of ZnO (nanoparticulate or bulk) results
from dissolution presumably as Zn2+.
Still, as Zn 2+ ions were released from the ZnO NPs even at the
lowest concentrations, the possibility that these ions are involved in
the genotoxic and cytotoxic effects observed in this study cannot be
excluded since experiments aimed at testing the potential of Zn 2+
ions to induce genotoxic damage, alterations in the cell cycle or apo-
ptosis on SHSY5Y cells were not performed.

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
V. Valdiglesias et al. / Environment International 55 (2013) 92100
5. Conclusions
Despite the fact that no cellular uptake was found, ZnO NPs induced
considerable cytotoxicity, including viability decrease, apoptosis and
cell cycle alterations, and different kinds of genetic damage, including
oxidative DNA damage, on human neuronal cells in a dose- and time-
dependent manner. Free Zn 2+ ions released from the ZnO NPs 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. Since neurotoxic effects of these NPs at different levels were dem-
onstrated in this study, further investigations are needed to develop the
necessary safety precautions and exposure limits for people working
with materials containing ZnO NPs and those who might be exposed
to them when they are implemented in commercial, industrial, and
medical applications.
Acknowledgments
This work was funded by the European Commission (ERA NET New
INDIGO Program, NanoLINEN 045-036-073, PIM2010ENI-00632) and
by Xunta de Galicia (EM 2012/079). G. Kili was supported by a fellowship
from the University of A Corua.
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