Mutagenesis vol. 28 no. 3 pp. 287–299, 2013
1093/mutage/get014
Advance Access publication 5 March 2013
Multiple cytotoxic and genotoxic effects induced in vitro by differently shaped copper
oxide nanomaterials
Sebastiano Di Bucchianico1,*, Maria Rita Fabbrizi1,
Superb K. Misra2,3, Eugenia Valsami-Jones2,3,
Deborah Berhanu2, Paul Reip4, Enrico Bergamaschi5 and
Lucia Migliore1
1
Department of Translational Research and New Technologies in Medicine
and Surgery, Medical Genetics, University of Pisa, Pisa, Italy, 2Department
of Earth Sciences, Natural History Museum, London, UK, 3School of
Geography, Earth and Environmental Sciences, University of Birmingham,
Edgbaston, UK, 4Intrinsiq Materials Ltd, Cody Technology Park, Ively Road,
Farnborough, Hampshire, UK and 5Department of Clinical and Experimental
Medicine, University of Parma, Italy
*To whom correspondence should be addressed. Tel: +39 050 2211029; Fax:
+39 050 2211034; Email: sebastiano.dibucchianico@for.unipi.it
Received on September 27, 2012; revised on December 19, 2012; accepted on
January 1, 2013
In nanotoxicology, the capacity of nanoparticles of the same
composition but different shape to induce cytotoxicity and
genotoxicity is largely unknown. A series of cytotoxic and
genotoxic responses following in vitro exposure to differently
shaped CuO nanoparticles (CuO NPs, mass concentrations
from 0.1 to 100 μg/ml) were assessed in murine macrophages
RAW 264.7 and in peripheral whole blood from healthy
volunteers. Cytotoxicity, cytostasis and genotoxicity were
evaluated by the colorimetric assay of formazan reduction
[3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-
mide (MTT)] and by the cytokinesis-block micronucleus
cytome (CBMN Cyt) assay. The comet assay was applied
for detecting DNA strand breaks and information on oxi-
dative damage to DNA (oxidised purines and pyrimidines).
The MTT assay revealed a decrease in cell viability in
RAW 264.7 cells and peripheral blood lymphocytes (PBL)
with significant dose–effect relationships for the different
CuO NP shapes. The comet assay revealed a dose-depend-
ent increase in primary DNA damage, and a significant
increase in oxidative damage to DNA was also detectable,
as well as increased frequency of micronuclei in binucleated
cells, often in a dose-related manner. Proliferative activity,
cytotoxicity and apoptotic markers showed a significant
trend in the two cell types. Finally, we have differentiated
clastogenic events from aneugenic events by fluorescence in
situ hybridisation with human and murine pancentromeric
probes, revealing for the first time characteristic aneugenic
responses related to the shape of CuO NPs and cell type.
Independently of size and shape, all CuO NPs revealed a
clear-cut cytotoxic and genotoxic potential; this suggests
that CuO NPs are good candidates for positive controls in
nanotoxicology.
Introduction
Nanomaterials may exhibit unusual interactions with cells
and tissues, thus necessitating studies to better understand
the different effects on biological systems relative to the
composition, size and shape of these emerging nanomaterials.
Likewise, it has been found that particles with dissimilar aspect
© The Author 2013. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
doi:10.
ratios have a differential impact on cellular function, including
cell proliferation, apoptosis, cytoskeleton formation, adhesion
and migration (1).
Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021
One of the most studied types of nanomaterials is CuO and
a number of studies have clearly demonstrated significant
cytogenotoxic responses both in vivo and in vitro, affecting the
normal functions of the nervous system (2). CuO nanoparti-
cles (NPs) can also be cytotoxic in a concentration- and time-
dependent manner and elicit permeability and inflammation
responses in human cardiac microvascular endothelial cells
in vitro (3). Moreover, CuO NPs induce genotoxic responses
through the p53 pathway in human pulmonary epithelial cells
A549 and induce oxidative stress in a dose-dependent way, as
indicated by depletion of glutathione and induction of lipid
peroxidation, catalase and superoxide dismutase (4). CuO
NPs, like many metal oxide NPs, exhibit low mutagenic poten-
tial toward Salmonella typhimurium. However, a dose-depend-
ent inhibition of Escherichia coli WP2 was found after CuO
NPs exposure at concentrations ranging from 100 to 1600 μg/
plate (5). In vivo studies in rats have shown hepatotoxicity
and nephrotoxicity and an integrated metabolomic analysis
suggested that mitochondrial failure, enhanced ketogenesis,
fatty acid β-oxidation and glycolysis contributed to the toxic-
ity induced by CuO NPs (6). Moreover, CuO NPs were found
to be inflammogenic to the lungs of rats and induced a unique
inflammatory footprint after both acute and chronic treatments.
Exposure to CuO NPs was severely associated with neutro-
philic/eosinophilic cytotoxic inflammation and increased lev-
els of interleukin (IL)-1β, macrophage inflammatory protein-2
(MIP-2), eotaxin and lactate dehydrogenase in bronchoalveo-
lar lavage fluid, 24 h after instillation (7). It was also shown
that CuO NPs were, by far, the most potent when compared
with several other metal oxide NPs in inducing DNA damage;
nevertheless, little is known about the comparative responses
of differently shaped CuO NPs (8).
The growing need for engineered nanostructured materials,
to meet the vast number of applications that exploit their
unique physical and chemical properties, requires the study
of their cytotoxic and genotoxic potential and necessitates an
improvement of current methods. One important aspect of the
cytogenotoxicity assays is the use of an appropriate NP-type
positive control (9). In the current OECD guidelines, there
is still no specific recommendation for NPs positive control.
A positive control is used to confirm the proper performance
of the assay, but must also include a control system for
nanosized compounds, so that the comparisons that are made
are relevant. Indeed, the lack of a nanoparticle-type positive
control questions the suitability of the tests to identify
nanomaterials with cytotoxic and genotoxic properties. ZnO
NPs have been often used as positive controls but they have
not always given unequivocal results, emphasising the need
for a more careful evaluation of ZnO NPs effects across a
spectrum of relevant cell types (10). Similar considerations
can be made for other types of NPs used as a positive control,
such as CdO, CeO2 and carbon black NPs.
287
S. Di Bucchianico et al.
In this article, we report on the cytotoxic and genotoxic
responses induced by CuO NPs of different morphologies in an
in vitro model of murine airway macrophages (RAW 264.7), a
commonly used macrophage model in toxicological studies on
nanomaterials. Airway macrophages are specialised for inter-
acting with inhaled particles and their exposure to NPs may
lead to distinct types of macrophage activation (11), which
account for different pathologic outcomes (12). We also used
human whole blood cultures from healthy volunteers, to mimic
possible interactions with blood cells, the major route for NPs
biodistribution (13). The aim of this study was to demonstrate
that, independently from geometrical characteristics, all CuO
NPs show positive responses making CuO a good positive con-
trol in nano-genotoxicological studies.
We used CuO NPs from an industrial partner (Intrinsiq
Materials Ltd and here named INT) and three CuO NPs dif-
fering in shapes and surface area (rods, spheres and spindles)
synthesised within the frame of the European Commission
Framework Programme 7 (FP7) NanoReTox project, to address
the environmental and human implications of increased expo-
sure to engineered NPs and to contribute to address major con-
cerns in nanotoxicity by specifically investigating a possible
correlation between physicochemical properties and toxicity.
Cytotoxic, primary and oxidative genotoxic effects of CuO
NPs were evaluated by the formazan reduction [3-[4,5-dimeth-
ylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)] assay
and the single-cell gel-electrophoresis (comet) assay in its clas-
sical version and in the modified one with the use of restric-
tion enzymes. A key mechanism thought to be responsible for
the genotoxic effects exerted by nanomaterials, in particular
by CuO NPs, involves an increased production of intracellular
reactive oxygen species (ROS). ROS-induced DNA damage is
typified by single- and double-strand DNA breaks that can be
detected by the comet assay. Thus, we applied a modification
of the assay, which allows to determine the presence of altered
purines, including 8-oxoguanine and oxidised pyrimidine,
using specific bacterial restriction enzymes, namely forma-
midopyrimidine-DNA glycosylase (Fpg) and endonuclease-
III (Endo-III) (14). Chromosomal aberrations were evaluated
using the cytokinesis-block micronucleus cytome (CBMN Cyt)
assays. In recent years, the micronucleus (MN) test has evolved
into a comprehensive approach aimed to evaluate at the same
time chromosome breakage, DNA misrepair, chromosome
loss, non-disjunction, necrosis, apoptosis and cytostasis (15).
Finally, we applied the fluorescence in situ hybridisation (FISH)
with both human and mouse pancentromeric probes to differ-
entiate aneugenic and/or clastogenic mechanisms involved in
micronuclei formation. Aneuploidy is a major cause of human
reproductive failure and an important contributor to cancer and
it is therefore important that any increase in its frequency due
to chemical exposures should be recognised and controlled (16)
especially for nanomaterials for which an adequate legislation
is still missing.
Materials and methods
Characterisation of CuO NPs
The main characteristics of the CuO NPs used in this study are summarised
in Table I. CuO powder (CuO INT) was obtained from a commercial source
(Intrinsiq Materials Ltd, Farnborough, UK) with the particle size specified by
the manufacturer in the range 10–100 nm. CuO INT was obtained by plasma
synthesis, whereas spheres, rods and spindles were obtained by reducing Cu2+
by OH− in presence of acetic acid, as described previously (17). X-ray dif-
fraction (XRD) was used for the identification of the crystal structure using
288
a Nonius PDS 120 powder diffraction system position sensitive detector.
Transmission electron microscopy (TEM) was used to image the particles and
assess the size and shape of the NPs and zeta potential to study the dispersion
of the NPs used. TEM imaging was done on a HITACHI H7100, operating at
100 kV. Zeta potential measurements were performed on the nanoparticle sus-
pension as well as the particles when suspended in cell culture media, using a
Malvern Zetasizer Nano ZS instrument (Malvern Instruments, UK).
Cell culture and exposure to CuO NPs
RAW 264.7 cells were purchased from the Istituto Zooprofilattico of Brescia
(Italy). The cells were cultured in minimal essential medium (CELBIO,
Milan, Italy) supplemented with 10%, heat inactivated, foetal bovine serum
(CELBIO), 1% penicillin/streptomycin (CELBIO) and 1% l-glutamine
Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021
(CELBIO). The cell cultures were maintained in a humidified atmosphere
(5% CO2) at 37°C. After 24 h of incubation, the cell cultures were treated
with different mass concentrations of CuO NPs (0.01–100 μg/ml) for all
cytotoxicity assays and from 0.1 to 10 μg/ml for the CBMN Cyt test and
the comet assay. The study was also performed on human peripheral blood
lymphocytes of two healthy male volunteers. Approximately 4–6 ml of blood
was drawn by venipuncture in Li-heparin vials, according to standard proce-
dure. The donors were chosen according to the following criteria: young age,
non-smokers, without pharmacological treatments for at least 3 weeks before
donation and without any radiological examination performed within the pre-
vious 3 months. Blood was drawn from the volunteers and 600 μl of whole
blood was used to prepare two paired independent lymphocyte cultures for
each subject in 4.7 ml of complete medium per culture. Standard medium was
prepared with RPMI 1640 (Gibco BRL, Italy) supplemented with 10% foetal
bovine serum (Gibco BRL), 1.5% phytohaemagglutinin (Gibco BRL) and 1%
penicillin–streptomycin (Gibco BRL). CuO INT powder was weighed and
suspended in the appropriate culture medium, then sonicated for 10 min in a
sonicating bath (FALC, Treviglio, Italy). CuO spheres, spindles and rods were
provided in aqueous suspensions by the Natural History Museum (NHM) of
London, UK, and stored at room temperature. Aliquots of the concentrated
aqueous suspension of CuO spheres, spindles and rods were directly added
to the culture medium (not exceeding 1% v/v) and sonicated for 10 min, to
obtain the final concentrations to be tested, evaluated by means of trypan blue
dye exclusion (standard method). All the experiments were performed within
7 days after the CuO NPs delivery from NHM to ensure the stability of the
suspension as indicated by the supplier.
MTT assay
Cytotoxicity was assessed by using MTT assay. Mitochondrial dehydrogenases
of viable cells reduce MTT to water insoluble blue formazan crystals that are
then solubilised by dimethylsulphoxide (DMSO); this assay thus indicates
cell mitochondrial activity impairment. RAW 264.7 cells were grown to sub-
confluence in 96-well plates before being exposed to 0.01, 0.1, 1, 10 and
100 μg/ml CuO NPs suspensions for 2 and 24 h. Hydrogen peroxide (H2O2,
10 μM) was used as a positive control. Whole blood cultures were carried out
as described above and treated with the same conditions of adherent cells.
We collected the cells (3000 rpm, 5 min) and resuspended them in 0.5 ml of
complete medium. Erythrocyte lysis buffer (3.5 ml) [155 mM NH4Cl, 10 mM
KHCO3, 1 mM Na2 ethylenediaminetetraacetic acid (EDTA), pH 7.4] was
added and after 1.5 min cells were collected (3000 rpm, 5 min). The cells were
washed in 3.5 ml of complete medium once and then pelleted (3000 rpm,
5 min). The obtained pellet was resuspended in 3.5 ml of complete medium and
100 μl of the cell suspension was put in a 96-well plate. After exposure of all
cell types (nine wells for each condition), 10 μl of a 5 mg/ml MTT solution was
added. After 3 h at 37°C, medium in adherent cell culture was then replaced by
100 μl of DMSO and mixed thoroughly to dissolve the formazan crystals; to
cell culture in suspension, 100 μl of DMSO was added without discarding the
medium. To limit potential interactions due to the possible presence of residual
NPs that could interfere with the assay, parallel cultures without cells (blank)
were used to take into account the absorption due to nanomaterials. Then,
absorbance was measured at 570 nm (630 nm background) and cell viability
was determined as a percentage of the negative control (unexposed cells)
minus the percentage of concurrent condition blank absorbance. IC50 values
(50% inhibitory concentration) were extrapolated graphically from the plotted
absorbance data.
Cytokinesis-block micronucleus cytome assay
The CBMN Cyt on whole blood treatments was performed according to the
procedure described by Migliore and co-authors (13,18). After 24 h of culture,
the cells were exposed to CuO NPs at concentrations ranging from 0.1 μg/ml
to 10 μg/ml. Mitomycin C (MMC; Kyowa Hakko Kogyo Co., Tokyo) (0.17 μg/
ml) was used as positive control. Cytochalasin B (6 μg/ml) was added after 44 h

to block the cytokinesis process and lymphocyte cultures were harvested after
72 h. Cells were then treated with an hypotonic solution (0.075 M KCl) to lyse
erythrocytes for 2 min, prefixed in 3:5 methanol:acetic acid, washed once with
methanol and subsequently fixed twice with a 6:1 methanol:acetic acid fixative
solution. Finally, the cell solution was dropped onto cold glass slides. The stain-
ing procedure was performed by immersing the air-dried slides in a 2% Giemsa
solution in Sorensen’s buffer (pH 6.8). To assess the genotoxic effects of CuO
NPs in RAW 264.7 cells, the CBMN Cyt assay was performed as follows.
Briefly, making the appropriate dilutions, a suitable quantity of cells (2 × 104
cells/cm2) was seeded into six-well plates and two paired independent cultures
for each concentration were prepared. After 24 h, the macrophages were treated
with CuO NPs as described earlier. MMC (0.17 μg/ml) was used as a positive
control. Forty-four hours after the initial preparation of the cultures, cytocha-
lasin B was added to each test well to a final concentration of 4 μg/ml to block
cytokinesis of dividing cells. Cell cultures were then harvested after 28 h and
treated as described earlier. Two thousand binucleated cells were examined for
each experimental point in a blind mode (1000 from each independent culture
replicate) using a Nikon Eclipse 800 optical microscope (final magnification
of ×400). The scoring criteria adopted by Fenech (15) were followed for each
endpoint. Briefly, we evaluated the binucleate micronucleated cells frequency
as number of binucleate cells containing one or more micronuclei per 1000
binucleated cells and the total number of micronuclei in 1000 binucleated cells.
Moreover, 500 cells were scored to evaluate the percentage of mono-, bi-, tri-
and multinucleated cells, and the CBPI (cytokinesis-block proliferation index)
was calculated as an index of cytotoxicity by comparing values in the treated
and control cultures. The CBPI indicates the average number of cell cycles per
cell during the period of exposure to cytoB and may be used to calculate cell
proliferation. Finally, other damage events were scored in once-divided binu-
cleated cells per 1000 cells, namely: (i) nucleoplasmic bridges, a biomarker of
DNA misrepair and/or telomere end-fusions and (ii) nuclear buds (NBUDs), a
biomarker of elimination of amplified DNA and/or DNA repair complexes. The
number of apoptotic and necrotic cells as defined by Fenech (15) and mitotic
figures per 500 cells were also evaluated.
Fluorescence in situ hybridisation
Separate slides from CBMN Cyt were used to achieve FISH with human
and murine pancentromeric probes (Kreatech Diagnostics, Netherlands).
Slides of treatments with 1 μg/ml of NPs, positive and negative control were
examined for the presence of centromeric spots in MN and were classified as
centromere positive (MN C+) and centromere negative (MN C−). Slides were
pretreated in 2× standard saline citrate (SSC)/0.5% Igepal, pH 7.0, at 37°C
for 15 min and dehydrated in a graded ethanol series (70, 85 and 100%);
slides and probes were co-denatured for 10 min at 75°C and overnight at
37°C on a humidified chamber. Post-hybridisation washing was performed
in 1× washing buffer (0.4 × SSC/0.3% Igepal) for 2 min at 72°C. Afterwards,
slides were washed in 2 × SSC/0.1% Igepal for 1 min at room temperature
and dehydrated in a graded ethanol series (70, 85 and 100%) for 1 min each.
The slides were counterstained with DAPI (0.1 μg/ml). For FISH analysis,
500 binucleated (BN) cells were scored per culture. The MN in BN were
examined using the same scoring criteria described previously for the CBMN
Cyt assay. The preparation was examined with a Nikon Eclipse 800 micro-
scope (final magnification of ×400) equipped with a 100W mercury lamp and
triple band-pass filters (DAPI/FITC/Texas Red or DAPI/FITC/Rhodamine)
are used to view multiple colours and single band-pass filters are used for
individual colour visualisation.
Comet assay and oxidative damage to DNA
The modified alkaline comet assay (19) was carried out on RAW 264.7 cell
cultures seeded at a concentration of 2 × 104 cells/cm2 in six-well plates. After
attachment, cells were treated with CuO NPs at different concentrations (see
above). In whole blood cultures, after 24 h from the beginning of culture,
the cells were exposed to the same concentrations of CuO NPs. In all cell
Table I. Characterisation of the various CuO NPs used in this study
Samples Initial form Size (nm)
CuO INT Powder 10–100 (polydisperse)
CuO spheres Suspension 7±1
CuO rods Suspension 7 ± 1 × 40 ± 10
CuO spindles Suspension 1200 ± 250 × 270 ± 50 × 30 ± 10
NA, not available.
a
Data taken from S. K. Misra, S. Nuseibeh, A. Dybowska, D. Berhanu, T. D. Tetley and E. Valsami-Jones, submitted for publication. Experiments were performed
in 1 mM NaNO3 medium.
Shape-specific CuO NP cytotoxicity and genotoxicity
cultures, 10 μM hydrogen peroxide was used as a positive control. At least two
independent experiments were performed for each concentration tested and
incubation was performed for 2 or 24 h. After treatment, the cell pellet was used
for the comet assay. Briefly, the slides were spread with 1% normal melting
agarose (Sigma–Aldrich, St Louis, MO, USA) in PBS (Sigma–Aldrich) and
left to solidify at 4°C. Cell pellets were suspended in 85 μl of 0.5% low-
melting agarose (Agarose wide range, Sigma–Aldrich) in PBS at 37°C and
two slides per culture were prepared. The cell suspension was dropped on top
of the first agarose layer, covered with a coverslip and allowed to solidify at
4°C. A final layer of 0.5% low-melting agarose was then added to the slide.
The lysis procedure was performed in cold lysis buffer (2.5 M NaCl, 100 mM
Na2 EDTA, 10 mM Tris, 10% DMSO and 1% Triton X-100, pH 10.0) for 1 h
at 4°C. The slides were then placed for 20 min in alkaline buffer (300 mM
Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021
NaOH, 1 mM Na2 EDTA, pH > 13.0) and electrophoresed for 20 min at 25 V
adjusted at 300 mA. The slides were neutralised in 0.4 M Tris buffer, pH 7.5,
for 5 min, twice and in methanol absolute for 5 min, once. Coded slides were
scored after staining with ethidium bromide (20 μg/ml) using a fluorescence
microscope (Nikon Eclipse E800) at ×200 magnification. The percentage of
DNA in the comet tail in a total of 100 randomly selected cells per sample (two
replicates, each with 50 cells per slide) was used as a measure of the amount
of DNA damage. Analysis was carried out by using a Comet Image Analysis
System, version 5.5 (Kinetic Imaging, Nottingham, UK). Results were reported
as percentage of tail DNA. To determine the presence of oxidised pyrimidine
and purine bases, specific bacterial enzymes, namely Endo-III and Fpg,
respectively, were used. Two independent experiments were performed at 2 and
24 h treatment. Before electrophoresis, the slides were washed with enzyme
buffer (3.72 × 10−3 g/ml Na2EDTA (Carlo Erba, Milan, Italy), 7.45 × 10−3 g/ml
KCl (Carlo Erba), 2.38 × 10−3 g/ml HEPES (Sigma–Aldrich) and 1 × 10−3 g/
ml BSA (Sigma–Aldrich) (only for the Fpg enzyme) three times for 15 min at
4°C. Next, 100 μl of either buffer alone (reference slides) or 1.5 × 10–6 g/ml of
Endo-III or Fpg (Trevigen, Gaithersburg, MD, USA) was applied to each slide,
which was subsequently covered with a coverslip and incubated at 37°C for
45 min (Endo-III) or 30 min (Fpg) in humidified chambers. After the treatment,
slides were placed in a horizontal electrophoresis chamber and electrophoresis
was performed as described earlier. To detect oxidised purines or pyrimidines,
slides were incubated without enzymes (i.e. only buffer) and compared with
those incubated with Fpg or Endo-III enzymes, respectively. To determine
the number of enzyme-sensitive sites, the difference between the value of the
percentage of tail DNA obtained after digestion with each enzyme and with the
buffer only was calculated.
Statistical analysis
All results are expressed in terms of the mean and standard error of the mean
for two independent duplicated experiments, except where it is differently indi-
cated. The statistical significance of the results was calculated using Student’s
t-test. The level of significance was set at P < 0.05. A linear regression equation
was used to calculate the effect of mass concentration on each endpoints. IC50
values are graphically interpolated from the dose–effect curves. All statistical
analyses were performed using STATGRAPHICS Plus (SGWIN, version 2.1).
Results
Characterisation of CuO NPs
Table I summarises the main characteristics of CuO NPs used
in this study. The sizes of the primary particles are measured
from the TEM images (Figure 1); INT NPs appear highly
polydispersed and particle sizes range from 10 to 100 nm
approximately, whereas the spheres, rods and spindles were
monodispersed with sizes of 7 ± 1 nm, 7 ± 1 × 40 ± 10 nm
and 1200 ± 250 × 270 ± 50 × 30 ± 10 nm, respectively. Zeta
Zeta potential (mV) Surface areaa (m2/g) Dissolutiona (%)
42 ± 1 NA 0.66
38.6 ± 0.2 60 0.7
47.4 ± 0.1 51 1
26.3 ± 0.8 18 3.5
289
S. Di Bucchianico et al.
Fig. 1. TEM images of (a) INT CuO, (b) CuO spheres, (c) CuO rods and
(d) CuO spindles.
potential indicates high suspension stability in deionised
water for INT (42 ± 1), spheres (38.6 ± 0.2), rods (47.4 ± 0.1)
except for spindles (26.3 ± 0.8). XRD patterns (Figure 2)
show that CuO spheres, spindles, rods and INT samples
contain tenorite copper oxide (ICDD 48-1548); no other
material is detected. To assess the stability of the nanoparticle
suspension in cell culture media, zeta potential analysis was
conducted. Particles at a concentration of 0.1 mg/ml were
added to RPMI medium and Dulbecco’s modified Eagle’s
medium and gently vortexed and pipetted for ensuring particle
dispersion. Zeta potential measurements showed very similar
values for all the four types of samples compared with the cell
culture medium alone (e.g. ZetaRPMI = –6.8 ± 0.1; ZetaRPMI+CuO rods
= –9.8 ± 0.4; ZetaRPMI+CuO spheres = –10 ± 1; ZetaRPMI+CuO spindles = –9 ± 1;
ZetaRPMI+CuO INT = –7 ± 0.4). However, on examining the
digital images of the suspension, the particles showed high
dispersability in both media, with the exception of CuO
INT samples that showed signs of particle sedimentation
indicating particle agglomeration. The CuO INT preparation
(pH 5.5 in water and 7.5 in medium) is far enough from its
Fig. 2. XRD pattern of all the types of CuO NPs used in this study. The patterns are shifted vertically to improve the clarity.
290
isoelectric point (between 9.0 and 10.0) to ensure a relatively
good colloidal stability (12). Data on surface area and percent
dissolution presented in Table I are largely discussed by S. K.
Misra, S. Nuseibeh, A. Dybowska, D. Berhanu, T. D. Tetley and
E. Valsami-Jones, submitted for publication, who performed
dissolution studies in 1 mM NaNO3 and also in serum-free cell
culture medium.
Cytotoxicity
The MTT assay was performed with 0.01, 0.1, 1, 10 and
100 μg/ml of CuO NPs but only the data obtained in 0.1, 1 and
Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021
10 μg/ml exposure are shown because the 0.01 dose was not
effective and the 100 dose was toxic for all tested NPs (percent
of viable cells <50).
CuO NPs exposure for 2 h at lowest tested concentrations
(Figure 3a) led to a reduction in cell viability more evident for
nanomaterials with smaller dimensions (spheres) in both cell
systems. The CuO spindles (the largest dimension), however,
generally showed a cytotoxicity trend in a dose-dependent
manner that was statistically significant for RAW 264.7 cells.
In peripheral blood lymphocytes, dose-dependent trends were
observed for the CuO spheres. After 24 h exposure (Figure 3b),
the two cell types showed a slightly different sensitivity only
to CuO spheres except for the highest dose of CuO rods and
CuO INT. Phagocytic cells showed a dose-dependent cytotox-
icity trend both in CuO spheres exposure, CuO spindles and
CuO rods, with spheres and rods treatments the most effec-
tive. The highest tested concentrations of spheres, spindles
and rods were the most effective in inducing cytotoxicity also
in exposed blood cultures. The IC50 values are summarised in
Table II highlighting the most effective response induced by
CuO spheres in RAW 264.7 (94.8 μg/ml) and in blood cultures
after 2 h exposure (22.6 μg/ml). Following 24 h exposure, the
most effective responses in RAW 264.7 were similarly induced
by CuO spheres and rods (9.9 and 10.2 μg/ml, respectively);
CuO spheres caused the most effective cytotoxicity in blood
cultures (12.1 μg/ml). The IC50 values bring out a slight sen-
sitivity of the phagocytic cells compared with lymphocytes.
Considering results obtained in RAW 264.7 after 24 h expo-
sures, it is possible to define the following cytotoxicity rank:
CuO spheres > CuO rods > CuO spindles > CuO INT, which
is quite the same rank of mass percent dissolution (3.5, 1, 0.66
and 0.7 %, respectively) but not similar to the size rank (7,
7 × 40, 1200 × 270 × 30, 10–100 nm, respectively) and avail-
able surface area (60, 51, 18, CuO INT m2/g) reported by S. K.
Misra, S. Nuseibeh, A. Dybowska, D. Berhanu, T. D. Tetley and
E. Valsami-Jones, submitted for publication. Due to increase in
 Shape-specific CuO NP cytotoxicity and genotoxicity

Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021

Fig. 3. Effects of CuO NPs treatments on the viability of RAW 264.7 cells and whole blood by the MTT assay. Cell viability was assessed by the MTT assay
and results are presented as a percentage of negative control viability. C+: 10 µM H2O2. Cells were treated with the indicated concentrations of CuO NPs for 2 h
(a) and 24 h (b). Statistically significant differences from the control were determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Data represent
the mean of two separate experiments and error bars represent the standard error of the mean. In RAW 264.7 (2 h treatment) CuO spindles showed a dose–effect
relationship (r = −0.92, R2 = 84%, P = 0.03) as well as CuO spheres in PBL (2 h exposure) (r = −0.97, R2 = 94.3%, P = 0.006). In RAW 264.7 (24 h treatment),
CuO spheres showed a dose–effect relationship (r = −0.94, R2 = 89.2%, P = 0.015) as well as CuO spindles (r = −0.92, R2 = 85.3%, P = 0.025) and CuO rods
(r = −0.92, R2 = 83.8%, P = 0.03) exposures.

size, we expect the surface area of INT and spindles to be lower
showing that the lower cytotoxicity can be linked to a lower
surface area, even if it has to be considered that the surface
area is not only dependent on the size but also on the porosity
of the NPs.
However, because the IC50 values for every NP tested
were similar, the relationship among dissolution, potential
agglomeration and cell viability was not directly proportional
to surface area. For example, CuO spindles (which dissolved
minimally and have the least surface area) were comparably
cytotoxic with respect to the other CuO NPs. This complex
bioreactivity is more evident in 24 h blood responses where
the rank of cytotoxicity is CuO spheres > CuO rods > CuO
spindles > CuO INT, confirming the major efficiency of CuO
spheres and rods to induce cell death. This situation could be
explained by the same order of mass percent dissolution and
size rank, taking into account the prominent phagocytic activity
of macrophages with respect to lymphocytes that could play

291
S. Di Bucchianico et al.

Table II. IC50 values of CuO NPs exposures in RAW 264.7 and PBL,
concentrations of CuO NPs suspensions. Despite the similar
graphically interpolated from linear regression equations of MTT data pattern of proliferation, the frequency of micronuclei is much
higher in phagocytic cells compared with lymphocytes except
RAW 264.7 PBL for exposure to CuO INT, where the highest tested concentra-
2 h (μg/ml) 24 h (μg/ml) 2 h (μg/ml) 24 h (μg/ml) tion induced a greater micronuclei formation in lymphocytes
and also caused a concurrent clear reduction of the CBPI
Spheres 94.8 9.9 22.6 12.1 (from 1.86 to 1.32). A linear relationship was found in the MN
Spindles 96.4 19.5 22.8 16.3 induction by CuO spheres and spindles treatments in RAW
Rods 195 10.2 29.7 15.2
INT 167 71.9 124.8 93.2 264.7 cells and by CuO spindles, rods and INT treatments in
lymphocytes.
There was a highly significant dose-dependent increase in

Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021

an important role in NP uptake. Coherently, the IC50 values
obtained in lymphocytes were generally higher than those
obtained in RAW 264.7. Results obtained with the MTT assay
were also confirmed by trypan blue dye exclusion assay (data
not shown).
Cytostasis and chromosomal damage
Table III summarises the results of cell proliferation and
cytostasis measured by scoring the CBPI and mitotic,
apoptotic and necrotic indices. All doses in both cell systems
reduce the proliferation index in a statistically significant
manner. A linear relationship was found in the CBPI of
CuO INT exposed RAW 264.7 cells, whereas exposed
lymphocytes to spindles and CuO INT revealed a decrease of
cell proliferation in a dose-related manner. Exposure to CuO
NPs caused apoptotic responses measured by apoptotic figures
scored in the CBMN Cyt assay. Programmed cell death was
increased to 2.5-fold (CuO spindles and rods) in macrophages
and up to 4.5-fold in lymphocytes (CuO INT), whereas severe
necrotic responses were observed in RAW 264.7 cells but
not in exposed whole blood. Consistent with the inhibition
of proliferation, an inhibition of mitotic index was observed
in both studied cellular systems. Cytostatic responses are not
directly related to considered physicochemical characteristics
of the NPs.
Figure 4 shows the micronuclei frequency obtained by treat-
ing RAW 264.7 cells and blood cultures with different mass
nucleoplasmic bridges frequency as a result of exposure to
CuO spindles, rods and INT in lymphocytes cultures, whereas
CuO spheres and rods treatments in RAW 264.7 reveal a trend
(P < 0.05 for all dose–response effects) (Figure 5). NBUDs,
MN-like bodies attached to the nucleus by a thin nucleoplas-
mic connection, are significantly generated only after RAW
264.7 cells exposure to NPs except CuO INT (Figure 6). Most
NBUDs in binucleate cells appear to originate from interstitial
or terminal acentric fragments, possibly representing nuclear
membrane entrapment of DNA that has been left in cytoplasm
after nuclear division, or excess DNA that is being extruded
from the nucleus (20). No significant NBUDs formations
were seen in lymphocytes except after CuO spheres and INT
exposure.
Clastogenic and aneuploidogenic effects of CuO NPs
Performing FISH analysis, we found a trend of MN induction
in response to NP exposures comparable to the results obtained
performing the CBMN Cyt assay (Table IV). All CuO NPs
induced an increased number of both MN C+ and MN C–
with respect to untreated control; however, it is possible to
observe that all treatments induced a significant increase in
aneuploidogenic response (MN C+) in both cell systems, with a
consistent different behaviour of CuO NPs in RAW 264.7 cell
treatments. In macrophagic cells, although the aneuploidogenic
events are predominant, CuO INT and spindles showed a major
degree of clastogenic events (MN C–) compared with spheres
and rods.

Table III. CBPI, mitotic, apoptotic and necrotic indices scored by CBMN Cyt assay

CBPI Mitotic index Apoptotic index Necrotic index

RAW 264.7 PBL RAW 264.7 PBL RAW 264.7 PBL RAW 264.7 PBL

C− 1.83 ± 0.02 1.86 ± 0.06 4.3 ± 0.2 4.9 ± 0.4 1.7 ± 0.2 0.7 ± 0.1 0.9 ± 0.1 0.6 ± 0.2
C+ 1.38 ± 0.07*** 1.40 ± 0.05*** 1.9 ± 0.3*** 2.3 ± 0.3*** 4.6 ± 0.5*** 2.2 ± 0.2*** 2.1 ± 0.2 1.9 ± 0.4
Spheres
0.1 1.64 ± 0.03** 1.59 ± 0.08*** 1.8 ± 0.2*** 3.9 ± 0.4* 3.0 ± 0.4*** 1.5 ± 0.2*** 1.9 ± 0.3** 0.8 ± 0.3
1 1.60 ± 0.02** 1.55 ± 0.07*** 1.7 ± 0.1*** 3.4 ± 0.3** 4.9 ± 0.7*** 1.6 ± 0.5** 2.9 ± 0.3*** 1.5 ± 0.1**
10 1.60 ± 0.02** 1.50 ± 0.06*** 1.6 ± 0.2*** 3.0 ± 0.2*** 2.6 ± 0.2** 1.7 ± 0.1*** 3.9 ± 0.3*** 1.3 ± 0.2*
Spindles
0.1 1.60 ± 0.03** 1.65 ± 0.04*** 1.8 ± 0.1*** 4.5 ± 0.5 3.4 ± 0.2*** 1.6 ± 0.5** 1.9 ± 0.1** 1.2 ± 0.4
1 1.53 ± 0.03*** 1.65 ± 0.03*** 1.5 ± 0.2*** 3.7 ± 0.7 3.7 ± 0.3*** 1.7 ± 0.3*** 4.7 ± 0.7*** 1.2 ± 0.2*
10 1.62 ± 0.06** 1.59 ± 0.04*** 1.6 ± 0.2*** 3.3 ± 0.3*** 4.3 ± 0.5*** 2.5 ± 0.4*** 5.3 ± 0.4*** 1.6 ± 0.3**
Rods
0.1 1.70 ± 0.03** 1.72 ± 0.03** 1.9 ± 0.1*** 4.1 ± 0.6 2.5 ± 0.4* 1.4 ± 0.2** 1.2 ± 0.3 1.1 ± 0.2*
1 1.70 ± 0.03** 1.75 ± 0.02* 1.8 ± 0.2*** 3.3 ± 0.4** 2.8 ± 0.7* 1.6 ± 0.4** 1.8 ± 0.4* 1.3 ± 0.2*
10 1.69 ± 0.08* 1.45 ± 0.08*** 1.9 ± 0.2*** 2.7 ± 0.2*** 4.3 ± 0.5*** 2.2 ± 0.2*** 2.3 ± 0.2** 2.2 ± 0.4***
INT
0.1 1.73 ± 0.05* 1.60 ± 0.03*** 2.2 ± 0.4*** 3.5 ± 0.5* 1.9 ± 0.5 1.8 ± 0.2** 0.7 ± 0.3 1.2 ± 0.2*
1 1.49 ± 0.05*** 1.67 ± 0.02*** 2.0 ± 0.2*** 2.3 ± 0.5*** 2.7 ± 0.2** 2.6 ± 0.5*** 1.7 ± 0.2* 1.4 ± 0.1**
10 1.35 ± 0.05*** 1.32 ± 0.01*** 1.1 ± 0.4*** 1.8 ± 0.2*** 4.2 ± 0.6*** 3.2 ± 0.4*** 2.5 ± 0.5*** 1.9 ± 0.2***

Data represent the mean ± SEM of two separate experiments. Statistically significant differences from the control were determined by Student’s t-test (*P < 0.05;
**P < 0.01; ***P < 0.001).

292
 Shape-specific CuO NP cytotoxicity and genotoxicity

Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021

Fig. 4. Micronuclei frequency determined by CBMN Cyt assay in RAW 264.7 and PBL cells from whole blood after CuO NPs exposure at indicated mass
concentrations (µg/ml). C+, 0.17 µg/ml mitomycin C. Statistically significant differences from the control were determined by Student’s t-test (*P < 0.05,
**P < 0.01, ***P < 0.001). Raw 264.7 CuO spheres treatment showed a dose–effect relationship (r = 0.96, R2 = 92.5%, P = 0.009) as well as the CuO spindles
exposure (r = 0.89, R2 = 79.6%, P = 0.042). PBL CuO spindles treatment showed a dose–effect relationship (r = 0.85, R2= 72.8%, P = 0.05) as well as both CuO
rods (r = 0.95, R2 = 90.4%, P = 0.045) and CuO INT (r = 0.91, R2 = 84.5%, P = 0.027) exposures.

Fig. 5. Nucleoplasmic bridges frequency determined by CBMN Cyt assay after CuO NPs exposure at indicated mass concentrations (microgram per millilitre).
C+, 0.17 µg/ml mitomycin C. Statistically significant differences from the control were determined by Student’s t-test (**P < 0.01, ***P < 0.001). RAW 264.7
CuO spheres treatment showed a dose–effect relationship (r = 0.87, R2 = 76.4%, P = 0.05) and CuO rods exposure (r = 0.99, R2 = 99.6%, P = 0.002). PBL CuO
spindles treatment showed a dose–effect relationship (r = 0.95, R2 = 90.1%, P = 0.012) as well as both CuO rods (r = 0.96, R2 = 91.3%, P = 0.044) and CuO INT
(r = 0.96, R2 = 92.2%, P = 0.04) exposures.

Primary and oxidative DNA damage
Figure 7 shows data obtained in evaluating the CuO NP-induced
DNA damage by the comet assay. Cells were exposed for 2 and
24 h, with the same range of CuO NPs concentrations used for
the CBMN Cyt assay. A significant increase in DNA migration
(i.e. primary DNA damage) was detected after 2 and 24 h
treatments.
Comparing the two cell systems, it is evident that they react
differently to NP treatments. Significant trends were found in
PBL (2 and 24 h), whereas the RAW 264.7 cells showed a dose-
dependent effect only after the short treatment. CuO INT expo-
sures were the most effective in inducing DNA fragmentation
after 2 h exposure both in RAW 264.7 and PBL (about 4.5-fold
and 6-fold higher than in untreated control, respectively), whereas
CuO INT and rods were the most effective after 24 h exposure.
The 2 and 24 h treatments were also used to investigate
oxidative damage to DNA, by using the repair enzymes Endo-III
(Figure 8) and Fpg (Figure 9). Both 2 and 24 h NPs treatments
demonstrated the induction of Endo-III sensitive DNA sites
(oxidised pyrimidine) in both cell systems. Specifically, after
2 h of treatment, RAW 264.7 and PBL showed linear dose–
effect curves with phagocytic cells particularly susceptible to
spindles and rods treatments, whereas the lymphocytes appear
to be generally more sensitive to 24 h CuO spheres exposures.
Fpg, instead, induced significant increases in strand breaks due
to oxidised purines at all concentrations tested at 2 h for RAW

293
S. Di Bucchianico et al.

Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021

Fig. 6. Nuclear bud frequency determined by CBMN Cyt assay after CuO NPs exposure at indicated mass concentrations (microgram per millilitre). C+, 0.17 µg/
ml mitomycin C. Statistically significant differences from the control were determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). RAW 264.7
CuO spindles treatment showed a dose–effect relationship (r = 0.91, R2 = 83%, P = 0.032). PBL CuO rods treatment showed a dose–effect relationship (r = 0.99,
R2 = 98.5%, P = 0.007).

Table IV. Percentage of centromere negative (MN C−) and centromere

attributable to any of the individual NPs characteristics. For
positive (MN C+) micronuclei in binucleated RAW 264.7 and PBL cells instance, despite the significant differences in solubility
(BNMN) incubated with 1 µg/ml of CuO NPs as evaluated with the CBMN between the four sets of CuO NPs (S. K. Misra, S. Nuseibeh,
Cyt assay in combination with fluorescence in situ hybridisation A. Dybowska, D. Berhanu, T. D. Tetley and E. Valsami-Jones,
Cell types Treatment % BNMN % MN C− % MN C+
submitted for publication), there is only a slight variation in
cytotoxic responses. Our studies were performed in 1 mM
RAW 264.7 Negative control 0.8 ± 0.1 61.4 ± 3.2 38.6 ± 3.6 NaNO3, but even in serum-free cell culture medium (DCCM-
MMC 3.8 ± 0.4 74.3 ± 2.6 25.4 ± 1.8 1) a significantly higher percentage dissolution was found for
Spheres 2.1 ± 0.1 23.7 ± 3.7 76.4 ± 2.8 all the nanoparticles, whereas the relative properties within
Spindles 1.9 ± 0.1 36.7 ± 3.4 63.3 ± 3.3
Rods 2.3 ± 0.3 30.4 ± 1.2 69.6 ± 1.6 the different shapes still persist (S. K. Misra, S. Nuseibeh,
INT 1.6 ± 0.2 46.2 ± 2.3 53.8 ± 1.8 A. Dybowska, D. Berhanu, T. D. Tetley and E. Valsami-Jones,
PBL Negative control 0.5 ± 0.1 68.6 ± 2.2 31.4 ± 1.4 submitted for publication).
MMC 4.6 ± 0.4 72.4 ± 4.2 27.6 ± 2.2 In terms of links with physicochemical properties, there
Spheres 0.8 ± 0.1 26.8 ± 2.2 73.2 ± 3.2
Spindles 0.7 ± 0.1 32.3 ± 1.8 67.7 ± 3.6 seems to be a better correlation between NPs available surface
Rods 1.2 ± 0.3 25.8 ± 3.6 74.2 ± 2.6 area and cytotoxicity (data not shown) as would be expected due
INT 1.1 ± 0.2 24.7 ± 2.3 75.3 ± 3.4 to the lower surface area of the larger particles. However, this
correlation is not significant and it is not discussed further here.
MMC, 0.17 µg/ml mitomycin C. Statistically significant differences from
the negative control were determined by Student’s t-test and all data result Moreover, we have also to take into account the relative differ-
P < 0.05 respect to relative control. ences in zeta potential that can lead to a higher agglomeration that
could retard the dissolution (21) and the diffusion of metal ions
(S. K. Misra, S. Nuseibeh, A. Dybowska, D. Berhanu, T. D. Tetley
264.7 and PBL. Moreover, 24 h of PBL treatment (CuO spheres and E. Valsami-Jones, submitted for publication). However, all
and rods) showed dose-dependent effects more than RAW264.7 CuO NPs used in this study showed strong cytotoxic responses,
cells (CuO spindles). CuO spheres treatments were the most independently from their differences in zeta potential.
effective in macrophages, whereas rods exposures were the Cytostatic responses, evaluated by scoring apoptotic,
most effective in lymphocytes. necrotic and mitotic figures as well as the proliferation index
in CBMN Cyt assay, could not be linked to a specific physico-
chemical property.
Discussion CuO NPs also induced a significant increase in MN fre-
Cytotoxic and genotoxic responses following in vitro quency in PBL and in RAW 264.7, and FISH analysis revealed
exposure to CuO INT and differently shaped CuO NPs (mass that all CuO NPs are able to induce a significant increase in
concentrations from 0.1 to 100 μg/ml) were assessed in murine malsegregation events. It is known that many nanomateri-
macrophages and in PBL. The MTT assay revealed a decrease als such as multi-walled carbon nanotubes, amorphous SiO2,
of cell viability in RAW 264.7 cells and PBL with significant TiO2 and CoCr can induce aneuploidy by mechanical interfer-
dose–effect relationships over the tested concentrations and ence with the cytoskeleton, causing chromosome missegrega-
with distinctive patterns for different CuO NPs; the comparison tion (22). Overall, this study provides the first evidence of
between cytotoxicity and physicochemical characteristics of the CuO NPs potential to induce clastogenic and aneugenic
NPs showed a differentiated bioreactivity, however, not directly events.

294
 Shape-specific CuO NP cytotoxicity and genotoxicity

Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021

Fig. 7. Primary DNA damage. Comet assay revealed an increase in primary DNA damage, with different sensitivity shown by cell types at 2 (above) and 24 h
(bottom) treatments. C+: 10 µM H2O2. CuO spheres treatment for 2 h in Raw 264.7 cells showed a dose–effect relationship (r = 0.95, R2 = 90.7%, P = 0.04) as
well as in CuO spindles (r = 0.99, R2 = 97.6%, P = 0.01), rods (r = 0.99, R2 = 97.4%, P = 0.01) and CuO INT (r = 0.99, R2 = 98.6%, P = 0.007) exposures. CuO
spindles (2 h treatment) showed in PBL a dose–effect relationship (r = 0.97, R2 = 93.6%, P = 0.007) as well as both CuO rods (r = 0.99, R2 = 99.3%, P = 0.0002)
and INT (r = 0.95, R2 = 89.9%, P = 0.05). Only CuO spindles RAW 264.7 (24 h treatment) continue to show a trend (r = 0.95, R2 = 89.9%, P = 0.05), whereas
in PBL (24 h treatment) a dose-dependent relationship was found for spheres (r = 0.98, R2 = 96%, P = 0.004), spindles (r = 0.93, R2 = 86.8%, P = 0.02) and rods
(r = 0.97, R2 = 94%, P = 0.007). Statistically significant differences from the control were determined by Student’s t-test (**P < 0.01, ***P < 0.001).

Moreover, there was a highly significant increase in nucleo-
plasmic bridges frequency indicating chromosome breakage
and fusions in both cell systems, whereas NBUDs were sig-
nificantly generated only in RAW 264.7 cells. The induction
of apoptosis and chromosomal rearrangements often in a dose-
dependent manner and specifically at low doses of NPs, accom-
panied by proliferation arrest at high concentrations, suggests
differential sensitivity of NPs concentrations independently
from cell types. A possible mechanism could be interpreted as
a status where cells tolerate DNA damage and gain resistance
to cell death at low dose exposures.
The comet assay also showed an increase in primary DNA
damage after 2 and 24 h treatments, whereas oxidative damage
to DNA was found with NPs shape-related trends.
Interestingly, for the RAW 264.7 cells the CuO INT NPs
caused the highest response in primary DNA damage scored
by the comet assay, but a more modest effect in terms of MN
frequency. The two assays have different sensitivity especially
with regard to the potential to induce missegregation as already
demonstrated by Hartmann and co-authors (23). In fact, geno-
toxic insults induced by aneugens are not readily detectable by
the comet assay and CuO INT NPs, the least aneugenic NPs
among those tested by us (as shown here by FISH analysis),
are the most effective in inducing DNA damage assessed by
the comet assay.
This study shows, therefore, that two sets of CuO NPs, syn-
thesised using very different protocols (lab aqueous synthesis
and commercial plasma synthesis) both can induce cytotoxic,
cytostatic and genotoxic effects. These findings are consist-
ent with a wide range of data available in the literature and
demonstrate that all CuO NPs, effectively independently from
their precise size and shape, clearly cause positive responses.

295
S. Di Bucchianico et al.

Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021

Fig. 8. Oxidative damage to DNA pyrimidines after 2 h (above) and 24 h (bottom) CuO NPs treatment in cell cultures. Endo III sensitive sites in treated,
untreated (C−) and positive control (C+: 10 µM H2O2) cultures are shown. Statistically significant differences from the control were determined by Student’s t-test
(*P < 0.05, **P < 0.01, ***P < 0.001). CuO spheres treatment for 2 h in RAW 264.7 showed a dose–effect relationship (r = 0.97, R2 = 94%, P = 0.03) as well
as CuO INT exposure (r = 0.98, R2 = 96.7%, P = 0.016). In 2 h whole blood treatment, a dose–effect relationship was found in spheres (r = 0.99, R2 = 98.4%,
P = 0.008), rods (r = 0.99, R2 = 98.6%, P = 0.007) and CuO INT (r = 0.98, R2 = 95.2%, P = 0.02). After 24 h treatment, CuO spheres and spindles showed a dose–
effect relationship in RAW 264.7 (r = 0.99, R2 = 98.8%, P = 0.006 and r = 0.96, R2 = 91.4%, P = 0.04, respectively) and also in PBL 24 h exposure a dose–effect
relationship was found for CuO spheres (r = 0.99, R2 = 98%, P = 0.01), rods (r = 0.99, R2 = 99.4%, P = 0.003) and CuO INT (r = 0.98, R2 = 96.6%, P = 0.02).

Among different metal oxide NPs for which a certain amount
of studies are available, CuO appears to trigger the highest
responses for inducing cytotoxicity, intracellular ROS genera-
tion and DNA damage (8). Karlsson and coworkers (8) showed
that the A549 cell viability determined after 18 h of exposure
to CuO NPs at doses of 40 μg/ml and 80 μg/ml was reduced
by 90 and 96%, respectively. These toxicity data were consist-
ent with our results and the results they obtained by the comet
assay using Fpg, demonstrating oxidative lesions induced in
DNA. We found effectively a clear induction of oxidative dam-
age to DNA detected not only by using FPg, an enzyme that
recognises and excises oxidised purines (including 8-hydroxy-
20-deoxyguanosine), but also by using Endo-III that recognises
and excises oxidised pyrimidines. Oxidative damage to DNA
seems to be the central element in the induction of the series of
multiple effects shown in this study. Consistent with the sup-
posed oxidative CuO NPs effects, the addition of the antioxi-
dant N-acetyl-cysteine has been shown to significantly protect
RAW 264.7 cells from the cytotoxicity induced by submaximal
doses of this nanomaterial (12).
Moreover, by evaluating their ability to cause cell death,
mitochondrial damage, DNA damage and oxidative DNA
lesions, NPs of CuO were found highly toxic when compared
with other metal oxide NPs and much more toxic compared
with CuO micrometric-sized particles (24). In human laryn-
geal epithelial cells (HEp-2), CuO induced the greatest amount
of cytotoxicity in a dose-dependent manner and was able to
overwhelm antioxidant defences (e.g. catalase and glutathione
reductase) compared with other oxides such as silicon oxide
and ferric oxide NPs. Moreover, a significant increase in the
level of 8-isoprostanes and in the ratio of GSSG to total glu-
tathione in cells exposed to CuO, together with a partial reduc-
tion of the cytotoxic effect when HEp-2 cells were co-treated
with resveratrol was observed (25).
Even in human A549 cells, CuO NPs were found to induce
oxidative stress in a dose-dependent manner indicated by

296
 Shape-specific CuO NP cytotoxicity and genotoxicity

Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021

Fig. 9. Oxidative damage to DNA purines after 2 h (above) and 24 h (bottom) CuO NPs treatment in cell cultures. Fpg sensitive sites in treated, untreated (C−) and
positive control (C+: 10 µM H2O2) cultures are shown. Statistically significant differences from the control were determined by the Student’s t-test (***P < 0.001).
CuO spheres treatment for 2 h in RAW 264.7 showed a dose–effect relationship (r = 0.95, R2 = 91%, P = 0.04) as well as spindles (r = 0.99, R2 = 97.4%,
P = 0.013), rods (r = 0.95, R2 = 90%, P = 0.04) and CuO INT (r = 0.95, R2 = 89.7%, P = 0.05) exposure. For 2 h whole blood treatments, a dose–effect
relationship was found in spheres (r = 0.98, R2 = 96%, P = 0.02), rods (r = 0.97, R2 = 93.5%, P = 0.03) and CuO INT (r = 0.97, R2 = 93.5%, P = 0.03). After 24 h,
only CuO spindles showed a dose–effect relationship in RAW 264.7 (r = 0.99, R2 = 97.3%, P = 0.01), whereas in PBL 24 h exposures, CuO spheres (r = 0.97,
R2 = 94%, P = 0.03) and rods (r = 0.99, R2 = 98.3%, P = 0.009) showed a dose–effect relationship.

depletion of glutathione and induction of lipid peroxidation,
catalase and superoxide dismutase (6). Moreover, that study
showed that CuO NPs induce significant effects on cell viabil-
ity, even at the lowest dose tested (10 μg/ml) and that A549 cell
viability is reduced to 48% at the highest concentration tested
(50 μg/ml) after 24 h of exposure.
The ability of the CuO NPs to cause cell death, DNA dam-
age and oxidative DNA lesions was evaluated by Wang and co-
authors (26) and one key mechanism proposed was the ability
of CuO NPs to damage mitochondria. CuO NPs showed signifi-
cant toxicity mediated by oxidative stress in both HepG2 cells
and catfish primary hepatocytes (26). The toxicity observed not
only highlights ROS-induced cell death but also was linked to
cell and mitochondrial membranes. Furthermore, high content
bright-field imaging demonstrated potent and dose-dependent
hatching interference in the zebrafish embryos compromising
the activity of the hatching enzyme, ZHE1 (27). This hypoth-
esis is based on the presence of metal-sensitive histidine in the
catalytic centre of this enzyme. Furthermore, complexation-
mediated leaching of CuO NPs by amino acids was identified
as the source of toxicity towards Escherichia coli showing that
the leached copper–peptide complex induces a multiple-fold
increase in intracellular ROS generation and reduces the frac-
tion of viable cells, resulting in the overall inhibition of bio-
mass growth (28).
NP cytotoxicity also depends on intracellular solubility as
demonstrated by a comparison of stabilised copper metal NPs
and degradable CuO NPs (29). Using copper as a representa-
tive example, Studer and collaborators (29) compared the cyto-
toxicity of copper metal NPs stabilised by a carbon layer with
copper oxide NPs using two different cell lines. Measuring the
intra- and extracellular solubility of the two materials in stand-
ard buffers, the authors explained the cytotoxicity difference
on the basis of altered copper release. In fact, the well-known
mechanism of intracellular dissolution (Trojan horse-type
mechanism) illustrates how NPs can bypass the protection of
mammalian cells against heavy metal ions and the dominating
influence of physicochemical parameters on the cytotoxicity of
a given metal (30). However, the electronic properties of the
oxide NPs, determined by their chemical composition, can be
modulated by crystalline structure, defect, size, shape and mor-
phology, and may play an important role in their cytotoxicity.

297
S. Di Bucchianico et al.
For instance, although no significant differences were observed
in the cytoxicity of spherical and sheet-shaped ZnO NPs on
BEAS-2B and RAW 264.7 cells, there were differences in their
cellular association and inflammatory potential (31). Even so,
ZnO NPs with different sizes and shapes can induce different
cytotoxic effects in A549 human lung epithelial cells as well as
different influences on the mitochondria activity and chemokine
production (32). Likely, the differential effects of NPs size and
shape on their cytotoxicity can also be closely linked to the cell
type. NPs with different specific surface area, different sizes and
different raw material but the same hydrodynamic diameter, dif-
ferentially disturbed the Caco-2 cell monolayer integrity, were
cytotoxic and triggered an increase of several transcripts cod-
ing for pro-inflammatory cytokines and chemokines; this dif-
ferential toxicity was only partly correlated with the release
of copper and with the shape of the NPs (33). For a number
of biomarkers, the biological responses are significantly more
important in presence of CuO NPs than in presence of soluble
Cu suggesting a specific nanoparticle effect (34). CuO NPs were
clearly observed in both the cell nucleus and the mitochondria
after being taken up by human pulmonary epithelial A549 cells
mainly through endocytosis, although part of the internalised
CuO NPs were dynamically excreted to the extracellular envi-
ronment (35). Intracellular localisation was also observed from
2 to 24 h of incubation with both rod-shaped and spherical CuO
NPs in HepG2 cells (36). In addition, numerous CuO NPs were
observed in dead A549 cells compared with viable cells, sug-
gesting that the difference in the amount of CuO NPs that are
absorbed into individual cells may determine their fate (37).
This interesting study by Hanagata and co-authors (37) also
showed that up-regulation of DNA damage-inducible 45 β and
γ genes expression was not observed with Cu ions released into
medium but was observed in cells exposed to CuO NPs, high-
lighting that CuO NPs themselves cause genotoxicity.
Other than size or shape, the response of NPs in the cells may
be directed by complications regarding dosage. For chemicals,
dosage simply implies molar concentration, whereas for NPs
the concentration can be viewed as a 2D entity, one compo-
nent being the atomic or molar density, the other being the size,
shape or other surface properties (38). Thus, a complex inter-
play prevails between size, shape, dissolution, zeta potential,
surface area and nature of surface functionalisation, as well as
the nature of protein corona that defines the biological identity
of NPs. Nevertheless, numerous controversies persist about the
biological impact of NPs physicochemical properties and fur-
ther studies are needed due to relatively few reports describing
the extents to which the NP properties influence their toxicity.
Overall, the data obtained here together with the available lit-
erature suggest that CuO NPs should be considered as a valuable
positive control for cytotoxicity and genotoxicity studies of nano-
structured materials, at least in in vitro studies with murine and
mammalian cells, due to the fact that they exert a clear cytotoxic
and genotoxic action, independent of their size and shape. For
instance, Rotoli and co-authors (12) observed that CuO NPs (the
same CuO INT used for our work) had very marked effects on
cell viability, comparable to that observed upon treatment with
Ni NPs, used as a positive control in comparative studies of metal
oxide NPs on human airway epithelial cells and macrophages.
However, although the primary role of oxidative stress mecha-
nisms and the contribution of aneugenic responses in cytotoxic-
ity and genotoxicity induced by CuO NPs is clear, it would need
to be combined with a better understanding of cellular uptake
levels, in particular in the function of different cell types.
298
Positive controls are needed to demonstrate the ability of
the cells used and the test protocols to detect compounds able
to induce adverse effects (39). Moreover, the appropriateness
of available testing systems for nanomaterials requires con-
firmation and standardisation (40). The availability of refer-
ence materials is important to function as a benchmark for
the adverse effects of materials with complex structures and
to compare toxicological data from various studies appropri-
ately (41).
Funding
Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021
European Community’s Seventh Framework Programme
(FP7/2007–2013) under grant agreement No CP-FP 214478-2
(project name NanoRetox; www.nanoretox.eu).
Conflict of interest statement: Paul Reip is an employee of Intrinsiq Materials
Ltd, which sourced (and also manufactures) the CuO used in this study.
References
1. Huang, X., Teng, X., Chen, D., Tang, F. and He, J. (2010) The effect of the
shape of mesoporous silica nanoparticles on cellular uptake and cell func-
tion. Biomaterials, 31, 438–448.
2. Xu, L. J., Zhao, J. X., Zhang, T., Ren, G. G. and Yang, Z. (2009) In vitro
study on influence of nano particles of CuO on CA1 pyramidal neurons of
rat hippocampus potassium. Curr. Environ. Toxicol., 24, 211–217.
3. Sun, J., Wang, S., Zhao, D., Hun, F. H., Weng, L. and Liu, H. (2011)
Cytotoxicity, permeability, and inflammation of metal oxide nanoparticles
in human cardiac microvascular endothelial cells. Cell Biol. Toxicol., 25,
333–342.
4. Ahamed, M., Siddiqui, M. A., Akhtar, M. J., Ahmad, I., Pant, A. B. and
Alhadlaq, H. A. (2010) Genotoxic potential of copper oxide nanoparticles
in human lung epithelial cells. Biochem. Biophys. Res. Commun., 396,
578–583.
5. Pan, X., Redding, J. E., Wiley, P. A., Wen, L., McConnell, J. S. and Zhang,
B. (2010) Mutagenicity evaluation of metal oxide nanoparticles by the bac-
terial reverse mutation assay. Chemosphere, 79, 113–116.
6. Lei, R., Wu, C., Yang, B., Ma, H., Shi, C., Wang, Q., Wang, Q., Yuan, Y. and
Liao, M. (2008) Integrated metabolomic analysis of the nano-sized cop-
per particle-induced hepatotoxicity and nephrotoxicity in rats: a rapid in
vivo screening method for nanotoxicity. Toxicol. Appl. Pharmacol., 232,
292–301.
7. Cho, W. S., Duffin, R., Poland, C. A., Howie, S. E., MacNee, W., Bradley,
M., Megson, I. L. and Donaldson, K. (2010) Metal oxide nanopar-
ticles induce unique inflammatory footprints in the lung: important
implications for nanoparticle testing. Environ. Health Perspect., 118,
1699–1706.
8. Karlsson, H. L., Cronholm, P., Gustafsson, J. and Möller, L. (2008)
Copper oxide nanoparticles are highly toxic: a comparison between
metal oxide nanoparticles and carbon nanotubes. Chem. Res. Toxicol., 21,
1726–1732.
9. Fubini, B., Ghiazza, M. and Fenoglio, I. (2010) Physico-chemical features
of engineered nanoparticles relevant to their toxicity. Nanotoxicology, 4,
347–363.
10. Hanley, C., Thurber, A., Hanna, C., Punnoose, A., Zhang, J. and Wingett,
D. G. (2009) The Influences of Cell Type and ZnO Nanoparticle Size on
Immune Cell Cytotoxicity and Cytokine Induction. Nanoscale Res. Lett., 4,
1409–1420.
11. Di Giorgio, M. L., Di Bucchianico, S., Ragnelli, A. M., Aimola, P., Santucci,
S. and Poma, A. (2011) Effects of single and multi walled carbon nanotubes
on macrophages: cyto and genotoxicity and electron microscopy. Mutat.
Res., 722, 20–31.
12. Rotoli, B. M., Bussolati, O., Costa, A. L. et al. (2012) Comparative effects
of metal oxide nanoparticles on human airway epithelial cells and mac-
rophages. J. Nanopart. Res., 14, 1069.
13. Migliore, L., Frenzilli, G., Nesti, C., Fortaner, S. and Sabbioni, E. (2002)
Cytogenetic and oxidative damage induced in human lymphocytes by plati-
num, rhodium and palladium compounds. Mutagenesis, 17, 411–417.
14. Collins, A. R., Oscoz, A. A., Brunborg, G., Gaivão, I., Giovannelli, L.,
Kruszewski, M., Smith, C. C. and Stetina, R. (2008) The comet assay: topi-
cal issues. Mutagenesis, 23, 143–151.

15. Fenech, M. (2007) Cytokinesis-block micronucleus cytome assay. Nat.
Protoc., 2, 1084–1104.
16. Parry, E. M., Parry, J. M., Corso, C. et al. (2002) Detection and characteri-
zation of mechanisms of action of aneugenic chemicals. Mutagenesis, 17,
509–521.
17. Zhu, J., Li, D., Chen, H., Yang, X., Lu, L. and Wang, X. (2004) Highly dis-
persed CuO nanoparticles prepared by a novel quick precipitation method.
Mater. Lett. 58, 3324–3327.
18. Migliore, L., Saracino, D., Bonelli, A., Colognato, R., D’Errico, M. R.,
Magrini, A., Bergamaschi, A. and Bergamaschi, E. (2010) Carbon nano-
tubes induce oxidative DNA damage in RAW 264.7 cells. Environ. Mol.
Mutagen., 51, 294–303.
19. Collins, A. R., Dusinská, M., Gedik, C. M. and Stĕtina, R. (1996) Oxidative
damage to DNA: do we have a reliable biomarker? Environ. Health
Perspect., 104(Suppl. 3), 465–469.20.
20. Lindberg, H. K., Wang, X., Järventaus, H., Falck, G. C., Norppa, H. and
Fenech, M. (2007) Origin of nuclear buds and micronuclei in normal and
folate-deprived human lymphocytes. Mutat. Res., 617, 33–45.
21. Peng, X., Palma, S., Fisher, N. S. and Wong, S. S. (2011) Effect of mor-
phology of ZnO nanostructures on their toxicity to marine algae. Aquat.
Toxicol., 102, 186–196.
22. Gonzalez, L., Decordier, I. and Kirsch-Volders, M. (2010) Induction of
chromosome malsegregation by nanomaterials. Biochem. Soc. Trans., 38,
1691–1697.
23. Hartmann, A., Schumacher, M., Plappert-Helbig, U., Lowe, P., Suter, W.
and Mueller, L. (2004) Use of the alkaline in vivo Comet assay for mecha-
nistic genotoxicity investigations. Mutagenesis, 19, 51–59.
24. Karlsson, H. L., Gustafsson, J., Cronholm, P. and Möller, L. (2009) Size-
dependent toxicity of metal oxide particles–a comparison between nano-
and micrometer size. Toxicol. Lett., 188, 112–118.
25. Fahmy, B. and Cormier, S. A. (2009) Copper oxide nanoparticles induce
oxidative stress and cytotoxicity in airway epithelial cells. Toxicol. In Vitro,
23, 1365–1371.
26. Wang, Y., Aker, W. G., Hwang, H. M., Yedjou, C. G., Yu, H. and Tchounwou,
P. B. (2011) A study of the mechanism of in vitro cytotoxicity of metal
oxide nanoparticles using catfish primary hepatocytes and human HepG2
cells. Sci. Total Environ., 409, 4753–4762.
27. Lin, S., Zhao, Y., Xia, T. et al. (2011) High content screening in zebrafish
speeds up hazard ranking of transition metal oxide nanoparticles. ACS
Nano, 5, 7284–7295.
28. Gunawan, C., Teoh, W. Y., Marquis, C. P. and Amal, R. (2011) Cytotoxic
origin of copper(II) oxide nanoparticles: comparative studies with micron-
sized particles, leachate, and metal salts. ACS Nano, 5, 7214–7225.
29. Studer, A. M., Limbach, L. K., Van Duc, L., Krumeich, F., Athanassiou,
E. K., Gerber, L. C., Moch, H. and Stark, W. J. (2010) Nanoparticle
Shape-specific CuO NP cytotoxicity and genotoxicity
cytotoxicity depends on intracellular solubility: comparison of stabilized
copper metal and degradable copper oxide nanoparticles. Toxicol. Lett.,
197, 169–174.
30. Xia, T., Kovochich, M., Liong, M., Mädler, L., Gilbert, B., Shi, H., Yeh, J.
I., Zink, J. I. and Nel, A. E. (2008) Comparison of the mechanism of toxic-
ity of zinc oxide and cerium oxide nanoparticles based on dissolution and
oxidative stress properties. ACS Nano, 2, 2121–2134.
31. Heng, B. C., Zhao, X., Tan, E. C., Khamis, N., Assodani, A., Xiong, S.,
Ruedl, C., Ng, K. W. and Loo, J. S. (2011) Evaluation of the cytotoxic and
inflammatory potential of differentially shaped zinc oxide nanoparticles.
Arch. Toxicol., 85, 1517–1528.
32. Hsiao, I. L. and Huang, Y. J. (2011) Effects of various physicochemical
characteristics on the toxicities of ZnO and TiO nanoparticles toward
Downloaded from https://academic.oup.com/mutage/article/28/3/287/1295389 by Universidade de Bras�lia user on 03 December 2021
human lung epithelial cells. Sci. Total Environ., 409, 1219–1228.
33. Piret, J. P., Vankoningsloo, S., Mejia, J. et al. (2012) Differential toxicity
of copper (II) oxide nanoparticles of similar hydrodynamic diameter on
human differentiated intestinal Caco-2 cell monolayers is correlated in part
to copper release and shape. Nanotoxicology, 6, 789–803.
34. Buffet, P. E., Tankoua, O. F., Pan, J. F. et al. (2011) Behavioural and bio-
chemical responses of two marine invertebrates Scrobicularia plana and
Hediste diversicolor to copper oxide nanoparticles. Chemosphere, 84,
166–174.
35. Wang, Z., Li, N., Zhao, J., White, J. C., Qu, P. and Xing, B. (2012) CuO
nanoparticle interaction with human epithelial cells: cellular uptake, loca-
tion, export, and genotoxicity. Chem. Res. Toxicol., 25, 1512–1521.
36. Piret, J. P., Jacques, D., Audinot, J. N. et al. (2012) Copper(ii) oxide
nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxi-
dative stress and induce pro-inflammatory response. Nanoscale, 4,
7168–7184.
37. Hanagata, N., Zhuang, F., Connolly, S., Li, J., Ogawa, N. and Xu, M. (2011)
Molecular responses of human lung epithelial cells to the toxicity of copper
oxide nanoparticles inferred from whole genome expression analysis. ACS
Nano, 5, 9326–9338.
38. Patra, H. K. and Dasgupta, A. K. (2011) Cancer cell response to nanopar-
ticles: criticality and optimality. Nanomedicine 2011 November 15 [Epub
ahead of print].
39. Donaldson, K., Poland, C. A. and Schins, R. P. (2010) Possible geno-
toxic mechanisms of nanoparticles: criteria for improved test strategies.
Nanotoxicology, 4, 414–420.
40. Warheit, D. B. and Donner, E. M. (2010) Rationale of genotoxicity testing
of nanomaterials: regulatory requirements and appropriateness of available
OECD test guidelines. Nanotoxicology, 4, 409–413.
41. Lai, D. Y. (2012) Toward toxicity testing of nanomaterials in the 21st cen-
tury: a paradigm for moving forward. Wiley Interdiscip. Rev. Nanomed.
Nanobiotechnol., 4, 1–15.
299