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Toxicology and Industrial Health

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Genotoxic and oxidative stress  The Author(s) 2015
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DOI: 10.1177/0748233715599472
tih.sagepub.com
oxide particles in Drosophila melanogaster

Erico R Carmona1, Claudio Inostroza-Blancheteau2,

Laura Rubio3 and Ricard Marcos3

Abstract
Zinc oxide nanoparticles (ZnONP) are manufactured on a large scale and can be found in a variety of consumer
products, such as sunscreens, lotions, paints and food additives. Few studies have been carried out on its
genotoxic potential and related mechanisms in whole organisms. In the present study, the in vivo genotoxic
activity of ZnONP and its bulk form was assayed using the wing-spot test and comet assay in Drosophila
melanogaster. Additionally, a lipid peroxidation analysis using the thiobarbituric acid assay was also performed.
Results obtained with the wing-spot test showed a lack of genotoxic activity of both ZnO forms. However,
when both particle sizes were tested in the comet assay using larvae haemocytes, a significant increase in DNA
damage was observed for ZnONP treatments but only at the higher dose applied. In addition, the lipid per-
oxidation assay showed significant malondialdehyde (MDA) induction for both ZnO forms, but the induction of
MDA for ZnONP was higher for the ZnO bulk, suggesting that the observed DNA strand breaks could be
induced by mediated oxidative stress. The overall data suggest that the potential genotoxicity of ZnONP in
Drosophila can be considered weak according to the lack of mutagenic and recombinogenic effects and the
induction of primary DNA damage only at high toxic doses of ZnONP. This study is the first assessing the
genotoxic and oxidative stress potential of nano and bulk ZnO particles in Drosophila.

Keywords
Comet assay, DNA damage, haemocytes, malondialdehyde, wing-spot test, ZnO

Introduction NM are commonly defined as engineering materi-

The development of the nanotechnology industry
implies the creation and use of new manufactured
nanomaterials (NM) with interesting physical–chem-
ical properties, such as high durability, mechanical
resistance, high conductivity and high chemical reac-
tivity. All of this makes NM attractive for different
areas ranging from industry and pharmaceutical appli-
cations to environmental remediation and biomedi-
cine. Until now, more than 1600 globally available
consumer products use manufactured NM (nanotech-
project.org), and predictions suggest that these nano-
based products will increase dramatically in the next
years (Fairbrother, 2010; Hansen et al., 2009). Hence,
the current human and environmental exposure to NM
will inevitably increase in the near future (Buzea
et al., 2007; Savolainen et al., 2010), requiring more
data on their potential secondary effects.
als with one or more dimensions having a size in the
range of 1–100 nm and include nanoparticles (NP),
nanofibres, nanotubes, composite materials and
1
Grupo de Genotoxicologı́a, Núcleo de Investigación en Estudios
Ambientales, Facultad de Recursos Naturales, Escuela de Medicina
Veterinaria, Universidad Católica de Temuco, Temuco, Chile
2
Núcleo de Investigación en Producción Alimentaria, Facultad de
Recursos Naturales, Escuela de Agronomı́a, Universidad Católica
de Temuco, Temuco, Chile
3
Grup de Mutagènesi, Departament de Genètica i de Microbiologia,
Facultat de Biociències, Universitat Autònoma de Barcelona,
Cerdanyola del Vallès, Barcelona, Spain
Corresponding author:
Erico R Carmona, Núcleo de Investigación en Estudios Ambientales,
Universidad Católica de Temuco, P.O. Box 15-D, Temuco 4813302,
Chile.
Email: ecarmona@uct.cl

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2 Toxicology and Industrial Health
nanostructured surfaces (Borm et al., 2006). Particles
in nanoscale show particular shape, large surface area
and surface reactivity. However, the same characteris-
tics mentioned above could contribute to human
health hazards and risks because they may also be
responsible for adverse biological effects (Xia et al.,
2009). To date, there is increasing evidence that NP
manifest increased toxicity compared to micrometric
particles of the same composition (Chibber et al.,
2013). Thus, several studies reveal that NP can induce
different adverse effects in biological systems, such as
oxidative stress, cytotoxicity, DNA damage, apoptosis
and inflammatory responses (Chibber et al., 2013; Cho
et al., 2010; Kumar and Dhawan, 2013). Considering
this background, an increasing number of toxicological
investigations are currently being carried out with NP
to determine their potential toxic effects and to eluci-
date human and environmental health risks (Dhawan
and Sharma, 2010).
Among the manufactured NM more widely pro-
duced are zinc oxide nanoparticles (ZnONP). These
may be found in a wide assortment of consumer prod-
ucts, such as sunscreens, cosmetics, paints, pigments,
food additives and disinfectants (Espitia et al., 2012;
Raj et al., 2012). Since ZnONP provide high trans-
parency in the skin and better protection from ultra-
violet (UV) solar radiation than bulk or micrometric
counterparts, this NM is being destined for use in
modern sunscreens and cosmetics (Osmond and
McCall, 2010). Thus, the probability of human and
environmental exposure to ZnONP is increasing, and
their potential adverse biological effects must be
adequately studied.
According to various authors, ZnONP can show
genotoxic potential in several cell types and organism
models, for example, it has been proven to induce
mutagenicity in bacterial assay systems (Kumar et al.,
2011a, 2011b). Likewise, in vitro studies indicate that
ZnONP can induce DNA breaks; micronuclei forma-
tion; and chromosomal breakage in nasal mucosa, epi-
dermal, neuronal and human blood cells (Bhattacharya
et al., 2014; Demir et al., 2014; Hackenberg et al.,
2011; Sharma et al., 2012b; Valdiglesias et al.,
2013). In addition, some in vivo studies with animals
have revealed genetic damage, apoptosis and tissue
inflammation associated with ZnONP exposure
(Nounou et al., 2013; Pasupuleti et al., 2012; Sharma
et al., 2012b). Nevertheless, negative results have also
been reported recently in the literature where ZnONP
were unable to exert genotoxic effects in several
in vitro and in vitro test systems (Kwon et al., 2014;
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Nam et al., 2013). All these suggest that the genotoxic
potential of ZnONP remains controversial.
Oxidative stress has been proposed as one of the
main mechanisms involved in toxic and genotoxic
manifestations induced by nanoscale particles (Shri-
vastava et al., 2014; Wang et al., 2013). Therefore,
oxidative stress analysis must be included in any gen-
otoxicity study carried out with NP to elucidate possi-
ble mechanisms related to induced genetic damage.
Several studies have demonstrated that ZnONP can
produce DNA damage mediated by the generation of
oxidative stress, as evidenced by an increase of reac-
tive oxygen species production, oxidative DNA dam-
age and depletion of antioxidant defence in diverse
types of cultured human cells (Alarifi et al., 2013;
Demir et al., 2014; Lin et al., 2009; Sharma et al.,
2009, 2012a). Such has also been reported for bacteria
cells where glutathione depletion with an associated
increase in free radicals, lipid peroxidation end prod-
ucts and lactate dehydrogenase activity demonstrate
that ZnONP produce oxidative stress leading to geno-
toxicity in Escherichia coli (Kumar et al., 2011b).
Similarly, some in vivo studies suggest that exposure
to ZnONP leads to an accumulation of NP in the liver
of mice and rats, causing DNA damage and apoptosis
as a result of oxidative stress (Nounou et al., 2013;
Sharma et al., 2012b). To date, studies evaluating oxi-
dative stress and DNA damage after ZnONP exposure
have not been carried out in Drosophila
melanogaster.
In nanotoxicology, in vitro models are usually
employed to evaluate and describe the toxicity of
NM. However, in vivo studies could be considered
much more interesting in terms of risk evaluation than
in vitro approaches with mammal or human cells
since they do not completely simulate the complex
cell–cell, cell–matrix interactions and hormonal effects
found in the in vivo systems (Chibber et al., 2013). In
this scenario, recent studies have demonstrated that
the fruit fly D. melanogaster offers several benefits
as an in vivo model for the study of dietary intake and
tissue distribution of nano-carbon–based materials
(Leeuw et al., 2007; Liu et al., 2009), the study of the
potential toxicity of metal and metal oxide-based NP
on reproduction and development (Gorth et al., 2011;
Philbrook et al., 2011; Pompa et al., 2011; Posgai
et al., 2011) and the study of genotoxicity after expo-
sure to silver, cobalt, gold, titanium, zirconium and
aluminium NP (Ahamed et al., 2010; Demir et al.,
2011, 2013; Sabella et al., 2011; Vales et al., 2012;
Vecchio et al., 2012). The aforementioned results
Carmona et al.
would reinforce the usefulness of the Drosophila
model as a first-tier in vivo test for genotoxicity test-
ing of NM.
In this context, the aim of the present study was to
explore the in vivo genotoxic activity and the levels
of oxidative stress associated with the exposure of
ZnONP as compared to the effects induced by its bulk
form using genotoxic and biochemical approaches in
D. melanogaster.
The wing-spot test was used as a short-test system
based on the loss of heterozygosity (LOH) in normal
genes, and the corresponding expressions of recessive
markers are called multiple wing hairs (mwh) and
flare-3 (flr3) in the wing blade of adult flies (Graf
et al., 1984). Thus, the induced genotoxic effects have
been microscopically observed as an increase in the
frequency of mutant clones markers (mwh or flr3) in
wing slides preparation. This assay can detect a wide
range of mutational events such as point mutations,
deletions, certain types of chromosome aberrations
(non-disjunction) and mitotic recombination (Graf
et al., 1984). It should be noted that the quantification
of mitotic recombination in somatic cells is consid-
ered a relevant process for genotoxicity screening
because aberrant recombination activity is commonly
associated with carcinogenesis (Luo et al., 2000;
Sengstag, 1994).
An in vivo comet assay was also applied using hae-
mocytes of D. melanogaster larvae to detect the induc-
tion of primary DNA damage (Carmona et al., 2011b;
Marcos and Carmona, 2013). Haemocytes of Droso-
phila, present in the haemolymph, may be directly
exposed to toxic substances circulating in the haemo-
lymph and are, therefore, an interesting target tissue
for genotoxicity assessment (Villela et al., 2006). The
comet assay is a sensitive tool for the detection of
several kinds of DNA damage, such as double- and
single-strand DNA breaks, DNA alkali-labile sites and
incomplete repair sites in individual cells (Tice et al.,
2000). This assay is a reliable technique to assess DNA
damage in different model organisms, including D.
melanogaster (Dhawan et al., 2009). The alkaline
comet assay version has been proven as a useful and
sensitive method for detecting DNA damage caused
by fullerenes, carbon nanotubes, quantum dots,
metal-based and metal oxide NP; therefore, it is highly
recommended for the genotoxic risk assessment of NM
(Karlsson, 2010).
Finally, to determine the induction of oxidative
stress in treated Drosophila larvae with ZnONP and
bulk forms, a lipid peroxidation assay was also
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3
performed. Lipid peroxidation is a consequence of
free radical attacks in the membrane system and
could be considered an indirect measure of oxidative
stress in the cells. This study employed the thiobar-
bituric acid reactive substance (TBARS) assay
described by Tironi et al. (2007). This biochemical
test is able to detect the extent of peroxidation of
lipids by measuring the malondialdehyde (MDA)
concentration in tissue extracts of exposed larvae.
MDA is one of the low-molecular-weight end prod-
ucts formed via the decomposition of certain primary
and secondary lipid peroxidation products (Halliwell
and Chirico, 1993).
Materials and methods
Chemicals
ZnONP (!100 nm average particle size, surface area
15–25 m2/g, >99% purity, CAS no. 1314-13-2, ref.
no. 544906), ZnO bulk (<5 mm, "99.0% purity, CAS
no. 1314-13-2, ref. no. 96479), ethyl methane sulpho-
nate (EMS, 100% purity, CAS no. 62-50-0), Triton X-
100, Trizma base, sodium hydroxide (NaOH), sodium
chloride (NaCl) and disodium salt of ethylenediami-
netetraacetic acid (Na2-EDTA) were obtained from
Sigma-Aldrich (St Louis, Missouri, USA); low-
melting-point agarose (LMA), normal melting point
agarose (NMA), phosphate-buffered saline (PBS)
and 4,6-diamidine-2-phenylindole (DAPI) were
obtained from Life Technologies Corporation
(Carlsbad, California, USA); phenylthiourea (PTU),
N-lauroylsarcosine sodium salt, potassium chloride,
trichloroacetic acid (TCA) and 2-thiobarbituric acid
(TBA) were obtained from Merck Company (Darm-
stadt, Germany).
ZnONP preparation
Various concentrations of ZnONP were prepared with
distilled water. Dispersion was carried out by sonica-
tion using an ultrasonic bath (37 kHz, Elmasonic S,
Elma, Germany) for 30 min at room temperature. ZnO
bulk was used to compare the genotoxic effects
between micrometric and nanoparticulated forms.
The compound was prepared with distilled water
and diluted through magnetic stirring for 10 min
at room temperature. Distilled water was used for
negative control, whilst the mutagenic agent EMS
was used as positive control in each experiment car-
ried out with both the wing-spot and comet assays.
4 Toxicology and Industrial Health
NP characterization
To confirm the physical characteristics of ZnONP, the
following techniques were performed: transmission
electron microscopy (TEM), dynamic light scattering
(DLS) and laser doppler velocimetry (LDV). TEM
was carried out with a JEOL JEM-2011 instrument
(Japan) to determine the size and shape of ZnONP
in dry form. DLS and LDV were performed with a
Malvern Zetasizer Nano-ZS zen3600 instrument
(UK) to measure the hydrodynamic diameter and zeta
potential in aqueous suspension, respectively. For
TEM analyses, ZnONP were measured at a concentra-
tion of 2.56 mg/ml. For DLS and LDV techniques,
ZnONP samples were measured at a concentration
of 10 mg/ml.
Strains
The following mutant Drosophila strains were used
for the wing-spot test: the multiple wing hairs strain
with the genetic constitution y; mwhj; and the flare-
3 strain with the genetic constitution flr3/ln (3LR)
TM3, Bds. The multiple wing hairs marker (mwh,
3–0.3) is a completely recessive homozygous viable
mutation, which is kept in homozygous condition. It
produces multiple trichomes per cell instead of the
normally unique trichome in the wing cells. The
flare-3 marker (flr3, 3–38.8) is a recessive mutation
that affects the shape of wing hairs, producing mal-
formed wing hairs that have a flare shape. Given their
zygotic lethality, flare alleles have to be kept in stocks
over balancer chromosomes carrying multiple inver-
sions and a dominant marker that is a lethal homo-
zygous (TM3, Bds). More detailed information on
genetic markers and descriptions of the phenotypes
are given by Lindsley and Zimm (1992).
In this study, the flr3 strain was also used for the
comet assay experiments with Drosophila haemo-
cytes. This strain was chosen according to the high
female fertility and low background level of DNA
damage in blood cells from untreated third instar lar-
vae. Both strains were cultured in glass bottles with
the standard medium for Drosophila (i.e. agar, corn
flour and yeast) at a temperature of 25 + 1# C and a
relative humidity of approximately 60%.
Wing-spot test
Virgin females of the flr3 strain were mated to mwh
males as previously described (Marcos and Carmona,
2013). Eggs from this cross were collected during 8 h
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periods in culture bottles containing the standard
medium. The resulting 3-day-old larvae (third instar
larvae) were then placed in plastic vials containing
4.5 g of Drosophila instant medium (Carolina Bio-
logical Supply, Burlington, North Carolina, USA)
prepared with 10 ml of various non-toxic concen-
trations of ZnONP and ZnO bulk (6, 12, 18 and
24 mM, respectively). Larvae were fed this medium
until pupation. The surviving adults were collected
and stored in 70% ethanol. Afterwards, their wings
were removed with fine tweezers and mounted in
Faure’s solution on microscope slides. The wings
were scored at 400$ magnification for the presence
of small single spots, large single spots and twin spots
(Figure 1). In each series, 60 wings were scored (from
30 individuals). Scoring of flies and data evaluation
were conducted following the standard procedures for
the wing-spot test, as used in recent investigations
(Arossi et al., 2010).
The conditional binomial test was applied to assess
differences between the frequencies of each type of
spot in treated and concurrent negative control with
significant levels ! ¼ " ¼ 0.05 (Kastenbaum and
Bowman, 1970). The multiple-decision procedure
was used to judge the overall response of an agent
as positive, negative or inconclusive (Frei and Würg-
ler, 1988). The treatment was considered positive if
the frequency of mutant clones in the treated series
was at least m (multiplication factor) times greater
than in the control series (Frei and Würgler, 1988).
Since small single spots and total spots have a com-
paratively high spontaneous frequency, m was fixed
at a value of 2 (testing for a doubling of the sponta-
neous frequency). For large single spots and twin
spots, which have a low spontaneous frequency, m
¼ 5 was used. The frequency of clone formation was
calculated, without size correction, by dividing the
number of mwh clones per wing by 24,400, which is
the approximate number of cells inspected in one
wing (Alonso-Moraga and Graf, 1989).
Comet assay
Third instar larvae at 72 + 4-h-old were placed in
plastic vials containing 4.5 g of Drosophila instant
medium prepared with 10 ml of various non-toxic
concentrations of ZnONP and ZnO bulk (6, 12 and
24 mM, respectively). Haemocytes from Drosophila
haemolymph were then collected according to the
method described by Marcos and Carmona (2013).
Briefly, chilled larvae at 96 + 4-h-old were removed
Carmona et al. 5

Figure 1. Typical manifestations of mutant spots affecting trichomes number and shapes on wing surface of Drosophila
melanogaster. (a) Small single mwh spot; (b) large single mwh spot; and (c) twin spot. Images were taken with $400
magnification.

from food media, washed in water, sterilized in 5%
bleach and dried. The cuticles from 50 larvae were
then disrupted with two fine forceps under a stereo-
scopic microscope. The haemolymph and circulating
haemocytes were directly collected into a drop of cold
PBS solution containing 0.07% PTU and separated in
1.5 ml microcentrifuge tube. Pooled haemolymph was
centrifuged at 300g for 10 min, the supernatant was
discarded, and the pellet was resuspended in 20 ml
of cold PBS.
The comet assay was performed as previously
described by Singh et al. (1988) with minor modifica-
tions. Cell samples (approximately 40,000 cells in 20
ml) were carefully resuspended in 75 ml of 0.75%
LMA and layered onto microscope slides pre-coated
with 1% NMA (dried at room temperature). Two gels
were mounted in each slide and covered with a cov-
erslip. Immediately after agarose solidification (for
10 min at 4# C), the coverslips were removed and the
slides were immersed in a cold fresh lysis solution
(2.5 M NaCl, 100 mM Na2-EDTA, 10 mM Tris, 1%
Triton X-100 and 1% N-lauroylsarcosine, pH 10) for
2 h at 4# CC in a dark chamber. To prevent additional
DNA damage, the following steps were performed
under dim light: the slides were placed for 25 min
in a horizontal gel electrophoresis tank filled with
cold electrophoretic buffer (1 mM Na2-EDTA and
300 mM NaOH, pH 13) to allow DNA unwinding.
Electrophoresis was carried out in the same buffer for
20 min at 25 V and 300 mA. Unwinding and electro-
phoresis processes were done at 4# C. After electro-
phoresis, slides were neutralized with two washes of

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6 Toxicology and Industrial Health
0.4 mM Tris (pH 7.5) for 5 min each. The slides were
stained with 20 ml of DAPI (1 mg/ml) per gel. The
images were examined at 400$ magnification with
a Nikon Eclipse E200 fluorescence microscope
(Japan), coupled with a CMOS digital camera. One
hundred randomly selected cells (50 cells on each one
of the two replicate slides) were analysed per treat-
ment. The percentage of DNA in the tail (%DNA tail)
was used to measure DNA damage since this is the
most widely used and recommended parameter for
comet data analysis (Kumaravel et al., 2009). The
%DNA tail was computed using CometScore version
1.5 image analysis system (TriTek Corp., Sumerduck,
Virginia, USA).
A generalized linear model (GLM) was used to
analyse differences in the %DNA tail with different
treatments and compounds. The conservative Scheffe
post hoc test was performed to compare negative con-
trols versus different treatments. The GLM is analo-
gous to traditional analysis of variance (ANOVA),
but it allows the use of non-parametric and heterosce-
dastic data, which was our case (Bolker et al., 2009).
Before analysis with GLM, the homogeneity of var-
iance and normality assumption of data were tested
with the Bartlett and Kolmogorov–Smirnov tests,
respectively. Results were considered statistically sig-
nificant at p ! 0.05. All data were presented as arith-
metic mean + standard error, and 95% confidence
intervals were constructed.
Lipid peroxidation assay
Third instar larvae of Drosophila (72 + 4-h-old) were
treated with three different concentrations of nano and
bulk-ZnO (6, 12 and 24 mM, respectively). Control lar-
vae received untreated instant medium for Drosophila
rehydrated with distilled water. Larvae were exposed
to the compounds during approximately 24 h. Lipid
peroxidation or TBARS assay was performed accord-
ing to the modified method of Tironi et al. (2007).
Approximately 0.5 g of larvae were used in each expo-
sure and control groups. Three replications and one
independent experiment were assessed. Control and
ZnONP-treated larvae were homogenized with 0.5%
w/v of TCA. The larvae homogenates were maintained
for 30 min in ice and then were filtered. A mixture of 0.5
ml filtered tissue homogenates and 0.5 ml of 0.5% w/v
TBA solution was incubated for 30 min to 70# C. The
absorbance was measured at 532 nm using a Thermo-
Scientific SpectronicGenesys 10 UV-visible (Vis)
scanning spectrophotometer (Madison, Wisconsin,
Downloaded from tih.sagepub.com at University of York on January 24, 2016
USA). TBARS levels were converted to MDA and
expressed in milligram of MDA/kilogram of sample
using a molar extinction coefficient of 1.56 $ 105 M&1.
ANOVA was used to analyse differences in MDA
levels with different treatments and compounds.
Tukey’s post hoc test was performed to compare neg-
ative controls versus different treatments and ZnO
particles. Results were considered statistically signif-
icant at p ! 0.05. All data were presented as arith-
metic mean + standard deviation.
Results
Physical characterization of ZnONP
TEM was used to characterize the morphology and
size of ZnONP. Most of the NP analysed were sphe-
rical, triangle, rod and rectangle shapes (i.e. hexago-
nal shapes) and showed low levels of agglomeration
(Figure 2(a)). The size of the ZnONP agglomerates
ranged from 25.06 to 360.97 nm, and the average +
SD size was 106.55 + 64.79 nm (Figure 2(b)). TEM
images and analyses of representative NP (n ¼ 200)
indicate that a significant number of ZnONP (40%
approximately) show sizes higher than 100 nm, dif-
fering from the manufacturer indications (less than
100 nm).
DLS and LDV techniques were used to measure
hydrodynamic diameter and zeta potential of ZnONP
in water suspension with previous dispersion by soni-
cation. The diameter average in water suspension was
different and higher than TEM analyses, reaching the
mean + SD values of 225.40 + 0.93 nm (Figure 3(a)).
Finally, the average + SD of zeta potential was
&21.00 + 0.80 for ZnONP (Figure 3(b)), indicating
a good stability and dispersion of this nano compound
in aqueous solution for genotoxic experiments with
Drosophila larvae.
The physical properties of ZnO bulk were described
in detail in a previous work by Lin et al. (2009). In
summary, most of the ZnO particles showed hexagonal
shapes. The primary size average was 4.2 mm (mea-
sured by TEM) and the hydrodynamic size was 9 mm
(with DLS methodology). The ZnO particles showed
a specific surface area of 8.61 m2/g Brunauer-
Emmett-Teller analysis (BET).
Toxicity of ZnO compounds
The different concentrations of nano and bulk forms of
ZnO used in the genotoxicity experiments with D. mel-
anogaster strains were selected according to previous
Carmona et al.
Figure 2. Physical characterization of ZnONP with TEM. (a) TEM image with high magnification (bar scales representing
200 nm) and (b) histogram showing the size distribution of ZnONP. ZnONP: zinc oxide nanoparticles; TEM: transmission
electron microscopy.
Figure 3. Characterization of ZnONP in water suspen-
sion. (a, b) Hydrodynamic size and zeta potential measured
with DLS and LDV techniques, respectively. ZnONP: zinc
oxide nanoparticle; DLS: dynamic light scattering; LDV:
laser doppler velocimetry.
toxicity and viability studies carried out in the labora-
tory. Hence, a range of doses from 6 to 24 mM was
established for both particle sizes of ZnO. Viability was
higher than 70% in all concentrations, except to 24 mM
where the viability was approximately 50%, and no dif-
ferences in toxicity were observed between nanosized
and bulk forms of ZnO for both mwh and flr3 strains
of D. melanogaster (data not shown). The main criterion
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7
to choose the final concentrations was the number of
emerging larvae and adults after treatments were high
enough to perform genotoxic experiments with wing-
spot and comet tests (Marcos and Carmona, 2013).
Genotoxicity analysis with the wing-spot test
Both sizes of ZnO were supplied to 72-h-old larvae (third
instar) at concentrations ranging from 6 mM to 24 mM.
The results indicate that no concentration of ZnONP and
ZnO bulk, administered by ingestion, induced signifi-
cant increases in the frequency of mutant spots (i.e. small
single, large single and twin spots), compared with the
negative control (Tables 1 and 2). In this study, the neg-
ative control frequencies (0.30 and 0.28) were in accor-
dance with the normal background range observed in our
laboratory and were not significantly different from pre-
vious results (Carmona et al., 2011a; Gürbüzel et al.,
2013). The positive control carried out with 1 mM of
EMS showed a clear response, and the mutant spot fre-
quencies scored were also in agreement with previous
and recent studies (Demir and Kaya, 2013; Gürbüzel
et al., 2012; Karadeniz et al., 2011), which supports the
validity of the negative results found for the nanoparticu-
lated and micrometric compounds of ZnO.
DNA damage evaluated with the comet assay
The results obtained in the comet assay to test the pri-
mary DNA damage (expressed as percentage of DNA
in tail) of NP and bulk compounds of ZnO on larval
8 Toxicology and Industrial Health

Table 1. Induction of mutant spots in Drosophila larvae treated with ZnONP.a

Small single Large single
spots (1–2 cells) spots (>2 cells) Twin spots Total mwh Total spots
(m ¼ 2) (m ¼ 5) (m ¼ 5) spots (m ¼ 2) (m ¼2) Frequency of clone
Compounds formation per
concentration (mM) N Fr D N Fr D N Fr D N Fr D N Fr D 105 cells
ZnONP
Control 15 0.25 1 0.02 1 0.02 17 0.28 18 0.30 1.22
6 10 0.17 & 3 0.05 i 1 0.02 & 14 0.23 & 15 0.25 & 1.02
12 13 0.22 & 1 0.02 & 0 0.00 14 0.23 & 15 0.25 & 1.02
18 20 0.33 i 0 0.00 0 0.00 20 0.33 i 20 0.33 i 1.35
24 15 0.38 & 3 0.05 i 1 0.02 & 19 0.32 i 19 0.32 i 1.31
EMS
1 162 2.70 þ 49 0.82 þ 33 0.55 þ 244 4.06 þ 260 4.33 þ 17.75
N: number of clones; Fr: frequency; D: statistical diagnosis according to Frei and Würgler (1988): þ: positive; &: negative; i: inconclusive;
m: multiplication factor; ZnONP: zinc oxide nanoparticles; EMS: ethyl methane sulphonate.
a
Results obtained from mwh/flr3 wings. Probability level ! ¼ " ¼ 0.05; 60 wings were analysed for each concentration (30 individuals).
Ultrapure water was used as negative control, and EMS as positive control.

Table 2. Mutant spots induced in Drosophila larvae treated with ZnO bulk.a
Small single Large single
spots (1–2 cells) spots (>2 cells) Twin spots Total mwh Total spots
(m ¼ 2) (m ¼ 5) (m ¼ 5) spots (m ¼ 2) (m ¼ 2) Frequency of clone
Compounds formation per
concentration (mM) N Fr D N Fr D N Fr D N Fr D N Fr D 105 cells
ZnO bulk
Control 15 0.25 1 0.02 1 0.02 17 0.28 17 0.28 1.14
6 12 0.20 & 2 0.03 i 1 0.02 & 15 0.25 & 16 0.26 & 1.06
12 14 0.23 & 1 0.02 & 1 0.02 & 16 0.26 & 16 0.26 & 1.06
18 14 0.23 & 2 0.03 i 0 0.00 16 0.26 & 16 0.26 & 1.06
24 18 0.30 i 2 0.03 i 0 0.00 20 0.33 i 20 0.33 i 1.35
EMS
1 195 3.25 þ 40 0.66 þ 34 0.56 þ 269 4.48 þ 284 4.73 þ 19.39
N: number of clones; Fr: frequency; D: statistical diagnosis according to Frei and Würgler (1988); þ: positive; &: negative; i: inconclusive;
m: multiplication factor; ZnO: zinc oxide; EMS: ethyl methane sulphonate.
a
Results obtained from mwh/flr3 wings. Probability level ! ¼ " ¼ 0.05; 60 wings were analysed for each concentration (30 individuals).
Distilled water was used as negative control, and EMS as positive control.

haemocytes of D. melanogaster are summarized in
Table 3. Both compounds were administered by feeding
during 24 + 4 h to third instar larvae in the same doses as
in the wing-spot test, ranging from 6 mM to 24 mM.
Concurrent negative and positive controls were also per-
formed for each comet experiment. Afterwards, the hae-
molymph was extracted from the larvae, and circulating
haemocytes were isolated by centrifugation for comet
testing and data analysis of primary DNA damage.
The data obtained indicate that the nanoparticu-
lated form of ZnO can induce significant increases
of DNA damage in larval hemocytes, but only at the
higher concentrations tested, that is, 24 mM (Figure 4).
Contrary to the results found with nanosized ZnO,
the treatments carried out with different concentra-
tions of micrometric ZnO did not induce significant
DNA damage in haemolymph cells of D. melanoga-
ster as compared to the negative control. The negative
and positive control values in %DNA in tail were in
agreement with the background range observed in
recent studies with the same test (Demir, 2012; Demir
and Kaya, 2013; Mukhopadhyay et al., 2004).
Lipid peroxidation of ZnONP in Drosophila
Third instar Drosophila larvae were exposed to nano
and bulk forms of ZnO by feeding during 24 + 4 h at

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Carmona et al. 9

Table 3. Primary DNA damage induction, measured as the

percentage of DNA in the tail, obtained in larval hemocytes
with the comet assay.
Compound 95% CI of %DNA
concentration (mM) %DNA in taila in tailb
Pooled negative control 11.41 + 0.58 10.28–12.54
Pooled positive control 21.61 + 0.93c 19.80–23.42
ZnONP
6 10.32 + 0.86 8.63–12.01
12 9.69 + 0.87 7.98–11.4
24 18.39 + 1.49c 15.47–21.31
ZnO bulk
6 12.82 + 0.99 10.87–14.77
12 11.30 + 0.90 9.54–13.06
24 10.75 + 0.94 8.91–12.59
Figure 5. Lipid peroxidation, measured as TBARS accumu-
CI: confidence interval; ZnONP: zinc oxide nanoparticles; EMS:
lation in Drosophila melanogaster when treated with various
ethyl methane sulphonate.
a
Mean + standard error based on experiments. concentrations of ZnONP and ZnO bulk. Bars show MDA
b
95% CI for % of DNA in tail. average (mg/kg FW) standard deviation (n ¼ 3). Different
c
p < 0.01 versus negative control. lowercase letters indicate differences between the same
treatments in the different concentrations (p ! 0.05).
Asterisks indicate significant differences (p ! 0.05) between
treatments at the same concentrations. ZnONP: zinc
oxide nanoparticle; ZnO: zinc oxide; MDA: malondialde-
hyde; TBARS: thiobarbituric acid reactive substance.

response relationship. When MDA induction was

compared between both ZnO forms at the same con-
centration, ZnONP induced higher MDA levels than
ZnO microparticles for all evaluated concentrations
(Figure 5, p ! 0.05).

Figure 4. Comet assay images showing genetic damage
induced by ZnONP in haemocytes of Drosophila ($400
magnification). ZnONP: zinc oxide nanoparticle.
concentrations ranging from 6 mM to 24 mM. After
the treatments, larval tissue of D. melanogaster was
used to evaluate the MDA marker for oxidative stress.
MDA values after ZnONP treatments increased sig-
nificantly at all concentrations in comparison with the
values observed in the negative control (p ! 0.05).
However, this increase in MDA values does not fol-
low a linear dose–response because a notable reduc-
tion of MDA concentration was observed at the
higher concentration of ZnONP evaluated (24 mM;
Figure 5). ZnO bulk showed a significant increase
of MDA levels in treated larvae with a linear dose–
Discussion
ZnONP is one of the manufactured NM more widely
produced and may be found in a wide variety of con-
sumer products available in the market, especially in
cosmetics and sunscreens. Thus, the probability of
human and environmental exposure to ZnONP is
increasing, and its genotoxic potential risk must be
exhaustively studied. Despite the high production and
wide use of ZnONP, there are few studies covering
their in vivo potential genotoxicity (Kumar and Dha-
wan, 2013).
In the present study, we used commercially avail-
able, manufactured ZnONP with the average particle
size, given by the manufacturer (Sigma-Aldrich), of
approximately 100 nm. This particle size was slightly
lower to that was obtained in the TEM analysis car-
ried out in this study, where ZnO nanopowder reached
an average of approximately 107 nm. According to
the DLS results, the hydrodynamic size of ZnONP

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10
was larger than TEM analyses, with an average of
approximately 225 nm, suggesting a certain grade of
agglomeration in aqueous media. These results are
in agreement with other similar in vivo genotoxic
studies, where ZnONP showed sizes greater than
200 nm in aqueous media and sizes less than 100
nm in dry state with TEM analyses (Nounou et al.,
2013; Sharma et al., 2012b). These differences are
commonly explained by the tendency of NP to
agglomerate in aqueous medium, making them larger
than in dry state (Dhawan and Sharma, 2010; Sharma
et al., 2012b). Taking into account the importance of
size in the biological properties of NP, the size char-
acterization by DLS technique may be considered
indispensable and more relevant compared to TEM
analyses, as it can measure size under conditions that
more closely resemble the exposure conditions in
Drosophila experiments (Sharma et al., 2009).
The zeta potential of ZnONP measured with
LDV technique reached an average of approximately
&21 in water suspension. This measure indicates the
moderate colloidal stability (i.e. resistance to agglom-
eration in water) of ZnONP, indicating suitable
exposure conditions for nanogenotoxic assays with
Drosophila.
To increase our knowledge of the potential geno-
toxic risk associated with ZnONP, we used two
well-known in vivo genotoxic assays using somatic
cells of Drosophila: the wing-spot test and the comet
assay on haemocytes. The wing-spot assay can detect
a wide range of mutational events, as well as mitotic
recombination, a relevant process related with carci-
nogenesis (Luo et al., 2000). In addition, this system
assay has already been demonstrated to be an excel-
lent tool to assess genotoxic effects of different NM,
including silver and cobalt NP (Demir et al., 2011;
Vales et al., 2012); titanium, zirconium and alumi-
nium metal oxide NP (Demir et al., 2013); and
multi-walled carbon nanotubes (de Andrade et al.,
2014; Machado et al., 2013).
The comet assay is one of the most widely
employed methods of in vivo genotoxicity screening
because it can be applied to any type of cell, it can
detect low levels of DNA damage and it can detect
different kinds of damage (Hartmann et al., 2003).
Thus, given these advantages, this assay is being rou-
tinely used to assess DNA damage of NM in different
cell and organism models (Karlsson, 2010), including
D. melanogaster (Sabella et al., 2011).
To the best of our knowledge, our results are the
first to be reported on the genotoxic and oxidative
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Toxicology and Industrial Health
stress assessment of ZnONP in Drosophila. The
results obtained with the wing-spot test after chronic
exposure to NP and microparticles of ZnO at the same
concentrations clearly indicate that both ZnO forms
were unable to induce genotoxic effects, as
an insignificant increase of mutant clone formations
were observed for all concentrations assayed in wing
preparations. Unlike the absence of genotoxicity
within the wing-spot test, the results obtained with the
in vivo comet assay in haemocytes of Drosophila
showed that ZnONP treatments can induce DNA
strand breaks in circulating blood cells, but at the
highest concentration tested. In addition, in the lipid
peroxidation assay, ZnONP showed higher MDA
induction than its bulk form, suggesting that DNA
damage observed with the comet assay could be
mediated by oxidative stress induction. The contra-
dictory results obtained in our study with both geno-
toxic tests in Drosophila could be explained in part
by the weak or low genotoxic potential of ZnONP
and/or the differences of genotoxic end points used
in the wing-spot and comet tests for measuring DNA
damage (mutation/recombination vs. primary DNA
damage).
In general, there are few genotoxic studies specifi-
cally for ZnONP, on whole organisms (i.e. in vivo
approaches). From the available literature, the geno-
toxicity of nanosized ZnO could be considered weak,
as mainly negative and weak genotoxic potential has
been reported in the literature (Chibber et al., 2013).
Although most of the in vitro studies have indicated
genotoxic effects associated with ZnONP exposure
(such as DNA strand breaks, micronuclei formation
and chromosomes breaks in human cells in culture
(Alarifi et al., 2013; Bhattacharya et al., 2014; Demir
et al., 2014; Hackenberg et al., 2011; Lin et al., 2009;
Sharma et al., 2009, 2012b; Valdiglesias et al., 2013)),
most of the studies carried out with bacteria indicate
negative or weak mutagenic effects (Kwon et al.,
2014; Nam et al., 2013; Pan et al., 2010). In addition,
recent in vivo studies with rodents indicate clearly that
ZnONP with different sizes (20 and 70 nm) and elec-
tric charges (i.e. anion and cationic NP) were unable
to induce DNA strand breaks and micronucleus for-
mation in rats and mice orally exposed at different
concentrations and at times of nanosized ZnO (Kwon
et al., 2014). In this context, the absence of genotoxic
effects found in this recent study agree with our neg-
ative data for the wing-spot test, suggesting that
ZnONP does not show significant genotoxic potential
on whole organisms.
Carmona et al.
The results obtained with the alkaline comet assay
in haemolymph cells of Drosophila indicate that
ZnONP exposure can induce genetic damage. Never-
theless, the low induction and the fact that this effect
was only observed at the highest concentration evalu-
ated does not support a genotoxic potential for
ZnONP. Similar results have been reported with the
same test system in whole animals and have shown
that ZnONP can exert DNA damage in kidney and
liver cells of mice (Sharma et al., 2012b) and induce
DNA fragmentation and DNA breakages in lung cells
of rats exposed orally to different concentrations of
ZnONP (Nounou et al., 2013). Likewise, another in
vivo study on earthworms (Eisenia fetida) indicates
that nanosized ZnO show significant primary DNA
damage in coelomocytes at the highest dose tested
(Hu et al., 2010). Thus, it seems that ZnONP is able
to induce primary DNA damage in different cells and
living organisms, but this happens under specific con-
ditions of toxicity (Magdolenova et al., 2014). This is
in agreement with our data from the comet assay.
Some studies indicate that ZnONP dissolution in
aqueous solutions, and the released Zn ions can play
a significant role in their toxicity (Wang et al.,
2014; Xia et al., 2008). However, other studies indi-
cate that the role of zinc (Zn) ions release in the toxi-
city of ZnO materials may be irrelevant. For example,
Alarifi et al. (2013) found that Zn ions can induce toxi-
city and DNA damage in human cells A375, but these
effects were less significant in comparison with similar
doses of ZnONP. Sharma et al. (2012a) suggested that
although the ZnONP released Zn ions into the media
test, this amount was insufficient to cause toxicity
in human liver cells. Similarly, Valdiglesias et al.
(2013) found low Zn ions release and the cytotoxicity
was restricted to high doses of ZnONP in mouse neu-
ronal cells. Finally, Lin et al. (2009) evidenced that the
Zn ions release in the media test was very low to induce
cytotoxicity in human lung epithelial cells.
Although the genotoxic effects of Zn ions was not
measured in our study with Drosophila, a previous
work indicated that the soluble ionic compound zinc
nitrate showed inconclusive results in the Drosophila
wing spot-test (Yeşilada, 2001). This data suggest that
Zn ions do not induce significant genotoxic effects in
somatic cells of Drosophila and support our conclu-
sions that the genetic damage of ZnONP observed
with the comet assay in Drosophila blood cells can
be related with the NP per se.
Oxidative stress mediated by ROS production and
depletion of the antioxidant barrier into the cells
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11
(e.g. superoxide dismutase and glutathione) have been
proposed as one of the main mechanisms involved in
toxic and genotoxic manifestations induced by nanos-
cale particles (Shrivastava et al., 2014; Wang et al.,
2013). NP can generate ROS in the cells that may
cause indirect oxidative damage to DNA through free
radical attack. ZnONP were found to generate free
radicals and oxidative stress, which lead to oxidative
DNA damage in vivo and suggest a probable mechan-
ism of genotoxicity in liver and lung cells of rodents
(Nounou et al., 2013; Sharma et al., 2012b). This
effect has also been observed in several studies with
human and mammals cells in culture, where ZnONP
exposure induced high ROS production; oxidative
DNA lesions; and depletion of glutathione, superox-
ide dismutase and catalase antioxidants (Alarifi
et al., 2013; Demir et al., 2014; Lin et al., 2009;
Sharma et al., 2012a; Valdiglesias et al., 2013). In our
study, we found that although ZnONP can induce
lipid peroxidation, this effect was also observed after
exposure to bulk ZnO, but at a lesser level. Thus, this
effect is not an intrinsic characteristic of ZnONP.
It is important to note that in our research we used
two genotoxic assays with different genetic end points
to assess the genotoxicity of nanoparticulated ZnO in
Drosophila. It is also important to state that the
genetic damage detected in the alkaline comet assay
is primary DNA damage that can be easily repaired
in the cells as to avoid mutations (Azqueta and Col-
lins, 2013; Bajpayee et al., 2013). On the contrary, the
Drosophila wing-spot test determines fixed DNA
damage (mutation and/or recombination) generated
after DNA lesions are misrepaired by DNA repair sys-
tems in somatic cells (Graf et al., 1984; Garcı́a Sar
et al., 2012; Garcia-Quispes et al., 2009). Therefore,
contradictory genotoxic effects observed between
wing-spot and comet assays could be explained by the
inability of the ZnONP to induce somatic mutation
(i.e. fixed genetic damage) in the wing cells of Droso-
phila. This would agree with the results of other
researchers using mice and rat models, where ZnONP
have failed to induce micronucleus formation follow-
ing validated and standard methods for genotoxicity
testing (Landsiedel et al., 2010; Kwon et al., 2014).
According to our results, we conclude that ZnONP
were unable to induce mutation and recombinational
events in somatic cells of Drosophila (absence of
fixed DNA damage). However, this NM can induce
primary DNA damage in haemolymph cells of Droso-
phila under a higher dose scenario, as evidenced with
the comet assay. As demonstrated, this genetic damage
12
could be mediated by oxidative stress. The overall
data suggest that the genotoxic potential of ZnONP
could be considered low or weak because it promotes
only primary genetic damage restricted to high-dose
exposures, which can be easily repaired. This study
demonstrates the usefulness of using more than one
genotoxic test in the evaluation of the genotoxic
potential of NM. Finally, it is important to remark that
this study is the first to assess the genotoxic effects
and oxidative stress of nano and bulk ZnO particles
in Drosophila.
Acknowledgements
We are grateful to MJ Rivadeneira, MM Carmona and M
Garrison for revising the English version of the manuscript.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest
with respect to the research, authorship and/or publication
of this article.
Funding
The author(s) disclosed receipt of the following financial
support for the research, authorship and/or publication of
this article: The authors thank the financial support given
by FONDECYT-CONICYT 11110181 project, Dirección
General de Investigación y Postgrado, Universidad Cató-
lica de Temuco, DGIP UCT CD 2010-01 project, and
MECESUP UCT 0804 project.
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