Akdeniz University, Faculty of Sciences, Department of Biology, 07058-Campus Antalya, TurkeyGrup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra,erdanyola del Vallès, 08193 Barcelona, SpainCIBER Epidemiología y Salud Pública, ISCIII, Madrid, Spain
i g h l i g h t s
Genotoxicity of amorphous silica (SAS) nanoparticles has been shown in Drosophila.Positive effects in the comet assay (with and without enzymes) were obtained.Oxidative DNA-damage induction was inversely associated to SAS size.No somatic and recombination mutations were obtained in the wing-spot test.No genotoxic effects were obtained with microparticulated silica dioxide.
r t i c l e i n f o
rticle history:eceived 6 March 2014eceived in revised form 1 July 2014ccepted 8 September 2014vailable online 28 September 2014
eywords:AS nanoparticles
a b s t r a c t
Although the use of synthetic amorphous silica (SAS) is steady increasing, scarce information exists onits potential health risk. In particular few and conflictive data exist on its genotoxicity. To fill in this gapwe have used Drosophila melanogaster as in vivo model test organism to detect the genotoxic activityof different SAS with different primary sizes (6, 15, 30 and 55 nm). The wing-spot assay and the cometassay in larvae haemocytes were used, and the obtained results were compared with those obtained withthe microparticulated form (silicon dioxide). All compounds were administered to third instar larvae atconcentrations ranging from 0.1 to 10 mM. No significant increases in the frequencies of mutant spots
were observed in the wing-spot assay with any of the tested compounds. On the other hand, significantdose-dependent increases in the levels of primary DNA damage, measured by the comet assay, wereobserved for all the SAS evaluated but mainly when high doses (5 and 10 mM) were used. These in vivoresults contribute to increase the database dealing with the potential genotoxic risk associated to SASnanoparticles exposure.
. Introduction
Nanotechnology industry is rapidly growing, due to the novel physicochemicalroperties of nanomaterials that are defined by their small size, with at least oneimension less than 100 nm. This supposes that such nanomaterials are increasinglypread into the environment and, in this way, human exposure certainly occurs. It
s assumed that the important biological reactivity of nanometals may also implyn increased toxicity, both systemic and specifically on the genetic material. Foruch reasons, nanotoxicology and nanogenotoxicology are extending as a novel field,
∗ Corresponding author at: Grup de Mutagènesi, Department de Genètica i deicrobiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus
e Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain. Tel.: +34 93 581 20 52;ax: +34 93 581 23 87.
looking for the potential toxic and genotoxic risk of nanomaterials as well as for theirmechanisms of action [1–6].
Synthetic amorphous silica (SAS) nanoparticles are used as a food additive inmany processed foods, as well as in pharmaceutical drug tablets, glass, electronics,and as hydrophobic anticancer drug [7]. With respect to SAS toxicity it is assumedthat it is mediated by inflammatory and oxidative stress mechanisms, as it has beenshown in both in vivo and in vitro [8–11], from the genotoxic point of view theobtained results are contradictory. When testing for primary DNA damage positiveeffects in the comet assay were obtained in human lung alveolar epithelial cells [12]and in human umbilical vein endothelial cells [13] but not effects were observedin mouse fibroblasts [14]. Similarly, in the micronucleus assay although positiveeffects were observed in mouse fibroblasts [15], negative results were also reportedin Balb/3T3 mouse fibroblasts [16]. With respect to in vivo approaches, studies using
the freshwater crustacean Daphnia magna and the larva of the aquatic midge Chi-ronomus riparius were unable to demonstrate the induction of genetic damage bySAS exposure using the comet assay. The only one study carried out with Drosophilamelanogaster showed that larval exposure to SAS supposes its internalizationthrough intestine track producing cellular stress and apoptosis in midgut cells [17].
Due to this lack of confirmatory results on the genotoxic potential of SAS expo-ure we have plan to use Drosophila to get further inside on its genotoxic risk. It muste pointed out that Drosophila is one of the most genetically and experimentallyccessible model organisms used in biology. It must also be stressed that about 75%f human disease genes have related sequences in Drosophila, suggesting that it canerve as an effective model to study the function of a wide array of genes involved inuman disease [18,19]. In addition to the technical advantages of using somatic cellss a target, such as scoring the effects on the exposed individuals without waitingor the two subsequent generations, somatic mutation inductions are directly linkedith cancer processes, which suppose a relevant role on human health [20]. Thus,rosophila is considered a very potent in vivo tool to detect the potential damagingffect of new environmental contaminants.
The advantages of Drosophila to detect potential genotoxicants have alreadyeen used to determine the potential risk of nanomaterials. In fact, this in vivo modelas already been used to evaluate the internalization of nanoparticles, as well as
ts cell uptake and tissue distribution [21,22]. In addition Drosophila has also beensed to determine the potential genotoxic harmful effects of different nanomaterials4,23–26].
In this context, in the present study we have use Drosophila to study the potentialenotoxicity of four different SAS characterized by their different sizes. SAS wasdministered to larvae and the targeted cells were those from the wing imaginalisk and haemocytes.
. Experimental
.1. Drosophila strains
For the Drosophila wing-spot test two D. melanogaster strainsere used: the multiple wing hairs strain with the genetic constitu-
ion mwh/mwh and the flare-3 strain with the genetic constitutionf flr3/In (3LR) TM3, Bds. More detailed information on genetic mark-rs and descriptions of the phenotypes is obtained in Lindsley andimm [27]. The studies were carried at the Akdeniz University andoth strains were kindly provided by Prof. R. Marcos (Universitatutònoma de Barcelona, Spain). The wild-type strain Oregon R+,roficient for all types of repair, was used for the comet assay. Thesetrains were cultured in bottles with standard Drosophila medium,t a temperature of 25 ± 1◦C and a relative humidity of ∼60%.
.2. Chemicals
Low melting-point agarose (LMA), normal melting-pointgarose (NMA), trisma base, ethidium bromide (EtBr), fluoresceiniacetate (FDA), N-lauroylsarcosine sodium salt solution, endonu-lease III (endo III), formamidopyrimidine DNA glycosilase (FPG),DTA disodium salt dehydrate, phosphate-buffered saline solu-ion without Ca+2, Mg+2 (PBS), HEPES, potassium chloride (KCl),ovine serum albumin (BSA), triton X-100, sodium chloride (NaCl),odium hydroxide (NaOH) and ethyl methanesulfonate (EMS) werebtained from Sigma Chemical Co. (St. Louis, MO).
Four different synthetic amorphous silica (SAS) nanoparti-les (LEVASIL®-types) with sizes 6, 15, 30 and 45 nm werebtained from H.C. Starck GmbH, Engineered Material SolutionsGoslar/Germany). The microparticulated form of silicon dioxideSiO2, CAS No: 7631-86-9) was provided by Sigma–Aldrich.
.3. Nanoparticles characterization
According to the manufacturer the physical characteristics ofhe different nanosized SAS are: for 6 nm, density (1.1 g/cm3), sur-ace area (min. 450 m2/g); for 15 nm, density (1.205 g/cm3), surfacerea (200 m2/g); for 30 nm, density (1.343 g/cm3), surface area100 m2/g) and for 55 nm, density (1.39 g/cm3) and surface area50 m2/g). We further characterized the selected SAS by using trans-
ission electron microscopy (TEM), dynamic light scat-tering (DLS)
nd laser Doppler velocimetry (LDV) methodologies. TEM method-logies were carried on a JEOL JEM-2011 instrument to determineize and morphology. DLS and LDV were performed on a Malvernetasizer Nano-ZS zen3600 instrument for the characterization of
Materials 283 (2015) 260–266 261
hydrodynamic size and zeta potential, for these measures SAS weredispersed in a 5% solution prepared with distilled water. For dis-persion, SAS were subjected to ultrasonication (S-250D, BransonSonifier) at 20 kHz for 16 min in an ice-cooled bath.
2.4. Drosophila wing-spot test protocol
To carry out the wing-spot assay virgin flr3 females were matedto mwh males, as previously described [4]. Eggs from this cross werecollected during 8-h periods in culture bottles containing standardfood medium. The resulting 3-day-old larvae were then transferredto plastic vials with 4.5 g of Drosophila instant medium (CarolinaBiological Supply Co., Burlington, NC) prepared with 9 mL of non-toxic concentrations of the four selected SAS (0.1, 1, 5 and 10 mM).Distilled water was used as negative controls and 1 mM EMS aspositive control. For each treatment five plastic vials were usedand 25 larvae per vial were included. Two replicated were doneby experiment. Larvae were fed on this medium until pupationand the emerged adults were conunted and stored in plastic vialswith 70% ethanol. After that he wings of the emerged adults wereremoved, mounted and scored for the presence of mutant clones. Ineach experiment we scored 80 wings (40 individuals). Wings werecarefully removed from adults and mounted in Faure’s solution onmicroscope slides and scored at 400× magnification for the pres-ence of spots. Single and large mwh or flr3 spots, as well as twinspots were recorded as previously reported [24,25].
2.5. Haemocytes collection and comet assay protocol
Larval haemocytes were collected according to Irving et al. [28]and Carmona et al. [29,30] with minor modifications. Third instarlarvae were extracted from the culture medium, washed, steril-ized with ∼5% sodium hypochlorite and dried with filter paper. Tocollect the haemolymph and circulating haemocytes, the cuticle ofeach larva was disrupted using two fine forceps, avoiding damageto internal organs. A total of 40–60 larvae per treatment were used.The comet assay was conducted as previously described by Singhet al. [31], with minor modifications. Cell samples (∼40,000 cells in20 �L) were carefully resuspended in 140 �L of 0.75% LMA preparedin PBS. The cells and agarose were gently mixed by repeated pipet-ting, and layered onto microscope slides pre-coated with 1% NMA(dried for 25 min). The slides were immediately covered with coverslips and kept on ice for 5 min to solidify the agarose. After solidi-fication, the cover slips were removed and 80 �L of molten 0.75%LMA prepared in PBS was spread on the slides. The slides were againcovered with cover slips and kept on ice for 5 min. Then, the coverslips were removed and the slides were immersed in cold, freshlymade lysis solution for 2 h at 4 ◦C in a dark chamber. To avoid addi-tional DNA damage, the next steps were performed under dim light.Slides were placed for 25 min in a horizontal gel-electrophoresistank filled with cold electrophoresis buffer to allow DNA unwind-ing. Electrophoresis was carried out in the same buffer for 20 min at25 V (1 V/cm) and 300 mA. After electrophoresis, slides were neu-tralized with three washes of 5 min in fresh chilled with 400 mMTris buffer (pH 7.5). The slides were stained with 50 �l of ethidiumbromide (EtBr) solution (60 �g/mL) for 10 min and covered with acover slip. For the visualizing of DNA damage, slides were exam-ined at 400× magnification using a fluorescence microscope (NikonEclipse E200) connected to a CCD camera and an image analysissystem (Comet assay IV version 4.11, Kinetic Imaging, UK). Ran-domly selected 100 cells (50 cells on each one of the two replicateslides) were analyzed per treatment. 4 mM EMS was used as posi-
tive control in the comet assay. The percentage of DNA in tail wasthe parameter used as a measure of DNA damage induction.
To determine the induction of oxidized bases the comet assaywas complemented with the use of FPG and endo III enzymes
Fig. 1. Viability results after the treatment of Drosophila melanogaster larvae withSAS (6, 15, 30 and 55 nm) and silica dioxide. Data correspond to the frequencyof emerged adult with respect to the seeded larvae. Chi-square test was used forsurvival statistical analysis. No significant differences were observed between treat-ments with respect to their respective controls.
0
20
40
60
80
100
Cel
l via
bilit
y (%
)
NC0.11510
Concentration (mM)
SiO2 SAS (6 nm) SAS (15 nm) SAS (30 nm) SAS (55 nm)
Compounds
Fig. 2. Viability results in haemocytes of Drosophila melanogaster obtained from lar-
was chosen for the genotoxicity studies with the tested SAS andwith the microparticulated silica dioxide. It must be indicated that10 mM is the highest recommended dose to be tested in absence
0
3
6
9
12
% D
NA
tail
NC
0.1
1
5
10
Concentration (mM)
*
**
*
*
*
SiO2 SAS (6 nm) SAS (15 nm) SAS (30 nm) SAS (55 nm)Compounds
62 E. Demir et al. / Journal of Haza
hat recognize oxidized purines and pirimidines, respectively. Thisffect was only analyzed with the highest concentration tested10 mM) of each SAS and the microparticulated form. To proceed,fter lysis slides were washed three times (5 min, 4 ◦C) in an enzymeuffer solution (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mLSA, pH 8.0). Then, aliquots of 100 �L of buffer containing the bac-erial enzymes endo III or FPG (enzyme concentration 1/1000) or nonzyme (control) were placed onto the agarose, sealed with a coverlass and incubated for 30 min at 37 ◦C. After enzyme treatments,ell samples were processed as in the standard alkaline comet assayrocedure.
.6. Statistical analysis
For the SMART assay, the conditional binomial test of Kasten-aum and Bowman [32] was applied to assess differences betweenhe frequencies of each type of spot in treated and concurrentegative control, with significance levels = = 0.05. The multipleecision procedure described by Frei and Würgler [33] was usedo judge the overall response of an agent as positive, weakly pos-tive, negative, or inconclusive. Then the data were also evaluated
ith Mann–Whitney–Wilcoxon nonparametric U test to excludealse positive and negative results [34], using P = 0.05 level of sig-ificance. As recommended, the treatment is considered effective
f the frequency of mutant clones in the treated series is at least (multiplication factor) times greater than in the control series.
ince small single spots and total spots have a comparatively highpontaneous frequency, m is fixed at a value of 2 (testing for a dou-ling of the spontaneous frequency). For the large single spots andhe twin spots, which have a low spontaneous frequency, m = 5 issed.
For the comet assay the statistical approach was the one-wayNOVA followed by Dunnett’s test, which was used to evaluate theignificance of the difference in DNA damage between the controlnd treated cultures. Results were considered statistically signifi-ant when the P-value was <0.05. Each compound was tested in twondependent experiments and a good concordance was observedetween these.
. Results
.1. SAS characterization
To further characterization of the selected SAS we used TEMo determine their size distribution and morphology. The obtainedesults are indicated as supplementary figures (Figs. S1–S4) show-ng that the majority of nanoparticles were in spherical shapend no marked agglomerations were observed following the dis-ersion protocol used. The observed average sizes agree with theeported by the supplier. The average hydrodynamic diameter andeta potential of the different SAS suspensions were determinedy DLS and LVD, respectively. DLS values were 6 ± 0.6, 15 ± 0.9,0 ± 0.4, 55 ± 0.7 for 6, 15, 30 and 55, respectively. The obtainedesults for Zeta potential were −45.1, −69.9, −54.1 and −54.3 mVor 6, 15, 30 and 55 nm, respectively (Figs. 1–4).
.2. Genotoxicity in the wing-spot test
To determine the range of concentrations to be used in the geno-oxicity studies, previous viability experiments were carried outnd the survival rates obtained are given in Fig. 1. As a toxicityeasure we determined the larvae-to-adult survival rate. When
he percentages of adult flies emerged from the different exper-ments were compared with those obtained in the control group97%), no significant differences were observed. Similar no toxicityffects were obtained with silica dioxide or in the positive control
vae treated with SAS (6, 15, 30 and 55 nm) and silica dioxide. Viability was measuredby using the FDA/EtBr staining technique. The statistical approach was the one wayANOVA followed by Dunnett’s test ( = 0.05).
(1 mM EMS) were a non significant decrease in the viability (87%)was observed. According to that, a range between 0.1 and 10 mM
Fig. 3. Genotoxicity of SAS (6, 15, 30 and 55 nm) in the comet assay after exposure todifferent doses. Damaged induction was measured as the percentage of DNA in tail.Results are expressed as mean and standard error of two independent experiments.*P < 0.05 when compared to negative control using one way ANOVA.
E. Demir et al. / Journal of Hazardous Materials 283 (2015) 260–266 263
0
3
6
9
12
15
Net
oxi
dativ
e da
mag
e
NC4 mM EMS6 nm15 nm30 nm55 nm
Endo III Fpg
Enzyme treatments
*
*
**
*
*
*
*
*
* *
Fig. 4. Net oxidative damage induction in haemocytes after SAS (6, 15, 30 and55 nm) exposure at the concentration of 10 mM during larvae stage. Effects inducedb(*
oswtqwcistwemi
3
dvFioobdsitiiowEa
waIrtiS
st
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a
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ined
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Res
ult
s
obta
ined
wit
h
mw
h/flr
3w
ings
.
, ion
(mM
)N
um
ber
ofw
ings
(n)
Smal
l sin
gle
spot
s(1
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cell
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(m
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Tota
l mw
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spot
s
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Tota
l sp
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2)
No.
Fr.
D
No
Fr.
D
No.
Fr.
D
No.
Fr.
D
No
Fr.
D
ater
80
14
(0.1
8)
3
(0.0
4)
1
(0.0
1)
18
(0.2
3)
18
(0.2
3)
80
218
(2.7
3)
+
82
(1.0
3)
+
18
(0.2
3)
+
310
(3.8
8)
+
318
(3.9
8)
+d
e72
15
(0.2
1)
i
2 (0
.03)
−
0
(0.0
0)
i
17
(0.2
4)
−
17
(0.2
4)
−80
16
(0.2
0)
i
2
(0.0
3)
−
0
(0.0
0)
i
18
(0.2
3)
−
18
(0.2
3)
−80
15
(0.1
9)
i
6
(0.0
8)
i
0
(0.0
0)
i
20
(0.2
5)
i
21
(0.2
6)
i80
18
(0.2
3)
i 2
(0.0
3)
−
0
(0.0
0)
i
20
(0.2
5)
i
20
(0.2
5)
i
;
Fr, f
requ
ency
;
D, s
tati
stic
al
dia
gnos
is
acco
rdin
g
to
Frei
and
Wü
rgle
r (1
988,
1995
);
+,
pos
itiv
e;
−,
neg
ativ
e;
i,
inco
ncl
usi
ve;
m, m
ult
ipli
cati
on
fact
or;
pro
babi
lity
leve
ls
˛
=
ˇ
=
0.05
. A
tota
l of 8
0
win
gs
wer
e
scor
ed
per
n.
y buffer exposure were subtracted from those obtained after enzyme treatmentsendo III and FPG). Data represent the average of two independent experiments.P < 0.05 when compared to negative control using one way ANOVA.
f marked toxicity. The results obtained in the wing-spot tests areummarized in Tables 1 and 2. Table 1 presents the results obtainedith the microparticulated form (silica dioxide) and, as observed,
his form was not able to induce significant increases in the fre-uency of any type of mutant sectors. When the different sized SASere evaluated in the wing-spot assay we obtained the results indi-
ated in Table 2. In this case, neither of the tested SAS was able ofncrease the spontaneous frequency of the different types of mutantpots. In spite of these negative findings the results obtained inhe concurrent positive control (1 mM EMS) were clearly positive,hich would confirm that the experiments were conducted prop-
rly and, as consequence, under our testing conditions the selectedaterials are unable to induce somatic mutation or recombination
n the wing imaginal disks of the treated larvae.
.3. Genotoxicity in haemocytes as detected by the comet assay
To confirm that no special toxicity was induced in haemocytesue to the larval treatments or by the isolation procedure, we pre-iously conducted a viability assay. This was carried out using theDA/EB viability approach and the results are presented in Fig. 2. Asndicated, no significant toxicity effects were observed for neitherf the SAS used or for the microparticulated silica dioxide. Effectsf positive control also produce non significant decreases in via-ility (88%, data not shown). When haemocytes were evaluated toetect the induction of primary DNA damage, the obtained resultshowed that all tested SAS were able to induce significant increasesn the levels of DNA breaks, at least at the highest concentrationsested (Fig. 3). Interesting, the microparticulated form (silica diox-de) was not able of inducing any increase in the percentage of DNAn the tail, which was the damage parameter used as biomarkerf effect. As expected, significant increases (10.48 ± 3.73; P < 0.05)ere observed for the positive controls carried out using 4 mM of
MS, which would support the validity of both the results observednd the protocols used.
To determine whether the induction of primary DNA damageas mediated by oxidative damage the effects of the highest dose
pplied (10 mM) was challenged using FPG and endo III enzymes.n this way, the presence of oxidized purines or pyrimidines are
ecognized and excised by FPG and endo III, respectively, increasinghe levels of DNA damage detected by the comet assay. In Fig. 4 wendicate the net oxidative values obtained for the different testedAS. The net values were calculated by subtracting the DNA damage Ta
ble
1W
ing-
spot
te
Com
pou
nd
con
cen
trat
Dis
till
ed
w1
mM
EMS
Sili
ca
dio
xi0.
1
1 5 10
No.
, nu
mbe
rco
nce
ntr
atio
264 E. Demir et al. / Journal of Hazardous
Tab
le
2W
ing-
spot
test
dat
a
obta
ined
afte
r
trea
tmen
t
wit
h
of
SAS
(6, 1
5,
30
and
55
nm
).
Res
ult
s
obta
ined
wit
h
mw
h/flr
3w
ings
.
Com
pou
nd
,co
nce
ntr
atio
n
(mM
)N
um
ber
ofw
ings
(n)
Smal
l sin
gle
spot
s(1
–2
cell
s)
(m
=
2)La
rge
sin
gle
spot
s(>
2
cell
s)
(m
=
5)Tw
in
spot
s
(m
=
5)
Tota
l mw
h
spot
s
(m
=
2)
Tota
l sp
ots
(m
=
2)
No.
Fr.
D
No.
Fr.
D
No.
Fr.
D
No.
Fr.
D
No.
Fr.
D
Dis
till
ed
wat
er
80
14
(0.1
8)
3
(0.0
4)
1
(0.0
1)
18
(0.2
3)
18
(0.2
3)1
mM
EMS
80
218
(2.7
3)
+
82
(1.0
3)
+
18
(0.2
3)
+
310
(3.8
8)
+
318
(3.9
8)
+SA
S
(6
nm
)0.
1
80
13
(0.1
6)
−
5
(0.0
6)
i
3
(0.0
4)
i
21
(0.2
6)
i
21
(0.2
6)
i1
80
14
(0.1
8)
−
0
(0.0
0)
−
1
(0.0
1)
i
15
(0.1
9)
−
15
(0.1
9)
−5
80
9
(0.1
1)
−
4
(0.0
5)
i
1
(0.0
1)
i
14
(0.1
8)
−
14
(0.1
8)
−10
80
12
(0.1
5)
−
1
(0.0
1)
−
1
(0.0
1)
i
14
(0.1
8)
−
14
(0.1
8)
−SA
S
(15
nm
)0.
1
80
18
(0.2
3)
i
2
(0.0
3)
−
1
(0.0
1)
i
21
(0.2
6)
i
21
(0.2
6)
i1
80
17
(0.2
1)
i
3
(0.0
4)
i
0
(0.0
0)
i
20
(0.2
5)
i 20
(0.2
5)
i5
80
21
(0.2
6)
i
1
(0.0
1)
−
0
(0.0
0)
i
22
(0.2
8)
i 22
(0.2
8)
i10
80
20
(0.2
5)
i
2
(0.0
3)
−
2
(0.0
3)
i
24
(0.3
0)
i 24
(0.3
0)
iSA
S
(30
nm
)0.
1
80
7
(0.0
9)
−
0
(0.0
0)
−
0
(0.0
0)
i
7
(0.0
9)
−
7
(0.0
9)
−1
80
12
(0.1
5)
−
2
(0.0
3)
−
0
(0.0
0)
i
14
(0.1
8)
−
14
(0.1
8)
−5
80
21
(0.2
6)
i
3
(0.0
4)
i
0
(0.0
0)
i
24
(0.3
0)
i
24
(0.3
0)
i10
80
13
(0.1
6)
−
3
(0.0
4)
i
2
(0.0
3)
i
18
(0.2
3)
−
18
(0.2
3)
−SA
S
(55
nm
)0.
1
80
13
(0.1
6)
−
4
(0.0
5)
i
1
(0.0
1)
i
18
(0.2
3)
−
18
(0.2
3)
−1
80
19
(0.2
4)
i
1
(0.0
1)
−
1
(0.0
1)
i
21
(0.2
6)
i
21
(0.2
6)
i5
80
16
(0.2
0)
i
5
(0.0
6)
i
0
(0.0
0)
i
18
(0.2
3)
i
21
(0.2
6)
i10
80
16
(0.2
0)
i
3
(0.0
4)
i
2
(0.0
3)
i
20
(0.2
5)
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Materials 283 (2015) 260–266
obtained in the treatments with enzyme buffer from those obtainedwhen FPG or endo III enzymes were used. As observed, all testedcompounds were able to induce significant increases in the levelsof oxidized DNA bases. Interestingly a clear effect of SAS size wasobserved with an indirect size–effect relationship.
4. Discussion
The interest of the present paper is that for first time Drosophilahas been used as an in vivo model organism to test the genotoxicpotential of SAS nanoparticles. Although larval exposure to SAS wasno able to induce somatic gene mutation or recombination, positiveeffects were obtained when looking for the induction of primaryDNA damage, as measured by the comet assay. In addition, sig-nificant induction of oxidative DNA damage was observed, suchinduction being indirectly related with SAS size. It must be pointedout that for the wing spot test we have used the standard (ST) strainsbut high bioactivation (HB) strains also exist [35,36]. Such strainshave shown to be mainly useful when compounds are metabolizedand the resulting metabolites pose genotoxic properties.
When testing for the toxicological properties of any compound,genotoxicity data is of paramount importance. The emergenceof novel compounds such as nanomaterials, with their particularphysico-chemical characteristics has supposed the appearance ofNanogenotoxicology as an emergent scientific field. Most of thestudies conducted in this area have been carried out using in vitroapproaches due to their different advantages, mainly simplicity.Nevertheless, in vivo studies are more relevant in terms of risk sinceaspects such as uptake, metabolism and repair in a whole organ-ism are taken into account. In this context we propose the use ofDrosophila, as a suitable in vivo model, due to its obvious advan-tages with respect to the in vitro studies and also with respect to themost complex in vivo approaches using whole mammal organisms.In fact, Drosophila has already been used to evaluate the internaliza-tion of nanoparticles and to solve questions concerning cell uptakeand live tissue distribution [21,22], and there is an increasing num-ber of toxicity studies with nanoparticles [4,37–40]. To get a moregeneral picture of the genotoxic potential associated to SAS expo-sure we have selected different sized SAS due to the well knownimportance of this physical characteristic as a factor modulatingthe biological reactivity of nanomaterials [41].
At present there is limited evidence suggesting whether ornot SAS are genotoxic, with contradictory results being reported[7,42]. In vitro studies conducted to detect chromosome damageinduction have reported positive effects for 80 nm SAS in 3T3-L1mouse fibroblasts in the micronucleus assay [15], but no effectswere observed in the same assay using Balb/3T3 mouse fibroblastsfor diameters ranging from 15 to 300 nm [16] and using humanlymphocytes for 15 and 55 nm SAS [11]. When the induction ofprimary DNA damage was assayed using the comet test negativeresults were reported in 3T3-L1 mouse fibroblasts [14] but posi-tive effects have been obtained in human lung alveolar (A549) cellswith 20 and 100 nm SAS [12], as well as in human umbilical veinendothelial cells [13]. In in vivo studies, although no effects wereobserved in the comet assay using Daphnia and Chironomus [43],small but reproducible effect were obtained in Wistar rats afterintravenous injections of 15 and 50 NM SAS in both the cometassay (in liver, lung and blood cells) and in the micronucleus assayin reticulocytes [11]. In this context, our positive results obtainedin the comet assay with haemocytes, where significant inductionof oxidative damage was observed, would reinforce the point of
view that indicates that genotoxic effects induced by SAS can bedue to the release of inflammatory cell-derivative oxidants [11].This inflammatory and oxidative stress response has been reportedby different authors [9,10,42]. In fact, our results match well with
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hose previously reported for Drosophila but looking for differentndpoints such as oxidative stress, heat shock genes expression,embrane destabilization, cellular internalization and apoptosis
17]. All these endpoints were affected after larvae exposure indi-ating that SAS can go through the intestinal barrier, be internalizedy the cells (midgut or haemocytes) and produce both intracellularroductions of ROS as well as oxidative DNA damage, as detectedy us in the comet assay.
SAS can enter into the cell and potentially bind to the DNAhosphate backbone [44] and generated increased reactive oxy-en species (ROS) levels [9] that have the capability to attack DNAia several different mechanisms to generate DNA strand breakssingle or double) and oxidatively induced base damage [45] whatan be identified by the comet assay. Our positive results obtainedn the comet assay would reinforce these proposed mechanisms ofction for SAS.
With respect to the findings showing that the induction of netxidative damage follow a indirect dose–response relationship, ouresults agree with those reported by Sergent et al. [46] in the humanpithelial intestinal HT-29 cells-line using the induction of �-H2Axoci, where although a rather genotoxic effect was observed, inverseose-dependent relationships were observed. This emphasizes the
mportance of size when testing the biological effects of nanoma-erials and the importance of include different sized compoundshen testing their potential harmful effects, including genotoxic-
ty.As overall our results show both the relevance of using
rosophila for testing the genotoxic potential of nanomaterials andhe fact that SAS can induce primary DNA damage, but only whenigh doses are used. Nevertheless, as primary DNA damage can beasily repaired, the fixed amount of genetic damage as gene or chro-osome mutation can be no relevant as indicated by the lack of
ffects observed in the wing-spot assay.
. Conclusions
To our knowledge this is the first study that uses Drosophila,s in vivo model organism, to evaluate the genotoxic potential ofynthetic amorphous silica (SAS). Four different primary sizes (6,5, 30 and 55 nm) were selected to determine the possible role ofize. The findings of this study can be summarized as follows.
. We have extended and confirmed the usefulness of Drosophila asan in vivo model to be used in the testing of the toxic/genotoxicproperties of nanomaterials.
. No differences in toxicity were observed between differentsized SAS although a slight direct dose–effect relationship wasdetected.
. When we used the wing-spot assay to determine genotoxic fixedeffects, resulting from both the induction of somatic mutationand recombination in the imaginal disk cells, no effects wereobserved.
. Nevertheless, when larvae’ haemocytes were used as a targetcells in the comet assay, to determine the induction of pri-mary DNA-damage, significant dose-dependent increases wereobserved for all the evaluated SAS, but mainly when high doseswere used.
. The use of enzymes detecting and excising oxidized DNA-baseshas permit us to detect that oxidative damage is an underlyingmechanism involved in the potential genotoxic risk of SAS.
. The use in parallel of the microparticulated form of silicon diox-ide have shown that it exhibits a different toxic and genotoxicpattern, showing less toxicity and no genotoxicity with respectto the tested SAS.
[
Materials 283 (2015) 260–266 265
7. Taking the results as a whole (wing spot and comet assays) wecan conclude that SAS are not genotoxic, unless used at very highdoses.
Acknowledgements
This investigation has been supported in part by the Scientificand Technical Research Council of Turkey (TUBITAK) (Project Num-ber: KBAG-112T677), Ankara, (Turkey), by The Scientific ResearchProjects Coordination Unit of Akdeniz University (Project Num-ber: 2012.01.0115.002), Antalya, (Turkey) and by the Generalitatde Catalunya (CIRIT, 2009SGR-725), Barcelona (Spain).
Appendix A. Supplementary data
Supplementary material related to this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2014.09.029.
References
[1] N. Singh, B. Manshian, G.J. Jenkins, S.M. Griffiths, P.M. Williams, T.G. Maffeis,C.J. Wright, S.H. Doak, NanoGenotoxicology: the DNA damaging potential ofengineered nanomaterials, Biomaterials 30 (2009) 3891–3914.
[2] R. Landsiedel, M.D. Kapp, M. Schulz, K. Wiench, F. Oesch, Genotoxicity inves-tigations on nanomaterials: methods, preparation and characterization of testmaterial, potential artefacts and limitations – many questions, some answers,Mutat. Res. 681 (2009) 241–258.
[3] J. Ai, E. Biazar, M. Jafarpour, M. Montazeri, A. Majdi, S. Aminifard, M. Zafari,H.R. Akbari, H.G. Rad, Nanotoxicology and nanoparticle safety in biomedicaldesigns, Int. J. Nanomed. 6 (2011) 1117–1127.
[4] E. Demir, F. Turna, G. Vales, B. Kaya, A. Creus, R. Marcos, In vivo genotoxic-ity assessment of titanium, zirconium and aluminium nanoparticles, and theirmicroparticulated forms, in Drosophila, Chemosphere 93 (2013) 2304–2310.
[5] E. Demir, D. Burgucu, F. Turna, S. Aksakal, B. Kaya, Determination of TiO2, ZrO2,and Al2O3 nanoparticles on genotoxic responses in human peripheral bloodlymphocytes and cultured embyronic kidney cells, J. Toxicol. Environ. Health A76 (2013) 990–1002.
[6] E. Demir, H. Akc a, B. Kaya, D. Burgucu, O. Tokgün, F. Turna, S. Aksakal, G. Vales,A. Creus, R. Marcos, Zinc oxide nanoparticles: genotoxicity, interactions withUV-light and cell-transforming potential, J. Hazard. Mater. 264 (2014) 420–429.
[7] D. Napierska, L.C. Thomassen, D. Lison, J.A. Martens, P.H. Hoet, The nanosilicahazard: another variable entity, Part. Fibre Toxicol. 7 (2010) 39.
[8] T. Kaewamatawong, A. Shimada, M. Okajima, H. Inoue, T. Morita, K. Inoue, H.Takano, Acute and subacute pulmonary toxicity of low dose of ultrafine col-loidal silica particles in mice after intratracheal instillation, Toxicol. Pathol. 34(2006) 958–965.
[9] W. Lin, Y.W. Huang, X.D. Zhou, Y. Ma, In vitro toxicity of silica nanoparticles inhuman lung cancer cells, Toxicol. Appl. Pharm. 217 (2006) 252–259.
10] C.M. Sayes, K.L. Reed, D.B. Warheit, Assessing toxicity of fine and nanoparti-cles: comparing in vitro measurements to in vivo pulmonary toxicity profiles,Toxicol. Sci. 97 (2007) 163–180.
11] T.R. Downs, M.E. Crosby, T. Hu, S. Kumar, A. Sullivan, K. Sarlo, B. Reeder, M.Lynch, M. Wagner, T. Mills, S. Pfuhler, Silica nanoparticles administered at themaximum tolerated dose induce genotoxic effects through an inflammatoryreaction while gold nanoparticles do not, Mutat. Res. 745 (2012) 38–50.
12] K. Bhattacharya, P.C. Naha, I. Naydenova, S. Mintova, H.J. Byrne, Reactive oxygenspecies mediated DNA damage in human lung alveolar epithelial (A549) cellsfrom exposure to non-cytotoxic MFI-type zeolite nanoparticles, Toxicol. Lett.215 (2012) 151–160.
13] J. Duan, Y. Yu, H. Shi, L. Tian, C. Guo, P. Huang, X. Zhou, S. Peng, Z. Sun, Toxiceffects of silica nanoparticles on zebrafish embryos and larvae, PLoS One 8(2013) e74606.
14] C.A. Barnes, A. Elsaesser, J. Arkusz, A. Smok, J. Palus, A. Lesniak, A. Salvati, J.P.Hanrahan, W.H. Jong, E. Dziubałtowska, M. Stepnik, K. Rydzynski, G. McKerr,I. Lynch, K.A. Dawson, C.V. Howard, Reproducible comet assay of amorphoussilica nanoparticles detects no genotoxicity, Nano Lett. 8 (2008) 3069–3074.
15] M.V. Park, H.W. Verharen, E. Zwart, L.G. Hernandez, J. van Benthem, A. Elsaesser,C. Barnes, G. McKerr, C.V. Howard, A. Salvati, I. Lynch, K.A. Dawson, W.H. deJong, Genotoxicity evaluation of amorphous silica nanoparticles of differentsizes using the micronucleus and the plasmid lacZ gene mutation assay, Nan-otoxicology 5 (2011) 168–181.
16] C. Uboldi, G. Giudetti, F. Broggi, D. Gilliland, J. Ponti, F. Rossi, Amorphous silicananoparticles do not induce cytotoxicity, cell transformation or genotoxicity
in Balb/3T3 mouse fibroblasts, Mutat. Res. 745 (2012) 11–20.
17] A. Pandey, S. Chandra, L.K. Chauhan, G. Narayan, D.K. Chowdhuri, Cellular inter-nalization and stress response of ingested amorphous silica nanoparticles inthe midgut of Drosophila melanogaster, Biochim. Biophys. Acta 1830 (2013)2256–2266.
19] L.T. Reiter, L. Potocki, S. Chien, M. Gribskov, E. Bier, A systematic analysis ofhuman disease-associated gene sequences in Drosophila melanogaster, GenomeRes. 11 (2001) 1114–1125.
20] S.R. Kennedy, L.A. Loeb, A.J. Herr, Somatic mutations in aging, cancer and neu-rodegeneration, Mech. Ageing Dev. 133 (2012) 118–126.
21] J.S. Yadav, M.P. Lavanya, P.P. Das, I. Bag, A. Krishnan, R. Leary, A. Bagchi, B. Jagan-nadh, D.K. Mohapatra, M.P. Bhadra, U. Bhadra, 4-N-pyridin-2-yl-benzamidenanotubes compatible with mouse stem cell and oral delivery in Drosophila,Nanotechnology 21 (2010) 155102.
22] J.S. Yadav, P.P. Das, T.L. Reddy, I. Bag, P.M. Lavanya, B. Jagannadh, D.K. Moha-patra, M.P. Bhadra, U. Bhadra, Sub-cellular internalization and organ specificoral delivery of PABA nanoparticles by side chain variation, J. Nanobiotechnol.9 (2011) 10.
23] M. Ahamed, R. Posgai, T.J. Gorey, M. Nielsen, S.M. Hussain, J.J. Rowe, Silvernanoparticles induced heat shock protein 70, oxidative stress and apo-ptosis in Drosophila melanogaster, Toxicol. Appl. Pharmacol. 242 (2010)263–269.
24] E. Demir, G. Vales, B. Kaya, A. Creus, R. Marcos, Genotoxic analysis of silvernanoparticles in Drosophila, Nanotoxicology 5 (2011) 417–424.
25] G. Vales, E. Demir, B. Kaya, A. Creus, R. Marcos, Genotoxicity of cobalt nanopar-ticles and ions in Drosophila, Nanotoxicology 7 (2013) 462–468.
26] G. Vecchio, A. Galeone, V. Brunetti, G. Maiorano, L. Rizzello, S. Sabella, R. Cin-golani, P.P. Pompa, Mutagenic effects of gold nanoparticles induce aberrantphenotypes in Drosophila melanogaster, Nanomedicine 8 (2012) 1–7.
27] D.L. Lindsley, G.G. Zimm, The Genome of Drosophila melanogaster, AcademicPress, San Diego, CA, 1992.
28] P. Irving, J.M. Ubeda, D. Doucet, L. Troxler, M. Lagueux, D. Zachary, J.A. Hoffmann,C. Hetru, M. Meister, New insights into Drosophila larval haemocyte functionsthrough genome-wide analysis, Cell Microbiol. 7 (2005) 335–350.
29] E.R. Carmona, A. Creus, R. Marcos, Genotoxic effects of two nickel-compoundsin somatic cells of Drosophila melanogaster, Environ. Mol. Mutagen. 718 (2011)33–37.
30] E.R. Carmona, A. Creus, R. Marcos, Genotoxicity testing of two lead-compoundsin somatic cells of Drosophila melanogaster, Environ. Mol. Mutagen. 724 (2011)35–40.
31] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quanti-tation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988)184–191.
32] M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical signifi-cance of mutation frequencies, Mutat. Res. 9 (1970) 527–549.
[
Materials 283 (2015) 260–266
33] H. Frei, F.E. Würgler, Statistical methods to decide whether mutagenicity testdata from Drosophila assays indicate a positive, negative, or inconclusive result,Mutat. Res. 203 (1988) 297–308.
34] H. Frei, F.E. Würgler, Optimal experimental design and sample size for thestatistical evaluation of data from somatic mutation and recombination test(SMART) in Drosophila, Mutat. Res. 334 (1995) 247–258.
35] U. Graf, N. van Schaik, Improved high bioactivation cross for the wing somaticmutation and recombination test in Drosophila melanogaster, Mutat. Res. 271(1992) 59–67.
36] B. Kaya, R. Marcos, A. Yanikoglu, A. Creus, Evaluation of the genotoxicity of fourherbicides in the wing spot test of Drosophila melanogaster using two differentstrains, Mutat. Res. 557 (2004) 53–62.
37] D.J. Gorth, D.M. Rand, T.J. Webster, Silver nanoparticle toxicity in Drosophila:size does matter, Int. J. Nanomed. 6 (2011) 343–350.
38] P.P. Pompa, G. Vecchio, A. Galeone, V. Brunetti, S. Sabella, G. Maiorano, A. Falqui,G. Bertoni, R. Cingolani, In vivo toxicity assessment of gold nanoparticles inDrosophila melanogaster, Nano Res. 4 (2011) 405–413.
39] R. Posgai, C.B. Cipolla-McCulloch, K.R. Murphy, S.M. Hussain, J.J. Rowe, M.G.Nielsen, Differential toxicity of silver and titanium dioxide nanoparticles onDrosophila melanogaster development, reproductive effort, and viability: size,coatings and antioxidants matter, Chemosphere 85 (2011) 34–42.
40] S.C. Silver Key, D. Reaves, F. Turner, J.J. Bangn, Impacts of silver nanoparti-cle ingestion on pigmentation and developmental progression in Drosophila,ATLAS J. Biol. 1 (2011) 52–56.
41] H. Johnston, D. Brown, A. Kermanizadeh, E. Gubbins, V. Stone, Investigating therelationship between nanomaterial hazard and physicochemical properties:Informing the exploitation of nanomaterials within therapeutic and diagnosticapplications, J. Control. Release 164 (2012) 307–313.
42] C. Fruijtier-Pölloth, The toxicological mode of action and the safety of syntheticamorphous silica-a nanostructured material, Toxicology 294 (2012) 61–79.
43] S.W. Lee, S.M. Kim, J. Choi, Genotoxicity and ecotoxicity assays using the fresh-water crustacean Daphnia magna and the larva of the aquatic midge Chironomusriparius to screen the ecological risks of nanoparticle exposure, Environ. Toxicol.Pharmacol. 28 (2009) 86–91.
44] M. Chen, A. von Mikecz, Formation of nucleoplasmic protein aggregates impairsnuclear function in response to SiO2 nanoparticles, Exp. Cell Res. 305 (2005)51–62.
45] M. Valko, C.J. Rhodes, J. Moncol, M. Izakovic, M. Mazur, Free radicals, metals
and antioxidants in oxidative stress-induced cancer, Chem. Biol. Interact. 160(2006) 1–40.
46] J.A. Sergent, V. Paget, S. Chevillard, Toxicity and genotoxicity of nano-SiO2
on human epithelial intestinal HT-29 cell line, Ann. Occup. Hyg. 56 (2012)622–630.
