genotoxicity of cobalt nanoparticles and ions in drosophila
TRANSCRIPT
Nanotoxicology, June 2013; 7(4):462–468© 2013 Informa UK, Ltd.ISSN: 1743-5390 print / 1743-5404 onlineDOI: 10.3109/17435390.2012.689882
Genotoxicity of cobalt nanoparticles and ions in Drosophila
Gerard Vales1,*, Esref Demir2,*, Bülent Kaya2, Amadeu Creus1,3, & Ricard Marcos1,3
1Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma deBarcelona, Campus de Bellaterra, Cerdanyola del Vallès, Spain, 2Department of Biology, Faculty of Sciences, Akdeniz University,Antalya, Turkey and 3CIBER Epidemiología y Salud Pública, ISCIII, Spain
AbstractNanogenotoxicology is an emergent area of research, relevantfor estimating the potential carcinogenic risk of nanomaterials.Since most of the approaches use in vitro studies, and neglectingthe whole organism limits the accuracy of the obtained results,we have used Drosophila melanogaster to study the possiblegenotoxic potential of cobalt nanoparticles (Co NPs). The wingsomatic mutation and recombination test has been the test ofchoice. This test is based on the principle that the loss ofheterozygosis and the corresponding expression of the suitablerecessive markers, multiple wing hairs and flare-3 can lead to theformation of mutant clone cells in growing up larvae, which areexpressed as mutant spots on the wings of adult flies. Co NPs, aswell as the ionic form cobalt chloride, were given to third instarlarvae through the food, at concentrations ranging from 0.1 to10 mM. The results obtained indicate that both cobalt forms areable to induce significant increases in the frequency of mutantclones. Although at low concentrations only Co NPs weregenotoxic, the level of genetic damage obtained at the highestdose tested of cobalt chloride (10 mM) showed a significanthigher increase in the frequency of total spots than thoseobserved after the treatment with cobalt nanoparticles. Asconclusion, our results indicate that Co NPs were able to inducegenotoxic activity in the wing-spot assay of D. melanogaster,mainly via the induction of somatic recombination. Thedifferences observed in the behaviour of the two selected cobaltforms may result from differences in the uptake.
Keywords: Genotoxicity, cobalt nanoparticles, Drosophilamelanogaster, wing-spot assay, somatic recombination
Introduction
Nanomaterials present interesting physicochemical proper-ties that are increasingly exploited by different research andeconomic fields. Among nanomaterials, different metalnanoparticles are already commercially available for severalapplications. This means that such nanomaterials are spread
into the environment and, in this way, human exposurecertainly occurs. The important biological reactivity of nano-metals, when compared with their corresponding bulk mate-rials, may also involve an increased toxicity. Thus, the ultrasmall size and unique properties of nanomaterials have leadto increasing concerns about their potential toxicologicalrisk. For that reason, nanotoxicology is increasing as a novelarea of research, looking for the potential toxicity of nano-materials as well as for their intrinsic mechanisms of action(Ai et al. 2011).
In addition to the general toxicological profile of nanoma-terials, it is important to obtain information on their potentialinteractions with DNA, due to the crucial role that geneticdamage may play in human health. It must be recalled thatgenotoxic damage is linked to cancer development aswell as toother adverse health effects, including fertility problems andgenetic disorders in subsequent generations, if germinal cellsare affected. Thus, genotoxicity studies of nanomaterials arerequired toobtainamore complete andcomprehensive viewofthe risks associated with nanomaterials exposure (Singh et al.2009; Landsiedel et al. 2009).
An important aspect of toxicity and genotoxicity studies isthe selection of the assay system. In vitro approaches withhuman cultured cells are often used because they couldreduce, refine and replace animal methods (Hartung et al.2004), and the use of alternative testing in nanotoxicologyhas been recently discussed (Hartung & Sabbioni 2011)Nevertheless, they do not completely mimicry what happensin the whole organism. To avoid the use of mammals,Drosophila appears as a good alternative organism. Thisin vivo eukaryotic model has been already used to evaluatethe internalization of nanoparticles and to solve openquestions concerning cell uptake and live tissue distribution(Yadav et al. 2010, 2011), and there is an increasing numberof nanoparticles toxicity studies using this organism(Gorth et al. 2011; Pompa et al. 2011; Posgai et al. 2011;Silver Key et al. 2011). In spite of its increasing use to solvespecific questions, only one very recent published study(Vecchio et al. 2012) has used Drosophila to evaluate the
Correspondence: Ricard Marcos, Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma deBarcelona, Campus de Bellaterra, Cerdanyola del Vallès, Spain. Tel: +34 935812052. Fax: +34 935812387. E-mail: [email protected]*The first two authors contribute equally to this work.
(Received 4 November 2011; accepted 26 April 2012)
Nan
otox
icol
ogy
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Rye
rson
Uni
vers
ity o
n 05
/08/
13Fo
r pe
rson
al u
se o
nly.
potential genotoxic harmful effects of nanomaterials. Wehave recently used Drosophila to determine the ability ofsilver nanoparticles to induce somatic mutation and recom-bination in Drosophila wing tissues. The genotoxic testingapproach is based on the loss of heterozygosity in normalgenes affecting the wing’s hairs and the correspondingexpression of two recessive markers, namely multiplewing hairs (mwh) and flare-3 (flr3), in the wing blade ofadult flies, which modify the structure of the normal hairs ofthe wings. Thus, the induced genetic effects are microscop-ically observed as a significant increase in the frequency ofmutant spots (mwh or flr) on the wing tissues (Demir et al.2011).
In this study, we have selected cobalt nanoparticles(Co NPs) because of its industrial uses. In spite of its use inbiology and medicine, data on the eventual genotoxic effectsof Co NPs are still missing or sparse. Ponti et al. (2009) haveshown that Co NPs are able to induce significant increases ofgenetic damage in Balb/3T3 cells, when using both themicro-nucleus and the comet assays for testing genotoxic effects. Inaddition, they also observed positive induction of morpholog-ical cell transformation. Interestingly, when they used cobaltions, no cell transformation effects were observed. Similareffects were previously obtained in the comet assay, but usinghuman lymphocytes (Colognato et al. 2008). Thus, to investi-gate the possible genotoxic potential risk associated with CoNPsexposure,wehaveevaluated its genotoxicity inDrosophilamelanogaster, bymeasuring the induction of mutant clones inthe wing blade tissues. The effects induced by Co NPs arecompared with those induced by the cobalt ionic form (cobaltchloride).
Materials and methods
StrainsTwo D. melanogaster strains have been used: the multiplewing hairs strain with the genetic constitution y; mwh j; andthe flare-3 strain with the genetic constitution, flr3/In (3LR)TM3, Bds. The two wing markers used are the multiple winghairs (mwh, 3–0.3), and the flare-3 (flr3, 3–38.8). Mwh is acompletely recessive homozygous viable mutation, whichis kept in homozygous condition. It produces multipletrichomes per cell instead of the normally unique trichome.Flr3 is a recessive mutation affecting the shape of wing hairs,producing malformed wing hairs that have the shape of aflare. Given their zygotic lethality, flare alleles have to bemaintained in stocks over balancer chromosomes carryingmultiple inversions and a dominant marker that is homo-zygous lethal (TM3, Bds). Detailed information on the othergenetic markers and descriptions of the phenotypes of thestrains used in this work are extensively given by Lindsley &Zimm (1992).
ChemicalsCo NPs were purchased from Sigma-Aldrich (St Louis, MO,USA). The physical characteristics of nanoparticles, accord-ing to the manufacturer, are: size (<50 nm), density (8.9 g/mL) and surface area (>15 m2/g). To confirm such char-acteristics, further characterization of Co NPs was carried out
by using transmission electron microscopy (TEM), dynamiclight scattering (DLS) and laser Doppler velocimetry (LDV)methodologies. TEM methodologies used were carried on aJEOL JEM-2011 instrument to determine size and morphol-ogy. DLS and LDV were performed on a Malvern ZetasizerNano-ZS zen3600 instrument for the characterization ofhydrodynamic size and zeta potential, for these measurescobalt nanoparticles were dispersed in 100% ethanol.
The different doses to be assayed in the genotoxicity studywere prepared by using a solution of 3% ethanol in distilledwater and sonication. Distilled water and 3% ethanol wereused as negative control, while ethyl methanesulfonate(EMS), at a dose of 1 mM, was used as a positive control.Cobalt nanoparticles were dispersed at the concentrationof 2.56 mg/mL and subjected to ultrasonication (S-250D,Branson Sonifier, USA) at 20 kHz for 16 min in an ice-cooledbath prior to addition to culture media.
Cobalt (II) chloride hexahydrate (CoCl2�6H2O, CAS No:7791-13-1) was also provided by Sigma-Aldrich and it wasdissolved in 3% ethanol in distilled water.
TreatmentsTo obtain transheterozygous individuals, virgin flr3 femaleswere mated to mwh males, as previously described(Rizki et al. 2006). Eggs from this cross were collected during8-h periods in culture bottles containing standard medium(maize flour, agar, sodium chloride and yeast). The resulting3-day-old larvae were then transferred to plastic vials with4.5 g of Drosophila instant medium (Carolina BiologicalSupply Co., Burlington, NC, USA) prepared with the differentconcentrations of Co NPs, or cobalt chloride. The foodmedium is dehydrated, and it is rehydrated with 10 mL ofthe different concentrations of the tested compounds. Sincelarvae are constantly dying the medium during all thedevelopment, the medium is homogenized and it is passingduring all the time through the larval digestive tract. Thelarvae were fed on this medium until pupation. Treatmentvials were maintained in a culture room at 25 �C, 75%humidity and a light/dark daily cycle of 12/12 h. Thesurviving flies were collected from the treatment vials andwere stored in 70% ethanol. Afterwards, their wings wereremoved and mounted in Faure’s solution (30 g of gum
Mwh clone Flr clone
Figure 1. View of a mwh (A) and flr3 (B) clones on the wing blade oftransheterozygous flies. Note the morphology of the normal hairs.
Co NP genotoxicity in Drosophila
Nan
otox
icol
ogy
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Rye
rson
Uni
vers
ity o
n 05
/08/
13Fo
r pe
rson
al u
se o
nly.
Arabic, 30 ml glycerol, 50 g chloral hydrate, and 50 mldistilled water) on microscope slides. The wings were scoredat 400X magnification for the presence of spots. Single mwhspots result from point mutation, recombination or smalldeletion of the wild type allele; on the other hand, single flr3
spots arise from small deletion of the wild type allele. Twinspots, consisting of both mwh and flr3 sub-clones, areoriginated exclusively from mitotic recombination betweenflr3 and the centromere (Graf et al. 1984). A view of the aspectof mutant clones is observed in Figure 1. In each series, weexamined 80 wings (40 individuals). The scoring of flies anddata evaluation were performed following the standardprocedures for the wing-spot assay, as used in recent inves-tigations (Carmona et al. 2008; Dihl et al. 2008).
Statistical analysisThe conditional binomial test of Kastenbaum & Bowman(1970) was applied to assess differences between the fre-quencies of each type of spot in treated and concurrentnegative control, with significant levels a = b = 0.05. The
multiple decision procedure described by Frei & Würgler(1988) was used to judge the overall response of an agentas positive, weakly positive, negative, or inconclusive. Asrecommended, we consider the treatment as positive if thefrequency of mutant clones in the treated series is at least m(multiplication factor) times greater than in the controlseries. Since small single spots and total spots have acomparatively high spontaneous frequency, m is fixed at avalue of 2 (testing for a doubling of the spontaneous fre-quency). For the large single spots and the twin spots, whichhave a low spontaneous frequency, m = 5 is used. Thefrequency of clone formation was calculated, without sizecorrection, by dividing the number of mwh clones per wingby 24,400, which is the approximate number of cellsinspected per wing (Alonso-Moraga & Graf 1989).
Results
In this study, we report the genotoxic activity of Co NPs andits ionic form. We used TEM to characterize the size and the
40
30
20
Fre
qu
ency
10
0 0 nm 20 nm 40 nm 60 nm
Diameter
80 nm 100 nm
Mean = 28.10Std. Dev. = 18.258
N = 200
Figure 2. Characterization of cobalt nanoparticles. Typical TEM images and size distribution histogram using such images.
G. Vales et al.
Nan
otox
icol
ogy
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Rye
rson
Uni
vers
ity o
n 05
/08/
13Fo
r pe
rson
al u
se o
nly.
morphology of Co NPs. The majority of nanoparticles werein spherical shape and important agglomerations weredetected following the dispersion protocol. Information onmean size and SD was calculated from measuring 200 iso-lated nanoparticles in random fields. The mean ± SD of CoNPs was 28.25 ± 18.18 (Figure 2). The average hydrodynamicdiameter in suspension was 288.20 nm and zeta potentialwas 20.6 mV.
Preliminary toxicity studies were carried out to define therange of doses to be tested in the genotoxicity studies.Toxicity was measured as the increase in the percentageof treated larvae that does not reach the adult stage. Co NPswere as toxic as cobalt chloride (result not shown) and,therefore, similar doses were selected for both cobalt com-pounds (0.5, 1, 5 and 10 mM). The selected highest dosesinduced a mortality lower than 75% in comparison withcontrol.
Table I shows the results obtained with the transheter-ozygous larvae treated with the different doses of Co NPs. Asindicated, this nanomaterial was administered to 3-day-old(third instar) larvae at doses ranging from 0.1 to 10 mM. Thetreatment was given to the larvae until they completeddevelopment. In this transheterozygous genetic background,single mutant spots results from both somatic mutation andsomatic recombinationmechanisms. Twin spots appear onlyas results of somatic mutations. Results indicate that Co NPsinduce significant increases in the frequency of small singlemwh spots and in the total mutant spots recorded, with adirect dose–response relationship. The induction of small or
large spots does not depend on the genotoxic potency of theagent but on the time that take to reach the cells target. Thiswould indicate that the progression of nanocobalt particlesto reach the wing imaginal disks is slow and only producegenetic damage in the final stages of the developmentoriginating, consequently, only small mutant spots. Signifi-cant increases were observed in the positive controls for alltypes of mutant clones recorded.
To get information on the mechanisms by which nano-cobalt induce mutant spots, we performed one experimentusing balanced heterozygous larvae. In this genotype,recombination is suppressed and only clones induced bysomatic mutation can be observed. The results of thisexperiment are summarized in Table II and, as observed,no significant induction of single or total mutant clones wasobserved. This would indicate that nanocobalt can exert itsgenotoxic effect mainly via somatic recombination. In spiteof this lack of significant effects, it must be pointed outthat the treatments induced a dose-dependent increase inthe frequency of the recorded mutant clones, but withoutattaining statistical significance.
To determine if the genotoxic potential of Co NPs differsfrom that of ionic form, an experiment using cobalt chloridewas carried out and the results are indicated in Table III. Asobserved, although no significant genotoxic effects wereobserved after treatments ranging from 0.1 to 5 mM, thehighest dose of cobalt chloride tested (10 mM) induced ahigh frequency of both single and total mutant spots. Thiswould indicate that high doses of ionic cobalt are muchmore
Table I. Wing-spot test data obtained after the treatment of larvae with cobalt nanoparticles. Results obtained with mwh/flr3 wings.Small single spots(1–2 cells) (m = 2)
Large single spots(>2 cells) (m = 5)
Twin spots(m = 5)
Total spots(m = 2)
Compounddose (mM) No Fr D No Fr D No Fr D No Fr D
Frequency of cloneformation per 105 cells
Distilled water 20 (0.25) 7 (0.09) 1 (0.01) 28 (0.35) 1.43
Ethanol (3%) 32 (0.40) i 5 (0.06) – 0 (0.00) i 37 (0.46) i 1.90
1 mM EMS 77 (0.96) + 25 (0.31) + 9 (0.11) + 111 (1.39) + 5.53
Nanocobalt
0.1 31 (0.39) i 4 (0.05) – 2 (0.03) i 37 (0.46) i 1.90
1 33 (0.41) + 6 (0.08) – 1 (0.01) i 40 (0.50) i 2.05
5 45 (0.56) + 3 (0.04) – 4 (0.05) i 52 (0.65) + 2.61
10 48 (0.60) + 7 (0.09) – 1 (0.01) i 56 (0.70) + 2.82
No, number; Fr, frequency; D, statistical diagnosis according to Frei & Würgler (1988); +, positive; –, negative, i: inconclusive;m, multiplicative factor; probability levels,a = b = 0.05; A total of 80 wings were scored per dose.
Table II. Wing-spot test data obtained after treatment of larvae with cobalt nanoparticles. Results obtained with mwh/TM3 wings.Small single spots(1–2 cells) (m = 2)
Large single spots(>2 cells) (m = 5)
Totalspots (m = 2)
Compounddose (mM) No Fr D No Fr D No Fr D
Frequency of cloneformation per 105 cells
Distilled water 20 (0.25) 4 (0.05) 24 (0.30) 1.23
Ethanol (3%) 19 (0.24) – 8 (0.10) i 27 (0.34) – 1.38
1 mM EMS 43 (0.54) + 15 (0.19) + 58 (0.73) + 2.97
Nanocobalt
0.1 16 (0.20) – 4 (0.05) – 20 (0.25) – 1.02
1 21 (0.26) – 3 (0.04) – 24 (0.30) – 1.23
5 21 (0.26) – 4 (0.05) – 25 (0.31) – 1.28
10 29 (0.37) i 3 (0.04) – 32 (0.40) i 1.64
No, number; Fr, frequency; D, statistical diagnosis according to Frei & Würgler (1988); +, positive; –, negative; i, inconclusive,m, multiplication factor; probability levels,a = b = 0.05; A total of 80 wings were scored per dose.
Co NP genotoxicity in Drosophila
Nan
otox
icol
ogy
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Rye
rson
Uni
vers
ity o
n 05
/08/
13Fo
r pe
rson
al u
se o
nly.
genotoxic than Co NPs. A comparison of the data obtainedwith Co NPs and cobalt chloride is shown in Figure 3.
Discussion and conclusions
This study shows the usefulness of Drosophila as a eukaryoticmodel for detecting the genotoxic potential of Co NPs and, byextension, for the testing of any nanomaterial, both for toxicity(Ahamed et al. 2010) and genotoxicity (Demir et al. 2011). Ourresults, showing that larvae exposure to Co NPs inducessomatic recombination in the wing imaginal disk cells sup-port not only Drosophila as a suitable biosystem but also thewing test as an easy method to detect in vivo genotoxicity. Inthis context it must be remembered that the quantification ofthe recombinagenic activity of a compound is of primaryimportance for genotoxicity screening (Graf & Würgler 1996),
since this event is strongly linked to carcinogenesis process(Sengstag 1994; Lupski 2007).
As far as we know, our study constitutes the first directgenotoxic evaluation for Co NPs in Drosophila. These resultsare interesting in terms of health risk because Drosophila isconsidered as a good health model system, since over 60% ofhuman disease genes have fly homologues, indicating that thefly response to physiological insults is comparable to humans(Schneider 2000; Koh et al. 2006; Marsh & Thompson 2006).Thiswouldreinforce theusefulnessof theDrosophilamodelasafirst tier in vivo test for nanoparticles toxicity. To supportour defence of the advantages of Drosophila in nanogenotox-icology, several studies have observed that, for different nano-materials, larval ingestion leads toan important systemicuptakeand tissue sequestration (Posgai et al. 2009), ensuring theirpotential effects.
Table III. Wing-spot test data obtained after treatment of larvae with cobalt chloride (CoCl2.6H2O). Results obtained with mwh/flr3 wings.Small single spots(1–2 cells) (m = 2)
Large single spots(>2 cells) (m = 5)
Twin spots(m = 5)
Total spots(m = 2)
Compounddose (mM) No Fr D No Fr D No Fr D No Fr D
Frequency of cloneformation per 105 cells
Distilled water 13 (0.16) 2 (0.03) 1 (0.01) 16 (0.20) 0.82
1 mM EMS 60 (0.75) + 25 (0.31) + 11 (0.14) + 96 (1.20) + 4.71
CoCl2.6H2O
0.1 15 (0.19) i 1 (0.01) i 0 (0.00) i 16 (0.20) – 0.82
1 21 (0.26) i 4 (0.05) i 0 (0.00) i 25 (0.31) i 1.28
5 19 (0.24) i 1 (0.01) i 1 (0.01) i 21 (0.26) i 1.08
10 85 (1.06) + 6 (0.08) i 4 (0.05) i 95 (1.19) + 4.87
No, number; Fr, frequency; D, statistical diagnosis according to Frei & Würgler (1988); +, positive; –, negative; i, inconclusive;m, multiplication factor; probability levels,a = b = 0.05; A total of 80 wings were scored per dose.
1.20
Cobalt chloride Cobalt nanoparticles
1.00
0.80
0.60
Fre
c. t
ota
l sp
ots
0.40
0.20
0.000 1
Dose (mM) Dose (mM)
5 100.1 0 1 5 100.1
Figure 3. Comparison of total spots obtained after the treatment with cobalt nanoparticles (A) and cobalt chloride (B).
G. Vales et al.
Nan
otox
icol
ogy
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Rye
rson
Uni
vers
ity o
n 05
/08/
13Fo
r pe
rson
al u
se o
nly.
Few studies have been conducted to evaluate thegenotoxicity of Co NPs and several of them have beendone with alloys containing cobalt, since these alloys arewidely used in orthopaedic implants. In these metal-on-metal implants, nanoparticles are generated (Doorn et al.1998). Thus Co-Cr alloys have shown to induce geneticdamage on human fibroblasts in vitro, inducing DNA breaksas detected by the comet assay, and aneuploidy using themicronucleus test (Papageorgiou et al. 2007). An elevatedfrequency of micronucleated cells were also observed in thestudy of Tsaousi et al. (2010) who also reported an increasedfrequency of gamma-H2AX foci after alloy treatment.
With respect to the studies carried out specifically with CoNPs, its in vitro genotoxic effects have been studied in Balb/3T3 cells by Ponti et al. (2009). In this study the effects of CoNPs are compared with those induced by cobalt ions (Co2+),as in our in vivo study. In general, they found genotoxicitywith both cobalt forms without large differences betweenthem for the micronucleus and the comet tests. Nevertheless,only Co NPs were able to induce morphological transforma-tion increasing the formation of type II foci. These differencesare attributed to the higher cellular uptake of Co NPs, whencompared to cobalt ions. This can be due to the fact thatnanoparticles interact with proteins present in the culturemedium and they are more readily taken up by the cells.
Similar results were also obtained by the same group in aprevious study comparing the same two types of cobalt(Colognato et al. 2008). They used human lymphocytecultures as target cells, and the comet assay and the micro-nucleus test as genotoxic assays. Although both cobalt formsinduced significant increases in the frequency of micronu-cleated cells, only Co NPs were able to induce significantincrement in DNA breaks, as measured by using the cometassay. A recent study (Jiang et al. 2011) using the comet assayin primary human T lymphocytes has shown the induction ofsignificant genotoxic effects of Co NPs, but no effects of cobaltions (cobalt chloride). This discrepancy with the results fromprevious investigations cannot lie in the differences in expo-sure times, since in this study the treatment lasts for 4 hwhile Ponti el al. (2009) and Colognato et al. (2008) used 2 htreatments. In addition, differences in concentration cannotbe assumed since Jiang et al. used a dose range of 10–30 mMin comparison with the 1–5 mM used by Ponti et al. Thus,specific differential uptake of T lymphocytes would explainthe observed differences.
Our studies show that Co NPs are more genotoxic at lowdoses than cobalt ions; nevertheless, at the highest dosetested, the genotoxic effects induced by cobalt chloride arehighest. Studies on the mechanisms underlying the geno-toxicity of cobalt support the view that most of the effects areinduced by oxidative damage (Lison et al. 2001) and that theinduced effects can be reverted by the addition of antiox-idants to the culture media (Zou et al. 2001).
The different activity of the different cobalt forms can bedue to the different uptake intensity observed, Co NPs beinguptaken with a highest efficiency than cobalt ions. Neverthe-less, internalized Co NPs could be dissolved producingcobalt ions (Co2+). This high efficiency of cobalt nanoparticlesto be uptaken can agree with the view of Limbach et al.
(2007), who proposed a Trojan horse-type mechanism toexplain the possible effects of nanoparticles due to their fastinternalization in the cell.
Our conclusion is thatCoNPs are able toproducegenotoxiceffects in an in vivo model as Drosophila, showing highergenotoxic potential than cobalt chloride at low doses tested.In addition, our results demonstrate that an important partof the observed effects are due to the induction of somaticmutation. In this point, it must be recalled that somaticmutation is an important event in carcinogenesis; thus,our results support the useful role of Drosophila when theeventual adverse health effects of nanoparticles are evaluated.
Acknowledgements
Gerard Vales is supported by a fellowship (PIF) fromthe Universitat Autònoma de Barcelona. Esref Demir issupported by a doctorate fellowship from the AkdenizUniversity and the Council of Higher Education (YÖK),Ankara (Turkey). This investigation has been supported inpart by the Generalitat de Catalunya (CIRIT, 2009SGR-725)and by theManagement Unit of Research Projects of AkdenizUniversity (Project ID: 2009.03.0121.004), Antalya (Turkey).
Declaration of interest
The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of the paper.
ReferencesAhamed M, Posgai R, Gorey TJ, Nielsen M, Hussain SM, Rowe JJ. 2010.
Silver nanoparticles induced heat shock protein 70, oxidative stressand apoptosis in Drosophila melanogaster. Toxicol Appl Pharmacol242(3):263–269.
Ai J, Biazar E, Jafarpour M, Montazeri M, Majdi A, Aminifard S, et al.2011. Nanotoxicology and nanoparticle safety in biomedical designs.Int J Nanomedicine 6:1117–1127.
Alonso-Moraga A, Graf U. 1989. Genotoxicity testing of antiparasiticnitrofurans in the Drosophila wing somatic mutation and recom-bination test. Mutagenesis 4(2):105–110.
Carmona ER, Kossatz E, Creus A, Marcos R. 2008. Genotoxic evaluationof two mercury compounds in the Drosophila wing spot test.Chemosphere 70(10):1910–1914.
Colognato R, Bonelli A, Ponti J, Farina M, Bergamaschi E,Sabbioni E, et al. 2008. Comparative genotoxicity of cobalt nano-particles and ions on human peripheral leukocytes in vitro. Muta-genesis 23(5):377–382.
Demir E, Vales G, Kaya B, Creus A, Marcos R. 2011. Genotoxic analysisof silver nanoparticles in Drosophila. Nanotoxicology 5(3):417–424.
Dihl RR, da Silva CGA, do Amaral VS, Reguly ML, de Andrade HHR.2008. Mutagenic and recombinagenic activity of airborne particu-lates, PM10 and TSP, organic extracts in the Drosophila wing-spottest. Environ Pollut 151(1):47–52.
Doorn PF, Campbell PA, Worral J, Benya PD, Mckellop HA,Amstutz HC. 1998. Metal wear particle characterization from metalon metal total hip replacements: transmission electron microscopystudy of periprostheic tissues and isolated particles. J Biomed MaterRes 42(1):1271–1286.
Frei H, Würgler FE. 1988. Statistical methods to decide whethermutagenicity test data from Drosophila assays indicate a positive,negative, or inconclusive result. Mutat Res 203(4):297–308.
Gorth DJ, Rand DM, Webster TJ. 2011. Silver nanoparticle toxicity inDrosophila: size does matter. Int J Nanomedicine 6:343–350.
Graf U, Würgler FE, Katz AJ, Frei HJ, Juon H, Hall CB, et al. 1984.Somatic mutation and recombination test in Drosophila melanoga-ster. Environ Mutagen 6(2):153–188.
Graf U, Würgler FE. 1996. The somatic white-ivory eye spot testdoes not detect the same spectrum of genotoxic events as the
Co NP genotoxicity in Drosophila
Nan
otox
icol
ogy
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Rye
rson
Uni
vers
ity o
n 05
/08/
13Fo
r pe
rson
al u
se o
nly.
wing somatic mutation and recombination test in Drosophilamelanogaster. Environ Mol Mutagen 27(3):219–226.
Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortaner S, et al.2004. A modular approach to the ECVAM principles on test validity.Altern Lab Anim 32(5):467–472.
Hartung T, Sabbioni E. 2011. Alternative in vitro assays in nanomaterialtoxicology. Wiley Interdiscip Rev Nanomed Nanobiotechnol3:545–573.
Jiang H, Liu F, Yang H, Li Y. 2011. Effects of cobalt nanoparticles onhuman T cells in vitro. Biol Trace Elem Res Epub ahead of print;PMID: 21968949.
Kastenbaum MA, Bowman KO. 1970. Tables for determiningthe statistical significance of mutation frequencies. Mutat Res9(5):527–549.
Koh K, Evans JM, Hendricks JC, Sehgal A. 2006. A Drosophila model forage-associated changes in sleep: wake cycles. Proc Natl Acad SciUSA 103(37):13843–13847.
Landsiedel R, Kapp MD, Schulz M, Wiench K, Oesch F. 2009. Geno-toxicity investigations on nanomaterials: methods, preparationand characterization of test material, potential artifacts and limit-ations-many questions, some answers. Mutat Res 681(2-3):241–258.
Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. 2007.Exposure of engineered nanoparticles to human lung epithelialcells: Influence of chemical composition and catalytic activity onoxidative stress. Environ Sci Technol 41(11):3791–3792.
Lindsley DL, Zimm GG. editors. 1992. The genome of Drosophilamelanogaster. San Diego, CA: Academic Press.
Lison D, De BoeckM, Verougstraete V, Kirsch-Volders M. 2001. Updateon the genotoxicity and carcinogenicity of cobalt compounds.Occup Environ Med 58(10):619–625.
Lupski JR. 2007. Genomic rearrangements and sporadic disease. NatGenet 39(7 Suppl):S43–S47.
Marsh JL, Thompson LM. 2006. Drosophila in the study of neurode-generative disease. Neuron 52(1):169–178.
Papageorgiou I, Brown C, Schins R, Sing S, Newson R, Davis S, et al. 2007.The effect of nano- and micron-sized particles of cobalt-chromiumallow on human fibroblast in vitro. Biomaterials 28(19):2946–2958.
Pompa PP, Vecchio G, Galeone A, Brunetti V, Sabella S,Maiorano G, et al. 2011. In vivo toxicity assessment of gold nano-particles in Drosophila melanogaster. Nano Res 4(4):405–413.
Ponti J, Sabbioni E, Munaro B, Broggi F, Marmorato P,Franchini F, et al. 2009. Genotoxicity and morphological transfor-mation induced by cobalt nanoparticles and cobalt chloride: an in
vitro study in Balb/3T3 mouse fibroblasts. Mutagenesis 24(5):439–445.
Posgai R, Ahamed M, Hussain SM, Rowe JJ, Nielsen MG. 2009. Inha-lation method for delivery of nanoparticles to the Drosophila respi-ratory system for toxicity testing. Sci Total Environ 408(2):439–443.
Posgai R, Cipolla-McCulloch CB, Murphy KR, Hussain SM, Rowe JJ,Nielsen MG. 2011. Differential toxicity of silver and titanium dioxidenanoparticles on Drosophila melanogaster development, reproduc-tive effort, and viability: size, coatings and antioxidants matter.Chemosphere 85(1):34–42.
Rizki M, Kossatz E, Velázquez A, Creus A, Farina M, Fortaner S, et al.2006. Metabolism of arsenic in Drosophila melanogaster and thegenotoxicity of dimethylarsinic acid in the Drosophila wing spot test.Environ Mol Mutagen 47(3):162–168.
Schneider D. 2000. Using Drosophila as a model insect. Nat Rev Genet1(3):218–226.
Sengstag C. 1994. The role of mitotic recombination in carcinogenesis.Critical Rev Toxicol 24(4):323–353.
Silver Key SC, Reaves D, Turner F, Bangn JJ. 2011. Impacts of silvernanoparticle ingestion on pigmentation and developmental pro-gression in Drosophila. ATLAS J Biol 1:52–56.
Singh N, Manshian B, Jenkins GJ, Griffiths SM, Williams PM,Maffeis TG, et al. 2009. NanoGenotoxicology: the DNA damag-ing potential of engineered nanomaterials. Biomaterials30(23-24):3891–3914.
Tsaousi A, Jones E, Case CP. 2010. The in vitro genotoxicity oforthopaedic ceramic (Al2O3) andmetal (CoCr alloy) particles. MutatRes 697(1-2):1–9.
Vecchio G, Galeone A, Brunetti V, Maiorano G, Rizzello L,Sabella S, et al. 2012. Mutagenic effects of gold nanoparticles induceaberrant phenotypes in Drosophila melanogaste. Nanomedicine8(1):1–7.
Yadav JS, Das PP, Reddy TL, Bag I, Lavanya PM, Jagannadh B, et al.2011. Sub-cellular internalization and organ specific oral delivery ofPABA nanoparticles by side chain variation. J Nanobiotechnol 9:10.
Yadav JS, Lavanya MP, Das PP, Bag I, Krishnan A, Leary R, et al. 2010.4-N-pyridin-2-yl-benzamide nanotubes compatible with mousestem cell and oral delivery in Drosophila. Nanotechnology21(15):155102.
Zou W, Yan M, Xu W, Huo H, Sun L, Zheng Z, et al. 2001. Cobaltchloride induces PC12 cells apoptosis through reactive oxygenspecies and accompanied by AP-1 activation. J Neurosci Res64(6):646–653.
G. Vales et al.
Nan
otox
icol
ogy
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Rye
rson
Uni
vers
ity o
n 05
/08/
13Fo
r pe
rson
al u
se o
nly.