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Mutation Research 724 (2011) 35– 40

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

journa l h omepage: www.elsev ier .com/ locate /gentoxC om mun i ty a ddress : www.elsev ier .com/ locate /mutres

enotoxicity testing of two lead-compounds in somatic cells of Drosophilaelanogaster

rico R. Carmonaa,1, Amadeu Creusa,b, Ricard Marcosa,b,∗

Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra,8193 Cerdanyola del Vallès, Barcelona, SpainCIBER Epidemiología y Salud Pública, ISCIII, Spain

r t i c l e i n f o

rticle history:eceived 9 February 2011eceived in revised form 13 April 2011ccepted 5 May 2011vailable online 30 May 2011

eywords:omet assay

a b s t r a c t

The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogasterby measurement of two different genetic endpoints. We used the wing-spot test and the comet assay.The comet assay was conducted with larval haemocytes. The results from the wing-spot test showedthat neither lead chloride, PbCl2, nor lead nitrate, Pb(NO3)2, were able to induce significant increasesin the frequency of mutant spots. In addition, the combined treatments with gamma-radiation andPbCl2 or Pb(NO3)2 did not show significant variations in the frequency of the three categories of mutantspots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indi-

rosophila melanogasteraemocytesead

ing-spot test

cate that the lead compounds tested do not interact with the repair of the genetic damage inducedby ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay withhaemocytes, Pb(NO3)2 was effective in inducing significant increases of DNA damage with a directdose–response pattern. These results confirm the usefulness of the comet assay with haemocytes asan in vivo model and support the assumption that there is a genotoxic risk associated with leadexposure.

. Introduction

Lead is a pollutant heavy metal that is considered as one ofhe most hazardous chemicals for human health [1]. Although leadmissions have declined in the last two decades, it is a persistentetal that can still be found in the environment, mainly in water,

ood, dust, soil, and in manufactured lead products [2]. Lead is aell-known toxin that can affect different organs and systems inumans, but its toxic effects are targeted mostly on the nervousystem, blood and kidney [3]. Moreover, children are particularlyusceptible to lead exposure due to high gastrointestinal uptakend their permeable blood–brain barrier [4].

Currently, lead compounds are classified as possibly car-

inogenic to humans mainly on the basis of experimentalnimal studies [5]. However, recent epidemiological studies showhat there is some evidence associating lead exposure with

∗ Corresponding author at: Grup de Mutagènesi, Departament de Genètica i deicrobiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus

e Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. Tel.: +34 93 581 2052;ax: +34 93 581 2387.

E-mail address: ricard.marcos@uab.es (R. Marcos).1 Present address: Facultad de Recursos Naturales, Escuela de Ciencias Ambien-

ales, Universidad Católica de Temuco, Chile.

383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.mrgentox.2011.05.008

© 2011 Elsevier B.V. All rights reserved.

increased risk of lung cancer, stomach cancer, and gliomas inhumans [6].

Even though the mechanism that underlies the genotoxicity oflead is not clear, two main indirect mechanisms have been pro-posed. First, lead compounds have shown to interfere with DNArepair since they act as co-mutagens with other mutagens, such asUV-radiation and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)[7] and, second, lead can alter chromosome segregation throughinteraction with cytoskeleton proteins [8].

Up to now, the genotoxic properties of different chemical formsof lead have been analyzed, evaluated and revised by differentauthors with diverse conclusions [9–11]. From the available data,it seems that lead compounds are weak mutagens in bacteria [7].However, in vitro studies in mammalian cells show that lead com-pounds can induce different genotoxic effects, such as aneugenicity,clastogenicity [8,12] and single-strand DNA breaks [13]. In addi-tion, several biomonitoring studies have revealed genetic damagein lead-exposed workers [14–18], suggesting a potential genotoxicrisk associated with lead exposure.

Drosophila melanogaster is an in vivo model organism commonlyemployed in genetic toxicology testing given its advantages: it

is easy to maintain in laboratory conditions; it presents a shortgeneration time, allowing a fast genotoxic evaluation; and it hasa metabolic activity capable of activating pro-mutagens and pro-carcinogens, analogous to that of the liver in mammals [19]. Thus,

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. melanogaster has been used basically in short-term tests for iden-ifying potential carcinogens and as a model organism to study the

echanisms of mutagenesis [20].To increase our knowledge about the potential genotoxic risk

ssociated with lead exposure, we have evaluated two inorganicead compounds, namely lead chloride, PbCl2, and lead nitrate,b(NO3)2, by use of two in vivo test systems for measuring DNAamage in somatic cells of D. melanogaster. On the one hand, wesed the wing-spot test (or wing SMART assay), a short-term testased on the loss of heterozygosity in normal genes and the cor-esponding expression of recessive markers, called multiple wingairs (mwh) and flare-3 (flr3), in the wing blade of adult flies. Thisssay can detect mitotic recombination and a diverse set of muta-ional events such as point mutations, deletions, and certain typesf chromosome aberrations [21]. In addition, the possible mod-lating effects of two lead compounds on the effects induced by

well-known genotoxic agent such as gamma-radiation was alsovaluated with the wing-spot test. On the other hand, the recentlyeveloped and adapted in vivo comet assay on circulating bloodells (or haemocytes) of D. melanogaster [22] was also performed inrder to test the genotoxicity of the two inorganic lead compounds.his assay is a simple and sensitive tool for the detection of lowevels of DNA damage and allows the detection of several kinds ofNA damage, such as double- and single-strand DNA breaks, DNAross-links, alkali-labile sites and delayed repair sites [23,24].

. Materials and methods

.1. Strains

Two D. melanogaster strains were used for the wing-spot test: the multipleing-hair strain with the genetic constitution y; mwh j; and the flare-3 strain with

he genetic constitution flr3/In (3LR) TM3, Bds . More detailed information on theenetic markers and descriptions of the phenotypes are given by Lindsley and Zimm25]. Both strains were initially provided by Prof. F.E. Würgler (University of Zürich,witzerland). The wild-type strain Oregon R+, proficient in all types of repair wassed for comet-assay experiments with haemocytes. These strains were cultured

n bottles with standard Drosophila medium, at a temperature of 25 ± 1 ◦C and aelative humidity of ∼60%.

.2. Chemicals

PbCl2 (CAS 7758-95-4, 98% purity), Pb(NO3)2 (CAS 10099-74-8, ≥99% purity),thyl methanesulfonate (EMS, CAS 62-50-0, 100% purity) and phenylthioureaPTU, CAS 103-85-5, ≥95% purity) were obtained from Sigma–Aldrich (Spain); low

elting-point agarose (LMA) and normal melting-point agarose (NMA) were fromibco BRL, Life Technologies (Paisley, UK); phosphate-buffered saline (PBS) and 4′ ,6-iamidine-2-phenylindole (DAPI) were from Sigma Chemical Co. (St. Louis, MO);-laurosylsarcosine sodium hydroxide and Triton X-100 were from Fluka ChemicalG (Buchs, Switzerland); sodium hydroxide was from Carlo Erba Reagenti (Milan,

taly); sodium chloride was from Panreac Química SA (Barcelona, Spain) and Trisuffer was from US Biochemical (Cleveland, OH).

.3. Drosophila wing-spot test protocol

Virgin females of the flr3 strain were mated to mwh males as previouslyescribed [26]. Eggs from this cross were collected during 8-h periods in culture bot-les containing standard medium. The resulting 3-day-old larvae were then placedn plastic vials containing 4.5 g of Drosophila instant medium (Carolina Biologicalupply, Burlington, NC) prepared with 10 mL of different non-toxic concentrationsf PbCl2 and Pb(NO3)2 (2, 4 and 8 mM, respectively). Larvae were fed on this mediumntil pupation.

For co-treatment experiments, larvae 48 ± 2 h old were collected and pre-reated with 2, 4, and 8 mM of PbCl2 and Pb(NO3)2, respectively, for 24 h; then theyere collected, washed and transferred to plastic vials for treatment with one dose of

amma-radiation (6 G�). After this treatment, larvae were transferred to new plasticials with the same concentrations of lead compounds as during the pre-treatment,ntil completion of development. The gamma-irradiation was performed in a 137Cs

rradiator Model IBL 437C (Schering Bio International, Germany), from the Technicalnit of Radiological Protection (UTPR) at the Universitat Autònoma de Barcelona.

The conditional binomial test of Kastenbaum and Bowman [27] was applied tossess differences between the frequencies of each type of spot in treated and con-urrent negative control, with significant levels ˛ = ˇ = 0.05. The multiple-decisionrocedure described by Frei and Würgler [28] was used to judge the overall responsef an agent as positive, weakly positive, negative, or inconclusive. The frequency of

search 724 (2011) 35– 40

clone formation was calculated, without size correction, by dividing the number ofmwh clones per wing by 24,400, which is the approximate number of cells inspectedin one wing [29].

The same statistical analysis was applied to test for possible interactionsbetween PbCl2 or Pb(NO3)2 and gamma-radiation.

2.4. Protocol for the comet-assay in haemocytes

Third-instar larvae (72 ± 4 h old) were placed in plastic vials containing 4.5 g ofDrosophila instant medium, prepared with the different concentrations of PbCl2 andPb(NO3)2. Immediately before use, the compounds were dissolved in distilled water.Larvae were fed on this medium during 24 ± 2 h. Distilled water and EMS were usedas negative and positive controls, respectively. All the experiments were performedat 25 ± 1 ◦C and at ∼60% relative humidity.

D. melanogaster haemocytes were collected according to Irving et al. [30] withminor modifications. Chilled larvae (96 ± 2 h old) were removed from food media,washed in water, sterilized in 5% bleach and dried. The cuticle from 40 to 60 lar-vae was then disrupted with two fine forceps. The haemolymph and circulatinghaemocytes were directly collected in cold PBS solution containing 0.07% PTU, andseparated in a 1.5-mL micro-centrifuge tube. Pooled haemolymph was centrifugedat 300 × g for 10 min at 4 ◦C; the supernatant was discarded and the pellet wasresuspended in 20 �L of cold PBS.

The comet assay was conducted as previously described by Singh et al. [31], withminor modifications. Cell samples (∼40,000 cells in 20 �L) were carefully resus-pended in 140 �L of 0.75% LMA, layered onto microscope slides pre-coated with150 �L of 1% NMA (dried at room temperature). Two gels were mounted on eachslide and covered with a coverslip. Immediately after agarose solidification (10 minat 4 ◦C), the coverslips were removed and the slides were immersed in cold, freshlymade lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% Triton X-100 and1% N-laurosylsarcosinate, pH 10) for 2 h at 4 ◦C in a dark chamber. Dimethyl sul-foxide (DMSO) was omitted from the lysis solution, because it has been consideredunnecessary for Drosophila tissues, and DMSO at low concentrations is cytotoxic inDrosophila [32,33]. To avoid additional DNA damage, the next steps were performedunder dim light. Slides were placed for 25 min in a horizontal gel-electrophoresistank filled with cold electrophoresis buffer (1 mM Na2EDTA, 300 mM NaOH, pH13) to allow DNA unwinding. Electrophoresis was carried out in the same bufferfor 20 min at 25 V and 300 mA. The unwinding and electrophoresis were done at4 ◦C. After electrophoresis, slides were neutralized with two washes of 5 min with0.4 mM Tris (pH 7.5). The slides were stained with 20 �L of DAPI (1 �g/mL) per gel.The images were examined at 400× magnification with a Komet 5.5 Image-AnalysisSystem (Kinetic Imaging Ltd., Liverpool, UK) fitted with an Olympus BX50 fluores-cence microscope equipped with a 480–550-nm wide-band excitation filter and a590-nm barrier filter. One hundred randomly selected cells (50 cells on each oneof the two replicate slides) were analyzed per treatment. The percentage of DNAin the tail (% DNA tail) was used to measure DNA damage, given its extensive andrecommended use for comet-data analysis [34,35].

The general linear model (GLM) was used to analyze significant differencesbetween results. The Fisher LSD test was performed to compare controls andtreatments in each experiment. Results were considered statistically significant atP ≤ 0.05. All data were presented in arithmetic mean ± standard error, and with 95%confidence intervals. Finally, data analyses were performed by use of Statgraphicsplus, version 5.1 (Statistical Graphics Corporation, 2001, Rockville, USA softwarepackage).

3. Results

3.1. Drosophila wing-spot test

The results obtained in the wing-spot tests with PbCl2 andPb(NO3)2 are summarized in Table 1. Both compounds were sup-plied to 72 h-old larvae (third instar) at concentrations rangingfrom 2 to 8 mM. The results indicate that, independent of theconcentrations of PbCl2 and Pb(NO3)2 applied, there were no sig-nificant increases in the frequency of any of the three categoriesof mutant spots recorded (i.e. small single, large single, and twinspots), compared with the negative control. In this study, the nega-tive control frequencies (0.58 and 0.70) were in good agreementwith the normal background range observed in our laboratory,and are not significantly different from previous results [36]. Onthe other hand, the EMS positive control showed a clear response,which supports the validity of the negative results found for the

two lead compounds tested.

The results obtained from co-treatment experiments with thelead compounds PbCl2 or Pb(NO3)2 and gamma-radiation aresummarized in Table 2. Results show that a dose of 6 G� of

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Table 1Wing-spot test results obtained after treatment with lead chloride (PbCl2) or lead nitrate (Pb(NO3)2).

Compound,concentration (mM)

Small single mwh spots(1–2 cells) (m = 2)

Large single mwh spots(>2 cells) (m = 5)

Twin spots (m = 5) Total mwh spots (m = 2) Frequency of cloneformation 105 cells

N◦ Fr D N◦ Fr D N◦ Fr D N◦ Fr D

PbCl20 22 0.55 1 0.03 0 0.00 23 0.58 2.382 17 0.43 − 2 0.05 − 0 0.00 − 19 0.48 − 1.964 14 0.35 − 2 0.05 − 2 0.05 i 18 0.45 − 1.848 10 0.25 − 2 0.05 − 0 0.00 − 12 0.30 − 1.23

Pb(NO3)2

0 27 0.68 1 0.03 0 0.00 28 0.70 2.872 10 0.25 − 1 0.03 − 1 0.03 − 12 0.30 − 1.234 12 0.30 − 2 0.05 i 1 0.03 − 15 0.38 − 1.568 12 0.30 − 2 0.05 i 2 0.05 i 16 0.40 − 1.64

EMS1 122 3.05 + 33 0.83 + 15 0.38 + 181 4.53 + 18.6

Results from mwh/flr3 wings. Ethyl methanesulfonate (EMS) was used as positive control. N◦ , number of spots; Fr, frequency; D: statistical diagnosis according to Frei and Würgler [28]: +, positive; −, negative; i, inconclusive; m,multiplication factor; probability levels ˛ = ˇ = 0.05. Forty wings were analyzed for each concentration (20 individuals).

Table 2Wing-spot data obtained after treatment with gamma-radiation alone and co-treatment with lead chloride (PbCl2) and lead nitrate (Pb(NO3)2).

Agents, concentration(mM) and dose (G�)

Small single mwh spots(1–2 cells) (m = 2)

Large single mwh spots(>2 cells) (m = 5)

Twin spots (m = 5) Total mwh spots (m = 2) Frequency of cloneformation 105 cells

N◦ Fr D N◦ Fr D N◦ Fr D N◦ Fr D

Control 22 0.55 1 0.03 0 0.00 23 0.58 2.382 PbCl2 + 6 � 14 0.35 − 49 1.23 + 29 0.73 + 92 2.30 + 9.434 PbCl2 + 6 � 17 0.43 − 43 1.08 + 19 0.48 + 79 1.98 + 8.118 PbCl2 + 6 � 17 0.43 − 41 1.03 + 23 0.58 + 81 2.03 + 8.32

Control 27 0.68 1 0.03 0 0.00 28 0.70 2.872 Pb(NO3)2 + 6 � 24 0.60 − 47 1.78 + 19 0.48 + 90 2.25 + 9.224 Pb(NO3)2 + 6 � 27 0.68 − 67 1.68 + 32 0.80 + 126 3.15 + 12.908 Pb(NO3)2 + 6 � 23 0.58 − 56 1.40 + 16 0.40 + 95 2.38 + 9.75

6 �-irradiation 24 0.60 − 45 1.13 + 16 0.40 + 85 2.13 + 8.73

Results from mwh/flr3 wings. N◦ , number of spots; Fr, frequency; D: statistical diagnosis according to Frei and Würgler [28]: +, positive; −, negative; i, inconclusive; m, multiplication factor; probability levels ˛ = ˇ = 0.05. Fortywings were analyzed for each concentration (20 individuals).

38 E.R. Carmona et al. / Mutation Re

Table 3Comet-assay results obtained after treatment of Drosophila haemocytes with leadchloride (PbCl2) or lead nitrate (Pb(NO3)2).

Compound, concentration (mM) % DNA tail a 95% CI of % DNA tail b

PbCl20 15.34 ± 0.80 14.20–16.482 14.12 ± 0.77 12.98–15.264 14.36 ± 0.84 13.22–15.508 14.65 ± 0.86 13.50–15.79EMS4 26.85 ± 0.84** 25.71–27.99Pb(NO3)2

0 15.07 ± 0.67 13.29–16.862 18.42 ± 0.79** 16.64–20.214 17.64 ± 0.86* 15.85–19.428 21.68 ± 1.16** 19.90–23.46EMS4 26.52 ± 0.98** 24.74–28.31

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a Mean ± standard error from three experiments (300 cells).b 95% confidence intervals of % DNA tail.** P < 0.01 versus control.

amma-radiation induced a clearly positive response, increasinghe overall frequency of total mutant spots. The combined treat-

ents of gamma-radiation and the three different concentrationsf PbCl2 and Pb(NO3)2, did not show a significant variation in therequency of mutant spots compared with the frequency inducedy gamma-radiation alone. Thus, these two lead-compounds do nothow a synergistic effect with gamma-radiation.

.2. Comet-assay with haemocytes

The results obtained in the comet-assay experiments to testhe genotoxicity of PbCl2 and Pb(NO3)2 on larval haemocytesf D. melanogaster are shown in Table 3. The compounds weredministered by feeding during 24 ± 2 h to larvae, 72 ± 4 h old,n concentrations ranging from 2 to 8 mM. Afterwards theaemolymph was extracted from the larvae, and the haemocytesere isolated for the comet assay.

The results indicate that none of the different treatments carriedut with the three concentrations of PbCl2 (2, 4, and 8 mM) induced

significant increase of DNA damage expressed in % of DNA tail onrosophila haemocytes, compared with the negative control. On

he other hand, the positive control with 4 mM of EMS showed aignificant increase of DNA damage, which supports the validity ofhe negative results observed in this study.

In contrast to the results found with PbCl2, the treatments per-ormed with the selected concentrations of Pb(NO3)2 (2, 4 and

mM) induced a significant increase of DNA damage in haemocytest all three concentrations tested, this increase showing a directose–response relationship. The positive values of DNA damagebtained with 4 mM of EMS support the validity of both the resultsbserved and the protocol used.

. Discussion

In the present study we used two well-known in vivo assayso measure the genotoxicity of two inorganic lead-compoundsn somatic cells of Drosophila. On the one hand, the wing-spotssay can detect a wide range of mutational events, as wells mitotic recombination, a relevant process for genotoxicitycreening because aberrant recombination activity is commonlyssociated with carcinogenesis [37]. In addition, given its versatil-ty it also has been applied to detect anti-genotoxic properties and

ther modulating effects of various chemicals and complex mix-ures [38,39]. Thus, the wing-spot test has demonstrated to be anxcellent tool for genotoxicity modulation studies of heavy metalsuch as selenium, arsenic and cadmium in Drosophila [40–42]. On

search 724 (2011) 35– 40

the other hand, the comet assay is one of the most widely employedtechniques for in vivo genotoxicity screening, because it can beapplied to any type of cell, it can detect low levels of DNA damageand different types of damage and only a small number of cells isrequired to carry out an experiment [43]. Thus, this test system hasbeen used in diverse model organisms, including D. melanogaster[44].

Prior to the genotoxicity experiments, we carried out severaltoxicity tests to select the doses to be used for the treatments withPbCl2 and Pb(NO3)2. Initially, the doses administered ranged from2 to 10 mM and, within this concentration range, an elevated toxic-ity was observed at 10 mM, reflected in both a reduced percentageof larvae developing into adults, and a significant delay in the timerequired for the larvae to develop into the adult stage (data notreported here). This suggests that both lead-compounds could dis-turb cell-division mechanisms and other cellular functions, as wepreviously found with mercury [36]. It should be noted that neitherPbCl2 nor Pb(NO3)2 showed differences in larval toxicity. As for bothcompounds a suitable larval viability (>50%) was obtained at 8 mM,the range of doses selected for genotoxicity testing was from 2 to8 mM. The criterion to choose these concentrations is based on tworeasons: first, the reduction in the percentage of developing treatedlarvae is a clear indication that the compounds affected the larvaeand, in addition, the number of emerging adults is high enough toscore the genotoxic effects.

The results obtained in the present study from in vivo exper-iments to assess the genotoxicity of PbCl2 and Pb(NO3)2 in theDrosophila wing-spot test show a negative response for all dosesapplied. These results could be partially explained by the weakmutagenic effects of lead, the chemical form evaluated, and the testsystem employed for measuring DNA damage. Nevertheless, giventhe low toxicity observed, lead compounds may not be showinggenotoxic effects in the wing-spot assay.

The genotoxic potential of lead has been previously studiedin a wide range of assay systems. The review of the literatureshows that lead compounds are weak mutagens [9]. However,in some recent studies positive results have been obtained withrespect to induction of chromosomal damage, such as clasto-genesis and aneuploidy in cultured mammalian cells [8,12]. Inaddition, micronuclei and sister-chromatid exchange have alsobeen reported in workers exposed to lead, suggesting an even-tual genotoxic risk in humans occupationally exposed to severallead compounds [14–16,18]. Therefore, the hypothesis that leadcan affect the integrity of chromosomes, leading to clastogenicityand aneuploidy, could be considered as a relevant mechanism forthe genotoxicity of lead. Nevertheless, if we accept the previouspremises, the negative results reported here are difficult to explain,since the wing-spot assay can detect aneuploidy [21] and, if thisoccurred, we would expect a significant induction of single spotscaused by non-disjunction.

The comparison with the available data about the genotoxic-ity of lead in Drosophila shows that our results are not so differentfrom those previously reported by other authors. For example, nei-ther organic nor inorganic compounds of lead induced aneugeniceffects in germ cells of Drosophila [45]. On the other hand, PbCl2and Pb(NO3)2 were negative in the somatic eye-colour test sys-tem [46], whereas in studies carried out in the wing-spot test, leadnitrate showed a weak positive response [47]. Hence, it seems thatthe genotoxic effects of lead compounds could be considered weakin D. melanogaster.

In the interaction studies carried out with lead and gamma-radiation, the two lead compounds evaluated did not show any

increase of the genotoxic effects induced by gamma-radiationalone, suggesting the absence of a co-mutagenic effect of lead withgamma-radiation. Even though there are no studies on the possiblegenotoxic modulation of lead in Drosophila, there are in vivo studies

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n humans assessing the effects of smoking and occupational leadxposure, where this metal does not increase the SCE frequencyn the lymphocytes of exposed workers [48]. We conclude thatlthough the single-strand breaks following irradiation were notffected by the concentration of lead in blood, the metal seemso sensitize the cells to damage induced by other genotoxicants.

oreover, Restrepo et al. [49] observed similar results, reportingo significant differences between single-strand breaks induced byombined treatment with lead and X-rays. This seems to indicatehat in in vivo studies lead does not show synergistic effects withther genotoxic agents, as opposed to the in vitro studies in mam-alian cells, where lead compounds can induce an enhancement

f genotoxicity in combination with a variety of DNA-damaginggents, suggesting an interaction with DNA-repair processes [7].ossible explanations for this difference are difficult to establishiven the scarce information about the in vivo modulating effectsf lead. However, it is important to note that the different mech-nisms associated with DNA repair can be activated according tohe specific damage induced. Hence, it would not be surprising tond synergistic effects against the action of certain genotoxins. Thisould provide information about the possible DNA-repair pathwaysctivated by lead compounds.

Currently, only few studies have evaluated the genotoxic effectsf lead by use of the alkaline comet assay. However, the availablenformation indicates that this method is suitable for detecting DNAamage induced by lead. Thus, the comet assay seems to have aigh sensitivity to measure DNA damage in blood cells of workersxposed to lead [17,50,51]. Moreover, this test system is capable ofetecting the in vivo genotoxic effects caused by Pb(NO3)2 in wholelood of mice [52], and in root cells of both lupin [53] and tobaccolants [54]. Given these results, the in vivo comet assay seems tohow a high sensitivity for the detection of DNA damage inducedy lead exposure.

The positive results of Pb(NO3)2 obtained with the in vivo cometssay in haemocytes of D. melanogaster are in agreement with theesults reported by other authors and suggest a certain genotoxicisk associated with some chemical forms of lead. At the same time,hese results contrast with those observed in the wing-spot assay,hich showed a negative response. But they support the idea that

he in vivo comet assay could be a more sensitive test system thanhe wing-spot assay.

onflict of interests statement

The authors declare to have no conflict of interests.

cknowledgements

E.R. Carmona thanks the valuable technical assistance from.A. García-Quispes, and the support from UAB-PIF Grant N◦ 409-

2-2/07. This investigation has been supported in part by theeneralitat de Catalunya (CIRIT, 2009SGR-725).

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