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Mutation Research 742 (2012) 31– 36

Contents lists available at SciVerse ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

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he use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detectomplex genotoxic mixtures in the coastal lagoon ecosystem

asquale Mosessoa,∗, Dario Angeletti a, Gaetano Pepea, Carlo Prettib, Giuseppe Nascetti a,affaela Bellacimaa, Roberta Cimmarutaa, Awadhesh N. Jhac

Dipartimento di Scienze Ecologiche e Biologiche, Università degli Studi della Tuscia, Largo dell’Università s.n.c., 01100 Viterbo, ItalyDipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti, Università degli Studi di Pisa, Viale delle Piagge, 2, 56124 Pisa, ItalySchool of Biomedical & Biological Sciences, University of Plymouth, Plymouth, PL48AA, UK

r t i c l e i n f o

rticle history:eceived 20 May 2011eceived in revised form 14 October 2011ccepted 17 November 2011vailable online 24 December 2011

eywords:editerranean Killifish

phanius fasciatusentinel organismcotoxicologyenetic toxicologyomet assaynvironmental pollutants

a b s t r a c t

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprin-odont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability ofthis fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxicmixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectivenessof the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) inducedby (a) in vivo exposure of individual fish to X-rays (dose, 3 Gy) and (b) following in vitro challenge oferythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strandbreaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circu-lating fish blood contained actively proliferating cells that could influence the extent of DNA damagein control (untreated) fish, we measured the number of “comets” positive for 5-bromo-2′-deoxyuridine(BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. withX-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) valuescompared with the relevant untreated controls, indicating the effectiveness of the comet assay in theerythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibodylabelling of erythrocytes indicated a very low percentage (5%) of “comets” positive for BrdU. Followingoptimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetellolagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically sig-

. Introduction

The lagoon and salt marshes listed in Annex I of the Euro-ean Council “Habitat” Directive 92/43/EEC [1] are ecologicallyunctional areas, in particular for migratory birds that inhabit

∗ Corresponding author at: Dipartimento di Scienze Ecologiche e Biologiche, Uni-ersità degli Studi della Tuscia, Largo dell’Università s.n.c., 01100 Viterbo, Italy.el.: +39 0761 357257; fax: +39 0761 357257.

E-mail address: mosesso@unitus.it (P. Mosesso).

383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.mrgentox.2011.11.018

© 2011 Elsevier B.V. All rights reserved.

especially in densely populated countries such as Italy, where urbansettlements can be very nearby. These impacts consist either inan excess of nutrients that promote eutrophication, resulting inthe deterioration of the aquatic environment [2,3,12], or the pres-ence of contaminants of urban, industrial and agricultural origin,a large proportion of which are potentially genotoxic and carcino-genic compounds [4,8]. Whilst exposure of aquatic organisms togenotoxic compounds could pose risk to human health via the foodchain, an ecological risk linked to the induction of transmittablemutations leading to loss of biodiversity, population reduction and

In recent years, the impact of chemical and physical con-taminants on the integrity and functionality of DNA has beeninvestigated in many organisms under laboratory and field

3 on Research 742 (2012) 31– 36

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Table 1Physico-chemical conditions determined in the four basins of the Natural Reserveof “Saline di Tarquinia” during collection of Aphanius fasciatus.

Basins

1 2 3 4

Salinity (‰) 36.0–46.0 35.0–50.0 38.0–88.0 42.0–140.0

2 P. Mosesso et al. / Mutati

onditions, by use of different biomarkers of exposure and effect,ncluding induction of DNA adducts, DNA strand-breaks, micronu-lei and chromosomal aberrations [8,9]. The recent regulatoryevelopments, for example, the Water Framework Directive (WFD)f the European Union (EU), emphasises the need to maintaincological quality of the hydrosphere, with particular emphasisn detection and impact of those contaminants that are carcino-enic, mutagenic and reprotoxic [10]. In this context, application ofiomarkers in native species is very useful to estimate early signsf damage, which allows the development of strategies to preventetrimental ecological consequences [8–11].

The single-cell gel electrophoresis (SCGE) assay (i.e. cometssay) has been successfully applied to the nucleated red bloodells of many fish species exposed to different genotoxic com-ounds and environmental stressors [8,9,12]. There are, however,nly few reports in the literature where this assay has been thor-ughly validated in native fish species and subsequently appliedor environmental monitoring purposes. The alkaline version ofhe comet assay (pH ≥ 13) [9,12] is usually employed becauset is a very sensitive method that detects a broad spectrum ofNA lesions, including DNA single- and double-strand breaks,lkali-labile sites and excision-repair events caused by simple andulky DNA adducts [13–21]. Additional advantages of this method

nclude the fact that in principle any type of cell at very smalluantities (<10,000) can be used. Furthermore, since the damage isbserved in interphase nuclei, factors such as the size and numberf chromosomes or the absence of cell proliferation – which oftenepresent limiting factors for determining genotoxic effects (e.g.CE, micronuclei, chromosome aberrations) in aquatic organisms –re completely bypassed by this method [8,9].

In view of the above information, in this study we evaluatedhe genotoxic effects of physical and chemical agents with differ-nt mechanisms of action (e.g. X-rays, restriction endonucleases)n the erythrocytes of cyprinodont fish, Aphanius fasciatus. We usedhe alkaline comet assay to assess its suitability in this sentinelrganism to detect complex genotoxic mixtures in coastal lagooncosystems. The Mediterranean Killifish, A. fasciatus is a cyprin-dont, living in coastal brackish waters, lagoons and salt marshes.t is distributed over the central and eastern Mediterranean coastalones [22,23]. This species is well suited to be used as “sentinelrganism” in these habitats for many reasons, including (a) its wideistribution in virtually every Mediterranean lagoon, often with a

arge population size; (b) it occupies these habitats during theirntire life cycle, from where it moves only in case of washes or sed-mentary events [24]; (c) it is present in habitats or micro-habitatsnder extreme conditions, often witnessed by these ecosystems.

. Materials and methods

.1. Study areas

Field sampling was conducted at two different sites: (1) saline ponds from theatural Reserve “Saline di Tarquinia” (Tarquinia, Latium, Italy) considered as a “ref-rence” site; (2) Orbetello lagoon (Tuscany, Italy), which lies in the heart of a denselyopulated area and is considered as significantly polluted site.

The Natural Reserve “Saline di Tarquinia” (Fig. 1A), located on the Tyrrhenianoast of central Italy was originally the site of a salt factory in which salt productionas terminated in 1997 [24,25]. The basins are in connection only with the sea andractically isolated from the inland by a canal that runs all around the area. Theifferent basins are characterised by a high variability of physico-chemical parame-ers and increasingly higher salt concentrations going from north to south (Table 1).ince 1980, the whole area is under protection, closed for the public and without anyecognised releases into the water resources of chemicals of domestic, agricultural orndustrial origin. In contrast, the Orbetello lagoon (Fig. 1B), located on the Tyrrhenian

O2 (mg/l) 3.4–11.5 4.5–9.9 4.0–9.8 2.2–8.5Temperature (◦C) 6.7–29.0 5.7–28.5 5.1–28.8 5.6–29.9

metals and metalloids [26,27], as well as moderate levels of polycyclic aromatichydrocarbons (PAHs) [27,28] and persistent organic pollutants (POPs) [29,30].

2.2. Collection and maintenance of the test organism

A total of 74 A. fasciatus fish (69 from the Natural Reserve “Saline di Tarquinia”and 5 from the Orbetello lagoon) were caught with appropriate nets. The lengthof these fish ranged between 21 and 59 mm (mean length, 23 mm). After capture,specimens were maintained in water collected from the sampling sites, which waskept oxygenated. Collected fish were transported to the aquarium facilities within2–4 h and maintained under standard conditions until used for the experiments.

2.3. Blood collection and setting of optimal conditions for the comet assay

The fish were sacrificed by means of a blow to the head. Approximately 10 �l ofpericardial blood was collected with a micropipette and diluted 1:1 (v/v) with PBS.Prior to the assay, the viability of the erythrocytes was assessed with trypan blue[12]. In all the experiments performed, cell viability was always above 80%, whichwas considered to be acceptable to perform the comet assay [32].

2.4. Single-cell gel electrophoresis (SCGE) or comet assay

The alkaline comet assay was performed according to the method of Mustafaet al. [12], with minor modifications. Briefly, 1 �l of each diluted blood sample wasmixed with 80 �l of 0.75% (w/v) low melting-point (LMP) agarose. Each mixture wasthen layered on microscope slides pre-coated with 1% (w/v) normal melting-point(NMP) agarose and immediately covered with a cover glass and kept for 10 minin a refrigerator to solidify. After gentle removal of the cover glass, a final layer of80 �l of 0.75% (w/v) LMP agarose was added to the slides, which were again cov-ered with a cover glass. After solidification at 4 ◦C in a refrigerator, the cover glasseswere removed and the slides were ready to be processed for the assay. Optimal con-ditions for lysis of cells and gel electrophoresis were found after several trials andestablished as follows: for cell lysis, slides were immersed in freshly prepared lysingsolution (2.5 M NaCl, 100 mM Na2-EDTA, 10 mM Tris, pH 10, with 10% DMSO and1% Triton X-100), overnight at 4 ◦C. Upon completion of lysis, slides were placed ina horizontal gel electrophoresis tank with freshly prepared alkaline electrophore-sis buffer (300 mM NaOH and 1 mM Na2-EDTA, pH ≥ 13) and left in the solution for10 min at 4 ◦C to allow DNA unwinding expression of the alkali-labile sites. Elec-trophoresis was carried out for 10 min at 4 ◦C, 30 V (1 V/cm), and 300 mA, with aBio-Rad power supply.

Following electrophoresis, the slides were neutralised in 0.4 M Tris buffer atpH 7.5 and air-dried at room temperature. Immediately before scoring, slides werestained with 12 �g/ml ethidium bromide (Boehringer Mannheim, Germany) andexamined at 400× magnification with an automatic image analyzer (Comet Assay III;Perceptive Instruments, UK) connected to a fluorescence microscope (Leitz Ergolux).To evaluate the amount of DNA damage, computer software generated differentparameters including Olive tail moment, tail length and tail DNA (%). These threeparameters showed similar levels of DNA damage but the results for tail DNA (%)was considered for presentation as a reliable parameter [36]. For each individualtreatment, a total of 100 randomly selected cells (50 cells from each of two replicateslides) were analysed from each individual.

2.5. Experiments performed under laboratory conditions with reference agents

The effectiveness of the comet assay with erythrocytes of A. fasciatus wasassessed in separate experiments by measuring the tail-DNA (%) values followingexposure to X-rays or restriction endonucleases, Eco-R1 and Fok-1 (Roche Diagnos-tics, GmbH). The proliferative status of fish blood following labelling of DNA withthe nucleotide analogue 5-bromo-2′-deoxyuridine (BrdU) and immunohistochem-ical detection, was performed to ensure that the tail-DNA (%) values reflected realgenotoxic damage and not transient DNA breaks at replication forks.

2.5.1. Evaluation of proliferation of erythrocytes by means of labelling with BrdU

water – collected from the control site or the reference site – containing 4.5 mg/mlBrdU (Sigma, St. Louis, MO, USA). At the end of the treatment, fish were sacrificedand approximately 10 �l pericardial blood were collected as described in Section 2.3,and processed for the comet assay. After the electrophoresis, the slides were washed

P. Mosesso et al. / Mutation Research 742 (2012) 31– 36 33

F the Natural Reserve “Saline di Tarquinia” located on the Tyrrhenian coast of central Italy,c Tyrrhenian coast at approximately 40 km north-east of the Natural Reserve “Saline diT

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Fig. 2. Immunohistochemical detection of cells (comets and nuclei) positive forBrdU, using anti-BrdU antibodies conjugated with FITC (green signal); counterstainwith ethidium bromide (red signal). (A) BrdU-positive comet (left side) and threeBrdU-negative nuclei (red signal) slightly displaced on the right side compared withthe comet. The presence of BrdU generates a green signal while unsubstituted DNAcounterstained by ethidium bromide emits a red fluorescence signal. Merging ofimages, green when positive for BrdU and red when negative generates a yellow sig-nal. This indicates that the cell is in the “S” phase of the cell cycle. In this case, part ofDNA fragments detected in the tail, could be attributed to DNA single-strand breaksgenerated at replication forks. (B) BrdU-negative comet indicated by the presence

ig. 1. Study areas for field sampling of Aphanius fasciatus: (A) Marine ponds from

onsidered as “reference” unpolluted site; (B) the Orbetello lagoon located on thearquinia”: a site with high anthropogenic impact.

n PBS three times over a period of 10 min and 50 �l of a solution containing approxi-ately 6 �g/ml anti-BrdU antibody conjugated with fluorescein isothiocyanate (FITC)

Phoenix Flow Systems, San Diego, CA, USA) diluted 1:20 in PBS with 0.1% bovineerum albumin (BSA), was layered over the cells and covered with a cover glass.he slides were incubated in a humidified chamber in the dark for 1 h. At the endf the incubation, the slides were washed with PBS three times over a period of0 min, dehydrated, dried and mounted in Vectashield (Vector Laboratories Ltd.,K) containing 12.0 �g/ml propidium iodide (PI) and an anti-fading agent. Fluores-ence microscopy was performed with a Leitz Ergolux microscope equipped withingle band-pass filters for the detection of FITC and EB signals. Images were cap-ured with a CCD camera mounted onto the microscope. An Isis digital imagingystem (MetaSystem GmbH, Altlussheim, Germany) was used for analysis. Afteruorescence in situ hybridisation (FISH) analysis with anti-BrdU antibodies conju-ated with FITC, actively and non-actively dividing cells appeared and were analysednder the microscope (Fig. 2).

.5.2. Irradiation of fish with X-raysIrradiation was carried out at room temperature with a Gilardoni X-ray gen-

rator (Como, Italy) operating at 250 kV and 6 mA at a dose-rate of 0.75 Gy/min.ish (n = 5) received a dose of 3 Gy in a glass container (base diameter 10 cm) con-aining a sufficient quantity of water. The delivered dose of X-rays was selectedased on previous studies with external X-ray and gamma-ray sources to induceytogenetic damage and mutations in different fish species. These studies havesed a dose between 325 R of X-rays [37] and 4.75 Gy of gamma-rays [38,39]. Theelected dose in the present study also aimed to ensure significant DNA damage inhe absence of marked toxicity. The experiment also included a concurrent nega-ive control (n = 5) maintained in an identical glass container as the one used for the-ray exposure. Immediately after irradiation, pericardial blood samples from both

reated and untreated animals were collected and processed for the alkaline cometssay as described in Sections 2.3 and 2.4.

.5.3. Treatment with restriction endonucleaseAn efficient method to selectively induce DNA double-strand breaks (DSBs) is

ased on the use of DNA restriction enzymes in mammalian cells [40,41]. In thistudy, we used the DNA restriction endonucleases (RE) Eco-RI and Fok-I (Roche Diag-ostic GmbH, Germany). Eco-RI recognises the sequence G/AATTC and generates

ragments with 5′-cohesive termini, while Fok-1 recognises the sequence GGATGN) 9/13 and generates fragments with 5′ ,3′-blunt termini. Following lysis of cells

n a humidified chamber at 37 ◦C in the dark for 1 h. Untreated controls were also

of an EB signal (red) and the absence of an FITC signal (green).

included and were treated with 100 �l of enzyme buffer only (100 mM Tris–HCl,pH 7.5, 50 mM NaCl, 10 mM MgCl2). The reaction was stopped by the addition

34 P. Mosesso et al. / Mutation Research 742 (2012) 31– 36

Table 2Mean tail DNA (%) values obtained under different protocol conditions. Standard deviations are shown in parentheses.

Parameters Time (min) of DNA unwinding (u) and electrophoresis (e)

+ 20(e) 20(u) + 10(e) 20(u) + 20(e)

(±3.05) 21.24 (±6.07) 23.11 (±6.18)

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Table 3Mean tail DNA (%) values obtained for the fish collected in ponds 1, 2, 3, and 4 of theNatural Reserve of “Saline di Tarquinia”. Standard deviations are in parentheses.

Basin (no. of individuals analysed)

1 (5) 2 (5) 3 (10) 4 (5)

Tail DNA (%) 8.50 (±0.72)B 10.65 (±6.23)B 9.12 (±2.13)B 11.19 (±2.26)B

Capital superscript letters (A, B) indicate comparison between mean values. Thesame letters indicate the lack of a statistically significant difference (p > 0.05).

Table 4Mean tail DNA (%) values obtained by pooling data from the 25 individual fish col-lected in ponds 1, 2, 3 and 4 of the Natural Reserve of “Saline di Tarquinia”. Thisgroup of 25 fish has been the base for our “negative” historical control. Standarddeviation (SD) and standard error (SE) are also indicated.

Mean values SD SE

Tail DNA (%) 9.72 3.20 0.64

Fig. 3. Mean values of tail DNA (%) from fish exposed to X-rays (3 Gy) and from

10(u) + 10(e) 10(u)

Tail DNA (%) 8.50 (±0.72) 10.33

ach treatment) were used for RE treatments. Five additional fish were used asntreated controls.

.6. Statistical analyses

Tail DNA (%) in control and exposed fishes were compared with Student’s t-test.he level of statistical significance was set at p < 0.05.

. Results and discussion

In this study, we initially evaluated under standard laboratoryonditions the genotoxic effects of reference agents with differ-nt mechanisms of action (i.e. X-rays and RE, Fok-I and Eco-RI)n the erythrocytes of the Killifish, A. fasciatus by means of theomet assay, in order to assess its suitability as a sentinel organ-sm to detect genotoxic compounds in coastal lagoon ecosystems.ue to variation in the response of different cell types from differ-nt species to comet-assay protocols [31–34], different trials wereerformed, with at least five individual fish for each trial. On theasis of the results obtained (Table 2), the following conditionsere selected to perform the assay in further studies: cell lysis was

onducted in freshly prepared lysing solution (2.5 M NaCl, 100 mMa2-EDTA, 10 mM Tris, pH 10, with 10% DMSO and 1% Triton X-100)vernight at 4 ◦C. Alkaline unwinding of DNA and expression of thelkali-labile sites was achieved in freshly prepared alkaline elec-rophoresis buffer (300 mM NaOH and 1 mM Na2-EDTA, pH ≥ 13)or 10 min at 4 ◦C. Electrophoresis was carried out at 4 ◦C for 10 min,0 V (1 V/cm), and 300 mA.

Although the values obtained for DNA damage (i.e. tail DNA %)ere in line with those found in previous studies with erythro-

ytes of fish [12,42], they were considered to be higher than valuessually reported for different mammalian cells exposed to genotox-

cants under in vitro and in vivo conditions. We therefore evaluatedhe proliferation potential of erythrocytes of A. fasciatus by labellingf DNA with the nucleotide analogue 5-bromo-2′-deoxyuridineBrdU). At least 200 cells for each individual were scored for theresence of actively dividing blood cells (BrdU-positive cells). Theumber of BrdU-positive cells did not exceed 5% for each fish (dataot shown), indicating that cells in S-phase did not contribute sig-ificantly to the relatively higher values of tail DNA (%) in therythrocytes of A. fasciatus, compared with values usually found inammalian cells. This would rather appear to be a specific feature

f the species suggesting that circulating erythrocytes in fish do notndergo mitotic division. To the best of our knowledge, use of anti-rdU staining in fish blood cells has not been applied before. The

ow number of mitotically dividing or active cells in the circulatingrythrocytes of fish reflects its insensitivity towards induction oficronuclei, a cell cycle-dependent phenomenon [9]. In this con-

ext, induction of micronuclei following intra-peritoneal injectionsf reference genotoxins (mitomycin C and cyclophosphamide) inathead minnow (Pimephales promelas), suggested that mitomycin

significantly induced micronuclei in erythrocytes from the spleenut not from the peripheral blood, suggesting that active divisionf erythrocytes does not take place in the circulating erythrocytes35]. This observation is in line with our study where anti-BrdU

Once the conditions for the comet-assay protocol were opti-ised, the influence of physico-chemical conditions (i.e. oxygen,

untreated controls. A total of ten fish (five fish for each treatment group) were used.Standard deviation values are also shown. ***Significantly different at p < 0.001.

salinity and temperature) in terms of spontaneous levels of DNAbreakage, were assessed in 25 individual fish collected over aperiod of three months (March–June 2010) from the reference site,Natural Reserve of “Saline di Tarquinia”, basins 1, 2, 3, 4 charac-terised by increasingly higher salinity and temperature movingfrom north to south (Fig. 1A). The results (Table 3) show that differ-ent physico-chemical conditions in the different ponds selected, didnot significantly influence the results. Pooled data from the 25 fishanalysed served in addition as “historical control” for the “referencesite” Natural Reserve of “Saline di Tarquinia” (Table 4). Values of tailDNA (%) for samples treated with X-rays (3 Gy) and with the restric-tion endonucleases Fok-I and Eco-RI are presented in Figs. 3 and 4,respectively.

Statistically significant increases for tail DNA (%) compared withthe relevant concurrent untreated controls were observed in alltreatment groups, indicating the effectiveness of the assay in theerythrocytes of A. fasciatus to detect different types of DNA lesion(i.e. SSB, DSB, base damage mainly induced by X-rays; blunt andcohesive DSB induced by restriction endonucleases Fok-I and Eco-RI, respectively). Whilst the use of restriction endonucleases (RE) to

P. Mosesso et al. / Mutation Res

Fig. 4. Mean values of tail DNA (%) from fish erythrocytes treated under in vitro con-ditions with: (a) restriction endonuclease Fok-1 (1200 U/ml), which recognises thesequence GGATG(N) 9/13 and generates fragments with 5′-blunt termini; (b) restric-tion endonuclease Eco-R1 (800 U/ml), which recognises the sequence G/AATTC andgenerates fragments with 5′-cohesive ends; (C) untreated controls. A total of tenfish (five for each treatment group) were used. Standard deviation values are alsoshown. ***Significantly different at p < 0.001.

Table 5Major contaminants found in the Orbetello lagoon and possible sources.

Pollution source Contaminants References

Orbetello urban area PAHs; trace elements [26–28]Former fertiliser factory PCDD/Fs; polychlorinated naphthalenes [29]Albegna river Organochlorine (OC) pesticides [29]Sewage-treatment plant Non-ionic surfactants; PCBs [29,30]

PAHs: polycyclic aromatic hydrocarbons; PCDD/Fs: polychlorinated dibenzodioxinsand dibenzofurans; PCBs: polychlorinated biphenyls.

Fig. 5. Mean values of tail DNA (%) in fish (n = 10; five per study site) collected fromthe Orbetello lagoon (Tuscany, Italy) considered to be a significantly polluted site,and in fish collected from Natural Reserve “Saline di Tarquinia” considered as ara

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eference site. Standard deviation values are also shown. ***Significantly differentt p < 0.001.

nowledge this is the first time that RE have been used effectively tonduce DNA damage in fish cells under the experimental conditions.

Results obtained under standard laboratory conditionsrompted us to apply this assay in the field assessing theegree of DNA breakage from five animals caught in the Orbetello

agoon (Tuscany, Italy) considered to be a significantly pollutedite [26–30]. In particular, the sampling sites used in this study areocated in an area influenced by different pollution sources, suchs a sewage treatment plant, former fertiliser production plantsnd the mouth of the pesticide-contaminated Albegna river [29].he main pollution sources and the major contaminants in therbetello lagoon are shown in Table 5. Fish (n = 5) collected from

he Natural Reserve “Saline di Tarquinia” considered as referenceite served as negative control. The results presented in Fig. 5how the mean values of tail DNA (%). Statistically significant

[

earch 742 (2012) 31– 36 35

In conclusion, the comet assay confirms to be an effective short-term assay for in vivo monitoring to determine potential genotoxiceffects of contaminants in aquatic species. Furthermore, standard-isation of the alkaline comet assay in the erythrocytes of thecyprinodont fish A. fasciatus, using reference genotoxic agents withdifferent modes of action, and ruling out the confounding effectsof any dividing cells by applying BrdU labelling, the study indicatesthis species to be a promising “sentinel organism” to detect geno-toxic pollutants in coastal lagoon ecosystems. Given that cometassay is helpful in detecting only generic genotoxic effects, thecomplexity of genotoxic responses, particularly of complex mix-tures would require adoption and applications of complementaryassays. Further work is therefore required to establish this speciesas model organism for environmental monitoring.

Acknowledgements

We wish to thank Ms. Gaia Vitale for her valuable help in datacollection.

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