Aquatic Toxicology 89 (2008) 188–196

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Aquatic Toxicology

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Cellular responses in the cyprinid Leuciscus cephalus from a contaminatedfreshwater ecosystem

G. Frenzilli a,∗, A. Fallenia, V. Scarcelli a, I. Del Bargaa, S. Pellegrinib, G. Savarinob, V. Mariottib,M. Benedetti c, D. Fattorini c, F. Regoli c, M. Nigroa

a Dipartimento di Morfologia Umana e Biologia Applicata, sezione di Biologia e Genetica, Università di Pisa, via Volta 4, 56126 Pisa, Italyb Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università di Pisa, via Roma 67, 56126 Pisa, Italyc Istituto di Biologia e Genetica, Università Politecnica delle Marche, via Ranieri, Monte D’Ago, 60100 Ancona, Italy

a r t i c l e i n f o

Article history:Received 11 March 2008Received in revised form 23 June 2008Accepted 25 June 2008

Keywords:BiomarkersEcotoxicologyChubBioaccumulation

a b s t r a c t

The response of wild chubs (Leuciscus cephalus) to chemical pollution was assessed in a metal contam-inated river (Cecina River, Italy) through a wide battery of biomarkers which included: Comet assaydetecting DNA strand breaks; diffusion assay for apoptosis induction; micronucleus test assessing chro-mosomal alterations; ethoxyresorufin O-deethylase (EROD) activity for the induction of cytochrome P4501A; acetylcholinesterase (AChE) activity responsive to pesticide exposure; vitellogenin gene expres-sion in males revealing estrogenic effects. Bioaccumulation of mercury, chromium and polycyclic aromatichydrocarbons (PAHs) was also determined. Levels of mercury and PAHs were higher in tissues of chubssampled from the most downstream station, reflecting an anthropogenic pollution of industrial origin.Otherwise, accumulation of Cr was quite similar in fish along the entire course of Cecina River confirminga natural origin due to local geochemical features. Biomarker responses revealed a significant increase of

Freshwater environmentapoptotic cells, DNA stand breaks and micronucleus frequency in chubs from the more impacted sites. Aslight EROD induction and AChE inhibition were only seen at the most downstream station demonstratinga limited impact due to PAHs and pesticides. On the other hand, the induction of vitellogenin gene in malechubs was measured in all the sites, suggesting a diffuse estrogenic effect. This study confirmed the utilityof large batteries of biomarkers in biomonitoring studies and the suitability of wild chub as bioindicator

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. Introduction

Rivers and lakes are receptacles for industry, agriculture, urbanater treatment plant wastes containing mixtures of well-knownollutants and unidentified/unregulated toxicants, whose biologi-al effects are hardly predictable (Ohe et al., 2004). The Europeannion highlighted the importance of water pollution for both pub-

ic health and ecosystem conservation within the Water Frameirective (2000/60 European Community), which demands a goodcological status for rivers by the 2015. In this respect, a mainhallenge for ecotoxicologists is to implement monitoring strate-ies to assess the biological impact of pollutant mixtures in river

asins, and to better characterize the ecological status of aquaticcosystems (Brack et al., 2007). The use of resident or translo-ated sentinel species appears as a valuable choice, especially whenhemical analyses are integrated with a wide battery of subcellu-

∗ Corresponding author. Tel.: +39 050 2219111; fax: +39 050 2219101.E-mail address: giada@biomed.unipi.it (G. Frenzilli).

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166-445X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2008.06.016

© 2008 Elsevier B.V. All rights reserved.

ar biomarkers, to evaluate the biological impact of pollution andhe mechanisms responsible for biological alterations (Regoli et al.,004; Nigro et al., 2006). Inventory-based chemical monitoring ofquatic environments is often restricted to the identification of aertain number of pollutants which does not provide informationn adverse effects to the biota (Livingstone, 1993). In this respect,he characterization of biological responses to pollutants in sentinelpecies has become a major issue in monitoring environmen-al quality and risk assessment. Among the numerous biologicalesponses proposed in the last three decades, those based on varia-ions at the molecular and cellular levels (or biomarkers) representhe earliest signals of environmental disturbance and have beenncreasingly applied in ecotoxicological investigations (Bayne et al.,985; Depledge, 1994; Lowe et al., 1995). Since individual biomark-rs cannot reflect the impairment of organism health conditions,

he use of a wide battery of responses is always recommendedhen assessing the biological impact of chemical pollutants (Regoli

t al., 2004).Multi-biomarker studies on freshwater species are still limited

nd, among these, the chub, Leuciscus cephalus, has been reported

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s a good model for monitoring the biological disturbance in Euro-ean rivers (Devaux et al., 1998; Viganò et al., 1998; Flammariont al., 2002a,b; Winter et al., 2004). This cyprinid is a common,on-migratory species that inhabits clean and polluted rivers inestern Europe (Flammarion et al., 2000) feeding on both benthic

nd water-column organisms, characterized by low fat content andigh tolerance to organic water pollution (Mancini et al., 2005).he use of L. cephalus as bioindicator has already been reported ineld studies assessing the environmental quality of inland watersifferently impacted by anthropogenic activities (Winter et al.,005; Randàk et al., 2006; Krca et al., 2007). Several biomark-rs have also been characterized in this species in response tohemical pollutants, including levels of DNA strand breaks (SB),thoxyresorufin O-deethylase (EROD) activity, polycyclic aromaticydrocarbon (PAH) metabolites in bile, DNA adducts, erythrocyteicronucleation, inhibition of acetylcholinesterase (AChE), activi-

ies of glutathione S-transferases (GST), vitellogenin gene inductionDevaux et al., 1998; Flammarion et al., 2000; Machala et al., 2001;lammarion et al., 2002a,b; Winter et al., 2005; Randàk et al., 2006;rca et al., 2007).

The aim of the present research was the application of the multi-arker strategy with L. cephalus as sentinel species for monitoring

he basin of the River Cecina (Tuscany, Italy), selected as one ofhe 15 pilot basins within the Water Framework Directive. Thisasin includes areas of high naturalistic and historical value, but

t is also impacted by industrial activities based on rock-salt min-ng, chloro-alkali production, and geothermal sources harvesting.n extensive chemical characterization of river sediments has beenecently carried out by the regional agency for environmental pro-ection (Cecina Pilot River Basin, Final Report). Levels and naturef pollutants are mainly due to metals deriving from industrialischarges which are largely removed or mitigated at present. Mer-ury concentrations in sediments normally range between 0.05 and.2 �g/g but elevated values (up to 4.5 �g/g) still persist in some hotpots along the middle river course, including two downstreamites investigated in the present research. Besides anthropogenicources, natural processes, namely the erosion of ophiolites, very

bundant rocks in the river basin, are likely responsible for thelevated levels of Cr in sediments with values ranging between0 and 640 �g/g (Cecina Pilot River Basin, Final Report). Limitedoncentrations of organic xenobiotics in river sediments have beenbserved with values of 0.008–0.3 �g/g for PAHs and always lower

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logy 89 (2008) 188–196 189

han 0.001 �g/g for PCBs (Cecina Pilot River Basin, Final Report). Ourrevious studies on the estuary of the Cecina River with the use ofmulti-biomarker approach on caged and native mussels (Nigro etl., 2006) showed a marked deterioration of environmental quality,igh metal levels and cellular alterations which were comparableo those reported in the highly industrialized harbour of GenovaRegoli et al., 2004).

In the present investigation, specimens of L. cephalus wereollected from different sites along the Cecina River basin andnalysed for both chemical contents in tissues (Hg, Cr, andAHs) and a battery of widely validated biomarkers. Genotoxi-ity responses included the Comet assay and the micronucleusest, detecting DNA strand breaks and chromosomal fragmenta-ion and/or chromosomal loss respectively (Al-Sabti and Metcalfe,995; Bolognesi et al., 1999; Grisolia and Starling, 2001; Jha,004); apoptosis induction was also tested by the diffusion assaySingh, 2000; Nigro et al., 2002; Frenzilli et al., 2004). As spe-ific biomarkers, the inhibition of AChE and the 7-ethoxyresorufin-deethylase activity, were selected for assessing potential expo-ure to organophosphorous-carbamate pesticides and PAH and/orCB-type pollutants respectively (Livingstone, 1993; Rickwood andalloway, 2004). Finally, the gene induction of vitellogenin in the

iver of male specimens was investigated as a typical responseoward exposure to xenoestrogens (Canapa et al., 2007).

The integration of chemical tissue levels, DNA and chromosomalamages, vitellogenin gene expression in males, EROD and acetyl-holinesterase activities were expected to reveal the presence andature of biological disturbance in the River Cecina basin and toalidate the use of a multi-biomarker approach in L. cephalus.

. Materials and methods

.1. Study area

Four sampling sites were selected in the River Cecina basin,ith different levels of chemical impact (Fig. 1). Two relativelyncontaminated locations were used as controls, Pavone (a trib-

tary of the River Cecina) and Berignone (on the upper course ofhe River Cecina). In these sites macro-invertebrate biodiversityas high (class 1 of the Extended Biotic Index) and Hg concen-

rations in sediments were lower than 0.1 mg/kg (Cecina Pilot Riverasin, Final Report). The two impacted sites were Saline di Volterra,

e Cecina River Basin.

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ownstream of the confluence of a heavily contaminated tributaryollecting industrial effluents (Hg sediment concentrations in theange of 1–5 �g/g) and Ponteginori located approximately 7 km fur-her downstream (Hg sediment concentrations 0.5–1 �g/g). Onlyow contamination by organic pollutants was measured in riverediments, PCBs being always lower than 0.001 �g/g in all theested sites, and PAHs ranging from 0.01 (Pavone site) to 0.78 �g/gSaline di Volterra site) (Cecina Pilot River Basin, Final Report).

.2. Sampling

Adult chubs (L. cephalus) were collected by hook and line inarch–April 2006. In order to minimise the interference of biomet-

ic variables on biomarker responses, specimens ranging between80 and 223 mm were selected. Age of fish was estimated to bebout 3 year according to Turin et al. (1994). After fishing, bloodas sampled from the caudal vein, and organisms were then sac-

ificed, weighed, measured and dissected. In the sampling period,hubs were mature and showed well-developed gonads, allowingn easy sex determination by gross morphology. Pieces of muscleissue, livers and brains were dissected, frozen in liquid nitrogen,nd maintained at −80 ◦C before metal determination and enzy-atic assays. The cephalic kidney was removed and smeared on

lass slides for micronucleus test; blood samples were kept on icend immediately processed for genotoxicity.

.3. Chemical analyses

Since an extensive chemical characterization of the River Cecinaas not the main objective of the present research only mercury,

hromium, and PAHs were selected for assessing the bioavailabil-ty in feral chubs. The two metals have been indicated as the

ain chemicals in sediments of the river basin, deriving fromoth anthropogenic and natural sources. Although PAHs were noteported in elevated concentrations, they still represent a poten-ially important class of chemicals released from anthropogenicctivities.

For trace metal analyses, muscle and liver tissues were dried at0 ◦C to constant weight, and digested with nitric acid and hydro-en peroxide (7:1) in a microwave digestor system (Mars II, CEMorporation). Quality assurance and quality control were tested byrocessing blank samples and reference standard materials (mus-el Tissue Standard Reference Material SRM 2977, National Institutef Standard and Technology). Metals were analysed by conven-ional atomic absorption spectrophotometry (AAS) with flamelesstomization and Zeeman effect for Cr, and by the cold vapourseneration technique for Hg (Regoli et al., 2004; Fattorini et al.,n press). The concentrations obtained for the standard reference

aterial were always within the 95% confidence interval of certifiedalues.

For PAHs, about 1 g wet liver tissues were extracted in 5 mL.5 M potassium hydroxide in methanol using a microwave sys-em at 55 ◦C for 15 min (Mars CEM, CEM Corporation, MatthewsC) according to Regoli et al. (2005). After centrifugation at000 × g for 5 min, the methanolic solutions were concentratedn a SpeedVac (RC1009; Jouan, Nantes, France) and purified witholid phase extraction (Octadecyl C18, 500 mg × 6 mL, Bakerbond;allinckrodt Baker, Phillipsburg, NJ, USA). A final volume ofmL was recovered with acetonitrile, and HPLC analyses werearried out with water–acetonitrile gradient and fluorimetric

etection. PAHs were identified by the retention time of appro-riate pure standard solutions (EPA 610 Polynuclear Aromaticydrocarbons Mix), and classified as low molecular weight

LMW: naphthalene, acenaphthene, fluorene, phenanthrene,nthracene) and high molecular weight (HMW: fluoranthene,

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logy 89 (2008) 188–196

yrene, benzo[a]antrhacene, chrysene, benzo[b]fluoranthene,enzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene,enzo[g,h,i]perylene). Quality assurance and quality control wereested by processing blank and references samples (mussel tissuesRM 2977, NIST), and concentrations obtained for the SRM werelways within the 95% confidence interval of certified value. Theater content in tissues was determined and concentrations of

AHs expressed as ng/g d.w.

.4. Genotoxic effects

The genotoxic effects were evaluated at molecular level, as DNAtrand breaks and apoptosis, by, respectively, the single cell gellectrophoresis (or Comet assay), and the diffusion assay; at chro-osomal level the frequency of micronuclei was assessed.The Comet assay was performed on peripheral erythrocytes

ccording to Frenzilli et al. (2004). Due to the high amount of alkali-abile sites in highly condensed chromatin of erythrocytes (Singht al., 1989; Frenzilli et al., 1999), the assay was carried out at pH2.1 to detect only single strand breaks (Tice et al., 2000). Ery-hrocytes were embedded in agarose (0.5% low melting agarose,MA) and layered on conventional slides, pre-dipped in 1% nor-al melting agarose (NMA) (Frenzilli et al., 2004). Cellular viability,

valuated by Trypan blue, always exceeded 95%. After at least 1 hn lysing solution (2.5 M NaCl, 10 mM Tris, 0.1 M EDTA, 1% Triton-100, and 10% DMSO, pH 10), slides underwent 10 min unwindingnd 10 min electrophoresis at 25 V, 300 mA, pH 12.1. Slides werehen neutralised with a 5 min rinse in 0.4 M Tris, put in 100% cold

ethanol for 3 min and allowed to dry. Slides were stained withthidium bromide and examined under epifluorescence at 200×agnification.For MN test, peripheral blood and cephalic kidney samples

ere smeared, air dried for 24 h at room temperature, fixed withethanol and 6% Giemsa stained for 20 min. A total of 2000/5000

rythrocytes per specimen were scored in coded slides (at least twolides) to determine the frequency of micronuclei according to theollowing criteria: micronuclei were defined as round structures,maller than 1/3 of the main nucleus diameter; micronuclei hado be on the same optical plan as the main nucleus but possessingistinguishable boundaries from it.

The DNA diffusion assay is a modified version of Comet assayetecting low molecular weight DNA which is lost from agaroseatrix during electrophoresis (Singh, 2000). Slides were prepared

s described before but electrophoresis was omitted. Apoptotic celluclei had a hazy or undefined outline without any clear boundaryue to nucleosomal-sized DNA diffusing into agarose and a diame-er 3 times over the mean nuclear diameter. Necrotic cell nucleiad a well-defined outer boundary of the DNA and a relativelyomogenous appearance (Singh, 2000).

.5. EROD activity

For the analysis of ethoxyresorufin O-deethylase individual liv-rs were homogenised (1:6, w:v) in 100 mM K-phosphate buffer pH.5, 0.15 M KCl and 1 mM ethylenediaminetetraacetic acid (EDTA)nd centrifuged at 12,000 × g for 15 min. The resulting super-atants (S9) were immediately incubated at 35 ◦C in a final volumef 1 mL containing 100 mM K-phosphate buffer pH 7.5, 4 �M 7-

mL acetone to stop the reaction (Regoli et al., 2003). Incubationixtures as above but stopped at time zero, were used as blank

alue and subtracted from the sample fluorescence. Fluorimetricnalyses (535/585 nm) were quantified by reference to resorufintandards (0.02–1 �M).

Toxicology 89 (2008) 188–196 191

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.6. Acetylcholinesterase activity

Acetylcholinesterase activity was determined in brain sam-les homogenised (1:1, w:v) in Tris–HCl 0.1 M, pH 7.2, sucrose.25 M and centrifuged for 10 min at 10,000 × g. Supernatantsere assayed with Ellman’s reaction at 412 nm (extinction coef-cient ε = 13.6 mM−1 cm−1), in Tris–HCl buffer 0.1 M, pH 7.0 with.5 mM acetylthiocholine and 0.2 mM 5,5-dithiobis-2-nitrobenzoiccid (Bocchetti and Regoli, 2006). Protein concentrations were mea-ured according to Lowry et al. (1951), using bovine serum albumineBSA) as standard.

.7. Vitellogenin gene expression

Thirty-nine specimens of L. cephalus, 32 males and 7 females, asositive controls, were analysed for Vitellogenin expression by RT-CR. Total RNA was extracted from liver tissues by the NucleoSpinNAII kit (Macherey-Nagel, Duren, Germany). RNA quantity andurity were measured by NanoDrop ND-1000 SpectrophotometerNanoDrop Technologies, Inc. Wilmington, Del, USA). RNA integrityas assessed by denaturing formaldehyde gel electrophoresis.

One microgram of total RNA from each sample was reverseranscribed with random and oligo-dT primers by using the Quan-iTect Reverse Transcription kit (Qiagen, Valencia, CA, USA). TheDNA samples were then amplified by using the Platinum TaqNA Polymerase (Invitrogen, Carlsbad, CA, USA) by a two-stepCR protocol: 30 s at 95 ◦C (denaturation step) and 30 s at 60 ◦Cannealing–extension step) per 35 cycles, preceded by 1 cyclet 95 ◦C per 2 min (hot-start DNA polymerase activation). Theeta-Actin gene transcript was utilized as housekeeping gene con-titutively expressed in liver. The used primer sequences (GenBanko. AB106873) were: VTGf: 5′ TGT GTG CTG ATG GTG TTC RG 3′

nd VTGr: 5′ GGC TCA ACA GAG TAG CAT TT 3′; beta-ActF: 5′ CAGGA GAA GAT GAC CCA GAT 3′ and beta-ActR: 5′ GAT ACC GCAGA CTC CAT ACC 3′. Each primer was used at a concentration of00 nM. The expected amplification products were 477 bp long forAct and 1081 bp long for Vtg. Sequences obtained from the clonesere analysed using an automated DNA sequencer (ABI PRISM 310,pplied Biopsystems) and were found to be identical.

.8. Statistical analysis

The level of DNA fragmentation obtained by Comet assay wasuantified by fluorescence microscopy and image analysis as theercentage of DNA migrating out of the nucleus (Komet 5.0 Soft-are, Kinetic Imaging Ltd.). Four slides per animal were prepared

two for Comet assay and two for diffusion assay), 50 cells per slideere scored and the mean was calculated. For statistical analysis

f genotoxicity data multifactor analysis of variance (MANOVA) or

ultiple regression analysis (MRA) were performed. In the apop-

otic diffusion assay, the percentage of cells with diffuse DNAnd a hazy outline were calculated from a total of 100 cells perpecimen. Data were analysed by MANOVA. Multiple range testMRT) was used (p < 0.05) to detect differences among the sam-

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able 1oncentrations of mercury, chromium, total polycyclic aromatic hydrocarbons (PAHs), loissues of chubs from different stations of the Cecina River basin

ite Hg (muscle) Hg (liver) Cr (liver)

avone 0.56 ± 0.12 (a) 0.32 ± 0.18 (a) 0.24 ± 0.0erignone 0.98 ± 0.43 (b) 0.2 ± 0.08 (ab) 0.27 ± 0.1aline di Volterra 1.48 ± 0.48 (c) 0.59 ± 0.21 (b) 0.28 ± 0.1onteginori 2.00 ± 0.53 (d) 1.1 ± 0.66 (b) 0.39 ± 0.1

ata are expressed as �g/g d.w. (mean values ± standard deviations, n = 5). Different lettetected.

ig. 2. DNA damage in erythrocytes of chubs from sampling sites along Cecinaiver. Data expressed as mean values ± standard deviations (n = 15). Different let-ers indicate significant difference between means after multiple range test. n.d.:ot detected.

ling sites. Results from chemical and biochemical analyses wereested by analysis of variance (ANOVA) to evaluate differencesetween sites (level of significance at p < 0.05). Post-hoc compar-

son (Newman–Keuls) was used to discriminate between means ofalues.

. Results

Mercury concentrations in muscle tissues ranged from.5 �g/g d.w. in chubs from control sites increasing downstream upo 2 �g/g d.w. in specimens collected from Ponteginori (Table 1).

ercury accumulation in the liver exhibited approximately theame trend, but concentrations were lower than in the muscle tis-ue (Table 1). Liver concentrations of chromium were similar insh from all the investigated sites, although higher average valuesere observed in organisms sampled at the most downstream loca-

ion (Ponteginori, Table 1). Significant differences were observedor PAHs with the highest concentrations in fish from Ponteginorihere HMW compounds contributed for almost 30% to the total

ontent (Table 1). Lower concentrations were measured in fish fromhe reference site of Pavone and, especially, in those from Salinei Volterra which also exhibited particularly low content of HMWydrocarbons (Table 1).

The highest level of DNA damage was observed in erythrocytesf fish sampled at Saline di Volterra and Ponteginori (Fig. 2), show-ng a percentage of DNA migrated in the Comet tail approximatelywice than in controls (p < 0.01).

The frequency of micronuclei in both mature and immature ery-hrocytes followed the same general trend exhibited by the DNA

igration in the Comet assay with higher values in chubs from

ownstream sites (Fig. 3). However, differences between sites wereot marked and, due to the elevated variability, not statisticallyignificant for kidney erythrocytes.

Apoptotic erythrocytes were rare in control fish (about 0.05%),hile greatly increased, by more than two orders of magnitude, in

w molecular weight (LMW) and high molecular weight (HMW) hydrocarbons in

Total PAHs (liver) LMW PAHs (liver) HMW PAHs (liver)

6 0.47 ± 0.06 (a) 0.33 ± 0.05 (a) 0.14 ± 0.01 (a)8 n.d. n.d. n.d.1 0.24 ± 0.01 (b) 0.21 ± 0.01 (b) 0.02 ± 0.01 (b)2 0.96 ± 0.19 (c) 0.68 ± 0.15 (c) 0.27 ± 0.05 (c)

ers indicate significant differences between sites (post-hoc comparison). n.d.: Not

192 G. Frenzilli et al. / Aquatic Toxicology 89 (2008) 188–196

Fig. 3. Frequency of micronuclei in circulating and immature erythrocytes of chubsfrom sampling sites along Cecina River and Pavone Stream. Data expressed as meanvalues ± standard deviations (n = 15). Different letters indicate significant differencebetween means after multiple range test.

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pecimens from the impacted site Saline di Volterra (6%) (Fig. 4).t Ponteginori the frequency of apoptotic cells was 0.25%, about 5

imes higher than in control specimens (Fig. 4).Multiple regression analysis showed a moderate but significant

orrelation between mercury concentrations in muscle tissue withNA migration (r = 0.66; R2 = 43.67; p < 0.001, Fig. 5) and micronu-leus frequency (r = 0.41; R2 = 16.61; p < 0.01, Fig. 6).

Liver EROD activity was more elevated in organisms from theost downstream sampling site (Ponteginori) than in fish from

aline di Volterra and control sites (Table 2), confirming the samerend observed for PAHs concentrations. Similarly, brain AChE activ-ty was significantly lower (about 30%) in chubs collected fromonteginori compared to the control site (Pavone) and Saline diolterra (Table 2).

ig. 5. Relationship between DNA damage and Hg concentrations in the liver ofhubs from sampling sites along Cecina River. r = 0.66; R2 = 43.67; p < 0.001.

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ig. 6. Relationship between micronucleus frequency and Hg tissue levels of chubsrom sampling sites along Cecina River. r = 0.41; R2 = 16.61; p < 0.01.

Qualitative evaluation of vitellogenin gene expression in maleshes was performed to reveal potential exposure to estrogenicubstances throughout the river basin. Eight mature male fish perite were analysed for vitellogenin mRNA; some mature femalesere also analysed as positive control (Fig. 7). Results indicated the

xpression of vitellogenin gene in the liver of approximately 50% ofhubs from the upstream (control) sites and of 80–90% of specimensollected from impacted sites. These indications point in light thattgn mRNA detected in males is most likely due to xenoestrogensr, at least to a combination of anthropogenic and natural causes.

. Discussion

Results of chemical analyses showed that mercury concentra-ions in fish muscle tissues were rather elevated (>0.5 �g/g d.w.)ven in control sites which are located upstream of the main sourcesf anthropogenic Hg pollution. Background levels in the muscle tis-ue of chubs from another Italian river (Tevere) ranged between.18 and 0.31 �g/g d.w. (Mancini et al., 2005); even lower concen-rations were measured in Leuciscus svallizi muscle from a Croatianiver, ranging from 0.078 to 0.190 mg/kg (Has-Schön et al., 2006).elatively high Hg levels in control chubs from Cecina River likelyeflect a naturally increased Hg background, related to the rich-ess of mineral deposit in Tuscany, or a diffuse Hg contaminationhroughout the basin of Cecina River.

Mercury bioavailability increased downstream, and the high-st tissue levels were measured in chubs from the impacted sites,aline di Volterra and Ponteginori, where Hg sediment concentra-ions ranged between 1 and 5 �g/g (Cecina Pilot River Basin, Finaleport).

Similar results were observed in the liver, although Hg levelsere typically lower than in muscles; such different distribution

f mercury in tissues is not surprising since liver concentrationsainly reflect short-term exposure from food intake, while lev-

ls in muscle tissue reflect the bioaccumulation over a wider timepan (Ausili et al., 2008). The higher concentrations of mercurybserved at Ponteginori compared to Saline di Volterra may beartly influenced by the different physical characteristics of the

able 2iver EROD activity and brain acetylcholinesterase in chubs from Cecina River basinmean values ± standard deviations, n = 5)

ite EROD pmol/min/mg AChE nmol/min/mg

avone 2.71 ± 1.33 (a) 161.90 ± 54.61(a)erignone 2.57 ± 0.22 (a) n.d.aline di Volterra 3.11 ± 0.81 (a) 142.71 ± 14.24 (a)onteginori 5.48 ± 0.99 (b) 112.52 ± 5.05 (b)

ifferent letters indicate significant differences between sites (p < 0.05)). n.d.: Notetermined.

G. Frenzilli et al. / Aquatic Toxicology 89 (2008) 188–196 193

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wo sites. At Saline di Volterra the river is shallow and the waterows fast, while at Ponteginori (7 km downstream) the river flowslowly and sedimentation is abundant: a higher bioavailability ofollutants associated with sediments could thus be hypothesizedt Ponteginori.

A more elevated chemical bioavailability in Ponteginori haslso been obtained for PAHs with tissue levels in chubs com-arable to those of moderately impacted organisms (Zorita etl., 2008). The higher prevalence of LMW compounds reflects aetroleum-derived origin but the significant contribution of HMWydrocarbons (almost 30% of total PAHs) also indicated the impor-ance of inputs from industrial, combustion-mediated processes.he limited hydrodynamic conditions of this site could furtherxplain the different accumulation of these chemicals in Pontegi-ori.

Chromium concentrations in fish livers were quite low and sim-lar throughout the investigated portions of the River Cecina. Suchesults suggest that the elevated natural levels of Cr in sediments,eriving from erosion of abundant Cr-rich ophiolites (Cecina Pilotiver Basin, Final Report), do not represent a particularly bioavail-ble form of the element.

Many authors have highlighted the importance of the use of aide battery of biomarkers when assessing the biological effects

n impacted environments since a single biomarker may not reflecthe health status of a sentinel species (Regoli et al., 2004). Thus, theoncurrent use of several biomarkers is important to minimise mis-nterpretation in cases of complex situations of pollution (Miniert al., 2000; Flammarion et al., 2002a; Linde-Arias et al., in press).isadvantages of multi-biomarker approach are mainly in terms ofosts. However, the use of a single biomarker might be acceptablehen the environment under study is known to be contaminated

y a well-defined chemical; in this case, a specific biomarker mighte effective for describing the level of environmental impact.

Among the set of biomarkers, the Comet assay has been usedor detecting DNA damage. The usefulness of this method for mon-toring the genotoxicity associated with river pollution has beenocumented by numerous papers in the last decades (Belpaeme

t al., 1998; Devaux et al., 1998; Mitchelmore and Chipman, 1998;renzilli et al., in press; Jha, 2008). In the present investigation theomet assay was performed at pH 12.1 instead of the commonlysed pH >13, which can reveal single-double strand breaks andlkali-labile sites. The pH > 13 was avoided because of the induc-

me2te

ctrophoretic separation (2% agarose gel). M: molecular marker GeneRulerTM 50 bp

ion of a large amount of single strand breaks associated withlkali-labile sites formed by functional condensation of chromatinn nucleated erythrocytes, which causes a marked DNA vulnerabil-ty and an elevated level of damage also in unpolluted specimens

hen slides are run at pH > 13 (Frenzilli et al., 1999); the mild alka-ine versions of the Comet assay is thus to be preferred for cellsaving a physiologically high condensation of chromatin (Singh etl., 1989; Tice et al., 2000; Frenzilli et al., in press).

The percentage of DNA migrated into the Comet tail significantlyiscriminated among sampling sites, with fish from the upstreamontrol stations showing a lower level of DNA fragmentation thanpecimens collected from the downstream anthropized sites. If lev-ls of DNA damage are expressed as Comet tail moment, the resultsbtained in specimens from Cecina River (control site: 0.51 ± 0.24;mpacted site: 2.17 ± 1.12) appear much lower than those reportedor chubs from the Rhõne watershed and Moselle River (controlite: 8.9 ± 4.3; impacted site: 23.1 ± 19.3) (Devaux et al., 1998;lammarion et al., 2002a). However, such differences are most likelyue to the Comet assay version rather than to actual differences ofnthropogenic disturbance. While the mild alkaline Comet assaypH 12.1) was used in our research, the alkaline version (pH > 13)as applied to chubs from the French rivers.

The micronucleus test in erythrocytes is widely used for thessessment of genotoxicity in fish species. However, micronucle-ted red blood cells are very rare, thus requiring thousands of cellso be scored to achieve statistically meaningful results. To over-ome this problem, hematopoietic tissues have been proposed asn alternative source of cells, namely immature erythrocytes, forhe assessment of micronucleus frequency (Hayashi et al., 1998). Inrder to compare these two approaches, both blood and cephalicidney smears were prepared and scored in our study. The fre-uency of micronuclei in both mature and immature erythrocytesollowed the general trend exhibited by the DNA migration in theomet assay with the lowest values of alteration in upstream con-rol sites. Absolute frequency and differences between control andmpacted sites were similar to those observed in L. cephalus fromhe Croatian River Sava (Kolak et al., 1999) but lower than values

easured in chub from the Po River, downstream of the conflu-nce with the highly polluted tributary Lambro River (Viganò et al.,002). In the latter river, the higher genotoxic impact can reflecthe more elevated contamination by PAHs, PCBs, DDTs and TCDDquivalents (Viganò et al., 2002).

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Our results showed that micronucleus frequencies were com-arable in immature fish erythrocytes and in circulating ones,lthough only the latter discriminated significantly between sites.n addition, the preparation of slides from cephalic kidney wasess rapid, requiring fish sacrifice: Besides, scoring slides was timeonsuming due to the abundance of tissue debris. Consequently,ccording to our results, the use of cephalic kidney for the evalua-ion of micronucleus frequency in fish species does not appear toffer any significant advantage in comparison with blood samples.

Multiple regression analysis showed a moderate but signifi-ant correlation between muscle mercury concentrations and DNAigration as well as between Hg levels and micronucleus fre-

uency. This suggests that at least part of the genotoxic impact onhubs might be due to Hg exposure. On the contrary, chromium andAHs exhibited different trends from DNA responses, with concen-rations being similar in all the stations (Cr) or more elevated onlyn Ponteginori (PAHs), and thus indicating a non-genotoxic effectf these chemicals for tissues of L. cephalus.

A very high DNA damage, which overwhelms the repair capacity,an lead to programmed cell death (apoptosis). Alternatively, thenduction of apoptosis in fish can result from the exposure to spe-ific pollutants such as the herbicides 2,4-D and butachlor (Ateeq etl., 2006). By omitting the electrophoresis run, slides prepared forhe Comet assay allow to discriminate apoptotic cells from dam-ged ones on the basis of the DNA diffusion pattern. Our resultshowed that erythrocytes from control fish were characterized byvery low frequency of apoptotic cells (0.05%), which increased bypproximately 5 fold at Ponteginori, and more than two orders ofagnitude in specimens from Saline di Volterra. Although the two

mpacted sites exhibited similar percentages of migrating DNA, theause of such a marked induction of apoptosis in Saline di Volterras not determined. However, these results warn against the riskhat massive apoptosis could lead to underestimate DNA damagey the Comet assay. In fact, the enzymatic DNA digestion typicalf the apoptotic process produces very small fragments (200 baseairs) which are lost by the gel during the electrophoresis and thusndetected by the Comet assay (Tice et al., 2000; Singh, 2000).

Molecular and cellular biomarkers are among the most sensitivend earliest responses to pollutants (Livingstone, 1993), allowing toetect biological alterations even at low levels of exposure. In thisespect, despite the chemical monitoring by the Regional Agencyor Environmental Protection revealed mostly inorganic (metal)ollution in sediments of the River Cecina basin, EROD and acetyl-holinesterase activities were measured as additional biomarkerso assess the potential impact of other lipophilic pollutants (dioxins,CBs, PAHs) and pesticides (carbamates, organophosphates). ERODctivities measured in chubs from the River Cecina were low (from.57 to 5.48 pmol/min/mg) and comparable to those measured inhubs from French unpolluted rivers (from 2 to 4 pmol/min/mg;lammarion et al., 1998; Flammarion and Garric, 1999). However,significant induction was observed in fish from the downstream

ite Ponteginori, where EROD activity was approximately doublehan in upstream sites. These data and the more elevated accumula-ion of PAHs in fish from this site would confirm the anthropogenicmpact in the area and/or the high bioavailability of pollutants,ue to sedimentation prevailing in this site. In addition, it shoulde noted that both estradiol in maturing females and the pres-nce of trace metals can inhibit cytochrome P450 (Regoli et al.,005; Benedetti et al., 2007), thus potentially influencing ERODctivities measured in chubs of the Cecina River. The activity of

rain acetylcholinesterase was higher in chubs collected from thepstream reference site and decreased moving downstream, withignificantly lower values in fish sampled at Ponteginori. Theseesults may suggest an exposure to pesticides from agriculturalractices which are abundant in the middle portion of the river

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ourse, despite recent studies indicated that acetylcholinesterasesan be modulated also by other classes of chemicals (Gorbi et al.,008).One of the most sensitive responses to estrogens in fish ishe expression of the gene encoding for vitellogenin in the liverissue. Vitellogenin is a phosphoglycolipoprotein normally synthe-ized only in female fish (Flammarion et al., 2000; Klaper et al.,006) being responsible for the production of the main precursor ofgg yolk. This protein can be produced in males when organisms arexposed to exogenous endocrine disruptors as synthetic estrogens,lkylphenolic compounds, phytoestrogens and some pesticides.or this reason, the presence of protein or the gene expression ofitellogenin in males is widely accepted as a possible indicator ofenoestrogenic pollution (Sumpter and Jobling, 1995; Flammariont al., 2000; Scott et al., 2005; Barucca et al., 2006). Moreover,easonal fluctuations of endogenous estrogens have been reportedGuerriero et al., 2005). Potentially, these natural fluctuations mightnfluence the expression of vitellogenin genes in males. However,pecimens studied were collected between March and April, whenstrogens in plasma of male chubs from Italian rivers have beenescribed at the lowest levels (Guerriero et al., 2005). Thus, it cane assumed that the vitellogenin gene expression observed in malehubs from Cecina River is likely due to xenoestrogens.

Our preliminary results detected vitellogenin mRNA in malehubs from all the sampling sites, suggesting the presence of a dif-used estrogenic effect along the course of the River Cecina, evenn upstream portions. As a working hypothesis, it is worthy to notehat some trace metals like cadmium and mercury have been sug-ested to exert estrogenic effects both in vitro experiments andn field conditions (Choe et al., 2003; Safe, 2003; Canapa et al.,007). Recent experimental studies demonstrated the estrogenic-

ike effects of mercuric chloride on MCF-7 human breast cancerells (Zhang et al., 2008) and a marked up-regulation of vitellogeninRNA in males of the fish fathead minnow (Pimephales promelas)

xposed to mercury (Klaper et al., 2006). More extensive researchctivities are needed to confirm these data and to characterize theotential contaminants responsible for the estrogenic effect in theiver Cecina.

. Conclusions

Overall the results obtained in this study confirmed that L.ephalus can be conveniently used for the assessment of river basinnvironmental quality based on multi-biomarker approach. Theumerous sources of natural variability and the elevated complex-

ty of contaminant mixtures in field conditions make it difficult tolearly associate biological effects to specific chemicals, nonethe-ess our results indicate a potential role of mercury for genotoxicffects and a more limited impact by PAHs. Preliminary results onitellogenin mRNA expression in males also indicate diffuse estro-enic responses in the river basin, suggesting the need of furthernvestigations.

cknowledgements

The author are indebted with Dr. C. Cuoco and A. Voliani for help-ng us in fish sampling and Dr. G. Benedettini from ARPAT (Tuscanyegional Agency for Environmental Protection) for constructive dis-ussions about the nature and status of impacts in the River Cecinaasin, with S. Calzolani for his technical contribution and with C.hezzani for his technical assistance in the imaging work.

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