Immunohistochemical and molecular biomarkers in Coris julis exposed to environmental contaminants

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Immunohistochemical and molecular biomarkers in Coris julis exposed toenvironmental contaminants

Salvatore Fasulo a,n, Angela Mauceri a, Maria Maisano a, Alessia Giannetto a, Vincenzo Parrino a,Florinda Gennuso b, Alessia D’Agata a

a Dipartimento di Biologia Animale ed Ecologia Marina, Universit �a delgi Studi di Messina, Salita Sperone 31, 98166 S. Agata, Messina, Italyb Agenzia Regionale per la Protezione dell’Ambiente (ARPA Sicilia), Corso Calatafimi 217, 90129 Palermo, Italy

a r t i c l e i n f o

Article history:

Received 18 March 2009

Received in revised form

9 September 2009

Accepted 20 December 2009Available online 2 February 2010

Keywords:

Biomarker

Coris julis

Immunohistochemistry

Cloning

FISH

a b s t r a c t

When a contaminant interacts with biotic components of a marine ecosystem, it causes a series of

changes that can compromise an entire community (Stebbing, 1985).

This present study wants to focus on changes in the gills of a bioindicator benthic organism, Coris

julis, collected in Milazzo (Messina, Italy), characterized by a strong anthropical impact), compared with

individuals from the control site (Marinello, Messina). RT-PCR has been used for both MT and HSP70,

and the respective mRNAs have been visualized by FISH. MT and HSP70 expression levels increased in

individuals collected in Milazzo. The presence of numerous apoptotic and proliferating cells and the

analysis of several neuronal markers by immunohistochemical method give information about the

adaptation to a heavy metal mixture. The obtained results show that, in stressed fishes, defensive

processes increase to maintain the normal functions of the organs more exposed to the action of

polluted substances.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

When a contaminant interacts with biotic components of anecosystem, it causes a series of changes at different levels ofstructural complexity. Starting with molecular damage, theseeffects can culminate in modifications of the structure of apopulation or an entire community (Stebbing, 1985). To determinethe mechanisms by which contaminants perturb animal commu-nities, an increasing number of ecotoxicologists have based theirstudies on the health status of ecosystems and their components,evaluating the early adverse effects caused by contaminants inorganisms in their natural environment (Depledge, 1994). Onevery useful approach involves the use of biomarkers. A biomarkeris defined as a change in a biological system related to exposure toenvironmental chemicals. The biomarker approach in determiningthe ecotoxicological status of a marine environment has been usedmany times, with many species (both vertebrates and inverte-brates) as the bioindicator (McCarthy and Shugart, 1990; Fossi,1998; Stronkhorst et al., 2003; Mauceri et al., 2005; Fasulo et al.,2008; Frenzilli et al., 2008).

The fish gill is an important organ that comes in contact withenvironmental pollutants and the major target organ for acutemetal toxicity in fishes (Van Heerden et al., 2004a, b; Mauceriet al., 2005; Pandey et al., 2008), which is then subjected to

damages that can be prevented or counteracted by an increasedrate of metal binding proteins synthesis, such as metallothionein(MT), characterized by low molecular mass (6.0–7.0 kDa) involvedin the sequestration and detoxification of heavy metals (Viarengo,1989). This organ represents multifunctional performing vitalfunctions such as respiration, osmoregulation, acid–base regula-tion and excretion (Laurent and Dunel, 1980; Evans et al., 2005).The heat shock proteins (HSP 70), together with MTs, play animportant role as molecular chaperones in both unstressed andstressed cells. In aquatic organisms, apoptosis and proliferatingcells have been used as biomarkers of exposure to heavy metals(Berntssen et al., 2001); in the gills, the control of proliferationand apoptosis are essential to ensure morphological and func-tional integrity of the gill tissue (Ferrando et al., 2005; Mauceriet al., 2005).

The purpose of this study was to investigate the stressresponses in the gills of the rainbow wrasse, Coris julis, living inthe natural environment of Milazzo (ME). A panel of biomarkersboth of exposure and effect types was used to assess the fish’sresponse to exposure to anthropogenic and industrial pollutionthat characterizes the environment. C. julis is a useful species forassessing seawater quality because it can accumulate contami-nants and it has physiological mechanisms that tend to offsetunfavorable environmental conditions by increasing tolerance topollutants and by developing specific cytoprotective proteins. It isparticularly suitable for this study because its feeding habits favorbio-accumulation of pollutants; its diet is based on zoobenthossuch as molluscs and benthic crustaceans (Pinnegar and

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Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.ecoenv.2009.12.025

n Corresponding author. Fax: +39 090 6765556.

E-mail address: [email protected] (S. Fasulo).

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Polunin, 2000). It also plays a key role in the food chain of thestudied area and thus it can provide information about a largespatial area. To assess the fish’s stress level and its potential forrecovery, expressions of the following biomarkers were evaluatedin samples of fish collected from Milazzo: calbindin, serotonin(5-HT), serotonin receptor (5-HT3), neuronal and inducible nitricoxide synthase (nNOS and iNOS), metallothioneins (MTs), heatshock proteins (HSPs) and cellular turnover biomarkers such asproliferating cell nuclear antigen (PCNA), FAS receptor andcaspase. Histochemical, immunohistochemical, molecular andfluorescence in situ hybridization (FISH) techniques were usedand all results were subjected to statistical analysis.

2. Materials and methods

2.1. Sampling

The specimens of C. julis were collected every month during the period April–

September 2005, at the depth of about 20 m using a bow net. To standardize the

sampling 180 individuals, for each site, of the same size and weight class (mean

size of 12 cm length and 10 g weight) and same sex (females), were selected. Adult

specimens were anesthesized with 0.01% ethyl 3-aminobenzoate methanesulfo-

nate (MS 222, Sigma-Aldrich). Thirty samples collected in Milazzo and other 30

collected in Marinello were used monthly to make the statistically important

below-mentioned analyses. In particular, 10 of these 30 individuals were used for

histological and immunohistochemical analysis, other 10 for biomolecular analysis

and the last ones for heavy metal analysis.

The fish-killing method used in this study followed the guidelines of animal

care and experimentation in compliance with the Italian National Bioethics

Committee (INBC).

2.2. Sample site

The harbor of Milazzo is a wide natural bay that runs along 15 km of the

northeastern coast of Sicily. It is characterized by low water exchange. Milazzo is

heavily industrialized, densely urban and experiences traffic from large tankers

that transport crude and refined oil to its refinery (Yakimov, 2005; Fig. 1). During

summer, the highest water temperature at the surface is 26 1C and at 20 m depth it

is 16 1C; salinity is 391/00 practical salinity units (PSUs; Table 1). The natural

reserve of Marinello (ME) was chosen as a reference site (i.e., control) for this study

(Sicily Region, 2002). The lagoon and coastal system of Marinello constitute a

wildlife reserve in the Sicilian region, which covers an area of 401.25 ha: 248.13 ha

of an integral reserve and 153.12 ha of a pre-reserve. It is unique in that it contains

many different environments within a small area. During summer at Marinello,

the highest surface water temperature is 24 1C and at 20 m depth it is 16 1C;

salinity is 36% PSU (Table 1). The concentrations of metals in the sediments

(in mg/kg of dry sample) are reported in Table 2.

2.3. Histological markers

Tissues of gill samples for histological assessment were removed from the gill

cavity, fixed in 4% paraformaldehyde in 0.1 M phosphate buffered solution

(pH 7.4) at 4 1C, dehydrated in ethanol and embedded in Paraplast (Bio-Optica,

Italy). Histological sections (5 mm thick) were glass-slide mounted and stained

with hematoxylin/eosin (Bio-Optica, Italy) to visualize typical morphological

features. The methods for polysaccharides (Periodic Acid Schiff (PAS) staining) and

Fig. 1. Locations of the sampling site: Milazzo (1) and Marinello (2).

Table 1Physico-chemical parameters, in the summer season, of both Marinello and

Milazzo environments.

Sampling area Milazzo Marinello

Temperature 1C 25.8 24

Conductivity (mS/cm) 56.6 57

Salinity (PSU) 39% 37%Oxygen (mg/l) 5.0 6.15

pH 8.13 8.09

Ammonium 10 (mg/l) 0.1 0.6

Free chlorine (mg/l) 0.03 0.03

Total chlorine (mg/l) o0.1 0.07

Fluorine 2 (mg/l) o0.1 o0.1

Total phosphate (mg/l) o0.1 0.1

Nitrites 2 (mg/l) o0.01 o0.01

Nitrites (mg/l) o0.01 o0.01

Ortophosphate (mg/l) o0.01 0.2

Potassium (mg/l) 27 27

Table 2Concentration of metals expressed in mg/kg of dry champion in sediments in

Marinello and Milazzo in the summer season.

Milazzo Marinello

Cd 24062 510

Co 37632 11565

Cr 43352 18015

Cu 45105 20970

Ni 36382 12505

Pb 52230 13705

Zn 107475 89255

Sn 89975 5200

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acid mucopolysaccharides (Alcian Blue staining, pH 2.5) were used to detect the

presence of mucous cells in the gill epithelium (Pearse, 1985).

2.4. Antisera and immunohistochemistry

Dewaxed sections were rinsed in PBS and treated with 3% H2O2 in PBS to stop

endogenous peroxidase activity for immunoperoxidase reactions. After incubation

with 10% normal goat serum for 30 min, they were incubated overnight in a moist

chamber at 4 1C with polyclonal antisera FAS, caspase, PCNA, calbindin, nNOS,

iNOS, 5-HT, 5-HT3 receptor, HSP70 and MT (concentrations and suppliers are

indicated in Table 3). After a rinse in PBS (10 min), the sections were incubated for

2 h at room temperature with a peroxidase or tetramethyl rodamine

isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) conjugated goat anti

rabbit/mouse IgG (Sigma, USA), diluted 1:100 in PBS. The peroxidase was stained

by a fresh solution of 3,3-diaminobenzidine tetrahydrochloride (DAB, tablet) and

H2O2 (0.001%) in PBS.

Negative controls for all immunohistochemical labeling were performed by

substitution of non-immune sera for the primary or secondary antisera. Specificity

of the labeling of some peptides was verified by incubating sections with

antiserum preabsorbed with the respective antigen (10–100 g/ml). The preabsorp-

tion procedures were carried out overnight at 4 1C.

2.5. RNA extraction and cDNA synthesis

Total RNA was extracted from the gills of specimens using TRIzol LS reagent

(Invitrogen) (Chomczynski and Sacchi, 1987). The cDNA was synthesized using

4 mg of total RNA, and oligo (dT)20 primer (150 pmol/ml) ( Invitrogen), with MMLV

reverse transcriptase (Invitrogen) following the manufacturer’s instructions. The

resulting reverse transcribed products were used for polymerase chain reaction

(PCR) amplification.

2.6. Polymerase chain reaction (PCR)

The sequences of the primers were based on MT conserved known regions

in Teleosts and were 5‘-GGAACCTGCAACTGCGGAG-3‘ for MT sense primer and

Fig. 2. (A)–(D) H/E and AB-PAS reaction. Gill of specimens collected from Marinello (A)–(C) compared with specimens from Milazzo (B)–(D) characterized by damaged

structure (B) and numerous mucous cells (D). Scale: bar 20 mm. Mean and standard deviation (SD) calculated from the number of positive cells to the AB-PAS reaction (E).

The values are statistically different (Po0.001) in both environments considered.

Table 3List of primari antibodies used in the present study. All antibodies were raised in

rabbit, except for the HSP70, MT, 5-HT and calbindin that were raised in mouse.

Antibodies and abbreviation Supplier Dilution

Fas ligand (Fas) Abcam (UK) 1:50

Proliferating cell nuclear antigen (PCNA) Abcam (UK) 1:50

Heat shock protein 70 kDa (HSP70) Abcam (UK) 1:100

Caspase MBL (USA) 1:25

Brain nitric oxide synthase (nNOS) Biomol (UK) 1:100

Inducible nitric oxide synthase (iNOS) Biomol (UK) 1:100

Metallothionein (MT) Dako (Denmark) 1:25

Serotonin (5-HT) Dako (Denmark) 1:25

Serotonin 5-HT3 receptor Sigma (USA) 1:100

Calbindin D-28K Sigma (USA) 1:100

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5‘-TCACTGGCAGCAGCTIGTGT-3‘ for MT antisense primer. H70pf2 sense primer

and H70pr2 antisense primer were used for HSP70 (Molina et al., 2000). The gene

of the actin (used as a positive control) of each examined organism has also been

amplified using sequence primers based on the actin cDNA sequence of C. julis to

amplify 500 bp. Oligonucleotides were synthesized and purified by MWG Biotech

AG. Polymerase chain reactions were performed using: 5 ml of 10� buffer, 0.25 ml

of 5 U/ml Dream Taq polymerase (Fermentas), 1 ml of 25 mM MgCl2, primers

(50 mM each), 2 ml of cDNA template, 1 ml of 10 mM dNTPs and Milli-Q water

(Millipore). The total reaction was performed in a 50 ml volume. The amplification

conditions required were as follows: 95 1C for 2 min, 35 cycles of [95 1C for 30 s,

57 1C for MT and 55 1C for HSP70 for 30 s, 72 1C for 1 min] and a final extension at

72 1C for 5 min. The Ep-Gradient Mastercycler (Eppendorf) was used. PCR products

were separated and analyzed by gel electrophoresis.

2.7. Cloning and sequencing of PCR products

PCR products were separated on 1% agarose gels and the region containing the

expected size fragment was sliced and purified using QIAquick Gel Extraction kit

(Qiagen). The products were ligated into the plasmid pGEM T Easy Vector

(Promega). Ligations were carried out in 10 ml reaction using 1 ml T4 DNA Ligase

(NEB), 1 ml buffer 10� , 50 ng vector and 30 ng fragment. The reaction mix was

incubated overnight at 4 1C and used to transform JM 109 competent Escherichia

coli following the manufacturer’s instructions. Transformed cells were plated on LB

plates supplemented with ampycilline (100 mg/ml), X-Gal (80 mg/ml) and IPTG

(0.5 mM). Plasmid DNA was purified, using GenElute Plasmid miniprep kit

(Sigma), and sequenced, using universal M13fw and M13rv primers and ABI

PRISM BigDye Terminator 3.1 Cycle Sequencing kit (PE Applied Bio-system).

2.8. Fluorescence in situ hybridization (FISH)

The expression of MT and HSP70 and their morphological localization were

investigated using in situ hybridization on histological sections (7 mm) from the

gills. Antisense 40 bp DNA probes were based on the MT and HSP70 cDNA

sequences of C. julis obtained by sequencing and were synthesized, 3‘ marked with

fluorescein and purified by MWG Biotech AG.

Tissue sections were deparaffinized in two changes of fresh xylene and

rehydrated in graded alcohols. Then the sections were postfixed in 4%

paraformaldehyde, 0.1 M PBS for 10 min and permeabilized for 15 min in Triton

X-100 0.3% PBS. After washing in PBS, the slides were incubated with pre-

hybridization buffer for 2 h at 37 1C. The slides were washed in 2� SSC buffer for

5 min, the hybridization buffer with fluorescent probe (final concentration

2000 ng/ml) was added to each tissue section and then they were incubated in

low light conditions in a sealed humidified chamber overnight at 37 1C. After

the hybridization, the slides were washed with a low-stringency washing buffer

(1� SSC and 10 mM DTT), and then with a high stringency washing buffer

(0.5� SSC and 10 mM DTT). Sections were mounted using a medium containing

an anti-fading agent and staining evaluated using an Axio Imager Z1 (Zeiss)

epifluorescence microscope. A control was carried out to determine that the probe

was only binding to RNA by digesting the tissue with RNases prior to hybridization

with the oligonucleotide probes (Fasulo et al., 2008).

2.9. Heavy metal analysis

Metal concentrations were determined by atomic absorption spectrometry

with a graphite furnace (model Perkin Elmer 4100ZL). Heavy metal analyses

(Pb, Cd, Cr, Zn, Cu) were performed on lyophilized tissues. Samples of gills were

digested with HNO3 in a high-pressure Teflon bomb. Digested samples were

transferred into polyethylene vessels and diluted 1:5 with ultra-pure water. The

method of additions was used and standards were prepared by serial dilution of

commercially available stock solutions to within the linear range of the respective

metals. Reagents were pro-analysis grade and the analytical procedure contained

simultaneously run reagent blanks. The accuracy of digestion and analytical

procedures was checked by routine determination of elements in standard

reference materials (SRM1577b, 1566a and 2711) from the National Institute of

Standards and Technology (Gaithersburg, USA) and (SRM1646) from the National

Bureau of Standards (Washington, USA). (Fasulo et al., 2008).

Fig. 3. Immunohistochemical labeling for FAS (A, B) for Caspase 3 (C, D) and for PCNA (E, F) of gill of specimens collected from Marinello and Milazzo, respectively.

Scale: bar 20 mm.

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2.10. Statistical analysis

Acid and neutral mucous cells, immunopositive cells quantification and FISH

analysis results of each specimen were performed by counting the positive cells

scored for field (40� magnification) with Automatic Measurement Programs of

Axio Vision release 4.5 (Zeiss) software and analyzing statistically with one-way

analysis of variance (ANOVA) using GraphPad (InStat) software. The intensities of

bands of MT and HSP70 were measured with Quantity One (BioRad) software and

analyzed statistically with GraphPad (InStat) software using one-way analysis of

variance (ANOVA). For heavy metal analysis, non-parametric Mann–Whitney

U-test was used to test differences between both groups. A P value less than 0.05

was considered to indicate statistical significance.

3. Results

3.1. Gill histology

The gills of C. julis from Marinello showed a typical organiza-tion as described below (Mauceri et al., 2005; Fig. 2A). Themucous cells were revealed by the Alcian Blue/PAS reaction(Fig. 2C). On the contrary, the structure of the gills of individualsfrom Milazzo was notably altered. A diffuse edema on bothfilaments and lamellae, telangiectasia, and exfoliated epitheliumwere frequent, and considerable hyperplasia and hypertrophy inthe lamellar epithelium were observed (Fig. 2B). In the branchialepithelium of Milazzo specimens, a high percentage of Blue-

stained mucous cells evidenced with Alcian Blue at pH 2.5 wasobserved; moreover, the PAS reaction revealed minor pink-stained neutral polysaccharides (Fig. 2D). The average of AB/PASpositive cells is in Fig. 2E.

3.2. Immunohistochemical analysis

Results for different tests are described below.Apoptosis: in the branchial epithelium of fishes from Marinello,

very few FAS (Fig. 3A) immunopositive and numerous Caspase3-immunopositive cells (Fig. 3C), distributed along the filament,were detected. Several FAS (Fig. 3B) and caspase immnupositivecells (Fig. 3D) were observed in the epithelium of the lamellae andthe apical filaments of specimens collected in Milazzo.

PCNA: in the branchial epithelium of Marinello fishes, PCNAimmunopositive cells were present in the lamellae (Fig. 3E). In thespecimens from Milazzo, numerous PCNA immunoreactive nucleiwere located mainly along the filament (Fig. 3F).

Calcium-binding proteins (calbindin CB D28K): in the specimensfrom Marinello, immunostaining for CB D28K clearly revealedimmunopositive cells in the lamellar epithelium (Fig. 4A). Theseimmunopositive cells were moderately observed in the specimensfrom Milazzo (Fig. 4B).

NOS: in the gills of specimens from Marinello, immunoposi-tivity for nNOS was limited to neuroepithelial cells (NECs)

Fig. 4. Immunoperoxidase reactions in paraffinate sections of branchial epithelium of C. julis collected from Marinello (A–C–E) and Milazzo (B–D–F): CB D28K (A and B),

nNOS (C and D) and iNOS (E and F). Scale: bar 20 mm

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distributed along the filament epithelium (Fig. 4C). In contrast, inthe gills of fishes collected from Milazzo, very few nNOSimmunopositive cells were distributed along the apical filamentepithelium (Fig. 4D). Few iNOS immunopositive cells were foundin the branchial epithelium of specimens from Marinello (Fig. 4E)with respect to specimens of Milazzo (Fig. 4F).

Serotonin: in the control site fishes, serotonin was found incells distributed on filament and lamellar epithelium and in a fewimmunoreactive nervous fibers (Fig. 5A). Immunopositivity wasnot observed for the 5-HT3 receptor (Fig. 5C). In the specimensfrom Milazzo, the few present serotonin immunopositive cellswere distributed on the lamellar epithelium (Fig. 5B), andnumerous 5-HT3 receptor immunopositive cells also werepresent on the lamellar epithelium (Fig. 5D)

HSP70 and MT: numerous HSP70 (Fig. 5F) and MT immunopositive (Fig. 5H) cells were present on the lamellar epitheliumof specimens from Milazzo; they were not present inthe branchial epithelium of the individuals from Marinello(Fig. 5E and G).

Immunopositive cells statistical analyses are presented in thegraphs of Fig. 6.

3.3. Amplification and sequencing of MT- and HSP70-specific

complementary DNA

RT-PCR products were characterized by electrophoresis onSYBR safe-stained agarose gel. Discrete bands of 156 bp for MTand 284 bp for HSP70 were visualized. The data reported

Fig. 5. Immunoperoxidase reactions for serotonin (A, B), 5-HT3 receptor (C, D), HSP70 (E, F) and MT (G, H) in specimens collected from Marinello (A–C–E) and Milazzo

(B–D–F).

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in Fig. 7A and B were normalized with the expression ofcytoplasmatic actin, which was expressed at basal levels both incontrol animals and in those from Milazzo. MT and HSP70 geneswere highly expressed in animals from Milazzo and wereexpressed to lower extent in the gills of control animals. MTand HSP70 band intensity differed significantly betweenMarinello and Milazzo animals (Po0.001; Fig. 7C and D).

PCR products were cloned and sequenced, and the partialnucleotide sequences of the MT and HSP70 cDNA (GenBankAccession Number EG649502 and ES702768, respectively)were submitted to GenBank and dbEST. The nucleic acid sequencesof the MT and HSP70 were compared with homologous genes ofother fish species. The highest degree of similarity was found withMT sequences of Sparus aurata (92%), and the highest levelof homology was found with HSP70 sequences of Dicentrarchuslabrax (94%).

3.4. In situ hybridization

In situ hybridization for the localization of MT and HSP70mRNA expression in paraffin-embedded sections confirmed themorphological and immunocytochemical results previously ob-tained. The signal for MT and HSP70 was absent in the gills ofMarinello specimens (Fig. 8A and C) but was clearly expressed inanimals from Milazzo (Fig. 8B and D), and the difference wasstatistically significant (Po0.001; Fig. 8E and F).

3.5. Metal analysis

The concentrations of metals in the sediments (in mg/kg of drysample) are higher in Milazzo than in Marinello.

Milazzo: Cd: 24062; Pb: 52230; Zn: 107475; Cu: 45105

Fig. 6. Mean and standard deviation (SD) of immunopositive cells scored for field (40� magnification) with Automatic Measurement Programs of Axio Vision release 4.5

(Zeiss) software and analyzing statistically with one-way analysis of variance (ANOVA) using GraphPad (InStat) software.

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Marinello: Cd: 510; Pb: 13705; Zn: 89255; Cu: 20970Fig. 9 shows concentrations of all metals detected in gills of C.

julis from both the environments considered. Levels of Cd foundin gills from polluted (Milazzo) and non-polluted (Marinello)environments showed different values ranging from 0.9 to1.01 mg/kg d.w. in polluted area and from 0.09 to 0.12 mg/kg d.w.in the control one. The difference is meaningful (Po0.001). Levels ofPb (1.3 mg/kg p.w. in Milazzo and 0.2 mg/kg p.w. in Marinello) andCr (2.12 mg/kg p.w. in Milazzo and was not detected in Marinello)were significantly higher (Po0.001 for both) in specimens fromMilazzo than in those from Marinello. Also levels of Zn (7 mg/kg p.w.in Milazzo and 4.3 mg/kg p.w. in Marinello) and Cu (1.7 mg/kgp.w.in Milazzo and 0.8 mg/kg p.w. in Marinello), in average, werehigher in polluted than in control environments.

4. Discussion

Numerous types of damage to branchial tissue have beendocumented both in aquatic organisms experimentally exposed totoxicants and in populations sampled from polluted environments(Giari et al., 2008; Mauceri et al., 2005). Hyperplasia with lamellarfusion, telangiectasia and edema with epithelial lifting dysfunctionof branchial respiratory and osmoregulatory processes (as docu-mented in the present survey) are typical gill lesions that occur inresponse to various types of xenobiotic substances and heavy metals(Sanchez et al., 1997; Mauceri et al., 2002). The present study showsthat heavy metal pollution in a natural environment could induce

different defensive responses as overproduction of alcianophilicmucous cells in the gills and an enhancement of the cellularturnover. The increase of proliferative and apoptotic cells in theepithelium of specimens from Milazzo suggests that more branchialdamages occur in fish from this site, which are more affected bytoxic substance exposure (Wendelaar Bonga and Van der Meij, 1989)than those from Marinello. The apoptotic signals were detected bythe presence of FAS and Caspase 3 pro-apoptotic protein immuno-positive cells. Caspase 3 is commonly defined as effector caspase andis known to be involved in both mithochondrial- and receptor-mediated pathways (Migliarini et al., 2005). High rates of apoptosiscombined with increased mitotic activity in the gills of fish stressedby chemical agents indicate high cellular turnover or acceleratedaging of the branchial epithelium cells during periods of stress(Wendelaar Bonga, 1997; Dang et al., 2000). This result, in theindividuals from Milazzo, indicates the occurrence of apoptoticprocesses in gills of fishes exposed to pollution (Migliarini et al.,2005). The presence of iNOS and MT immunopositive cells and theMT and HSP70 mRNA expression in chloride cells of the branchialepithelium in specimens from Milazzo have demonstrated that thegills of this species have a ready detoxification response. Theseresults also indicate that the presence of mixture of pollutants insediments and gills could induces oxidative stress that inhibitscalcium cellular homeostasis (through inhibition of pumps andbinding Ca2+ protein such as calbindin D28K); for example,increased waterborne zinc specifically disrupts Ca2+ uptake acrossthe gills (Hogstrand and Wood, 1996), leading to hypocalcemia,which may culminate in the death of fish within a few days,

Fig. 7. Typical experiments (RT-PCR) for MT and HSP70. Electrophoresis on 1% agarose gel stained with SYBR Safe. (A) In order, MT RT-PCR products of C. julis collected from

Marinello; MT RT-PCR products of C. julis collected from Milazzo; 50 bp molecular weight marker (gene Ruler Fermentas); actin RT-PCR products of C. julis collected from

Marinello and actin RT-PCR products of C. julis collected from Milazzo. (B) HSP70 obtained from gill RNA of C. julis. In order, HSP70 RT-PCR products of C. julis collected

from Marinello; HSP70 RT-PCR products of C. julis collected from Milazzo; 50 bp molecular weight marker (gene Ruler Fermentas); actin RT-PCR products of C. julis

collected from Marinello and actin RT-PCR products of C. julis collected from Milazzo. (C, D) Mean and standard deviation (SD) calculated from the ratio of MT or HSP70 to

actin band intensity. The values are statistically different (Po0.001) for MT and for HSP70 (P40.05) from the corresponding controls according to the one-way analysis of

variance (ANOVA) test.

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depending on the concentration. However, this assumptions requirefurther studies.

The lower presence of immunopositive nNOS cells in thespecimens from Milazzo is also significant. Our observation suggeststhat NO may be acting as a paracrine signaling molecule to regulatethe gill vascular tone (Mauceri et al., 1999; Hyndman et al., 2006).Serotonin is a neurotransmitter involved in different physiologicaland behavioral roles such as the regulation of blood flow, cellular

proliferation and apoptosis, gastrointestinal motility and in gills actson the contraction and vascular tone (Azmitia, 2001).

In C. julis from Marinello, serotonin was commonly immuno-detected in branchial neuroepithelial cells (NECs), which areindicative of an O2 sensitive chemoreceptor; in contrast, serotoninwas absent in the specimens from Milazzo. These differences inthe serotoninergic NEC distribution appear to reflect chemor-eceptive roles related to hypoxia tolerance and might beconsequent to heavy metal exposure (Ferrando et al., 2005).

The different roles played by serotonin are mediated by specificreceptors (Hoyer et al., 1994). In particular, the 5-HT3 receptor is anion channel receptor. Behavioral, neurochemical and electrophysio-logical investigations indicate that this receptor modulates dopamineneurone activity (Barnes and Sharp, 1999). The presence of 5-HT3

receptors in the branchial filaments in the specimens from Milazzoseems to regulate the response to hypoxia, dopamine neurone activityand the neurotransmitter release (Barnes and Sharp, 1999).

The results obtained show that, in stressed fishes, thedefensive processes increase to maintain the normal functionsof organs more exposed to the action of polluted substances.

5. Conclusion

The results of this study illustrate that in fish stressed by heavymetal exposure, defensive processes increase to maintain the normal

Fig. 8. In situ hybridization with fluorescent probe (FISH) for MT and HSP70 on histological sections (7 mm) from the gills of C. julis collected in Marinello and in Milazzo.

Positive cells on all of the epithelium in specimens from Milazzo (B for MT and D for HSP70). Absence of positive cells in specimens from Marinello (A for MT and C for

HSP70). Scale: bar 20 mm. Mean and standard deviation (SD) calculated from the number of positive cells to the FISH reaction. The values for MT (E) and HSP70 (F) mRNA

are statistically different (Po0.001).

Fig.9. Mean concentration 7SD (mg/kg p.w.) of Cr, Pb, Cd, Zn and Cu detected in

gills of C. julis from polluted (Milazzo) and control (Marinello) environments.

Significant difference from control (Po0.01).

S. Fasulo et al. / Ecotoxicology and Environmental Safety 73 (2010) 873–882 881

Author's personal copyARTICLE IN PRESS

functions of those organs most exposed to the action of toxicsubstances. Exposure to toxicants, in samples from the pollutedenvironment (Milazzo), was correlated with changes in the morphol-ogy of the gill epithelium (e.g., increasing density of mucous cells,hyperplasia with lamellar fusion, telangiectasia) and with alteredbranchial activities (e.g., induction or inhibition of some peptides orneurotransmitters). The presence of numerous apoptotic and pro-liferating cells and proteins that have chelating, cytoprotective anddetoxifying roles (e.g., MT, iNOS and HSP70) is likely associated with aphysiological adaptation to stress due to a heavy metal mixture ratherthan a specific response to toxicants.

These results suggest that changes in morphology, cellularturnover and staining of several neuronal markers might provideuseful informations about environmental conditions. Monitoringthese changes (i.e., using them as specific biomarkers) might be afeasible way to assess the health of the environment, but thisshould be supplemented with a more complex set of analyses.

Care in data interpretation is required, however, since otherenvironmental factors might be involved, and additional researchis necessary to understand the effect of each heavy metal found invarious concentrations in the natural environments.

This study confirms that C. julis can be used successfully as anindicator species for monitoring the presence of contaminants.

Acknowledgments

This research was supported by Arpa Sicilia in 2005.

References

Azmitia, E.C., 2001. Modern views on an ancient chemical: serotonin effects on cellproliferation, maturation and apoptosis. Brain. Res. Bull. 56, 413–424.

Barnes, N.M., Sharp, T., 1999. A review of central 5-HT receptors and their function.Neuropharmacology 38, 1083–1152.

Berntssen, M.H.G., Aspholm, O.O., Hylland, K., Wendelaar Bonga, S.E., Lundebye,A.K., 2001. Tissue metallothionein, apoptosis and cell proliferation responsesin Atlantic salmon (Salmo salar L.) parr fed elevated dietary cadmium. Comp.Biochem. Physiol. C 128 (3), 299–310.

Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation byacidguanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem.162, 156–159.

Dang, Z.C., Lock, R.A.C., Flik, G., Wendelaar Bonga, S.E., 2000. Na+/K+-ATPaseimmunoreactivity in branchial chloride cells of Oreochromis mossambicusexposed to copper. J. Exp. Biol. 203, 379–387.

Depledge, M.H., 1994. Genotypic toxicity: implications for individuals andpopulations. Environ. Health Perspect. 102 (Suppl. 12), 101–104.

Evans, D.H., Piermarini, P.M., Choe, K.P., 2005. The multifunctional fish gill:dominant site of gas exchange, osmoregulation, acid–base regulation, andexcretion of nitrogenous waste. Physiol. Rev. 85, 97–177.

Fasulo, S., Mauceri, A., Giannetto, A., Maisano, M., Bianchi, N., Parrino, V., 2008.Expression of metallothionein mRNAs by in situ hybridization in the gills of Mytilusgalloprovincialis, from natural polluted environments. Aquat. Toxicol. 88, 62–68.

Ferrando, S., Ferrando, T., Girosi, L., Mauceri, A., Fasulo, S., Tagliafierro, G., 2005.Apoptosis, cell proliferation and serotonin immunoreactivity in gut of Lizaaurata from natural heavy metal polluted environments: preliminaryobservations. Eur. J. Histochem. 49 (4), 331–340.

Fossi, M.C. 1998. Biomakers: strumenti diagnostici e prognostici di saluteambientale. Ecotossicologia (UTET) Ed. Vighi e Bacci, 237.

Frenzilli, G., Falleni, A., Scarcelli, V., Del Barga, I., Pellegrini, S., Savarino, G.,Mariotti, V., Benedetti, M., Fattorini, D., Regoli, F., Nigro, M., 2008. Cellularresponses in the cyprinid Leuciscus cephalus from a contaminated freshwaterecosystem. Aquat. Toxicol. 89, 188–196.

Giari, L., Simoni, E., Manera, M., Dezfull, B.S., 2008. Histo-cytological responses ofDicentrarchus labrax (L.) following mercury exposure. Ecotoxicol. Environ. Saf.70, 400–410.

Hyndman, K.A., Choe, P.K., Havird, G.J., Rose, R.E., Piermarini, P.M., Evans, D.H.,2006. Neuronal nitric oxide synthase in the gill of the killifish, Fundulusheteroclitus. Comp. Biochem. Physiol. B 144 (4), 510–519.

Hogstrand, C., Wood, C.M., 1996. The physiology and toxicology of zinc in fish. In:Taylor, E.W. (Ed.), Toxicology of Aquatic Pollution. Cambridge UniversityPress, Cambridge, pp. 61–84.

Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J.,Saxena, P.R., Humphrey, P.P., 1994. International Union of Pharmacologyclassification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol.Rev. 46 (2), 157–203 Review.

Laurent, P., Dunel., S., 1980. Morphology of gill epithelia in fish. Am. J. Physiol.Regul. Integr. Comp. Physiol. 28 (3), 147–159.

Mauceri, A., Fasulo, S., Ainis, L., Licata, A., Lauriano, E.R., Martinez, A., Mayer, B.,Zaccone, G., 1999. Neuronal nitric oxide synthase (nNOS) expression in theepithelial neuroendocrine cell system and nerve fibers in the gill of the catfish,Heteropneustes fossilis. Acta Histochem. 101, 437–448.

Mauceri, A., Tigano, C., Ferrito, V., Barbaro, B., Calderaio, M., Ainis, L., Fasulo, S.,2002. Effect of natural confinement on the gill cell types and bony elements ofLebias fasciata (Teleostei, Cyprinodontidae): a morphological and immuno-histochemical analysis. Ital. J. Zool. 69, 195–203.

Mauceri, A., Fossi, M.C., Leonzio, C., Ancora, S., Minniti, F., Maisano, M., Lo Cascio, P.,Ferrando, S., Fasulo, S., 2005. Stress factors in the gills of Liza aurata(Perciformes, Mugilidae) living in polluted environments. Ital. J. Zool. 69(72), 285–292.

McCarthy, J.F., Shugart, L.R., 1990. Biomarkers of Environmental Contamination..Lewis Publishers, Boca Raton, USA 3-14.

Migliarini, B., Campisi, A.M., Maradonna, F., Truzzi, C., Annibaldi, A., Scarponi, G.,Carnevali, O., 2005. Effects of cadmium exposure on testis apoptosis in themarine teleost Gobius niger. Gen. Comp. Endocrinol. 142, 241–247.

Molina, A., Biemar, F., Muller, F., Iyengar, A., Prunet, P., Maclean, N., Martial, J.A.,Muller, M., 2000. Cloning and expression analysis of an inducible HSP70 genefrom tilapia fish. FEBS Lett. 474 (1), 5–10.

Pandey, S., Parvez, S., Ahamd, A.R., Ali, M., Kaur, M., Hayat, F., Ahmad, F., Raisuddin,S., 2008. Effects of exposure to multiple trace metals on biochemical,histological and ultrastructural features of gills of a freshwater fish, Channapunctata Bloch. Chem. Biol. Interact. 174, 183–192.

Pearse, A.G.E., 1985. Histochemistry, Theoretical and Applied Analytical Techno-logyChurchill Livingstone, Edinburgh.

Pinnegar, J.K., Polunin, N.V.C., 2000. Contributions of stable-isotope data toelucidating food webs of Mediterranean rocky littoral fishes. Oecologia 122,399–409.

Stronkhorst, J., Arlese, F., van Hattum, B., Postma, J.F., de Kluijver, M., Den Besten,P.J., Bergman, M.J.N., Daan, R., Murk, A.J., Vethaak, A.D., 2003. Environmentalimpact and recovery at two dumping sites for dredged material in the NorthSea. Environ. Pollut. 124, 17–31.

Sanchez, J.G., Speare, D.J., Johnson, G.J., 1997. Morphometric and histochemicalassessment of the branchial tissue response of rainbow trout, Oncorhynchusmykiss (Walbbaum), associated with chloramines-T treatment. J. Fish Dis. 20,375–381.

Stebbing, N., 1985. Therapeutic considerations for use of immunomodulators inthe treatment of ataxia–telangiectasia. Kroc Found. Ser. 19, 339–352.

Van Heerden, D., Tiedt, L.R., Vosloo, A., 2004a. Gill damage in Oreochromismossambicus and Tilapia sparrmanii after short-term copper exposure. Int.Cong. Ser. 1275, 195–200.

Van Heerden, D., Vosloo, A., Nikinmaa, M., 2004b. Effects of short-term copperexposure on gill structure, metallothionein and hypoxia-inducible factor-1a(HIF-1a) levels in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 69,271–280.

Viarengo, A., 1989. Heavy metals in marine invertebrates: mechanisms ofregulation and toxicity at the cellular level. CRC Crit. Rev. Aquat. Sci. 1,295–317.

Wendelaar Bonga, S.E., 1997. The stress response in fish. Physiol. Rev. 77,591–625.

Wendelaar Bonga, S.E., Van Der Meij, C.J.M., 1989. Degeneration and death, byapoptosis and necrosis, of the pavement and chloride cells in the gills of theteleost Orechromis mossambicus. Cell Tissue Res. 255, 235–243.

Yakimov, M.M., Denaro, R., Genovese, M., Cappello, S., D’Auria, G., Chernikova, T.N.,Timmis, K.N., Golyshin, P.N., Giluliano, L., 2005. Natural microbial diversity insuperficial sediments of Milazzo Harbor (Sicily) and community successionsduring microcosm enrichment with various hydrocarbons. Environ. Microbiol.7, 1426–1441.

S. Fasulo et al. / Ecotoxicology and Environmental Safety 73 (2010) 873–882882