Effects of “in vivo” exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax)

Aquatic Toxicology 105 (2011) 688–697

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Effects of “in vivo” exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax) Elena De Domenico a , Angela Mauceri a , Daniela Giordano b , Maria Maisano a , Giuseppina Gioffrè a , Antonino Natalotto a, Alessia D’Agata a, Margherita Ferrante c, Maria Violetta Brundo d, Salvatore Fasulo a,∗ a

Department Animal Biology and Marine Ecology, University of Messina, Viale Stagno D’Alcontres 31, 98166 Messina, Italy Institute for the Coastal Marine Environment, National Council of Research - Spianata S. Raineri 86, 98122 Messina, Italy c Department Hygiene and Public Health “G.F. Ingrassia”, University of Catania, Via S. Sofia 87, 95123 Catania, Italy d Department Animal Biology “M. La Greca”, University of Catania, Via Androne 81, 95124 Catania, Italy b

a r t i c l e

i n f o

Article history: Received 15 April 2011 Received in revised form 29 August 2011 Accepted 30 August 2011 Keywords: Toxic sediment exposure Teleost Gills Metals Biomarkers

a b s t r a c t Aquatic ecosystems are affected by all the impacts generated by a variety of anthropogenic activities present along coastal environments. The sediment compartment is the final receptor of water-insoluble pollutants, acting both as a sink and as a source of pollutants to the water column, and affecting both nektonic and benthic organisms. The aim of this study is to assess the impact of metals in the sediments collected from two sites in the petrochemical area between Augusta and Priolo (SR, Sicily, Italy) on gills of Dicentrarchus labrax. This was done to enhance the scarce knowledge on the bioavailability of metals bound to sediment and their capacity to interact with the bioindicator species. Various sublethal endpoints were assessed such as histopathological lesions, metallothioneins (MTs) and molecules involved in the homeostasis pathways by immunolocalization and RT-PCR. In the specimens exposed to sediments, the data suggested a reduction of gill cell membrane permeability, which could result in altered osmotic balance and gas exchange. Further, an increase of MT expression was detected, consisted the involvement of this protein in detoxification of toxic non-essential metals. The findings of this study demonstrate that a subchronic test, conducted by using sensitive and sublethal endpoints, in combination with chemical analyses, is a powerful tool for early identification of environmental hazards associated with contaminated sediments. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Aquatic environments, especially marine coastal areas and brackish inland waters, are greatly impacted by a variety of anthropogenic activities such as urban development. Because of a growing demand to reconcile development needs with conservation, it is important to further develop the use of techniques based on biological responses to pollution as a tool for assessing the quality of the environment (Fasulo et al., 2010a). In order to screen for potential toxicity of complex mixtures of chemicals present in environmental samples, short-term in vivo bioassays have been developed for ecotoxicological studies. Bioassays provide detailed information on the estimation of

∗ Corresponding author. Tel.: +39 090391435; fax: +39 0906765556. E-mail address: [email protected] (S. Fasulo). 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.08.026

total biological activity of chemicals and of potential hazards of impacts of contaminants, and for this reason they can be considered as an extension of chemical analyses (Vondracek et al., 2001). For example, sediment toxicity bioassays are instruments employed to evaluate the bioavailability of pollutants in sediments and their ecotoxicity on marine organisms. This kind of bioassay is performed by exposing the organisms to sediment samples collected in situ, and after a period of exposure the biological responses are measured (Morales-Caselles et al., 2006). Sediment toxicity studies are significant for two main reasons. First, several studies have demonstrated that sediments can absorb persistent and toxic chemicals to levels many times higher than water column concentrations, so that the sediment may become sufficiently polluted to disrupt natural biological communities (Adams et al., 1992; DelValls et al., 2002; Tolun et al., 2001). Second, metals and organic contaminants present in the sediment can accumulate in different organs and tissues and adversely affect

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fish health, predominantly targeting the gills, liver, spleen, kidney, reproductive organs and skin (Fasulo et al., 2008; Lauren et al., 1990). Fish gills are prime targets of toxic chemicals in the environment. They are the first organs to come in contact with environmental pollutants, and their xenobiotic detoxification systems are not as robust as that of liver (Oliveira et al., 2009; Pandey et al., 2008). Fish gills serve a variety of physiological functions, including respiratory gas exchange, osmoregulation, nitrogen excretion and acid–base regulation (Wendelaar Bonga, 1997). These different functions explain the structural complexity of this organ (Evans et al., 2005; Wilson and Laurent, 2002). They consist of several distinct cell types like specialized ion-transporting cells, the chloride cells (CCs)—also called mitochondria-rich cells (Brunelli et al., 2010). CCs are large round or ovoid cells rich in mitochondria and Na+ /K+ -ATPase, reflecting their extraordinary power of active ion transport (Hirose et al., 2003). Many authors consider the expression and activities of the Na+ /K+ -ATPase as strong indicators of osmoregulatory capabilities of fish, particularly in the sea-bass (Giffard-Mena et al., 2007; Varsamos et al., 2005). Since the movement of salt and water is inextricably linked, the evaluation of aquaporin expression in fish flanked with Na+ /K+ -ATPase was also investigated in several studies (Brunelli et al., 2010; Tse et al., 2006). Aquaporins (AQPs), indeed, are a family of water-specific channel proteins that regulate the water movement across the cell membranes and allow the transport of water and other solutes such as glycerol and/or urea, in the presence of osmotic or concentration gradients (Borgnia et al., 1999). The numerous functions of the gills require control and coordination by a complex web of neural, endocrine, and paracrine signaling pathways (Evans et al., 2005). Anatomical and histochemical studies have demonstrated that the bulk of autonomic neurotransmission in fish gills is attributed to cholinergic and adrenergic mechanisms (Mauceri et al., 2005; Zaccone et al., 2006), but more recent evidence indicates that nonadrenergic–noncholinergic (NANC) nerve fibers – such as those that utilize amine, peptide, or nitric oxide signals – may also play an important role. Little is known about the NANC neurotransmission, and studies on neuropeptides and NOS (nitric oxide synthase) are very fragmentary in the gills. In pioneering studies, Mauceri et al. (1999), demonstrated the presence of nNOS, the neuronal isoform of the enzyme involved in the production of nitric oxide (NO), in dispersed and clustered cells, and a well-developed system of nitrergic nerve fibers in gills of Indian catfish (Heteropneustes fossilis). NO is involved in the regulation of a variety of processes, e.g. oxygen homeostasis. Recent studies in mammals demonstrated that NO is involved in oxygen sensing and regulation of anaerobic energy production during oxygen-deficient states such as hypoxia (Benamar et al., 2008; Berchner-Pfannschmidt et al., 2007). While the important role of NO is well known for mammalian models, its involvement in metabolic regulation of lower vertebrate is still unknown. In the present study, specimens of sea bass (Dicentrarchus labrax), the most important marine non-salmonid species to be commercially cultured in Mediterranean areas (FAO, 2003) and key species for toxicological testing/evaluation as recommended by the Italian Environmental Protection Agency, were exposed to two sediments from the industrial area between Augusta and Priolo (SR, Sicily, Italy). The objective was to determine the adverse effects on gills associated with the contaminants bound to sediments. The investigated area has become one of the largest and most complex petrochemical sites in Europe, because of its several oil refineries, chemical plants, mineral deposits, a military base and many other industrial installations (Ausili et al., 2008).

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Table 1 Concentration of metals in the sediments A and B expressed in mg kg−1 dry weight.

Vanadium Chrome Manganese Cobalt Nickel Copper Zinc Arsenic Cadmium Antimony Lead Mercury Iron a

A

B

51.43 50.60 193.00 3.89 21.65 14.99 57.05 5.78 0.12 0.05 11.21 17.63a 18512.0

0.39 5.66 298.00 1.80 5.08 2.21 14.25 4.66 0.06 0.08 4.30 0.27 6329.0

All values that exceed SQG.

Specifically, the aim of this work was to assess the impact of metals bound to sediments coming from a highly contaminated coastal environment by means of chronic partial exposure toxicity tests (7 days) with D. labrax juveniles, in order to enhance the scarce knowledge on the bioavailability of metals and their capacity to interact with the bioindicator species.

2. Materials and methods 2.1. Sampling and metal determination The present study was carried out by using sediment samples collected from two sites in the petrochemical area between Augusta and Priolo. Two different sampling stations were chosen according to their geographical location, in relation to their closeness to the industrial center and the consequent potential rate of pollution. The sites were called: A (37◦ 13 12.56 N; 15◦ 12 15.69 E) close to the pollution sources and B (37◦ 12 0.04 N; 15◦ 13 36.8 E) located off the industrial belt and potentially the less contaminated. Sediment samples were collected with a stainless steel grab sampler. The top 5 cm oxic layer of the sediment was scooped with a plastic spoon, stored in double-layer polythene bags, and kept at 4 ◦ C for physico-chemical analysis. For the lab tests, 1 g of each sea sediment sample was mineralized in a microwave system Ethos TC (Milestone S.r.l., Italy) after tissue digestion using a heated mixture of strong acids. The method for sea sediments requires a digestion solution prepared with 6 ml of HNO3 65% (Carlo Erba) and 2 ml of HCl 37% (Carlo Erba) with a 50 operation cycle at 200 ◦ C. After mineralization, ultra pure water (Merck) was added to the samples up to 20 ml. They were then divided into two aliquots, each of 10 ml: one for Hg measurement and one for the other metals. The samples for Hg analyses were oxidized with 5% potassium permanganate (KMnO4 ), then neutralized with 1.5% hydroxylamine hydrochloride (NH2 OH HCl). This strong oxidation allows the total conversion of organic Hg into inorganic Hg. The test was performed with a FIAS 100 (Perkin-Elmer, USA) using the cold vapor capture technique. Quantification of the other metals was determined by an ICP-MS Elan 6100 DRC-e (Perkin-Elmer, USA). Analytical blanks were run in the same way as the samples and concentrations were determined using standard solutions prepared in the same acid matrix. Standards for the instrument calibration were prepared on the basis of mono-element certified reference solution ICP Standard (Merck). Such analyses allowed to characterize the concentrations of metals present in the A and B sediments (Table 1).

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Table 2 Details of primary antibodies used. Antigen

Supplier

Dilution

Animal source

Na+ /K+ -ATPase Water channel aquaporin 3 (AQP3) Neuronal nitric oxide synthase (nNOS) Metallothionein (MT)

Hybridoma Bank, IA, USA Sigma–Aldrich, St. Louis, MO, USA Biomol, Milan, Italy Dako, Denmark

1:100 1:100 1:100 1:50

Mouse Rabbit Rabbit Mouse

2.2. Fish maintenance and holding conditions D. labrax specimens, employed in this study, were provided by Acqua azzurra Factory Fish-Farming of Pachino (SR, Sicily, Italy) and acclimated to laboratory conditions for 60 days. During this period, fish were maintained in previously set-up 150-L capacity aquaria with seawater and equipped with filter and oxygenation systems. During the acclimation period, salinity (35‰), density (1.027–1.028 g/cm3 ), temperature (18–24 ◦ C) and nitrite and nitrate concentration were measured and kept constant (dissolved oxygen 8–8.6 mg/L; hardness 100 mg CaCO3 /L and absence of heavy metals). For the entire duration of the experiment, the animals were maintained under a natural light/dark cycle and fed every second day with commercial fish food. Animal maintenance and experimental procedures were in accordance with the Guide for Care and Use of Laboratory Animals (European Communities Council Directive 1986). 2.3. Exposure to contaminated sediments Fishes were exposed to the two sediments (A and B) collected from the petrochemical area between Augusta and Priolo, under the experimental conditions described above. The control group (n = 12) was maintained under the same conditions except for the source of the sediment, which was collected from the natural Reserve of Marinello (ME, Sicily, Italy; 38◦ 8 24.4 N; 15◦ 3 24.5 E), which served as the reference site. Fish of comparable body sizes (5 ± 1 g) were randomly assigned to the various exposure tanks containing 12 fishes each. For each test, 2 L of sediment were placed in 15 L polyvinyl tanks with 12 L of clean seawater and allowed to settle for 48 h prior to the start of the assays. Assays were performed in triplicate, and were maintained under constant aeration. Twenty-five percent of the total water volume was changed weekly. Sampling times were scheduled for days 2 (T2) and 7 (T7) from the beginning of the experiment. At each sampling time, four animals from each tank were anaesthetised with 2–4 g/L tricaine methane sulphonate (MS 222, Sandoz, Sigma, St. Louis, MO, USA), killed by spinal cord transection and measured for standard length and total wet weight. The gills were excised for the analyses of the different biomarkers. Animal manipulation was performed according to the Ethical Committee recommendations. 2.4. Histology and immunohistochemistry Morphological staining was carried out on histological sections (5 ␮m thick) using haematoxylin/eosin (H/E, Bio-Optica) (Mazzi, 1977). Sections were prepared from paraffin-embedded tissues. Sections from the same samples were also used for immunodetection of biomarkers with an immunofluorescence method (Mauceri et al., 1999). Non-specific binding sites for immunoglobulins were blocked by incubations for 1 h with normal goat serum (NGS) in PBS (1:5). The sections were incubated overnight in a humid chamber at 4 ◦ C with the following primary antibodies: anti-Na+ /K+ -ATPase ␣ and ␤ subunits, anti-AQP3, anti-nNOS and anti-MT, as listed in Table 2. After a rinse in PBS for 10 min, the sections were

incubated for 2 h at room temperature with fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG (Sigma) and tetramethyl rhodamine isothiocyanate (TRITC) conjugated goat anti-mouse IgG (Sigma). Labeling specificity of the peptides was verified by incubating sections with antiserum pre-absorbed with the respective antigen (10–100 g/ml). The pre-absorption procedures were carried out overnight at 4 ◦ C. Negative controls for immunohistochemical labeling were performed by substitution of blank sera (without antibodies) for the primary antisera. In order to identify five fields with the largest number of immunostained cells, a first observation of the tissue sections under a 20× objective was carried out. Then using 40× oil-immersion objective, the counting of the immunopositive cells was performed in each one of these fields. All observations were made with a Zeiss Axio Imager Z1 epifluorescence microscope (Carl Zeiss AG, Werk Göttingen, Germany), equipped with an AxioCam camera (Zeiss, Jena, Germany) for the acquisition of images. 2.5. RNA extraction and polymerase chain reaction (PCR) Total RNA was extracted from the gills of specimens using TRIzol LS reagent (Invitrogen, Carlsbed, CA, USA) (Chomczynski and Sacchi, 1987). Electrophoresis using 1.2% agarose gel under denaturating condition and quantification by Thermo Scientific NanoDropTM 2000 were performed to verify RNA quality. Ratios of absorbance 260/280 nm greater than 1.9 were considerate indicative of the high purity of RNA. The cDNA was synthesized using 4 ␮g of total RNA and oligo (dT)20 primer (150 pmol/␮l) (Invitrogen), by M-MLV reverse transcriptase (Invitrogen) as prescribed by the manufacturer’s instructions. An aliquot of 1 ␮l of the resulting cDNA was amplified in the PCR reaction. Specific primers were based on the AQP3 and MT cds of D. labrax. Primer sequences used for mRNA expression analyses of genes are listed in Table 3. The ␤-actin housekeeping gene of each examined organism was amplified using primers based on the actin cDNA sequence of D. labrax to obtain a fragment of about 700 bp. PCR was carried out using 2.5 ␮l of 10× buffer, 0.5 ␮l of 5 U/␮l BIOTAQ DNA Polymerase (Bioline), 2 ␮l of 50 mM MgCl2 , primers (50 ␮M each), 1 ␮l of cDNA template, 0.5 ␮l of 100 mM dNTPs, and Milli-Q water (Millipore, Vimodrone MI, Italy). The total reaction was performed in a 25 ␮l volume. The PCR program, used to amplify fragments of AQP3 and MT, consisted of an initial denaturation of 2 min at 95 ◦ C followed by 35 cycles of denaturation for 30 s at 95 ◦ C, annealing for 30 s at 55.2 ◦ C for AQP3 (56 ◦ C for MT), extension for 3 min at 72 ◦ C and a final extension period of 10 min. To perform the reaction Ep-Gradient Mastercycler (Eppendorf, Milano, Italy) was utilized. Table 3 Sequences of the primers. Gene

Forward primer

Reverse primer

AQP3 MT ACTIN

5 -GTTCTTCCAGATCCGTCACC-3 5 -GGAACCTGCAACTGCGGAG-3 5 -ATGAAGCCCAGAGCAAGAGA-3

5 -CTTCTTTGGAGTTGTCGTTGC-3 5 -TCACTGGCAGCAGCTCGTGT-3 5 -GGAAGGAAGGCTGGAAGAG-3

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Fig. 1. Haematoxylin and eosin (H–E) staining on histological sections of Dicentrarchus labrax gills. (a) H/E in control specimens showing a regular gill epithelium. (b) H/E in individuals exposed to A sediment for 48 h. Note the spiked secondary lamellae (arrow) and hyperplasia of filament. (c) H/E in specimens exposed to A sediment for 7 days. Note the epithelial cell hyperplasia. (d) H/E in specimens exposed to B sediment for 48 h showing the shortening of the lamellae. (e) H/E in specimens exposed to B sediment for 7 days showing the partial fusion of adjacent lamellae (asterisk). Scale bar = 20 ␮m.

2.6. Statistical analyses

3.2. Gill morphology

Immunohistochemical results were performed using fully automatic AxioVision Release 4.5 software, that facilitates counting of the positive cells. The intensities of band of MT and AQP3 were measured with Quantity One software (BioRad, Marnes-laCoquette, France). The analyses were carried out on each specimen tested. All obtained data were statistically processed with ANOVA system followed by Tukey’s post hoc test by GraphPad Instat software (GraphPad Software, La Jolla, CA, USA).

The general structure of the gills in control specimens showed a typical organization as described by Evans et al. (2005) (Fig. 1a). At T2 sampling time, both the treated groups presented hyperplasia of the gill filament, shortening of secondary lamellae, severe edema and cellular necrosis. In the group exposed to the A sediment, spiked secondary lamellae were also observed (Fig. 1b). After 7 days, in the specimens exposed to the A sediment the general morphology of gills was maintained, while in the specimens treated with the B sediment, damage was aggravated with fusion of secondary lamellae, loss of structure at several points and prominent hemorrhage (Fig. 1c).

3. Results 3.3. Induction of MTs 3.1. Analyses of sediments Metal content was measured in the sediments by ICP-OES and ICP-MS, except for mercury which content was measured by FIAS. The A sample is characterized by higher concentrations of all the metals except manganese than B sample, as shown in Table 1. The most relevant pollutant is Hg with a concentration very high compared to the international sediment quality guidelines (SQGs) that give chemical contaminant levels associated with biological effect (DelValls et al., 2004). The contaminants that exceed SQGs are highlighted in Table 1.

Immunohistochemical analysis in the branchial epithelium of control fish showed a reduced number of MT-immunopositive cells localized on the lamellae (Fig. 2a). In the specimens of the tanks with polluted sediments, at the first sampling time T2, numerous MT-immunopositive cells were revealed. These immunopositive cells were distributed regularly both along the filament and lamellae (Fig. 2b). After 1 week of treatment, there was an increase in the number of immunopositive cells in specimens taken from the tank with the A sediment, when compared with the T2 sampling time. In

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Fig. 2. Micrographs of histological gill sections labeled with MT-antibodies. (a) Control specimens showing no MT-immunopositive cells. (b) Specimens exposed to A sediment for 48 h revealing numerous MT-immunopositive cells (arrow) regularly distributed on the gills. (c) Specimens exposed to A sediment for 7 days showing a slight decrease in positive cells, mainly distributed along the filament. (d) Specimens exposed to B sediment for 48 h displaying numerous MT-immunopositive cells mostly along the lamellae. (e) Specimens exposed to B sediment for 7 days displaying immunoreactivity inhibition. Scale bar = 20 ␮m.

the tissue of the specimens exposed to the B sediment, a small decrease in the number of immunopositive cells, with respect to the T2 sampling time, was observed (Fig. 2c). Results of the RT-PCR indicated that bands of roughly 150 bp were amplified. The analyses of band intensity of MT gene in the specimens under treatment, showed significant induction at both T2 and T7 time periods in comparison to specimens from the control tank.

3.4. AQP3 and NA+ /K+ -ATPase AQP3 and Na+ /K+ -ATPase double-immunolabeling revealed the presence of numerous positive cells for both antibodies in the specimens from the control tank (Fig. 3a). At T2 in the organisms exposed to the polluted sediments, an inhibition of immunoreactivity for AQP3 was recorded in respect to the control. However, the immunoreactivity for Na+ /K+ -ATPase was quite similar to control. Immunopositive cells were regularly distributed along the main gill filament and in the interlamellar areas, but were not found in the lamellar epithelium. The pattern of AQP3 and Na+ /K+ -ATPase

distribution was quite similar in both the specimens exposed to the sediments and in those from the control tank (Fig. 3b). After a week from the beginning of the experiment, in the specimens exposed to the A sediment no significant differences were found in the number of AQP3 and Na+ /K+ -ATPase positive chloride cells (Fig. 3c). There was evidence of an increase of immunoreactivity for Na+ /K+ -ATPase in the specimens exposed to the B sediment, but there was a decrease in the immunoreactivity of AQP3 with respect to the control (Fig. 3d). Concerning the RT-PCR results of AQP3 gene at T2 sampling time, a band of major intensity was visualized in the specimens coming from the A tank compared with the individuals from the B tank, where the band appeared less intense than control. After 1 week, all the treated specimens showed significant induction with respect to specimens from the control tank.

3.5. nNOS The nNOS labeling showed a small number of immunopositive cells along the filament, while few nitrergic varicose fibers were

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Fig. 3. Immunocolocalization of Na+ /K+ -ATPase and AQP3 in the gills of Dicentrarchus labrax. (a) Specimens of control group displaying immunopositive cells both for Na+ /K+ -ATPase (green channel) and AQP3 (red channel) distributed along the filament. The colocalization of Na+ /K+ -ATPase and AQP3 is visible as yellow labeling due to the overlapping of the two channels (arrow). (b) Sea bass sampled from A tank at T2 showing a higher number of AQP3-immunopositive cells detectable as green label (arrowhead). (c) Sea bass from A tank at T7 showing a number of Na+ /K+ -ATPase immunopositive cells (asterisk) higher than AQP3-immunopositive cells, resulting in a preponderance of red signal. (d) Specimens exposed to B sediment at T2 revealing along the filament a great number of cells immunopositive to both the antibodies. (e) Specimens exposed to B sediment at T7 showing a noticeable decrease of AQP3-immunopositive cells and a great number of Na+ /K+ -ATPase immunopositive cells. Scale bar = 20 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

detected on the lamellae in the specimens from the control tank (Fig. 4a). After 48 h of treatment nNOS-positive fibers on the lamellae and immunoreactive neuroepithelial cells (NECs) distributed along the gill filament were present in the specimens exposed to sediments from both contaminated sites (Fig. 4b). There was a higher number of immunopositive NECs in the treated specimens than in control specimens, but this trend was greater for the T2 sampling time than the T7 sampling time. Few nitrergic varicose fibers were also found in treated specimens at T7 (Fig. 4c and d). 3.6. Statistical analyses Values of mean and standard deviation of both immunohistochemical and molecular analyses were calculated by statistical analyses. The differences between the two treated and control groups at T2 and T7 were considered significant at p < 0.05, p < 0.01 and p < 0.001. The values of mean ± S.D. were reported on graphics in Fig. 5.

4. Discussion The data obtained in this work provide a general framework of the health status of sea bass juveniles exposed to sediments collected from a natural environment, the industrial area of Priolo, which is strongly impacted by the presence of refineries, chemical industries, mineral deposits, a military base and many other industrial plants. The metal concentrations of the sediments used in this study were compared to the international sediment quality guidelines and found to be lower than the threshold values, except for the T-Hg concentration in the sediment collected in the area closer to the coast, which falls into the class of heavily polluted sediments in accordance with the data obtained by Ausili et al. (2008). Metals and organic contaminants present in the sediment are readily accumulated by aquatic organisms (Fasulo et al., 2010b; Livingstone, 1993; Mauceri et al., 2005). Fish gills are highly sensitive to metal exposure, since the absorption takes place primarily through this organ (Brunelli et al., 2011; Fasulo et al., 2010a), and the gill filaments and lamellae provide a very large surface area

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Fig. 4. Immunohistochemical labeling for nNOS in gills of Dicentrarchus labrax. (a) Control specimens showing few NECs (asterisk) along the filament and presence of fibers on the lamellae (arrow). (b) Specimens exposed to A sediment at T2 displaying a great amount of positive cells (asterisk) along the filament and fibers (arrow) along the lamellae. (c) Specimens exposed to A sediment at T7 revealing immunoreactivity inhibition of both nNOS-immunopositive fibers and cell. (d) Specimens exposed to B sediment for 48 h displaying a slight increase of positive cells (asterisk) distributed along the gill filament. (e) Specimens exposed to B sediment for 7 days showing few nitrergic varicose fibers (arrow) on the lamellae. Scale bar = 20 ␮m.

for direct and continuous contact with contaminants (MoralesCaselles et al., 2006). The histopathological analysis performed in this study indicated that there were serious lesions, such as hyperplasia of the gill filament, shortening of secondary lamellae and severe hemorrhagic phenomena after 2 days of exposure, followed by an increasing damage with fusion of secondary lamellae and loss of structure at several points in the specimens exposed to the B sediment. In contrast, the specimens exposed to the A sediment displayed a recovery of the histomorphological status of gills. Changes in gill morphology due to exposure to metals are found to be a compensatory response to keep metals from entering through gill cells (Evans, 1987). Fernandes et al. (2007) state that lamellar fusion and hyperplasia could be protective effects for diminishing the amount of vulnerable gill surface area.

However, most of the histopathologic lesions are non-specific and may not be attributed exclusively to metal exposure, and therefore only indicative of the general quality of the environment (Au, 2004). The gills have a large superficial area through which gaseous exchanges between the blood and the external medium take place (Newstead, 1987). The thin epithelial layer that covers the secondary lamellae represents the largest site for gaseous exchanges. The chloride cells, responsible for ionic exchanges, are usually concentrated in the primary lamellae, but are also distributed among the secondary lamellae under conditions of low ionic concentrations as demonstrated by Brunelli et al. (2010) and Fracacio et al. (2003). By co-localization of AQP3 and Na+ /K+ -ATPase in the specimens exposed to contaminants, a time-dependent inhibition of

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Fig. 5. Mean and standard deviation of immunopositive cell number and band intensity. Graphics show the results of the quantification of immunopositive cells and band intensity. The statistical analyses were carried out on sea bass gill samples collected at T2 and T7 from the tanks with A and B sediments. Asterisks indicate significant differences of the biomarker expression for each treated group at the two sampling times in respect to the control (***p < 0.001, **p < 0.01 and *p < 0.05).

AQP3 and increase of Na+ /K+ -ATPase were detected. Even if RTPCR results of AQP3 gene showed a partial increase, it is known that the expression of the transcripts may be transient and not always correspondent to protein content (Gornati et al., 2004). Studies have shown that exposure to metals can affect osmoregulation in fish, inhibiting the AQP3 water channel and Na+ /K+ -ATPase ionic pump. In particular, Savage and Stroud (2007) argue that the effect of mercury on AQP3 inhibition is consistent with a covalent modification of a cysteine within the selectivity filter region of the channel pore, resulting in a steric block. The increase of Na+ /K+ -ATPase immunopositive cells observed in this study could be explained by an increase of the chloride cell (CC) density, which could result from increase of CC proliferation (Wong and Wong, 2000), since it is known that Na+ /K+ pump is localized within these specialized branchial ion transporting cells (Cutler et al., 2000). Although the movement of salt and water are inextricably linked, the main route for water transport (either paracellular or transcellular) and the mechanisms controlling the acquisition, retention and excretion of water at the molecular level have still to be characterized (Cutler et al., 2000). However, the findings in the present study suggest that it is possible that heavy metals influence water permeability of fish gills, which could lead to an altered osmotic balance. Besides changes in aquaporin and Na+ /K+ -ATPase expression, the results of this study indicated perturbations of nNOS expression. The induction of NOS could be attributable to a local

inflammatory reaction or a stimulation of the stress response because, together with biogenic amines and cytokines, induction of nNOS is thought to represent a putative stress response (Kergosien and Rice, 1998; Smith et al., 2000). Furthermore, the expression of nNOS in cells adjacent to CCs suggests that NO may act as a paracrine signaling molecule to regulate gill ion transport, as well as gill perfusion, in fishes (Brunelli et al., 2011; Hyndman et al., 2006; Mauceri et al., 2002). Thus, nNOS may have an essential function in the regulation of basal blood vessel physiology influencing the oxygen intake in the gills. Hansen and Jensen (2010) highlighted the preference of goldfish to defend intracellular NO homeostasis during hypoxia, by observing tissue NO metabolites largely maintained at their tissue-specific values under hypoxia, pointing at nitrite transfer from extracellular to intracellular compartments and cellular NO generation from nitrite. However, few studies on the effects of pollutants on NO function have been carried out previously, hence additional experiments are necessary to determine the exact nature of these responses and the way in which NOS activity responds to chronic sublethal pollutant exposure (Smith et al., 2000). A further defensive mechanism against metal exposure, as indicated in this paper, was the induction of MTs. These metal-binding proteins play an important role in metal metabolism or detoxification of heavy metals (Fasulo et al., 2008; Wu et al., 2007), and their induction has been proposed as useful biomarker for ecotoxicological studies on aquatic animals (Alvarado et al., 2007). The results obtained from MT-immunodetection on gill tissues and the

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