Comparative Biochemistry and Physiology, Part C 169 (2015) 7–15

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Neurotoxicological effects on marine mussel Mytilus galloprovincialis caged at petrochemical contaminated areas (eastern Sicily, Italy): 1 H NMR and immunohistochemical assays Tiziana Cappello, Maria Maisano ⁎, Alessia Giannetto, Vincenzo Parrino, Angela Mauceri, Salvatore Fasulo Department of Biological and Environmental Sciences, University of Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy

a r t i c l e

i n f o

Article history: Received 29 October 2014 Received in revised form 17 December 2014 Accepted 23 December 2014 Available online 6 January 2015 Keywords: 1 H NMR metabolomics Caged mussels Cholinergic system Dopaminergic system Environmental pollution Gills Immunohistochemistry Serotoninergic system

a b s t r a c t The neurotoxicological potential of environmental pollution, mainly related to petrochemical activities, was investigated in marine mussel Mytilus galloprovincialis. Bivalve mollusks, particularly mussels, are widely used as sentinel organisms in biomonitoring studies for assessing the impact of anthropogenic contaminants. The gills, mainly involved in nutrient uptake, digestion, gas exchange and neuronal signaling, are the first organ to be affected by pollutants present in the external environment, and therefore were selected as the target organ for this study. Mussels from an aquaculture farm were caged at a highly polluted petrochemical area and a reference site along the Augusta coastline (eastern Sicily, Italy) for one month. A battery of biomarkers indicative of neuronal perturbations was applied on gills in order to investigate on the serotonergic (i.e. serotonin, 5-HT, and its receptor, 5-HT3R), cholinergic (i.e. acetylcholine, acetylcholinesterase, AChE, and choline acetyltransferase, ChAT), and dopaminergic systems (i.e. tyrosine and tyrosine hydroxylase, TH). Overall, impairment in the normal ciliary motility was found in mussels caged at the polluted site. Alterations in serotoninergic and cholinergic systems were revealed, with enhancement of dopaminergic neurotransmission resulting in a cilio-inhibitory effect. However, the over-expression in 5-HT3R and ChAT at cellular level may indicate an adaptive response of mussels to recover a regular physiological activity in gills. To our knowledge, this is the first study that uses 1 H NMR and immunohistochemical assays. Their concurrent use demonstrated to be sensitive and effective for assessing environmental influences on the health status of aquatic organisms, and thus suitable to be applied in ecotoxicological studies. © 2015 Elsevier Inc. All rights reserved.

1. Introduction It is generally recognized that the quality of coastal marine environments is greatly affected by a variety of anthropogenic activities. These activities result in the release of a mixture of contaminants into the aquatic ecosystems, and pose a threat to both animal and human health. In order to assess the harmful effects of environmental pollutants, field studies have been conducted on aquatic organisms (Cajaraville et al., 2000; Matozzo et al., 2005; Mauceri et al., 2005; Rank et al., 2007; Viarengo et al., 2007; Sureda et al., 2011; Cravo et al., 2012; Fasulo et al., 2012a, 2012b; Kwon et al., 2012), used as biological indicators of the health status of aquatic environments. Bivalve mollusks, particularly mussels, are widely used in biomonitoring studies because they are sedentary and sessile filter-feeders, having wide geographical distribution, ability to accumulate toxic chemicals, and suitability for caging experiments at field sites (Viarengo et al., 2007; Ausili et al., 2008; Fasulo et al., 2012b; Cappello et al., 2013a, 2013b). The use of caged mussels from a single population with uniform starting ⁎ Corresponding author. Tel.: +39 090 391435; fax: +39 090 6765556. E-mail address: [email protected] (M. Maisano).

http://dx.doi.org/10.1016/j.cbpc.2014.12.006 1532-0456/© 2015 Elsevier Inc. All rights reserved.

size/age, allows the assessment of the combined effects of environmental pollutant mixtures at locations where suitable natural populations are not available. Therefore the mussel caging, as an in situ approach, has become a recognized tool in ecotoxicological studies (Rank et al., 2007; Ausili et al., 2008; Tsangaris et al., 2010; Cappello et al., 2013a, 2013b). In our previous studies (Fasulo et al., 2012b; Cappello et al., 2013a, 2013b), we presented evidence of the adverse and compromising effects of petrochemical environmental pollution on mussel Mytilus galloprovincialis caged at the “Augusta–Melilli–Priolo” industrial site (eastern Sicily, Italy). It is one of the largest and most complex petrochemical sites in Europe, with very high levels of Hg and polycyclic aromatic hydrocarbons (PAHs) in sediments (Di Leonardo et al., 2007, 2008). After caging the mussels, we found high concentrations of naphthalene and fluoranthene, indicative of pyrolytic origin of the PAHs, and benzo(a)pyrene and dibenzo(a,b)anthracene, indicative of urban and industrial contamination, in mussel digestive gland tissue (Fasulo et al., 2012b). As biological effects of petrochemical pollution, we found severe morphological damage in the mussel digestive gland (Fasulo et al., 2012b) and gills (Cappello et al., 2013b), as well as metabolic disturbance investigated by proton nuclear magnetic resonance (1H NMR) spectroscopic analysis. In detail, mussel digestive

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Fig. 1. Map showing location of the mussel caging sites, Priolo (37°12′10″N; 15°13′44″E) and Vendicari (36°47′35″N; 15°08′52″E).

glands showed changes in metabolites involved in energy and lipid metabolism (Fasulo et al., 2012b), and activation of detoxification and antioxidant defense mechanisms, namely cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and catalase (CAT) as oxidative stress index (Cappello et al., 2013a). The changes observed in the 1H NMR metabolomic profile of gills suggested alteration in osmotic regulation, energy metabolism and neurotransmission (Cappello et al., 2013b). The gills of marine mollusks, mainly involved in nutrient uptake, digestion, gas exchange and neuronal signaling, have been used in ecotoxicological studies as indicators of aquatic pollution (Gregory et al., 2002; Gagné et al., 2007; Fasulo et al., 2008; Zhang et al., 2011; Ciacci et al., 2012; Cappello et al., 2013b). Gill activity is regulated by sympathetic and parasympathetic innervations of the autonomic nervous system running through the branchial connective tissue (Catapane et al., 1974), with various neurotransmitters participating in regulation of ciliary beating (Stefano, 1990). Serotonin, or 5-hydroxytryptamine (5-HT), involved in the serotonergic system, plays a cilioexcitatory activity in the gills of lamellibranchiates (Gosselin, 1961; Carroll and Catapane, 2007). The molluskan gill

Table 1 Details of primary antibodies. Antigen

Animal source

Supplier

Dilution

Serotonin (5-HT)

Mouse

1:50

Serotonin receptor (5-HT3R) Acetylcholine esterase (AChE) Choline Acetyltransferase (ChAT) Tyrosine hydroxylase (TH)

Rabbit

Dako Cytomation, Milan, IT Sigma-Aldrich, St. Louis, MO, USA Chemicon International, Temecula, CA, USA Abcam, Cambridge, UK Sigma-Aldrich, St. Louis, MO, USA

1:100

Mouse Rabbit Mouse

1:100 1:50 1:250

contains significant amounts of endogenous 5-HT, which produces prompt, sustained, and reversible stimulation of ciliary beat frequency, and the magnitude of the response is graded over a wide range of 5-HT levels (Gosselin, 1961). Impairment in the serotoninergic system may occur after exposure to toxicant compounds, as observed in gills of the mussel M. galloprovincialis exposed to chromium and copper, resulting in increase of the 5-HT-stimulated adenylate cyclase activity in vivo and over-expression of 5-HT receptors (Fabbri and Capuzzo, 2006). Acetylcholine is a neural transmitter used in efferent nervous systems, and it is synthesized in the cytoplasm of cholinergic neurons by the enzyme choline acetyltransferase (ChAT) and split into choline and acetate in cholinergic synapses and neuromuscular junctions by acetylcholinesterase (AChE) (Matozzo et al., 2005). AChE is essential for the normal functioning of the central and peripheral nervous system (Lionetto et al., 2013). The measurement of AChE activity is a validated biomarker of neurotoxic compounds in aquatic organisms (Cajaraville et al., 2000; Matozzo et al., 2005; Ciacci et al., 2012; D'Agata et al., 2014). AChE activity is directly inhibited by pollutants like organophosphate and carbamate pesticides (Lionetto et al., 2013), even though its responsiveness to other chemicals, such as heavy metals and hydrocarbons, has been demonstrated (Rank et al., 2007). Tyrosine is a non-essential amino acid that serves as substrate precursor for the synthesis of catecholamines, which include adrenaline, noradrenaline, and dopamine. The conversion of tyrosine into catecholamines is catalyzed by the enzyme tyrosine hydroxylase (TH), involved in the adrenergic–dopaminergic system that, in Mytilus, has an inhibitory effect on lateral cilia beating (Zhu et al., 2005). TH is a mixed-function oxidase that uses molecular oxygen and tyrosine as its substrates, and biopterin as its cofactor (Shiman et al., 1971). TH activity may be affected by toxic compounds present in the environments, as reported by Ciacci et al. (2012) with a noticeable dose-dependent increase of TH in M. galloprovincialis exposed to Cr(VI), resulting in an enhancement of dopaminergic neurotransmission. The aim of this study was to assess the neurotoxic potential of environmental pollution, mainly related to Hg and PAHs, in marine

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mussel M. galloprovincialis caged in an eastern Sicily petrochemical area. In order to extend our previous 1H NMR findings (Cappello et al., 2013b), a battery of biomarkers indicative of neuro-endocrinal perturbations was applied on mussel gills in order to investigate on the (i) serotoninergic (i.e. 5-HT and its receptor, 5-HT3R), (ii) cholinergic (i.e. acetylcholine, AChE and ChAT), and (iii) dopaminergic systems (i.e. tyrosine and TH). The environmental metabolomics, based on NMR spectroscopy, is a qualitative and quantitative approach that can be used to accurately determine metabolite concentration with a very high sensitivity (Hines et al., 2007; Espina et al., 2009; Kwon et al., 2012). To our knowledge, this is the first study that has used 1H NMR measurements of neurotransmitter concentrations in combination with immunohistochemical assays. 2. Materials and methods 2.1. Study area As reported previously (Ausili et al., 2008; De Domenico et al., 2011, 2013; Fasulo et al., 2012b), the petrochemical area of “Augusta–Melilli– Priolo” was declared a “site of national interest” by the Italian Ministry of Environment (Law No. 426/98; Ministerial Decree of 10.01.2000) owing to the high level of pollution, mainly due to Hg and PAHs, and subsequent risk for human health. Conversely, the natural reserve of Vendicari (southeastern Sicily) was considered as a reference site not known to be impacted by petrochemical contamination (Fig. 1). Physical and chemical parameters of water (i.e. temperature, salinity, pH, dissolved oxygen) were measured at both sampling sites at the cage deployment depth (8 m) three times, as reported in Cappello et al. (2013a). 2.2. Experimental design The experimental design was performed as detailed in our previous papers (Fasulo et al., 2012b; Cappello et al., 2013a, 2013b). Briefly, mussels M. galloprovincialis (mean shell length: 6.1 ± 0.54 cm) were purchased in October 2009 from an aquaculture farm in Goro (Ferrara, Italy), named Consortium of Fishermen. After maintaining in aerated seawater in the laboratory for one week, mussels were transplanted to the two selected sites at 8 m depth in stainless steel cages (about 200 specimens at each site) for 30 days. The translocation period of one month allows the adaptation of the organisms to the new environmental conditions, and it is used in a number of national and international Mussel Watch programs (i.e. RAMOGE). From each area, twelve male individuals, sexed by microscopic observation of gonad tissue, of comparable body length and mass (6.6 ± 0.46 cm shell length; 27.8 ± 3.2 g wet weight) were selected randomly and sacrificed. Gill samples were rapidly excised and flash-frozen in liquid nitrogen, then transferred to the laboratory and stored at −80 °C prior to the NMR analysis. Further, small pieces of each dissected tissue were taken for morphological and immunohistochemical analyses.

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Fig. 2. Western blot (WB) of proteins from (A) whole cell lysates and (B) mussel gill tissue extracts. SKBR-3, HeLa and PC-12 whole cell lysates were used as WB positive control for AChE, ChAT and TH, respectively.

phosphate, pH 7.0, containing 1 mM 2,2-dimethyl-2-silapentane-5sulfonate (DSS) (Sigma-Aldrich Co) as chemical shift standard. Fifty microliters of each resuspended sample was then pipetted into 4 mm-diameter zirconia rotors. NMR analyses were performed using a Bruker Avance-700 NMR spectrometer operating at 6000 Hz at 298 K, and equipped with a High Resolution-Magic Angle Spinning (HR-MAS) probe used for our convenience. One-dimensional (1-D) 1H NMR spectra were obtained as previously described (Cappello et al., 2013b), and manually phased, baseline-corrected, and calibrated (DSS at 0.0 ppm) using XWIN-NMR software (version 3.5; Bruker). The peaks of interest (i.e. serotonin, acetylcholine and tyrosine) within the 1H NMR spectra were identified and quantified using Chenomx Profiler, a module of Chenomx NMR Suite software (version 5.1; Chenomx Inc., Edmonton, Canada), which uses the concentration of a known DSS signal to determine the concentrations of individual metabolites (Kwon et al., 2012). 2.4. Immunohistochemical analysis Gill tissues of twelve mussels from each sampling site were fixed in 4% paraformaldehyde (Immunofix, Bio-Optica Milano, Italy) for 4 h, and then rinsed in 0.1 M phosphate buffered saline (PBS, pH 7.4). After dehydration in a graded series of ethanol and embedding in Paraplast (Bio-Optica Milano, Italy), 5 μm thick histological sections were cut with a rotary automatic microtome (Leica Microsystems, Wetzlar, Germany), mounted on glass slides and processed for indirect immunofluorescence method (Mauceri et al., 1999) for localization of 5-HT and its receptor, 5-HT3R, AChE and ChAT, and TH. Briefly, non-specific binding sites for immunoglobulins were blocked with normal goat serum (NGS) in PBS (1:5) by incubation for 1 h. The sections were then incubated overnight at 4 °C in a humid chamber in the presence of the primary antisera (dilutions and suppliers as indicated in Table 1). After a rinse in PBS for 10 min, binding sites of the primary antibodies were visualized by corresponding fluorescein isothiocyanate

2.3. 1H NMR-based metabolomic analysis Polar metabolites were extracted from gills (n = 12 per site) using a “two-step” methanol/chloroform/water procedure (Wu et al., 2008), as previously described (Cappello et al., 2013b). Briefly, the gill tissue (ca. 100 mg) was homogenized in 4 ml/g of cold methanol and 0.85 ml/g of cold water by an Ultraturrax homogenizer. The homogenates were transferred to glass vials, and 4 ml/g chloroform and 2 ml/g water were added. Samples were vortexed and centrifuged for 5 min at 2000 g at 4 °C. The upper methanol layer with the polar metabolites was transferred to glass vials, dried in a centrifugal vacuum concentrator (Eppendorf 5301) and stored at −80 °C. Prior to NMR analysis, the dried polar extracts were resolvated in 100 μl of D2O (Armar AG, Döttingen, Switzerland) buffered in 240 mM sodium

Fig. 3. 1H NMR measurements of serotonin (mM) from mussel gill tissue, expressed as means ± standard deviation (n = 12). Asterisks indicate significant differences relative to control (**p b 0.005).

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Table 2 1 H chemical shift and percent changes in neurotransmitter concentrations between polluted and reference site caged mussel groups, presented together with their significance (Student's t-test). Letters in parentheses denote the peak multiplicities: s, singlet; d, doublet; t, triplet; dd, doublet of doublet; m, multiplets. Metabolites (Chemical formula)

Chemical shift and peak shape (ppm)

Changes (%)

p-Value

Serotonin (C10H12N2O)

3.10(t), 3.30(t), 6.87(dd), 7.09(d), 7.28(s), 7.41(d) 2.15(s), 3.20(s), 3.75(t) 3.04(m), 3.18(m), 3.93(m), 6.89(d), 7.19(d)

42% ↓

b0.005

64% ↓ 35% ↓

b0.05 b0.05

Acetylcholine (C7H16NO2) Tyrosine (C9H11NO3)

(FITC)-conjugated goat anti-rabbit IgG (Sigma), and tetramethylrodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG (Sigma), diluted 1:50 for 2 h at room temperature. Positive controls for labeling specificity of each peptide were performed 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. In addition, negative controls were also performed by substitution of non-immune sera (without antibodies) for the primary antisera. All observations were made on five fields of one section per sample using a 40× oil-immersion objective with a motorized Zeiss Axio Imager Z1 epifluorescence microscope (Carl Zeiss AG, Werk Göttingen, Germany),

Fig. 4. Immunohistochemical investigation of the serotoninergic system in mussel gills. (A) Mussels caged at the reference site showed an intense 5-HT positivity in cells and fibers, while (B) individuals caged at Priolo displayed positive fibers. Scale bars, 20 μm. (E) Mean and standard deviation (S.D.) of 5-HT positive cells. Asterisk indicates significant differences relative to control (*p b 0.0001). (C) Control mussels displayed numerous 5-HT3R immunolabeled fibers along the branchial epithelium and very few positive cells, while (D) mussels caged at the pertrochemical site showed numerous positive cells and hemocytes, and few fibers. Scale bars, 20 μm. (F) Mean and standard deviation (S.D.) of 5-HT3R positive cells. Asterisk indicates significant differences relative to control (*p b 0.0001).

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equipped with an AxioCam digital camera (Zeiss, Jena, Germany) for the acquisition of images. Sections were imaged using the appropriate filters for the excitation of FITC (480/525 nm) and TRITC (515/590 nm), and then processed by using AxioVision Release 4.5 software (Zeiss). 2.5. Western blotting The specificity of the antibodies selected for this study was Western Blot (WB) tested in marine mussels. According to the manufacturer's recommendation, the antibodies suitable for use in WB analysis were only those against AChE, ChAT, and TH. SKBR-3, HeLa and PC-12 whole cell lysates were used as WB positive control for anti-AChE, anti-ChAT and anti-TH antibodies, respectively, as suggested by the antibody suppliers. Gill tissues were homogenized in a lysis buffer (20 mM Tris-HCl, 2% SDS, 5 M NaCl, 1 M MgCl2, 1 mM EDTA, 1 mM EGTA, pH 7.4) containing protease inhibitor mixture (Sigma-Aldrich) for 10 min at 50 vibrations/s using a TissueLyser LT bead mill (Qiagen) with 3.2 mm stainless steel beads, and centrifuged at 3000 g at 4 °C. The protein concentration of the supernatants was determined by using BCA Protein assay (Thermo scientific). Samples (40 μg total protein) were separated by SDS-PAGE (12%) and after electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane 0.45 μm (BioRad), successively stained with Ponceau red. After blocking the membrane in 5% bovine serum albumin in TPBS (PBS pH 7.4, 0.1% Tween 20), the blot was incubated overnight at 4 °C with appropriate polyclonal antibodies against different proteins (1 μg/ml). The blot was then washed and incubated with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to peroxidase (Sigma-Aldrich St. Louis, MO, USA). Antibody binding was detected by chemiluminescence staining using the ECL detection kit (BioRad). 2.6. Statistical analysis The concentrations of the three neurotransmitters of interest, i.e. serotonin, acetylcholine and tyrosine, were expressed in mM as means ± standard deviation (S.D.), and Student's t-test was applied to determinate significant differences between the two caged mussel groups induced by environmental pollutants, by using the GraphPad software (Prism 5.0, San Diego CA, USA). For 1H NMR-based metabolomic analysis, statistical significance was accepted at p b 0.05. Quantification of immunoreactive cells was performed by counting the number of positive cells on five fields per section using Axio Vision Release 4.5 software (Zeiss, Göttingen, Germany). All the obtained data were statistically processed with one-way analysis of variance (ANOVA) using the GraphPad software (Instat, La Jolla, CA, USA), applying Student's two-tailed t-test for unpaired data. For immunohistochemical analysis, statistical significance was accepted at p b 0.0001.

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fibers (Fig. 4B). Statistical analysis of immunohistochemical results for 5-HT (p b 0.0001) is shown in Fig. 4E. The immunodetection of the serotonin receptor in mussels caged at the reference site revealed a number of intensely immunolabeled fibers along the branchial epithelium and very few positive cells (Fig. 4C). On the contrary, the gills of mussels caged at the petrochemical site showed numerous cells, hemocytes underlying the gill epithelium, and few fibers immunopositive to 5-HT3R (Fig. 4D). Statistical analysis of immunohistochemical results for 5-HT3R (p b 0.0001) is shown in Fig. 4F. 3.2. Cholinergic system Proton NMR-based metabolomic analysis showed a significant decrease of 64% in acetylcholine concentration (p b 0.05) in gills of mussels transplanted at Priolo relative to mussels caged at the reference site (Fig. 5). Proton chemical shift and percent change in acetylcholine level are reported in Table 2. The immunohistochemical investigation of AChE in mussels caged at the reference site showed a high number of immunopositive cells, regularly distributed along the gill epithelium, and no AChE immunopositive fibers (Fig. 6A). Conversely, in gills of mussels transferred to the polluted area few cells limited to the area in which the structural integrity was maintained were immunopositivite to AChE. However, a great number of AChE-immunopositive hemocytes distributed along the epithelium was noticed (Fig. 6B). Statistical analysis of immunohistochemical results for AChE (p b 0.0001) is shown in Fig. 6E. By using the antibody directed against ChAT, a low number of cells and several fibers lining the branchial epithelium were found to be immunopositive in mussels caged at the reference site (Fig. 6C). On the contrary, in mussels caged at the petrochemical site a very high number of ChAT-immunopositive cells, mostly frontal cells situated at the apex of the gill filament, were observed (Fig. 6D). Statistical analysis of immunohistochemical results for ChAT (p b 0.0001) is shown in Fig. 6F. 3.3. Dopaminergic system Data from 1H NMR-based metabolomic analysis reported that tyrosine significantly declined by 35% (p b 0.05) in gills of mussels caged at the polluted site relative to mussels caged at the reference site (Fig. 7). Proton chemical shift and percent change in tyrosine level are reported in Table 2. In mussels caged at the reference site few TH-immunopositivite cells were detected along the gill epithelium (Fig. 8A), while in the gills of individuals acclimatized in Priolo a number of TH-immunopositive cells and hemocytes were observed, resulting in an intense TH immunoreactivity (Fig. 8B). Statistical analysis of immunohistochemical results for TH (p b 0.0001) is shown in Fig. 8C.

3. Results 3.1. Serotoninergic system A quantitative comparison of 1-D 700 MHz 1H NMR spectra of gill tissue extracts from the mussel M. galloprovincialis revealed a significant 42% depletion in serotonin level (p b 0.005) in mussels caged at the petrochemical area in respect to mussels caged at Vendicari (Fig. 3). Proton chemical shift and percent change in serotonin concentration are reported in Table 2. In the gill tissue of specimens caged at Vendicari an intense 5-HT immunoreactivity was seen in a number of ciliated cells and several fibers (Fig. 4A). In contrast, the expression of 5-HT in the gills of individuals acclimatized in Priolo was found almost solely along the

Fig. 5. 1H NMR measurements of acetylcholine (mM) from mussel gill tissue, expressed as means ± standard deviation (n = 12). Asterisk indicates significant differences relative to control (*p b 0.05).

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3.4. Western blotting

4. Discussion

Immunoreaction with AChE antibody revealed the predicted band of 68 kDa both in the SKBR-3 extract and mussel gills. Specific ChAT immunoreactive bands of molecular mass of 70 kDa, corresponding to the expected size for ChAT, were observed in both HeLa and mussel gill extracts. Finally, the antibody against TH in the PC-12 whole cell lysate revealed the presence of the predicted band of approximately 60 kDa. A similar band was visualized in mussel gill extract. The WB results for AChE, ChAT and TH are reported in Fig. 2.

The neurotoxicological potential of environmental pollution, mainly related to petrochemical activities, was assessed in the marine mussel M. galloprovincialis caged in an eastern Sicily industrial area. The gills, mainly involved in nutrient uptake, digestion, gas exchange and neuronal signaling, were chosen as the target organ. Evidences of metabolomic disturbances in neurotransmission were previously reported in Cappello et al. (2013b), therefore the neuronal perturbations on the serotoninergic, cholinergic, and dopaminergic

Fig. 6. Immunohistochemical investigation of the cholinergic system in mussel gills. (A) Mussels caged at the reference site showed a high number of AChE-immunopositive cells regularly distributed along the gill epithelium, while (B) in mussels caged at Priolo AChE-immunopositivity was reduced, and numerous hemocytes were observed. Scale bars, 20 μm. (E) Mean and standard deviation (S.D.) of AChE positive cells. Asterisk indicates significant differences relative to control (*p b 0.0001). (C) Control mussels displayed few ChAT-immunolabeled cells and several fibers along the gill epithelium, while (D) mussels caged at Priolo showed numerous positive cells mostly at the apex of the gill filament. Scale bars, 20 μm. (F) Mean and standard deviation (S.D.) of ChAT positive cells. Asterisk indicates significant differences relative to control (*p b 0.0001).

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systems were examined more closely, by the use of 1H NMR and immunohistochemical assays. The environmental metabolomics is a well-established technique based on the identification of low molecular weight endogenous metabolites within a biological system (i.e. cells, tissues, organs or whole individuals), whose production and levels vary in response to environmental stressors, diseases or exposure to toxicants (Viant et al., 2003; Iacono et al., 2010; Liu et al., 2011; Zhang et al., 2011; Fasulo et al., 2012b; Kwon et al., 2012; Cappello et al., 2013b). In this study, 1H NMR-based metabolomics measurements of the metabolites of interest from mussel gill extracts, allowed the successful investigation on the changes in neurotransmitter levels between mussels caged in the reference site and those transplanted in the polluted area in response to environmental stress. The significant decline in serotonin in gills of mussels caged at the petrochemical area relative to mussels caged at the reference site was revealed both by 1H NMR and immunohistochemical analyses. As a matter of fact, these results are consistent with the impairments in energy metabolism highlighted in our previous work (Cappello et al., 2013b), in which we supposed the occurrence of glycogenolysis that may be associated with compensatory mechanisms elicited by mussels in response to compromised gill ciliary activity, likely due to interferences of environmental pollutants with the serotoninergic system. In fact, it is well-known that serotonin is involved in hormonal and neuronal pathways, and plays a key role in cilioexcitatory activity, reproductive success and regulating food intake in invertebrates (Gosselin, 1961; Stefano, 1990). Noteworthy, a concomitant 5-HT3R over-expression at cellular level in mussels caged at the polluted area was detected, while in mussels caged at Vendicari solely nerve fibers were 5-HT3R immunopositive. The rise of 5-HT3R could be related to an adaptive response mediated by paracrine signaling activities in order to recover a regular physiological activity in gills. Indeed, it has been reported that gill epithelial cells containing the lateral cilia present 5-HT receptors to increasing the ciliary beating rate, if activated (Carroll and Catapane, 2007). Fabbri and Capuzzo (2006), after exposing mussel M. galloprovincialis to Cr(VI) and Cu(II), found increased 5-HTstimulated adenylate cyclase activity in vivo, and suggested the possibility that metal accumulation in mussel gills might induce the overexpression of 5-HT receptors. Further evidences of alterations in serotonin signaling pathways were provided by the significantly up-regulated transcription of 5-HT receptor in both sexes of M. galloprovincialis exposed to 0.1, 1, and 10 μg Cr(VI) L-1 animal−1 for 96 h, with males showing more sensitivity to metal treatment than females (Ciacci et al., 2012). Quantitative 1H NMR analysis showed a 64% significant decrease in the concentration of acetylcholine, a cholinergic neurotransmitter, in gills of mussels caged at Priolo in respect to mussels caged at Vendicari. In studies of ecotoxicology, measurements of AChE activity have been used to test for neurotoxicity in aquatic organisms since it can be inhibited by the presence of contaminants in complex mixtures (Matozzo et al., 2005; Cajaraville et al., 2000; Tsangaris et al., 2010; Cravo et al., 2012; D'Agata et al., 2014), by pollutants like organophosphate and carbamate pesticides (Lionetto et al., 2013), or heavy metals and hydrocarbons (Rank et al., 2007; Ciacci et al., 2012). As expected, in mussels caged at the petrochemical site of Priolo AChE immunopositivity was reduced, and limited almost solely to hemocytes. In marine mussels, hemocytes possess a complex cell signaling network with high homology with that of vertebrates, that allows them to modulate their own functions (Humphries and Yoshino, 2003; Franzellitti and Fabbri, 2013). However, little it is known on the physiological roles of these signaling pathways in mussel hemocytes. Also, the presence of ChAT, which enzymatic action results in synthesis of acetylcholine, implicating a negative correlation with AChE, was found noticeably increased. As a matter of fact, the inhibition of AChE and increment in ChAT are followed by accumulation of acetylcholine. However, in the present study the opposite result of

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reduced acetylcholine was found in gills of mussels transferred at the petrochemical site for one month, likely to be ascribed to an ongoing acetylcholine synthesis process. Similar findings have been showed by Zhang et al. (2011), which reported a decline in acetylcholine in gills of adult Manila clams Ruditapes philippinarum after a 24 h exposure to both low (10 μg L−1) and high (40 μg L−1) doses of Cu. The concentration of tyrosine was found significantly reduced by 35% in mussels caged at the petrochemical area in respect to mussels caged at the reference site. In the same individuals, the presence of the enzyme tyrosine hydroxylase (TH), which serves for conversion of tyrosine into catecholamines, was significantly induced in mussel gills. This data is consistent with the depletion of tyrosine, which is negatively correlated to the elevation of TH. Previous experimental studies have reported a noticeable dose-dependent increase of TH in the gills of M. galloprovincialis exposed to 0.1, 1, 10 μg Cr(VI) L− 1 animal−1 for 96 h (Ciacci et al., 2012). The increase in TH may result in the enhancement of dopaminergic neurotransmission that has an inhibitory effect on lateral ciliary activity of mussel gills (Carroll and Catapane, 2007). Zhu et al. (2005) demonstrated that tyrosine and tyramine may augment endogenous ganglionic morphine and dopamine levels in Mytilus edulis under in vitro and in vivo experiments, with the involvement of CYP2D6 and TH in the process, resulting in a cilioinhibitory effect.

5. Conclusions This study provides evidence that environmental pollution, mainly related to petrochemical activities, adversely affects the neurotransmission system in caged M. galloprovincialis individuals, resulting in impairment in the normal ciliary motility, and thus in feeding efficiencies. 1H NMR-based environmental metabolomics offers an alternative method of measuring metabolite concentration. Indeed, the high sensitivity of 1H NMR allowed measurement of changes in neurotransmitter levels, namely serotonin, acetylcholine and tyrosine. The depletion in neurotransmitter concentrations was supported by evidence of neuronal perturbations in mussel gills. In detail, alterations in serotoninergic and cholinergic systems were revealed, together with enhancement of dopaminergic neurotransmission, resulting in a cilio-inhibitory effect. However, the concomitant over-expression in serotonin receptors and choline acetyltransferase at cellular level may indicate an adaptive response mediated by paracrine signaling activities in order to recover a regular physiological activity in gills. Therefore, the concurrent use of 1 H NMR and immunohistochemical assays demonstrated to be sensitive and effective for assessing environmental influences on the health status of aquatic organisms, and thus suitable to be applied in ecotoxicological studies.

Fig. 7. 1H NMR measurements of tyrosine (mM) from mussel gill tissue, expressed as means ± standard deviation (n = 12). Asterisks indicate significant differences relative to control (*p b 0.05).

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Fig. 8. Immunohistochemical investigation of the dopaminergic system in mussel gills. (A) Control mussels showed a slight TH-immunopositivity in cells of the gill epithelium, while (B) in mussels caged at Priolo an intense immunoreactivity of TH was revealed, with a number of positive cells and hemocytes. Scale bars, 20 μm. (C) Mean and standard deviation (S.D.) of TH positive cells. Asterisk indicates significant differences relative to control (*p b 0.0001).

Acknowledgments This research was supported by a National Interest Research Project (PRIN 2007-20079FELYB).

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