Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Marine Environmental Research xxx (2013) 1e9

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Effects of environmental pollution in caged mussels (Mytilus galloprovincialis) Tiziana Cappello a, Maria Maisano a, *, Alessia D’Agata a, Antonino Natalotto a, Angela Mauceri a, b, Salvatore Fasulo a, b a b

Department of Animal Biology and Marine Ecology, University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy Centro Universitario CUTGANA, Via Terzora 8, 95027 San Gregorio di Catania, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 April 2012 Received in revised form 5 December 2012 Accepted 26 December 2012

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated. The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Environmental toxicology Coastal waters Eastern Sicily Mytilus galloprovincialis Digestive gland Biomarker Cytochrome Glutathione S-transferase Catalase

1. Introduction Coastal environments are heavily influenced by human activities, especially the highly productive zones such as harbours. These are generally enclosed areas characterized by poor water quality, reduced oxygen in the water column, and low biodiversity (GuerraGarcía and García-Gómez, 2005). Owing to the limited hydrodynamics on the inside portions of harbours and to the intense anthropogenic impact, which can be associated with release of environmental contaminants into the aquatic ecosystem, concerns have been raised over the potential health risks to humans and aquatic organisms. The “Augusta-Melilli-Priolo” industrial area has been chosen as an example of an anthropogenically impacted coastal marine environment for this study. It extends 20 km along the Augusta costal area (eastern Sicily, Italy) and is one of the largest and most complex petrochemical industry sites in Europe since it is characterized by oil refineries, chemical plants, mineral

* Corresponding author. Tel.: þ39 090 391435; fax: þ39 090 6765556. E-mail address: [email protected] (M. Maisano).

deposits, a military base and many other industrial installations (Ausili et al., 2008). Mercury (Hg) and polycyclic aromatic hydrocarbons (PAHs) are found in excessive concentrations (ICRAM, 2005). Levels of these contaminants exceed national and international regulatory guidelines, as reported in recent studies on sediments collected from the coastal zone of Augusta (Di Leonardo et al., 2008, 2007). Pollutant mixtures (heavy metals, drugs, PAHs, polychlorinated biphenyls PCBs) can induce toxic effects at different biological levels (e.g. biochemical, molecular, cellular, physiological). Since changes at the organism level lead to changes at the population and community levels, biomarkers can be used as early warning signals of environmental disturbance (Walker et al., 2006). In environmental monitoring studies, mussels, particularly the genus Mytilus, are widely used as sentinel organisms (Bebianno et al., 2007; Ciacci et al., 2012; Fasulo et al., 2008, 2012; Fernández et al., 2012; Hellou and Law, 2003; Manduzio et al., 2004; Shaw et al., 2011; Sureda et al., 2011; Viarengo et al., 2007). This is because of their wide geographical distribution, ability to tolerate a range of environmental conditions, accumulate toxic chemicals, and suitability for caging experiments at field sites

0141-1136/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2012.12.010

Please cite this article in press as: Cappello, T., et al., Effects of environmental pollution in caged mussels (Mytilus galloprovincialis), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2012.12.010

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T. Cappello et al. / Marine Environmental Research xxx (2013) 1e9

(Andral et al., 2004; Fasulo et al., 2012; Roméo et al., 2003; Tsangaris et al., 2010; Viarengo et al., 2007). The use of transplanted mussels originating from a non-polluted area allows comparison of control organisms with those caged in potentially polluted sites, and allows more control over the experiment than collection of native individuals. In addition, using caged mussels from a single population minimizes confounding factors such as the age and reproductive status of the organisms that influence both contaminant bioaccumulation and biomarker responses. Thus, a more accurate assessment of the real biological effects of pollutant exposure is possible, providing an early sign of impaired health of the ecosystem (Andral et al., 2004; Regoli, 2000; Tsangaris et al., 2010; Viarengo et al., 2007). The digestive gland of molluscs has been known as a target organ for pollution effects because it accumulates contaminants and plays a major role in xenobiotic metabolism (Rajalakshmi and Mohandas, 2005). It is also involved in immune defense, detoxification and in homeostatic regulation (Marigomez et al., 2002; Moore and Allen, 2002), and therefore exposure to contaminants may lead to its histopathological alteration (Fasulo et al., 2012; Garmendia et al., 2011). The mechanism of detoxification of organic pollutants consists of two steps essential to modify the toxicity of lipophilic xenobiotics, converting them to water-soluble and easily excretable metabolites (Kleinow et al., 1987; Livingstone, 1993). Phase I is usually catalysed by cytochrome P450 (CYP)-dependent monooxygenases, and represent the introduction of functional groups. The phase I metabolites can then act as substrates for phase II enzyme reactions, also known as conjugation pathways (Buhler and Williams, 1988; Daly, 1995). Hence, induction and/or inhibition of certain enzymes may indicate presence of biologically significant levels of xenobiotics, and thus enzyme measurement is strongly recommended in environmental monitoring programs (Bebianno et al., 2007; Bucheli and Fent, 1995; Fasulo et al., 2010; Iacono et al., 2010; Sureda et al., 2011). In particular CYP4Y1, a gene belonging to the CYP4 subfamily, was recently identified in the digestive tissues of Mytilus galloprovincialis, and its expression was inhibited after exposure to beta-naphthoflavone, a typical hydrocarbon inducer of cytochrome P450 (Snyder, 1998), and degraded oil (Snyder et al., 2001). Different isoforms of glutathione S-transferase (GST) have been studied in the blue mussel Mytilus edulis (Fitzpatrick and Sheehan, 1993). GST is involved in phase II of biotransformation, and thus in the detoxification of many environmental chemicals by catalyzing the conjugation of glutathione (GSH) to electrophilic compounds (e.g. epoxides of PAHs), rendering them less reactive and more water-soluble (Cheung et al., 2001; Pan et al., 2009). Another important mechanism of toxicity associated with xenobiotic products is oxidative damage (Sanchez et al., 2005). The cellular defense system against oxidative damage comprises enzymatic antioxidant defenses such as catalase (CAT), which is an oxidoreductase enzyme that catalyzes the conversion of hydrogen peroxide into water and oxygen. It can be used as a biomarker of oxidative stress, although it does not respond specifically to a group of pollutants. Indeed, CAT can be induced by a wide range of contaminants, including organic xenobiotics, heavy metals and PAHs (Altenburger et al., 2003; Livingstone, 2001; Roméo et al., 2003; Sureda et al., 2011). The aim of this study was to assess the biological effects of environmental pollution, mainly related to the presence of PAHs, in an eastern Sicily petrochemical area in caged mussel M. galloprovincialis. In particular, phase I and phase II biotransformation (CYP4Y1 and GST, respectively) and antioxidant (CAT) enzymes were evaluated, as well as the content of PAHs in digestive glands of mussels.

2. Materials and methods This study was conducted according to the guidelines for the protection of animal welfare, in compliance with the Italian National Bioethics Committee (INBC). 2.1. Study area The “Augusta-Melilli-Priolo” industrial area, chosen as polluted site for this study, has been classified as a “site of national interest” by the Italian Ministry of Environment (D.M. 10.01.2000; L. 09.12.1998) due to the high level of pollution and subsequent risk for human health. By contrast, the Natural Reserve of Vendicari, established as a wildlife reserve in the southernmost part of the east coast of Sicily in 1984, was chosen as a reference site not impacted by petrochemical contamination. It covers an area of 1512 ha (575 ha of core reserve and 937 ha of buffer zone) and its biological importance is based on the presence of different biotopes, e.g. rocky and sandy coastlines, Mediterranean shrub, both salt and fresh water marshes (Fig. 1). At both sampling sites, water physicoechemical parameters (i.e. temperature, salinity, pH, dissolved oxygen) were measured at the deployment depth (8 m) three times, at the beginning of the experimental study before cage placement (mid October 2009), two weeks after and then at the end of the 30-day deployment period before retrieving the cages, using a portable instrument (Multi 340i/SET, WTW WissenschaftlichTechnische Werkstätten GmbH, Weilheim, Germany). Data are reported as mean  standard deviation (S.D.) in Table 1. 2.2. Experimental design Mussels, M. galloprovincialis (6.1  0.54 cm shell length), were purchased in October 2009 from a consortium of fishermen of Goro (Ferrara, Italy), which physicoechemical parameters have been previously reported (Fasulo et al., 2008). Mussels were maintained for one week in aerated seawater in the laboratory and then transplanted to the two selected sites for 30 days in stainless steel cages (one cage containing about 200 specimens at each location) covered with a net to guarantee free circulation of seawater and to protect mussels from predation. Cages were immersed by scubadiving at 8 m depth below the surface both in Priolo (371201000 N; 15130 4400 E) and Vendicari (36 470 3500 N; 15 080 5200 E). The mussels were retrieved after 4 weeks by diving. From each area, fifteen 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. Digestive gland samples were rapidly excised and flash-frozen in liquid nitrogen for chemical, molecular and enzymatic measurements, then transferred to the laboratory and stored at 80  C prior to analysis. Further, small pieces of each dissected tissue were taken for histological and immunohistochemical analyses. 2.3. PAHs analysis in digestive gland For PAHs analysis, approximately 3 g of three pooled samples (each with tissues of 5 specimens) per site were weighted, homogenized using an Ultraturrax homogenizer and then saponified with 10 ml of 1 M KOH in an ethanol solution for 3 h at 80  C. Then 20 ml of cyclohexane were added and samples mixed by an orbital agitator for 10 min using dark glassware (Dafflon et al., 1995). The hexanic phase was recovered and the polar mixture washed once with cyclohexane and then discharged. The extracts were filtered, concentrated under a nitrogen gas stream to about 1 ml, and the concentrated extract was removed with a pasteur pipette and loaded into a Varian Bond Elut C18 cartridge 12 ml, previously

Please cite this article in press as: Cappello, T., et al., Effects of environmental pollution in caged mussels (Mytilus galloprovincialis), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2012.12.010

T. Cappello et al. / Marine Environmental Research xxx (2013) 1e9

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Fig. 1. Map depicting location of the consortium of fishermen (Goro) and mussel caging sites (Vendicari and Priolo).

conditioned. The eluates were dried under a nitrogen stream and dissolved in 1 ml of acetonitrile (ACN) before the analysis. The concentrations of the following sixteen PAHs identified by the EPA as priority pollutants, naphthalene (NA), acenaphthylene Table 1 Mean (S.D.) of water physicoechemical parameters of Vendicari and Priolo. Sampling area

Vendicari

Temperature ( C) Salinity (PSU) pH Oxygen (mg/1)

23.4 37.6 8.0 4.8

   

0.5 0.1 0.1 0.2

Priolo 22.5 38.2 7.9 3.7

   

0.6 0.2 0.1 0.3

(ACY), acenaphthene (AC), fluorene (FL), phenanthrene (PHE), anthracene (AN), fluoranthene (FA), pyrene (PY), benzo(a)anthracene (BaA), chrysene (CH), benzo(b)fluoranthene (BbF), benzo(k) fluoranthene (BkF), benzo(a)pyrene (BaP), dibenzo(a,h)anthracene (DahA), benzo(g,h,i)perylene (Bghi) and indeno(1,2,3-cd)pyrene (IP), were determined. Quantitative analysis of PAHs was carried out with a Varian high-performance liquid chromatography (HPLC) equipped with a 20 ml loop and a fluorescence detector (FLD ProStar 363). The software used was Star Chromatography Workstation version 5.2 (Varian, Palo Alto, CA). The analytical method involved a mobile phase consisting of H2O/ACN 50% for 5 min, which achieved 100% ACN in 5 min with

Please cite this article in press as: Cappello, T., et al., Effects of environmental pollution in caged mussels (Mytilus galloprovincialis), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2012.12.010

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a flow of 1 ml/min. The UV determination was performed at 255 nm, while the fluorescence (FL) detection was conducted with six different excitation/emission wavelengths. The National Institute of Standards and Technology (NIST) Standard Reference Material SRM 1647c, consisting of an acetonitrile solution of sixteen PAHs (target compounds), was used as a calibration mixture. Percent recovery and matrix interference was assessed with reference M. galloprovincialis tissue. The external standard multipoint calibration technique was used to determine the linear response interval of the detector and in all cases, regression coefficients were higher than 0.996 for all the analytes detected by UV, and higher than 0.989 for all the analytes detected in FL. 2.4. Histological analysis For histological assessment, digestive gland tissues of 15 mussels from each sampling site were fixed in 4% paraformaldehyde in 0.1 M phosphate buffered solution (pH 7.4) at 4  C, dehydrated in ethanol and embedded in Paraplast (Bio-Optica, Italy). Histological sections, 5 mm thick, were cut with a rotary automatic microtome (Leica Microsystems, Wetzlar, Germany), mounted on glass slides and stained with Hematoxylin/Eosin (Bio-Optica, Italy) to assess morphological features. Observations were made on five fields of one section per sample using a 40 oil-immersion objective with a motorized Zeiss Axio Imager Z1 microscope equipped with an AxioCam digital camera (Zeiss). 2.5. Immunohistochemical analysis

were performed to verify RNA quality. Ratios of absorbance at 260/ 280 nm of greater than 1.9 were considerate as indicative of the high purity of RNA. 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. The sequences of the CYP4Y1 primers, designed on the sequences present in Genebank (n AF072855), were 50 -AGGCTTTCACCAGTTCC30 for the sense primer and 50 -TCCGGCAGAAATGGAGTAAA-30 for the antisense primer, amplifying a 172 bp sequence. Actin (used as a positive control) of each examined specimen was amplified usingsequence primers based on the actin cDNA sequence of M. galloprovincialis to obtain a fragment of 200 bp. Oligonucleotides were synthesized and purified by MWG Biotech AG. Polymerase chain reactions were performed using: 2.5 ml of 10 buffer, 0.13 ml of 5 U/ml Poly Taq polymerase (Invitrogen), 0.8 ml of 50 mM MgCl2, primers (50 mM each),1 ml of cDNA template, 0.5 ml of 10 mM dNTPs and Milli-Q water (Millipore). The total reaction was performed in a 25 ml volume. The amplification conditions were as follows: 95  C for 2 min, 35 cycles of [95  C for 30 s, 56  C for 30 s, 72  C for 30 s] and a final extension at 72  C for 5 min. The Ep-Gradient Mastercycler (Eppendorf) was used. RT-PCR products were separated and analyzed by electrophoresis on SYBR Safe-stained agarose gel. The band intensity of CYP was measured with Quantity One software, and then normalized with the expression of cytoplasmatic actin. 2.7. Enzyme analysis Digestive glands of 10 mussels per sampling site were homogenized in 1:10 (w/v) 100 mM TriseHCl buffer, pH 7.5. Homogenates were then centrifuged at 9000 g at 4  C for 15 min (Manduzio et al., 2004). After centrifugation, supernatants were collected and immediately used for the determination of CAT activity. All assays were performed in duplicate and results were based on the total protein amount, estimated using the Pierce BCA Protein Assay Kit (Thermo Scientific). CAT activity (mK/mg protein) was measured by the method of Aebi (1984) based on the decomposition of hydrogen peroxide, using a UV Mini 1240 spectrophotometer (Shimadzu, Milano, Italy).

On histological sections of digestive gland tissues from 15 mussels per sampling site, an indirect immunofluorescence method was applied for localization of the CYP4 isoform, part of a sequence that is unique for the CYP4-family (Simpson, 1997) and confirmed in M. galloprovincialis CYP4Y1 (Jonsson et al., 2006), and GST. Paraffin wax-embedded sections were dewaxed and rehydrated by conventional protocols (Mauceri et al., 1999). After several rinses in Phosphate Buffered Saline (PBS), sections were then incubated overnight at 4  C in a humid chamber for immunofluorescence labeling with the pre-diluted rabbit polyclonal antiCYP4 antibody (kindly provided by Prof. Anders Goksøyr, University of Bergen, NO) and with a rabbit polyclonal anti-GST antibody (Sigma, Saint Louis, Missouri, US) diluted 1:100 (Ciacci et al., 2012; Fasulo et al., 2010; Jonsson et al., 2006). Binding sites of the primary antibodies were visualized by corresponding fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma), diluted 1:100 for 2 h at room temperature. Negative controls for the immunohistochemical labeling were performed by substitution of non-immune sera for the primary or secondary 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 microscope equipped with an AxioCam digital camera (Zeiss). Sections were imaged using the appropriate filter setting for the excitation of FITC (480e525 nm), and then processed by using AxioVision 4.5 (Zeiss) software.

Immunoreactive cell quantification was performed by counting the number of positive cells on five fields per section using Axio Vision Release 4.5 software (Zeiss, Göttingen, Germany). The band intensity of CYP was measured with Quantity One software (BioRad, Marnes-la-Coquette, France), while a statistical package (SPSS 12.0 for Windows) was used for the enzymatic analysis. Results were expressed as mean  S.D. All the obtained data were statistically processed with one-way analysis of variance (ANOVA) using Graph Pad software (Instat, La Jolla, CA, US), applying Student’s two-tailed t-test for unpaired data, and statistical significance was accepted at P < 0.0001 for immunohistochemical analysis, P < 0.001 for molecular data, and P < 0.05 for enzymatic results.

2.6. Molecular analysis of CYP4Y1

3. Results

Total RNA was extracted from the digestive glands of 10 specimens per sampling site using TRIzol LS reagent (Invitrogen, Carlsbad, California, US) (Chomczynski and Sacchi, 1987). Electrophoresis using 1.2% agarose gel under denaturating condition and quantification by Thermo Scientific NanoDropÔ 2000

3.1. PAHs content

2.8. Statistical analysis

The recovery for PAHs molecules containing from 2 to 5 condensed rings was from 90% to 97%, while for the remaining, the recovery was from 99% to 100%.

Please cite this article in press as: Cappello, T., et al., Effects of environmental pollution in caged mussels (Mytilus galloprovincialis), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2012.12.010

T. Cappello et al. / Marine Environmental Research xxx (2013) 1e9 Table 2 PAHs concentrations (mean  S.D.) in pooled samples of digestive glands (mg/g wet wt; n ¼ 3). PAHs

Priolo

Vendicari

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Dibenzo(a,h)anthracene Benzo(g,h,i)perylene Indeno(1,2,3-cd)pyrene

0.912 ± 0.038 0.011  0.007 0.007  0.002 0.009  0.005 <0.006 <0.006 0.187 ± 0.012 0.008  0.004 <0.006 n.d. 0.012  0.007 0.008  0.002 0.060 ± 0.017 0.925 ± 0.028 <0.006 <0.006

n.d n.d n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

PAHs with high values are shown in bold. n.d. ¼ not detectable.

PAH concentrations for all analytes in the digestive gland samples from the reference site were lower than the instrument detection limit. By contrast, samples from Priolo showed elevated levels of PAHs, mainly naphthalene (0.912 mg/g) and fluoranthene (0.187 mg/g) among light PAHs, and benzo(a)pyrene (0.06 mg/g) and dibenzo(a,b)anthracene (0.925 mg/g) among high molecular weight PAHs, as reported in Table 2. 3.2. Histological observations The digestive gland of M. galloprovincialis caged in the reference site (Fig. 2A) showed the typical organization of the digestive diverticula of bivalves, as described by Owen (1970). In contrast, in mussels from the polluted area the histological integrity of the digestive gland tissue was poor (Fig. 2B). The tissue appeared severely damaged, and massive haemocytic infiltration was observed among digestive tubules. 3.3. Cytochrome CYP4 isoform In the digestive gland tissue of control specimens an intense positive immunoreactivity for cytochrome P450 4 (CYP4) was seen along the epithelium, consisting of digestive and basophilic cells, which lines the digestive tubules (Fig. 3A). In contrast, a drastic

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CYP4-inhibition was found in the digestive gland of individuals acclimatized in Priolo (Fig. 3B). Statistical analysis of immunohistochemical results (P < 0.0001) is shown in Fig. 3C. RT-PCR products showed discrete bands of 172 bp for CYP4Y1. The expression of cytoplasmatic actin was expressed at basal levels both in control animals and in those from Priolo. CYP4Y1 gene was highly expressed in specimens caged in the unpolluted site, whereas it was expressed at a lower level in the digestive glands of mussels caged at the industrial area. CYP4Y1 band intensity differed significantly between animals transferred to the two selected sites (P < 0.001; Fig. 3E). 3.4. Glutathione S-transferase Immunohistochemical labeling of glutathione S-transferase revealed no positive cells in the digestive glands of specimens from the reference site (Fig. 4A). In contrast, digestive glands from specimens caged in the petrochemical area of Priolo showed a highly positive GST immunostaining (Fig. 4B). Statistical analysis of immunohistochemical results (P < 0.0001) is shown in Fig. 4C. 3.5. Catalase Significantly greater CAT activity was recorded in mussels caged at the site influenced by anthropogenic activities (238 mK/mg protein) compared to the reference site (139 mK/mg protein) (P < 0.05; Fig. 5). 4. Discussion Biological effects of environmental pollution, mainly related to the presence of PAHs, were assessed in mussel M. galloprovincialis caged in an eastern Sicily petrochemical area. The use of mussel caging demonstrated to be an effective and useful tool for assessing the environmental quality status and biological effects induced by xenobiotics (Andral et al., 2004; Regoli, 2000; Roméo et al., 2003). The digestive gland, a primary organ for bioaccumulation and involved in pollutant detoxification and homeostasis maintenance (Marigomez et al., 2002; Moore and Allen, 2002), was chosen as target organ for this study. Chemical analyses performed in mussel digestive gland tissue revealed higher levels of PAHs in individuals caged in the

Fig. 2. (AeB) Hematoxylin and Eosin (H&E) staining in the digestive gland of Mytilus galloprovincialis caged in the reference site (A) compared with those transferred to the polluted area (B), which displayed severe histopathological alterations and relevant aggregations of haemocytes (arrows) among digestive tubules. Scale bars, 20 mm.

Please cite this article in press as: Cappello, T., et al., Effects of environmental pollution in caged mussels (Mytilus galloprovincialis), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2012.12.010

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Fig. 3. (AeB) Immunohistochemical labeling for CYP4 in mussel digestive glands. (A) Mussels caged in the reference site displayed an intense immunoreactivity along the epithelium lining the digestive tubules (arrows), while (B) individuals caged in Priolo showed a drastic CYP4 inhibition. Scale bars, 20 mm. (C) Mean and standard deviation (S.D.) of immunopositive cells. Significant difference (P < 0.0001) between control and polluted site is indicated by an asterisk. (D) Electrophoresis on SYBR Safe-stained agarose gel with, from right to left, CYP4Y1 RTePCR products of M. galloprovincialis (n ¼ 10) caged in Vendicari and Priolo, respectively; 500 bp molecular weight marker (gene Ruler Fermentas); actin RTePCR products of M. galloprovincialis caged in Vendicari and Priolo, respectively. (E) Mean and standard deviation (S.D.) of the ratio between CYP4Y1 and actin band intensity in polluted and control environment. Significant difference (P < 0.001) between control and polluted site is indicated by an asterisk.

petrochemical area of Priolo compared to those acclimatized in the Natural Reserve of Vendicari, the reference site. In detail, high concentrations of naphthalene and fluoranthene, consistent with a dominantly pyrolytic origin of PAHs, and benzo(a)pyrene and dibenzo(a,h)anthracene indicative to urban and industrial contamination, were recorded. These findings provide thus an assessment of contaminant levels entering the food chain, and confirm

the presence of PAHs in the industrial area investigated. PAHs considered in this study are mentioned as generic carcinogenic PAHs (2006), and/2006), and are frequently monitored according to recommendations by the EU Scientific Committee for Food (SCF), the European Union (EU), and the US Environmental Protection Agency (EPA). In reference to PAHs found with the highest values in this study, according to IARC (International Agency for Research on

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Fig. 4. (AeB) Immunohistochemical labeling for GST in mussel digestive glands. (A) In mussels caged in the reference site no immunopositive cells were detected, while (B) individuals caged in Priolo showed a highly positive GST immunostaining in secernent and absorbent digestive gland cells (arrows). Scale bars, 20 mm. (C) Mean and standard deviation (S.D.) of immunopositive cells. Significant difference (P < 0.0001) between control and polluted site is indicated by an asterisk.

Cancer) classification for carcinogenicity, benzo(a)pyrene belongs to group 1 (carcinogenic to humans), dibenzo(a,h)anthracene to group 2A (probably carcinogenic to humans), naphthalene to group 2B (possibly carcinogenic to humans), and fluoranthene to group 3 (not classifiable as to its carcinogenicity to humans). As a consequence of PAHs presence in the polluted area, digestive glands from mussels caged in Priolo displayed significant histological lesions with altered diverticula morphology and conspicuous haemocytic infiltration, which might result in impairment of its metabolic activities, as reported for other organs of M. galloprovincialis (Ciacci et al., 2012). Previous studies have provided evidence of haemocytic infiltration in response to exposure to hydrocarbons (Cajaraville et al., 1990), which was interpreted as a repair process following tissue damage (Garmendia et al., 2011).

Fig. 5. Catalase (CAT) activities (mK/mg protein) in the digestive gland of mussels Mytilus galloprovincialis (n ¼ 10) caged in Vendicari and Priolo. Results are reported as mean and standard deviation (S.D.). Significant difference (P < 0.05) between control and polluted site is indicated by an asterisk.

The cytochrome P450 family, belonging to phase I (functional reactions) and GST, involved in phase II (conjugative reactions) of the biotransformation process, are the main enzymes used as biomarkers of the detoxification of organic pollutants (Regoli et al., 2002). In this study, phase I and phase II detoxification mechanisms were thus investigated by evaluating CYP4 and GST expression, respectively, in digestive gland of M. galloprovincialis as indicators of the biotransformation response to PAHs exposure. In particular CYP4, where expression is known to be inhibited by PAHs (Snyder, 1998, 2001), was found to be down-regulated both at the enzymatic and protein level in the digestive tissue of stressed organisms. CYP4Y1 mRNA levels were significantly reduced. This result is consistent with the 30% fold decrease in CYP4Y1 mRNA observed in digestive gland of M. galloprovincialis after two days of exposure to the hyrocarbon beta-naphthoflavone, as reported by Snyder (1998). Furthermore, a large number of cells were GSTimmunopositive in the digestive gland of mussels caged at the polluted site, which could indicate xenobiotic exposure resulting in increased oxidative stress. These results show a direct relationship between the high concentrations of PAHs in mussel digestive gland tissues and the activation of the detoxification process, suggesting that PAHs may alter the activity of phase I and phase II biotransformation enzymes (Bebianno et al., 2007). Similar results were observed by Sureda et al. (2011) in mussel M. galloprovincialis subjected to contamination by the oil spill from the Don Pedro ship in the Ibiza Harbour. The activity of catalase, a primary enzyme in antioxidant defense system and often one of the earliest antioxidant enzymes to be induced, has also been investigated in the present study. Mussels caged in Priolo presented significantly higher levels of CAT activity in the digestive gland compared to those caged at the reference site, indicative of oxidative stress. Considering the correlation between

Please cite this article in press as: Cappello, T., et al., Effects of environmental pollution in caged mussels (Mytilus galloprovincialis), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2012.12.010

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the increase in catalase activity and high levels of PAHs measured in the mussel digestive gland tissue, it is hypothesised that the induction of antioxidant enzymes is a protective response to the oxidative stress associated with a high bioavailability of hydrocarbons in the coastal marine environment investigated. This is in accordance with previous field studies that have reported increasing CAT activity values in relation to PAH and PCB accumulation in mussel tissues (Cheung et al., 2001; De Luca-Abbott et al., 2005; Fernández et al., 2010; Porte et al., 1991). Nevertheless, antioxidant enzymes are general stress biomarkers that are induced by a wide range of environmental contaminants capable to enhance the formation of reactive oxygen species (ROS) such as PAHs, PCBs, heavy metals, organic xenobiotics, and pesticides (Cajaraville et al., 2000; Khessiba et al., 2005; Livingstone, 2001; Regoli and Principato, 1995; Roméo et al., 2003; Sureda et al., 2006). Hence, CAT induction may reflect an adaptive behavior to ROSinducing environmental contaminants, representing an important early indicator of oxidative stress (Cossu et al., 1997). However, further research is needed to investigate other antioxidant parameters. 5. Conclusion Data reported in this study revealed that the “Augusta-MelilliPriolo” industrial area, which is contaminated by PAHs, provokes marked changes in the digestive gland morphology, as well as an increase in the detoxification and antioxidant defense mechanisms in caged M. galloprovincialis individuals, indicative of oxidative stress. Thus, phase I and phase II biotransformation (CYP4Y1 and GST, respectively) and antioxidant (CAT) enzymes demonstrated to be reliable biomarkers for the assessment of anthropogenic effects on aquatic organisms. References Aebi, H.E., 1984. Catalase. In: Bergmeyer, H.U. (Ed.), Methods in Enzymatic Analysis. Verlag Chemie, Basel, pp. 273e286. Altenburger, R., Segner, H., Van dar Oost, R., 2003. In: Douben, P.E.T. (Ed.), PAHs: An Ecotoxicological Perspective. Wiley, Chichester, England, pp. 147e171. Andral, B., Stanisiere, J.Y., Sauzade, D., Damier, E., Thebault, H., Galgani, F., Boissery, P., 2004. Monitoring chemical contamination levels in the Mediterranean based on the use of mussel caging. Mar. Pollut. Bull. 49, 704e712. Ausili, A., Gabellini, M., Cammarata, G., Fattorini, D., Benedetti, M., Pisanelli, B., Gorbi, S., Regoli, F., 2008. Ecotoxicological and human health risk in a petrochemical district of southern Italy. Mar. Environ. Res. 66, 217e219. Bebianno, M.J., Lopes, B., Guerra, L., Hoarau, P., Ferreira, A.M., 2007. Glutathione Stranferases and cytochrome P450 activities in Mytilus galloprovincialis from the South coast of Portugal: effect of abiotic factors. Environ. Int. 33, 550e558. Bucheli, T.D., Fent, K., 1995. Induction of cytochrome-P450 as a biomarker for environmental contamination in aquatic ecosystems. Crit. Rev. Env. Sci. Tec. 25, 201e268. Buhler, D.R., Williams, D.E., 1988. The role of biotransformation in the toxicity of chemicals. Aquat. Toxicol. 11, 19e28. Cajaraville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C., Viarengo, A., 2000. The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. Sci. Total Environ. 247, 295e311. Cajaraville, M.P., Diez, G., Marigomez, I., Angulo, E., 1990. Responses of the basophlic cells of the digestive land of mussels to petroleum hydrocarbon exposure. Dis. Aquat. Org. 9, 221e228. Ciacci, C., Barmo, C., Gallo, G., Maisano, M., Cappello, T., D’Agata, A., Leonzio, C., Mauceri, A., Fasulo, S., Canesi, L., 2012. Effects of sublethal, environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis. Aquat. Toxicol. 120, 109e118. Cheung, C.C., Zheng, G.J., Li, A.M., Richardson, B.J., Lam, P.K., 2001. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis. Aquat. Toxicol. 52, 189e 203. Chomczynski, P., Sacchi, N., 1987. Single-step method of Rna isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem. 162, 156e159. Cossu, C., Doyotte, A., Jacquin, M.C., Babut, M., Exinger, A., Vasseur, P., 1997. Glutathione reductase, selenium-dependent glutathione peroxidase, glutathione levels, and lipid peroxidation in freshwater bivalves, Unio tumidus, as

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Please cite this article in press as: Cappello, T., et al., Effects of environmental pollution in caged mussels (Mytilus galloprovincialis), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2012.12.010