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Marine Environmental Research 91 (2013) 52e60

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Marine Environmental Research

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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

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

a r t i c l e i n f o

Article history:Received 5 April 2012Received in revised form5 December 2012Accepted 26 December 2012

Keywords:Environmental toxicologyCoastal watersEastern SicilyMytilus galloprovincialisDigestive glandBiomarkerCytochromeGlutathione S-transferaseCatalase

* Corresponding author. Tel.: þ39 090 391435; fax:E-mail address: mmaisano@unime.it (M. Maisano

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

a b s t r a c t

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed inmussels 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 his-topathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1(CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity ofcatalase (CAT) as oxidative stress index, were evaluated.

The results show a direct correlation between the high concentrations of PAHs in digestive glands ofmussels 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 showthe enhancement of the detoxification and antioxidant defense systems. The mussel caging approach andselected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects onaquatic organisms.

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1. Introduction

Coastal environments are heavily influenced by human activ-ities, especially the highly productive zones such as harbours. Theseare generally enclosed areas characterized by poor water quality,reduced oxygen in the water column, and low biodiversity (Guerra-García and García-Gómez, 2005). Owing to the limited hydrody-namics on the inside portions of harbours and to the intenseanthropogenic impact, which can be associated with release ofenvironmental contaminants into the aquatic ecosystem, concernshave been raised over the potential health risks to humans andaquatic organisms. The “Augusta-Melilli-Priolo” industrial area hasbeen chosen as an example of an anthropogenically impactedcoastal marine environment for this study. It extends 20 km alongthe Augusta costal area (eastern Sicily, Italy) and is one of thelargest and most complex petrochemical industry sites in Europesince it is characterized by oil refineries, chemical plants, mineral

þ39 090 6765556.).

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deposits, a military base and many other industrial installations(Ausili et al., 2008). Mercury (Hg) and polycyclic aromatic hydro-carbons (PAHs) are found in excessive concentrations (ICRAM,2005). Levels of these contaminants exceed national and interna-tional regulatory guidelines, as reported in recent studies on sedi-ments collected from the coastal zone of Augusta (Di Leonardoet al., 2008, 2007).

Pollutant mixtures (heavy metals, drugs, PAHs, polychlorinatedbiphenyls PCBs) can induce toxic effects at different biologicallevels (e.g. biochemical, molecular, cellular, physiological). Sincechanges at the organism level lead to changes at the population andcommunity levels, biomarkers can be used as early warning signalsof environmental disturbance (Walker et al., 2006).

In environmental monitoring studies, mussels, particularly thegenus Mytilus, are widely used as sentinel organisms (Bebiannoet 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 totolerate a range of environmental conditions, accumulate toxicchemicals, and suitability for caging experiments at field sites

T. Cappello et al. / Marine Environmental Research 91 (2013) 52e60 53

(Andral et al., 2004; Fasulo et al., 2012; Roméo et al., 2003;Tsangaris et al., 2010; Viarengo et al., 2007). The use of transplantedmussels originating from a non-polluted area allows comparison ofcontrol organisms with those caged in potentially polluted sites,and allows more control over the experiment than collection ofnative individuals. In addition, using caged mussels from a singlepopulation minimizes confounding factors such as the age andreproductive status of the organisms that influence both con-taminant bioaccumulation and biomarker responses. Thus, a moreaccurate assessment of the real biological effects of pollutantexposure is possible, providing an early sign of impaired health ofthe 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 targetorgan for pollution effects because it accumulates contaminantsand plays a major role in xenobiotic metabolism (Rajalakshmi andMohandas, 2005). It is also involved in immune defense, detox-ification and in homeostatic regulation (Marigomez et al., 2002;Moore and Allen, 2002), and therefore exposure to contaminantsmay lead to its histopathological alteration (Fasulo et al., 2012;Garmendia et al., 2011).

The mechanism of detoxification of organic pollutants consistsof two steps essential to modify the toxicity of lipophilic xenobi-otics, converting them to water-soluble and easily excretable me-tabolites (Kleinow et al., 1987; Livingstone, 1993). Phase I is usuallycatalysed by cytochrome P450 (CYP)-dependent monooxygenases,and represent the introduction of functional groups. The phase Imetabolites can then act as substrates for phase II enzyme re-actions, also known as conjugation pathways (Buhler andWilliams,1988; Daly, 1995). Hence, induction and/or inhibition of certainenzymes may indicate presence of biologically significant levels ofxenobiotics, and thus enzyme measurement is strongly recom-mended 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 belongingto the CYP4 subfamily, was recently identified in the digestive tis-sues of Mytilus galloprovincialis, and its expression was inhibitedafter exposure to beta-naphthoflavone, a typical hydrocarboninducer 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 bio-transformation, and thus in the detoxification of many environ-mental chemicals by catalyzing the conjugation of glutathione(GSH) to electrophilic compounds (e.g. epoxides of PAHs), render-ing them less reactive andmore water-soluble (Cheung et al., 2001;Pan et al., 2009).

Another important mechanism of toxicity associated withxenobiotic products is oxidative damage (Sanchez et al., 2005). Thecellular defense system against oxidative damage comprisesenzymatic antioxidant defenses such as catalase (CAT), which is anoxidoreductase enzyme that catalyzes the conversion of hydrogenperoxide into water and oxygen. It can be used as a biomarker ofoxidative stress, although it does not respond specifically to a groupof pollutants. Indeed, CAT can be induced by a wide range of con-taminants, 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 ofenvironmental pollution, mainly related to the presence of PAHs, inan eastern Sicily petrochemical area in caged musselM. galloprovincialis. In particular, phase I and phase II bio-transformation (CYP4Y1 and GST, respectively) and antioxidant(CAT) enzymes were evaluated, as well as the content of PAHs indigestive glands of mussels.

2. Materials and methods

This study was conducted according to the guidelines for theprotection of animal welfare, in compliance with the Italian Na-tional Bioethics Committee (INBC).

2.1. Study area

The “Augusta-Melilli-Priolo” industrial area, chosen as pollutedsite 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 riskfor human health. By contrast, the Natural Reserve of Vendicari,established as a wildlife reserve in the southernmost part of theeast coast of Sicily in 1984, was chosen as a reference site notimpacted by petrochemical contamination. It covers an area of1512 ha (575 ha of core reserve and 937 ha of buffer zone) and itsbiological importance is based on the presence of different bio-topes, e.g. rocky and sandy coastlines, Mediterranean shrub, bothsalt and fresh water marshes (Fig. 1). At both sampling sites, waterphysicoechemical parameters (i.e. temperature, salinity, pH, dis-solved oxygen) were measured at the deployment depth (8 m)three times, at the beginning of the experimental study before cageplacement (mid October 2009), twoweeks after and then at the endof the 30-day deployment period before retrieving the cages, usinga portable instrument (Multi 340i/SET, WTW Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany). Data arereported as mean � standard deviation (S.D.) in Table 1.

2.2. Experimental design

Mussels, M. galloprovincialis (6.1 � 0.54 cm shell length), werepurchased in October 2009 from a consortium of fishermen of Goro(Ferrara, Italy), which physicoechemical parameters have beenpreviously reported (Fasulo et al., 2008). Mussels were maintainedfor one week in aerated seawater in the laboratory and thentransplanted to the two selected sites for 30 days in stainless steelcages (one cage containing about 200 specimens at each location)covered with a net to guarantee free circulation of seawater and toprotect mussels from predation. Cages were immersed by scuba-diving at 8 m depth below the surface both in Priolo (37�1201000N;15�1304400E) and Vendicari (36�4703500N; 15�0805200E). The musselswere retrieved after 4 weeks by diving. From each area, fifteenmaleindividuals, sexed by microscopic observation of gonad tissue, ofcomparable 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 inliquid nitrogen for chemical, molecular and enzymatic measure-ments, then transferred to the laboratory and stored at�80 �C priorto analysis. Further, small pieces of each dissected tissuewere takenfor 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, ho-mogenized using an Ultraturrax homogenizer and then saponifiedwith 10 ml of 1 M KOH in an ethanol solution for 3 h at 80 �C. Then20 ml of cyclohexane were added and samples mixed by an orbitalagitator for 10 min using dark glassware (Dafflon et al., 1995).

The hexanic phase was recovered and the polar mixture washedonce with cyclohexane and then discharged. The extracts werefiltered, concentrated under a nitrogen gas stream to about 1 ml,and the concentrated extract was removed with a pasteur pipetteand loaded into a Varian Bond Elut C18 cartridge 12 ml, previously

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

T. Cappello et al. / Marine Environmental Research 91 (2013) 52e6054

conditioned. The eluates were dried under a nitrogen stream anddissolved in 1 ml of acetonitrile (ACN) before the analysis.

The concentrations of the following sixteen PAHs identified bythe EPA as priority pollutants, naphthalene (NA), acenaphthylene

Table 1Mean (�S.D.) of water physicoechemical parameters of Vendicari and Priolo.

Sampling area Vendicari Priolo

Temperature (�C) 23.4 � 0.5 22.5 � 0.6Salinity (PSU) 37.6 � 0.1 38.2 � 0.2pH 8.0 � 0.1 7.9 � 0.1Oxygen (mg/1) 4.8 � 0.2 3.7 � 0.3

(ACY), acenaphthene (AC), fluorene (FL), phenanthrene (PHE),anthracene (AN), fluoranthene (FA), pyrene (PY), benzo(a)anthra-cene (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 carriedout with a Varian high-performance liquid chromatography (HPLC)equipped with a 20 ml loop and a fluorescence detector (FLD Pro-Star 363). The software used was Star Chromatography Work-station version 5.2 (Varian, Palo Alto, CA).

The analytical method involved a mobile phase consisting ofH2O/ACN 50% for 5 min, which achieved 100% ACN in 5 min with

T. Cappello et al. / Marine Environmental Research 91 (2013) 52e60 55

a flow of 1 ml/min. The UV determination was performed at255 nm, while the fluorescence (FL) detection was conducted withsix different excitation/emission wavelengths.

The National Institute of Standards and Technology (NIST)Standard Reference Material SRM 1647c, consisting of an acetoni-trile solution of sixteen PAHs (target compounds), was used asa calibrationmixture. Percent recovery andmatrix interferencewasassessed with reference M. galloprovincialis tissue.

The external standard multipoint calibration technique wasused to determine the linear response interval of the detector andin all cases, regression coefficients were higher than 0.996 for allthe analytes detected by UV, and higher than 0.989 for all theanalytes detected in FL.

2.4. Histological analysis

For histological assessment, digestive gland tissues of 15 mus-sels from each sampling site were fixed in 4% paraformaldehyde in0.1 M phosphate buffered solution (pH 7.4) at 4 �C, dehydrated inethanol and embedded in Paraplast (Bio-Optica, Italy). Histologicalsections, 5 mm thick, were cut with a rotary automatic microtome(Leica Microsystems, Wetzlar, Germany), mounted on glass slidesand stained with Hematoxylin/Eosin (Bio-Optica, Italy) to assessmorphological features.

Observations were made on five fields of one section per sampleusing a 40� oil-immersion objective with a motorized Zeiss AxioImager Z1 microscope equipped with an AxioCam digital camera(Zeiss).

2.5. Immunohistochemical analysis

On histological sections of digestive gland tissues from 15mussels per sampling site, an indirect immunofluorescencemethod was applied for localization of the CYP4 isoform, part ofa sequence that is unique for the CYP4-family (Simpson, 1997) andconfirmed in M. galloprovincialis CYP4Y1 (Jonsson et al., 2006), andGST. Paraffin wax-embedded sections were dewaxed and rehy-drated by conventional protocols (Mauceri et al., 1999). After sev-eral rinses in Phosphate Buffered Saline (PBS), sections were thenincubated overnight at 4 �C in a humid chamber for immuno-fluorescence labeling with the pre-diluted rabbit polyclonal anti-CYP4 antibody (kindly provided by Prof. Anders Goksøyr, Univer-sity 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 primaryantibodies were visualized by corresponding fluorescein isothio-cyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma), diluted1:100 for 2 h at room temperature.

Negative controls for the immunohistochemical labeling wereperformed by substitution of non-immune sera for the primary orsecondary antisera.

All observations were made on five fields of one section persample using a 40� oil-immersion objective with a motorized ZeissAxio Imager Z1 microscope equipped with an AxioCam digitalcamera (Zeiss). Sections were imaged using the appropriate filtersetting for the excitation of FITC (480e525 nm), and then processedby using AxioVision 4.5 (Zeiss) software.

2.6. Molecular analysis of CYP4Y1

Total RNA was extracted from the digestive glands of 10 speci-mens per sampling site using TRIzol LS reagent (Invitrogen, Carls-bad, California, US) (Chomczynski and Sacchi, 1987).Electrophoresis using 1.2% agarose gel under denaturating condi-tion and quantification by Thermo Scientific NanoDrop� 2000

were performed to verify RNA quality. Ratios of absorbance at 260/280 nm of greater than 1.9 were considerate as indicative of thehigh purity of RNA. The cDNA was synthesized using 4 mg of totalRNA, and oligo(dT)20 primer (150 pmol/ml) (Invitrogen), withMMLVreverse transcriptase (Invitrogen) following the manufacturer’sinstructions. The resulting reverse transcribed products were usedfor polymerase chain reaction (PCR) amplification.

The sequences of the CYP4Y1 primers, designed on the sequencespresent in Genebank (n� AF072855), were 50-AGGCTTTCACCAGTTCC-30 for the sense primer and 50-TCCGGCAGAAATGGAGTAAA-30 for theantisense primer, amplifying a 172 bp sequence. Actin (used asa positive control) of each examined specimen was amplified using-sequence primers based on the actin cDNA sequence ofM. galloprovincialis to obtain a fragment of 200 bp. Oligonucleotideswere synthesized andpurified byMWGBiotechAG. Polymerase chainreactions were performed using: 2.5 ml of 10� buffer, 0.13 ml of 5 U/mlPoly Taq polymerase (Invitrogen), 0.8 ml of 50 mM MgCl2, primers(50mMeach),1ml of cDNAtemplate, 0.5ml of 10mMdNTPs andMilli-Qwater (Millipore). The total reactionwas performed in a 25 ml volume.The amplification conditions were as follows: 95 �C for 2 min, 35 cy-cles of [95 �C for 30 s, 56 �C for 30 s, 72 �C for 30 s] andafinal extensionat 72 �C for 5 min. The Ep-Gradient Mastercycler (Eppendorf) wasused. RT-PCR products were separated and analyzed by electro-phoresis on SYBR Safe-stained agarose gel. The band intensity of CYPwasmeasuredwithQuantityOne software, and thennormalizedwiththe expression of cytoplasmatic actin.

2.7. Enzyme analysis

Digestive glands of 10 mussels per sampling site were homog-enized in 1:10 (w/v) 100 mM TriseHCl buffer, pH 7.5. Homogenateswere then centrifuged at 9000 g at 4 �C for 15 min (Manduzio et al.,2004). After centrifugation, supernatants were collected andimmediately used for the determination of CAT activity. All assayswere performed in duplicate and results were based on the totalprotein amount, estimated using the Pierce BCA Protein Assay Kit(Thermo Scientific). CAT activity (mK/mg protein) was measured bythemethod of Aebi (1984) based on the decomposition of hydrogenperoxide, using a UV Mini 1240 spectrophotometer (Shimadzu,Milano, Italy).

2.8. Statistical analysis

Immunoreactive cell quantification was performed by countingthe number of positive cells on five fields per section using AxioVision Release 4.5 software (Zeiss, Göttingen, Germany). The bandintensity of CYP was measured with Quantity One software (Bio-Rad, Marnes-la-Coquette, France), while a statistical package (SPSS12.0 for Windows) was used for the enzymatic analysis. Resultswere expressed as mean � S.D.

All the obtained data were statistically processed with one-wayanalysis of variance (ANOVA) using Graph Pad software (Instat, LaJolla, CA, US), applying Student’s two-tailed t-test for unpaired data,and statistical significance was accepted at P < 0.0001 for immu-nohistochemical analysis, P < 0.001 for molecular data, andP < 0.05 for enzymatic results.

3. Results

3.1. PAHs content

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

Table 2PAHs concentrations (mean � S.D.) in pooled samples of digestive glands (mg/g wetwt; n ¼ 3).

PAHs Priolo Vendicari

Naphthalene 0.912 ± 0.038 n.dAcenaphthylene 0.011 � 0.007 n.dAcenaphthene 0.007 � 0.002 n.d.Fluorene 0.009 � 0.005 n.d.Phenanthrene <0.006 n.d.Anthracene <0.006 n.d.Fluoranthene 0.187 ± 0.012 n.d.Pyrene 0.008 � 0.004 n.d.Benzo(a)anthracene <0.006 n.d.Chrysene n.d. n.d.Benzo(b)fluoranthene 0.012 � 0.007 n.d.Benzo(k)fluoranthene 0.008 � 0.002 n.d.Benzo(a)pyrene 0.060 ± 0.017 n.d.Dibenzo(a,h)anthracene 0.925 ± 0.028 n.d.Benzo(g,h,i)perylene <0.006 n.d.Indeno(1,2,3-cd)pyrene <0.006 n.d.

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

T. Cappello et al. / Marine Environmental Research 91 (2013) 52e6056

PAH concentrations for all analytes in the digestive gland sam-ples from the reference site were lower than the instrumentdetection limit. By contrast, samples from Priolo showed elevatedlevels 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) anddibenzo(a,b)anthracene (0.925 mg/g) among high molecular weightPAHs, as reported in Table 2.

3.2. Histological observations

The digestive gland ofM. galloprovincialis caged in the referencesite (Fig. 2A) showed the typical organization of the digestivediverticula of bivalves, as described by Owen (1970). In contrast, inmussels from the polluted area the histological integrity of thedigestive gland tissue was poor (Fig. 2B). The tissue appearedseverely damaged, and massive haemocytic infiltration wasobserved among digestive tubules.

3.3. Cytochrome CYP4 isoform

In the digestive gland tissue of control specimens an intensepositive immunoreactivity for cytochrome P450 4 (CYP4) was seenalong the epithelium, consisting of digestive and basophilic cells,which lines the digestive tubules (Fig. 3A). In contrast, a drastic

Fig. 2. (AeB) Hematoxylin and Eosin (H&E) staining in the digestive gland ofMytilus galloproarea (B), which displayed severe histopathological alterations and relevant aggregations of

CYP4-inhibition was found in the digestive gland of individualsacclimatized 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 levelsboth in control animals and in those from Priolo. CYP4Y1 gene washighly expressed in specimens caged in the unpolluted site,whereas it was expressed at a lower level in the digestive glands ofmussels caged at the industrial area.

CYP4Y1 band intensity differed significantly between animalstransferred to the two selected sites (P < 0.001; Fig. 3E).

3.4. Glutathione S-transferase

Immunohistochemical labeling of glutathione S-transferaserevealed no positive cells in the digestive glands of specimens fromthe reference site (Fig. 4A). In contrast, digestive glands fromspecimens caged in the petrochemical area of Priolo showeda 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 cagedat the site influenced by anthropogenic activities (238 mK/mgprotein) compared to the reference site (139 mK/mg protein)(P < 0.05; Fig. 5).

4. Discussion

Biological effects of environmental pollution, mainly related tothe presence of PAHs, were assessed in mussel M. galloprovincialiscaged in an eastern Sicily petrochemical area. The use of musselcaging demonstrated to be an effective and useful tool for assessingthe environmental quality status and biological effects induced byxenobiotics (Andral et al., 2004; Regoli, 2000; Roméo et al., 2003).The digestive gland, a primary organ for bioaccumulation andinvolved in pollutant detoxification and homeostasis maintenance(Marigomez et al., 2002; Moore and Allen, 2002), was chosen astarget organ for this study.

Chemical analyses performed in mussel digestive gland tissuerevealed higher levels of PAHs in individuals caged in the

vincialis caged in the reference site (A) compared with those transferred to the pollutedhaemocytes (arrows) among digestive tubules. Scale bars, 20 mm.

Fig. 3. (AeB) Immunohistochemical labeling for CYP4 in mussel digestive glands. (A) Mussels caged in the reference site displayed an intense immunoreactivity along the epi-thelium 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.) ofimmunopositive 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 bandintensity in polluted and control environment. Significant difference (P < 0.001) between control and polluted site is indicated by an asterisk.

T. Cappello et al. / Marine Environmental Research 91 (2013) 52e60 57

petrochemical area of Priolo compared to those acclimatized in theNatural Reserve of Vendicari, the reference site. In detail, highconcentrations of naphthalene and fluoranthene, consistent witha dominantly pyrolytic origin of PAHs, and benzo(a)pyrene anddibenzo(a,h)anthracene indicative to urban and industrial con-tamination, were recorded. These findings provide thus an assess-ment of contaminant levels entering the food chain, and confirm

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

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) in-dividuals caged in Priolo showed a highly positive GST immunostaining in secernent and absorbent digestive gland cells (arrows). Scale bars, 20 mm. (C) Mean and standarddeviation (S.D.) of immunopositive cells. Significant difference (P < 0.0001) between control and polluted site is indicated by an asterisk.

T. Cappello et al. / Marine Environmental Research 91 (2013) 52e6058

Cancer) classification for carcinogenicity, benzo(a)pyrene belongsto group 1 (carcinogenic to humans), dibenzo(a,h)anthracene togroup 2A (probably carcinogenic to humans), naphthalene to group2B (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 significanthistological lesions with altered diverticula morphology and con-spicuous haemocytic infiltration, whichmight result in impairmentof its metabolic activities, as reported for other organs ofM. galloprovincialis (Ciacci et al., 2012). Previous studies have pro-vided evidence of haemocytic infiltration in response to exposureto hydrocarbons (Cajaraville et al., 1990), which was interpreted asa repair process following tissue damage (Garmendia et al., 2011).

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

The cytochrome P450 family, belonging to phase I (functionalreactions) and GST, involved in phase II (conjugative reactions) ofthe biotransformation process, are the main enzymes used as bio-markers of the detoxification of organic pollutants (Regoli et al.,2002). In this study, phase I and phase II detoxification mecha-nisms were thus investigated by evaluating CYP4 and GST expres-sion, respectively, in digestive gland of M. galloprovincialis asindicators of the biotransformation response to PAHs exposure. Inparticular CYP4, where expression is known to be inhibited by PAHs(Snyder, 1998, 2001), was found to be down-regulated both at theenzymatic and protein level in the digestive tissue of stressed or-ganisms. CYP4Y1 mRNA levels were significantly reduced. Thisresult is consistent with the 30% fold decrease in CYP4Y1 mRNAobserved in digestive gland of M. galloprovincialis after two days ofexposure to the hyrocarbon beta-naphthoflavone, as reported bySnyder (1998). Furthermore, a large number of cells were GST-immunopositive in the digestive gland of mussels caged at thepolluted site, which could indicate xenobiotic exposure resulting inincreased oxidative stress. These results show a direct relationshipbetween the high concentrations of PAHs in mussel digestive glandtissues and the activation of the detoxification process, suggestingthat PAHs may alter the activity of phase I and phase II bio-transformation enzymes (Bebianno et al., 2007). Similar resultswere observed by Sureda et al. (2011) in mussel M. galloprovincialissubjected to contamination by the oil spill from the Don Pedro shipin the Ibiza Harbour.

The activity of catalase, a primary enzyme in antioxidant de-fense system and often one of the earliest antioxidant enzymes tobe induced, has also been investigated in the present study. Musselscaged in Priolo presented significantly higher levels of CAT activityin the digestive gland compared to those caged at the reference site,indicative of oxidative stress. Considering the correlation between

T. Cappello et al. / Marine Environmental Research 91 (2013) 52e60 59

the increase in catalase activity and high levels of PAHsmeasured inthe mussel digestive gland tissue, it is hypothesised that the in-duction of antioxidant enzymes is a protective response to theoxidative stress associated with a high bioavailability of hydrocar-bons in the coastal marine environment investigated. This is inaccordance with previous field studies that have reportedincreasing CAT activity values in relation to PAH and PCB accumu-lation 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 areinduced by awide range of environmental contaminants capable toenhance the formation of reactive oxygen species (ROS) such asPAHs, 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 ROS-inducing environmental contaminants, representing an importantearly indicator of oxidative stress (Cossu et al., 1997). However,further research is needed to investigate other antioxidantparameters.

5. Conclusion

Data reported in this study revealed that the “Augusta-Melilli-Priolo” industrial area, which is contaminated by PAHs, provokesmarked changes in the digestive gland morphology, as well as anincrease in the detoxification and antioxidant defense mechanismsin caged M. galloprovincialis individuals, indicative of oxidativestress. Thus, phase I and phase II biotransformation (CYP4Y1 andGST, respectively) and antioxidant (CAT) enzymes demonstrated tobe reliable biomarkers for the assessment of anthropogenic effectson aquatic organisms.

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