susceptibility to oxidative stress of mussels (mytilus galloprovincialis) in the venice lagoon...

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Marine Pollution Bulletin 50 (2005) 1548–1557

Susceptibility to oxidative stress of mussels(Mytilus galloprovincialis) in the Venice Lagoon (Italy)

Daniela M. Pampanin a,*, Lionel Camus b, Alessio Gomiero a, Ilenia Marangon a,Elisa Volpato a, Cristina Nasci a

a Institute of Marine Science, ISMAR-CNR, Castello 1363/a, Venice, Italyb RF-Akvamiljø, Mekjarvik 12, 4070 Randaberg, Norway

Abstract

The aim of this study was to evaluate the susceptibility to pollutant mediated oxidative stress of the Mediterranean mussel Myti-

lus galloprovincialis in the Venice lagoon (Italy).In June 2003, mussels from a farm were transplanted to eight sites in the lagoon for five weeks. Oxidative stress responses were

measured by: (i) total oxyradical scavenging capacity (TOSC) assay, for an overall evaluation of the oxidative stress response capa-bility; (ii) catalase (CAT), as a key enzyme involved in the antioxidant defence system; (iii) malondialdehyde (MDA), as an indicatorof lipid peroxidation, to evaluate an oxidative damage; (iv) metallothioneins (MTs), as they play a role in the antioxidant defence.

The TOSC analysis revealed a reduced capability to eliminate: (i) peroxyl radical in mussels transplanted at Palude della Rosa,Valle Millecampi and Chioggia; (ii) hydroxyl radical at Campalto and Valle Millecampi; (iii) peroxynitrite at Valle Millecampi.

Inhibition in CAT activity, observed in all the monitored sites, confirms the presence of an oxidative pressure in transplantedmussels.

In addition, Pearson correlation analysis was performed in order to observe possible links between the various parameters. ThePCA was a powerful tool to discriminate impacted sites, suggesting that the mussels transplanted throughout the Venice lagoon weresubjected to different levels of oxidative pressure. Furthermore, it provided an easy and useful tool to summarize the obtainedresults.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Mussel; Oxidative stress; TOSC; Catalase; Malondialdehyde; Metallothionein; Venice Lagoon

1. Introduction

Since the discovery of the importance of radical reac-tions both in normal biological processes and in toxicitymechanisms of many xenobiotics, there is an increasingconcern about prooxidant and antioxidant processes(see the review of Livingstone, 2001). Antioxidants rep-resent the cellular defence mechanisms which counteract

0025-326X/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2005.06.023

* Corresponding author. Present address: RF-Akvamiljø, Mekjarvik12, 4070 Randaberg, Norway. Tel.: +47 51875502; fax: +47 51875540.

E-mail address: [email protected] (D.M. Pampanin).

toxicity of reactive oxygen species (ROS), these mecha-nisms have been extensively investigated in sentinelorganisms such as marine mussels (Winston et al.,1990; Viarengo et al., 1991a,b; Livingstone et al., 1992;Regoli, 1998; Camus et al., 2004). Responsiveness ofantioxidants to pollutants is difficult to predict and ahigh degree of variability has been reported as a func-tion of class of chemicals, kind of exposure, phase ofthe biological cycle (Livingstone, 2001). Nevertheless,laboratory and field studies indicated that variations inthe levels or activities of antioxidants are potential bio-markers revealing a contaminant-mediated biological ef-fect on the organisms (Porte et al., 1991; Ribera et al.,

mailto:[email protected]

Fig. 1. Location of the sampling stations in the Venice lagoon.1 = Palude della Rosa, 2 = Campalto, 3 = Tresse, 4 = Sacca Sessola,5 = S. Pietro in Volta, 6 = Valle Millecampi, 7 = Ca Roman,8 = Chioggia.

D.M. Pampanin et al. / Marine Pollution Bulletin 50 (2005) 1548–1557 1549

1991; Livingstone, 1991; Livingstone et al., 1995; Regoliand Principato, 1995; Sole et al., 1996; Camus et al.,2004).

The analysis of the total oxyradical scavenging capac-ity (TOSC) has been recently demonstrated as a reliabletool for quantitatively assessing the biological resistanceto toxicity of different forms of ROS, including peroxylradicals, hydroxyl radicals and peroxynitrite decomposi-tion products (Winston et al., 1998; Regoli and Win-ston, 1999). Catalase (CAT) is a commonly studiedantioxidant enzyme involved in the initial anti-oxidativemechanism and widely used as a biomarker in mussel(Cajaraville et al., 2000; Khessiba et al., 2001; Nasciet al., 2002; Lau and Wong, 2003; Romeo et al.,2003). It reduces H2O2, produced by the superoxide dis-mutase enzyme (SOD), to produce water and oxygen.Moreover, high catalase activity is found in inverte-brates, confirming its important role in antioxidantdefence in aquatic invertebrates (Livingstone et al.,1992). Malondialdehyde (MDA) is a naturally occurringproduct of lipid peroxidation and prostaglandin biosyn-thesis that is mutagenic and carcinogenic. It reactswith DNA to form adducts (Marnett, 1999). It isparticularly interesting to associate the study of antiox-idant molecules (such as catalase and metallothione-ins) to an oxidative damage assay, such as MDAconcentration.

Although the physiological role of metallothioneins(MTs) is still under debate, a fundamental involvementin essential and non essential metal pathways is certain(Roesijadi, 1992; Bebianno and Langston, 1992; Viar-engo and Nott, 1993; Langston et al., 1998). MTs occurmainly in cytosol and are characterised by high cysteincontent, low molecular weight, heat-stability and astrong affinity for binding metals such as Ag, Cd, Cu,Hg and Zn. Moreover, MTs could protect cells fromoxidative stress not only by acting as an oxyradical scav-enger (Andrews, 2000; Colangelo et al., 2004), but alsothrough metal binding/release dynamics, as suggestedby Viarengo et al. (2000).

The Mediterranean mussel is widely distributed in theVenice lagoon (Italy). The Venice lagoon is a shallowcoastal basin with a surface area of about 550 km2, con-nected with the sea by three sea inlets (Lido, Malamoccoand Chioggia). Inorganic and organic micro-pollutantsenter the lagoon from industrial point sources (PortoMarghera industrial area), municipal wastewater dis-charges (Venice and Mestre urban areas), agriculturaldrainage, atmospheric deposition and illegal dumping(UNESCO, 2000).

A transplantation experiment was performed at thislocation, since this strategy allowed the comparison ofchemical and biological properties of samples whichhave been collected from one population and whichhave been exposed to different environmental conditions(de Kock and Kramer, 1994).

The aim of this study is to gain knowledge on thesusceptibility to oxidative stress of the mussel speciesMytilus galloprovincialis in the Venice lagoon ecosystemlooking at various biomarkers involved in this stressmechanism. Moreover, it is important to observe thetransplantation procedure adopted that is proposed asa useful tool for environmental biomonitoring. TOSC(for an overall evaluation of the oxidative stress responsecapability), CAT (enzyme involved in the antioxdantdefence system), MDA (representative of an oxidativedamage) and MTs (as they play a role in the antioxidantdefence) were then measured.

2. Materials and methods

2.1. Transplantation procedure

In June 2003 mussels (Mytilus galloprovincialis ) ofstandardized shell size (4.5 ± 0.5 cm), purchased froma mussel farm, located at Alberoni, considered as a cleansite (Pampanin et al., 2004) were transplanted to eightsites throughout the Venice Lagoon (from North toSouth): Palude della Rosa, Campalto, Tresse, Sacca Ses-sola, S. Pietro in Volta, Valle Millecampi, Ca Roman(reference site) and Chioggia (Fig. 1). Mussels weretransported in cold boxes from the farm to the boatand, after a first sorting, they were divided into groups(of about 100 individuals each), placed in cages consti-tuted by polyethylene netting and immersed subtidallyat all sites. The transplantation procedure is the same

1550 D.M. Pampanin et al. / Marine Pollution Bulletin 50 (2005) 1548–1557

used in other studies (Pampanin et al., 2004). After fiveweeks of exposure, mussels were collected and trans-ported to the laboratory in cold boxes for the biologicalassays. Digestive glands were dissected and immediatelyfrozen in liquid nitrogen and stored at �80 �C beforeanalysis.

2.2. TOSC assay

The method was based on Winston et al. (1998)and Regoli and Winston (1999), except that bufferswere adjusted for marine invertebrates. Digestiveglands of mussels were homogenised (1:4) in KH2PO4

buffer (100 mM, pH 7.5) containing NaCl (2.5%). Thehomogenate was centrifuged at 100000·g for 1 h, andcytosolic fractions were aliquoted and stored at�80 �C. Peroxyl radicals are generated by the thermalhomolysis of 2-2 0-azo-bis-(2 methyl-propionamidine)-dihydrochloride (ABAP) at 35 �C. The iron-ascorbateFenton reaction was used for hydroxyl radicals, whileperoxynitrite was generated from 3-morpholinosydno-mine (SIN-1), a molecule that releases concomitantlynitric oxide and superoxide anion, which rapidly com-bine to form HOONO.

Peroxyl, hydroxyl and peroxynitrite radicals can oxi-dize the substrate KMBA to ethylene gas which is mea-sured with gas chromatography. With these assayconditions, the various oxyradicals induce a comparableyield of ethylene in the control reaction, thus the relativeefficiency of cellular antioxidants is compared by theirability to counteract a quantitatively similar prooxidantchallenge (in terms of KMBA oxidation). Ethylene pro-duction was monitored for 96 min with a gas-chromato-graphic analysis (Hewlett Packard HP 5890 series II)equipped with a Supelco SPB-1 capillary column and aflame ionization detector (FID).

The data acquisition system was run by the softwareMillenium32� (Waters).

Each analysis required the measurement of control(no antioxidant in the reaction vial) and sample reac-tions (biological fluid in the vial). In the presence of anti-oxidant, ethylene production from KMBA was reducedquantitatively and higher antioxidant concentrations re-sulted in longer periods in which ethylene formation wasinhibited relative to controls. By plotting the absolutevalue of the difference between the ethylene peak areasobtained at each time point for the sample and controlreaction it is possible to quantify whether the oxyradicalscavenging capacity of the solution is changed. The areaunder the kinetic curve was calculated mathematicallyfrom the integral of the equation that best defines theexperimental points for both the control and samplereactions. TOSC is then quantified according to theequation:

TOSC ¼ 100� ðIntSA=IntCA�100Þ

where IntSA and IntCA are the integrated areas fromthe curve defining the sample and control reactions,respectively.

The TOSC values were expressed as TOSC unit permg proteins. Total protein content was determined byBradford method (Bradford, 1976).

2.3. Catalase activity

Catalase (CAT, EC 1.11.1.6) activity was performedas described by Livingstone et al. (1992).

Digestive glands were homogenised (1:4) in Tris–HClbuffer (10 mM, pH 7.6) containing sucrose (0.5 M) andKCl (0.15 M). Samples were subsequently centrifugedto obtain a soluble fraction (500·g for 10 min at 4 �C,10000·g for 40 min at 4 �C).

CAT activity was determined by the decrease inabsorbance at 240 nm, due to the H2O2 consumption(ext. coeff. 40 M�1 cm�1) in the following reaction (Aebi,1974):

2H2O2 !CAT2H2OþO2

The activity was evaluated using the formula:

½CAT� ¼ ðODS=eH2O2Þ�f =mg proteins

where ODS is the optical density of the sample, eH2O2the

extinction coefficient of H2O2 (40 M�1 cm�1) and f thedilution factor.

The results were expressed as mmol/min/mg proteins.Total protein content was determined by Bradfordmethod (Bradford, 1976).

2.4. Malondialdehyde evaluation

Lipid peroxides, derived from polyunsaturated fattyacid, are unstable and decompose to form a complexseries of compounds. These include reactive carbonylcompounds, of which the most abundant is MDA. Mea-surement of MDA is widely used as an indicator of lipidperoxidation. The MDA assay is based on the reactionof the chromogenic reagent N-methyl- 2-phenylindole(NMPI) with MDA at 45 �C, giving rise to a chromo-phore with absorbance at 586 nm (Esterbauer et al.,1991).

Digestive glands were rapidly rinsed in a cold NaClsolution (0.9%), homogenised (1:2) in Tris–HCl buffer(20 mM, pH 7.4) containing b-mercaptoethanol (0.01%)and then centrifuged at 10000·g for 20 min at 4 �C.The resulting supernatant was used for determinationof MDA content, after incubation at 45 �C, for 60 min,in NMPI solution (650 ll 10.3 mM NMPI, 100 ll H2O,100 ll sample, 150 ll HCl 37%).

A calibration curve was prepared using MDA stan-dard as tetramethoxypropane (TMOP), because MDAis not stable.


MDA concentration was calculated according to thefollowing equation:

½MDA� ¼ ½ðODS � bÞ=aÞ�f �where ODS is the optical density of the sample, b and a

are derived from the standard curve and f is the dilutionfactor.

The results were expressed as nmol/g of tissue.

2.5. Metallothionein content

MT content was evaluated in digestive gland homo-genates according to a spectrophotometric methoddescribed by Viarengo et al. (1997).

This method consists of the ethanol/chloroform frac-tionation of the cytosolic fraction, to obtain a partiallypurified fraction of these proteins. MT concentrationis quantified by evaluating the SH residue content by aspectrophotometric method, using Ellman�s reagent(DTNB: 5,5 dithiobis 2 nitrobenzoic acid).

Tissues were homogenised (1:3) in Tris Buffer (Tris20 mM, 0.5 M sucrose, pH 8.6) containing antiproteo-litic substances (leupeptin and phenylmethylsulphonylfluoride) and b-mercaptoethanol (0.01%). The solublefractions containing MTs were obtained by centrifugingthe homogenate at 10000·g for 30 min at 4 �C.

The supernatant was then treated with cold absoluteethanol and chloroform to allow the protein precipita-tion. Finally, MT content was spectrophotometricallydetermined using DTNB reaction.

A standard curve was built using GSH (SigmaG4521), where the absorbance at 412 nm is functionof GSH concentration (nmol/ml) (y = bx + c); the MTvalues were calculated following the equation

MTs ¼ ðððABS MT412=bÞ=21Þ�8600Þ�f ;where 21 is the number of cystein residues of musselMT, 8600 is the molecular weight of mussel MTs, f isthe dilution factor. The results were expressed as lg/gof tissue.

2.6. Statistical analysis

Statistical analyses were performed using JMPv3.2.6., SAS Institute, Inc., Cary, NC, USA. Dunnett�stest was used for testing change of analysed parametersbetween the reference site (Ca Roman) and the other la-goon sites. Data are plotted as mean and standard error(mean ± s.e.). A Pearson�s linear correlation was calcu-lated (n = 8, p > 0.05) using the statistical softwarepackage STATISTICA 5.0 package.

Principal Component Analysis (PCA) was used todiscriminate between the transplantation sites, sixvariables were taken into consideration: TOSC resultstowards peroxyl, towards hydroxyl and towards per-

oxynitrite separately, CAT, MDA and MTs (STATIS-TICA 5.0 package).

3. Results

3.1. TOSC assay

Fig. 2 illustrates the TOSC results of the three ana-lysed oxyradicals: peroxyl (ROO�), hydroxyl (�OH) andperoxynitrite (HOONO). After five weeks of exposure,significant TOSC reduction (p < 0.05) was measuredwhen compared to the reference site (Ca Roman) forperoxyl (Palude della Rosa, S. Pietro in Volta, ValleMillecampi and Chioggia), for hydroxyl (Campalto,S. Pietro in Volta and Valle Millecampi), and forperoxynitrite (Valle Millecampi).

A positive correlation was occurred with peroxyl andCAT (0.42), whereas a negative correlation was seen be-tween this radical and MDA (�0.56) and MTs (�0.49)(Table 1). Regarding hydroxyl, a positive correlation isfound with peroxynitrite (0.82), CAT (0.39) and MTs(0.47); TOSC for peroxynitrite significantly correlatedonly with hydroxyl (0.82).

3.2. Catalase activity

CAT activity ranged from 0.010 to 0.043 mmol/min/mg proteins (Fig. 3a). A significant inhibition (p <0.001) of CAT activity was observed in organisms trans-planted to all sites compared with the reference site (CaRoman). The minimum level of activity was measured atCampalto and Valle Millecampi.

For this enzyme activity, a positive correlation wascalculated with peroxyl (0.42) and hydroxyl (0.39), anda negative correlation with MDA (�0.52) (Table 1).

3.3. Malondialdehyde evaluation

MDA concentration varied from 39.84 to 81.33nmol/mg proteins (Fig. 3b). Significantly higher(p < 0.05) levels of MDA were found at Palude dellaRosa, Sacca Sessola and S. Pietro in Volta comparedto the reference site.

MDA was negatively correlated with peroxyl (�0.56)and CAT (�0.52) (Table 1).

3.4. Metallothionein content

MT content ranged from 29.13 to 69.50 lg/g of tissue(Fig. 3c). No significant differences were found betweenthe exposed sites and the reference site. Maximum con-centration of MTs was found at Valle Millecampi, whilethe minimum one was found at Campalto. A positivecorrelation occurred with hydroxyl (0.47) and a negativecorrelation with peroxyl (�0.49) (Table 1).

ROO.

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Campalto Tresse Sacca Sessola S.Pietro inVolta

ValleMillecampi

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.OH

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HOONO

0

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ValleMillecampi

Cà Roman Chioggia

TO

SC u

nit/m

g pr

otei

nsT

OSC

uni

t/mg

prot

eins

TO

SC u

nit/m

g pr

otei

ns

* *

*

Fig. 2. Total oxyradical scavenging capacity (TOSC) towards peroxyl (ROO�), hydroxyl (�OH) and peroxynitrite (HOONO) of digestive glandcytosolic of mussels (Mytilus galloprovincialis) after five weeks of transplantation at various sites throughout the lagoon of Venice. Values areexpressed as TOSC unit/mg proteins (mean ± s.e., n = 5). Asterisks indicate significant difference (Dunnett�s test) between exposed and reference site(Ca Roman) (*p < 0.05).


3.5. Principal component analysis (PCA)

The data matrix for the PCA was constructed of 48elements (6 variables · 8 sites), each variable was calcu-lated from the mean value for the individual index.

Factor 1, explained 36% of the total variance, ischaracterized mainly by high loading of the variableTOSC towards �OH and CAT, and factor 2 (34% of ex-plained variance) by high loading of TOSC towardsHOONO.

A slight spatial separation between the eight contam-inated sites and the reference site (Ca Roman) was evi-dent (Fig. 4).

4. Discussion

The studied sites represent various environmentalconditions and are differently affected by pollutants.The diffused xenobiotic contamination in the lagoon is

Table 1Results of the linear correlation of Pearson between total oxyradicalscavenging capacity (TOSC), catalase (CAT), malondialdehyde(MDA) and metallothioneins (MTs), statistical software packageStatgraphics 5.0 (STSG) (n = 8, *p > 0.05)

ROO� �OH HOONO CAT MDA MTs

ROO� 1.00 0.10 0.04 0.42 �0.56 �0.49�OH 0.10 1.00 0.82* 0.39 �0.17 0.47HOONO 0.04 0.82* 1.00 0.02 0.28 0.16CAT 0.42 0.39 0.02 1.00 �0.52 �0.01MDA �0.56 �0.17 0.28 �0.52 1.00 �0.09MTs �0.49 0.47 0.16 �0.01 �0.09 1.00


known (Volpi Ghirardini et al., 1999; Fossato et al.,2000; Nasci et al., 2002) and a ‘‘clean site’’ has not yetbeen adequately identified. Nevertheless Ca Romanwas selected as a reference site, due to its position closeto the lagoon inlet of Chioggia, where mussel farms arelocated. This area is known to be not directly affected byanthropogenic activity, because of the distance fromthe industrial area (Marghera), from the urban area ofVenice city and finally from the agricultural activity ofthe mainland.

The TOSC results reveal the overall capacity of mus-sels to cope with oxidative stress exposure. After fiveweeks of exposure, mussels show a reduced capabilityto eliminate: peroxyl in mussels transplanted to threesites (Palude della Rosa, S. Pietro in Volta, ValleMillecampi and Chioggia) and hydroxyl (at Campalto,S. Pietro in Volta and Valle Millecampi). Molluscstransplanted to Valle Millecampi show a low capabilityto scavenge all three oxyradicals indicating an intenseoxidative stress exposure which may be due to the runoff from the inner land (draining basin), especially agri-cultural pollutants (i.e. pesticides). The TOSC reductionfor hydroxyl at Campalto may be associated with pollu-tant exposure, in particular PCBs, PAHs, Pb and Zn,originating from an industrial landfill located in thisarea (Volpi Ghirardini et al., 1999). With regard toChioggia, a small urban centre located in the south partof the lagoon, the TOSC reduction for peroxyl could bethe result of the presence of contaminants associatedwith domestic sewage and intense harbour activities.These findings support the study of Nasci et al. (2002)which reported that molluscs from this area suffer fromoxidative stress. In the north part of the lagoon, Paludedella Rosa is known as a site with relatively low contam-ination, influenced by inland activities and agricul-tural activity. A previous study utilizing the clamTapes philippinarum (Nasci et al., 2000), demonstratedthe presence of biochemical responses (CAT, SOD) oforganisms toward oxidative stress, supporting the find-ing of this study in TOSC reduction for peroxyl.

The ecological relevance of sub-cellular biological re-sponses is arguably greater when they are associatedwith adverse effects at higher levels of biological organi-

sation: cellular and organism level (Depledge, 1994).Therefore at biochemical level, the inhibition of theCAT activity observed in all sites compared with refer-ence confirms the presence of an oxidative stress thatmay affect mussels throughout the Venice lagoon. Otherstudies have demonstrated that when invertebrates aresubjected to oxidative stress, a series of defence mecha-nisms start to protect the organism (Winston and DiGiulio, 1991; Regoli and Principato, 1995). One of thefirst effects of increased oxidative stress is the productionof a low molecular weight scavenger (Livingstone,2001). Nevertheless, when this stress increases, an inhibi-tion of enzyme activity, such as CAT, has been found(Regoli and Principato, 1995). When the stress levelsare very high, a decrease in TOSC values occurs, indicat-ing the difficulty that the organisms have in defendingagainst oxidative stress. The inhibition/decrease inCAT activity in molluscs after five weeks of transplanta-tion represents an indication of diffuse oxidative stresspressure throughout the Venice Lagoon, which is severeonly at Valle Millecampi, as revealed by the TOSC data,where all three TOSC species are affected. Reducedcapability in neutralizing various ROS and simultaneousinhibition of CAT, as shown in this study, is found in ex-posed animals characterized by an increased susceptibil-ity to oxidative stress disease (Regoli, 2000; Regoli et al.,2002; Nesto et al., 2004).

The toxicity effect of ROS can produce various dam-ages to the cell, such as DNA damage, lipid peroxida-tion and lysosomal alteration (Viarengo et al., 1989;Winston et al., 1996; Regoli, 1998; Regoli, 2000; Frenz-illi et al., 2001). Lipid peroxidation is a well-knownmechanism of cellular injury in vertebrates and inverte-brates, and is an indicator of an oxidative damage incells and tissues. Therefore, measurement of MDA iswidely used as an indicator of lipid peroxidation(Wheatley, 2000). MDA can react readily with aminogroups on proteins and other molecule to form a varietyof adducts (Esterbauer et al., 1991; Marnett, 1999). Anincrease in MDA concentration is found in three sites:Palude dell Rosa, Sacca Sessola and S. Pietro in Volta.The inverse correlation between MDA content and theactivity of catalase suggests its importance in protectingthe cell from membrane damage. In fact an effectiveCAT control will end up with a low MDA level and viceversa (Lau and Wong, 2003).

Here, it is important to mention that the initial prod-ucts of polyunsaturated fatty acid oxidation are peroxylradicals. When a cell or a tissue is not capable of pre-venting oxidative damage, an increase of lipid peroxida-tion, measured as an increase in MDA, causes a decreasein TOSC value for peroxyl (Marnett, 1999). A negativecorrelation between MDA and peroxyl is observed inthis study.

The MT content in mussel transplanted for five weeksto the Venice lagoon is below values reported in earlier

0.00

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0.06

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S.Pietro inVolta

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CATALASE ACTIVITY

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tissu

eµg

/g o

f tis

sue

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ol/m

in/m

g pr

otei

ns

(a)

(b)

(c)

Fig. 3. (a) Catalase activity (expressed as mmol/min/mg), (b) malodialdehyde (as nmol/g tissue) and (c) metallothioneins content (as lg/g of tissue)in digestive gland of mussels (Mytilus galloprovincialis) after five weeks of transplantation at various sites throughout the lagoon of Venice(mean ± s.e., n = 3). Asterisks indicate significant difference (Dunnett�s test) between exposed and reference site (Ca Roman) (*p < 0.05; ***p < 0.001).


environmental biomonitoring programs from the Medi-terranean Sea (Viarengo et al., 1997; Petrovic et al.,2001). The antioxidant role of MTs has been evaluatingin other studies. Viarengo et al. (1999) showed that theseproteins are effective in protecting cells and the entireorganism against oxidative stress in mussel. That varia-tions in antioxidant enzyme activities parallel the MTcontent, may indicate a response to oxidative stress.

Nevertheless, it must be considered that MT contentof mussel tissue seems to be only �transiently� alteredby oxidative stress (Cavaletto et al., 2002). It can thenbe postulated that MT levels increased following thefirst week of transplantation and recovered basal valuesby the time of sampling (five weeks). It would have beeninteresting to monitor MT levels throughout the trans-plantation period every week. Nevertheless, the positive

Fig. 4. Principal component analysis (PCA), obtained by biplot technique, applied to mean data for each index in the eight sampling site.


correlation between MT content and hydroxyl radicalssupports the idea that this radical is preferably scav-enged by these metal binding proteins (Viarengo et al.,2000).

The PCA was a good tool, being able to discriminateimpacted sites compared with a reference, suggestingthat the mussels transplanted throughout the Venicelagoon were subjected to different levels of oxidativestress. It provided an easy and useful tool to summarizethe biomarker results, as previously shown by others(Romeo et al., 2003; Nesto et al., 2004).

5. Conclusions

Four oxidative stress biomarkers characterised thesusceptibility responses to oxidative stress of musselstransplanted to all the eight sites of the Venice lagoon.The TOSC results revealed the overall capacity of theorganisms to cope with varying degrees of oxidativestress exposure and were capable of discriminating be-tween sites. A decrease in CAT activity in all the studiedsites confirmed the use of this enzyme as a early warningbiomarker of oxidative stress, representing a sensitive re-sponse of the mussel antioxidant defence system. TheMDA content, based on the reaction of the chromogenicreagent N-methyl- 2-phenylindole (NMPI), seems to bea sensitive method to evaluate an oxidative stress dam-age in mussels. Sufficient separation of the lagoon siteswas obtained and a negative correlation with peroxylradical and catalase activity was found. Similar MT re-sponse in all lagoon sites after five weeks of transplanta-tion did not provide new information about the role ofthese proteins in oxidative stress, although the exact rea-son was not defined.

Acknowledgements

This study was funded by the Venice Water Author-ity through Consorzio Venezia Nuova (ICSEL Project).Additional funding was provided by the Norwegian Re-search Council under the program ‘‘Marine Resource,Environment and Management’’, project No146478/120 and by the Norwegian marine research laboratoryRF-Akvamiljø.

We would like to thank Emily Lyng for revising theEnglish.

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