Ecotoxicological effects of metal pollution in two mollusc species from the Spanish South Atlantic...

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Environmental Pollution -- (2005) ---e---

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Ecotoxicological effects of metal pollution in two molluscspecies from the Spanish South Atlantic littoral

V. Funes a, J. Alhama a, J.I. Navas b, J. Lopez-Barea a, J. Peinado a,*

a Department of Biochemistry and Molecular Biology, University of Cordoba, Campus de Rabanales,

Edificio Severo Ochoa, 14071 Cordoba, Spainb CIFPA ‘‘Agua del Pino’’, IFAPA, Consejerıa de Innovacion, Ciencia y Empresa,

Junta de Andalucıa. Aptdo. 104, 21071 Huelva, Spain

Received 7 January 2005; accepted 13 May 2005

Oysters adapt to metal pollution while mussels are more sensitive.

Abstract

Metal accumulation and some of their biochemical effects have been studied in oysters (Crassostrea angulata) and mussels(Mytilus galloprovincialis) of the South Atlantic Spanish littoral. Especial attention has been paid to antioxidant defences and

oxidative damage to biomolecules. Deep differences in the response of oysters and mussels to metal pollution were found. Oysters,with the higher metal loads of both species, showed increased antioxidant defences, and less extensive oxidative damage. In contrast,mussels, which accumulated much lower metal concentrations, showed clear increases in oxidized biomolecules, in agreement withtheir low increases in the antioxidant defence mechanisms. Our results suggest that mussels are more sensitive and less well adapted

to metal pollution, probably explaining their absence in the most contaminated studied site, Mazagon. We conclude that oysters canbe used as more sensitive bioindicator of pollution in the South Spanish littoral, and as a suitable model to study the adaptation tometal pollution.

� 2005 Elsevier Ltd. All rights reserved.

Keywords: Oyster; Mussel; Metal pollution; Antioxidative defence; Oxidative damage

1. Introduction

Bivalve molluscs are useful organisms for monitoringtrace metal pollution in coastal environments, since they

Abbreviations: BCS, bathocuproine disulphonate; CAT, catalase;

DTPA, diethylenetriaminepentaacetic acid; 8-oxodG, 7,8-dihydro-8-

oxodeoxyguanine; DTNB, 5,5#-dithiobis-2-nitrobenzoic acid; EDTA,

ethylenediaminotetraacetic acid; GPX, glutathione peroxidase; GST,

glutathione-S-transferase; MDA, malondialdehyde; MT, metallothio-

nein; PMSF, phenylmethylsulphonylfluoride; ROS, reactive oxygen

species; SOD, superoxide dismutase.

* Corresponding author. Tel.: C34 57 218317; fax: C34 57 218592.

E-mail address: [email protected] (J. Peinado).

0269-7491/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2005.05.016

accumulate high metal levels in their tissues, providingan indication of environmental pollution (Regoliand Orlando, 1994). Studies have been carried out atvarious levels to identify alterations that could serve asuseful biomarkers of exposure and effect to contami-nants. Often, field results differ from those obtainedunder laboratory conditions, since organisms chronicallyexposed to polluted environments have acquired com-pensatory adaptive mechanisms (Lopez-Barea, 1995;Regoli and Principato, 1995; Giguere et al., 2003).

The Spanish South Atlantic littoral, and particularlythe coast of Huelva, is an intensive area for productionof bivalve molluscs. Nevertheless, high levels of metalsand organic pollutants are detected in water, sediments,

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2 V. Funes et al. / Environmental Pollution -- (2005) ---e---

and animals, since Huelva littoral receives contaminantsfrom several sources (Fig. 1). The Odiel and Tinto riversdrain the Iberian Pyrite Belt, one of the largest sulphidedeposits in the world. Mining activities in this area areknown from the sixth century BC and have left a largequantity of mine tailings, from which metal-rich acidicleachate and eroded material are washed into the rivers.As a result, the rivers are highly acidic (pH 2.2e3.6) andhave millimolar sulphate and iron concentrations andmicromolar cobalt, copper, manganese and zinc concen-trations (Elbaz-Poulichet et al., 1999; Rodrıguez-Ortegaet al., 2002; Braungardt et al., 2003; Morillo et al., 2004).In addition, numerous chemical industries related topetrochemistry, and the production of fertilizers, sul-phuric acid, titanium oxide, cellulose and copper, arelocated in Huelva Estuary. The dominant winds in thisarea are south-westerly, generating a current parallel tothe coastline (known as the longshore drift) with highcapacity for carrying matter (Fig. 1). As a result, Cu andZn concentrations at Huelva littoral show the followinggradient: Isla Cristina ! Punta Umbrıa ! Mazagon(Morillo et al., 2004). Previous studies showed that fishand bivalve molluscs living in Punta Umbrıa andMazagon accumulate metals and organic compounds(Rodrıguez-Ariza et al., 1992, 1993). Oysters collectedfrom the Huelva Estuary accumulated copper in so highconcentrations that their tissues showed a greenishcolour (Sarasquete et al., 1997).

Copper (Cu) and zinc (Zn) are essential traceelements found in a variety of cells and tissues.

SevillaHuelva

Mz

Industrialareas

PU

Odi

el R

iver

Tinto

Riv

er

Atlantic Ocean

Iberian Pyrite Belt

IC

Fig. 1. Map of southwest Spain showing the localization of the

sampling sites at Huelva littoral. A thick black line shows the

Portuguese-Spanish border; black lines, the Odiel and Tinto Rivers

flowing from the Iberian Pyrite Belt (IPB) to form Huelva Estuary;

small black dots, the sampling sites: Isla Cristina (IC), Punta Umbrıa

(PU) and Mazagon (Mz); black rectangles, location of industrial areas.

A thick dark arrow shows the longshore drift.

However, when present in excess Cu and Zn can betoxic and cause several biochemical effects (Viarengo,1985; Gaetke and Chow, 2003). Particularly, transitionmetals catalyse the generation of reactive oxygen species(ROS) that damage oxidatively various biomolecules(Sies, 1986). For instance, fatty acids generate by-products such as malondialdehyde (MDA) (Gutteridge,1984; Brown and Kelly, 1996). In addition, 7,8-dihydro-8-oxodeoxyguanine (8-oxodG) is one of the multipleoxidative causes of damage induced in DNA byhydroxyl radical. This DNA damage can lead tonecrosis, apoptosis, or heritable mutations, and there-fore has the potential to impact individuals as well aspopulations (Kuchino et al., 1987; Halliwell, 1993).

Aerobic organisms have several defence lines fromoxidative stress. Primary antioxidant enzymes, such assuperoxide dismutase and catalase, detoxify ROS.Glutathione peroxidase also reduces reactive lipidhydroperoxides, avoiding the formation of malondial-dehyde (Flohe, 1989). Glutathione-S-transferases (GST)conjugate electrophilic compounds to GSH; in addition,some GST forms play significant roles in the de-toxification of the reactive products from lipid perox-idation (Jensson et al., 1986; Pickett, 1989). Antioxidantenzymatic defences are useful biomarkers of pollutionby metals that generate oxidative stress in marineorganisms (Rodrıguez-Ariza et al., 1992, 1993; Rodrı-guez-Ortega et al., 2002; Romero-Ruiz et al., 2003).Taurine is a ubiquitous sulphur amino acid present inhigh concentrations in bivalve molluscs, especially inoysters (Yoshikawa et al., 1997). Its functions have beenmore extensively studied in mammals and includemembrane stabilization, neurotransmission, and show-ing an antioxidant role (Palmi et al., 1999; Obrosovaet al., 2001). Taurine also forms stable ternary com-plexes with copper and dipeptides at physiological pH(O’Brien et al., 1999). Metallothioneins, small inducibleproteins that bind metals with their cysteine residues, areuseful metal-pollution biomarkers (Viarengo et al.,2000). Biological functions of metallothioneins includehomeostasis and sequestration of both essential andnonessential metals, detoxification of metals and scav-enging of free radicals (Bremner and Beattie, 1990;Viarengo et al., 2000).

In the present work, we have studied the status of theSpanish South Atlantic littoral to assess biologicaleffects of metal contaminants on two species of bivalvemolluscs, oysters (Crassostrea angulata) and mussels(Mytilus galloprovincialis). Both species are differentlydistributed along the Huelva littoral; since oysters areregularly distributed, while mussels are absent from themost polluted site studied, Mazagon. The relationshipsbetween metal levels, antioxidant defences, and oxida-tive damage were studied to establish the distinctadaptive responses showed by oysters in comparisonto mussels.


3V. Funes et al. / Environmental Pollution -- (2005) ---e---

2. Materials and methods

2.1. Reagents

Malondialdehyde bis(dimethyl acetal), 2#-deoxygua-nosine, bathocuproine disulphonate (BCS), diethylene-triaminepentaacetic acid (DTPA), RNAse A, proteinaseK, nuclease P1, dinitrofluorobenzene, taurine, phenyl-methylsulphonylfluoride (PMSF), 5,5#-dithiobis-2-nitro-benzoic acid (DTNB), leupeptin, NADPH, GSH,cytochrome c, xanthine, xanthine oxidase and chlorodi-nitrobenzene were purchased from Sigma (St Louis,USA). DNA-grade phenol-saturated solution was fromAmresco (Solon, USA); EDTA, cumene hydroperoxideand metal standards were obtained from Merck(Darmstadt, Germany); hydrogen peroxide was fromPanreac (Barcelona, Spain); PTFE filters were from Lida(Wisconsin, USA). Chelex 100 resin and the Bradfordbased protein assay were purchased from Bio-Rad(Barcelona, Spain). Pure 8-oxodG was a generous giftfrom Professor J. Westendorf, University of Hamburg,Germany. All other chemicals used were reagent quality.

2.2. Animals and sampling areas

Oysters (Crassostrea angulata) and mussels (Mytilusgalloprovincialis) were collected with low tide level inspring (MayeJune 2000) at three sites from the SouthAtlantic Spanish littoral, Isla Cristina, Punta Umbrıaand Mazagon (Fig. 1). Animals were sent at 4 �C to thelaboratory, where the shells were washed, opened anddiscarded. Oysters were occasionally dissected, the gillsbeing separated from the rest of the body; haemolymphwas obtained by aspirating from the heart witha micropipette, and haemocytes were separated bycentrifugation (1000 ! g for 5 min). Mussels were notdissected and the study was carried out with the wholeanimal. For biochemical studies, soft tissues were frozenat �80 �C, ground in a mortar with liquid N2 and keptat �80 �C until used.

2.3. Metal analysis

Ground samples (0.25 g) were dried at 100 �C for12 h, digested with concentrated HNO3 (0.2e0.4 ml),and treated with 30% H2O2 (1.5e2 ml) in Teflonreactors (12.5 ! 3.5 cm) using a microwave oven.Reactors were subsequently allowed to cool for at least30 min. Finally, bidistilled water (10 ml) was addedbefore metal analysis using a flame atomic spectropho-tometer model 3100 (Perkin Elmer). Metal content wasestimated using metal standard solutions (1000 ppm) in0.05 M nitric acid.

2.4. HPLC equipment

HPLC separations were carried out using a liquidchromatograph fitted with a solvent delivery module126, an injection valve 210A, a 20 ml injection loop andan analogue interface 406 (all from Beckman, SanRamon, CA, USA). The system was controlled witha Deskpro 386/20e computer (Compaq, Houston, TX,USA) using System Gold 6.0 software (Beckman).Malondialdehyde, taurine and deoxyguanosine werequantified using a diode array detector (model 168;Beckman). A Coulochem EC detector (ESA, Bedford,MA, USA), fitted with a guard cell 5020 and ananalytical cell 5011 was used for 8-oxodG quantifica-tion. Separation of dG and 8-oxodG was achieved ina Spherisorb ODS2 column (250! 4 mm, 3 mm par-ticles; Supelco, Bellefonte, PA, USA). For taurine andmalondialdehyde assays, samples were separated ona Supelcosil LC-18 column (250 ! 4.6 mm, 5 mmparticle; Supelco).

2.5. Cell-free extract preparation

All steps were carried at 4 �C. Pooled ground animalsor tissues (2e3 g) were disrupted in a T25 Ultraturraxapparatus (Janke and Kunkel, Staufen, Germany) in10 mM potassium phosphate buffer (pH 7.5, 4 ml/g)containing 1 mM EDTA, and 1 mM PMSF. Homoge-nates were centrifuged for 1 h at 31 000 ! g, and thesupernatants dialysed in PD-10 columns (Pharmacia)previously equilibrated in the above buffer.

2.6. Biochemical determinations

Enzymatic activities were assayed spectrophotomet-rically (Rodrıguez-Ortega et al., 2002). Catalase (CAT)was assayed at 240 nm with 20 mM H2O2. Superoxidedismutase (SOD) was assayed at 550 nm with 48 mMxanthine, 0.2 U xanthine oxidase, and 19.2 mM cyto-chrome c. Glutathione peroxidase (GPX) was measuredat 340 nm with 1 mM GSH, 2 mM cumene hydroper-oxide, 0.24 U glutathione reductase, 0.15 mM NADPH,and 2 mM cumene hydroperoxide (total GPX) or0.15 mM H2O2 (Se-GPX). Protein determination wasmade according to Bradford (1976).

Malondialdehyde (MDA) was extracted and analysedas described by Lepage et al. (1991) after separation byRP-HPLC and detection at 535 nm. MDA content wasestimated using malondialdehyde bis(dimethyl acetal)as standard. For MDA extraction, 25 mM EDTA wasadded, although no increase in MDA content wasdetermined in its absence.

For 8-oxodG quantification, DNA was isolatedbasically as described by Shigenaga et al. (1994). Pre-viously, conditions for DNA extraction had to beoptimized, since artefactual oxidation was observed due



to the high metal loads in oysters from the more pollutedsites. Different metal chelating agents were tested, in-cluding EDTA, DTPA, Chelex 100 resin (results notshown) and BCS. The lowest oxidation during DNAisolation, monitored by 8-oxodG content, was obtainedwith BCS. Fig. 2 shows the effect of BCS concentration inthe homogenization and extraction buffers for DNAisolation, on the 8-oxodG/dG ratio. Extremely highamounts of this oxidized DNA form were obtained inthe absence of metal chelant, while much lower 8-oxodGcontents were determined with increasing BCS concen-trations. No appreciable decrease in 8-oxodG was foundabove 15 mM BCS in the extraction steps; thus, 15 mMBCS was routinely added to the homogenization andextraction buffers. After DNA extraction and hydrolysis,deoxynucleosides were analysed by RP-HPLC with UVand electrochemical detection as previously described(Alhama et al., 1998; Rodrıguez-Ariza et al., 1999).Results are expressed as the ratio of 8-oxodG/105 dGresidues, based on calibration curves obtained with dGand authentic 8-oxodG.

Taurine was extracted and analysed as describedPolanuer et al. (1994) with slight modifications. Groundtissue was disrupted in Milli-Q water (2 ml/g) containing15 mM bathocuproine disulphonate, heated at 100 �Cfor 2 h, and centrifuged at 15 000 ! g for 3 min. Afterderivatization of the supernatant with dinitrofluoroben-zene, taurine content was assayed by RP-HPLC withdetection at 350 nm.

Metallothionein was analysed by the colorimetricassay of the sulphydryl content of denatured and metalfree protein with DTNB as reported by Viarengo et al.(1997).

0.00001

0.0001

0.001

0.01

151050 20 25

8-o

xod

G/d

G

BCS concentration (mM)

Fig. 2. Protective antioxidative effect of bathocuproine disulphonate

(BCS) in nuclear DNA isolated from Mazagon oysters. Different BCS

concentrations were added to the homogenization and DNA

extraction buffers, and the ratio 8-oxodG/dG was determined in

DNA. Data show the mean values G SD of three independent

extractions.

2.7. Statistical analysis

The numbers show the mean G SD of at least threeindependent extractions. Statistical significance of thedifferences compared with reference site of Isla Cristinawas determined by the Student t-test and is shownas)p ! 0.05;))p ! 0.01;)))p ! 0.001. Normality ofdata and homogeneity of variance were previouslychecked. Correlation coefficients were calculated by themethod of parametric lineal correlation (Pearson).Instat� software, version 2.01/93 (Graphpad, SanDiego,CA, USA) was used in both cases.

3. Results

The quality of bivalve-producing areas of SouthSpanish littoral is periodically checked by analysingmetals and organic contaminants in water, sedimentsand in soft tissues of several bivalves using establishedprotocols. To add further insight to these routinemonitorizations, oysters (Crassostrea angulata) andmussels (Mytilus galloprovincialis) were sampled inspring 2000 at three sites of the Huelva littoral (Fig. 1),and the levels of two metals and of a battery ofbiochemical biomarkers of pollution were assessed inparallel in both organisms. Copper and zinc levelsdetected in water were higher in Punta Umbrıa (8 mgCu/L and 21 mg Zn/L), and Mazagon (11 mg Cu/L and46 mg Zn/L) sites in comparison to Isla Cristina (1.8 mgCu/L and 14 mg Zn/L), selected as a suitable referencesite (Consejerıa deMedio Ambiente, Junta de Andalucıa,personal communication). Table 1 shows the contents ofcopper and zinc determined in soft tissues of bivalvemolluscs. Oysters had much higher metal loads thanmussels, ranging between 30e52 fold for copper and22e30 fold for zinc in the whole body. Metal determina-tion was also carried out in two oyster tissues, gills andhaemocytes. As previously found in whole oyster bodies,the concentration of Cu and Zn was always higher inboth tissues in animals from Punta Umbrıa andMazagon sites compared to those from Isla Cristinareference site. The contents of Cu (5642 mg/g) and Zn(15 494 mg/g wet weight) found in haemocytes fromMazagon oysters were specially significant, since theycorresponded to 15.5- and 14.4-fold increases, respec-tively, relative to the reference site. The high metalcontent found in haemocytes explains the characteristicgreenish colour in their tissues and viscera (Han andHung, 1990; Lee et al., 1996; Sarasquete et al., 1997; Linand Hsieh, 1999) from oysters collected in Punta Umbrıaand Mazagon. With regard to mussels, animals collectedin Punta Umbrıa accumulated 5.2- and 2.9-fold morecopper and zinc contents than those from the referencesite. It is worthwhile to indicate that mussels were notfound in the Mazagon site.



Table

1

Copper

andzinccontents

inbivalvemolluscsfrom

differentsamplingsites

Samplingsite

Oysters

Mussels

Whole

body

Gills

Haem

ocytes

Whole

body

Cua

Zn

Cu

Zn

Cu

Zn

Cu

Zn

Isla

Cristina

153G

46b(1.0)c

553G

110(1.0)

223G

7(1.0)

892G

49(1.0)

365G

17(1.0)

1077G

99(1.0)

5G

0.4

(1.0)

25G

1(1.0)

Punta

Umbrıa

1352G

125(8.8))

))d

2240G

301(4.0))

))

1729G

24(7.8))

))

3103G

115(3.5))

))

1274G

274(3.5))

))

1831G

431(1.7))

26G

3(5.2))

))

73G

1(2.9))

))

Mazagon

1611G

57(10.5))

))

3016G

131(5.5))

))

1907G

138(8.6))

))

4275G

33(4.8))

))

5642G

2906(15.5))

15494G

8885(14.4))

n.d.e

n.d.

aMetalcontentisexpressed

asmg/g

wet

weight.

bData

show

themeanG

standard

deviationofthreemetaldeterminations.

cRatiobetweenmetalcontentin

each

samplingsite

relativeto

thereference

site

(IslaCristina).

dStatisticalsignificance

ofthemetalcontentin

thedifferentsamplingsitesrelativeto

thevalues

ofeach

metalatthereference

area;)p!

0.05;)))p!

0.001.

eMusselswerenotfoundin

Mazagon(n.d.,notdetermined).

The levels of some antioxidant enzymatic activitieswere also measured in oysters and mussels (Fig. 3) toassess some biological responses to metal pollution,focusing particularly on antioxidative defences andon oxidative damage to several biomolecules. Oystersfrom Mazagon showed significantly higher activities ofCAT, SOD, total-GPX and GST, compared with thosefrom Isla Cristina reference site; Se-GPX activity wasalso increased, but not significantly. In relation tomussels, only GST activity rose significantly ( p ! 0.05)in Punta Umbrıa while the differences in the otherbiomarkers were much smaller.

Fig. 4 shows the levels of taurine and metallothioneindetermined in oysters and mussels of the sites studied.Oysters from both polluted sites contained significantlyhigher levels of taurine (w3-fold) than referenceanimals. On the contrary, in mussels from the pollutedsite the concentration of taurine showed a significantdecrease. The higher metal content of bivalve molluscsfrom Punta Umbrıa and Mazagon was further con-firmed by analysing the metallothionein content (Fig. 4).Both oysters and mussels from Punta Umbrıa andMazagon displayed significantly higher metallothioneinlevels than animals from the reference site. It should benoticed the higher increase in MT content in musselsfrom Punta Umbrıa relative to Isla Cristina (3.9-fold),compared to the limited although significant incrementdetected in oysters. Owing to their inducibility bymetals, metallothioneins have been proposed as bio-markers of the metallic contamination of the environ-ment (Cosson, 2000). The higher activities ofantioxidant enzymes in Mazagon oysters and the higherlevels of taurine and MT in oysters from Punta Umbrıaand Mazagon, while only MT and GST increased inPunta Umbrıa mussels, suggested that oysters could bebetter protected than mussels from the oxidative stressassociated to metal pollution. Absence of mussels at themost polluted site studied, Mazagon, could support thisidea.

In consequence, we assayed oxidative damage toimportant biomolecules, such as lipids and DNA, bydetermining the levels of MDA and 8-oxodG. Fig. 5shows the MDA level and 8-oxodG DNA contentdetermined in whole oyster and mussel bodies. BothMDA and 8-oxodG decreased significantly in oystersfrom Punta Umbrıa and Mazagon sites relative to IslaCristina, suggesting a better protection of oysters withhigher metal loads against oxidative damage to bothlipids and DNA. The opposite response was found inmussels that showed higher oxidative damage tobiomolecules in Punta Umbrıa, a result that couldexplain the absence of mussels from Mazagon, a sitewhere the metal load of oysters was significantly higher(Table 1).

Finally, a statistical analysis was carried out tocorrelate metal contents and the response of biochemical



IC PU Mz IC PU0

100

200

300

0

20

40

60

***

0

50

100

150

0

50

100

150

200

0

50

100

150

200

250

Oysters Mussels

GS

T(m

U/m

g p

rote

in)

*

SeG

Px

(mU

/mg

pro

tein

)to

tal G

Px

(U/m

g p

rote

in)

**

***

*

**

SO

D(m

U/m

g p

rote

in)

Cat

alas

e(U

/mg

pro

tein

)
biomarkers in bivalve molluscs (Table 2). In oysters,a positive and significant correlation was obtainedbetween all the studied biomarkers involved in thedefence against oxidative stress, both enzymatic (CAT,SOD, GPX, GST) and not enzymatic (taurine andMTs), and the Cu and Zn contents. On the contrary,a significant negative correlation was obtained betweenbiomarkers indicative of oxidative damage to biomole-cules (MDA and 8-oxodG) and both metals. A differentcorrelation pattern was observed in mussels, since GST,Se-GPx and MTs showed a positive significant correla-tion with Cu and Zn. Nevertheless, unlike oysters,a positive and significant correlation was obtained inmussels between metals and MDA content, whilea negative significant correlation was obtained betweentaurine content and Cu and Zn levels.
4. Discussion

The measurement of the biological effects of chemicalpollutants has become of major importance for theassessment of the quality of the coastal environment(Gray, 1992). Marine bivalve molluscs, especiallymussels and oysters, have gained a worldwide impor-tance as bioindicators of marine and estuarine pollution(Goldberg et al., 1978). In the present study, oysters andmussels have been used as sentinel organisms to assessthe metal pollution status of the South Atlantic Spanishlittoral. At Huelva littoral a metal contaminationgradient is present, being Mazagon the more pollutedarea (Fig. 1). When molluscs are exposed at their naturalhabitats to metal pollution, processes of bioaccumula-tion and subsequent biological effects can be expected tooccur. Metal bioaccumulation capacity depends onspecies, and particularly oysters collected at Huelvalittoral had much higher metal contents than musselsdue to their well-known bioconcentration capacity(Kwon and Lee, 2001; Liang et al., 2004), to the pointthat their tissues had a greenish appearance. Thiscolouration is probably due to metal accumulation inhaemocytes, that are distributed by the haemolymphthroughout the entire organism (Sarasquete et al., 1997).Establier (1972) found in Punta Umbrıa oysters a coppercontent (1657 mg/g whole body) similar to that de-termined by us (1342), indicating that oysters of thisarea are living in a chronic metal-polluted environment.

A number of biochemical pollution biomarkers wereanalysed in oysters and mussels to assess the effect of Cu

Fig. 3. Antioxidative enzymatic activities in oysters (Crassostrea

angulata) and mussels (Mytilus galloprovincialis) from different

sampling sites. Activities of catalase (CAT), superoxide dismutase

(SOD), total (total-GPX) and selenium-dependent glutathione perox-

idase (Se-GPX) and glutathione S-transferase (GST) were assayed.

Data show the mean values G SD (n Z 3).



and Zn metals. Since the formation of ROS has beenproposed as the basis of copper toxicity (Gaetke andChow, 2003), biomarkers responsive to oxidative stresshave been used in the present study. While many studieshave been carried out in relation to the effect of metalson molluscs, the toxicity of metals can be under-estimated when short exposure tests are performed,especially if they are shorter than the life span of the testorganism (Giguere et al., 2003). We now demonstratethat in natural habitats, oysters are better adapted tocopper and zinc pollution than mussels. Despite theirmuch higher accumulation of Cu and Zn, oysters aremore efficiently protected from oxidative stress thanmussels. Antioxidant enzymatic activities, and taurineand metallothionein levels were higher in pollutedoysters, explaining their lower oxidative damage(MDA and 8-oxodG contents) according to the pro-tective role of such defences from the deleterious effectsof ROS (Sies, 1986). In close parallel, Chamaelea gallinaclams chronically exposed to contaminants releasedfrom Huelva and Guadalquivir Estuaries also showedhigher antioxidant and glutathione-related activities,and lower MDA content (Rodrıguez-Ortega at al.,2002). Similarly, three species of bivalve molluscs(Chamaelea gallina, Ruditapes decussatus, and Crassos-trea gigas) from contaminated areas of the SouthAtlantic Spanish littoral contained fewer genotoxins ofoxidative type than animals from less polluted areas(Rodrıguez-Ariza et al., 1992). The behaviour of themussels was markedly different in the present study; aninsufficient response of their antioxidant systems in-duced a precarious state and impaired their ability toneutralize ROS and to prevent oxidative damage tobiomolecules. In a comparative study, an increase ofMDA levels was noticed for the gills and the digestivegland of mussels (Mytilus edulis) exposed for 21 days tocadmium, silver or mercury whereas a decrease of MDAlevels was observed in the gills of Crassostrea gigasoysters (Geret et al., 2002).

Antioxidant enzymatic activities can explain part ofthe special protection of oysters against metal-inducedoxidative stress. Antioxidant properties have been alsoassigned to MTs mainly derived from sulphydrylnucleophilicity, but also by binding transition metalsdisplaying Fenton reactivity (Fe, Cu) that reducesoxidative stress (Bremner and Beattie, 1990; Viarengoet al., 2000). Oyster extracts contain high levels of aminoacids, taurine being the most abundant, and displayradical-scavenging activity, although the compoundsresponsible of this effect have not been definitivelyidentified (Yoshikawa et al., 1997). Nevertheless, it hasbeen proven that taurine binds metals (O’Brien et al.,1999) and that Zn and Cu complexation with taurineoccurs in polluted oysters (Howard and Nickless, 1977).

Owing to their inducibility by certain metals, metal-lothionein contents have been proposed as biomarkers

of the metallic contamination of the environment(Cosson, 2000). In the present study the MT levels weresignificantly correlated with Zn and Cu contents in bothmussels and oysters, validating the use of MT levelsas biomarkers responsive to metals in field conditions.Although it is generally assumed that MTs play a centralrole in cellular metal homeostasis, their levels do notexplain the different metal bioaccumulation capacity ofboth mollusc species. All aquatic invertebrates take upand accumulate high levels of trace metals, although thesubsequent body concentrations show wide variabilityacross metals and invertebrate taxa, even living in thesame habitat (Rainbow, 2002). Tolerance depends onthe ability to regulate the heavy metal concentration inthe cell and to accumulate excess metal in non-toxicforms (Viarengo and Nott, 1993; Rainbow, 2002). Threeheavy metal homeostasis mechanisms have been identi-fied in marine invertebrate cells, showing varyingdegrees of effectiveness in different organisms: (1)binding to metallothioneins, (2) compartmentalizationwithin lysosomes, and (3) formation of insolubleprecipitates such as Ca/Mg concretions or Ca/S granules(Viarengo and Nott, 1993). Fe, Cu and Zn granules havebeen observed in the wall of the heart of Crassostrea

0

3

6

9

12

Tau

rin

e(µ

mo

l/mg

pro

tein

)

*

*

IC PU MZ IC PU0

20

40

60

80

**

**

***

***

Oysters Mussels

Met

allo

thio

nei

n(µ

g/m

g p

rote

in)

Fig. 4. Taurine and metallothionein levels in bivalves from different

sampling sites. Data show the mean values G SD (n Z 3).



angulata from different Southwest Spanish coasts, inbrown cells, as well as in granular amoebocytes, thatincrease in green specimens (Sarasquete et al., 1997).Oliver et al. (2003) have demonstrated not only greaterhaemocyte numbers but also a higher bactericidalcapacity in oyster haemocytes from metal contaminatedsites that may provide an elevated level of defenceagainst bacterial challenge.

Oysters accumulate metals at very high concentra-tions without apparent signs of any ill or detrimentaleffects (Lin and Hsieh, 1999). This ecological advantagecould be due to the fact that oysters have very efficientantioxidative defence mechanisms and, possibly, a highercapacity to held metals in detoxified form. On thecontrary, mussels have lower defence mechanismsagainst metal oxidative challenge and toxicity, explain-ing their absence from the most polluted habitat.

5. Conclusion

Exposed bivalves (oysters and mussels) were collectedfrom their natural habitat along a metal contaminationgradient. Oysters accumulated higher amounts of metaland, even so, they were found in the more polluted

20

10

0

30

40

50

Mal

on

dia

ldeh

yde

(nm

ol/m

g p

rote

in)

8-o

xod

G/1

05 d

G

IC PU MZ IC PU0

2

6

4

** **

**

*** ***

**

Oysters Mussels

Fig. 5. Malondialdehyde level and DNA content of 8-oxodG in bivalves

fromdifferent sampling sites. Data show themean values G SDof three

(for MDA) or six to nine (for DNA) independent extractions.

Table

2

Correlationcoeffi

cients

betweencopper

andzinccontentandthebiochem

icalbiomarkersanalysedin

bivalvemolluscs

Oysters

Mussels

Oysters

Mussels

Cu

Zn

Cu

Zn

Cu

Zn

Cu

Zn

GST

0.743)))

0.714)))

0.810)))

0.816)))

Taurine

0.935)))

0.770)))

�0.787)))

�0.793)))

Se-GPX

0.742)))

0.331)))

0.490)

0.492)

MT

0.892)))

0.914)))

0.977)))

0.989)))

Total-GPX

0.854)))

0.617)))

0.057ns

0.057ns

MDA

�0.972))

�0.963)))

0.920)))

0.926)))

SOD

0.627)))

0.490)))

0.045ns

0.045ns

8-oxodG

�0.703))

�0.653)))

0.020ns

0.020ns

CAT

0.848)))

0.627)))

0.228ns

0.230ns

Coeffi

cients

werecalculatedbythemethodofparametriclinealcorrelation(Pearson).Statisticalsignificance:ns,notsignificant;

)p!

0.05;

))

p!

0.01;

)))p!

0.001.



environments, unlike mussels. The present study dem-onstrates that the combined increase of antioxidantdefences and metal stabilization by complexation pro-tect oysters from stress provoked by metal-pollutedenvironments. In consequence, oysters can be chosen asmodel sentinel organisms for adaptation studies, whilemussels, more sensitive to pollution, can be more usefulas indicator organisms.

Acknowledgement

This work was funded by the Spanish Ministry ofScience and Technology (grants IFD1997-0610-CO3-01/AMB and REN2002-04366-CO2).

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