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2008-CBP-Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed

Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed to polychlorobiphenyls and metals

Melissa Faria a ,Luis Carrasco b ,Sergi Diez b ,c ,Maria Carmen Riva a ,Josep Maria Bayona b ,Carlos Barata b ,?

a Laboratory of Environmental Toxicology (UPC),CN 150Km 14.5,Terrassa 08220,Spain

b Department of Environmental Chemistry,IDAEA,(CSIC),Jordi Girona 18,Barcelona 08034,Spain c

Environmental Geology Department,ICTJA-CSIC,Lluis Soléi Sabarís,s/n,Barcelona 08028,Spain

a b s t r a c t

a r t i c l e i n f o Article history:

Received 20June 2008

Received in revised form 28July 2008Accepted 29July 2008Available online xxxx Keywords:

Oxidative stress

Dreissena polymorpha Methyl mercury Mercury PCB

Cadmium EROD

Contaminant related changes in behavioral,phase I and II metabolizing enzymes and pro-oxidant/antioxidant processes in the freshwater mussels Dreissena polymorpha exposed to metals and PCBs were assessed.Behavioral and biochemical responses including ?ltering rates,key phase I,II and antioxidant enzymes and levels of metallothioneins,glutathione,lipid peroxidation and DNA strand breaks were determined in digestive glands of mussels after being exposed to sublethal levels of mercury chloride,methyl mercury,cadmium and Aroclor 1260during 5days.In 7out of 12responses analyzed,mussels showed signi ?cant differences across treatments.Unusual properties of measured ethoxyresoru ?n-O -deethylase (EROD)activities indicated that mussels lack an inducible CYP1A enzymatic activity.Despite of using similar exposure levels,inorganic and organic mercury showed different biomarker patterns of response with methyl mercury being more bio-available and unable to induce metallothionein proteins.Mussels exposed to Cd presented higher levels of metallothioneins and an enhanced metabolism of glutathione,whereas those exposed to Aroclor showed their antioxidant glutathione peroxidase related enzyme activities inhibited.Although there was evidence for increased lipid peroxidation under exposure to inorganic and organic mercury,only mussels exposed to Aroclor had signi ?cant greater levels than those in controls.

?2008Elsevier Inc.All rights reserved.

1.Introduction

Aquatic organisms are currently being exposed to multiple chemical contaminants with different mechanisms of toxicity,each contributing to a ?nal overall adverse effect.Consequently,in ecological quality monitoring programs,the integration of chemical data with biological responses (biomarkers)is strongly recommended to characterize effects of contaminants to organisms (den Besten,1998;Clements,2000).Biomarkers can offer a more complete and bio-logically more relevant information on the potential impact of toxic pollutants on an organism's health (van der Oost et al.,2003).Fur-thermore,the use of a large set of biochemical responses may allow us to identify potential hazardous contaminants in the ?eld.This approach has been successfully used mostly in ?sh and bivalve marine mollusks (see reviews of Di Giulio et al.,1995;Livingstone,2001;van der Oost et al.,2003)and include biochemical response that are related with the metabolism and toxicity modes of action of contaminants.

Due to its abundance in many European and American ?uvial habitats,its relative long life span and great ability to bio-concentrate toxic chemicals,the freshwater bivalve zebra mussel (Dreissena

polymorpha )has been used extensively as sentinel species to monitor persistent organic contaminants and metals (Kraak et al.,1991;De Lafontaine et al.,2000;Binelli et al.,2001;Camusso et al.,2001;Berny et al.,2002).More recently,the use of biochemical responses of D.polymorpha has also allowed to detect speci ?c biological response to particular contaminants or environmental pressures (De Lafontaine et al.,2000;Lecoeur et al.,2004;Minier et al.,2006;Binelli et al.,2005,2006a,b;Giamberini and Cajaraville,2005;Ricciardi et al.,2006;Voets et al.,2006;Marie et al.,2006;Osman et al.,2007;Guerlet et al.,2007;Binelli et al.,2007,2008).Nevertheless,the above mentioned studies have been focused in most occasions on just a few biochemical responses and restricted to few contaminant substances,thus their use as diagnosis toxicity tools of ?eld populations exposed to multiple pollutants is limited and can be misleading.Indeed contradictory results exist within zebra mussel biomarker studies and between them and those conducted with other related bivalve species.For example in laboratory exposures,Lecoeur et al.(2004)reported that in zebra mussel metallothionein proteins were induced by Cd but not by Cu,whereas in an early ?eld work De Lafontaine et al.(2000)found a positive relationship between metallothionein levels and Cu.Moreover,De Lafontaine et al.(2000)and Binelli et al.(2005,2006a,b;2007)reported measurable and inducible CYP1A-ethoxyresoru ?n-O -deethylase (EROD)like activity in zebra mussels collected from polluted sites or exposed in the laboratory to polychlorobiphenyls (PCBs),polybrominated diphenyl

Comparative Biochemistry and Physiology,Part C xxx (2008)xxx –xxx

?Corresponding author.Tel.:+34934006100;fax:+34932045904.E-mail address:cbmqam@cid.csic.es (C.Barata).

CBC-07411;No of Pages 8

1532-0456/$–see front matter ?2008Elsevier Inc.All rights reserved.doi:10.1016/j.cbpc.2008.07.012

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology,Part C

j o u r n a l h o m e p a g e :w ww.e l s ev i e r.c o m /l o c a t e /c b p c

ARTICLE IN PRESS

ethers(PBDEs),dichlorodiphenylthrichlor-oethanes(DDTs)and hexa-chlorobenzene,whereas in other bivalve species this activity was seldom detected(Livingstone et al.,1990a;Porte et al.,1995,2001;Soléand Livingstone,2005).

The aim of this study is to use a large set of biomarkers to identify speci?c and distinctive patterns of responses of D.polymorpha individuals to known contaminants.The biomarkers included the xenobiotic metabolizing phase I and II enzyme responses EROD and glutathione S-transferase,respectively(van der Oost et al.,2003); glutathione(GSH)levels and glutathione reductase,which aids maintenance of GSH levels recycling oxidized glutathione(Canesi et al.,1999);metallothionein proteins as a marker for metal exposure (Amiard et al.,2006);antioxidant enzymes involved in detoxifying reactive oxygen species such as superoxide dismutase(SOD EC1.15.1.1 converts O2·?to H2O2),catalase(CAT EC 1.11.1.6—reduces H2O2to water),glutathione peroxidase(GPX EC1.11.1.9—detoxi?es H2O2or organic hydroperoxides)and markers of oxidative tissue damage (lipid peroxidation and DNA strand breaks)(Halliwell and Gutteridge, 1999).The selected contaminants included the metals cadmium, mercury in its inorganic and organic forms,which are known to alter GSH and metallothionein levels in mussels(Canesi et al.,1999;De Lafontaine et al.,2000;Lecoeur et al.,2004)and a PCB mixtures, Aroclor1260,which has been reported to induce EROD activity in zebra mussel(Binelli et al.,2006a).The chosen contaminants may co-occur together in large amounts in heavy industrialized rivers and lakes and are susceptible to be bioaccumulated by mussels(Mikac et al.,1985;Rutzke et el.,2000;Hanari et al.,2004).Furthermore,by including different forms of mercury it will be possible to test if metal speciation matters.So far in common(Mytilus galloprovincialis)there is evidence that mercury speciation affected differently glutathione metabolism(Canesi et al.,1999),but there is little or no information in other mussel species nor on other biomarkers(Diez et al.,2008).

2.Materials and methods

2.1.Chemicals

The following chemicals were used for laboratory exposures: Aroclor1260(technical PCB mixture,Supelco,Bellefonte,USA), mercury chloride(HgCl2;99%purity,Sigma-Aldrich),methyl mercury chloride salt(CH3ClHg,Pestanal-Riedel-de Ha?n,Sigma-Aldrich, Seelze,Germany)and cadmium(CdCl2,99%purity,Sigma-Aldrich).

Homogenization along with enzymatic activities,lipid peroxidation, DNA damage and protein assays was preformed with dithiothreitol (DTT);phenylmethanesulfonly?uoride(PMSF);ethylenediamine-tetra-acetic acid,disodium,salt,dihydrate(EDTA);hydrogen peroxide(H2O2); xanthine oxidase(EC 1.2.4.22);superoxide dismutase(EC 1.15.1.1); xanthine;β-nicotinamide adenine dinucleotide2′-phosphate reduced tetrasodium salt(NADPH);oxidized glutathione(GSSG);reduced glutathione(GSH);cumene hydroperoxide(CHP);sodium azide,1-chloro-2,4-dinitrobenzene(CDNB);glutathione S-transferase(EC 2.5.1.18);monochlorobimane(mCB);resoru?n ethyl ether(7-ethoxyr-esoru?n);resoru?n sodium salt;sodium hydroxide,2,6-di-tert-butyl-4-methylphenol(BHT);1-methyl-2-phenylindole;1,1,3,3-tetramethoxy-propane(TMP);sodium dodecyl sulfate(SDS);bisBenzimide H33258 (Hoescht dye);deoxyribonucleic acid sodium salt from calf thymus,type 1,?bres andγ-globulins from Sigma-Aldrich(St.Louis,MO,USA).All other chemicals were analytical grade and were obtained from Merck (Darmstadt,Germany).

2.2.Experimental animals and culture conditions

Following Binelli et al.(2006a),zebra mussels(D.polymorpha, Bivalvia:Dreissenidae)2cm long(shell length)tied on rocks,were collected from Riba-Roja reservoir in May2006by a scuba diver at3–5m depth and transported to the lab.Riba-Roja reservoir is localized in Ebro River,NE Spain and can be considered a reference site with low pollution levels(http://oph.chebro.es/DOCUMENTACION/Calidad/ CalidadDeAguas.html).Mussels attached to rocks were rinsed and introduced into glass aquaria at a density of0.5L per individual (approx.)and maintained under constant oxygenation N90%,tem-perature(20°C)and photoperiod(14h;10h;light:dark).Animals were cultured in local?eld collected water,which was progressively replaced by arti?cial ASTM hard water,and fed with a suspension1:1 of algae Scenedesmus subspicatus and Chlorella vulgaris(106cells/mL, daily).The medium was renewed every other day for10days to allow the acclimatization of animals.After this period,300mussels with similar length(2cm long)were selected for the experiments.They were gently cut off from rocks,placed on sheets of glass suspended in glass30L aquaria?lled with of20L medium and maintained5days further in the same conditions described above,but exposed to the different treatments.Only specimens able to re-attach themselves by the byssus on the sheet glass were used in the experiments,the test medium was changed daily and mussels were fed adding food only2h before water renewal.

2.3.Experimental design

Five different treatments were performed:control(no exposure); Aroclor1260(150ng/L);mercury chloride(40μg/L total ion Hg), methyl mercury chloride(40μg/L total ion Hg)and cadmium(34μg/L total ion Cd).Exposure levels were selected since are known to alter biomarker responses in mussels(i.e.metallothioneins,glutathione metabolisms and EROD activities;Canesi et al.,1999;Lecoeur et al., 2004;Binelli et al.,2006a).Stock solutions of cadmium and inorganic mercury(expressed as total ion concentrations)were prepared by adding analytical reagent grade salts to2L deionized water(Milli-Q; 18MΩcm?1resistivity)and sonicated during1h.Nominal test concentrations were subsequently prepared by adding aliquots of each metal stock solution to the aquaria?lled with20L of ASTM. Concentrated(20×)aqueous solutions of methyl mercury and PCB mixtures were prepared by adding appropriate amounts in acetone (HPLC grade;b0.5mL/L)to a borosilicate glass2L bottle container and allowing the acetone to completely evaporate with a stream of N2, leaving behind a crystalline residue on the glass.Then the glass container was?lled with the appropriate volume of ASTM hard water, sonicated during1h and mixed at20°C on an orbital shaker for1day. By using this procedure organic contaminants are dissolved directly in water without the aid of a carrier and hence the use of a solvent control is not needed(Barata and Baird,2000).Nominal test con-centrations were then subsequently prepared by adding the whole content of the solutions to the20L tanks pre-?lled with18L of ASTM hard water to provide a total volume of20L.

2.4.Chemical analyses

The analysis of contaminants were restricted to their tissue levels in whole mussel samples collected at the end of exposures using groups of four to ten organisms.Levels of Cd were determined following the methods of Barata et al.(2005).Freeze-dried organisms were digested in concentrated nitric acid and hydrogen peroxide using Te?on bombs at 90°C overnight.Within each digestion series,appropriate blanks with no animals and samples of similar weight of a certi?ed reference material(lobster hepatopancreas,Tor1,National Council of Canada, Ottawa)were also subject to the same procedure to account for background contamination levels and to validate the entire procedure. Cooled digested samples were diluted to a standard volume with deionized water.Trace Cd analyses were determined using a Perkin Elmer model Elan6000inductively coupled plasma mass spectrometer (ICP-MS).Calibration standards and a reagent blank were analyzed with every ten samples to monitor signal drift.In every instance,the signal typically changed by3–5%throughout an analytical run.Additionally,

2M.Faria et al./Comparative Biochemistry and Physiology,Part C xxx(2008)xxx–xxx

rhenium was used as an internal standard to correct for any non-spectral interference.Detection and quanti?cation limits of Cd were set to 0.01μg/g d.w.

Analysis of total mercury was performed following Diez et al. (2007)using the Advanced Mercury Analyzer AMA-254(Leco Corp., Altec,Praha,Czech Republic).The AMA instrument is based on sample catalytic combustion,preconcentration by gold amalgamation,ther-mal desorption and atomic absorption spectrometry(AAS).Freeze-dried mussel samples were precisely weighed(10to50mg)in a nickel boat on an analytical balance(Mettler-Toledo AT200,Columbus,OH, USA)and placed into the instrument which is automatically intro-duced into the AMA.The entire analytical procedure was validated by analyzing total mercury in dog?sh muscle CRM DORM-2obtained from the National Research Council Canada(NRCC),Ottawa,Canada. Analyses of CRM at the beginning and end of each set of samples (usually10)ensured that the instrument remained calibrated during the course of the study.Detection and quanti?cation limits were calculated from blank measurements;these values were0.2and 0.7ng/g d.w.of mercury,respectively.

For PCBs analysis,2g samples of zebra mussel were spiked with known amounts of isotopically labeled13C12-PCBs,homogenized with anhydrous sodium sulfate and extracted with hexane–dichloro-methane(1:1)in a Soxhlet apparatus for24h.Extracts were then clean-up with?orisil and concentrated before analysis.The PCB determinations were based on US EPA Method1668(US EPA,1997)by an isotope-dilution high-resolution mass spectrometric analysis.The puri?ed PCB extracts were analyzed by high resolution gas chromato-graphy–high resolution mass spectrometry on an AutoSpec-Ultima mass spectrometer(Micromass,Manchester,UK)coupled to a GC 8000series gas chromatograph(Carlo Erba Instruments,Milan,Italy) and equipped with a CTC A200S autosampler.A DB-5(J&W Scienti?c, Folsom,CA,USA)fused-silica capillary column(60m×0.25mm i.d., 0.25μm?lm thickness)was used with helium as carrier gas at a linear velocity of35cm/s at100°C.The temperature was raised from90°C (held for1min)to180°C(held for1min)at a rate of20°C per min, and then from180°C to300°C(held for10min)at a rate of3°C per min,using the split-less injection mode.High resolution gas chromatography–high resolution mass spectrometry operating con-ditions were:ion source and interface temperatures,250°C and 275°C,respectively;ionization energy,35eV electron ionization mode,and trap current300μA.The resolving power was kept at 10,000(10%valley de?nition).Veri?cation of resolution in the working mass range was obtained by measuring per?uorokerosene reference peaks.For high resolution gas chromatography–high resolution mass spectrometry analysis in the selected ion monitoring mode the two most abundant isotope peaks from the molecular ion region of each PCB homologue were used.The accuracy and precision of the system were evaluated by the use of a certi?ed reference material issued by NIST(SRM2977Mussel Tissue)and by periodical international intercalibration exercises(i.e.Quasimmeme).Recovery of internal standards in all samples was within90–95%and the limits of detection for each resolved congener were0.01ng/g f.w.

2.5.Biochemical and behavioral analysis

Following5days of exposure,50animals were sacri?ced and after rapid dissection,ten pools of?ve digestive glands each were dissected and immediately frozen in liquid nitrogen and stored at?80°C. Preliminary studies were performed in order to optimize the methodol-ogy involving the use of zebra mussel digestive glands,and to assure that the?ltering,enzyme,GSH,lipid and DNA assays were carried out within the linear range of calibration standards,enzyme concentration vs. reaction rate,and time vs.reaction rate.For the DNA damage and the majority of biomarkers including antioxidant enzymes,GST and GSH determination,pools were homogenized in a1:5weight:volume ice cold phosphate buffer100mM pH7.4containing150mM KCl and1mM EDTA.An aliquot of each homogenate was separated for DNA damage quanti?cation.The remaining homogenates were further centrifuged at 10000g for10min and the supernatants were immediately used for biochemical determinations.Metallothionein levels(MT)were deter-mined in the cytosolic digestive gland fractions and EROD activity in both the10000g and microsomal fractions,respectively,which were prepared by a modi?cation of the method of Jewell and Winston(1989). Brie?y,digestive gland pools were homogenized in1:3w/v cold100mM phosphate buffer(pH7.4)containing150mM KCl and1mM EDTA and supplemented with1mM dithiothreitol(DTT),0.1mM phenylmetha-nesulfonly?uoride(PMSF).Homogenates were centrifuged at10000g for30min.An aliquot of each homogenate was then separated for 10000g EROD assessment and the remaining10000g supernatant was further centrifuged at100000g for60min to obtain the cytosolic and microsomal fractions,respectively.Microsomal pellets were resus-pended in a small volume of100mM phosphate buffer(pH7.4) containing1.0mM EDTA,20%w/v glycerol,1mM DTT,0.1mM PMSF. Cytosolic and microsomal proteins were measured by the Bradford method(Bradford,1976)usingγ-globulin as standard.

CAT measurements were carried out using a spectrophotometer Cecil-CE9200(Cambridge,England)at25±0.5°C,whereas all the rest of the biomarkers were determined using a Multi-Detection Micro-plate Reader,BioTek?(Vermont,USA).Assays were run at least in duplicate for CAT and quadruplicate for the rest of the biomarkers.

EROD activity was determined in the10000g and microsomal fraction using a96-well microplate reader with modi?cations according to Burke and Mayer(1974).Essentially,10μL of microsomes were incubated at30°C for60min in a?nal volume of300μL containing100mM phosphate buffer,pH7.4,0.25mM NADPH,and 4.15μM7-ethoxyresoru?n.The increase in?uorescence resulting from 7-hydroxyresoru?n formation was measured at an excitation/emis-sion wave length of530nm:590nm.Quanti?cation was achieved using a standard curve of7-hydroxyresoru?n.Enzyme activity ethoxyresoru?n-O-deethylase(EROD)was expressed as fmol of reso-ru?n min?1mg?1of total assay proteins.

CAT activity was measured by the decrease in absorbance at 240nm due to H2O2consumption(extinction coef?cient40M?1cm?1) according to Aebi(1974).The reaction volume and reaction time were 1mL and1min,respectively.The reaction solution contained80mM phosphate buffer,pH6.5and50mM H2O2(Ni et al.,1990).SOD activity was determined indirectly with modi?cations according to McCord and Fridovic(1969)based on the measurement of the degree of inhibition of the reduction of cytochrome c by O·2?released by the xanthine oxidase/xanthine reaction.SOD units were determined using a standard curve of0–1.5SOD units/mL.The reaction contained 50mM phosphate buffer,pH7.8,0.1mM EDTA,23μM xanthine, 1.7mU/mL xanthine oxidase and10μM cytochrome c.Reactions were measured during2min at550nm,25°C.Final results were normalized by tissue total protein content and expressed as U/mg of total protein. GST activity towards1-chloro-2,4-dinitrobenzene(CDNB)was mea-sured as described by Habig et al.(1974).The reaction mixture contained 100mM phosphate buffer(pH7.5),1mM CDNB and1mM GSH.The formation of S-2,4dinitro phenyl glutathione conjugate was evaluated by monitoring the increase in absorbance at340nm during5min. Activity calculation was determined using GST's extinction factor coef?cient of9.6mM?1cm?1.Glutathione peroxide activities were measured according to Paglia and Valentine(1967)modi?ed by Lawrence and Burk(1976)and adapted to a96-well microplate reader. The reaction mixture contained100mM phosphate buffer(pH7.5), 2mM GSH,2U glutathione reductase,0.12mM NADPH,sodium azide (0.5mM),0.2mM H2O2and3mM cumene hydroperoxide(CHP).GPX activity was monitored by following the decrease in NADPH concentra-tion(at340nm),which is consumed during the generation of GSH from oxidized glutathione(extinction coef?cient6.2cm?1M?1),using H2O2 (Se dependent activity)and cumene hydroperoxide(total GPX)as substrate.Glutathione reductase(GR)activity was determined

3

M.Faria et al./Comparative Biochemistry and Physiology,Part C xxx(2008)xxx–xxx

according to Carlberg and Mannervik(1985)adapted to a96-well microplate reader,by following the oxidation of0.07mM NADPH consumed by glutathione reductase enzyme present in the sample in order to reduce the0.7mM of oxidized glutathione(GSSG)during2min at340nm.Activity calculation was determined using the enzymes extinction factor coef?cient of6.2cm?1mM?1.Reduced glutathione (GSH)quanti?cation was adapted to zebra mussel digestive gland according to Kamencic et al.(2000),who developed an alternative to the known chromatographic(HPLC)technique by using a monochlorobi-mane(mCB)approach.The new technique is simpler than the HPLC method and consists of adding0.1mM of mCB along with1U/mL of GST to each sample.Then the GSH present in the cells forming a GSH–mCB complex is measured?uorometrically using an excitation/emission wave length of360nm:460nm,after an incubation period of60min at room temperature and protected from light.The total content of GSH was then extrapolated from a GSH standard curve determined under the same experimental conditions as the samples.Results are then expressed as nmol per g of tissue wet mass.

Lipid peroxidation was determined according to Esterbauer et al. (1991).Lipid peroxides,derived from polyunsaturated fatty acid,are unstable and decompose to from a complex series of compounds.These include carbonyl compounds,of which the most abundant is mal-ondialdehyde(MDA).Measurement of MDA is widely used as an indicator of lipid peroxidation.The MDA assay is based on the reaction of the chromogenic reagent1-methyl-2-phenylindole with MDA at45°C, giving rise to a chromophore with absorbance at586nm.Digestive glands were homogenized(1:2.5)in phosphate buffer(0.1M,pH7.4) containing150mM KCl,1mM EDTA and0.012%of the antioxidant BHT and then centrifuged at10000g for30min at4°C.The resulting supernatant was used for determination of MDA content,after incubation of200μL of sample in650μL methanol/1-methyl-2-phenylindole in acetonitrile(5mM,?nal concentration),150μL of HCl 37%and12μl of BHT1%at45°C,for40min.Absorbance was read at 560nm vs.a standard solution of1,1,3,3-tetramethoxypropane(TMP) treated similarly.MDA content in each sample was extrapolated from the standard curve.The?nal results were normalized by grams equivalents of assay tissue wet weight and thus expressed as nmol/g of tissue.

DNA strand breaks were quanti?ed according to De Lafontaine et al. (2000)using the DNA alkaline precipitation assay(Olive,1988).Tissue homogenates(50μL)were incubated in500μL of a SDS solution(2%) containing50mM sodium hydroxide(NaOH),10mM Tris and10mM (EDTA)and500μL of0.12M of potassium chloride(KCl)at60°C for 10min.Samples were cooled to4°C for15min in order to precipitate SDS-associated nucleoproteins and genomic DNA.The mixture was further centrifuged at8000g for4min to enhance the precipitation process.Levels of single and double-stranded DNA remaining in the supernatant were labeled by mixing50μL of supernatant with200μL of 1μg/mL Hoechst dye(bisBenzimide H33258,Sigma-Aldrich)in0.1M phosphate buffer(pH7.4).The resulting complex was then measured using an excitation/emission wave length of360/450nm.Blanks contained identical constituents without homogenate and calf thymus DNA standard was used in order to extrapolate DNA concentration.The ?nal results were expressed asμg of DNA damaged per g assay equi-valent tissue wet weight.

Finally,as a short term measure of individual level toxicological responses zebra mussel?ltration rates were measured using the method described by Coughlan(1969),based on the loss of neutral red dye particles from the water column due to?ltration by the mussels. Immediately following exposures,?ve mussels from each treatment were placed in200mL beakers(1per beaker)containing100mL of neutral red solution(1mg/mL)and left protected from light for2h.Just prior to placing the mussels in the solution an aliquot of water was removed from each beaker representing the initial concentration,C0. After2h mussels were removed and the remaining solution(Ct)along with the initial aliquot(C0)were acidi?ed to pH5with HCl.Neutral red concentrations were determined by measuring the optical density at 530nm.Standards of neutral red were measured along with samples and used to generate a standard curve from which dye concentration could be extrapolated.Filtration rate(f in mL/animal/h)calculation was determined by Coughlan(1969)equation:f=[M/nt]log(C0/Ct);where M is the volume of the test solution,n the number of mussels used,t the time in h,C0and Ct the initial and?nal concentration of the dye.

2.6.Data analysis

All measurements were replicated at least?ve times and the results reported as means±SE.Analyses of variance(one-way ANOVA) followed by Tukey's post-hoc multiple comparison tests were performed to compare toxicant dependent effects with control treatments on each behavioral and biochemical parameters studied. Signi?cant differences were established at P b0.05.Prior to any analysis,data were log transformed to meet ANOVA assumptions of normality and variance homoscedasticity(Zar,1996).

3.Results and discussion

Despite that exposure levels of the studied contaminants in water were quite high,measured residue levels in the soft tissues of exposed mussels after5days(Table1)were of similar order of magnitude to those reported in contaminated sites:Cd in zebra mussels from Lakes Erie and Ontario(3.4–10μg/g d.w.;Rutzke et el.2000),total mercury in M.galloprovincialis from Kastela bay,eastern Adriatic(100μg/g d.w.; Mikac et al.,1985)and PCBs in zebra mussels from Great Lakes (2920ng/g f.w.;Hanari et al.,2004).Interestingly,despite of being dosed at equivalent concentrations,total mercury ion levels in whole mussels were equivalent to those of Cd in individuals exposed to CdCl2and HgCl2 but over four fold higher in mussels exposed to methyl mercury.This data shows that methyl mercury is the highly toxic form of mercury that readily bioaccumulates in?sh and other biota and con?rms the well-established fold difference in bioavailability between the two mercury forms(Sjoblom et al.,2000;Diez et al.,2007).

Mean±SE clearance rates of zebra mussels during exposures were 7.75±0.88mL/h/individual and did not vary signi?cantly across treatments(Table1,ANOVA,P b0.05).Bivalves need to open their valves to facilitate the free circulation of water through their gills allowing them to respire and feed(Jorgensen,1996).There is also reported information that metals and organic contaminants alter the behavior of bivalve mollusks by changing the valve movement and reducing ?ltration rates(Kraak et al.,1994,1997;Duquesne et al.,2004;Cooper et al.,2006;Anguiano et al.,2007).Here we showed that despite that the measured contaminant levels in mussels were in the reported top range for contaminated?eld sites,they were still too low to affect zebra mussel behavioral responses,thus indicating that under the whole experiment, contaminant uptake rates were maximal(Cooper et al.,2006).

From the eleven biochemical responses analyzed,in seven of them mussels showed signi?cant differences across treatments.Mussels exposed to cadmium and Aroclor1260showed greater EROD like activity than those of controls and those exposed to inorganic mercury (HgCl2),which were marginally but not signi?cantly lower than controls.These results support previous?ndings obtained with zebra

Table1

Feeding rates and contaminant levels in whole mussel tissues(mean and standard error, SE;number of pools=3)

Treatment Filtering rates

(mL/h/individual)

Total Cd

(μg/g d.w.)

Total Hg

(μg/g d.w.)

Aroclor1260

(ng/g f.w.) Control8.24±0.550.29±0.020.24±0.0410.3±1.3 CdCl27.22±0.9544.20±2.10

HgCl28.85±1.0442.70±0.66

CH3ClHg7.29±0.46196.05±21.18

Aroclor7.38±1.281863.7±268.5 Metal and Aroclor1260levels are given in dry(d.w.)and fresh weight(f.w.),respectively.

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mussels exposed to Aroclor and co-planar PCBs(Binelli et al.,2006a) but disagree with most studies performed with other invertebrate and vertebrate species since a priori metals should inhibit rather than induce the activity of EROD(Whyte et al.,2000).In mollusks, cytochrome P450and associated mixed function oxidase components of phase I have been mainly localized in microsomes of the digestive gland(Livingstone et al.,1989).A putative cytochrome CYP1A1-like enzyme in Mytilus edulis with unusual properties as indicated by its NADPH-independent metabolism were partially puri?ed by Porte et al.(1995)but it was not possible to measure its related EROD like enzymatic activity(personal communication).In relation to this and to better characterized the above mentioned unusual Cd effects on EROD activity,the assay was repeated in microsomal,cytosolic and 10000g fractions of digestive glands of unexposed mussels.The results obtained showed four fold lower EROD activity in microsomes (mean±SE,89.8±1.02fmol/min/mg prot)than in the cytosolic(350.3±35.1fmol/min/mg prot)and10000g fraction(335.4±32.1fmol/min/ mg prot),thus indicating that this activity cannot be considered a truly related CYP1A one.This?nding agrees with previous studies performed in other mollusk(Sole and Livingstone,2005)and suggests that caution should be consider in those studies that used EROD activity in zebra mussels as surrogate of CYP1A or mixed function related enzymes(De Lafontaine et al.,2000;Binelli et al.,2005,2006a,b;2007).

Conjugation of GST activities was unaffected by CdCl2,HgCl2and Aroclor1260and marginally inhibited by CH3ClHg(Fig.1),thus denoting its marginal role in detoxifying these contaminants.Phase II enzymes can play an important role in homeostasis as well as in detoxi?cation and clearance of many xenobiotic compounds.GST is considered an important phase II enzyme for conjugation of electrophilic compounds and metabolites from phase I metabolism, and usually in laboratory and?eld studies is considered a sensitive biomarker of exposure to a broad range of contaminants in mussels (Cheung et al.,2002,2004;Damiens et al.,2007).The observed mismatch between our results and those reported in other species may be related to species or tissue speci?c patterns of responses.For example GST conjugating activities were positively and non-related with PAHs and PCBs levels in the mussel Perna viridis and M.galloprovincialis,respectively(Cheung et al.,2004;Bebianno et al.,2007).Additionally,Canesi et al.(1999)and Osman et al. (2007)reported that the GST activities in the gills of mussels exposed to metals and persistent organic pollutants were more responsive than those measured in digestive glands or in the rest of the tissues.

Metallothionein levels were higher in mussels exposed to Cd and

mercury chloride and not induced by methyl mercury nor by Aroclor 1260(Fig.1).In zebra mussel Cd is known to induce the gene expression and protein metallothionein levels(Engelken and Hildebrandt,1999; Lecoeur et al.,2004;Marie et al.,2006)and in other mollusks inorganic mercury is also known to induce metallothionein proteins(Amiard et al., 2006).Unfortunately there is no reported information on the ability of methyl mercury to induce metallothionein proteins in mollusks but studies performed in?sh indicate that organic mercury forms are unable to induce metallothioneins(Schlenk et al.,1997;Diez et al.,2008).

The production of reactive oxygen species as a side product of electron transfer during phase I metabolism of organochlorine con-taminants(Livingstone,2003)or redox cycling of metals(Stohs and Bagghi,1995)is a common pathway of toxicity.In costal mussels, metallothioneins and antioxidant enzymes play an effective antioxidant role by scavenging preferentially hydroxyl radicals(Viarengo et al.,1999, 2000).In our study all contaminants increase the activity of SOD,which can be considered the?rst line of antioxidant enzymatic defense against ROS,whereas only mussels exposed to Cd and mercury chloride showed enhanced levels of metallothioneins(Figs.1,2),thus indicating the preferential greater role of SOD detoxifying ROS.Conversely,the activities of the second line of defense against ROS,CAT and Se dependent GPx,which detoxify H2O2to H2O,were unaffected and inhibited by most of the studied contaminants,respectively(Fig.2).There is reported evidence that antioxidant enzyme activities such as CAT and GPx may decrease under the production of ROS.For example an excess of superoxide radicals not detoxi?ed by SOD may directly inhibit CAT(Kono and Fridovich,1982)and if inter-converted to the highly reactive hydroxyl radical,may also be able to react with proteins inhibiting other antioxidant enzymatic activities like GPx(Halliwell and Gutteridge,1999).It is also important to notice that CAT and GPx have complementary roles in H2O2detoxi?cation(Halliwell and Gutteridge, 1999),having different subcellular localizations such as peroxisomal, mitochondrial and cytosolic fractions(Livingstone et al.,1992),target molecules(reduction of H2O2by CAT and reduction of both H2O2and toxic hydroperoxides by GPx),and sensitivities across tissues and mussel species(De Luca-Abbott et al.,2005).Furthermore,the fact that antioxidant responses to contaminant exposure are usually transient in mussels(Livingstone et al.,2003)and that CAT and GPx showed the highest af?nities for high and low H2O2levels,respectively(Halliwell and Gutteridge,1999),may have contributed to the observed distinct responses of CAT and GPx activities across the studied contaminants.

Of particular interest are the results obtained for glutathione(GSH) and its related enzymes(GPx,GR).GSH levels decreased in mussels exposed to methyl mercury,were unaffected by inorganic mercury(Hg SO4)and increased in individuals exposed to Cd and Aroclor1260 (Fig.2).In common mussels(M.galloprovincialis)exposed to

similar Fig.1.Phase I and II ethoxyresoru?n-O-deethylase like(EROD)and glutathione S-transferase(GST)activities,respectively,and levels of MT in mussels exposed to the studied compounds.Results are depicted as the mean and standard error(SE;number of pools n=5).Letters denote homogeneity among exposure treatments at P b0.05following ANOVA and Tukey's post hoc multiple comparison tests.

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concentrations of mercury,Canesi et al.(1999)also reported that inorganic and methyl mercury forms did not change and decrease the levels of GSH in the digestive gland,respectively.Recently,Lehmann et al.(2007)and Osman et al.(2007)also found increased levels of GSH in Asiatic clams (Corbicula ?uminea )and in zebra mussels exposed to Aroclor 1260and sediment extracts contaminated with PCBs and polycyclic aromatic hydrocarbons,https://www.doczj.com/doc/357785256.html,rmation regarding the effects of Cd on GSH levels in mussels is scarce but in ?eld collected ?oater mussels (Pyganodon grandis ),Giguerèet al.(2003)showed a positive relationship between the content of Cd of the cytosolic fraction containing GSH and the concentrations of free Cd ion in water.Therefore,the results depicted in this and other studies indicate that in mussels the tripeptide GSH could be involved in the detoxi ?cation of metals and organic persistent organic contaminants.

Cytosolic GSH can be complexed with heavy metals or in combina-tion with antioxidant enzymes detoxify ROS produced by both metals and organic contaminants (Canesi et al.,1999;Lehmann et al.,2007).Except under Cd exposure,enzymatic glutathione related activities were unaffected (GST,GR)or inhibited (GPX)by the studied contaminants,thus suggesting that the observed effects on GSH levels could not be a consequence of its use in enzymatic activities but rather due to effects on the rate-limiting enzyme of its biosynthesis,the γ-glutamyl-cysteine synthetase (Canesi et al.,1999).Notice,however,that under Cd exposure,glutathione reductase activity,which converts oxidized glutathione (GSSG)to its reduced form (GSH),increased signi ?cantly (Fig.2),thus both GR and γ-glutamyl-cysteine synthetase enzymes may have contributed to maintain high levels of GSH and hence facilitated the detoxication of https://www.doczj.com/doc/357785256.html,rmation regarding GR responses across metals in mussels is scarce but in ?oater mussels (P.grandis )collected across a Cd,Zn and Cu gradient,Giguère et al.(2003)reported a positive relation-ships between GR activity and body burdens of Cu,thus indicating that redox cycling metals may trigger this enzyme.

Failure of antioxidant defenses to remove exogenous ROS produced by redox cycling chemicals either by being inhibited by those compounds or overwhelmed by an excess ROS,will disrupt the balance between the antioxidant/pro-oxidant system within the organisms leading to oxidative damage (Livingstone,2003).In this study oxidative tissue damage was evaluated determining lipid peroxidation measured as MDA and DNA strand breaks.There was evidence for increased lipid peroxidation measured as MDA for mercury chloride,methyl

mercury

Fig.2.Activities of superoxide dismutase (SOD),catalase (CAT),total (GPX Tot)and Se dependent glutathione peroxidase (GPX-Se),glutathione reductase (GR)and levels of glutathione (GSH),lipid peroxidation and single DNA strand breaks in mussels exposed to the studied compounds.Results are depicted as the mean and standard error (SE;number of pools n =5).Letters denote homogeneity among exposure treatments at P b 0.05following ANOVA and Tukey's post hoc multiple comparison tests.

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and Aroclor1260,although only for the last compound observed levels were signi?cantly increased.Effects on DNA strand breaks were less apparent being only marginally increased under exposure to mercury chloride and Aroclor1260(Fig.2).High activities of SOD and levels of GSH and metallothioneins found in mussels exposed to the studied chemicals may have prevented ROS to react with lipids and DNA molecules,thus minimizing lipid peroxidation and DNA strand breaks. For Aroclor1260,however,pro-oxidant effects may have overwhelmed the observed antioxidant defenses leading to signi?cantly increased levels of lipid peroxidation.Although the studied chemicals are known to cause lipid peroxidation and DNA damage in mussels(Livingstone et al.,1990a,b;De Lafontaine et al.,2000;Giguère et al.,2003;Akcha et al.,2004;Cheung et al.,2002;Rocher et al.,2006;Binelli et al.,2007), disparity of results suggests that the ef?ciency of antioxidant defensive systems to remove ROS and prevent oxidative stress varies across species.In addition our results agree with previous studies indicating that lipid peroxidation responses are more sensitive to pro-oxidant conditions than DNA damage based markers(Rocher et al.,2006), probably due to the high ef?ciency of cell DNA repair systems(Pruski and Dixon,2002).

Since many environmental contaminants susceptible of being accumulated such as PCBs and metals promote oxidative stress altering biotransformation routes,antioxidant and metal defensive cell systems, these processes should be considered together using a multi-biomarker assessment approach.Phase I and phase II metabolizing enzymes, metallothioneins,antioxidant enzyme responses and glutathione metabolism related markers have been studied in many mussel species and their relationships between response and contaminant exposure relatively well established.However,their function in detoxi?cation processes motivates continued research on the potential use of these biomarkers in monitoring programs.Nonetheless,the results reported in this and other studies indicate that biomarker responses are transient and variable for different species and chemicals(Livingstone et al., 1990a,b;De Lafontaine et al.,2000;Livingstone,2003;Giguère et al., 2003;Akcha et al.,2004;Cheung et al.,2002;Rocher et al.,2006). Indeed,in?eld studies,higher,equal or lower activities of these biomarkers have been observed in polluted compared to cleaner areas (Regoli et al.,1998;Narbonne et al.,1999;De Lafontaine et al.,2000; Porte et al.,2001,Livingstone,2003;Akcha et al.,2004;Pampanin et al., 2006).Thus,in order to credibly use these biomarkers as reliable indicators of exposure and toxic effects of contaminants,controlled laboratory experiments are needed to elucidate biomarker patterns of response against different toxicants and species,and to assess uncovered mechanisms of action(Livingstone,2003).In this study, despite of using similar exposure levels,mercury chloride and methyl mercury showed different biomarker patterns of response.For methyl mercury the observed difference might be related to its greater bio-availability and the apparent inability of metallothionein proteins to detoxify it,whereas for PCB mixtures pro-oxidant effects of Aroclor1260 may have resulted in higher levels of lipid peroxidation and DNA damage.

Acknowledgments

This study was funded by the Ministrerio de Medio Ambiente and Agencia Catalana del Aigua projects MOVITROF and041/SGTB/2007/1.1. References

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