Effects of the arbuscular mycorrhizal symbiosis on protein expression in the leaves of an elite...

27th ESCPBnew

CongressAbstracts

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY

VOLUME 157A, NUMBER 1/Suppl. SEPTEMBER 2010 ISSN: 1095-6433

CBP27th Congress of the newEuropean Society of Comparative Biochemistry and Physiology,

Alessandria, Italy, September 5–9, 2010

Biological effects of climatic changes and pollution: from biomarkers to system biology

3

157A

/1SC

omparative B

iochemistry and Physiology, Part A

Vol. 157A

/1S(2

010) S

1–

S58

EL

SEV

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cba157-1S cover5 7/21/10 10:41 AM

INDEXED/ABSTRACTED IN: CABS (Current Awareness in Biological Sciences), Curr. Cont. ASCA, Aqua Abstr., Curr. Cont./Life Sci., Curr. Cont. ISI/BIOMED Database, Reference Update, Curr. Cont. SCISEARCH Data. Also covered in the abstract and citation

database SCOPUS®. Full text available on ScienceDirect®

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGYAims and scope of Comparative Biochemistry and Physiology

The Journal publishes original articles emphasizing comparative and environmental aspects of the physiology, biochemistry, molecular biology, phar-macology, toxicology and endocrinology of animals. Adaptation and evolution as organizing principles are encouraged. Studies on other organisms will beconsidered if approached in a comparative context.

Part A. Molecular and Integrative Physiology. This section deals with molecular, cellular, integrative, and ecological physiology. Topics include bioener-getics, circulation, development, excretion, ion regulation, endocrinology, neurobiology, nutrition, respiration, and thermal biology. Study on regulatorymechanisms at any level of organization such as signal transduction and cellular interactions and control of behaviour are encouraged.

Part B. Biochemistry and Molecular Biology. This section covers biochemical and molecular biological aspects of metabolism, enzymology, regulation,nutrition, signal transduction, promoters, gene structure and regulation, metabolite and cell constituents, macromolecular structures, adaptational mechanismsand evolutionary principles.

Part C. Toxicology and Pharmacology. This section is concerned with chemical and drug action at different levels of organization, biotransformation ofxenobiotics, mechanisms of toxicity, including reactive oxygen species and carcinogenesis, endocrine disruptors, natural products chemistry, and signal trans-duction. A molecular approach to these fields is encouraged. Measured rather than nominal exposure concentrations of toxicants must be reported wheneverpossible. For water-borne exposures of aquatic organisms, reporting of detailed chemistry data for the exposure waters is encouraged. When reporting dataobtained from bioassays (e.g., LC50 tests), raw data (i.e., the value of the measured biological response variable(s) for each treatment and each observationtime) should be submitted as online supplementary material.

Part D. Genomics and Proteomics. This section covers the broader comprehensive approaches to comparative biochemistry and physiology that can begenerally termed as “-omics”, e.g., genomics, functional genomics (transcriptomics), proteomics, metabolomics, and underlying bioinformatics. Papersdealing with fundamental aspects and hypotheses in comparative physiology and biochemistry are encouraged rather than studies whose main focus ispurely technical or methodological.

Editors-in-ChiefT.P. Mommsen P.J. WalshUniversity of Victoria, Canada University of Ottawa, Canada

Editorial Secretariat: CBP Editorial Office, University of British Columbia, Vancouver, Canada.E-mail: [email protected]; Managing Editor: Leslie Lightheart.

Reviews Editor

Associate Editors

International Editorial Board

Nancy D. DenslowUniversity of Florida, Gainesville, FL, USA

Yoshitaka FukadaUniversity of Tokyo, Japan

Anthony J. HulbertUniversity of Wollongong, NSW, Australia

Dietmar KültzUniversity of California, Davis, CA, USA

Mark A. SheridanNorth Dakota State University, Fargo, ND, USA

Michael C. ThorndykeKristineberg Marine Research Station,Fiskebäckskil, Sweden

Dick J. van der HorstUniversity of Utrecht, The Netherlands

Aldo ViarengoUniversità del Piemonte Orientale,Alessandria, Italy

B.M. Barnes, University of Alaska,Fairbanks, USA

N.J. Bernier, University of Guelph,ON, Canada

J.E.P.W. Bicudo, University of Sao Paulo, Brazil

F. Bozinovic, Pontificia Univ. Catolicade Chile, Santiago, Chile

L.T. Buck, University of Toronto,ON, Canada

K.G. Burnett, College of Charleston,SC, USA

L.E. Burnett, Grice Marine Laboratory,Charleston, SC, USA

N.R. Bury, King’s College London, UK

M.E. Chamberlin, Ohio University,OH, USA

A.S. Clare, Newcastle University, UKJ.M. Conlon, United Arab Emirates

University, U.A.E.S. Currie, Mount Allison University,

NB, CanadaS.L. Edwards, Appalachian State

University, NC, USAM.R. Elphick, Queen Mary, University

of London, UKE. Fabbri, Univ. di Bologna, ItalyG. Flik, University of Nijmegen,

The NetherlandsF. Geiser, University of New England,

Armidale, NSW, AustraliaA.G. Gibbs, University of Arizona,

AZ, USAK.M. Gilmour, University of Ottawa,

ON, Canada

C.N. Glover, University of Canterbury,New Zealand

G.G. Goss, University of Alberta,Alberta, Canada

J.B. Graham, Scripps Institute of Oceanography, La Jolla, CA, USA

M. Grosell, University of Miami, FL, USA

H. Guderley, Univ. Laval, PQ, CanadaR.P. Henry, Auburn University,

AL, USAM. Hermes-Lima, Universidade de

Brasília, BrazilJ.W. Hicks, University of California,

Irvine, USAC. Hogstrand, King’s College

London, UKP.M. Hopkins, University of Oklahoma,

OK, USAA.Y.K. Ip, National University of

Singapore, SingaporeC.J. Kennedy, Simon Fraser University,

BC, CanadaO. Koizumi, Fukuoka Women’s

University, JapanG. Krumschnabel, Innsbruck

Medical University, Innsbruck,Austria

G. Laverty, University of Delaware,DE, USA

J.-S. Lee, Hanyang University, Seoul,South Korea

H. Lillywhite, University of Florida,Gainesville, USA

D.L. MacLatchy, University of NewBrunswick, NB, Canada

G.B. McClelland, McMasterUniversity, Ontario, Canada

C.D. Moyes, Queen’s University,Kingston, ON, Canada

D.L. Mykles, Colorado StateUniversity, Fort Collins, USA

Y.V. Natochin, Russian Academy ofSciences, St. Petersburg, Russia

C.A. Navas, Univ. de Sao Paulo, SP, Brazil

S.C. Nicol, University of Tasmania,Hobart, Australia

B. Pinshow, Ben Gurion Universityof the Negev, Israel

J.E. Podrabsky, Portland StateUniversity, OR, USA

H-O. Poertner, Alfred Wegener Inst.for Polar and Marine Research,Bremerhaven, Germany

M. Samaja, University of Milan, ItalyP.J. Schwarzbaum, University of

Buenos Aires, ArgentinaH. Segner, University of Berne,

SwitzerlandT. Serebrovskaya, Bogomoletz

Institute of Physiology, Kiev,Ukraine

H.A. Shiels, University ofManchester, UK

V.A. Shuvalov, Russian Academy ofSciences, Moscow, Russia

B.D. Sidell, University of Maine,ME, USA

K.A. Sloman, University ofPlymouth, UK

B.C. Small, United States Departmentof Agriculture, MS, USA

J.E.G. Smits, University ofSaskatchewan, SK, Canada

G.N. Somero, Stanford University,Pacific Grove, CA, USA

T. Suzuki, Kochi University, JapanY.V. Syniuk, Ternopil State

Pedagogical University, UkraineM. Takahata, Hokkaido University,

Sapporo, JapanA. Takemura, University of the

Ryukyus, JapanY. Takei, University of Tokyo,

JapanM. Ugrumov, Russian Academy of

Sciences, Moscow, RussiaG. Van Der Kraak, University of

Guelph, ON, CanadaV. Vieira, University of St Andrews, UKS. Watabe, University of Tokyo,

JapanP.C. Withers, University of Western

Australia, AustraliaK. Yoshimura, University of

Maryland, MD, USAT. Zenteno-Savín, Centro de

Investigaciones Biológicas delNoroeste, La Paz, México

Z.Q. Zhou, Peking UniversityCollege of Life Sciences, China

G. NilssonUniversity of Oslo, Norway

cba157-1S cover5 7/21/10 10:38 AM

ACBPCOMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY

M o l e c u l a r & I n t e g r a t i v e P h y s i o l o g y

Volume 157A/1S (2010)

Editors

T.P. MOMMSEN and P.J. WALSH

Molecular &IntegrativePhysiologyCBP

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY

157A/1S (2010)

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27th Congress of the newEuropean Society of

Comparative Biochemistry and Physiology

Alessandria (Italy) — Sept. 5–9, 2010

Biological effects of climatic changes and pollution:

from biomarkers to system biology

Abstracts Index

Keynote Lectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S1

Environmental Genomics and system toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S3

Toxicity mechanisms of organic xenobiotic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S8

Pollutant-induced genotoxicity and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S12

Pollution and environmental changes: From biomarkers to the physiological status of the organisms . . . . . . . . . . . . . . . . . . . . . . . . S15

Biomarker in aquatic organisms from protists to top predators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S22

Cell signalling and endocrine disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S29

Toxicity in soil organisms and role of environmental changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S32

Mechanisms of homeostasis and toxicity of heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S35

Effects of environment changes and pollution on Artic and Antarctic organisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S40

Environmental proteomics and biomarker discovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S42

Environmental metabolomics novel biochemical approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S49

Cellular reaction and modelling of cell processes in response to pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S51

Biological effects of Nanoparticles mixture toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S53

Role of biossays and community studies in the understading of biological effects of pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S55

27th ESCPBnew CongressNew European Society for Comparative Physiology and Biochemistry

BIOLOGICAL EFFECTS OFCLIMATIC CHANGES AND POLLUTION:

FROM BIOMARKERS TO SYSTEM BIOLOGY

Alessandria, Italy - 5-9 September 2010

The year 2010 is the international year for studies on biodiversity and therefore our meeting concerning climatechanges and their biological effects will represent a central point in the European and international scientific debate onthis topic. In fact from their starting activity, the ESCPB scientists have been involved in research programmes devotedto understand biochemical and physiological processes from protozoa to vertebrate and to utilize this basic researchresults to clarify both the adaptation mechanisms to environmental changes and/or the toxicity pathways of chemicalpollutants.

Considering the hypothesis that pollution interaction with climate changes might generate the condition for biodiversitydecline, it seems of exceptional interest acquiring knowledge about the newest scientific developments concerning thesublethal effects of the different classes of chemicals under different environmental conditions.

The 27th ESCPB Congress is organised in sessions concerning effects of natural and anthropogenic environmentalfactors on living organisms.

I hope to meet in Alessandria not only scientists but also environmental managers in particular from environmentalagencies: it is time to transfer the information from the scientific congresses to the application of advancedbiomonitoring studies to save our planet for future generation!

Prof. Aldo Viarengo

CommitteesInternational advisory board - Augustine Arukwe -- Norwegian University of Science & Technology, Norway- Mohamed El Banni -- Higher Institute of Biotechnologies, Monastir University, Tunisia - Janina Barsiené -- Institute of Ecology of Nature Research Centre, Lithuanua - Maria Joao Bebianno -- University of Algarve Campus de Gambelas, Portugal - Ronny Blust -- University of Antwerp, Belgium - Miren P. Cajaraville -- University of the Basque Country, Spain - Kevin Chipman -- University of Birmingham, United Kingdom - John Craft -- Glasgow Caledonian University, United Kingdom - Reinhard Dallinger -- University of Innsbruck, Austria - Elena Fabbri -- University of Bologna, Italy - M. Cristina Fossi -- University of Siena, Italy - Stephen George -- University of Stirling, United Kingdom - Anders Goksoyr -- University of Bergen, Norway - Tom Hutchinson -- CEFAS, United Kingdom - Bjorn Munro Jenssen -- Norwegian University of Science and Technology, Norway- Angela Koehler -- Alfred Wegener Institute, Germany - Kari Lehtonen -- Finnish Environment Institute Marine Centre, Finland - Teresa Lettieri -- ISPRA, Italy - Cristina Miceli -- University of Camerino, Italy - Christophe Minier -- University of Le Havre, France - Michael N. Moore -- Plymouth Marine Laboratory, United Kingdom - Cristina Nasci – Thetis Spa, Italy- Cinta Porte Visa -- CSIC, Barcelona, Spain- Phil Rainbow -- Natural History Museum, United Kingdom - Mauro Rebelo -- Institute of Biophysics Carlos Chagas Filho, Brazil- Francesco Regoli -- Polytechnic University of Marche, Italy - Trifone Schettino -- University of Lecce, Italy - Daniel Schlenk -- University of California Riverside, USA- Helmut Segner -- University of Bern, Switzerland - David Sheehan -- University College Cork, Ireland - David Spurgeon -- Centre for Ecology & Hydrology Maclean Building, United Kingdom - John J. Stegeman -- Woods Hole Oceanographic Institute, USA- Claus Svendsen -- Centre for Ecology and Hydrology, United Kingdom - Rita Triebskorn -- University of Tuebingen, Germany - Ron Van der Oost -- DWR Waterproof, Netherland - Roy Weber -- University of Aarhus, Denmark - Richard Winn -- Warnell School of Forestry and Natural Resources, USA

Local organizing committee Maurizio TOMADA, Susanna SFORZINI, Caterina OLIVERI, Lara M.V. BOATTI, Laura OLIVERI,Alessandro NEGRI, Ilenia SAGGESE, Alessandro DAGNINO, Francesco DONDERO, Aldo VIARENGO

Sponsors

27th Congress of the newEuropean Societyof Comparative Biochemistry and Physiology


Biological effects of climatic changes and pollution:From biomarkers to system biology

KEYNOTE LECTURES

Marine ecosystem response to multiple climatic and anthropogenicdrivers: A modelling approach

J.I. Allen (Plymouth Marine Laboratory, Prospect Place, The Hoe,Plymouth PL1 3DH, UK)

Marine ecosystems are sensitive to the impacts of both climaticchange and the direct effects of human activity. Predictive models thatconsider the full range of drivers (e.g. Climate and Ocean Acidification,Pollution, Fishing, Invasive Species) are required to elucidate theresponses of the marine ecosystem in a holistic manner, rather thandriver by driver as has been done in the past. One of the challenges is tointegrate the response of ecosystems tomultiple drivers across a rangeof trophic levels by developing coupled models to assess keyvulnerabilities and risks of global change for the marine ecosystem.A focus for this work is MSFD which distinguishes 11 qualitativedescriptors that together determine the characteristics of GoodEnvironmental Status (GES). Underpinning this activity is the require-ment for a range of scenarios which take account of the impacts ofmultiple climatic and anthropogenic drivers on marine ecosystems.Recently the FP7 MEECE has simulated present and future primaryproduction states in several regions and this is being expanded toinclude simulations with coupled plankton fish models along with theeffects of pollution and ocean acidification. Implementation of suchmodels requires addressing challenges such as the choice of modelstructure, scaling processes from physiology to functional types to fish,the ecosystem model sensitivity to changes in the physical environ-ment, new methods for the evaluation and comparison of ecosystemand biogeochemistry models and the quantification of simulationuncertainty. These points are illustrated with examples.

doi:10.1016/j.cbpa.2010.06.002

Molecular integration: How we accommodate multiple changes

J.J. Stegeman (Woods Hole Oceanographic Institution, USA)

We live in an environment changing physically, chemically, andbiologically. Day by day, season by season, the environment changesin predictable ways. While the fact and often direction of change arepredictable, the magnitude of change often is not. What are theconsequences of change that is extreme, either in magnitude or intime? Integration of physical, chemical and biological changes governthe nature of life on Earth. Such integration must be accomplishedthrough the mechanisms by which organisms adapt. How are thebiochemical and physiological mechanisms of adaptation to differentchanges integrated? Do these mechanisms and integration differ withspecies, lifestyle and habitat? Do unusual or inexorable changes set anew baseline upon which the predictable changes are superimposed?What are the determining properties of genes and proteins? Thesequestions are the heart and soul of comparative biochemistry andphysiology. Examples of the intimate intersection of physics,chemistry and biology, can be seen in gene family evolution andprotein function, and interaction (defense genes, regulatory genes),steroid responses, NfKB, and other genes of adaptation; new genes,new controls, new patterns. How do these operate in environmentsthought to be constant, such as the deep sea, as compared to en-vironments where physical change is extreme, such as the inter-tidalrocky shore? Our expanding view of new processes governing geneexpression, and systems biology offer a rich field for exploring howthese mechanisms participate in fitness, adaptive and adverseresponses, which may help us to detect and discern the significanceof change. (Support: NIH R01ES015912).

doi:10.1016/j.cbpa.2010.06.003

The origins of evolutionary innovation

A. Wagner (Dept. of Biochemistry, University of Zurich, Switzerland)

Life can be viewed as a four billion year long history of innovations.These range from dramatic macroscopic innovations like the

evolution of wings or eyes, to a myriad molecular changes that formthe basis of macroscopic innovations. We know many examples ofinnovations — qualitatively new phenotypes that can provide a

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A

j ourna l homepage: www.e lsev ie r.com/ locate /cbpa

Comparative Biochemistry and Physiology, Part A 157 (2010) S1–S2

1095-6433/$ – see front matter

http://dx.doi.org/10.1016/j.cbpa.2010.06.002


http://www.sciencedirect.com/science/journal/01678809

critical advantage in the right environment— but have no systematicunderstanding of the principles that allow organisms to innovate.Most phenotypic innovations result from changes in three classes ofsystems: metabolic networks, regulatory circuits, and protein orRNA molecules. I will discuss evidence that these classes of systems

share two important features that are essential for their ability toinnovate.

doi:10.1016/j.cbpa.2010.06.004

S2 Abstracts / Comparative Biochemistry and Physiology, Part A 157 (2010) S1–S2

27th Congress of the newEuropean Society ofComparative Biochemistry and Physiology

Alessandria (Italy) — Sept. 5-9, 2010

Biological effects of climatic changes pollution:From biomarkers to system biology

Session 1: Environmental Genomics and system toxicology

INVITED LECTURE

1.Approaches to interpretation of ‘Omics data: Identification ofresponses in European Flounder populations sampled from siteswith different levels of environmental pollutants

S. George (University of Stirling, UK); G. Stentiford (CEFAS,Weymouth,UK); K. Chipman, M. Viant, F. Falciani (University of Birmingham, UK)

Application of functional genomics techniques potentially providesan opportunity to gain a better insight into the toxicological effects ofenvironmental pollutants and permit development of more objectivecriteria for assessment of adverse health effects. The datasets obtainedare extremely complex and rarely provide unambiguous informationon toxic mechanisms; moreover, in field sampled animals interpreta-tion is made even more difficult due to the many variables, includingphysical differences in sites, age and sex differences betweenindividuals, potential genetic differences in the populations, as wellas differences in feeding, disease andparasite status.We collectedmaleEuropean flounder (13-25 cm) from two Irish Sea sites, two UK NorthSea estuaries and three German North Sea sites. General healthmeasures were recorded, transcriptomic (using the GENIPOL floundercDNA array) and metabolomic (1H–NMR) analyses of livers andanalyses of protein biomarkers were conducted. Chemical analyses ofsediment and tissues, histopathology, parasitological and moleculargenetic analyses of individual fish were also carried out.

Here we describe results obtained from a number of computa-tional approaches for analysis of the datasets. Comparison of livergene expression profiles of field sampled fish with predictive generesponses obtained from laboratory treatment of fish with proto-typical pollutants produced reasonable correlations between theexpression profiles from the different sites and chemical analyses ofsediment contaminants inferring biological availability of selectedcontaminants. Application of a genetic algorithm (GALGO) to thetranscriptomic data using these pollutant responsive genes produced

a model where only 17 genes were capable of distinguishing andpredicting the site of origin of the fish also inferring that geneexpression profiles were indicative of contaminant exposure. Use of asystems biology approach employing a network inference algorithmin a force driven layout (as implimented in the software Cytoscape)identified a network integrating mRNA, metabolite measurements,physiological indicators and parasite infections. This model identifiedmolecular networks which were diagnostic of nematode and copepodinfections, tissue abnormalities and indices of fish health.

doi:10.1016/j.cbpa.2010.06.006

ORAL PRESENTATIONS

2.Molecular mechanisms of tributyltin-induced pathogenesis inthicklip grey mullets Chelon labrosus

O. Diaz de Cerio, I. Rojo, U. Izagirre, M.P. Cajaraville, I. Cancio (Zoologyand Animal Cell Biology, University of the Basque Country, Bilbao,Basque Country, Spain)

Organotin compounds such as TBT are highly persistent organicpollutants that bioconcentrate in marine biota, behaving as potentendocrine disruptors. In mammals, TBT has been described as anenvironmental obesogen, as it contributes to adipocyte differentia-tion and lipid accumulation. With the aim of studying the molecularmechanisms that elicit TBT-induced pathogenesis in fish, thicklip greymullets Chelon labrosus, were exposed to low (10 ng/L) and high(500 ng/L) TBT concentrations for 1, 7 and 21 days and hepaticlysosomal membrane stability (LP), neutral lipid accumulation andgene transcription profiles were studied. Previously, the multitissuenormalized transcriptome of mullet was sequenced by 454 pyrose-quencing obtaining 126 Mb of sequence information. After assemblyand annotation, 8330 different gene signatures were spotted in a








high-density oligonucleotide microarray. Distinctive gene transcrip-tion profiles were recorded depending on the concentration of TBTand time of exposure. Functional pathway analysis revealed that earlyresponses (500 ng/L) were characterized by genes related toresponse to organic substances and to oxidative stress, glucosemetabolic processes and apoptosis. After 21 days of exposureenriched GO terms in both concentrations were related to lipidhomeostasis with a significant regulation of steroid metabolicprocess, lipid biosynthetic and transport processes and of theandrogen receptor signaling pathway. Oxidative phosphorylationwas strongly regulated in all cases. Neutral lipid accumulationmeasured through Oil-Red-0 histochemistry did not show anytreatment related effect due to individual variability, but LP clearlyidentified a stress response in individuals exposed for 21 days.Transcription profiles indicate that TBT pathogenesis might involvealterations of lipid metabolic pathways that should be confirmed infuture lipidomic studies. Funded: Basque Government (GIC07/26-IT-393-07 and AE-2008-1-1).

doi:10.1016/j.cbpa.2010.06.007

3.Systems biology assessment of binary mixture effects in the socialamoeba Dictyostelium discoideum

F. Dondero, A. Negri, L. Boatti, M. Marsano, M. Bellone, A. Viarengo(University of Piemonte Orientale, DiSAV, Italy); G. Bloomfield, R. Kay(MRC, UK); A. Ivens (The Wellcome Sanger Institute, UK); D. Sato,M. Tomita (Keio University, Japan)

Wepresent a systems toxicologyassessmentof the interactive effectsof a binary mixture of Nickel and the organophosphate pesticideChlorpyrifos in the unicellular eukaryote Dictyostelium discoideum.

Whilst Dictyostelium amoebae showed a high tolerance to theheavy-metal, theexposure to theorganophosphate led to a suddenburstof toxicity comprised into a very narrow range. Interestingly, Chlorpyr-ifos toxicity significantly decreased when sub-lethal Nickel amountswere added to the medium. To underpin the molecular mechanisms atthe basis of such interaction, we implemented an assessment based ontranscriptomics, proteomics, metabolomics and physiomics.

Transcriptomic analysis (DNA-microarray and quantitative-PCR)highlighted the involvement of calcium-signalling and energymetabolism disorders in Chlorpyrifos toxicity. Moreover, the mRNAlevels of several genes usually involved in detoxification of xenobio-tics, such as ABC-transporters and GSTs, were affected. This molecularsignature was suppressed in mixture-exposed amoebae where thefingerprint of Ni was dominant over that of Chlorpyrifos.

Proteome changes were assessed by means of bidimensional-electrophoresis and ESI-q-TOF mass-spectrometry analysis. Theinvolvement of proteins related to energetic pathways (transketolase,succinate-dehydrogenase, pyruvate-hydratase, all overexpressed)wasconfirmed alongwith the capability of Ni to revert the pesticide effects.

Metabolomic profiling (capillary electrophoresis-TOF analysis)identified and quantified the absolute levels of about 120 cellularmetabolites. In particular, a dramatic impairment of the energeticcharge, glutathione and aminoacid levels was observed in Chlorpyr-ifos exposed amoebae. Also in this case, non-toxic Ni amounts wereable to ameliorate when not abolish the effects of the pesticide.

Further analyses are in progress to identify the key-note cellulareffectors responsible for Chlorpyrifos toxicity and Ni cyto-protection.

This research was granted by the 6th EU-FW-Project-NoMiRACLE.

doi:10.1016/j.cbpa.2010.06.008

4.Systems biology investigation of the mechanisms of brominatedflame retardant neurotoxicity

C. Hogstrand, T.S. Carroll, J.D. Rasinger, V. Reffatto (King's CollegeLondon, UK); A.K. Lundebye, M. Haave (National Institute of Nutritionand Seafood Research, Norway); R. Tassinari, I. Altieri, F. Maranghi,G. Moracci, P. Patriarca, A. Mantovani, A. Menditto, A. Macrì (IstitutoSuperiore di Sanità, Italy)

Fish with high fat content tend to accumulate lipophiliccontaminants, such as brominated flame retardants (BFR) and aretherefore a source of BFR exposure to wildlife fish predators as well asto the human consumer. We have used a systems biology approach tostudy the effects in of two BFRs that may be found at elevatedconcentrations in salmon. Effects of BDE-47 and HBCD wereinvestigated in juvenile female balb/c mice fed either of the toxicantsfor 28 days as a part of a salmon-based diet. Abundance profiles ofmRNA and protein were measured in brain along with serum 17ß-oestradiol, testosterone and thyroid hormone levels, and histologicalmetrics on several tissues. In addition, transcriptome profiling wascarried out on neuroblastoma (N2) cells exposed to BDE-47 or HBCD.Results showed that both BFRs induced changes in gene expressionand protein abundance in brain. Whilst differences in gene andprotein expression profiles elicited by BDE-47 and HBCD did exist,many of the regulated genes and proteins were shared. Histopatho-logical aberrations were observed in thyroid, spleen, thymus andliver, but not in brain, adrenals or uterine tissues. Dietary exposure toBDE-47 and HBCD also showed disturbances in serum thyroidhormone levels. Gene expression in cultured N2A cells exposed toBDE-47 or HBCD confirmed bias of regulated genes involved in braindevelopment, neurodegenerative disease, calcium homeostasis, thyr-oid hormone and retinoid regulation, and cytoskeleton structuremaintenance.

doi:10.1016/j.cbpa.2010.06.009

5.Temporal study of the expression of sex-specific genes in themantle of common mussel (Mytilus edulis)

S. Anantharaman, J. Craft (Biological & Biomedical Sciences, GlasgowCaledonian University, Glasgow G4 0BA, UK)

Mytilus species are frequently used as sentinels for the assessmentof environmental health. Sex of the mussel or stage in thereproductive cycle is rarely considered in such studies but bothparameters are likely to influence biological responses to pollutionand climatic stressors. A temporal study to evaluate the reproductivestages of mussel over a year was conducted using the relativeexpression of gender-specific genes. A qPCR assay was used tomeasure male-specific vitelline coat lysin (VCL) and female-specificvitelline envelope receptor for lysin (VERL) in the mantle of Mytilusedulis. Histology on the mantle sections was further conducted tooperate as a physiological anchor for the qPCR results. Mussels werecollected over a 13-month period from the same location on the Firthof Clyde and the sex of these animals could be assigned by qPCR in>90% of the animals even during the regression and recrudescence ofgonads. The results showed that VCL and VERL transcripts werehighest in February-March 2009, decreased to a minimum in July-August 2009 before increasing again from September – October 2009and reaching a new peak in February-March 2010. The histology ofmantle tissue from each animal matched sex established by qPCR andwith the stage of egg and sperm development. The results provide a


firm basis for the inclusion of gender and stage of the reproductivecycle in evaluating mussel responses to environmental stressors andmay account for some of the inter-individual variation that com-plicates interpretation of such studies.

doi:10.1016/j.cbpa.2010.06.010

POSTER PRESENTATIONS

6.Transcriptome analysis in Hepa cells exposed to BaP as aprerequisite for a cell-wide understanding of BaP-cell interactions

D.J. Madureira (Swiss Federal Institute of Technology Zürich, Switzer-land); J. Michaelson, A. Beyer (Biotechnology Center TU Dresden,Germany); F. Herden, S. Trump, M. von Bergen, S. Attinger, I. Lehmann(Helmholtz Centre for Environmental Research, Germany); K. Schirmer(Swiss Federal Institute of Aquatic Science and Technology, Switzerland)

The aim of the systems biology initiative, “From contaminantmolecules to cellular response: system quantification and predictivemodel development”, is to build a model of interactions between cellsand the toxic chemical Benzo-a-Pyrene (BaP). Part of this projectaims to identify the regulation of genes by BaP in a time andconcentration-dependent manner and relate the regulation tocellular distribution of BaP and the physiologic or toxicologic cellularresponse.

In a first step, Hepa1c1c7 cells were exposed to two concentrations(50 nM and 5 µMBaP), and four time points (2, 4,12 and 24 hours), forcomprehensive transcriptome analysis using Affymetrix exon arrays.Analysis showed a total of 1637 genes to be significantly regulatedeither by concentration or by time and concentration. About 89regulated genes are transcription factors, raising the possibility oftranscription factor cross-talk in BaP signaling. Genes related totranscription/translation, cell adhesion, DNA repair and cell cycle areamong the regulated genes. Some of the robustly regulated genes arethose encoding for cytochrome P450 enzymes (cyp1a1 and cyp1b1)and the TCDD-inducible poly(ADP-ribose) polymerase, Tiparp. Theseare known targets of the aryl hydrocarbon receptor (AhR), which isactivated by BaP. To start to mechanistically link these regulations inthe transcriptome to the cellular phenotype, we compare the changesin the transcriptome to that identified for the proteome and investigatehow the molecular changes translate to altered cellular functions andtoxicity in response to BaP.

doi:10.1016/j.cbpa.2010.06.011

7.Application of SSH andQ-PCR to investigate genes transcriptionallyupregulated in Mytilus edulis after exposure to styrene

O. Diaz de Cerio, M.P. Cajaraville (Dept. Zoology and AnimalCell Biology, University of the Basque Country, Bilbao, BasqueCountry, Spain); E. Hands, J. Humble, J.A. Craft (Dept. Biological andBiomedical Sciences, Glasgow Caledonian University, Glasgow, UK); I.Cancio (Dept. Zoology and Animal Cell Biology, University of theBasque Country, Bilbao, Basque Country, Spain)

The analysis of the genes regulated by exposure to spilledchemical substances could help to elucidate adaptatory, compensa-tory and toxicity pathways that participate in recovery of homeostasisor pathogenesis in marine biota. With the aim of analyzingthe transcription response in the mussel Mytilus edulis against the

corrosive and putatively carcinogenic hydrocarbon styrene, a forwardsubtracted library was produced. Mussels were exposed to styrene(3 mg/l) for 3-days. For the suppression subtractive hybridization(SSH) female mussels were selected by histology and digestive glandmRNA was isolated. After a partial sequencing of the resulting librarythat contains 1440 clones, 287 sequence-fragments were obtained.BlastN analysis using the Mytibase database allowed identification of42% of these sequences, although 28% belonged to unknown proteins.Additionally 19% of the sequences were identified through BlastN andBlastX against NCBI database. Genes related to the immune systemand proto-oncogenes such as c-myc and ras were identified. It isnoteworthy that 15 sequences were related to chitin metabolism(chitinase-1) and 3 to the metabolism of beta-1-3-glucan. A fragmentbelonging to a nuclear receptor with high homology to theecdysozoan E75 nuclear receptor (NR1DEF) was also present. E75regulates chitin metabolism in ecdysozoa and previous musselmicroarray studies have demonstrated that chitin metabolism genesare highly regulated under chemical exposure. Together with beta-1,3-glucan metabolism proteins, chitinases participate in extracellulardigestion processes that might be altered by styrene exposure. Thetranscription levels of all these genes will be quantified through Q-PCR. Funded: Spanish MEC (CTM2006-06192/MAR) and BasqueGovernment (GIC07/26-IT-393-07).

doi:10.1016/j.cbpa.2010.06.012

8.Development of a web-based system for the collection, analysisand data mining of ecotoxicological related data: A startingproject

L. Boatti, F. Dondero, A. Viarengo (University of Piemonte Orientale,DiSAV, Italy); F. Mignone (University of Milan, Italy)

Variations in environmental parameters, such as the presence ofchemical pollutants, or climate changes can induce organismresponses at the cellular and molecular level. Beside more traditionalanalysis system like evaluation of biomarkers, researchers cannowadays benefit of high throughput technologies (such as micro-array, deep sequencing, 2-DE/NMR spectrometry) that allow verylarge scale investigation. Data deriving from these technologies offersnapshots of the changes induced by the presence of environmentalvariations, nonetheless they are poorly informative if consideredseparately and do not offer an exhaustive overview of the real cellularresponse.

Moreover management, analysis and interpretation of the massivedatasets obtained from high throughput technologies can be quitechallenging. Finally it would be very useful to compare the biologicaleffects induced by the same environmental stress on differentorganisms. Indeed the mechanisms of response to environmentalchanges are usually conserved during evolution thus making appro-priate and beneficial to develop tools that allow the comparison ofresponses between different organisms to detect recurring features.

To try to fill this gap here we propose a project (already funded byItalian Ministers of education, university and research – MIUR) thataims to the development of a centralized resource for the collection,analysis and integration of ecotoxicology related high throughputdata. "Aggregated" information will be accessible by means of simpleweb based queries and supported by effective and simple graphicrepresentations to facilitate the understanding of biological responsemechanisms.

doi:10.1016/j.cbpa.2010.06.013

S5Abstracts / Comparative Biochemistry and Physiology, Part A 157 (2010) S3–S7

9.Expression and diversity of AMP and other immune-relatedmolecules in M. galloprovincialis

L. Varotto, U. Rosani, S. Domeneghetti, G. Procopio (University ofPadova, Italy); C. Manfrin, M. Gerdol, A. Pallavicini (University ofTrieste, Italy); P. Venier (University of Padova, Italy)

Anthropogenic activities, accidental events and global climatetrends currently threaten the marine biological resources as theyimpose functional adjustments to the biota, change the ecologicalniches and lead to significant ecosystem changes. In the watercolumn or sediments, bivalves interact with a variety of microorgan-isms which include commensal, opportunistic species and potentialpathogens. Ofter acting together with other detrimental factors,microscopic agents can divert energy from growth and reproductionto the defence reactions, and finally overwhelm the host defenseswith onset of diseases and mortality outbreaks in the natural andfarmed stocks. Mussels of the genus Mytilus commonly appear moreresilient than other bivalves to the environmental variation, and morelikely act as pathogen reservoir for the animal species sharing thesame habitat. In the frame of the European Integrated projectIMAQUANIM, standard and advanced sequencing is confirmingabundance and molecular diversity of the antimicrobial peptideprecursors expressed in farmed, wild and immunostimulatedmussels. Transcripts denoting pathogen recognition receptors alsoshowed significant molecular diversity, namely sequences containingC1q, C-type-lectin or fibrinogen-like domains. Overall, gene expres-sion profiling and massive sequencing progressively unveil thecomplexity of the innate defences of the Mediterranean mussel.

doi:10.1016/j.cbpa.2010.06.014

10.Screening neoplastic haemocytes of two Mytilus species forchanges in gene expression

A. Muttray (University of British Columbia, Canada); F. Dondero(University of Piemonte Orientale, DiSAV, Italy); E. Vassilenko(University of British Columbia, Canada); A. Negri (University ofPiemonte Orientale, DiSAV, Italy); S. Baldwin (University of BritishColumbia, Canada); A. Viarengo (University of Piemonte Orientale,DiSAV, Italy)

Marine mussels Mytilus trossulus and M. edulis are used forenvironmental monitoring of estuaries worldwide. Both species aresusceptible to a leukemia-like disease of the haemocytes calledhaemic neoplasia. The etiology of the disease in mussels is unknown,but investigations have indicated high temperature stress, transmis-sible particles, and pollution stress. Interestingly, the prevalence ofhaemic neoplasia is much higher in M. trossulus than in M. edulis,when both species are caged at the same location. Previous studiesfound that the p53 tumor suppressor gene family is involved in thedisease, but no further genes outside this network were investigatedat the molecular level. The objective of this study was therefore toinvestigate global differences in gene expression between healthyand neoplastic animals, as well as between the two species, using amussel cDNA-microarray encompassing 1.7 K sequences.

By means of dual color competitive hybridizations, we obtained24 differentially expressed genes, all up-regulated in M. edulisneoplastic haemocytes. These genes included ribosomal, mitochon-drial, structural and extracellular matrix protein genes as well as the“lethal giant larvae” (lgl) gene, a tumor suppressor gene first isolatedfrom Drosophila. Preliminary data showed that this gene was also

differentially expressed in M. trossulus along with another foursequences involved in cell-cell adhesion and gap junction formation.Lgl is also involved in regulating epithelial cell polarity, the loss ofwhich is a hallmark of cancer, and has been implicated intumorigenesis in Drosophila. Further microarray and quantitativePCR analysis are in progress to clarify the possible role of theidentified genes in haemic neoplasia formation.

doi:10.1016/j.cbpa.2010.06.015

11.Environmental pollutants and non-coding telomeric RNA levels inChironomus riparius (Diptera)

R. Planelló, J.L. Martínez-Guitarte, G. Morcillo (Biología y ToxicologíaAmbiental, Facultad de Ciencias UNED, Madrid, Spain)

The importance of non-coding RNAs (ncRNAs) has been increas-ingly recognized recently. They are highly abundant and involved in agreat variety of processes that control gene expression at differentlevels in eukaryotic genomes. They have also been related with thecellular stress response induced by heat shock and anoxia. Althoughsome environmental contaminants are able to induce the stressresponse by increasing the levels of heat shock proteins, little is knownabout their potential effects on non-coding RNAs. Chironomus ripariustelomeric RNA is a class of ncRNA transcribed from repetitive telomericDNA that is activated under heat-shock conditions. We have selecteddifferent compounds with known endocrine-disrupting activity invertebrates (Bisphenol A, Cadmium chloride, Benzyl Butyl Phthalateand Nonylphenol) to analyse their effects on the structure andtranscriptional activity of telomeric sequences. The effect on telomericregions was visualized in polytene chromosomes from salivary glandcells using immunocytochemistry with specific fluorescent probes toDNA/RNA hybrids. RT-PCRwas used to evaluate the effects of acute andshort-term exposure to the pollutants on the expression levels oftelomeric RNA as well as hsp70. The results show an increase in thelevels of telomeric and hsp70 transcripts induced by the treatmentswith a different susceptibility to each toxicant tested.

Acknowledgements: Funded by MICINN, Spain, Project ref:CTM2009-07189.

doi:10.1016/j.cbpa.2010.06.016

12.Environmental biomonitoring of Zoarces viviparus in combina-tion with large scale gene expression profiling

N. Asker, E. Kristiansson, D.G.J. Larsson, L. Förlin (University ofGothenburg, Sweden)

The eelpout (Zoarces viviparus) inhabits the coastal waters ofNorthern Europe and has been part of the environmental monitoringof the Swedish coastal areas for several years. In the search for newbiomarkers and understanding the molecular mechanism behindphysiological changes we now combine classical biomonitoring of theeelpout with large scale gene expression profiling. We have recentlysequenced the eelpout liver transcriptome and the sequence datahave been used to design and construct a oligonucleotide microarray.This eelpout microarray contains several categories of genes that areof particular interest for ecotoxicological research, including 45different cytochrome P450 variants. Also heat shock proteins (e.g.HSP 70, HSP 90 alpha and beta) and genes related to oxidative stress(e.g. superoxide dismutase and glutathione peroxidase) are present,


as well as several known biomarkers, such as vitellogenin, the zonapellucida proteins and metallothionein. Here we present data from amonitoring study performed on eelpout captured along the Swedishcoast. We have combined traditional monitoring measurements withlarge scale gene expression analysis. As the eelpout lives relativelystationary we can link the microarray data and observed physiolo-gical responses to the environmental situation where the fish wascaught. Furthermore, the viviparity of the eelpout gives the uniqueopportunity to associate the effects of pollutants to individualreproductive performance, including the development of embryosand fry.

doi:10.1016/j.cbpa.2010.06.017

13.Biomarkers and transcription level of cancer-related genes incockles Cerastoderma edule from Galicia (NW Spain) withdisseminated neoplasia

P. Ruiz, A. Orbea, M.P. Cajaraville (University of the Basque Country,Spain); S. Díaz, M.J. Carballal, A. Villalba (Centro de InvestigaciónsMariñas, Spain)

Disseminated neoplasia is a pathological condition reported forseveral species of marine bivalves throughout the world, but itsaetiology has not yet been satisfactorily explained. It has beensuggested that chemical contamination could be a factor contributingto neoplasia. A high prevalence of disseminated neoplasia has beenreported in Galician cockles, a dominant species in many Galicianbivalve beds with an important economical value. The aim of thepresent study was to compare cell and tissue biomarkers and thetranscription level of cancer-related genes in cockles affected bydisseminated neoplasia with those of healthy cockles in relation tochemical contaminant burdens. For this, 186 cockles were collectedfrom a natural bed in Cambados (Ria de Arousa) in May 2009.Disseminated neoplasia was diagnosed in histological sections and inhaemolymph samples. We measured lysosomal membrane stability(LMS) as general stress biomarker and micronuclei (MN) frequencyas marker of genotoxicity. A histopathological analysis of digestivegland, gonad and gill, and chemical analysis were also carried out.The prevalence of disseminated neoplasia was 12.36% in the studiedlocality and 3 degrees of intensity were distinguished. No significantdifferences in metal content, non specific inflammatory responsesand parasites were observed between healthy and affected cockles.LMS was reduced significantly in cockles affected by disseminatedneoplasia, which indicates a poorer health condition than healthycockles. Healthy and affected animals presented very low frequenciesof MN when compared with other species in laboratory and fieldstudies and no significant differences in MN were observed between

groups. The frequency of binucleated hemocytes was statisticallysignificantly higher in neoplastic cockles than in healthy cockles. PCR-based screening of cockle cDNA with degenerate primers resulted inthe amplification, cloning and sequencing of cDNA fragments codingfor tumour suppressor protein p53 (199 bp) and ras (341 bp) protein.No significant differences were observed in the transcription level ofp53. In conclusion, cockles affected by disseminated neoplasia suffera general stress situation and further studies are in progress toelucidate mechanisms of carcinogenesis in this species. Study fundedby Spanish MEC (project CANCERMAR, CTM2006-06192 and pre-doctoral fellowship to P. Ruiz) and Basque Government through agrant to consolidated research groups (GIC07/26-IT-393-07).

doi:10.1016/j.cbpa.2010.06.018

14.Physiological and transcriptomic responses of fathead minnowexposed to dietary or aqueous copper combined with heat stress

P. Couture, D. Lapointe, F. Pierron (Institut National de la RechercheScientifique - Centre Eau Terre Environnement, Québec, Canada)

The effects of copper (Cu) on aquatic biota have been extensivelystudied. Contaminant effects occurring at the molecular or geneexpression levels are more sensitive and generally precede effects atthe population level, providing an early warning signal of toxic risk.Our study examined the additivity of heat and Cu stresses on geneexpression and physiological responses in fatheadminnows. Fishwereexposed to environmentally relevant dietary or aqueous Cu concen-trations for 28 days at two different temperatures, the higher oneexpected to cause a heat stress. At the end of the experiment, musclesamples were taken for gene expression, metal and enzyme analyses.The expression of ldh, cat and cco-1 geneswas higher in fish exposed todietary Cu alone compared to unexposed fish at 20 °C. In contrast,therewas a decrease in cs and sod-1 gene expression in fish exposed tohigh temperature (32 °C) alone or in combination with aqueous Cucompared to unexposed fish. Overall, the physiological and transcrip-tomic responses were strongerwith dietary than aqueous Cu exposureat either temperature tested, but heat exacerbated the transcriptomicresponse to dietary Cu exposure. There was no correlation betweengene expression and the activity of corresponding enzymes, high-lighting that the two approaches provide complementary information.This research will contribute to our understanding of the combinedeffects of contaminants and stressful environmental conditions such asheat stress, which is likely to increase in frequency in a context ofglobal warming.

doi:10.1016/j.cbpa.2010.06.179





Session 2: Toxicity mechanisms of organic xenobiotic compounds

INVITED LECTURE

1.Cellular fate of organic compounds in marine invertebrates

A. Köhler (Alfred Wegener Institute für Polar und Meeresforschung/IMARE Institute for Marine Resources, Germany)

Organic compounds are introduced into the oceans in the range of upto millions of tons per year. Major sources are polycyclic aromatichydrocarbons, persistent organic pollutants and microplastics, acciden-tally released from oil platforms, industrial discharges and plastic litter. Inorder to estimate the toxic potential on cell and tissue function in marinebiota we were prompted to score available techniques and to developnovel approaches to visualise organic compounds in cells and organs. Bluemussel was chosen as a well established global bioindicator forexperimental exposure studies to implement the technologies at thelight- (fluorescence/epipolarisation) and electronmicroscopic level. Es-tablished direct imaging of autofluorescence of oil components andreflection of microplastic materials as recently discovered in our laballowed precise localisation of accumulation sites in cell types of tissues ofblue mussel. Exposure to microplastics caused granuloma formation indigestive gland and a pronounced immune response in blood. For thesubcellular localisation of organic compounds in cell organelles we havedeveloped a combinational protocol using antibodies directed againstchemicals togetherwith amultiple signal amplification system (catalyzedsignal amplification/gold-substituted silver-intensified peroxidase). Withthis approach we detected the oil component phenanthrene and thepolychlorinated biphenylmixture Aroclor 1254 at their subcellular sites ofaccumulation by transmission electron microscopy. The endo-lysosomalsystem of digestive cells and mitochondria of epithelial cells appeared tobe the preferred accumulation sites for the organic compounds studied.Microscopic visualisation and, thus, localisation of organic compounds inrelation to cell and tissue pathologies open new insights into the targetsand mechanisms of their toxicity.

doi:10.1016/j.cbpa.2010.06.020

ORAL PRESENTATIONS

2.Tissue-specific signaling: Comparison of AhR-dependent geneexpression profiles in immune organs and liver of rainbow trout

A. Möller, C. Largiader, H. Segner (University of Bern, Switzerland)

Xenobiotic activation of the aryl hydrocarbon receptor (AhR)pathway exerts multi-faceted influences on an exposed organism. Infish, research to date has focused mainly on toxic effects related to theAhR pathway in liver tissue. Here, we investigate AhR signaling inimmune cells of rainbow trout, Oncorhynchus mykiss. Several studieshave shown immunosuppression and increased pathogen suscept-ibility in fish exposed to AhR-activating chemicals such as dioxins andpolycyclic aromatic hydrocarbons (PAHs). In mammals, a modulationof immune processes is evident. However, there is no correspondingknowledge available for fish. Previous studies of our group demon-strated the presence of inducible expression of the AhR-regulatedcytochrome P4501A, CYP1A, in immune cells of rainbow trout,suggesting functional AhR signaling and xenobiotic metabolism. Inthis study, we investigated primary cultures from head kidney andspleen, as well as from the liver (as a reference). As a measure of BaPeffect strength, induction of EROD and CYP1A mRNA levels wasdetermined. Effect thresholds of both parameters were similar inimmune and liver cells, whereas induction amplitude in liver cellswas by an order of magnitude higher than in leucocytes. Transcrip-tional expression profiles were analyzed using an optimized rainbowtrout microarray. Liver and immune cells showed a number ofcommon genes modulated by BaP exposure, however, the latter cellsalso displayed immune-specific gene responses. These findingsprovide a basis to reveal mechanisms being involved in the PAHimmunosuppressive effects in rainbow trout.

doi:10.1016/j.cbpa.2010.06.021









3.Phase I biotrasformation enzymes to PAHs contamination inCommon sole (Solea solea, Linnaeus, 1758)

A. Trisciani, I. Corsi, C. Della Torre, G. Perra, S. Focardi (Department ofEnvironmental Science, University of Siena, Italy)

The release of polycyclic aromatic hydrocarbons (PAHs) in marinecoastal environment due to oil refinery activities can causedetrimental effects in living organisms. These effects can be inferredfrom measurements of contaminant-selective induction of biotrans-formation enzymes. The aim of the present study was to evaluate theresponsiveness of Common sole (Solea solea) liver phase I enzymes toPAHs at transcriptional and post-transcriptional levels in the field.Soles were collected in 2007 from three sites with different degrees ofPAHs contamination: one site characterized by petroleum hydro-carbon contamination (high-impact site HIS), and the other two atincreasing distances from HIS, moderate-impact site (MIS) andreference area (Ref). Sixteen PAHs (EPA) were determined insediments and fish fillets; EROD, BaPMO enzymatic activities andtranscriptional levels of cyp1a were measured in liver of soles.

Levels of PAHs in sediments reflected the oil pollution gradient ofthe area, with weak correspondence in fish fillets. EROD, BaPMOactivities were significantly induced in HIS respect to the MIS and Refwhile no differences of cyp1a transcription were observed in samplesfrom HIS and MIS compared to those from Ref.

As reported in previous studies, EROD and cyp1a follow differentphysiological principles, mRNA may have a shorter half-time than thecorresponding protein. In the natural environment a direct, straight-forward relation between the concentration of an inducing chemicaland the cyp1a response is not always to be expected, because anumber of confounding factors may overlay the induction response.

doi:10.1016/j.cbpa.2010.06.022

4.Expression profiling of endocrine-disrupting compounds using ashrimp (Crangon crangon) cDNA microarray developed throughsuppression subtractive hybridization PCR

Y. Verhaegen, K. Parmentier, J. Robbens (Institute for Agricultural andFisheries Research (ILVO), Belgium); G. Smagghe (Ghent University,Belgium);W. De Coen (University of Antwerp, Belgium); K. Cooreman(Institute for Agricultural and Fisheries Research (ILVO), Belgium)

Many environmentally important contaminants tend to accumu-late in marine sediments and related benthic biota, thereby inducingpotential hazardous biological effects. Over the past few years, newresearch techniques have enabled a detailed assessment of pollution-related genetic responses. To date, however, the use of pollution-related marker genes in international marine monitoring programshas been relatively limited. We believe that the brown shrimpCrangon crangon can act as an important sentinel species in suchprograms because it is an ubiquitous and often dominant species inEuropean estuaries and coastal zones. Furthermore, pollutantbioaccumulation in brown shrimp is high and comparable to theaccumulation in benthic predatory fish. As crustacean developmentand reproduction tend to be very sensitive towards endocrinedisrupting compounds (EDCs), sex-related genes from C. crangoncan act as important biomarkers to indicate the presence of suchchemicals. Therefore, suppression subtractive hybridization-PCR(SSH-PCR) was applied on male and female shrimp to derive a setof gender-specific genes. Of the 690 sequenced gene fragments, 280unique unknown sequences were derived. The other gene fragments

corresponded to 80 known unique genes, including genes encodingcuticular and cytoskeleton-related proteins (actins, myosin, cathe-psins,…), energy metabolism-related proteins (cytochrome c oxidase,ATP synthase, sugar binding proteins,…), antimicrobial proteins(crustin, carcinin,…) and reproduction-related proteins (vitellogenin,male reproductive-related protein a,…). In a final series of experi-ments, the laboratory effects of acute and subchronic exposures toseveral intriguing marine contaminants on the expression levels ofthese genes were studied using a custom-made microarray.

doi:10.1016/j.cbpa.2010.06.023

5.Accumulation patterns, biotransformation enzymes, and in vitrobiotransformation of polychlorinated biphenyls in several speciesof sea turtle

K.L. Richardson, D. Schlenk (University of California, USA)

Polychlorinated biphenyls (PCBs) are toxic, persistent, ubiquitous,halogenated contaminants. While well studied in mammals, little isknownabout thepotential health impacts of halogenated compounds onreptiles. Because toxicity can be mediated by biotransformation,assessing biotransformation provides a deeper understanding of thepotential negative health impacts. In mammals, biotransformation ofindividual PCB congeners is mediated by different Cytochrome P450isoforms (CYP), based on the structure of the congener. Two classes ofpolychlorinated biphenyl metabolites have been identified in biota andhave been implicated in various modes of endocrine disruption. Thisresearch aims to identify potential impacts of PCBs on the health of seaturtle populations by assessing accumulation of PCB congeners, expres-sion of CYP enzymes, and in vitro biotransformation of representativePCBs in the livers of four species of sea turtle—loggerhead (Carettacaretta), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), andhawksbill (Eretmochelys imbricate). PCBs accumulated in the livers of seaturtles, with levels ranging from 5 to 25 ng/g, and grouping individualPCB congeners into structure–activity based groupsprovided insight intometabolic pathways. Westerns blots, using antibodies raised againstrainbow trout (Oncorhynchus mykiss) CYPs, revealed the presence of seaturtle hepatic proteins which crossed reacted with anti-CYP2K1, but notanti-CYP1A. In vitro incubations with 2,2′,5,5′-tetrachlorinated biphenyl(PCB 52) and liver microsomes from sea turtles indicated formation ofhydroxy-tetrachlorinated biphenyl by liquid chromatography/massspectroscopy, while incubations with 3,3'4,4’-tetrachlorinated biphenyl(PCB 77) did not reveal metabolites. PCB 52-hydroxylation ratescorrelated with CYP2K1-like expression (r2=0.86, p<0.001). Takentogether, this research provides critical insight on reptilian detoxification(and bioactivation) pathways and potential health impacts of haloge-nated compounds on endangered sea turtle populations.

doi:10.1016/j.cbpa.2010.06.024


6.Organochlorine compounds in swordfish (Xiphias gladius) fromthe Mediterranean Sea

C. Burroni, S. Corsolini, S. Focardi, M.C. Fossi (Department ofEnvironmental Science “G. Sarfatti”, University of Siena, Italy)

The Mediterranean swordfish (Xiphias gladius) is an intensivelyfished species of high commercial value. Due to the great use for





human consumption, it is important to evaluate the presence of toxiccontaminants in their fillets. Mono and non-ortho polychlorobiphe-nyls (PCBs), hexachlorobenzene (HCB), hexachlorocyclohexane iso-mers (HCHs), p,p′ and o,p′ isomers of DDT, DDE, and DDD,polychlorodibenzo-dioxins and -furans (PCDDs/DFs) were detectedin all samples of liver and muscle tissues of male and femalespecimens. GC–ECD and GC–MS analyses revealed higher concentra-tions of ∑PCBs in liver samples of males (64±19 ng/g wet wt) andfemales (∑PCBs (47±45 ng/g wet wt). The ∑PCB concentrationsfound in samples of the fillet were 21±13 ng/g wet wt in males and16±16 ng/g wet wt in females. p,p′-DDE was the most abundantisomer of ∑DTTs in all the samples; HCH was always below 0.1 ng/gwet wt. PCDD/Fs were below the limits of detection (LOD=0.1 pg/gwet wt). Toxic Equivalents (TEQs) and the Tolerable Weekly Intake(TWI) were evaluated for mono-, non-ortho PCB congeners andPCDD/DF congeners. The presence of toxic contaminants in Medi-terranean swordfish should be monitored regularly due to thecommercial value of this species and its large use in human diet.

doi:10.1016/j.cbpa.2010.06.025

7.The MRP(ABCC)-mediated MXR phenotype is differentiallyexpressed in subpopulations of mussel hemocytes

D. Rioult, J. Pasquier, C. Boulangé-Leconte,M.Marin, F. Durand, C.Minier,F. Le Foll (University of Le Havre, France)

The multixenobiotic resistance (MXR) phenotype is known to bedifferentially expressed among cells composing a particular tissue.This variability is recognized as an obstacle for accurate monitoring ofthe resistance properties of tissues or organs. The mussel hemocyteshave been shown to display a MXR phenotype that can be induced bychemical treatment or pollutant exposure. Nevertheless, the musselhemocytes do not form a homogeneous population and, in this study,different subpopulations have been characterized based on Hemaco-lor staining, cell morphology (complexity and volume) determinedby flow cytometry and phagocytic activity. Accordingly, threesubpopulations were identified as eosinophilic granulocytes, hyali-nocytes and basophils. The MXR phenotype was then assessed byflow cytometry using calcein as fluorescent dye and a variety ofpharmacological blockers known as drug efflux inhibitors invertebrates. Results revealed that mussel hemocytes were particu-larly sensitive to the inhibitor MK571 suggesting that the monitoredMXR activity was carried out by an ATP-binding cassette (ABC)transporter phenotypically related to the mammalian ABCC family.Among subpopulations, the basal fluorescence level of eosinophilsloaded with calcein was significantly lower than those of othersubpopulations and showed a particular sensitivity to the MK571inhibitor. Hemocytes from mussels collected in polluted area wereable to significantly reduce (by two third) their dye content whencompared to hemocytes from mussel of reference site. Flowcytometry analysis revealed that hemocytes from mussels collectedin polluted area had 27% of their population which is less sensitive toMK571 efflux inhibition. These results thus showed that the MXRphenotype is related to pollution exposure and mediated bytransporters of the ABCC-type that are mainly expressed in eosino-philic granulocytes.

doi:10.1016/j.cbpa.2010.06.026

8.Differential effects of tributyltin (TBT) and dibutyltin (DBT) in theinhibition of the mitochondrial MgATPase in the digestive glandof Mytilus galloprovincialis

S. Nesci, V. Ventrella, F. Trombetti, M. Pirini, A.R. Borgatti, A. Pagliarani(University of Bologna, Italy)

Organotin compounds and especially TBT, widely exploited in thepast in antifouling paints, are still a matter of global concern, due totheir persistence and bioaccumulation properties in spite of bans ontheir use. Mitochondria are a preferred target of organotins. FOF1ATPases are blocked by TBT which interacts with FO subunit. DBT isgenerally considered as a far less effective toxicant. However inbivalve haemocytes, the DBT inhibition on Ca-ATPase and immuno-toxicity efficiency were both higher than TBT.

In mussel midgut gland mitochondria both the oligomycin-sensitive Mg-ATPase activity (OS Mg-ATPase) and the oligomycin-insensitive Mg-ATPase (OI MgATPase) were spectrophotometricallyevaluated in the presence of increasing TBT and DBT concentrations.The OS Mg-ATPase activity was inhibited by micromolar concentra-tions of both TBT and DBT individually tested and even more stronglyby the latter. The main difference between the two organotin effectsrelies into the inhibition curve shape. While TBT inhibition curvedisplayed a complex hormetic profile tentatively ascribed to multipleTBT-enzyme interactions, the enzyme inhibition curve by DBT washyperbolic. IC50 values were 0.32 and 0.19 μM respectively. At higherthan 5 mM TBT concentrations a striking rise in the OI Mg-ATPasewas observed; conversely DBT did not affect the OI Mg-ATPase. Thedifferent behaviour could be related to the different interactionkinetics with crucial ATPase domains. TBT acts as a non-competitiveinhibitor of the ATP substrate and is likely to interact with FO subunit,thus blocking proton flux as shown in bacteria. Conversely DBTbehaves as an incompetitive inhibitor of the ATP and as a competitiveinhibitor of Mg ion and may block the catalytic F1 subunit. Organotinbinding sites still remain to be defined. At least in some cases, thiolsor di-thiols on the enzyme structure are involved in organotin effects.

doi:10.1016/j.cbpa.2010.06.027

9.Transient inhibition of cyp1a gene transcription and EROD activityby musk xylene exposure in two fish cell lines PLHC-1 and RTG-2

M.L. Monti, C. Della Torre, I. Corsi, S. Focardi (Department ofEnvironmental Sciences “G. Sarfatti”, Università di Siena, Italy)

Musk xylene (MX) is one of the most used synthetic nitromuskfragrance in personal care products. The high release in the aquaticenvironment together with the evidence of bioaccumulation inaquatic organisms suggest that this chemical could be an actualconcern for toxicological consequences. Nevertheless no much dataare available on cellular pathways and mechanisms of toxicity inaquatic organisms. The aim of the present study was to investigatethe capability of MX to interfere with phase I of biotransformation bylooking at cyp1a gene transcription and related enzyme activity intwo fish cell lines as PLHC-1 and RTG-2. A dose-dependent (2, 4,20 μM) and time-dependent (6, 12 and 24 h) exposure experimentshave been performed testing MX alone and combined with a knownCYP1A inducer as B(a)P. A transient inhibition of cyp1a genetranscription was observed after 6 h in both cell lines which seemsto recover dose-dependently after 24 in RTG-2 and less markedly inPLHC-1. EROD activity did not show clear effect after 6 h while





resulted significantly inhibited in both cell lines after 24 h. A commonpathway of inhibition at gene level could be thus hypothesised inboth cell lines after 6 h of exposure confirmed by EROD activityinhibition at 24 h. The potential involvement of MX metabolites incyp1a gene recovery after 24 h of exposure could be hypothesized.

Results of the time dependent experiment also suggest a differentpathway of gene regulation for MX compared to B(a)P.

doi:10.1016/j.cbpa.2010.06.028






Session 3: Pollutant-induced genotoxicity and cancer

INVITED LECTURE

1.Setting anchor in a sea of biomarkers — The role of diseaseassessment in marine monitoring

G.D. Stentiford, S.W. Feist (Centre for Environment, Fisheries andAquaculture Science (Cefas), Weymouth, UK)

Assessment of ‘Good Ecological Status’ (GES) in European marinehabitats is implied in the draft of the EU Marine Strategy FrameworkDirective (MSFD). Under the MSFD, European Member States mayapply a set of assessment tools to provide evidence of GES. For thispurpose, it is apparent that individual assessment tools will requireappropriate quality assurance measures to ensure cross-MemberState comparability of data. Such quality assurance systems arealready in place (albeit on a voluntary basis) for some biomarkers(e.g. fish disease under the BEQUALM programme), but even wherethese are applied, recent work from our laboratory has demonstratedhow greater effort is required to ensure accurate measurement ofdata from particular field sites. Such efforts include the collection ofbasic life history trait data from sentinel species (e.g. age, size andsex) alongside specific biomarker data — all of which may alter theinterpretation of evidence collected and therefore the outcome of anassessment tool that aims to utilize the data in a managementcontext. This presentation will demonstrate the application of asimple assessment tool for fish diseases (including liver cancer) tomarine sites in UK waters and further will demonstrate how somekey life history variables (such as age) have the capacity to act asconfounding factors for population disease prevalence. We utilize thisadditional life history data to reveal subtle differences in response(e.g. in the age at disease onset) observed at geographically distinctmarine sites and to provide some recommendations for qualityassured data collection from biological effects monitoring surveys.Since under the MSFD, greater efforts will be required to integratedata pertaining to specific biomarkers with that for chemical and

ecological variables, it will be necessary for experts involved withthese disciplines to engage in discussions to formulate meaningfulways for such integration and to provide well grounded assessmenttools to define thresholds for GES in European waters.

doi:10.1016/j.cbpa.2010.06.030

ORAL PRESENTATIONS

2.The micronucleus test as a biomarker of genomic damage: Thevalidation process in environmental animals

C. Bolognesi (Environmental Carcinogenesis Unit, National CancerResearch Institute, Genoa, Italy)

Genotoxic compounds are the components of complex environ-mentalmatrices, that can have adverse health effects on humans and onindigenous biota. The application of genotoxicity biomarkers in sentinelorganisms, allows for the assessment of mutagenic hazards and/or forthe identification of the sources and fate of the contaminants.Micronucleus (MN) test as an index of accumulated genetic damageduring the lifespan of the cells, is one of themost promising techniquesto identify integrated response to the complexmixture of contaminants.MN assay, originally developed with mammalian species, is todaywidely applied in a large number of environmental species. Nuclearanomalies, chromatin buds, invaginations, and vacuoles are alsoconsidered as biomarkers of genetic events. Interspecies and inter-individual variability in spontaneous MN frequency, related to differ-ences in metabolic competency and DNA repair mechanisms as well asin the MN expression, has been observed, impairing the use of somespecies in environmental programs. The validation process of the test insentinel species involves the identification of the target tissues for thegenotoxic effects through the evaluation of the kinetics of cytogenetic








alterations under controlled conditions. Sensitivity and specificity of theassay, in revealing the exposure to the main classes of genotoxicpollutants, have to be determined. Sources of variability that mayinfluence the expression of the cytogenetic damage such as climatechange, nutritional status, and reproductive status have to be evaluatedin field experiment.

doi:10.1016/j.cbpa.2010.06.031

3.Genotoxicity and genetic changes in RTG-2 fish cell line uponexposure to benzo[a]pyrene and ethyl methanesulfonate detectedby the Comet assay and AFLP marker analysis

M. Šrut, A. Štambuk, G.I.V. Klobučar (Department of Zoology, Facultyof Science, University of Zagreb, Croatia)

Genotoxicity is often one of the earliest signs of toxicant impact andis frequently assessed using in vitro models in biomonitoringprogrammes. In this study Comet assay and amplified fragment lengthpolymorphism (AFLP) were applied to assess DNA damage in RTG-2fish cell line after 3 days of exposure to a concentration range of modelgenotoxic agents (benzo[a]pyrene (B[a]P) and ethylmethanesulfonate(EMS)). Quantitative modifications arising in AFLP profiles as ameasure of DNA effects were analyzed in order to establish permanentgenetic changes arising from mutation events (e.g. rearrangements,pointmutations, small inserts or deletions of DNA) induced by toxicantexposure. Significant induction of DNA damage measured by theComet assay was noticed in RTG-2 cells after 3 days of B[a]P treatmentat all concentrations used (0.1 µM, 1 µM, 5 µM, 10 µM). After 3 days ofrecovery all the values returned to the control level. In contrast, 3 daysof exposure to EMS (10 µM, 50 µM,100 µM, 500 µM,1000 µM) inducedsignificant DNA damage only at the highest concentration used and3 days of recovery resulted in more pronounced genotoxic effect. Thechanges occurring in AFLP profiles of RTG-2 cells following bothtoxicant treatments included loss of normal bands and appearance ofnew bands at higher toxicant concentrations in comparison to theband profile of the control. Our results indicate that the AFLP methodcould be a useful biomarker for detection of permanent genotoxicinfluence of toxicant exposure and encourage the use of Comet assayon fish cell lines as a versatile tool for genotoxicity assessment.

doi:10.1016/j.cbpa.2010.06.032

4.Use of the Comet and micronucleus assays for in vivo genotoxicityassessment in the coelomocytes of the earthworm Eisenia andrei

S. Sforzini, I. Saggese, L. Oliveri, A. Viarengo (University of PiemonteOrientale, DiSAV, Italy); C. Bolognesi (Environmental CarcinogenesisUnit IST, Genova, Italy)

The assessmentof genotoxic effects causedbyenvironmental stressorsrepresents an essential part of ecotoxicology because of its relevance incarcinogenesis. Many POPs are genotoxicant, i.e. they can alter both thestructure and integrity of DNA, either directly or indirectly. The Cometassay is a method now widely used on different vertebrates andinvertebrates to detect DNA damage (e.g. single- and double-strandbreaks, alkali-labile sites, oxidative DNA base damage, etc.) in individualcells. Notwithstanding, the micronucleus test has emerged as one of themore powerful methods for assessing chromosome damage (bothchromosome loss and chromosome breakage) accumulated during life-span of the cell. In this study, earthworms of the species Eisenia andrei

were exposed to artificial standard soils contaminated with environmen-tally relevant concentrationsof2,3,7,8 tetrachlorodibenzodioxin(1×10−4,1×10−5, 2×10−3 mg/kg) and benzo[a]pyrene (0.1, 10, 50 mg/kg) for 10and 28 days. The DNA damage, using both the neutral and alkalineversions of the Comet assay, was assessed in earthworm coelomocytes inthe different experimental conditions. Moreover, on these same cells, thepresence of cytoplasmic micronuclei (MNi) was also studied bycytochemical staining of DNA by the fluorescent dye DAPI. The resultsobtained by the Comet assay showed, only in alkaline condition,significant effects at the different concentrations of the chemicals utilisedand the times of exposure. Induction of MNi in coelomocytes, identifiedaccording to standardized criteria, was demonstrated, indicating asignificant increase in MNi frequency particularly relevant in animalsexposed to the higher concentrations of both chemicals. This studyrepresents the first step of validationwork of this test on earthworm cells.

doi:10.1016/j.cbpa.2010.06.033

5.Potential genotoxic effects induced by different superficialmorphologies and different dimensions of amorphous silicapowders evaluated in murine alveolar macrophages (Raw 264.7)cell lines

P. Guidi, V. Scarcelli, M. Bernardeschi, P. Lucchesi, M. Nigro, G. Frenzilli(University of Pisa, Italy)

No sufficient evidence exists about the health hazards caused bynanosized amorphous silica, nor as regard its superficial conformity.

The aim of the study was to investigate the potential genotoxiceffects of silica particles with different superficial morphologies(mesoporous and dense spheres), and dimensions (250 and 500 nm)by in vitro experimental models. Pure quartz, vitreous silica andimogolite (1 nm diameter) were also investigated. Murine alveolarmachrophages (Raw 264.7) cell lines have been used as representativeof occupational and environmental exposures. Genotoxicity wasevaluated by Comet Assay (4 and 24 h exposure) and MicronucleusTest. Cytotoxicity was tested using Trypan Bluemethod. Cell lines havebeen treated with 5–10–20–40–80 µg/cm2 of different silica-basedcompounds. The same doses of exposure were tested byMicronucleustest. MMSwas used as positive control in Comet assay,Mytomicin C formicronucleated cells. Comet assay results showed that the particlesuperficial morphology had an effect on DNA integrity, as mesoporousones induced higher damage level with respect to the densenanoparticles. On the contrary, particle dimension did not seem tohave any effect. Clear dose–effect relationships were not detected, butthe highest dose of exposure induced significant DNA fragmentation inpowders tested. An increase in micronucleated cells was observed atintermediate doses of imogolite. Cell suspensions exposed to thelowest dimension of MCM-41 show a particular distribution of theseparticles inside the cell, with respect to the one across the slide. Adeeper analysis of this phenomenon is in progress (TEM observation).

doi:10.1016/j.cbpa.2010.06.034


6.Genotoxicity of marine sediment samples assessed by the Cometassay on PLHC-1 fish hepatoma cell line

M. Šrut, A. Štambuk, S. Kralj, G.I.V. Klobučar (Department of Zoology,Faculty of Science, University of Zagreb, Croatia); L. Traven, V. Mićović


(Department of Environmental Medicine, Medical Faculty, Universityof Rijeka, Croatia/Teaching Institute of Public Health of the Primors-kooranska County, Croatia); R. Žaja (Laboratory for MolecularEcotoxicology, Ruđer Bošković Institute, Croatia)

Aquatic sediments are the main sink for anthropogenic pollutantswhich are known to induce versatile toxic effects in marine andfreshwater organisms, among which the genotoxic ones are con-sidered to be of particular significance. It is therefore important toinvestigate negative effects of such contaminated sediments on biotasince this kind of information can serve as a warning of possibleimpacts on the entire aquatic environment. The main goal of thisstudy was to test the usefulness of the Comet assay in the PLHC-1hepatoma fish cell line as a tool for detecting the presence ofgenotoxic compounds in contaminated marine sediments. For all ofthe analysed sediment extracts a concentration dependent genotoxiceffect was observed. The sediment with the highest observedgenotoxic potential was additionally extracted using various solventsin order to test which class of compounds, according to their polarity,is mostly responsible for the observed genotoxic effect. Non-polarsolvents (cyclohexane and dichloromethane) yielded stronger geno-toxic effect but the highest level of DNA damage was determined afterexposure to sediment extract obtained with the solvent mixturedichloromethane/methanol which extracts a wide range of contami-nants. Our results indicate that the PLHC-1 cell line is a suitable invitro model in sediment genotoxicity assessment and encourage theuse of fish cell lines as versatile tools in ecogenotoxicology.

doi:10.1016/j.cbpa.2010.06.035

7.First assessment of DNA damage in Mediterranean pelagic speciesloggerhead turtle and swordfish: Application of comet assay anddiffusion assay in frozen total blood

I. Caliani, S. Casini, T. Campani, M. Giannetti, L. Marsili, M.C. Fossi(University of Siena, Italy)

The aim of this study was to apply and validate the comet assay anddiffusion assay on frozen total blood to assess the DNA damage inswordfish (Xiphias gladius) and loggerhead turtle (Caretta caretta).Untilnow, a limited number of studies were published utilizing these assayswith marine species in comparison to those using fresh water species.This may be due to logistical problems associated with performing thetechniques at sea. Before applying the genotoxicity biomarkers inswordfish and loggerhead turtle in order to develop the frozenconservation methodology, we performed the tests in parallel on freshand cryopreserved blood ofDicentrarchus labrax. The total blood sample

collectedwasmixedwith RPMI 1640 andDMSOand immediately storedin −80 °C. We observed no statistically significant differences in thelevel of DNA damage between fresh and frozen blood samples. Theprotocol was then applied to blood samples of the two speciesinvestigated. In sea turtles, we applied the technique on fresh wholeblood too and the results showedDNA fragmentation values in linewiththose of the frozen blood. Results were compared with those of ENAassay, a genotoxicity biomarker validated for the first time in thesespecies. This study strongly suggests that comet and diffusion assays infrozen whole blood can be utilized, in combination with ENA assay, toassess genotoxic damage in top predators or long living species exposedto potential toxicological risk.

doi:10.1016/j.cbpa.2010.06.036

8.Potential genotoxicity of nano-sized TiO2 particles in isolatedbottle-nose dolphin (Tursiops truncatus) and human leukocytes

M. Bernardeschi, P. Guidi, V. Scarcelli, P. Lucchesi, M. Nigro, G. Frenzilli(University of Pisa, Italy)

Many studies on NPs toxicity have been focused on their potentialeffects on human health, but scarce information is available on thesusceptibility of endangered species, such as marine mammals.Titanium dioxide (TiO2) engineered NPs are used in a wide range ofproducts and processes, and they can reach the aquatic environmentfrom different sources. This study is aimed to assess the profitabilityof isolated leukocytes for evaluating the potential genotoxic effects ofNPs on toothed cetaceans and human lymphocytes. The bottle-nosedolphin Tursiops truncatus was selected as study species beingcommonly reared in captivity. Blood samples from four males and afemale specimen were achieved by the Adriatic SeaWorld “Oltre-mare” (Riccione, Italy). Human blood samples were taken fromhealthy male volunteers (24–30 years old), and processed as follows.Leukocytes were isolated by the lyses procedure and exposed in vitroto TiO2 (rutile and anatase) NPs. Three experimental times ofexposure (4, 24, 48 h) and three doses (20, 50, 100 µg/ml) weretested. Genotoxicity was assessed by the comet assay, detecting DNAstrand breaks. Cell viability was also assessed by the Trypan Blue test.In order to assess aggregation, particles were observed with a Jeol 100SX transmission electron microscope (TEM). Results of the cometassay showed that both the crystalline forms of TiO2 induced DNAfragmentation on a group basis.

doi:10.1016/j.cbpa.2010.06.037

S14 Abstract / Comparative Biochemistry and Physiology, Part A 157 (2010) S12–S14




Session 4: Pollution and environmental changes: From biomarkers to the physiologicalstatus of the organisms

INVITED LECTURE

1.Ecotoxicological implications of global environmental change

M.H. Depledge (European Centre for Environment and Human Health,Peninsula Medical School, UK)

Climate change has been identified as the greatest threat tohuman health and wellbeing in the 21st century. Clearly, it will alsobe a key driver of ecological change and will be responsible for thelosses of numerous species and changes in the distributions of others.Anthropogenic greenhouse gas pollution can be regarded therefore,as being of ecotoxicological significance. In this paper some of thelikely effects of climate change on pollutant exposure and effects willbe discussed. These include effects on bioavailability, uptake rates andbody burdens of contaminants, altered physiological and behaviouralresponses, population and community effects, and changes inecosystem structure and function. Climate change will also bediscussed in the context of other global environmental changes thatwill be occurring simultaneously in the coming decades. This raisessome contentious issues as to whether non-climatic global changesare likely to be greater relevance to the ecotoxicological research. Forexample, will the ecotoxicological impacts of an additional 3 billionhumans added to the world population by 2050, overwhelm effectsthat might be generated by climate change? Or will technologicaldevelopments and changes in human lifestyles be particularlyrelevant? The possibility of interactions among these and otheraspects of global environmental change on ecotoxicological processeswill be discussed.

doi:10.1016/j.cbpa.2010.06.039

ORAL PRESENTATIONS

2.Biomarkers as tools to assess the health status of organisms inpolluted environments

R. Triebskorn (University of Tübingen and Transfer Center forEcotoxicology and Ecophysiology, Rottenburg, Germany); H.-R.Köhler (University of Tübingen, Germany)

During the last two decades, interest in using biomarkers orbioindicators as monitoring tools to assess environmental pollu-tion has steadily increased. This is based on the fact, thatbiomarkers are suitable not only for delivering information onthe health status of exposed organisms (biomarkers of effect), butalso on the quality and/or quantity of the exposure situation(biomarkers of exposure). Thus, biomarkers can be used either asmeasures of toxic effects or as fingerprints for chemical exposure.Depending on the character of biomarkers selected for applicationin a biomarker battery, the evaluator will receive more data oneither adverse effects in organisms or on the exposure situationitself. In our laboratory, several cellular and biochemical biomar-kers are established for fish and invertebrate species. Usingexamples for biomarker application in large field surveys theiradvantages, disadvantages, their potential, and their limits for theassessment of environmental pollution will be discussed in thispresentation.

doi:10.1016/j.cbpa.2010.06.040









3.Are Damsels in Distress? Combined effects of chlorpyrifos andtemperature stress on the tropical damselfish Acanthochromispolyacanthus from the Great Barrier Reef (Australia)

E. Botté (James Cook University, Australia; Australian Institute ofMarine Science, Australia); A. Negri, S. Codi King (Australian Instituteof Marine Science, Australia); M. Gagliano (University of WesternAustralia, Australia); C. Smith-Keune, D. Jerry (James Cook University,Australia)

Tropical reefs are facing multiple stressors worldwide. Along theNorthern Great Barrier Reef (GBR) of Australia, seasonal floods due toheavy rains induce terrestrial run off, potentially spreading agricul-tural chemicals into the GBR inshore reefs. Meanwhile episodes ofhigh temperature have become more frequent on the GBR the lastfifteen years.

With this in mind, we measured the impact of exposure to theorganophosphate chlorpyrifos (CPF) and of temperature stress, sepa-rately and in combination, on juveniles of the damselfish Acanthochro-mis polyacanthus from the Indo-Pacific. We assessed the musclecholinesterase (ChE) activity, hepatic glutathione-S-transferase (GST)activity, coenzyme Q (CoQ) redox balance and gene expression.

We found that GST was not affected by any of the treatments.However, exposure to 10 μg/L of CPF induced a 50% inhibition of ChEactivity after 96 h and an increase in the antioxidant form of CoQ after6 h. Temperature increases of 4 °C and 6 °C compared to control(28 °C) decreased ChE activity by 50% after 96 h but did not affectCoQ redox balance. Combining both temperature and CPF exposuredid not induce additive or synergistic effects on the stress indicatorsinvestigated.

Our results suggest that A. polyacanthus is quickly reacting to CPFexposure and might be well armed against temperature-inducedoxidative stress. The strong impairment of ChE with both stressorssuggests potential damages on the nervous system of the fish. Furtheranalyses involving gene expression should deepen our understandingof A. polyacanthus defence mechanism against physical and chemicalstress.

doi:10.1016/j.cbpa.2010.06.041

4.Cell and tissue level responses to gradual temperature raising indigestive gland of mussels from sites with different levels ofenvironmental stress

M. Múgica, M. Ortiz-Zarragoitia, U. Izagirre, I. Marigómez (CBET ResGrp, Dept. Zoology & Cell Biology, Univ. Basque Country, Bilbao,Basque Country, Spain)

Research on the biological responses to changes in temperature, amajor environmental stressor in marine ecosystems, is gainingrelevance as a result of global climate change. In this context, theeffects of gradual temperature raising was investigated undercontrolled laboratory conditions in marine mussels, Mytilus gallopro-vincialis, collected from two sites in the Bay of Biscay with differentlevels of environmental stress in July and November 2009 and March2010: Mundaka (reference site) and Arriluze (leisure harbour at theindustrial area). At each season, mussels were kept in each own'sseawater and acclimated to laboratory conditions at local seawatertemperature (20 °C in July, 16 °C in November and 12 °C in March) for5 d. Isochrysis sp. was provided as food. In the 3 experimental sets,temperature was raised 1 °C per day for the first 8 d and then keptconstant for the following 19 d. Cell and tissue level biomarkers were

recorded in the digestive gland, including lysosomal size (volumedensity of lysosomes; VvLYS) and membrane stability (labilizationperiod; LP), intracellular neutral lipid accumulation (VvNL), cell typereplacement (VvBAS), epithelial thinning (mean-luminal-radius-to-mean-epithelial-thickness ratio; MLR/MET) and tissue integrity indigestive gland tissue (connective-to-diverticula ratio; CTD). Histol-ogy of gonad was assessed as supporting parameter. Markeddifferences in the recorded cell and tissue biomarkers were foundbetween Mundaka and Arriluze as well as between their responsive-ness to gradual temperature raising. Interactions between tempera-ture and the health status of mussels and their consequences forbiomarker based marine pollution monitoring programs are dis-cussed. Acknowledgements: Funded by Basque Government (ETOR-TEK2007 K-Egokitzen and Consolidated Res Grp Grant 2007-12).

doi:10.1016/j.cbpa.2010.06.042

5.Monitoring coastal water ecological status through early signalsof biological effects — an integrated approach

C.D.S. Pereira, A. Cesar, R.B. Choueri (Universidade Santa Cecília,Brazil); M.G.M. Catharino, S. Taniguchi, M.C. Bicego, M.B.A. Vascon-cellos, E.C.P.M. Sousa (Universidade de São Paulo, Brazil); A.C.D. Bainy(Universidade Federal de Santa Catarina, Brazil); A.A. Mozeto(Universidade Federal de São Carlos, Brazil); M.L. Martin-Díaz,T.A. Del Valls (Universidad de Cádiz, Spain)

The São Paulo coast (Brazil) represents one of the most importantmetropolitan regions of South America, which has been consideredranging from low to highly pollute. This study aimed to provide a one-year long biomonitoring in this coastal zone and, for this purpose,biomarkers and bioassays were employed simultaneously with con-taminant body burden analysis in a weigh-of-evidence approach. Thebrown mussel Perna perna was selected to be transplanted from afarming area (Cocanha— Caraguatatuba) to four possibly polluted sites:Engenho D'Água, São Sebastião Oil Terminal, Palmas Island and Itaipu,comprising a 200 km coastal area to be monitored. After 3 months ofexposure in each season (winter 2005, spring 2005, summer 2006 andautumn 2006) mussels were recollected and DNA damage, lipidperoxidation, lysosomal membrane stability and embryo–larval devel-opment were analyzed. The whole tissue concentrations of polycyclicaromatic hydrocarbons andnon essentialmetalswere also analyzed andlinked to biological responses by multivariate analysis. Biomarkers andbioassays exhibited most significant alterations all year long in musselstransplanted to the Oil terminal and Itaipu, where bioaccumulation oforganic and inorganic compounds was also detected. This studyemploying caged mussels under field conditions produced a rapid andsensitive assessment, pointing out critical areas where adverse effectscould be related to chronic contamination. Variations according toseasons, presence of emergent pollutants, as well as difficulties incorrelating biological responses and physiochemical analysis in fieldstudies are also discussed, considering the applicability of this approachas descriptor of ecological status.

doi:10.1016/j.cbpa.2010.06.043

6.Biological and biochemical responses of Carcinus maenas: Aconsequence of pharmaceutical exposure

G.V. Aguirre-Martínez, M.J. Salamanca, T.A. Del Valls, M.L. Martín-Díaz(University of Cádiz, Spain)


Due to the potential risk associated to the presence of pharma-ceuticals in aquatic environments, a 28 day bioassay was carried outwith the species Carcinus maenas with the aim to evaluate biologicaland biochemical effects caused by four widely used pharmaceuticals.Crabs were exposed under laboratory conditions to concentrations ofcaffeine (stimulant), carbamazepine (anticonvulsant/mood stabiliz-ing), ibuprofen (anti-inflammatory) and novobiocin (antibiotic). Seawater was spiked with DMSO, caffeine, carbamazepine, ibuprofen andnovobiocin (0.1–50 μg/L). Biological response was evaluated inhaemolymph applying neutral red retention time (NRRT), biochem-ical responses were studied in gill, hepatopancreas, muscle and gonadtissues using a battery of biomarkers, including; ethoxyresorufin O-diethylase (EROD), dibenzylflourescein dealkylase (DBF), glutathioneS-transferase (GST), glutathione peroxidase (GPx), lipid peroxidation(LPO) and DNA damage. Chosen responses were observed differentlyfrom control when increasing the concentration of pharmaceuticals.NRRT in crabs exposed to environmental concentrations of pharma-ceuticals were diminished by 50%. EROD and DBF activity weresignificantly induced by caffeine and carbamazepine in muscle andhepatopancreas (p<0.05); GST was significantly activated byibuprofen and carbamazepine in gill, hepatopancreas and muscle athigh concentrations (p<0.05). All tissues evidenced GPx and LPO(p<0.05). Crabs exposed to caffeine, Ibuprofen and novobiocin pre-sented DNA damage mainly in hepatopancreas (p<0.05). Resultsreveal that environmental concentrations of pharmaceuticals have aneffect in the biological and biochemical responses studied. The meth-odology utilized is a suitable tool to assess pharmaceutical toxicity inaquatic environment.

doi:10.1016/j.cbpa.2010.06.044

7.Effects of Vibrio challenge on digestive gland biomarkers andgene expression in Mytilus galloprovincialis: Bacterial infection asa possible confounding factor in understanding biomarkerresponses

L. Canesi, C. Barmo, R. Fabbri, L. Vergani, G. Gallo (Dipartimento diBiologia, Università di Genova, Italy); C. Ciacci (DISUAN, Università“Carlo Bo” di Urbino, Italy); P. Roch (JRU Ecosystèmes Lagunaires,CNRS-niversité de Montpellier 2, France)

Biomarkers are worldwide utilised to identify stressful conditionsinduced by environmental perturbations in marine bivalves. Inaddition to many environmental factors other than pollution (season,reproduction, and food availability), also host responses to naturalbacteria and parasites may potentially affect biomarker responses.Despite the fact that bivalves can accumulate huge numbers ofbacteria, in particular marine Vibrios, no data are so far available onthe possible effects of bacterial challenge at the tissue level, wherebacterial infection may cause stressful conditions, in particularoxidative stress due to inflammatory processes. Data are herereported on digestive gland biomarkers in mussels (Mytilus gallopro-vincialis) injected with either heat-killed V. splendidus LGP32 or V.anguillarum (ATCC19264) at 3, 6 and 24 h p.i. Lysosomal biomarkersand activities of catalase and Glutathione S-transferase (GST) wereevaluated, as well as the expression of catalase, GST-π andmetallothioneins (MT10 and MT20) by RT-q-PCR. Both Vibriosinduced lysosomal membrane destabilisation and increased theactivities of antioxidant enzymes; however, V. splendidus inducedup-regulation of antioxidant gene expression, whereas a generaldownregulation or no effect was observed with V. anguillarum. Thelack of this response was reflected in stronger oxidative stressconditions in mussels challenged with V. anguillarum, as indicated by

higher lipofuscin accumulation at longer times p.i. The resultsrepresent the first data on biomarker responses to bacterial challengein bivalve tissues, and indicate that lysosomal and oxidative stressbiomarkers could be usefully applied in order to monitor earlychanges in the physiological status of bivalves induced by bacterialinfection. Moreover, the results support the hypothesis that in theseorganisms responses to bacteria may be taken into account wheninterpreting biomarker data in field studies.

doi:10.1016/j.cbpa.2010.06.045


8.Seasonal changes in basal and stress-induced cortisol levels in thesubterranean rodent Ctenomys talarum: Differences betweensexes and reproductive condition

F. Vera, R.R. Zenuto, C.D. Antenucci (Universidad Nacional de Mar delPlata, CONICET, Argentina)

The activation of the hypothalamic-pituitary-adrenal (HPA) axis isone of the hallmarks of the stress response in vertebrates. The resultingsecretion of glucocorticoids (GCs) is used to monitor stress in naturalpopulations because they reflect physiological adjustments to changingenvironmental conditions, intra and inter-specific interactions andhuman disturbance. Despite changes in HPA activity influence survivaland reproduction; studies on GCs are scarce in free-ranging animals.Here, we evaluated seasonal changes in basal and stress-induced cortisollevels in Ctenomys talarum, to assess differences between sexes andreproductive condition, and their utility as indicators of stress. Duringreproductive and non-reproductive seasons we obtained, in the field,baseline blood samples and also subjected other animals to immobiliza-tion to obtain cortisol levels in response to stress. We also determinedleukocyte profiles to address the significance of seasonal changes incortisol levels. Both sexes had higher basal plasma cortisol levels duringthe breeding season, but cortisol levels were 2–3-fold higher in femalesthan males during the breeding cycle. In spite of their high basal cortisolconcentration during the reproductive season, females respond to acutestress reaching very high cortisol concentrations after the treatment inthis season. These results suggest physiological differences in the HPAaxis between sexes and show that adjustments in the output of thisneuro-endocrine axis occur seasonally concurrently with changes inreproductive activity.We show thatGC concentrations should be coupledwith the determination of other stress indicators (leukocyte profiles) toallow a broad understanding of seasonal variation in HPA axis activity.

doi:10.1016/j.cbpa.2010.06.046

9.Morphometric characteristics and nuclear marker Me 15/16analyses of mussel Mytilus galloprovincialis populations alongthe eastern Adriatic coast

B. Hamer, M. Korlević, V. Nerlović (Ruđer Bošković Institute, Centerfor Marine Research, Rovinj, Croatia); E. Durmiši (Juraj DobrilaUniversity of Pula, Croatia)

Mussels Mytilus spp. belong to a group of key species in marine,coastal ecosystems. This work demonstrated the dominant presenceand morphological variations (shell shape) of Mediterranean musselM. galloprovincialis along eastern Adriatic coast (22 sites), fromLimski kanal to Dubrovnik. Morphometric characteristics of sampled


mussel specimens were inside normal values for M. galloprovincialis:height/length 55.89±5.08% and width/length 38.76±3.42%. Differ-ent alleles coding an adhesive protein of the byssus were observed inthe population of mussels in Croatian Adriatic Sea. Only 2 specimensamong the 110 analysed were found that showed a heterozygotegenotype: one with an E (M. edulis) allele and one with a T (M.trossulus) allele, all others were identified as homozygotes for the G(M. galloprovincialis) allele. The presence of the E allele at lowfrequency in M. galloprovincialis mussels can simply be explained byintrogression, as M. edulis and M. galloprovincialis are known tohybridise and exchange genes in the European contact zone. Thepresence of the T allele is very surprising. Shell height/length valuesof heterozygote mussels: GE (h/l 46.90%, w/l 38.45%) and GT (h/l48.91%, w/l 39.11%) were closer to M. edulis (h/l 44.50±2.26%, w/l35.38±2.00%), while the width/length values were typical for M.galloprovincialis. Further analyses using different genetic markers anddetailed shell morphometric measurements on larger numbers ofspecimens are needed to clarify the processes underlying introgres-sion in Mediterranean mussels.

doi:10.1016/j.cbpa.2010.06.047

10.Lysosomal responses to intertidal exposure in Mytilus edulis fromIcelandic coast

V. Moschino (Institute of Marine Sciences, ISMAR-CNR, VeneziaItaly); H.P. Halldorsson (Sudurnes University Research Centre,University of Iceland, Sandgerdi, Iceland); L. Da Ros (Institute ofMarine Sciences, ISMAR-CNR, Venezia Italy)

Lysosomal alterationsmeasured in bivalve digestive cells are generalstress biomarkers which, although widely used to detect consequencesof pollutant exposure on organism well-being, may reveal negativeeffects of natural environmental factors such as temperature, foodavailability and tides. It is also known that tidal fluctuations are arelevant source of variability for intertidal individuals which can exhibitdifferences in metabolic and digestive processes compared to subtidalorganisms. The aim of the present study was to investigate the possibleeffects of intertidal exposure on lysosomes of Mytilus edulis from thesouth-west coast of Iceland.Mussels from a clean sitewere transplantedin the intertidal and subtidal zones at the contaminated site of Reykjavikand at the reference site of Hvalfjordur, and then sampled after 3, 6 and8 weeks. Lipofuscin and neutral lipid accumulation, lysosomal mem-brane stability and lysosomal structural changes were histochemicallyevaluated. In general, lysosomal parameters, except for neutral lipidswhich were absent in most individuals, showed slight differencescomparing intertidal and subtidal samples from each studied site after 3and 6 weeks; higher differences were observed after 8 weeks, particu-larly at Reykjavik.Moreover, a decrease in thewell-being ofmusselswasapparentwith theextentof the transplantationperiod. Lastly, organismsfrom Reykjavik generally showed larger and most abundant lysosomes,as well as lower membrane stability than those from Hvalfjordur.

doi:10.1016/j.cbpa.2010.06.048

11.Macrophysiology of subterraneanmammals. Ctenomys: Amodel toassess the effect of global warming on physiology and distribution

C.D. Antenucci, F. Luna (Universidad Nacional de Mar del Plata,CONICET, Argentina)

Macrophysiology aims to explain how physiological traits areaffected by environmental variability over large geographic distances.The knowledge of physiological strategies to cope with long-termenvironmental and geographical changes is a key for conservationbiology. Thermal physiology influences ecological and evolutionaryanimals´ success, and represents a valuable tool to understand theeffect of these changes on animal distribution. We are unable to knowthe effect of global warming on species survival but, we can usespecies that underwent similar selection pressures as a proxy.Subterranean rodents are an excellent model to evaluate the effectof extreme conditions on physiology and distribution. Its evolutionwas linked to an aridization period (late Cenozoic), following by apeak in genus diversification during a warm period (Pliocene).

Ctenomys is exposed to extreme ambient temperatures (Ta) whenforage, undergo overheating during digging and is widely distributed.We assess the effect of Ta on energetics and thermogenesis atmolecular and organismic levels. We also explored which are thefactors affecting metabolic scope and thermal balance. Finally, weinvestigated if large scale environmental patterns modulate thermalbiology. Ta was not a good predictor of basal and maximummetabolicrate. Molecular and physiological patterns of thermoregulatory heatproduction were species-specific. In smaller species, non shiveringthermogenesis was the main mechanism, whereas shivering was inbigger species. Biogeographical factors that influence surface-dwell-ing species have no effect on Ctenomys. We illustrate how macro-physiology could provide insights into energetics within Ctenomys,and their consequence on species distribution in a changing world.

doi:10.1016/j.cbpa.2010.06.049

12.Melatonin, vasotocin and isotocin as biomarkers of the conditionof fish

E. Kulczykowska (Pomeranian University, Poland, Institute of Ocea-nology PAS, Sopot, Poland); M. Gozdowska (Institute of OceanologyPAS, Poland); J.A. Martos-Sitcha (Universidad de Cádiz, Instituto deCiencias Marinas de Andalucía, CSIC, Puerto Real, Spain); H. Kalamarz,M. Nietrzeba (Institute of Oceanology PAS, Poland); J.M. Mancera(Universidad de Cádiz, Puerto Real, Spain); G. Martínez-Rodríguez(Instituto de Ciencias Marinas de Andalucía, CSIC, Puerto Real, Spain)

The environmental changes interfere with vertebrate endocrinesystems. The endocrine biomarkers are used to indicate the conditionof both free-ranging and farmed animals. The aim of this study was toshow an impact of environmental changes on arginine vasotocin(AVT), isotocin (IT) and melatonin (MEL) in selected fish species. AVTand IT are neurohormones synthesized in hypothalamic nuclei andreleased to the blood stream in the neurohypophysis. Changes inhypothalamic, pituitary and plasma AVT and IT concentrations werefound in many fish species subjected to different type of stress, i.e.confinement, disturbance, high density, food deprivation or osmotic.An effect of stress on nonapeptides' synthesis and release depends onthe type of stress stimuli. MEL, synthesised mainly in pineal organ, is awell-known scavenger of free radicals and serves as a sensitive, first-line defensive molecule in the protective mechanism of organisms.Thus alterations in pineal and plasma MEL levels could be usefulbiochemical markers of exposure to stress. A role of AVT, IT andMEL instress axis of fish has been suggested. AVT, IT and MEL can beconsidered as hormonal markers of the internal state of the individualand thus seems to be good candidates for welfare indicators.

doi:10.1016/j.cbpa.2010.06.050


13.First attempts in evaluating acidification effects on physiologicalresponses in Mytilus galloprovincialis

A. Chinellato, M. Munari, V. Matozzo, M. Bressan, M.G. Marin(University of Padova, Italy)

Increasing CO2 atmospheric concentration produced by humanactivities is responsible for both global warming and progressiveacidification of oceans. Ocean acidification may affect calcareousstructures of organisms, and modify their physiological performance,in particular survival, growth, feeding, and physiological pH. Adults ofthe mussel Mytilus galloprovincialis were used to investigate thecumulative effects of pH, temperature, and salinity, as predicted inpossible climate change scenarios, on physiological parameters, such asclearance and respiration rates. An experimental flow-through systemwas setup to test simultaneously effects of different temperatures (22and 28 °C) and pH (8.1, 7.7, and 7.4). Three experimentswere performedat 28, 34, 40 psu salinity, and physiological responses of mussels weremeasured after 7 days exposure. At 28 psu, the highest temperature(28 °C) affected negatively mussel clearance rate, at each pH valuetested. Respiration rate increasedwith decreasing pH, reaching a peak at7.4 pH and 28 °C. At 34 psu,mussels exposed at 7.7 pHand22 °C showedamarked increase in clearance rate, respiration rate remainingat controlvalues. An opposite situation was observed at 28 °C. Increasedrespiration rates were also observed at 7.4 pH and 28 °C. Lastly, at40 psu, the highest clearance rate was recorded at 8.1 pH and 22 °C,whereas the highest respiration rate was observed at 7.4 pH and 28 °C.Overall, results obtained demonstrated that among salinities, 28 and40 psu seem to discriminate better differing experimental conditionstested, the former in termsof clearance rate, the latter of respiration rate.Acknowledgements: This work was funded by the Italian Ministry forEnvironment, Land and Sea (IMELS) in the frame of the CIRCLE-MEDProject “The integrated impacts ofmarineacidification, temperature andprecipitation changes on bivalve coastal biodiversity and fisheries: howto adapt (ACIDBIV)”.

doi:10.1016/j.cbpa.2010.06.051

14.Combined effects of temperature, salinity and pH on immuneparameters in the clam Chamelea gallina

M. Munari, A. Chinellato, V. Matozzo, M. Bressan, M.G. Marin(University of Padova, Italy)

The role of haemocytes in immune responses in bivalves is widelydemonstrated. Immunosuppression due to stressful environmentalconditions can strongly affect survival and growth of marine bivalves.In a global climate change scenario, the evaluation of possible effectsof variations in environmental parameters on immune responses ofbivalves deserves particular attention. In this context, adults of theclam Chamelea gallinawere used to investigate the cumulative effectsof pH, temperature, and salinity on clam haemocyte parameters [totalhaemocyte count (THC), endocytotic activity, and haemolymphlysozyme activity]. An experimental flow-through system was setupto test simultaneously effects of different temperatures (22 and28 °C) and pH (8.1, 7.7, and 7.4). Three experiments were performedat 28, 34, 40 psu salinity, and immune parameters of clams weremeasured after 7 days exposure. At 28 psu, THC decreased markedlywith increasing temperature at each pH values. Endocytotic activityincreased with increasing temperature and decreasing pH. At 34 psu,THC increased with increasing temperature and decreasing pH,except at 7.4 pH. Endocytotic activity showed the highest values at

7.7 pH and 28 °C, and 7.4 pH and 22 °C. At 40 psu, both THC andendocytotic activity decreased markedly with increasing temperatureand decreasing pH. Overall, results obtained demonstrated that bothimmunomarkers allow to highlight stress conditions in clams, andindicate differing immunomodulation patterns, on the basis of theexperimental conditions tested at least.

Acknowledgements: This work was funded by the Italian Ministryfor Environment, Land and Sea (IMELS) in the frame of the CIRCLE-MED Project “The integrated impacts of marine acidification,temperature and precipitation changes on bivalve coastal biodiversityand fisheries: how to adapt (ACIDBIV)”.

doi:10.1016/j.cbpa.2010.06.052

15.HSP70 response in Mediterranean mussels exposed to theantibiotic oxytetracycline

S. Buratti, S. Franzellitti, P. Valbonesi, E. Fabbri (CIRSA, University ofBologna, Ravenna, Italy); L. Boatti, A. Viarengo (University ofPiemonte Orientale, DiSAV, Italy)

Heat shock proteins (HSP) are highly conserved intracellularproteins expressed in all living organisms from prokaryotes toeukaryotes, and grouped according to their molecular weight (e.g.the HSP70 family). They are also distinguished into constitutiveisoforms (heat shock cognate, HSC) expressed at physiologicalconditions serving as molecular chaperones, and inducible isoforms(heat shock protein, HSP) over-expressed under stressful conditionswith a cytoprotective role. Although the term HSP specifically refersto heat shock, the accumulation of these proteins is not increasedonly by heat. A variety of stimuli increases their concentrations,probably acting through several mechanisms; however, proteotoxi-city remains at the moment the sole common factor at the cellularlevel. Pharmaceutical residues were shown to survive conventionalwater-treatment processes, thus contaminate aquatic ecosystems as aresult of very widespread human and veterinary usage. Weinvestigated whether environmental concentrations of oxytetracy-cline (OTC) may represent a stress factors for mussels Mytilusgalloprovincialis and elicit the HSP response. OTC is a broad-spectrumantibiotic active against a wide variety of bacteria, which works byinterfering with the ability of bacteria to produce essential proteins.Its presence in the marine environment has been recently reported.Mussels exposed at 16 °C for 4 days to 5 concentrations of OTC (from0.1 to 1000 μg/L) showed an increase in the inducible HSP70 proteinexpression, with a maximum effect of about 185% vs control valuesreached at 1 μg/L OTC and slightly decreasing thereafter. Theconstitutive isoform expression was not changed after OTC exposure.Further effects at 20 and 24 °C are currently under investigation.

The present research is carried out in the framework of 7FP-EUMEECE.

doi:10.1016/j.cbpa.2010.06.053

16.Are complex approaches a real advantage in the assessment ofecotoxicological effects in real scenarios?

L. Guilhermino (ICBAS-Institute of Biomedical Sciences of Abel Salazar,University of Porto, Portugal; CIIMAR-Centre of Marine and Environ-mental Research, Laboratory of Ecotoxicology, University of Porto,Portugal); L. Guimarães (CIIMAR-Centre of Marine and EnvironmentalResearch, Laboratory of Ecotoxicology, University of Porto, Portugal)


Assessing the adverse effects of complex mixtures of environ-mental contaminants on autochthonous populations of estuarineorganisms and identifying their causes are difficult tasks for fourmain reasons: (i) the several contaminants present usually interactamong them producing different compounds making difficult toestablish cause–effect relationships; (ii) toxicological interactionsmay occur, including antagonistic actions resulting in non-measur-able effects; (iii) contaminants may decrease the water quality actingindirectly; and (iv) variations in biological parameters in response tonatural fluctuations of both abiotic factors and physiological altera-tions use to act as important confounding factors. Thus, in the lastyears, monitoring approaches including a high number of differenttypes of parameters (e.g. biological endpoints, chemical analysis,abiotic factors, among others) have been used. But, do they bring realadvantages relatively to more simple approaches based only in asmall number of parameters? This question is discussed using twocase studies done in estuaries of the NW Portuguese coast, includingbiological parameters (morphometric parameters, condition indexesand several biomarkers) chemical analysis (metals and PAHs), waterquality variables and other abiotic factors, and multivariate statisticalanalysis.

This study was conducted in the scope of the projects RAMOCS(ERA-AMPERA/0001/2007) EU ERA-Net AMPERA (ERAC-CT2005-016165), and hrough the project RAMOCS (ERA-AMPERA/0001/2007) and CRABSTHEMES, (FCOMP-01-0124-FEDER-007383, fundedby the Portuguese Foundation for the Science and Technology andEU-FEDER funds.

doi:10.1016/j.cbpa.2010.06.054

17.Evaluation of antibiotic and hormonal residues in edible animaltissues and their physiological responses in human

T.A.M. Msagati (Department of Chemical Technology, Faculty ofScience, University of Johannesburg, South Africa)

A variety of antibiotics, antimicrobial agents and hormonalsubstances are used widely in poultry and animal husbandryindustries as prophylactic and therapeutic agents to promote healthand productivity of animals (fattening). In most cases, these drugagents have been reported to be abused by farmers for fattening oftheir animals in which case residues of the same are found in theanimal products available for human consumption, e.g. animal tissueand milk. The mechanism of fattening is believed to be due to theantibiotics' ability to alter the normal, non-pathogenic flora of the gutresulting into changes associated with beneficial effect on digestiveprocesses as well as the utilization of nutrients in feed. For exampleantibiotics added to the animal feed are said to be capable of alteringthe activities of microbes and hence prevent some loss to microbialfermentation thus enhance uptake of nutrients.

However, there are concerns about residues of the antibiotics andtheir metabolites that may get into animal food products such as milkor in the meat products of animals.

A study has been conducted to evaluate the physiological changesassociated with the various drug residues that may be found inanimal products. The results from exposure of a number of hormones,antibacterial and antibiotics on the interaction with plasma proteinsand serum are reported. The drugs investigated include beta lactamsand sulfonamides.

doi:10.1016/j.cbpa.2010.06.055

18.Biotests and biomarkers in model or feral fish: What is the betterapproach to evaluate effects of micropollutants in small streams?

D. Busch, A. Henneberg (University of Tübingen, Germany); R. Triebskorn(University of Tübingen, Transfer Center for Ecotoxicology andEcophysiology, Rottenburg, Germany)

Micropollutants like pesticides, industrial chemicals, pharmaceu-ticals or human care products, are not completely removed by routinetreatment. In 2010, several sewage treatment plants in SouthernGermany will therefore be equipped with an addition of cleaningstage using an activated carbon filter. The focus of our study is thesewage treatment plant Langwiese located at the Schussen streamwhich empties into the Lake Constance. It was our aim to evaluate thesuitability of biotests and biomarker studies in feral (Alburnoidesbipunctatus and Leuciscus cephalus) and model fish (Danio rerio) forthe assessment of the health status of fish in the Schussen river priorto sewage treatment plant extension. For this study, the Argen streamserved as a control. Liver, gill, kidney and gonads of feral fish wereexamined histologically and the hsp70 level of these organs wasdetermined. With the water and the sediment, embryo tests with D.rerio were performed and the hsp70 level was also determined inexposed fish embryos. In all investigated organs of feral fish, thehistopathological studies revealed slight to severe impairment inthose fish sampled at the sites below the sewage treatment plant. Thehsp70 levels in these fish confirmed the impaired health status ofthese fish. The results of the embryotests (survival, heartbeats, andhatching success) and the determination of hsp70 levels in D. rerioshowed only significant difference between upper stream and estuaryof Schussen after exposure to sediment. These preliminary studymade evident, that the health situation of fish in the river Schussen isdifferent upstream and downstream the sewage treatment plantLangwiese. Biomarkers in feral fish were more suitable to elucidatethis difference than responses in the model fish D. rerio.

doi:10.1016/j.cbpa.2010.06.056

19.Mytilus galloprovincialis shell as an indicator of environmentalconditions

B. Hamer (Ruđer Bošković Institute, Center for Marine Research,Rovinj, Croatia); T. Kanduč (Jožef Stefan Institute, Ljubljana, Slovenia)

Fluctuations in environmental conditions (including changingclimate) can produce a broad range of cellular responses andorganisms adaptations. It is therefore necessary to take this intoaccount during the evaluation of the data and results of biotests andbiomarker analyses. Mussel Mytilus galloprovincialis was tested as anindicator of environmental temperature and salinity conditions atinvestigated locations along the entire CroatianAdriatic coast. The δ18Oand δ13C were determined in shells in order to trace geochemicalconditions in water. Obtained values were correlated with measuredseawater temperature and salinity average values over the entireinvestigated period (1998–2007). While salinity is relatively constantin the open sea, with typical values in the northern and southernAdriatic of 37.65±0.40 and 38.37±0.19 psu respectively (derivedfrom average monthly values in the 1998–2007 period), it varies inintertidal zones, estuaries, close to under-sea freshwater springs andduring rainy days in closed lagoons (4–37 psu). At the same time,seawater temperatures showed some intra-seasonal differences inaddition to strong seasonal variation. Salinity and temperaturevariations at investigated locations showed freshwater influx which


was also reflected in lower δ18O and δ13C values of mussel shellcarbonates. According to the measured variables of seawater tem-perature, salinity and δ18O and δ13C, three groups of locations in theinvestigated area (25 locations) can be assigned: those with strong tomedium freshwater influence (9), those with minor freshwaterinfluence (6) and those of the marine environment (10).

doi:10.1016/j.cbpa.2010.06.057

20.Biological effects of palytoxin-like compounds from benthicmicroalga Ostreopsis ovata: A multibiomarkers approach withthe mussels Mytilus galloprovincialis

S. Gorbi, R. Bocchetti, A. Binelli, C. Totti, E. Cucchiari, S. Accoroni,F. Raffaelli, L. Nanetti, A. Vignini, L. Mazzanti, F. Regoli (MarchePolytechnic University, Italy); P. Ciminiello, E. Fattorusso (Universityof Naples Federico II, Italy); S. Bacchiocchi, T. Graziosi, R. Orletti(Regional Veterinary Institute of Umbria and Marche, Italy)

In the Adriatic Sea, the benthic dinoflagellate Ostreopsis ovatacaused intense blooms associated to human intoxication and to deathof marine organisms since 2006. The aim of the present study was anecotoxicological approach to investigate the biological effects inducedby the palytoxin-like compounds, produced by O. ovata, in a typicalmarine sentinel species, the mussel Mytilus galloprovincialis. Musselswere collected along the rocky shores of Conero Riviera (Ancona)during the algal blooms in summer 2009, while in laboratoryconditions organisms were exposed to different algal concentrations(from 10,000 to 200,000 cells l−1), for different periods (from 1 to4 weeks), and at different temperature (18 and 25 °C). The totalcontent of palytoxin-like compounds in the O. ovata cells, wasdetermined by liquid chromatography coupled to mass spectrometry(LC/MS); the presence of the algal toxin in mussels tissues wasindirectly assessed by the mouse bioassay and haemolysis neutraliza-tion assay, while analyzed biomarkers included oxidative stressresponses, cellular alterations, peroxidation processes, nervous im-pulses transmission and physiological indices. As from our resultssignificant alterations of Na+,K+-ATPase activity, immunological andlysosomal responses were observed in mussels exposed to O. ovataboth in field and laboratory experiments, while markers of oxidativestress showed more limited variations. Although several parametersevidenced dose and time dependent variations, mussels exposed to O.ovata at the higher temperature showed a more elevated mortality,confirming a key role of temperature in mediating responsiveness ofthese organisms toward palytoxin-like toxicity.

doi:10.1016/j.cbpa.2010.06.058

21.The slime mold Dictyostelium discoideum as a test organism tomeasure soil pollution effects: From cellular biomarkers toparameters indicative of organism fitness

A. Rodríguez-Ruiz, I. Marigómez (Ekoiz-Berrilur Res Consortium,CBET Res Grp, Dept. Zoology and Animal Cell Biology, University of theBasque Country, Bilbao, Basque Country, Spain); L. Boatti, F. Dondero,A. Viarengo (University of Piemonte Orientale, DiSAV, Italy)

In previous studies, a battery of early response biomarkers(endocytic capability and lysosomal membrane stability) andecotoxicological high level endpoints (cell viability and replication)has been applied in amoebic D. discoideum for toxicity testing offreshwater and soil elutriates. Likewise, the DDDC (D. discoideumdevelopmental cycle) assay has been recently launched for solidphase toxicity testing in soils. Briefly, triggered by starvation, amoebiccells aggregate forming multicellular organisms that undergo adevelopmental cycle, differentiating into slugs and thereafter intofruiting bodies that wait to favorable nutritional and environmentalconditions to release spores that germinate into unicellular amoebas.Adverse environmental conditions can arrest or slow down this cycle,which is tested by the DDDC assay as a measure of the capacity of thisorganism to respond to environmental challenges (fitness). For thispurpose, checkpoints have been identified (aggregation-to-slug, slug-to-culmination and culmination-to-mature fruiting body) and theirprogressing has been quantified by several indices based on directmicroscopical examination and counting. In addition, the success inproducing fruiting bodies is also quantified. Alterations in the lifecycle and/or the output of fruiting bodies would be indicative of soilhealth impairment. In the present investigation, both the elutriateand the solid phase assays were applied to a soil artificially pollutedwith a mixture of Zn, Cd and diesel oil (freshly polluted and afterweathering). Results revealed significant effects in biomarkers as wellas ecotoxicological high level endpoints and organism fitness. It isconcluded that the combination of this multi-approach (elutriatesversus solid phase; from cellular biomarkers to parameters indicativeof organism fitness) is a promising cost effective strategy to identifyand quantify the effects of pollutants, even in mixtures, in soils.

Acknowledgements. Funded by Basque Gov (ETORTEK BERRILUR II;CTP UE09+/58).

doi:10.1016/j.cbpa.2010.06.059


27th Congress of the NewEuropean Society ofComparative Biochemistry and Physiology



Session 5: Biomarker in aquatic organisms from protists to top predators

INVITED LECTURE

1.Assessment of toxic effects ofmetal nanoparticles using biomarkersand in vitro toxicity tests

M.P. Cajaraville (Dept. Zoology & Animal Cell Biology, School ofScience & Technology, University of the Basque Country, Sarriena z/g,E-48940, Leioa, Basque Country, Spain)

Due to the great increase in the use of nanomaterials for a varietyof biomedical, domestic and industrial applications, the expectedinput of nanoparticles (NP) and other nanomaterials in the aquaticenvironment is expected to rise in the following years. In spite of this,there is only limited information on the fate, distribution and toxicityof NPs to aquatic organisms. In the framework of the EU-fundedproject Nanoretox, the aims of our research group were to investigatethe bioavailability of metal NPs in mussels Mytilus galloprovincialisand zebrafish Danio rerio and to determine their possible adverseeffects on the same target organisms. For this, a two-tiered strategywas developed combining both in vitro and in vivo studies. In vitrotechniques provide a quick and reproducible tool for the screening ofnanoparticle toxicity. In step 1, cytotoxicity of a variety of NPs at awide range of concentrations was tested in isolated musselhemocytes and gill cells using neutral red uptake and MTT assaysand LC50s were calculated. In step 2, sublethal concentrations belowthe LC25 were selected to investigate in vitro uptake and reactivity ofNPs and to discover putative mechanisms of toxicity in both celltypes. In vivo studies with mussels comprised short-term 1–3 daysexperiments to determine bioavailability and lysosomal membranestability as a general indicator of health (step 1) and 21 daysexperiments to assess adverse effects using a battery of molecularand cellular biomarkers (step 2). Similarly, studies with zebrafishstarted with short-term embryo toxicity tests (step 1) followed by21 days experiments (step 2) in case significant toxicity was found instep 1. The developed strategy will be illustrated with results






obtained with CuO NPs, in comparison with bulk CuO and ionic Cu,and results obtained with different TiO2 NPs compared to thecorresponding bulk forms.

Funded by EU 7th FP (project Nanoretox), Spanish Ministry ofScience and Innovation (project Nanocancer) and Basque Govern-ment through a grant to consolidated research groups (GIC07/26-IT-393-07).

doi:10.1016/j.cbpa.2010.06.061

ORAL PRESENTATIONS

2.Inter- and intraspecies differences in biomarker responses andcontaminant levels in two mysticete species (Balaenopteraphysalus and Balaenoptera edeni) of Gulf of California (Mexico)and Mediterranean Sea

M.C. Fossi, S. Maltese, L. Mazzi, D. Coppola, S. Casini, C. Panti, L. Marsili(University of Siena, Italy); J. Urban, C. Torres (Universidad Autónomade Baja California Sur, Mexico); L. Rojas-Bracho (Instituto Nacional deEcologia, Mexico); B. Jimenez, J. Muñoz (CSIC, Spain)

Inter- and intra-species differences in biomarker responses andcontaminant levels were investigated in twomysticete species, finwhale(Balaenoptera physalus) and the unexplored species Bryde whale(Balaenoptera edeni) of Gulf of California (Mexico) and fin whale ofMediterranean Sea, using skin biopsy as diagnostic tool. We developed anon lethal “multi-trial-biomarker-tool”, combining protein (western blotof CYP1A1, CYP2B) and gene expression biomarkers (qRT-PCRof CYP1A1,ER, AhR, and E2F-1) with analysis of OCs, PAHs and PBDEs. In the firstphase of the project we explored the level and effects of contaminants inskin biopsies of the two species of Gulf of California in comparison to finwhale ofMediterraneanSea. In the second, in-vitro, phaseweapplied this



approach to fin whale biopsy slices treated with mixtures of OCs andPBDEs in order to explore the different toxicological effects ofcontaminants. This “multi-trial-diagnostic-tool”, applied to skin biopsies,underlined differences in contaminant levels and biomarker responsesbetween the two species ofGulf of California andalsobetween the two finwhale populations. Higher levels of OCs and PAHs were detected in thezooplankton-eating species (fin whale) in comparison to the fish-eatingspecies (Bryde whale); on the opposite, higher levels of CYP1A1 andCYP2B were detected in the fish-eating species, similar to odontocetespecies. The interspecies investigation showed the presence of a higher“toxicological stress” in the Mediterranean fin whale population, high-lightedbywarning signals suchasCYP1A1 induction andup-regulationofERα and E2F-1 genes, combined with a lack of CYP2B induction in fieldand in vitro experiments.

doi:10.1016/j.cbpa.2010.06.062

3.Non-vertebrate animals have only one gene of the p53 familywhich is most similar to vertebrate p63

M. Štifanić, A. Baričević, R. Batel (Ruđer Bošković Institute, Croatia)

Genes of the p53 family are known to be critical regulators of thecell cycle. They have already been established as possible biomarkers.Elaborate regulation mechanisms result in numerous mRNA andprotein isoforms being expressed from each gene of the family. Arelatively recent discovery of p53 paralogs (p63 and p73) and highsimilarity of isoforms expressed from all three p53 family genescaused confusion in their nomenclature in non-vertebrate species.We analyzed all molluscan p53-similar sequences found in theGenBank database and compared their sequences and genomicstructures to p53 family genes from other animals. Our results leadto the conclusion that invertebrates have only one gene of the p53family which radiated into three genes only in vertebrates. Thisancestral (molluscan) p53 family gene shows to be most similar top63 in vertebrates. Furthermore, our analyses indicate p53 familygenes to be single-copy genes thus increasing their value as potentialbiomarkers.

doi:10.1016/j.cbpa.2010.06.063

4.Pathogenic infection attenuates the response of the estrogenicbiomarker, vitellogenin, in rainbow trout

R. Burki, T. Wahli, H. Segner (University of Bern, Switzerland);P. Burkhardt-Holm (University of Basel, Switzerland); C.E. Rexroad,S. Afanasyev, M. Antikainen, A. Krasnov (AKVAFORSK, Norway)

In the environment, aquatic organisms are exposed multipleinstead of single stressors. During recent years, considerable progresshas been achieved in assessing combination effects of chemicalmixtures. For instance, it has been demonstrated that the induction ofvitellogenin (VTG) in (male) fish – an established biomarker ofexposure to estrogen-active compounds – responds additively tocombinations of estrogen-active compounds, while simultaneousexposure to ligands of the estrogen receptor and of the arylhydro-carbon receptor has an antagonistic effect on VTG induction. Incontrast to the progress in understanding combination effects ofchemical mixtures, approaches to assess the combined effects ofchemical and physical and/or biological stressors are less developed.Here, we study the combined impact of a chemical and a biological

stressor on the biomarker VTG in rainbow trout. To this end, juvenilefish were exposed to two doses of the prototypic estrogen-activecompound, 17-beta-estradiol (E2) in combination with the fish-pathogenic parasite Tetracapsuloides bryosalmonae, the etiologicalagent of the proliferative kidney disease (PKD). The VTG responsewas assessed at the mRNA level using qRT-PCR; in parallel, the livertranscriptomic response to the single stressors and their combina-tions was examined. Combined chemical/biological exposure resultedin a significant inhibition of both the rate and the magnitude of theVTG response, while neither the parasite nor the fish response to theparasite was affected by the presence of E2. The observed attenuationof the VTG biomarker response in diseases may have implications forthe utility of this marker in environmental scenarios with lowestrogenic exposure.

doi:10.1016/j.cbpa.2010.06.064

5.Assessing the effects of the selective serotonin reuptake inhibitorfluoxetine on Carcinus maenas using locomotor behaviour andbiomarkers as effect criteria

S.R. Mesquita, L. Guilhermino, L. Guimarães (University of Porto,CIIMAR/ICBAS, Portugal)

The serotonin reuptake inhibitor drug fluoxetine is currently found ineffluent and surface waters. Considering its antidepressant and othereffects in humans, biochemical, physiological and behaviour alterationsare also expected to occur inwild organisms exposed to the substance. Inthe present study, intermoult Carcinus maenas were exposed for sevendays to five fluoxetine concentrations ranging from 0.49 to 750 μg/L in alaboratorial bioassay. The following endpointswere assessed at the end ofthe test: spontaneous locomotor behaviour (SLB) using the classical open-field test; epidermis chitobiase activity (CTB), muscle cholinesterase(ChE), lactate dehydrogenase (LDH) and NADP+-dependent isocitratedehydrogenase (IDH) activities; the activities of the enzymes glutathioneS-transferases (GST), glutathione peroxidase (Gpx), glutathione reductase(GR), and the levelsof total glutathiones (TG)and lipidperoxidation (LPO)in the hepatopancreas. Animals exposed to 120 and 750 μg/L spentsignificantly more time moving (TM) in the open-field and crossed ahigher number of squares (NS) than controls. ChE, GST and GR activities,andTG levels,werealso significantly increased in crabsexposed to120and750 μg/L. Interestingly, significant correlations were found between TMand ChE (38.6%), TM and TG (31.7%), NS and ChE (47.1%), and NS and TG(34.4%). These results show that SLB was a sensitive endpoint regardingfluoxetine exposure, suggesting that bioassays based on itmay be suitabletools to assess the toxicity of environmental contaminants to C. maenas.This work was supported by national-FCT and EU-FEDER funds throughthe project CRABTHEMES (FCOMP-01-0124-FEDER-007383).

doi:10.1016/j.cbpa.2010.06.065


6.A non-lethal multi-biomarker approach to investigate theecotoxicological status of Mediterranean loggerhead sea turtle(Caretta caretta, Linneo, 1758)

S. Casini, I. Caliani, L.Marsili,M.Giannetti, S.Maltese, S. Ancora,N. Bianchi,C. Panti, T. Campani, L. Carletti, D. Coppola,M.C. Fossi (University of Siena,Italy); A. Cañadas (Alnitak, Spain); M. Parga (Submon, Spain)


In this study we present a non-lethal multi-biomarker metho-dological approach developed and applied to study the ecotoxico-logical status of the Mediterranean population of loggerhead seaturtle (Caretta caretta, Linneo, 1758). In the last 8 years more than120 specimens of C. caretta were sampled for blood, skin biopsiesand carapax sub-samples in several Italian rescue centers and duringa survey in Spain. Blood lymphocytes and skin biopsy slices treatedwith different contaminants (OCs, PAHs and PBDEs) showedinduction of cytochrome P450 1A (WB) and this biomarker ofexposure to liposoluble contaminants was subsequently applied toC. caretta sampled in the different areas. Oxidative stress responseswere evaluated on plasma samples by developing a plasma lipidperoxidation (LPO) test which showed a correlation to CYP1A.Potential genotoxic effects were investigated by developing cometassay and ENA (erythrocytic nuclear abnormalities) test in wholeblood samples. Biomarkers of hepatic stress such as ALT AST and γ-GT were measured in plasma. Possible estrogenic effects wereinvestigated by measuring plasma levels of vitellogenin by ELISAand WB, including investigation of females during eggs deposition.Butyrylcholinesterase activity was detected in plasma as biomarkerof neurotoxicity. The possible relation between biomarkers andcontaminant levels in blood (PAHs and OCs) and carapax (traceelements) was investigated. Biomarker and contaminant levelresults were also elaborated by classes of age. The informationobtained in this research represents a strong contribution for thedevelopment of a complete protocol of analysis for the monitoringand protection of this endangered species, highly stressed inMediterranean Sea.

doi:10.1016/j.cbpa.2010.06.066

7.Characterization of cholinesterase activity in different tissues ofAnguilla anguilla

P. Valbonesi, M. Mattioli, T. Rossi, F. Brunelli, E. Fabbri (Interdept.Centre for Environmental Science Research, University of Bologna,Ravenna, Italy)

European eel (Anguilla anguilla) is in a dangerous state of decline.The causes are still largely unknown, however pollution wasaddressed as one of the possible reasons, hampering the extremeperformance eel have to produce during transoceanic migration.Sublethal exposure to anti-cholinesterase (ChE) chemicals may affectneurotransmission, respiration, feeding, etc., ultimately compromis-ing a successful migration. Inhibition of ChE activity would be asuitable biomarker for assessing early exposure to pesticides orherbicides, and may be used for diagnosis to evaluate pollution-related pathophysiological alterations. We characterized the ChEactivity in different tissues of A. anguilla in the presence of acetyl-(ASCh), proprionyl- (PSCh) and butyryl- (BSCh) thiocholine sincedifferent enzyme forms may be present, i.e. acetylcholinesterase(AChE), and the pseudocholinesterases proprionyl- (PChE) andbutyryl- (BChE) cholinesterase. The highest ChE activity was foundin the presence of ASCh in brain, muscle and liver (about 66.5, 41.7and 9.49nmol min−1mg protein−1, respectively), followed by PSChand to a lesser extent by BSCh. ASCh hydrolysis was significantlyinhibited by the specific inhibitor eserine at nM concentration in alltissues. The presence of different ChEs in tissues from A. anguilla wasconfirmed by using selective inhibitors for AChE (BW284c51) andBChE (iso-OMPA). BW284c51 strongly reduced ASCh hydrolysis,while iso-OMPA was without effect in all cases, indicating that AChEis the predominant ChE form in these tissues. Further experimentsare in progress to characterize ChEs in eel plasma, that can be used as

a nondestructive means of predicting adverse effects in living animalspotentially exposed to pesticides or herbicides.

doi:10.1016/j.cbpa.2010.06.067

8.Biochemical biomarkers in Nile tilapia (Oreochromis niloticus)exposed to diesel and biodiesel

L. Nogueira, A.L.M. Sanches, V.C. Ferrizzi, A.B. Moreira, E.A. Almeida(IBILCE, São Paulo State University, Brazil)

Biodiesel production has increased around the world as analternative renewable fuel for petroleum-derived diesel. Apart fromeconomical advantages, it can be supposed that biodieselwould be lesstoxic to different environments compared to petroleum diesel, sincemost of the carcinogenic polycyclic aromatic hydrocarbons (PAHs) areabsent in biodiesel. However there are scarce works concerned to testthe effects of biodiesel in aquatic species. In this work we wereinterested to compare the responses of classical biochemical biomar-kers (the activities of EROD, GST, SOD, CAT, GPx, and lipid peroxidationlevels) in gills and liver of Nile tilapias exposed to 0.01 and 0.1 mL/L ofpure petroleum diesel (B0), pure biodiesel (B100) and petroleumdiesel mixed to 5 and 20% of biodiesel (B5 and B20). The level of 1-hydroxypyrene in the bile was also evaluated. EROD activity wasinduced in a dose–response manner according to increasing propor-tion of petroleum diesel in the mixture, while GST decreased in fishexposed to B100. SOD and CAT were unchanged in gills, but showed acomplex response in liver according to treatment, which was alsoobserved for lipid peroxidation levels andGPx activity in both liver andgill. Indeed, fish exposed to 0.01 mL/L of B5 and B20 presented higherlevels of 1-hydroxypyrene in the bile compared to those fish exposedonly to B0, suggesting that the presence of biodiesel on petroleumdiesel increases the rate of PAH absorption in fish, or the rate of itsexcretion in the bile. (Financial support: FAPESP-2006/03873-1).

doi:10.1016/j.cbpa.2010.06.068

9.MICA expression as toxicological stress marker in fibroblast cellcultures of cetaceans

L. Marsili, S. Maltese, L. Carletti, D. Coppola, S. Casini, M.C. Fossi(University of Siena, Italy)

The skin is an important tissue of the immune systemand contributesto this biological structure by acting not only as a protective physicalbarrier, but also as a target for immune components thatmount the initialdefense against invading pathogens, noxious stimuli, and residentneoplastic cells. However, little is known about immune cells in dolphinskin. The genes encoding MICA and MICB are found within the MajorHistocompatibility Complex. Although MIC products have been found invarious cells/tissues, the current consensus is that MIC genes are mainlyexpressed in gastrointestinal epithelium, endothelial cells and fibroblasts.MIC molecules are considered to be stress-induced antigens that arerecognized by cytotoxic Tcells and natural killer (NK) cells, which play animportant role in the surveillance of transformed infected and damagedcells. The aimof this studywas to evaluate theMICAprotein expression infibroblast cell cultures of cetaceans (skin biopsies of free-rangingspecimens and skin samples of stranded cetaceans dead from 12–24 h).Here we present the immunofluorescence technique in culturedfibroblasts used for qualitative and quantitative evaluations of MICAexpression, induced by treatment with organochlorine compounds


(DDTsandPCBs), polybrominateddiphenyl ethers (PBDEs)andpolycyclicaromatic hydrocarbons (PAHs), as toxicological stress marker of theimmune system of different species of cetaceans odontocetes (Stenellacoeruleoalba, Delphinus capensis, Physeter macrocephalus) and mysticetes(Balaenoptera physalus, Balaenoptera edeni).

doi:10.1016/j.cbpa.2010.06.069

10.Impact of pollution on the development of glass eels (Anguillaanguilla) entering three NW Portuguese estuaries

L. Guimarães, C. Gravato, J. Santos, M. Faria, A. Alves (University ofPorto, CIIMAR, Portugal); L. Guilhermino (University of Porto, CIIMAR/ICBAS, Portugal)

European eel (Anguilla anguilla) stocks have strongly decreased overthe past decades, culminating with the inclusion of this species in theHELCOM Red list and the CITES Convention, as protection measures.Exposure to pollution has been indicated as one possible cause for thisdecline. The present study investigated the physiological and healthstatus of glass eels, at their entrance and after five months ofdevelopment in estuaries with different levels and types of contamina-tion. The Fulton's condition index and several biomarkers involved inphysiological processes vital for the survival and/or performance of theanimals were used to evaluate their health condition: the activities ofthe enzymes cholinesterases (ChE), lactate dehydrogenase (LDH),sodium/potassium ATPase (Na+/K+-ATPase), glutathione S-trans-ferases (GST), the total (TG), reduced (GSH) and oxidised glutathione(GSSG) levels, and theextent of lipid peroxidation (LPO). Glass eelswerecollected during November 2005 andMarch 2006 in theMinho estuary(reference), and the polluted Lima and Douro estuaries. Several waterquality parameters were also measured (e.g. temperature, dissolvedoxygen, salinity, conductivity, pH, and nutrient concentrations). Aredundancy analysis (RDA) was used to integrate all data. The resultsindicate that glass eels arriving to the three different estuaries were atsimilar physiological and health conditions. However, after 5 monthsdeveloping in the polluted estuaries they were already smaller thanthose from the reference estuary, and exhibited a decline in biomarkersof neurotransmission, energy metabolism, and antioxidative defences.The RDA allowed depicting patterns of biological effects and theinfluence of water quality parameters on these responses.

Work supported by national-FCT and FEDER-EU funds through theproject EELEANORA (POCTI/BSE/47918/2002).

doi:10.1016/j.cbpa.2010.06.070

11.Evaluation of the effects of nonylphenol in fourth-instar larvae ofChironomus riparius (Diptera)

P. Martinez-Paz, M. Morales, J.L. Martínez-Guitarte, G. Morcillo(Biología y Toxicología Ambiental, Facultad de Ciencias UNED,Madrid, Spain)

4-Nonylphenol (NP) is a breakdown product of nonylphenolethoxylates (NPE), used as surfactants in the industry. They appear insediments and waste water. NP mimics the action of the femalehormone estrogen in mammals and there are different studies thatreport chronic and acute effects of it in aquatic organisms. The midgeChironomus is widely used in aquatic toxicology as an appropriate testspecies. The present study was undertaken to determine the earlymolecular and subcellular effects of NP in Chironomus riparius larvae.

The effects of a wide range of NP treatments (at 24h and differentconcentrations) were examined in the expression of selected genes.Transcription levels were analyzed by semi-quantitative RT-PCR. Theanalysis included stress-induced genes, such as HSP70, and hormone-related genes, such as the ecdysone receptor gene (EcR) andultraspiracle (Usp). Furthermore, the activity of detoxificationenzyme Glutathione-S-transferase (GST) was evaluated. Our studyprovides evidence for selective changes induced by NP exposure. Themost remarkable ones were an upregulation of stress-related genesand an increase of the GST activity. In contrast, EcR and Usp do notshow transcriptional changes.

Acknowledgements: Funded by MICINN, Spain, Project ref:CTM2009-07189.

doi:10.1016/j.cbpa.2010.06.071

12.Application of a biomarker–transcriptomics approach utilizingmussels in the evaluation of the water quality in a marine coastalarea of Medio Campidano (Sardinia, Italy)

A. Negri, F. Dondero, A. Dagnino, L. Boatti, S. Sforzini, F. Capri, C. Oliveri,A. Viarengo (University of Piemonte Orientale, DiSAV, Italy); L.A. Chessa(ISE-CNR, Istituto per lo studio degli Ecosistemi, Sassari, Italy)

A systems toxicology approach based on combination of biomar-kers and transcriptomics analyses was used to assess the occurrenceof a stress syndrome in mussels caged in proximity of a decommis-sioned mine site in marine coastal area of Medio Campidano(Sardinia, Italy). Mussels (M. galloprovincialis) were caged for 28 daysin front of the Piscinas stream, supposed to drain pollutants from theabandoned mine sites (PIS), and in a reference site (CTR). The healthstatus of transplanted mussels was determined involving a battery ofnine biomarkers (stress, exposure and genotoxicity biomarkers) andapplying an Expert System of Classification. Gene expression profileswere evaluated using a cDNA-microarray encompassing 1.7K se-quences. The expression of mt10 and mt20 genes was furtherinvestigated in quantitative PCR analysis. Results showed a mildstress in the animals sampled at PIS: data show significant alterations(Mann–Whitney U-test, p<0.05) in Lysosomal-Membrane-Stability,Neutral-Lipid and Metallothionein content, suggesting a moderatestress induced by heavy metals exposure. The transcriptomic analysisdid not show the modulation of genes involved in detoxificationprocesses, but we found two putative sequences implicated inantibacterial humoral response and other genes involved in devel-opmental processes. RT-Q-PCR data showed a stimulated transcrip-tion of the mt20 gene in mussels from PIS. Finally, data onbioaccumulation indicated very high levels of Pb (12 fold) and othermetals at a lower extent (Fe, Mn and Cd) in the animals sampled atPIS, while PAHs were below the detection levels. This complex ofdata seems to indicate a possible risk for both humans and theecosystem mainly related to the leakage of metals – mainly Pb – fromthe decommissioned site.

doi:10.1016/j.cbpa.2010.06.072

13.Investigating drug-metabolising cytochrome P450 (cyp) isoformsin a fish hepatocyte model

J. Corcoran, A. Lange, C.R. Tyler (University of Exeter, UK); M.J. Winter(Brixham Environmental Laboratory, AstraZeneca, UK)


Pharmaceuticals have been detected globally in waste-watereffluent, surface waters, and in some cases in groundwater anddrinking waters too, and although the concentrations are relativelylow, unlike for other pollutants, these compounds are specificallydesigned to alter physiological function in vertebrates. Many drugtargets are conserved across animal phyla, including the cytochromeP450 enzymes involved in drugmetabolism.With a desire to develop asensitive and robust screen for compounds that modulate cypexpression, we investigated the expression and responses of themajordrug-metabolising cyp isoforms, cyp1a, cyp2k and cyp3a in a primaryhepatocyte culture from common carp (Cyprinus carpio). Constitutiveand a constant level of expression occurred for all three target genes inthe hepatocytes for up to 96h in culture. Induction of cyp1a by β-naphthoflavone (βNF) and cyp2k and cyp3a by rifampicin (RIF) wasconcentration-dependant, and comparable to that reported formammalian hepatocytes. Using a time-course study, maximal induc-tion occurredwith cyp1a after 24 h exposure toβNF 10 μM(∼50-fold);cyp2k and cyp3a after 72 h exposure to RIF 10 μM (∼21-fold and ∼8-fold, respectively). Furthermore, these induction responses for cyp2kand cyp3a were inhibited in a concentration-dependant manner byketoconazole (KET). In contrast, cyp1a induction (stimulated via βNF)was not inhibited at all byα-naphthoflavone (αNF), (which does so inmammalian hepatocytes) and this may represent divergence in cypregulation betweenmammals and fish. So far, our studies are showingthat the fish hepatocyte model is proving to be a sensitive andreproducible system for screening compounds that modulate cypfunction. We are now screening a suite of environmentally-relevantpharmaceuticals to investigate the wider applicability of the testsystem.

doi:10.1016/j.cbpa.2010.06.073

14.Development and application of molecular biomarkers forecotoxicological monitoring in Stenella coeruleoalba (Cetacea,Odontoceti)

C. Panti, G. Spinsanti, L. Marsili, F. Frati, M.C. Fossi (University of Siena,Italy)

Most xenobiotic compounds are widespread in the environmentand can affect animal health at different biological levels since theytend to be persistent in the environment and biota being thereforeaccumulated in the organisms. Mediterranean cetacean odontocetesare potentially at serious to hazard, due to exposure to POPs(persistent organic pollutants), PBDEs (polybromodiphenyl-ethers)and PAHs (polycyclic aromatic hydrocarbons).

In the present study, the response of gene expression biomarkerhas been evaluated in Mediterranean striped dolphin (Stenellacoeruleoalba) skin biopsies or slices of skin biopsies exposed todifferent doses and different mixtures of toxic compounds. The mRNAlevels of five putative biomarker genes (AHR, CYP1A, E2F-1, ER-α, andHSP70) were measured by quantitative Real Time PCR (qRT-PCR). Anaccurate selection of control genes was previously carried out in alltissues analyzed to ensure the reliability of our data. The “gene-expression biomarkers” exhibit a sensitive response in all tissuesanalysed, confirming the molecular approach as a suitable tool in thiskind of studies.

The specimens, coming from two regions of Mediterranean Seaand a contiguous area with different toxicological impacts (PelagosSanctuary, Ionian Sea, and Strait of Gibraltar), were analyzed by qRT-PCR. The response of most biomarker genes tested reflects thecontamination levels of the three regions showing a differentialresponse to different classes of environmental contaminants across

Mediterranean basin. In addition, an in vitro assay was developed toshow the different susceptibilities of cell cultures and slices fromstriped dolphin skin biopsies exposed to different classes ofcompounds.

doi:10.1016/j.cbpa.2010.06.074

15.Ecotoxicological status of Tursiops truncatus in the MediterraneanSea and in the Gulf of California (Sea of Cortez-Mexico) using skinbiopsy as diagnostic tool

S. Maltese, L. Marsili, M. Bartolini, D. Coppola, S. Casini, M.C. Fossi(University of Siena, Italy); R. De Stephanis (CIRCE, ES, Spain);C. Fortuna, S. Canese, G. Lauriano (ISPRA, Italy); D. Holcer (BlueWorld Institute of Marine Research and Conservation, HR, Croatia);J. Urban (University of Baja California Sur, La Paz, Mexico)

The main objective of this study was to apply a sensitive non-lethal “multi-trial-diagnostic-tool” in skin biopsy of common bot-tlenose dolphin (Tursiops truncatus), combining molecular biomar-kers, such as two isoforms of the cytochrome P450 (CYP1A1 e CYP2B)with analysis of lipophilic contaminants, such as hexachlorobenzene(HCB), dichlorodiphenyltrichloroethane (DDTs), polychlorobyphenils(PCBs) and polycyclic aromatic hydrocarbons (PAHs), to evaluate thetoxicological status of this odontocete in 4 Mediterranean areas (Isleof Vis; Isle of Lampedusa; Isle of Asinara; and Strait of Gibraltar) andin the Sea of Cortez (Mexico). These surveys allowed to estimate the“toxicological stress” of the specimens of the different populations, inparticular considering that the Sea of Cortez (Mexico) is a remotearea and therefore exposed to a significantly lower anthropic pressurethan the Mediterranean Sea. The results of this study reveal that thebottlenose dolphin population most affected by OCs and PAHscontamination is the one sampled in the Strait of Gibraltar. In thespecimens of this area the highest levels of CYP1A1 were also found.Lower levels of OCs were found in the specimens sampled in the Seaof Cortez where, however, the highest levels of CYP2B were detected.The lowest average values of PAHs were measured in skin biopsies ofthe specimens sampled in the Adriatic area and in the area of the Isleof Asinara. In conclusion this preliminary data suggest the potentialapplication of the proposed diagnostic approach as a powerfulmethod to define the toxicological status of this marine top predatorand suggest the application of this methodology in the future EUMarine Strategy.

doi:10.1016/j.cbpa.2010.06.075

16.Application of a battery of stress biomarkers to assess the qualityof coastal and estuarine environments on the cosmopolitanmarine protozoa Euplotes crassus (Dujardin, 1841)

A. Gomiero, L. Boatti, A. Dagnino, A. Viarengo (University of PiemonteOrientale, DiSAV, Italy); C. Nasci (Thetis S.p.A, Venezia, Italy)

Estuarine and coastal sediments represent the final destination ofthousands of organic and inorganic pollutants which are continu-ously discharged into the environment as result of industrial,agricultural and domestic activities. It has been observed that thepollutant concentration in sediments and sediment's interstitialwater can be more than 10–100 times higher than the one presentin the overlying water column. Due to the complexity of thisenvironmental compartment sole chemical characterization often


fails to correctly estimate the toxicity of such complex matrix.However, a multi-disciplinary, chemical–biological approach canovercome this lack providing useful information for identifying thosesituations which require a close investigation at an early stage. Onthis context, the possibility of utilizing Euplotes crassus, an interstitialmarine protozoa, was examined by applying a battery of testsincluding cell viability and replication as typical ecotoxicologicalhigh-level endpoints. This study was also addressed to detect moresensitive sublethal early-warning responses utilising biological para-meters such as lysosomal membrane stability and endocytotic rate,which are typical stress biomarkers in eukaryotic cells. The sensitivityof E. crassus assay has been assessed within a range of organic (B(a)pyrene) and inorganic (Hg and Cu) chemical concentrations and theirmixtures. Effects of binary mixtures of chemicals were predictedusing the Concentration Addiction model (CA). Further investigationswere addressed to the effect of three different temperatures onchemicals toxicity.

A field application was also performed aiming to validate theproposed bioassay. Four sampling sites within the Venice Lagoonwere chosen according to expected differences among anthropicpressure levels. The results demonstrate the high sensitivity of E.crassus to the organic and inorganic chemicals and that theseorganisms are suitable for the evaluation of marine sediment toxicity.

doi:10.1016/j.cbpa.2010.06.076

17.Application of Neutral Red Retention Assay in the clam Ruditapesphilippinarum and the crab Carcinus maenas as a screening toolfor sediment quality assessment in marine environment

S. Buratti, E. Fabbri (CIRSA, University of Bologna, Italy); C.D.S. Pereira(Santa Cecilia University, Brazil); J. Ramos-Gómez, T.A. Del Valls,M.L. Martín-Díaz (University of Cádiz, Spain)

Dredged material can cause several negative impacts to theecosystems. Its management is a key issue for the protection of theaquatic environments. Sediment quality guidelines alone are not ableto estimate the potential adverse effects that contaminants bound tosediment can cause. The in situ approach using caged bioindicatorspecies has been chosen lately as a new sensitive tool for theassessment of dredged material. In a tier testing approach, NeutralRed Retention Assay (NRRA) has been applied as a screening tool inorder to detect adverse changes in health status associated tocontaminated environments. Nevertheless, to the authors' knowl-edge, little is known about the application and validation of NRRA insediment bioindicator species and under field conditions.

Caged Ruditapes philippinarum and Carcinus maenas were exposedduring 28 days to 3 potentially contaminated sites in the AlgecirasBay (SW Spain) and to a reference site in the Cadiz Bay (SW Spain).Health status of exposed organisms was measured through NRRA inhaemolymph samples.

No significant differences of Neutral Red Retention Time (NRRT)were measured neither in clams nor in crabs over time in theorganisms from control site (70±10min).

However significant differences (p<0.05) were found at the endof the exposure period in clams and crabs exposed to contaminatedsediments from Algeciras Bay, showing a decrease of NRRTs from 30to 70% compared to the control organisms.

The results obtained confirmed the use of NRRA as a suitable andsensitive method to be used in the assessment of sediment quality.

doi:10.1016/j.cbpa.2010.06.077

18.Biomarkers employment to show no effects of toxic diatoms oncopepods?

R. Minutoli, C. Brugnano (Department of Animal Biology and MarineEcology, University of Messina, Italy); S. Casini, G. Mori, G. Zagami(Department of Environmental Sciences “G. Sarfatti”, University ofSiena, Italy)

A new interesting research topic is the effect of toxic algae oncopepods. There are in literature many evidences of reduction in eggproduction and viability, in fecundity and hatching success, withabortions, birth defections, abnormal teratogenic nauplii, poor post-embryonic larval development and high mortality, in these crusta-ceans fed on toxic diatoms. It was already demonstrated that somephytoplanktonic species produce during digestion several polyunsa-tured aldehydes (PUA), secondary metabolites, used as chemicaldefence against predation. Our previous and current studies carriedout on the benthopelagic calanoid copepod Pseudocyclops xipho-phorus collected in Faro Lake, Messina, South Italy, showed that thisspecies is very less sensitive to diatom secondary metabolites thanother tested copepod species reported in literature. In this study weinvestigated the hypothesis of implication of Mixed FunctionOxidases System (MFO) in P. xiphophorus in detoxification processes,that transform aldehydes and possibly decrease their toxic effect. Theresults of a Western Blot analysis showed an induction of CYP1A-likeprotein in P. xiphophorus specimens fed on the toxic diatomSkeletonema marinoi. The benzo[a]pyrene monooxygenase test(BPMO) showed a higher activity in P. xiphophorus fed on S. marinoiwith respect to the control sample.

doi:10.1016/j.cbpa.2010.06.078

19.Battery of biomarkers (GST, GPx, CAT and AChE): A standardizationof sampling procedures in mussels, Mytilus galloprovincialis

L. Vidal-Linan (Universidade de Vigo, Spain); J. Bellas (InstitutoEspañol de Oceanografía, Centro Oceanográfico de Vigo, E-36200Vigo, Spain); R. Beiras (Universidade de Vigo, Spain)

In recent years molecular biomarkers have been incorporated tomarine pollution monitoring programmes. Biomarkers are impor-tant tools for revealing biological effects of contaminants in marineorganisms. These sub-organismic changes occurring at cellular,biochemical, molecular, or physiological levels, provide earlywarning signals of significant biological effects preceding those thatoccur at higher levels of biological organization such as population,community or ecosystem. However, such biological responses can beinfluenced by natural factors and this basal variability needs to bedistinguished from the effects caused by pollutants. Therefore, theaim of this work was to standardize an appropriate samplingprocedure for a battery of biomarkers in a wild population ofmussels (Mytilus galloprovincialis), dealing with various relevantaspects such as: (a) the calculation of minimum sample sizerequired to estimate the population mean with a fixed error marginat a given confidence level; (b) the selection of the specific organ ortissue where the enzymatic activity is higher for each biomarker; (c)the influence of the tidal height on the basal enzymatic activity; and(d) the effect of temperature on each enzymatic activity. A suite ofbiomarkers, among the most widely used, including antioxidantenzymes: Catalase (CAT) and Glutathione peroxidase (GPx); a phaseII detoxification enzyme: Glutathione S-Transferase (GST), and the


neurotransmitter catabolism enzyme Acetylcholinesterase (AchE),were considered for this work.

doi:10.1016/j.cbpa.2010.06.079

20.Expression of VTG and CYP1A biomarkers in Zosterisessorophiocephalus sampled in different areas of the Venice Lagoon

E. Negrato, F. Pascoli, D. Bertotto, G. Radaelli (University of Padua,Italy); F. Maradonna, G. Gioacchini, O. Carnevali (Polytechic Universityof Marche, Italy)

The Venice Lagoon is a transitional environment on the Adriaticcoast influenced by such human activities as agriculture, industry andtourism. Several studies carried out in the last years have demon-strated the presence of a wide range of contaminants in the VeniceLagoon. Biomonitoring in the lagoon environment plays an importantrole in strategies and actions to identify, control and reduce theenvironmental threats. Biomarkers monitored in this study are VTG

(Vitellogenin) and CYP1A. VTG is produced in the liver of sexuallymature female fish in response to endogenous estrogens. CYP1Arepresents the most intensively studied P450 gene in fish and itsexpression is induced by a wide variety of lipophilic contaminants. Inthe present study we used Western blot and Real Time PCRtechniques to detect the expression of VTG and CYP1A in plasmaand liver of a goby fish, Zosterisessor ophiocephalus, sampled indifferent areas of the Venice Lagoon, possibly interested by differentpollution entities. Our results evidenced that VTG expression wasdetectable in adult male of Z. ophiocephalus collected from thedifferent sites evidencing high toxicological risk probably due toendocrine disrupting chemicals. Moreover, the highest levels ofCYP1A were observed in animals sampled at the Porto Marghera siteshowed that this site, which is influenced by the presence of theindustrial area and the city of Venice, is the most highly impactedarea of the lagoon. VTG and CYP1A induction in fish can be usefulbiosensors for environmental pollution monitoring.

doi:10.1016/j.cbpa.2010.06.080





Session 6: Cell signalling and endocrine disruption

INVITED LECTURE

1.Endocrine disruption in mussels: Approaches and perspectives

C. Porte (Environmental ChemistryDept., IDAEA–CSIC, Barcelona, Spain)

It is now generally accepted that invertebrates are susceptible tothe action of endocrine disrupters. However, progress on under-standing the endocrine disruption phenomena has been hamperedby the lack of detailed knowledge on invertebrate endocrinology.Focusing on mussels, although key steps of steroidogenesis leadingto androgens or estrogens have been described, the role of steroidhormones is still under debate and the knowledge of the endocrinesystem is fragmentary. Therefore, identifying endocrine alterationsas a consequence of exposure to pollutants is not an easy task. Thiswork will focus on the marine mussel Mytilus galloprovincialis andthe zebra mussel Dreissena polymorpha, and will show annualvariations of sex steroid levels (free and esterified) along thereproductive cycle of D. polymorpha and their relationship withdifferent stages of sexual maturation. The activity of key enzymaticpathways involved in steroid synthesis, such as 17β-hydroxysteroiddehydrogenases (17β-HSDs) that catalyze the formation of testos-terone from androstenedione and acylCoA: testosterone acyltrans-ferases (ATAT) that catalyze the esterification of testosterone withfatty acids will also be assessed along the reproductive cycle. Finally,some evidence of steroid metabolism modulation in musselsfollowing exposure to environmental relevant concentrations oftestosterone and estradiol as well as pollutants such as nonylphenoland tributyltin, will be discussed.

doi:10.1016/j.cbpa.2010.06.082

ORAL PRESENTATIONS

2.Alteration of gene expression by UV-filters ethyl-hexyl-4-trimethoxycinnamate (EHMC) and benzophenone-4 (BP4) inzebrafish (Danio rerio) determined by microarrays and qPCR

S. Zucchi, A. Ieronimo (University of Applied Sciences NorthwesternSwitzerland (FHNW), School of Life Sciences, Muttenz, Switzerland);K. Fent (University of Applied Sciences Northwestern Switzerland(FHNW), School of Life Sciences, Muttenz, Switzerland); (SwissFederal Institute of Technology (ETH), Department of EnvironmentalSciences, Zürich, Switzerland)

UV-filters are heavily used in sunscreens, personal care productsand in the protection of materials, but still little is known about theiradverse effects to aquatic life. Several UV-filters detected in surfacewaters showed hormonal activities in vitro, but their moleculareffects remain elusive. The elucidation of mechanisms of action onthe sex hormone system (e.g. steroidogenesis) is of major interestdue to consequences on fertility and reproduction. In our study weanalysed the molecular effects of two major UV-filters, ethyl-hexyl-4-trimethoxycinnamate (EHMC) and benzophenone-4 (BP4) in zebra-fish (Danio rerio) adults and in larval stages. Global gene expressionanalysis was performed in zebrafish whole body using microarraysand selected genes were analysed by qRT-PCR analysis. The effects ofBP4 was analysed by qPCR in several tissues of adult male fish and infish larvae. Most of the target genes, including vitellogenin 1,vitellogenin 3, ERalpha, ERbeta1, ERbeta2, hydroxysteroid-17β dehy-drogenase-3, androgen receptor, cyp19a and cyp19b were altered byboth UV-filters. Genes involved in steroidogenesis altered in wholebody (microarray data) were also found by qPCR to be altered in liver,brain and testis of adults and larvae. Gene expression alterationsvaried in different tissues; however, we observed a relationshipamong mRNA responsiveness of selected genes in the whole body








with liver, testis and brain, suggesting that microarray results reflectthe overall alteration of gene expression based on individualcontributions of different tissues. Some similarities in gene expres-sion alteration were observed with EHMC and BP4. The resultsobtained from the microarray and qPCR analysis indicate that bothUV-filters alter hormonal pathways in zebrafish.

doi:10.1016/j.cbpa.2010.06.083

3.Differential sensitivity of mussel tissues to pharmaceuticalexposures: Putative relationship with specific pathways ofinteraction

S. Franzellitti, S. Buratti, E. Fabbri (Universityof Bologna, Italy);A. Capuzzo(University of Ferrara, Italy)

Propranolol is a beta-adrenergic receptor-blocker widely used incardiovascular diseases therapies, and detected in aquatic systems atconcentrations up to 56 ng/L. Due to similarities between human andother vertebrate beta-adrenergic receptors, consequences of beta-blocker exposure in vertebrate wildlife, such as fish may be expected.Instead, little is known about the presence and function of thesereceptors in invertebrates, and possible effects of beta-blockers arethen unpredictable.

In a preliminary study [Fabbri et al. (2009) Comp. Biochem.Physiol 154A:S15] we showed that mussel exposure to 0.3 and 30 μg/L of (−)-propranolol caused a stress syndrome and alteration of cellsignaling. Here we assessed whether propranolol in the chemicalform and concentrations encountered in coastal environmentsinduces detectable responses in mussels.

Mussels (Mytilus galloprovinicialis) were exposed for 7 days to fiveconcentrations of (±)-propranolol, namely 0.3, 3, 30, 300, 30,000 ng/L;the three lowest concentrations represented the environmental range.Different endpoints related to the beta-adrenergic pathway and to thestress response were evaluated in digestive gland and mantle, as thetissues showing the highest responsiveness in preliminary trials. BothcAMP levels and PKA activity were reduced in digestive gland, whileincreased in mantle. Similar expression patterns were observed for theMXR-related genes, a suite of transporters involved in xenobioticmetabolism. The opposite responses of the tissues may result fromdifferently expressed receptor types, suggesting the occurrence ofdifferent interaction pathways. Significant effects were observed atconcentrations as low as 0.3 ng/L, indicating that (±)-propranolol atenvironmental levels affects physiological pathways in mussels.

doi:10.1016/j.cbpa.2010.06.084

4.Nuclear receptor targets for endocrine disrupting compounds

M. Rusten, R. Lille-Langøy, R. Male, A. Goksøyr (University of Bergen,Norway); M. Milnes (San Diego Zoo, USA); B. Blumberg (University ofCalifornia, Irvine, USA)

Nuclear receptors (NRs) are ligand activated transcription factorsthat convert extracellular signals into transcriptional responses. In ourproject we are focusing on the nuclear NR1I2 steroid and xenobioticreceptor (SXR)/pregnane X receptor (PXR) system, known to beinvolved in metabolism and elimination of both xenobiotics andendobiotics. SXR, being activated by an unusually wide range ofcompounds also exhibit large species differences in ligand specificity.

We have cloned NR1I2-like genes from various vertebrate andurochordate species, including Arctic and North Atlantic species suchas the polar bear, glaucous gull, Atlantic cod, Atlantic salmon, andOikopleura dioica, as a basis for comparative functional studies of ligandspecificity and target gene regulation. In another part of the project weare focusing on how SXR integrates ligand activation and intracellularsignaling pathways into transcriptional responses. Posttranslationalmodification of SXR and SXR-associated proteins has been shown toaffect important biological functions such as DNA binding, RXRheterodimerization and subcellular localization. Kinases, such as thecyclic AMP-dependent protein kinase (PKA) have been reported toaffect the activity of SXR by altering its phosphorylation status.Interestingly, the PKA-signaling pathway has been shown tomodulateSXR activity in a species-specific manner. By using a combination ofspecific kinase activators and inhibitors we are trying to dissect whatsignalingpathways are activated byenvironmental pollutants andhowit affects SXR dependent transcription in three well characterizedmodel systems: humans, mice and zebrafish.

The study is supported by a Norwegian Research Council grant,Environment 2015 project 181888.

doi:10.1016/j.cbpa.2010.06.085

5.Characterization of multiple vitellogenins and their transcripts inthe liver of sand goby (Pomatoschistus minutus)

J.L. Humble, J.A. Craft (Biological and Biomedical Sciences, GlasgowCaledonian University, Glasgow, UK); O. Diaz de Cerio (Zoology andAnimal Cell Biology, University of the Basque Country, Spain);M. Saaristo(Bio-and Environmental Sciences, University of Helsinki, Finland);K. Lehtonen (Finnish Environment Institute, Marine ResearchCentre, Helsinki, Finland); K. Lindström (Environmental and MarineBiology, Åbo Akademi University, Turku, Finland); I. Cancio (Zoologyand Animal Cell Biology, University of the Basque Country, Spain)

The sand goby is a small, marine, benthic teleost, which inhabitsthe coastal waters of Europe. The sand goby has been used as a modelspecies for the investigation of oestrogenic endocrine disruption.

The primary objective of this study was to sequence all the sandgoby vitellogenin (Vtg) cDNAs and design homologue-specific mRNAexpression assays to investigate the effects of oestrogens upon Vtgexpression in both genders of sand goby.

Suppressive subtraction hybridisation was used to generate acDNA library enriched for genes whose expression is altered by 17-alpha-ethinyl-oestradiol (EE2). We have sequenced 267 clones andBLAST searches identified 72.3% of these to be vitellogenins. Thevitellogenin sequences matched most closely with Vg-530 or Vg-320of Acanthogobius flavimanus or vitellogenin B of Pagrus major. Thesegenes were used as anchors for sequence assembly, which confirmedthat the Vtg fragments belong to three Vtg gene homologues.Sequence-specific primers were derived and qPCR assays weredeveloped to quantify vitellogenin transcripts.

Intrinsic levels of vitellogenin mRNAs in female sand goby livervaried among vitellogenin type (VtgA=VtgC=5×VtgB) and tracelevels were observed inmale sand goby liver. Exposure to EE2, inducedvitellogeninmRNA inmale fish and also raised the quantity of each Vtgtranscript in liver of female fish by 3-fold. This study is the first toutilize qPCR to quantifymultiple Vtg transcripts in amarine teleost andthese assays will provide a tool to scrutinize the upregulation ofmultiple Vtgs during oogenesis and oestrogenic endocrine disruption.

doi:10.1016/j.cbpa.2010.06.086



6.Testing the ‘obesogen’ hypothesis: Direct effects on of Bisphenol A(BPA) on lipid accumulation in rat hepatocytes

E. Grasselli, L. Vergani, A. Voci, G. Gallo, L. Canesi (Department ofBiology, University of Genoa, Italy)

Obesity and related disorders, such as fatty liver disease, areepidemic in western countries. An association between exposure toenvironmental chemicals and development of obesity has beenproposed; according to the ‘Environmental Obesogen’ hypothesis,obesogens are chemicals that affect lipid homeostasis to promoteadipogenesis and lipid accumulation. Potential candidates arealkylphenols, phthalates and organotin compounds.

Bisphenol A (BPA), a component of different plastics and epoxyresins, including those used for the lining of food and beveragecontainers, is considered as a potential endocrine disruptor withestrogenic properties. Recent evidence indicates that BPA can also actas an obesogen by promoting adipogenesis. However, little is knownon the direct effect of BPA on lipid homeostasis in the hepatocytes.

In this work, the effects of BPA on lipid accumulation wereinvestigated in rat hepatocyte cell lines. In both MH1C1 and Faohepatoma cells BPA exposure induced lipid accumulation, asevaluated by Oil-Red-O staining. In Fao cells, 1 μM BPA induced upto a +60% increase in triglyceride content. This effect wasaccompanied by down-regulation of Peroxisome Proliferation Acti-vated Receptors PPARα and PPARγ, that play a key role in regulationof lipid metabolism. Moreover, down-regulation of the antioxidantgenes catalase and superoxide dismutase-SOD was observed. Theresults support the hypothesis that BPA is able to directly affect lipidhomeostasis and redox balance in the hepatocytes. The effects of BPAon activities and expression of enzymes crucial in metabolicpathways (glycolysis, fatty acid oxidation, lipid droplet formation)are under investigation.

doi:10.1016/j.cbpa.2010.06.087

7.Endocrine disrupting effects of cadmium in the ovary of the lizardPodarcis sicula

P. Simoniello, F. Trinchella, R. Scudiero, S. Filosa, C.M. Motta(Department of Biological Sciences, University Federico II, Naples,Italy)

Cadmium (Cd) is a highly polluting heavy metal known to havedeleterious effects on the reproductive organs and the developingembryo. Cd in fact is a strong endocrine disruptor that interferes withfunctioning of endogenous receptors and hormones causing detri-mental effects on offspring production and survival.

In spite of the wide number of studies carried out in laboratorymammals, data on Cd effects on gonadic tissues, fertility andreproduction of reptilian vertebrates are still limited. For this reasonwe decided to investigate cadmium effects on the ovary of the lizardPodarcis sicula.

Results indicate that Cd treatment induces severe damages infollicles and, in particular, in the oocytes: their cytoplasm becomesvacuolated, and the zona pellucida (ZP) appears disorganised. ZP has

a fundamental role in sperm recognition and binding; therefore, ZPanomalies may have severe consequences on fecundity. In effect,treated animals show a significantly reduce clutch size and a low rateof embryo survival with respect to controls.

Cd in lizard ovary stimulates oogonial proliferation and oocyterecruitment. Interestingly, the supernumerary oocytes formed afterCd treatment become apoptotic so that the number of primaryfollicles does not increase. The mechanisms triggering such massivedegeneration are still unknown. A first hypothesis is that theformation of extra oocytes would activate the endogenous mechan-ism controlling germ cell number; an alternative hypothesis is that Cdhas a direct pro-apoptotic effects on zygotene–diplotene oocytes.Being this true, Cd in germ cells would exert a two-fold effect: wouldinduce proliferation in oogonial stem cells and death in zygotene-diplotene differentiated oocytes. Indeed, Cd in lizard seems to actboth as oestrogen mimetic, increasing follicular cell proliferation, andgonadotropin mimetic, increasing oocyte recruitment.

doi:10.1016/j.cbpa.2010.06.088

8.Estrogenic effects of alkylphenol isomers in vitro and in vivo inzebrafish

E. Puy-Azurmendi, M. Ortiz-Zarragoitia, M.P. Cajaraville (University ofthe Basque Country, Bilbao, Basque Country, Spain); A. Olivares, B.Piña (IDAEA-CSIC, Barcelona, Spain)

Commercial alkylphenols are complex isomer mixtures that canbe individually present in the environment, showing xenoestrogenicactivity. The aim of this study was to establish the estrogenic potencyof some alkylphenol isomers in comparison to the commercialnonylphenol mixture in vitro and in vivo. In vitro an estrogenreceptor-based recombinant yeast assay was used to test theestrogenicity of specific alkylphenol isomers (22octyphenol, 33octy-phenol, 22nonylphenol, 33nonylphenol, and 363nonylphenol) andcommercial nonylphenol. The EC50 were in the range of 0.6–7.7 mg/L.Both octylphenol isomers and 363nonylphnol exhibited higherestrogenic activity than commercial nonylphenol. For in vivoexperiments, one day postfertilization (dpf) zebrafish embryos wereexposed to commercial nonylphenol (50, 250 and 500 μg/L),363nonylphenol and 33octylphenol (50 μg/L), 17β-estradiol (10 and100 ng/L) and DMSO for 4 weeks. After exposure fish were main-tained for 2 weeks in clean water in order to evaluate recovery.Samples were taken after 3, 7, 28 and 42 dpf for histology andvitellogenin, estrogen receptor, retinoid X receptor and cyp19a2aromatase transcription level analyses. Gonad tissue was notobserved at 28 dpf, while at 42 dpf oocytes were observed in 31%of animals. Different transcription levels were observed among33octyphenol, 363nonylphenol and commercial nonylphenol at 1and 4 weeks. Upregulation of vtg by 33octyphenol suggests that thisis the most estrogenic compound tested. In vitro and in vivo resultsshowed a similar estrogenic potency for tested compounds.

Supported by UNESCO Chair of UPV/EHU (project SICAES,UNESCO07/09) and Basque Government through a grant to con-solidated research groups (GIC07/26-IT-393-07).

doi:10.1016/j.cbpa.2010.06.089



Alessandria (Italy) – Sept. 5-9, 2010


Session 7: Toxicity in soil organisms and role of environmental changes

INVITED LECTURE

1.Comparing the toxicity of a pharmaceutical (ivermectin) in thefield with effects observed a single-, two- and multi-specieslaboratory tests

J. Jensen (Aarhus University, National Environmental ResearchInstitute, Silkeborg, Denmark); J. Römbke (ECT OekotoxikologieGmbH, Flörsheim/Main, Germany)

Soil organisms interact in foodwebs thatplaya crucial role in thebio-geochemical cycling of carbon andmineral nutrients. Nevertheless, verylimited knowledge is present on how pollutants impact speciesinteractions in terrestrial ecosystem. Because the function of individualsoil organisms is most meaningful when seen as part of the entire foodweb it is important to improve our measurement and understanding ofhow hazardous substances interact with species at a higher level oforganisation. Important features of food webs are described aspredatory-prey relationships, competition for resources, beneficialmutualistic interactions and commensalism. To evaluate the abovementioned species interactions under controlled conditions, semi-fieldsystems have been used to simulate real field situations. Thispresentation compares the effects of the antiparasitic ivermectinobserved with soil dwelling microarthropods (primarily the springtailFolsomia fimetaria) exposed in single- two- and multi-species testsystems with the effects observed in a field study with dung from cattlemedicated with ivermectin. The springtails were significantly strongeraffected by ivermectin in two- and multi-species systems compared tothe effects observed when exposed alone. No significant effects wereobserved in the field study at soil concentrations markedly higher thanthe levels causing substantial effects in the laboratory.

doi:10.1016/j.cbpa.2010.06.091

ORAL PRESENTATIONS

2.Ultra-high Throughput TRANScryptOME Sequencing of Enchy-traeus crypticus – Innovative Tool for Stress Response Assessment

M.P.C. Ferreira (CESAM Dpt. Biology, University of Aveiro, Portugal /Dpt. Animal Ecology, Vrije Universiteit Amsterdam, Netherlands);D. Roelofs, C.A.M. van Gestel (Dpt. Animal Ecology, Vrije UniversiteitAmsterdam, Netherlands); M.J.B. Amorim, A.M.V.M. Soares (CESAM &Dpt. Biology, University of Aveiro, Portugal)

Enchytraeids are an ecologically relevant functional group of the soilfauna,beingdistributedgloballyandpresent inmost soils; these importantmembers of the foodweb also play a role in organicmatter decompositionand in structuring the soil. Enchytraeus crypticus (Oligochaeta) is a testspecies frequently used in standard soil toxicity (OECD 220, 2004; ISO16837, 2005). In the present project we aim to study the transcriptome ofthis species.Ultra-high throughput transcriptomesequencingusing theGSFLX System has a great potential for gene expression profiling. In order toassess the highly diverse and rich transcriptomic information, theexperimental design comprises a wide variety of exposure conditions.Our samples include different life stages (adults, juveniles and embryos),different temperatures and soil moisture regimes and a series of distincttoxicants to which E. crypticus are exposed at different concentrations. Allsamples will be tagged with multiplex identifiers in order to distinguishtreatments and allow bioinformatic analysis of each stressor separately.Five environmental contaminants with distinct modes of action weretested for their toxicity to E. crypticus in standard natural LUFA 2.2 soil.EC50s for the effect on enchytraeid reproduction for carbendazim,cadmium, phenanthrene, di-chloroaniline and penta-chloroaniline were0.004, 35, 144, 100 and 274mg a.i./kg soil DW, respectively. With theTRANScryptOME project we aim at studying E. crypticus gene expressionpatterns underlying distinct environmental stress conditions.

doi:10.1016/j.cbpa.2010.06.092









3.Ecotoxicological evaluation of urban pollution with the land snailCornu aspersum

S. Gorbi, M. Mezzelani, A. Notti, D. Fattorini, R. Bocchetti, M. Benedetti,F. Regoli (Università Politecnica delle Marche, Ancona, Italy)

Atmospheric pollution from vehicular traffic is a matter ofgrowing interest in urban areas. Although guidelines indicate limitsfor several parameters, the real toxicological impacts remain largelyunexplored in field conditions. This work aimed to validate anecotoxicological approach to evaluate both bioaccumulation andtoxicological effects caused by airborne pollutants using the land snailCornu aspersum as sentinel species. Organisms were caged in severalsites within the city and provincial territory of Ancona, chosen withdifferent characteristics in terms of anthropogenic or urban devel-opment, traffic intensity and typology; translocation experimentswere carried out for 4 weeks in 2 different seasonal periods.Bioaccumulation of trace metals and polycyclic aromatic hydrocar-bons (PAH) in tissues of snails was integrated with the analyses ofseveral molecular and biochemical responses reflecting the inductionof detoxification pathways or the onset of cellular toxicity. Suchbiomarkers included levels of metallothioneins, activity of biotrans-formation enzymes (EROD, ECOD), peroxisomal proliferation, oxida-tive stress responses, accumulation of lipid peroxidation products,lysosomal destabilization and loss of DNA integrity. Obtained resultsrevealed a marked accumulation of metals and/or PAHs in organismsmaintained in more trafficated sites but, quite unexpectedly, even ahilly urban park resulted affected by the transport of airbornepollutants. Bioaccumulation results were supported by data fromautomatic monitoring stations, and the appearance of severalbiological alterations in caged snails confirmed the cellular reactivityof these chemicals. Impairment of antioxidant defences, lysosomaldysfunction and loss of DNA integrity allowed to discriminate siteswith more evident bioaccumulation of pollutants, indicating thepossibility of toxicological effects with some concern also for humanhealth. The overall results of this study suggested the potential utilityof C. aspersum as sentinel organism for monitoring the bioavailabilityand biological impact of atmospheric pollution in urban areas.

doi:10.1016/j.cbpa.2010.06.093

4.Further examination of toxicity profiles in contaminated soils posingpotential risk to the environment: multiple endpoint bioassays

A. Rodríguez-Ruiz (Ekoiz-errilur Res Consortium, CBET Res Grp, Dept.Zoology and Animal Cell Biology. University of the Basque Country,Basque Country, Spain); L. Boatti (University of Piemonte Orientale,DiSAV, Italy); M. Soto (Ekoiz-errilur Res Consortium, CBET Res Grp,Dept. Zoology and Animal Cell Biology, University of the BasqueCountry, Basque Country, Spain); A. Viarengo (University of PiemonteOrientale, DiSAV, Italy); I. Marigómez (Ekoiz-Berrilur Res Consortium,CBET Res Grp, Dept. Zoology and Animal Cell Biology. University ofthe Basque Country, Basque Country, Spain)

Basque environmental administration, inspired by the Ducthsystem, has established Intervention Values (A, B and C) for pollutantsin soil. A-values correspond to baseline reference values whereas C-values are those levels of pollutants in soil that pose an unacceptablerisk for life. B-values indicate that there exists a potential risk andtherefore further examination of toxicity is required before interven-tion measures are decided/designed. Presently, the toxicity profiles oftwo chronically polluted soils from Basque Country which contained

various metals at B-values were examined: an ancient iron mine inZugaztieta (ZU) and an industrial ruin pollutedwith hydrocarbons andmetals in Legazpi (LG). Standard (E. fetida acute toxicity andreproduction tests, plant -lettuce and radish- seed germination androot elongation test, and Microtox) and novel (D. discoideum develop-mental cycle, mortality, replication, endocytic rate and lysosomalmembrane stability) toxicity assays were applied for toxicity profilingto either solid phase or elutriates. According to the results, verydifferent sensitivities were found for the different tests. Overall,whereas ZU soil showed very little or no toxicity, LG soil resulted to betoxic for almost all the toxicity assays, which revealed that for similarB-values the risk posed to the environment and therefore the need forintervining may be very different. In conclusion, toxicity assaysprovides additional reliable information for decision makers andenvironmental managers that go beyond the limited environmentalsignificance of the criteria used to establish Intervention Values, whichare purely based on the concentrations of chemicals and theoreticalapproaches to their toxicity based on bibliographical data. Individualassessment of each soil toxicity (based on toxicity profiling) isrecommended for soil management.

AcknowledgementsThanks are due to D Castillo-Sañudo (UPV/EHU) for technical

assistance. Funded by Basque Gov (ETORTEK BERRILUR II; CTPUE09+/58) and Spanish MICINN (CGL2006-06154).

doi:10.1016/j.cbpa.2010.06.094

5.Application of biomarkers in Lumbricus terrestris for pollutionsoil monitoring

M.G. Lionetto, A. Calisi, T. Schettino (University of Salento, Lecce, Italy)

As recently recognised the measurement of biochemical andcellular responses to pollutants (i.e. biomarkers) on living organismsin the soil has become of major importance for the assessment of thequality of terrestrial ecosystems. The aim of the preset work was toanalyze a suite of cellular and biochemical biomarkers suitable forassessing the pollutant induced stress syndrome in the earthwormLumbricus terrestris chosen as soil bioindicator organism. Severalbiomarkers were studied: granulocyte morphometric alteration,lysosomal membrane stability, tissutal metallothionein concentra-tion, acetylcholinesterase (AChE) activity, micronuclei frequencies.Biochemical and cellular responses were related to whole organismendpoints (grow and reproduction rates).

The study was carried out in standardised laboratory conditionsand further validated in field conditions. In the laboratory studyearthworms were exposed to copper sulphate and methiocarb in thesoil, as representative models of heavy metals and organic xenobio-tics. In the field study L. terrestris specimens were sampled in foursites in the Salento Peninsula (South of Italy) exposed to differentanthropogenic impacts.

The cellularandmolecular biomarkers showedsignificant changes inresponse to both contaminant exposures. They corresponded toalteration in whole organism endpoints such as individual grow andreproduction rates and proved to be suitable for assessing stresssyndrome in the field exposure experiment. In particularmorphometricalterations of granulocytes proved to be a suitable biomarker ofpollutant effect to be included in a multibiomarker strategy. It providesa sensitive generalized response to pollutants that can integrate thecombined effect of multiple contaminants present in the soil.

doi:10.1016/j.cbpa.2010.06.095



6.Potential toxicicty and genotoxicity of soils around a highimpacted site

A. Calisi, M.G. Lionetto, E. Erroi, T. Schettino (University of Salento,Lecce, Italy)

The importance of earthworms in testing the adverse effects ofchemicals on soil biota has been recognised by various environmentalorganisations and resulted in a set of standard toxicity tests. Recently,there is growing interest in the use of biomarkers as a newcomplementary approach providing more information about the stressresponse of the organisms to pollutants.

The aim of the present work was to apply an integrated approachof toxicity tests (mortality and reproduction rates) and genotoxicbiomarkers (micronuclei and comet assay) on the bioindicatororganism Eisenia foetida in the assessment of arsenic contaminationrisk around a national high impacted site (Termo-Electric Central,Cerano, Brindisi, Italy). The animals were exposed to soil samplesobtained from 20 sites in the area around the Central. The exposurewas carried out for 2 weeks (acute toxicity test) and for 4 weeks forthe reproduction test. The adults were then removed from the soiland the newborns were counted after further 4 weeks. On the adultsexposed for 4 weeks micronuclei and comet assay were performed oncoelomatic cells.

Obtained results excluded the presence of acute toxicity in all thesampled sites but revealed the presence of relevant chronic toxicity insome sites. Micronuclei and comet assay data showed a highsignificant correlation with chronic toxicity test and with arsenicconcentration in the soil.

The study suggests the applicability of the proposed integratedapproach of genotoxicity biomarkers and chronic toxicity test in theassessment of arsenic risk contamination in the soil.

doi:10.1016/j.cbpa.2010.06.096

7.Use of the earthworm Eisenia andrei as model organism for soiltoxicity assessments

S. Sforzini, A. Dagnino, I. Saggese, L. Oliveri, A. Negri (University ofPiemonte Orientale, DiSAV, Italy); L. Canesi (University of Genoa,Italy); A. Viarengo (University of Piemonte Orientale, DiSAV, Italy)

Earthworms (such as Eisenia sp., Lumbricus sp.) are extensivelyused as model organisms to assess soil toxicity. Standard toxicologicalend points (such as mortality and reproduction rate) are generallyutilised to evaluate the possible effects at the organism/populationlevel. More recently, a battery of biomarkers suitable for provingpollutant-induced physiological changes at different levels of func-tional complexity (molecule/cell/tissue) has been developed on theearthworm Eisenia andrei. The set of biomarkers included Ca2+-ATPase (PMCA) activity, lysosomal membrane stability (LMS),lipofuscin and neutral lipid lysosomal content, lysosome/cytoplasmratio and DNA damage. In this study, the biomarker battery wasutilised to assess the toxicity of soils sampled in Campania andPiemonte Regions (Italy) characterized by different levels and typesof pollutants (PCBs, dioxin, heavy metals). The presence of organicpollutants in the earthworms' tissues was detected by immunohis-tochemistry utilising chemicals-specific antibodies. The resultsshowed that, although no mortality was detected, the sublethal endpoints utilised were able to demonstrate a stress response in theearthworms. The data clearly indicated that LMS and PMCA activitywere more sensitive early warning biomarkers and that the effects atthe tissue level (such as lysosome/cytoplasm ratio) were capable offollowing the evolution of the stress syndrome. The integration of thebiomarkers data utilising the Earthworm Expert System, recentlydeveloped, was able to objectively rank the health status of theorganisms into five levels, from A (healthy) to E (pathological stress).

doi:10.1016/j.cbpa.2010.06.097





Session 8: Mechanisms of homeostasis and toxicity of heavy metals

INVITED LECTURE

1.Heavy metals toxicity at very low, yet environmentally relevantdoses: The cases of humanmercurial contaminations through fishconsumption, uranium in drinking water, and gold nanoparticlesin aquatic systems

J.P. Bourdineaud (University of Bordeaux, CNRS, Arcachon MarineStation, France)

In 2005, 84% of Wayana Amerindians living in the upper marshes ofthe Maroni River in French Guiana presented a hair mercury concentra-tion exceeding the limit set up by theWorld HealthOrganization (10 μg/g). To determine whether this mercurial contamination was harmful,micehave been feddiets prepared by incorporation ofmercury-pollutedfish from French Guiana. A vegetarian diet containing as little as 0.1% ofmercury-contaminated fish (5 ng ofmethylmercury/g of food) was ableto trigger in mice, after only three months of exposure, disorderspresenting all the hallmarks of mercurial contamination.

Anthropogenic contributions of uranium (U), originating from thenuclear fuel cycle or military activities, enhance U concentrations interrestrial and aquatic ecosystems above the naturally occurringbackground levels. The provisional drinking water guideline value ofthe WHO being 15 μg U/L, we exposed zebrafish to 30 μg/L ofwaterborne U for 28 days. Histological analyses revealed an alterationof muscle and brain tissues, along with a genotoxic impact.

Gold nanoparticles (AuNPs) are used in the treatment of tumors,and drugs containing AuNPs are available on the market. However,little is known about their potential effects on aquatic biota. Weinvestigated on zebrafish the impact of AuNPs of two sizes (10 and50 nm) at a daily dose of 40 ng AuNPs/fish through food in order tomimic part of the food web. After 60 days of exposure, we foundimpacts on mitochondrial activity, DNA damages and genetic re-sponse to oxidative and general stress in different organs.

doi:10.1016/j.cbpa.2010.06.099

ORAL PRESENTATIONS

2.Structural and functional differentiation of gastropodmetallothioneins has been achieved by evolution of metal-specificisoform families

R. Dallinger, M. Höckner, M. Egg (University of Innsbruck, Austria);Ò. Palcios, M. Capdevila (Autonomous University of Barcelona,Spain); A. Pagani, S. Atrian (University of Barcelona, Spain)

Pulmonate gastropods handle metal exposure and metabolismthrough metallothioneins (MTs), a family of proteins binding metalions by complex formation. In contrast to most other MTs, pulmonateMT isoforms can be isolated from preparations in vivo as homo-metallic complexes loaded either with Cd2+ (CdMT) or Cu+ (CuMT).

Here we show by phylogenetic analyses that metal specificity is anintrinsic property shared by all pulmonate MTs, featuring three isoformfamilies: two of them are metal-specific (CdMT and CuMT), whereas athird isoform type (Cd/CuMT) exhibits promiscuous bindingof both, Cd2+

and Cu+. The three pulmonate MT isogenes exhibit a strictly metal-specific transcription pattern, with only the CdMTs being inducible by Cdexposure. The two metal-specific MT isoforms from Roman snail (Helixpomatia)were expressed recombinantly.Mass spectrometric and spectro-polarimetric characterizations of the recovered metal complexes con-firmed that metal specificity of Roman snail CdMT and CuMT has a truestructural basis, yielding homometallic complexes for either of the twoisoforms onlywith their cognatemetal ions (i.e. Cd2+ or Zn2+with CdMTand Cu+ with CuMT). Upon transformation in MT-deficient yeastknockout cells, the two MT isoforms of Helix pomatia exhibited afunctional behaviour dictated by their metal-specific binding preferences.Thusmolecular evolution in pulmonates has led to a complete separationof MTs with respect to their metal binding preferences and metabolism.

This study has been supported by grants from the Austrian ScienceFoundation and the Spanish Ministerio de Ciencia y Tecnología.

doi:10.1016/j.cbpa.2010.06.100









3.Nuclear proteome changes in Dictyostelium discoideum duringadaptations to mercury

L. Boatti, F. Marsano, A. Negri, A. Viarengo (University of PiemonteOrientale, DiSAV, Italy)

Living organisms respond to adverse environmental conditionsactivating a number of molecular mechanisms which, in turn, allowthem to adapt to the new environmental conditions. Such mechan-isms are the outcome of a tight cross-talk among several differentcellular components (ranging from signal transduction to the controlof gene expression) which are so far poorly characterized and mostlyunknown.

This work aims to deepen the analysis of changes in the nuclearproteome of a model organism, the social amoeba Dictyosteliumdiscoideum in relation to exposure to a heavy metal widelydistributed in soil and waters: mercury.

The nuclear protein modifications represent a crucial point in theactuation of stress response, as can be seen as a hub between signaltransduction events and realization of new gene expression/proteinsynthesis, phenomena which in turn may lead to the establishment ofdetoxificationandadaptationprocesses tonewenvironmental conditions.

The proteins extracted from nuclei of control's cells and treatedwith mercury were labelled using Isotope-coded protein label (ICPL)and then analysed by LC–ESI MS/MS. We have identified about 100peptides, their main post-translational modifications, and quantita-tive variations induced by mercury treatment on different proteinssuch as histones and other proteins that should play a role in thenuclear processes. Histones – between the nuclear proteins – arehighly conserved and they do not play simply a structural role, butpost-translational modifications at specific aminoacid residues cangreatly influence gene expression.

These data represent a link between genomics and proteomicsand provide information on the regulation of gene expression inDictyostelium exposed to mercury.

doi:10.1016/j.cbpa.2010.06.101

4.Dynamic transcriptomic profiles of zebrafish gills in response tozinc exposure

C. Hogstrand, D. Zheng, P. Cunningham (King's College London, UK);P. Kille, G.P. Feeney (Cardiff University, UK); R.D. Handy (University ofPlymouth, UK)

Zinc is an essential micronutrient and an aquatic pollutant ofconcern. The fish gill is an important locus for zinc toxicity as well asfor acclimation to elevated zinc. We exploited the tractability of thezebrafish gill to study time-dependent regulatory networks of gene-expression leading to zinc acclimation. Fish were exposed to zinc inwater (260 μg L−1) and diet (2023 mg zinc kg−1) and compared tofish kept at control condition ([Zn(II)] water: 16 μg L−1, diet:233 mg kg−1). Gill tissues were harvested at five time points up to14 days and transcriptome changes analysed in quintuplicate withresults anchored to physiological variables. In total, 525 genes wereregulated (1.8 Fold-Change, 10% FDR) by zinc exposure. The numberof regulated genes increased up to day seven and declined thereafter.Many genes displayed cyclic expression patterns, typical for homeo-static control mechanisms. Annotation enrichment analysis revealedstrong overrepresentation of developmental genes and of transcrip-tion factors, such as steroid hormone receptors. Early regulation ofgenes was coordinated by a few key transcription factors and

followed by hedgehog and bone morphogenic protein signalling.The results suggest that zinc exposure reactivated developmentalpathways in the gill and stimulated stem cell differentiation, aresponse likely reflecting gill remodelling in response to exposure.This provides insight to the mechanisms of zinc toxicity andacclimation as well as roles of zinc in biology. The study alsohighlights the importance of temporal transcriptomics analysis inorder to resolve the discrete elements of biological processes, such aszinc acclimation.

doi:10.1016/j.cbpa.2010.06.102

5.Effects of hexavalent chromium in Mytilus galloprovincialis

L. Canesi, C. Barmo, R. Fabbri, S. Olivieri, G. Gallo (Dipartimento diBiologia, Università di Genova, Italy); L. Boatti, F. Dondero, A. Viarengo(University of Piemonte Orientale, DISAV, Italy)

Hexavalent chromium Cr(VI) is an important contaminantreleased from both domestic and industrial effluents, representingthe predominant chemical form in aquatic ecosystems. Cr(VI) is amodel oxidative toxicant due to its intracellular reduction to thebiologically active Cr(III) form and ROS production. Moreover, recentevidence suggests that chromium may interfere with the endocrinesystems, in particular acting as a ‘metalloestrogen’.

In this work, the effects of Cr(VI) were investigated in the marinebivalve Mytilus galloprovincialis. Mussels were exposed to Cr(VI) for96 h (0.01–100 μg/animal/L) and different endpoints were evaluatedin different tissues. The highest concentration induced severe stressconditions in the animals. At lower concentrations significantchanges in lysosomal biomarkers in both hemocytes and digestivegland were observed. Cr(VI) also induced stimulation of antioxidantenzyme activities (catalase and GST) and lysosomal lipofuscinaccumulation, indicating oxidative stress conditions. Moreover,changes in PES (Protein Expression Signature) in hemolymph serum,as evaluated by 2D electrophoresis were observed. Interestingly, inmale digestive gland, Cr(VI) induced significant changes in the levelof transcripts for Mytilus Estrogen Receptors (MeERs) and serotonin(5-HT) receptors, as evaluated by RT-Q-PCR, supporting the hypoth-esis that this metal may have significant endocrine disruptingproperties in marine bivalves.

The results demonstrate that environmental concentrations of Cr(VI) induce a wide range of responses in mussel tissues, withconcentration-, tissue- and sex-related differential effects.

Supported by the Italian Ministry of Research (PRIN2007) and byFondazione CARIGE (prot. 2009.0659-42).

doi:10.1016/j.cbpa.2010.06.103

6.Trace metals modulate gene expression and efflux activity of ABCtransport proteins in the PLHC-1 fish cell line

C. Della Torre (Department of Environmental Sciences, “G. Sarfatti”Università di Siena, Italy); R. Zaja, J. Loncar (Laboratory for MolecularEcotoxicology, Division for Marine and Environmental Research,Ruđer Bošković Institute, Zagreb, Croatia); S. Focardi (Department ofEnvironmental Sciences, “G. Sarfatti”Università di Siena, Italy); T. Smital(Laboratory for Molecular Ecotoxicology, Division for Marine andEnvironmental Research, Ruđer Bošković Institute, Zagreb, Croatia);I. Corsi (Department of Environmental Sciences, “G. Sarfatti”Università di Siena, Italy)


The aim of this study was to investigate the interaction of selectedtoxic heavy metals with fish ABC transport proteins using PLHC-1 fishcell line as in vitro model. Cells were exposed for 24 h to 0.3–3 μM ofCdCl2, HgCl2, As2O3, Sb2O5 or K2Cr2O7, and the expression of a series ofABC genes was then determined by qRT-PCR: abcc1-5 and abcc10(encoding for Multidrug Resistance Associated Proteins, MRPs); abcb1(P-glycoprotein; P-gp) and abcg2 (Breast Cancer Resistance Protein;BCRP). Abcb1, abcc2, abcc3 and abcc4 genes were dose-dependentlyup-regulated by As2O3, K2Cr2O7 and HgCl2. HgCl2 was the mosteffectivemetal, resulting in the 16-fold increase in expression of abcc2,followed by abcc3 (4-fold vs. control, unexposed cells) and abcc4 (2.5-fold). K2Cr2O7 significantly up-regulated abcb1 (5.3-fold), abcc2 (3.4-fold) and abcc3 (2.8-fold), while As2O3 up-regulated abcc2 (3.3-fold).In addition, transport activity of corresponding proteins was evaluatedupon exposure to selected metals in the wild type (PLHC-1/wt) and P-gp over expressing doxorubicin resistant cells (PLHC-1/dox), usingaccumulation assay with monochlorbimane and Calcein-AM as modelsubstrates, respectively. All metals showed a significant inhibition ofboth P-gp andMRP related transport activities, with IC50 values below1 μM. In conclusion, our results indicate that (1) specific fish ABCtransporters may be actively involved in cellular defense against toxicheavy metals, and (2) some of the tested heavy metals effectivelyinhibit ABC protein(s) transport activity.

doi:10.1016/j.cbpa.2010.06.104

7.Marisa topless: What platinum may bring about

H.-R. Köhler, R. Osterauer, L. Marschner, M. McKee, O. Betz (Universityof Tübingen, Germany); M. Gerberding (Max-Planck-nstitute forDevelopmental Biology, Tübingen, Germany); B. Sawasdee (Univer-sity of Tübingen, Germany); P. Cloetens (European SynchrotronRadiation Facility Grenoble, France); N. Haus, B. Sures (University ofDuisburg-ssen, Germany); R. Triebskorn (University of Tübingen,Germany; Transfer Center for Ecotoxicology and Ecophysiology,Rottenburg, Germany)

An external calcareous shell characterizes the archetypal body planof conchiferan molluscs. Nevertheless, shell internalization into thebody has evolved independently in a number of taxa, including somegastropods. In this class, the developmental process of torsion, aprocess in gastropod ontogenesis that rotates the visceral body 180°relative to the larval headfoot region in counter-clockwise direction isregarded as a hallmark associatedwith a newanatomical configurationwhich is present in extant non-heterobranch gastropod species(formerly known as ‘prosobranchs’), predominantly bearing externalshells. Short-term platinum exposure uncouples at least two of theprocesses associated with torsion during the embryonic developmentof the freshwater snail Marisa cornuarietis (Ampullariidae). Eventhough the anus of the Pt-treated snails is located anteriorly (whichcorresponds to the archetype), the gill and the designated mantletissue remains in a posterior location and thus prevents the formationof an external shell. Similar to the situation in the belemnites, aphragmocone-like internal shell which persists across the lifetime isinduced even after pulse treatment with Pt for just one to two daysduring embryogenesis. We observed the effect of induced snail-slugconversion also in the pulmonate Planorbarius corneus. Althoughwedonot claim to artificially repeat exactly what has evolved in squids andslugs our findings demonstrate that selective alteration of embryonickey processes can result in fundamental body plan changes.

doi:10.1016/j.cbpa.2010.06.105


8.Cadmium affects nematocytes discharge and cell volume regulation

A. Marino, R. Morabito, G. La Spada (University of Messina, Italy)

Cnidarians viability depends on some physiological features suchas discharge capability and cell volume regulation. Discharge is anexocytosis process consisting in the rapid eversion of the tubule andrelease of toxins stored in the nematocyte, the highly specialized celltypical of Cnidarians. Both mechanoreceptors and chemoreceptorsare needed to perform this peculiar response. With regard to cellvolume regulation, essential for cell survival, isolated nematocytescan regulate their volume in hyposmotic conditions (RegulatoryVolume Decrease, RVD) activating different channels and cotran-sporters. Therefore we aimed to verify the effect of Cadmium, atoxicant accumulated in the environment due to industrial activities,on Cnidarians viability by investigating nematocytes discharge andcell volume regulation. Experiments were performed on acontia(mesenterial filaments bearing nematocytes) of Calliactis parasitica(Anthozoa) collected in the Strait of Messina (Italy). Discharge testshave been set up by a non-vibrating test probe on tissue, with orwithout Cadmium, to verify discharge response under both mechan-ical and chemical stimulations. The number of discharged nemato-cytes was then counted. Cell volume regulation was studied onnematocytes isolated from acontia by 605 mM SCN—plus 0.01 mMCa2+ application and submitted to 35% hyposmotic shock withCadmium. Results showed a significant impairment of dischargeresponse under chemico-physical stimulation and an inhibition ofRVD capability. Taken together these preliminary findings show thatthe survival of the entire animal may be affected by exposure toCadmium and suggest Cnidarians as indicators of water quality.

doi:10.1016/j.cbpa.2010.06.106

9.Cadmium impairs RVD in the digestive cells of Mytilusgalloprovincialis

C. Faggio, A. Torre, D. Alberghina, G. Piccione, F. Trischitta (Universitàdi Messina, Italy)

It is known that most cells are able to regulate their volume inanisotonic conditions. When exposed to hyposmotic medium theyrapidly swell and thereafter exhibit a regulatory volume decrease(RVD), due to a net loss of intracellular solutes followed by an obligedwater efflux. In this work we evaluated if the hepatopancreas cells ofMytilus galloprovincialis are able to perform RVD under hypotonicshock and if heavy metal pollution can affect this homeostaticresponse. The ability to perform RVD was evaluated by videometrictechnique in isolated cells both in the absence and in the presence ofcadmium, one of the major metal contaminants in the aquaticenvironment.

We found that 56% of cells exposed to hypotonic solution (π from1100 mOsm to 750 mOsm) were able to regulate cell volume, while44% of cells rapidly swell by about 15% of their original volume andmaintained the value attained throughout the observation period inhypotonic conditions. However in the presence of inhibitors ofvarious transport pathways, known to be involved in RVD, cellvolume showed a significantly larger increase. These results suggestthat most digestive cells have the ability to perform RVD while asmaller number have a partial capacity to perform RVD, avoiding alarge increase in cell volume. Preincubation with CdCl2 (1 μM) for 30,


75 and 120 min produced effects similar to those observed in thepresence of the transport inhibitors, while cells preincubated with10 μM CdCl2 did not tolerate the hypotonic stress, after 75 min ofpreincubation they exhibited a progressive swelling and lost theirintegrity. These results suggest that cadmium impairs RVD by alteringmechanisms responsible of the homeostatic response.

doi:10.1016/j.cbpa.2010.06.107

10.Pollutants effects on ROS production and DNA damage of Mytilusgalloprovincialis hemocytes: Role of NHE, PKC and cAMP

S. Koutsogiannaki, M. Kaloyianni (Aristotle University of Thessaloniki,Greece)

Hemocytes are the main cell effectors of immune responses ofMytilus galloprovincialis. Reactive oxygen species (ROS) represent oneof the main immune mechanisms in invertebrates and also serve asintracellular signaling molecules. However, excess of ROS seems to beimplicated in cytotoxicity and mutagenic damage. In the presentstudy it was of interest to investigate the effects of cadmium and 17β-estradiol on ROS production, as measured by DCFDA and DHE andDNA damages, as measured by comet assay inMytilus galloprovincialishemocytes. The implication of Na+/H+ exchanger (NHE), proteinkinase C (PKC) and cAMP in these processes was also studied. Ourresults suggest that both cadmium (5 μM) and 17β-estradiol (25 nM)triggered an increase in ROS production as well as in DNA damage inisolated hemocytes compared to control cells. Pre-incubation ofhemocytes with either cariporide (20 nM), a Na+/H+ exchangerinhibitor or GF-109203X (10 μΜ), an inhibitor of all PKC isoforms, orGO6976 (500 nM), an inhibitor of alpha- and beta-PKC isoforms(PKCα, PKCβΙ, and PKCβΙΙ) counteracted cadmium and 17β-estradiolalone effects. Pre-incubation with forskolin (10 μM), an adenylylcyclase (AC) activator enhanced cadmium and 17β-estradiol effectson ROS production. Our results suggest that cadmium and 17β-estradiol enhance ROS production and induce DNA damage in Mytilusgalloprovincialis hemocytes via a signaling pathway that involvesNHE, PKC and cAMP.

doi:10.1016/j.cbpa.2010.06.108

11.Cadmium toxic effects in the liver of the wall lizard Podarcis sicula

P. Simoniello, F. Trinchella, C.M. Motta, S. Filosa, R. Scudiero(Department of Biological Sciences, University Federico II, Naples,Italy)

Cadmium (Cd) is a widespread metal highly toxic to organs,particularly to the liver and kidney where it accumulates. To preventdamage, cells respond to cadmium exposure by increasing theexpression of stress-response proteins, such as metallothionein (MT).

Cytological/molecular data on cadmium effects in reptiles arescanty, so we decided to carry out a multidisciplinar investigation todetermine the influence of intraperitoneal and dietary cadmiumexposure on Podarcis sicula liver.

Cadmium accumulation was measured by atomic absorptionspectrometry. MT gene induction was determined by dot blot analysison total RNA. Results demonstrate a significant Cd uptake, in whichpatterns vary with exposure route. After a single intraperitonealtreatment, concentrations of Cd and MT transcripts are positivelycorrelated; following a dietary treatment, no correlation is present

between Cd accumulation and the amount of MT mRNA, that doesn'tchange.

Cytological analyses revealed that Cd induces oedema andextracellular matrix production. In hepatocytes it causes swellingand alteration in lipid/sugar metabolism. The severity of responses issimilar in all samples indicating an apparent independence fromcadmium concentrations. Finally, no occurrence of necrosis/apoptosisis found, even with high cadmium concentrations.

Together, results demonstrate that in Podarcis Cd is toxic to theliver by acting on the osmotic balance, metabolism, and proteinexpression, and that it interferes with cell structure/function even atlow concentrations. Results also indicate that liver parenchymadoesn't recover rapidly from an occasional contamination and thatresponses aren't dose and time dependent.

In conclusion, our data indicate that lizard liver cannot be used asbiomarker of cadmium pollution.

doi:10.1016/j.cbpa.2010.06.109

12.Health state of mollusc productive sites: Biochemical, physiologi-cal and molecular markers

G. Baccarani, S. Manente, S. Chiesa, L. Gobbo, F. Minello, C. Bettiol,G. Minervini, E. Mane, G. Bordignon, G. Ravagnan, E. Argese (Ca'Foscari University of Venice, Italy)

One of the main priorities of the European Union political agendaconcerns the tracking, management and control of fisheries resources.

As most coastal zones, Venice Lagoon is subject to the deposit ofsedimentological materials, in form of inorganic and organic detritus,nutrients and heavy metals, and there is also a very strong anthropicpressure; therefore, analysis and monitoring of production sites havea great importance in fisheries management and control.

Aim of the present study is to identify and validate biochemical,physiological and molecular markers that could be utilized forassessing the state of health of productive sites, as well as to identifynew ones.

A multidisciplinary approach has been developed to define andcharacterize both the populations of bivalve mollusc growing up insome aquaculture lagoon sites and the best sites as well.

Model organisms for the research are Tapes philippinarum and Tapesdecussatus, two species of bivalvemollusc of theVeneridae family,whichhappens to have a great socio-economical importance: as a conse-quence, fishing and production sites have to be carefully controlled.

The main focus of this study centers on inorganic pollution,specifically heavy metals bioaccumulation and storing in differenttissues; the evaluation of metallothioneins induction has proved veryhelpful in determining the quality of old and new aquaculture sites;in addition, molecular markers have been used to look for a molluscbivalve sub-population identification.

doi:10.1016/j.cbpa.2010.06.110

13.Sub-lethal effects of copper combined with temperature stress inthe marine mussel Mytilus galloprovincialis

A. Dagnino, S. Sforzini, L. Boatti, F. Capri, C. Oliveri, A. Negri, F. Dondero,A. Viarengo (University of Piemonte Orientale, DiSAV, Italy)

The intertidal bivalves Mytilus spp. have been used in biomonitor-ing of marine ecosystems for over 30 years. Water temperature


fluctuations are typical of Mediterranean Sea environments and itmight represent a confounding factor for the correct assessment ofthe mussel health status and the use of mussels as bioindicators.

To date, very little is known about the interaction of heat withpollutants for what concern the cellular responses of mussels. Withthe aim to clarify some of these aspects, we carried out short termexposure of Mytilus galloprovincialis specimens to five copperconcentrations (2.5–40 μg/L) within a temperature gradient rangingfrom 16 °C to 24 °C.

Core biomarker responses, i.e. lysosomal membrane stability(LSM), neutral lipid (NP) and lipofuscin content, were examined inthe digestive tissue along with a battery of molecular assays,including DNA-microarray and real time quantitative PCR.

Our results showed significant lysosomal destabilization andaccumulation of NP and lipofuscin along with the raise of pollutantconcentration. However, in general effects of temperature weremostly confined to molecular changes and did not affect musselphysiological functions.

These data seem to indicate that marine inter-tidal organismssuch as mussels are suddenly able to adapt to temperature changesconfirming their primary role in biomonitoring programs as robustbioindicators of chemical contaminations.

The present research is carried out in the framework of 7FP-EUMEECE.

doi:10.1016/j.cbpa.2010.06.111

14.Sex-dependent differences in response to Cd exposure and musselacclimatization effects on mussel cells in vitro

A. Katsumiti, M.P. Cajaraville (University of the Basque Country, Spain)

Due to the extensive use of bivalve mollusks such as musselsas sentinel species in environmental risk assessment, there is aconsiderable interest in the development of in vitro models in thisspecies. Mussel gill cells and hemocytes are primary targets for theaction of environmental pollutants. In the present work, sex-dependent differences in the sensitivity of hemocytes and gill cellsexposed to Cd (0.002, 0.02, 0.2, 2, 20, 200 and 2000 mg/L) weretested. In addition, the effect of acclimatization on cell viability ofmussel hemocytes and gill cells was assessed. Sex-dependentdifferences in the sensitivity of hemocytes or gill cells to cadmiumwere not observed. In hemocytes and gill cells isolated from bothmales and females, neutral red uptake and MTT mitochondrialactivity were reduced significantly at Cd concentrations of 2 mg/L andabove. Cytotoxicity was dose-dependent and LC50s for Cd weresimilar in cells isolated from males and females with the two assaystested. Acclimatization exerted effects only on hemocytes, viabilityincreasing at 2 days and progressively decreasing afterwards. Thisincrease can be viewed as an activation of cell metabolism and/or cellproliferation occurring in parallel with mussel acclimatization to thenew environment with different water conditions and food supply.Due to the fact that the epithelial gill cells are directly in contact withthe external environment, constantly adapting to changes in theexternal environment, it appears that gill cells are more resistant thanhemocytes to changes associated to the acclimatization of mussels.

Funded by the Spanish Ministry of Environment (project VertiTox,ref 018/RN08/02.1) and the Basque Government through a grant toconsolidated research groups (GIC07/26-IT-393-07).

doi:10.1016/j.cbpa.2010.06.112





Session 9: Effects of environment changes and pollution on Artic and Antarctic organisms

INVITED LECTURE

1.Effects of environmental changes and pollution on Arctic andAntarctic organisms

B.M. Jenssen (NorwegianUniversityof Science andTechnology,Norway)

Arctic and Antarctic ecosystems are threatened by anthropogenicactivity at increasing rates. Global warming, persistent organic pollutants(POPs), exploitation of marine resources and petroleum activity arecurrently the major threats to these fragile ecosystems. Pollution istransported to Polar environments via long-range atmospheric transport,and the rate of global warming has been claimed to be very high in theArctic. POPs identified in Polar ecosystems originate from the industria-lized and heavily populated areas of the Earth. Many of these POPs haveendocrine disrupting properties. They may therefore interfere withphysiological adaptations of Polar organisms to their extreme environ-ments and to environmental change. Even though environmental levels ofPOPs generally are much lower in Polar environments than in the moredensely populated areas on Earth, levels of POPs in Polar top-predators arevery high. This is due to the biomagnification of these compounds. Highlevels of POPs have been associated with endocrine disruption in severalendemic Arctic top predators. The thyroid and the reproductive hormonesystems seem to be particularly susceptible to endocrine disruption. Thethyroid hormone system is involved in several physiological processes thatare important for evolutionary adaptation and acclimatization to environ-mental changes. Thyroid hormones are also involved in growth anddevelopment, and in cognitive and behavioural processes. Even relativelylow levels of POPs have been associatedwith thyroid hormone disruption.Endocrine disruptive effects at the organismal level may cascade up thebiological system, resulting in reduced abilities of Polar ecosystems torespond adequately to other anthropogenic environmental changes.

doi:10.1016/j.cbpa.2010.06.114

ORAL PRESENTATIONS

2.Characterization of new biomarkers from the Antarctic ciliateEuplotes focardii by a transcriptomic approach

S. Pucciarelli, R.R. Devaraj, S. Barchetta, Ting Yu, A. La Terza, C. Miceli(University of Camerino, Italy)

The stable cold Antarctic coastal seawaters host a rich variety ofeukaryotic microbes, a large fraction of which is represented byspecies of ciliates, in particular Euplotes. Among the Euplotesspecies isolated from Terra Nova Bay, E. focardii shows strictlypsychrophilic and stenothermal phenotypes, including optimalsurvival and multiplication rates at 4–5 °C, that denote an ancientcolonization of the Antarctic environment. The successful coloni-zation of the Antarctic habitat by E. focardii should be related tothe evolution of macromolecules able to function at low tempera-tures. To understand the molecular basis responsible for cold-adaptation, we started the characterization of the genome andtranscriptome from E. focardii. At present we sequenced∼15 Gbases, representing about 7200 genes. To predict thefunction of these sequences, we performed a blastx batchoperation with Standalone BLAST. To verify if this stenothermalorganism constitutively induces genes involved in the thermalresponse, we selected the sequences that gave hits to stressproteins from the transcriptome blastx result file. We found fourdifferent transcripts corresponding to Heat Shock Proteins (HSPs)70, and two corresponding to the Heat Shock transcription Factors(HSF). A deeper analysis revealed the presence of transcripts thatshared similarity to metallothioneins, DNA damage repair factors,proteins induced by oxidative and osmotic stresses, and proteinsresponding to centrifugal and shear forces. Moreover, we alsofound transcripts that blasted with proteins responding to generalstresses. We are currently investigating the expression variation ofthese genes determined by environmental stresses. These results








obtained from the genome and transcriptome of E. focardii openthe way to the characterization of new biomarkers that can beused for biomonitoring the effects of pollutants and thermal stresson Antarctic organisms.

doi:10.1016/j.cbpa.2010.06.115

3. Associative interactions of complex mixtures of organohalogencompounds and circulating testosterone in polar bears (Ursusmaritimus)

B.M. Jenssen, G.D. Villanger, E.I. Smette (Norwegian University ofScience and Technology, Norway); R.J. Letcher (Carleton University,Canada); C. Sonne, R. Dietz (University of Aarhus, Denmark)

In natural environments animals are exposed to, and bioaccumu-late, a wide range of different man-made contaminants, includingorganohalogenated compounds (OHCs). Many of these contaminantshave been shown to have endocrine disrupting (ED) effects. The aimof the present study was to examine associative interactions ofcomplex mixtures of organohalogen compounds and circulatingtestosterone in adult and juvenile male polar bears (Ursus maritimus).

The study was conducted on 14 adult and 19 subadult male polarbears from the East-Greenland population. The samples werecollected from legally hunted polar bears by indigenous hunters,and concentrations of a range of OHCs and polybrominated biphenylswere analysed in blubber. Concentrations of testosterone wereanalyzed in whole blood samples following extraction. The effectsof the complex mixtures of the OHCs in the two groups of male polarbears (adults and subadults) on testosterone were modelled usingprojection to latent structures (PLS: Simca-P, Umetrics, Umeå,Sweden). The results showed that the composition of OHCs differedbetween the two groups of male polar bears. Furthermore, the PLSmodels showed that different compounds explained the variation inblood testosterone concentrations in subadult and adult males. Thus,the effects of ED chemicals on testosterone encompass a complexdynamic process that is dependent of both the composition of thecomplex mixture of contaminants and the physiology of the animal.

The present study is part of the International Polar Year projectIPY-BearHealth, and is financed in part by the Norwegian ResearchCouncil.

doi:10.1016/j.cbpa.2010.06.116



Alessandria (Italy)—Sept. 5–9, 2010


Session 10: Environmental proteomics and biomarker discovery

INVITED LECTURE

1.Environmental proteomics and biomarker discovery

D. Sheehan (University College Cork, Ireland)

Environmental toxicology represents a complex triangular inter-play between anthropogenic chemicals, the environment (or com-partments thereof) and biota. The biomarker approach involvesidentifying molecules capable of responding to anthropogenicchemicals (e.g. xenobiotics) by up- or down-regulation with a viewto using these to assess overall environmental quality. Proteins, whichare readily assayable (e.g. by ELISA or enzymatic activity) have oftenbeen used as biomarkers of choice. Examples include stress-response(e.g. hsps), antioxidant (e.g. catalase) and detoxification (e.g. GSTs, P-450 activities) proteins. As the main agents of biological change,proteins are also important toxicity targets. The traditional biomarkerapproach is essentially "hypothesis-led" i.e. most biomarker proteinsmight be expected a priori to be influenced by xenobiotics by virtue oftheir known biological function. Proteomics offers an alternative tothe traditional biomarker approach in that it makes possible adiscovery-led trawl through the entire complement of proteins of acell, tissue or organism with the potential, ideally, for unbiaseddiscovery of new biomarkers including the potential to elucidatetoxicity mechanisms. This provides an alternative and complemen-tary route to biomarker discovery. The workhorse proteomicstechnique is a two dimensional electrophoresis (2DE) and thispresentation describes how protein expression changes and post-translational modifications can be applied to ecotoxicology investiga-tions. More recently, we have complemented 2DE with gel-freeproteomics approaches in which proteins carrying particular chemi-






cal lesions are enriched on chemically functionalised beads. Despitebottlenecks and limitations of proteomics, it does offer a robust routeto both biomarker discovery, elucidation of toxicity mechanisms andidentification of toxicity targets.

doi:10.1016/j.cbpa.2010.06.118

ORAL PRESENTATIONS

2.

Concentration–response concept in ecotoxicoproteomics: Anoptimised design for the detection of robust chemical stressassociated protein markers: Effects of different concentrations ofphenanthren to the proteome of Danio rerio embryos

U. Gündel (Dept. Bioanalytical Ecotoxicology, UFZ, Germany); S. Kalkhof,M. vonBergen, R. Altenburger, E. Küster (Proteomics, UFZ, Leipzig)

Ecotoxicoproteomics studies have the potential for the detectionand quantification of chemical stress related proteins which mightserve as novel biomarkers and provide insights into mechanisms oftoxicity. As studies based on 2-DE are comparatively elaborate, time-consuming and expensive, the finding of robust biomarkers islimited by the numbers of replicates, statistical methods and also bythe experimental design. For these reasons, many publishedecotoxicoproteomics studies are confined to one or two testedconcentrations. As reactions at the molecular level, however, areexpected to be a function of exposure concentration, the analysis offew predefined concentrations might mislead the conclusionsdrawn.



In the present study an optimised experimental design for anecotoxicoproteomics study was tested for detection of contaminantinduced protein regulation which includes an analysis of concentra-tion dependence without increasing the replicate number andexperimental effort. Danio rerio embryos were exposed to sixdifferent concentrations [0.049 µg/L (1% of LC50) to 0.345 µg/L(LC20)], selected according to the concentration–response relation-ship of apparent lethal effects, of phenanthren, a well studied modelPAH. Proteomics analyses were done by 2DE-DIGE. Analyses wereperformed using Pavlidis Template Matching followed by signifi-cance testing. Expression profiles of 713 signals were studied. Aboutone third of the protein spots could be detected to show robustreactions correlating well with stressor intensity. Hereby, 55proteins spots showed significant changes already at EC01. Interest-ingly, 29 proteins significantly reacted at very low concentrationsand showed different expression profiles at lower and higherconcentrations. Significantly regulated protein spots were identifiedby mass spectrometry.

In this study it could be shown that increasing the number ofmeasured concentrations instead of performing higher replicatenumbers in 2DE experiments allowed the robust detection of stressmarkers.

doi:10.1016/j.cbpa.2010.06.119

3.Screening for cellular targets of chemicals with cDNA phagedisplay

B. Van Dorst, J. Mehta, E. Rouah-Martin (University Antwerp, Belgiumand Institute for Agricultural and Fisheries Research, Belgium); W. DeCoen, R. Blust (University Antwerp, Belgium); J. Robbens (UniversityAntwerp, Belgium and Institute for Agricultural and FisheriesResearch, Belgium)

cDNA phage display is a powerful proteomics tool to screen forcellular proteins with affinity for targets, like proteins and chemicals.The cDNA phage display technique is frequently used in drugdevelopment to screen for cellular target of drugs, however inenvironmental toxicology cDNA phage display remains unexplored.Because of the high potential of cDNA phage display in environmentaltoxicology, we introduced cDNA phage display in the environmentaltoxicology field, to screen for cellular target of chemical compounds.The knowledge of the interactions between chemical compounds andcellular proteins can provide important complementary informationto toxicogenomic studies in understanding the mechanism of actionof chemical compounds.

In our study, the potential of cDNA phage display as a novel tool inenvironmental science is demonstrated with 17β estradiol (E2). cDNAphage display was used to screen for cellular targets of E2 and two sofar unknown cellular targets were selected. Surface plasmonresonance (SPR) was used to confirm and characterize the E2bindings affinity of these cellular targets, by determining the Kdvalues. The Kd values in the low nM range verify that the selectedcellular proteins are indeed binding proteins for E2. The identificationof these two cellular targets of E2, is valuable to have a morecomplete understanding of the mechanisms of action of E2. Our studydemonstrates the potential of cDNA phage display in environmentaltoxicology, as a fast and cost effective alternative for the identificationof cellular targets, which is crucial to unravel the mechanism of actionof chemical compounds.

doi:10.1016/j.cbpa.2010.06.120

4.The role of oxidative stress in setting thermal tolerance limits inMytilus

L. Tomanek (California Polytechnic State University, USA)

Increasing temperatures accelerate the flux through the electrontransport chain and thereby increase the production of reactive oxygenspecies (ROS). We tested the role of temperature in causing oxidativestress in mussel species (genusMytilus) that differ in thermal toleranceby comparing their proteomic response to acute heat stress. Aquantitative proteomic analysis, using 2D gel electrophoresis and massspectrometry, showed that the two congeners, the warm-adapted M.galloprovincialis and the cold-adapted M. trossulus, show broadsimilarities in their response to acute exposure to 24 °C, 28 °C and32 °C and a 24 h recovery at 13 °C (control).However, changes inproteinlevels suggest that the highest exposure temperature causes a switchfrom ROS-generating NADH-producing pathways to NADPH-producingpathways that can scavenge ROS in the cold- but not thewarm-adaptedspecies. Reduced levels of oxidative stress proteins in the cold-adaptedM. trossulus accompany these changes, suggesting that a limitedresponse to ROS production may cause these changes. Lower levels ofa NAD-dependent deacetylase (sirtuin-2), an indicator of cellularlifespan, in M. trossulus suggest that these changes may in part becaused bymodifications in the acetylation status of proteins. Additionalinterspecific differences in levels of proteins involved in molecularchaperoning, proteolysis and cytoskeleton suggest several new hypoth-eses on cellular processes limiting thermal tolerance.

doi:10.1016/j.cbpa.2010.06.121

5.Fish peptidome patterns can distinguish from exposure toantropogenic pollution

I. Apraiz, J. Lind, L. Käll (Stockholm University, Sweden); J. Bergquist(Uppsala University, Sweden); S. Cristobal (Stockholm University,Sweden)

Plasma from fish contains a complex mixture of proteolyticallyderived peptides that could be used to define a peptide profile. Thispeptidome patterns are correlated with biological events that affect theentire organism. In this project, fish plasma from Baltic flounder(Platichthys flesus) was collected in five sampling sites around the BalticSea. Tenmicroliters of the samplewas processed following a solid phaseextraction coupled to MALDI-TOF mass spectrometry strategy forobtaining a polypeptide profile that would be capable of distinguishingfish collected at the different sites. MS analysis was carried out on aBruker Ultraflex II TOF/TOF, on the mass range of 1280–20,137m/zwithion suppressionup to1250 Da.MIB-HIC8 fishplasmaspottedwithDHAPyielded the greatest number of polypeptide peaks on the MS analysis.Plasma from five individuals at each stationwasprocessed 10 timeswiththe MIB-HIC8 in order to obtain experimental replicates. Three spectraof 500 shots each (acquired as five times 100 shots) were acquired foreach experimental replicate. The raw spectra were interpreted usingsignal processing algorithms to define suitable features. These featureswill be used later to compare measured peptidomes and to derivedifferential “biomarker” panels, defining the meaningful differencesbetween data sets, through appropriate statistical analysis. Therefore,peptide profiling could provide a simple, scalable, and automaticplatform for the identification, and characterization of aquatic organ-isms exposed to environmental stressors such as pollutions.

doi:10.1016/j.cbpa.2010.06.122


6.Does global warming modulate susceptibility to pollutants in asentinel fish species, Cottus gobio?Clues hold by enzymati sub-proteomic approaches

J. Dorts, P. Kestemont, M. Dieu, M. Raes, F. Silvestre (University ofNamur, Belgium)

The globally averaged surface temperature is projected to increaseby 1.4 to 5.8 °C over the period 1990 to 2100, with similar increases inthe temperature of aquatic ecosystems. Whilst fish are expected to beamongst the most affected animals by global warming there is littleempirical evidence to suggest precisely what such effects might be.Many freshwater species are subjected to polluted environments aswell, and increased temperature could modulate their susceptibilityto contaminants.

The aim of the present study was to investigate whetheracclimation to elevated temperature may lower or enhance theability of C. gobio, an ecologically relevant fish species, to toleratesubsequent cadmium exposure.

Fish were acclimated to 3 different temperatures (15.1, 18.1, and21.1 °C) for 28 days, and subsequently exposed to 1 mg Cd/L for4 days. The effects of heat acclimation and subsequent Cd exposurewere investigated using enzymatic and sub-proteomic approaches.

First of all, exposure to Cd significantly decreased (p<0.05) theactivity of the lactate dehydrogenase (LDH) in gills of fish acclimatedto either 15 or 18 °C, while an acclimation to 21 °C suppressed thisinhibitory effect. Secondly, using a sub-proteomic analysis by 2DDIGE, 54 branchial protein spots displayed significant interactioneffects of temperature and cadmium (p<0.05). The molecularmechanisms by which acclimation to elevated temperature maymodulate the ability of C. gobio to tolerate subsequent Cd exposureare discussed regarding ongoing proteins identification by MS/MS.

The present investigation provides data indicating that globalwarming, in the range predicted by scientists, could modulate theability of poikilotherms to tolerate chemical insults by acting as stressfactor synergists/antagonists.

doi:10.1016/j.cbpa.2010.06.123

7.iCod: Integrative environmental genomics of cod (Gadus morhua);the proteomics approach

O.A. Karlsen,M. Brattås, S. Bjørneklett, K. Berg, I. Eidhammer, A. Goksøyr,University of Bergen, Norway); V. Zgoda (Orekhovich Institute ofBiomedical Chemistry RAMS, Russia)

The aim of this project is to develop a deeper understanding oftranscriptional and translational responses of the cod genome toenvironmental insults, and integrating these into mechanistic in-sights using a systems biology approach. The project is a first steptowards a systems biology understanding of genomic responses toenvironmental stress in the Atlantic cod (Gadus morhua), animportant species in both North-Atlantic fisheries and aquaculture.One of the current ongoing proteomic projects of iCod targets theeffects of methylmercury (MeHg) exposure on cod. MeHg is a highlyneurotoxic and ubiquitous environmental contaminant that humansare being exposed to through consumption of contaminated fish andseafood. Two-dimensional gel electrophoresis combined with MALDI-ToF mass spectrometry is used to identify differentially expressedproteins in the brain and liver proteomes of cod exposed to MeHg.Among differentially regulated proteins we observe members offunctional classes such as cell integrity, biotransformation and redox

reactions. The differentially regulated proteins are candidates for newand more sensitive biomarkers for MeHg exposure for use inenvironmental monitoring and risk assessment. Annotation andexpression studies of cytochromes P450 (CYP) present in cod arealso in progress. MALDI-ToF- and ORBITRAP mass spectrometry areused to identify CYPs isolated from liver microsomes of β-naptho-flavone treated cod. Further, genome mining is used in mapping CYPspresent in the cod genome.

Funded by the Norwegian Research Council (project 192441/I30).Thanks to the Genofisk Consortium and the Cod Genome SequencingProject Team for sharing data in advance of the public release of thecod genome data.

doi:10.1016/j.cbpa.2010.06.124

8.In vivo experiments for the evaluation of protein expressionprofiles in Zebra mussels (Dreissena polymorpha) exposed tobenzo[α]pyrene

C. Riva, A. Binelli, F. Rusconi, G. Colombo, A. Pedriali, R. Zippel, A. Provini(University of Milan, Italy)

It is recently established that the use of proteomic techniques canbe an useful tool in the biomonitoring programs to assess the waterquality since these methodologies could be applied to obtain proteinexpression signatures of the pollutant exposure that could be furtherused for the validation of new biomarkers. Despite the fact that themussel Dreissena polymorpha is considered a reference model forfreshwater ecosystems, the investigation of proteomic approach inthis organism has not been deeply investigated. To this end, severalzebra mussel specimens were exposed for 7 days to two differentconcentrations (0, 1 and 2 μg L−1) of benzo[α]pyrene (B[α]P) whichis well known in inducing DNA damage and alteration in antioxidantdefenses. Changes in protein expression profiles were investigated incytosolic fractions of gills from control/exposed male and femalemussels by 2-DE electrophoresis. We evaluated an overall change inexpression profile of 25 proteins in exposed mussels, 13 and 12 ofwhich were respectively over and under expressed. Spots of interestwere manually excised and analyzed by a MALDI-TOF/TOF massspectrometer. The most significant identified altered proteins arerelated to oxidative stress, signal transduction, cellular structure andgeneric metabolism. This preliminary study shows the utility ofproteomic approach also in ecotoxicological studies, even if musselsare poorly represented in sequence databases. Further studies on theidentified proteins are however needed in order to provide crucialinformation and to understand possible mechanisms of action of B[α]P in this biological model.

doi:10.1016/j.cbpa.2010.06.125

9.A proteomic approach to study the influence of climate change inmarine organisms—Hydrothermal vents as a model of unstableenvironments

R. Company (Universityof Algarve, Portugal);O. Antúnez, A. Torreblanca(University of Algarve, Portugal); M.P. Cajaraville (University of theBasque Country, Spain); M.J. Bebianno (University of Algarve, Portugal)

Climate change is one of the most important challenges in sciencenowadays, mainly because it affects the majority of ecosystems and lifeforms, however the exact long term consequences are still very much


unknown. Climate change may influence the occurrence, dispersion andtoxicity of contaminants present in the marine environment and it islikely that organisms will be increasingly exposed to higher levels ofstress inducers. In this context, deep-sea hydrothermal vents, emanatinga wide range of toxic compounds (metals, polycyclic aromatic hydro-carbons, methane, and sulphide) can be considered a model of unstableenvironments. Mussels Bathymodiolus azoricus live in three major ventsites (Menez-Gwen, Lucky Strike and Rainbow) with markedly differentwater chemistry,whichreflects on their oxidative status. It is aimed touseproteomic tools to identify the effects of extreme environmentalconditions comparable to climate change alterations on the proteomeof B. azoricus. Results show that 31 proteins had different expressionprofiles among vent sites and both cluster and principal componentanalysis confirm a clear separation of samples between sites. Amongidentified proteins by mass spectrometry are antioxidant defenceenzymes (catalase), metabolism proteins (S-adenosylhomocysteinehydrolase) and structural proteins (actin). Environmental proteomicscan evaluate the effects of environmental changes by detecting complexchanges in protein expression profiles among hydrothermal ventorganisms subjected to increasing load of toxic compounds and oxidativestress caused by fluctuating water chemistry conditions and contami-nants.Nevertheless, proteomics showsanenormouspotential to evaluateclimate change effects on non-vent marine organisms as well.

doi:10.1016/j.cbpa.2010.06.126


10.Depletion of abundant proteins from Bothrops jararaca snakeplasma using chromatography for proteomics analysis

A.M. Tanaka-Azevedo, G.L. Moreira (Laboratório de Fisiopatologia,Instituto Butantan, São Paulo, Brazil)

Plasma, the best known but perhaps the most complex biofluid,contains thewidest dynamic range of cellular protein species in the body.The 22 most abundant proteins constitute more than 99% of the mass oftotal plasma proteins. Therefore, depletion of the high abundant proteinsbecomes a critical step in the plasma proteome profiling, especiallywhenthe limit peak capacity and low dynamic range of mass spectrometryanalysis occur with protein identification. HiTrap Blue HP column isknown for human albumin purification. So, the aim of this work was todeplete the most abundant protein, albumin, from the Bothrops jararacasnake plasma, with the intention of extending the dynamic range ofproteome detection for greater coverage and diversity of proteinidentifications. In a one-step process, albumin was partially removed byaffinity chromatography using aHiTrap BlueHP column and the collectedfractionswere analyzed by 1D and 2DSDS-PAGE. The results showed thatalbuminwas depleted around 40% by 1D SDS-PAGE, while analysis by 2Dresulted in a lower number of spots in the depleted plasma than nondepleted plasma, 56 and 91, respectively. We concluded that HiTrap BlueHP column is effective to deplete plasmatic albumin from B. jararacasnake plasma; however, it depletes other proteins too. The perspective ofthis project is to identify spots’ proteins in order to analyze whether thispurification step is as necessary as it is for human plasma. These resultsare important considering the limitation of materials, such as snakeplasma and venom, and could also be useful to deplete highly abundantproteins from other animals’ plasmas.

Supported by: FAPESP (08/08140-8, 09/03484-3, 09/54708-9 and09/15573-0).

doi:10.1016/j.cbpa.2010.06.127

11.Fourier transform ion cyclotron resonance mass spectrometry as atool for exploring viperid plasma proteomes

A.M. Tanaka-Azevedo (Laboratório de Fisiopatologia, InstitutoButantan, São Paulo, Brazil); K.F. Grego (Laboratório de Herpetologia,Instituto Butantan, São Paulo, Brazil); A.S. Tanaka (Departamentode Bioquímica, UNIFESP-EPM, São Paulo, Brazil); K. Morais-Zani(Laboratório de Fisiopatologia, Instituto Butantan, São Paulo, Brazil);C. Tokarski, C. Rolando (Chimie Organique et Macromoléculaire etProtéomique, Modifications Post-traductionnelles et Glycobiologie,Université des Sciences et Technologies de Lille, France)

Proteome analyses of plasma identify thousands of proteins thatcan explain the relation among snake venom, snake plasma and evenwith other animal plasmas. Its combination with unmatched massresolution,mass accuracy, and exceptional detection sensitivity,makesthe Fourier Transform Ion Cyclotron Resonance Mass Spectrometry(FTICR-MS) a powerful new technique for high-throughput proteinanalysis. This work shows the use of 1D gel electrophoresis of Bothropsjararaca plasma followed by slice-by-slice analysis of the proteinssubjected to digestion followed by Fourier transform ion cyclotronresonance (FTICR). The analysis of the most concentrated gel slicesamples by FTICR identified 7 proteins, which presented similarity tothe following snake proteins: precursor C3 complement from Najanaja, complement protein C3-2 and venom factor from Austrelapssuperbus, depleting complement factor from Bungarus fasciatus, serumalbumin andphospholipase A2 inhibitor 1 precursor from Trimeresurusflavoviridis, anti-hemorrhagic precursor and metalloproteinase inhi-bitor from B. jararaca. Our results confirmed that FTICR-MS is well-suited for high-throughput snake plasma proteome analysis.

Supported by: FAPESP (06/61735-4, 08/08140-8, 09/54708-9 and09/03484-3).

doi:10.1016/j.cbpa.2010.06.128

12.Circadian rhythms of oxidative stress markers in Ovis aries, Bostaurus and Equus caballus

G. Piccione, S. Casella, S. Marafioti, A. Torre, C. Faggio, F. Trischitta(University of Messina, Italy)

Circadian rhythmicity is an inherent property of living systemsand constitutes an essential part of their internal and externaltemporal order. Changes in circadian rhythms of oxidative stressmarkers were studied to evaluate the possible involvement of thecircadian organization of light/dark cycles on the fluctuation of redoxstate into three different species. The experimental groups consistedof 5 Ovis aries, 5 Bos taurus and 5 Equus caballus. On all animals, keptunder natural photoperiod (sunrise 05:10/sunset 20:45), bloodsamples were collected on the same days and with the sameprocedures every 4 h over a 48 h period, starting at 8:00 on day 1and finishing at 8:00 on day 3. Obtained sera after centrifugationwere immediately analyzed by means of a UV spectrophotometer forthe assessment of reactive oxygen metabolites (dRoms), antioxidantbarrier (Oxy-ads) and thiol-antioxydant barrier (SHp). Two-waymeasures ANOVA showed a significant effect of time and differentspecies (P<0.0001) and the trigonometric statistical model of thesingle cosinor procedure indicated the existence of a circadianrhythm of parameters. The oxidative stress parameters showed amesor value statistically significant in all studied species (P<0.001),a nocturnal acrophase in sheep and diurnal acrophase in cattle andhorse. The amplitude was significant on Oxy-ads in horse vs sheep


and cattle (P<0.05) instead the robustness was significant on dRomsin horse vs cattle and on SHp in all studied species (P<0.05). Inconclusion, we can claim that there is a synergism between oxidativestress markers and the circadian rhythm of anti-oxidant power indifferent species.

doi:10.1016/j.cbpa.2010.06.129

13.Protein expression profiles in Mytilus galloprovincialis exposed toa combination of contaminants

V.L. Maria, T. Gomes, M.J. Bebianno (University of Algarve / CIMA,Faro, Portugal)

The knowledge of aquatic organisms responses exposed con-currently to more than one toxic contaminant is a hard task inecotoxicology evaluation. Chemical interactions of mixtures can beperceived when the observed effect is bigger than expected (morethan additive), similar (additive) or less than predicted (less thanadditive). Copper (Cu) and benzo(a)pyrene (BaP) are stable andpersistent environmental contaminants with several chronicsublethal effects recognized in marine invertebrates, namelymussels.

The purpose of this research was the assessment of theinteractive toxicity of BaP (10 μg/L) and Cu (10 μg/L) in a binarymixture using the observed toxicity of the single metal and organiccompound species and of their binary mixture. This toxicity wasevaluated by the proteomic analysis in order to convey futureidentification of proteins suitable for biomarkers of metal andorganic compounds mixtures. Polypeptides of the cytosolic frac-tion of gill were separated by 2D-SDS PAGE with immobilized pHgradient in the first dimension. The protein expression profiles(PEPs) obtained by staining with silver were digitalised andanalysed with PDQuest software.

In the gill, a number of significant differences were identified inPEPs between the selected exposure conditions (BaP, Cu and thebinary mixture). Specific and common protein expression signatureswere identified for the three treatments.

doi:10.1016/j.cbpa.2010.06.130

14.Selection of high-affinity aptamers for chloramphenicol

J. Mehta, B. Van Dorst, E. Rouah-Martin (University Antwerp, Belgiumand Institute for Agricultural and Fisheries Research, Belgium); W. DeCoen, R. Blust (University Antwerp, Belgium); J. Robbens (UniversityAntwerp, Belgium and Institute for Agricultural and FisheriesResearch, Belgium)

Chloramphenicol, an effective antibiotic, has lost favour due to itsserious side effects such as aplastic anemia, bone marrow suppressionand leukaemia. The health and environmental concerns of chloram-phenicol are a result of their physicochemical properties which aredirectly derived from their structures. To detect these 3D structures, weengineered specific DNA aptamers that recognize their targets primarilyby shape and hence the name, ‘aptus’, whichmeans ‘to fit’. Aptamers arebeing increasingly used as the recognition element in biosensors. Theyoffer advantages over antibodies, since they are very stable, cost-effective, animal-friendly and bind tightly to their targetswithout losingtheir efficiency, even in complex matrices.

In vitro selections were used to identify chloramphenicol bindingaptamers. A stringent selection protocol was employed, in order to gethighly specific and sensitive aptamers. Selections were conducted byimmobilising chloramphenicol onto magnetic beads and incubatingthem with a random oligonucleotide library consisting of approxi-mately 1015molecules. The boundDNAwas eluted, amplified andusedas an input for the next selection round. At the eighth round, aptamerswith desired affinity (nanomolar range) were formed. The secondarystructure analysis of the different aptamers obtained, revealed thediversity amongst them while also showing some distinct character-istics common to all. The selected aptamerswere further characterisedwith spectroscopy, whereby their affinity constants were determined.The highest binding aptamer will be used to develop a biosensor thatallows easy detection of chloramphenicol in food samples such asmilk.In view of the unprecedented advantages brought by aptamers, weexpect aptamer-based antibiotic biosensors to find broad applicationsin environmental and food monitoring.

doi:10.1016/j.cbpa.2010.06.131

15.Maldi TOF profile of Mytilus galloprovincialis haemolymphproteins

L. Perić, E. Robotti, L. Boatti, F. Marsano, A. Negri, E. Marengo, A. Viarengo(University of Piemonte Orientale, DiSAV, Italy)

Mussels (Mytilus galloprovincialis) are sentinel species routinelyused for monitoring the health status of coastal waters. Mussel'scirculatory fluid (haemolymph) contains secreted proteins as well asproteins coming from the damaged tissue, which could be useful asindicators of risk associated with exposure to environmentalcontaminants.

In this work, we developed a simple procedure for rapid screeningof mussel's haemolymph proteins based on the matrix assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and chemometric data analysis. To overcome the difficultiesrelated to relatively high dilution of proteins and the presence ofsalts, an easy and reproducible technique of solid-phase proteinextraction was applied, followed by direct elution of sample on theMALDI-target plate. After preliminary optimization of analyticalparameters, a sensitive MALDI TOF mass-spectrometry was capableto effectively resolve the low molecular weight haemolymph proteinsthanks to their rapid ionization in the <20 kDa m/z region. Finally,MALDI MS peptide profiling was applied to haemolymph samples ofcopper-exposed mussels to detect lost and/or gained ions within theMALDI spectra and variations of ions intensity upon treatment. Thepossibility to use the developed method to monitor the changes inmussel's haemolymph peptides/proteins profile and to identify newbiomarkers related to pathological states of organisms is discussed.

doi:10.1016/j.cbpa.2010.06.132

16.Differential protein expression in Corbicula fluminea exposed to amixture of pharmaceutical products

F. Geret (LaboratoireGEODE,UMRCNRS5602, CentreUniversitaire Jean-François Champollion, Place de Verdun, Albi, France); T. Gomes(University of Algarve, CIMA, Campus de Gambelas, Faro, Portugal);P.Marty (Laboratoire GEODE, UMRCNRS 5602, Centre Universitaire Jean-François Champollion, Place de Verdun, Albi, France); M.J. Bebianno(University of Algarve, CIMA, Campus de Gambelas, Faro, Portugal)


Pharmaceutical products are a major group of chemical com-pounds that are continuously released into the environment. They areemerging pollutants or emerging environmental contaminants andtheir amount is steadily rising. Numerous studies show thepersistence of these pharmaceutical residues in surface waters. Aprogram called “Health and the Environment” (PNSE) started inFrance in 2004 to address the issue of these emerging pollutants andinclude pharmaceutical products mainly aspirin, diclofenac, carba-mazepin, paracetamol, metformin, and atenolol since 2008.

The aim of the study is to assess the effect of a mixture of pharma-ceutical products (aspirin (200ng L−1), ibuprofen (600ng L−1), diclofe-nac (380ng L−1), paracetamol (36mg L−1), metformin (735ng L−1),naproxen (185ng L−1) and atenolol (35ng L−1)) on freshwater molluscCorbicula fluminea exposed to concentrations present in French riversusing a proteomic approach.

Protein expressionprofiles (PEPs) in the freshwater clamgills exposedto the mixture of the pharmaceutical products (3 and 14 days) werecompared tounexposedones. Cytosolic fractionsof gillswere analyzedbytwo-dimensional (2D) electrophoresis (pH 4–7). On average, about 1000protein spots were resolved after silver staining. Changes in proteinexpression profiles were quantitatively detected in gills. Pharmaceuticalproducts induce major changes in tissue-specific protein expressionprofiles in gills. After 3 days of exposure, 15 proteins where up-regulatedin clam gills when compared to unexposed ones, three of them up-regulated 5-fold while the other tenwere up-regulated 4-fold. Moreoverfive proteins were down-regulated 5-fold when compared to controls.However, after 14 days of exposure, a total of 28 proteins were down-regulated (nine 10-fold and nineteen 5-fold)when compared to controls.Moreover, eight proteins were up-regulated (5-fold) and three 10-fold.The intensity of two up-regulated proteins increases with the time ofexposure (PM=33.1 KD and PI=5.26; PM=88.5 KD, PI=6.6). Someproteins were only present in unexposed clams (especially, protein withPM=30.9 KD and PI=5.71)while others only appeared in exposed ones(especially, protein with PM=30.54 KD and PI=5.59). The alteredprotein spots were excised, digested and analyzed by MALDI-TOF-MS.

doi:10.1016/j.cbpa.2010.06.133

17.Identification of a 2-cys peroxiredoxin in the extreme halophyteArthrocnemum macrostachyum

A. Trotta, F. Marsano, A. Antonacci (University of Piemonte Orientale,DiSAV, Italy); S. Redondo-Gomez, E. Figueroa (Universidad de Sevilla,Spain); R. Barbato (University of Piemonte Orientale, DiSAV, Italy)

Halophytes are plants belonging to phylogenetically unrelatedfamilies, able to tolerate environmental salt concentrations which arelethal for the greatest part of the world's flora. Salt stress leads tophysiological water deficit, limited CO2 assimilation and overreduc-tion of the electron transport chain. The same holds true inhalophytes when grown in limiting NaCl concentrations. Thus, inglycophytes as in halophytes, high salt growing conditions lead tophotooxidative stress, which results in ROS generation. Thiol reducingsystems regulate the activity of target proteins by change in the redoxstate of thiol groups. One of the terminal targets in this network is the2-cys Prxs which reversibly shifts between the oxidized dimeric formand a reduced oligomeric form. Prxs have a physiological role notonly in detoxifying H2O2 but also in redox sensing and in signallingpathways modulation. In this work we report the identification bytetramethylbenzidine/H2O2 staining method and mass spectrometryof a 2-cys Prx isolated from thylakoids of the extreme halophyteArthrocnemum macrostachyum grown in different external NaClconcentration (i.e. 0 g/l; 5 g/l; 10 g/l; 30 g/l; and 60 g/l). The activity

of 2-cys Prx is strongly related to the external NaCl concentration,suggesting a role in adaptation to different salt concentrations.

doi:10.1016/j.cbpa.2010.06.134

18.Effects of low pH on gill protein expression in two fish species,stickleback (Gasterosteus aculeatus) and cod (Gadus morhua)

J. Sturve, F. Gunnarsson, L. Förlin (University of Gothenburg, Sweden)

Climate changes are not the only consequence of increasing CO2

concentration in the atmosphere. The increase in atmospheric CO2

concentration increases the partial pressure of CO2 (pCO2) in surfacewaters, reducing the pH of all open waters leading to oceanacidification and hypercapnia. Long term effects of ocean acidificationon marine habitats and organisms are not fully investigated,especially not physiological effects on adult fish. Proteomic 2D-gelelectrophoresis (2D-GE) is a good approach for comparative analysesgiving a specific 2D pattern of expressed proteins. 2D-GE was used toidentify differences of pH acclimatization in the gills of two marinefish species, cod (Gadus marhua) and stickleback (Gasterosteusaculeatus), kept in pH 7.7 or 8.1 for three weeks. These species werechosen since they are adapted to different fluctuations in the abioticenvironment, the stickleback being more used to changes in theenvironment. The 2D protein expression pattern gave significantlyregulated spots (three in cod and two in stickleback) in low pHexposure groups and four of the protein spots were significantlymatched with mass spectrometry identification. The spots detectedwere: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) andhemoglobin beta chain in cod and annexin max3 and proteasomesubunit N3 in stickleback. These results show that lower pH leads to apossible stress forcing the fish to up regulate proteins in order tomeet new challenges. Additionally, SELDI analysis will be used inorder to investigate if the magnitude of protein regulation differsbetween the investigated species.

doi:10.1016/j.cbpa.2010.06.135

19.Nonylphenol in marine organisms in North American estuaries:Trophic chain accumulation and proteomic responses

L. Tomanek, J.M. Diehl, S.E. Johnson (California Polytechnic StateUniversity San Luis Obispo, USA); K. Xia (Mississippi State ChemicalLaboratory, USA); S.J. Teh (University of California at Davis, USA)

We characterized the presence of the endocrine disruptor 4-nonylphenol (NP) within Morro Bay, California, and four other westcoast estuaries in North America. Our goals were to determine howNPaccumulates along the trophy chain within a small estuarineecosystem, identify sources, and explore a suggested link with tissuepathologies of the arrow goby, Clevelandia ios. Concentrations of NP inseawater, sediment, organisms, and household products, were deter-mined byGC–MS, and C. ios tissueswere examined histopathologicallyto characterize gonadal tumors and liver pathologies. Proteomicstudies were conducted using gel electrophoresis and mass spectro-metry. NP concentrations increased from seawater (<1 ppb) toestuarine sediment (38 ppb, dw), and continued to increase withtrophic position for molluscs, fish, sea otters and marine mammals.Gobies and/or molluscs from the five estuaries had similarly high NPlevels for all species regardless of nearby urbanization. A common


input is wastewater, suggested by excessive NP in septic sludge(5000 ppm dw), likely originating from toilet paper where NP rangesbetween 50 and 2790 ppb (ww). Based on histopathology, 29% offemale C. ios from Morro Bay have gonadal tumors, and livers of bothgenders display glycogen depletion and macrophage aggregates,indicative of environmental contaminants. Tumors and most liverpathologies are absent in C. ios from British Columbia, Canada,coinciding with a restricted range of NP in fish tissues. A proteomicanalysis in response to laboratory exposure showed pronounced andlong-term (60 days) changes in the abundance of C. ios liver proteinsinvolved in energy metabolism and oxidative stress.

doi:10.1016/j.cbpa.2010.06.136

20.Differentially expressed proteins improved the tolerance toarsenic in Pteris vittata colonized by arbuscular mycorrhizal fungi

E. Bona, C. Cattaneo, F. Marsano, G. Lingua, P. Cesaro, M. Cavaletto,G. Berta (University of Piemonte Orientale, DiSAV, Italy)

Pteris vittata can tolerate soil As concentration up to 1500 ppmand rapidly accumulates the metalloid in its fronds. However, itstolerance to As has not been completely explored. Arbuscularmycorrhizal (AM) fungi colonize the root of most terrestrial plants,including ferns. It has been observed that plants growing on arsenicpolluted soils are usually mycorrhizal.

The aimof the presentworkwas to study the effects of the AM fungiGlomus mosseae and Gigaspora margarita on P. vittata plants treatedwith As. The interest was focused on protein expression in the fronds.

For proteomic analysis, leaf proteins were extracted by the TCA-acetone method and separated by 2DE. Spots of interest wereidentified by MS/MS analysis.

Image analysis showed that As treatment, in the absence of AMsymbiosis, induced the up-regulation of 19 proteins and the down-regulation of 36 proteins. Arsenic predominantly affected proteinsbelonging to the photosynthesis and carbon fixation group.

In G. mosseae plants treated with As, 26 proteins disappeared,5 were down-regulated and 40 proteins were up-regulated withrespect to As plants; in G. margarita plants treated with As, 25proteins disappeared, 13 were down-regulated and 30 were up-regulated with respect to As plants.

The proteomic approach has been extremely useful in underlyingthe specificity of each AM fungal symbiosis in the arsenic metabolismof P. vittata. The fern fronds displayed different protein profiles,reflecting different molecular strategies adopted to tolerate/detoxifyarsenic.

doi:10.1016/j.cbpa.2010.06.137

21.Effects of the arbuscular mycorrhizal symbiosis on proteinexpression in the leaves of an elite poplar clone grown on heavymetal polluted soil

V. Todeschini, E. Bona, F. Marsano, C. Cattaneo, G. Berta, G. Lingua,M. Cavaletto (University of Piemonte Orientale, DiSAV, Italy)

Arbuscularmycorrhizal (AM) fungi establish amutualistic symbiosiswith the roots of most plant species. While receiving photosynthates,they improve themineral nutrition of the plant and can also increase itstolerance towards some pollutants, like heavy metals.

In this work a clone of Populus alba L., previously selected for itstolerance to copper and zinc, was used to investigate the effects of thesymbiosis with the AM fungus Glomus intraradices on the leaf proteinexpression. Poplar leaf sampleswere collected from plantsmaintained ina glasshouse on polluted (copper and zinc contaminated) or unpollutedsoil, after four, six and sixteen months of growth. For the proteomeanalysis, poplar leaves were ground in liquid nitrogen and proteinswere extracted with TCA/acetone precipitation. All samples wereseparated by 2-DE using linear pH 3–10 and 4–7 IPG strips. Comparativeanalysis was performed with Same Spot software and results werestatistically analysed byANOVA. Identificationswere carriedout by nano-LC ESI Q-TOF MS/MS, followed by searching the NCBInr database.

At each harvest, about 400 proteins were reproducibly separated.At the first harvest the most relevant effect on protein modulationwas exerted by the AMF, at the second one by the metals, and at thelast one by both treatments. The modulated proteins concern variousmetabolic activities, most notably photosynthesis, sugar and glu-tathione metabolism, oxidative damage and protein synthesis.

doi:10.1016/j.cbpa.2010.06.138





Session 11: Environmental metabolomics novel biochemical approaches

INVITED LECTURE

1.A metabolomic investigation to identify molecular changesinduced in roach after exposure to the anti-androgen organopho-sphate pesticide fenitrothion

A.D. Southam, A. Hines, M.R. Viant (University of Birmingham, UK);A. Lange, C.R. Tyler (Exeter University, UK); E.M. Hill (SussexUniversity, UK)

Xenobiotic-induced endocrine disruption and toxicity in wildfish populations are of major international concern. There is acritical need to discover biomarkers that can be measured rapidlyto determine the causative class(es) of pollutants and informdirectly on reproductive fitness. This presentation will describe ametabolomic study investigating the effects of the anti-androgen,fenitrothion, on a freshwater fish species, the roach (Rutilusrutilus). Sexually mature male fish were exposed to 2, 20 and200 μg/L of fenitrothion for 28 days and their tissues harvested forhistology and metabolomics. Non-targeted nuclear magneticresonance (NMR) and mass spectrometry (MS)-based metabolo-mics were used to identify changes to the polar metabolome inliver and testes tissue, whilst liquid chromatography MS was usedto investigate sex steroids in testes tissue. Univariate statisticaltesting within the non-targeted datasets (with false discoverycorrection, FDR<10%) revealed a greater change to the metabo-lome of liver tissue (9.9% of NMR bins and 5.8% of MS peaks weresignificant) compared to testes tissue (0% of NMR bins and 1.6% ofMS peaks were significant). This indicated that fenitrothionexerted a greater metabolic effect in the liver compared to thetestes. Specific metabolic alterations included highly significant(FDR<10%) changes to creatine (NMR: p=4.5×10− 7; MS:p=1.9×10−5) and phosphocreatine (NMR: p=4.3×10−4; MS:p=1.3×10−3) levels in liver tissue only. Also, N-acetyl-phenyla-lanine levels were significantly increased as fenitrothion dose was

increased in both liver (p=1.3×10−3) and testes (p=3.8×10−4).A significant decrease in cortisone (p=0.004) and a near signifi-cant increase in 11-hydroxyandrostenedione (p=0.057) wereobserved as fenitrothion dose increased, confirming an endocrinedisruptive effect.

doi:10.1016/j.cbpa.2010.06.140

ORAL PRESENTATIONS

2.Seasonal variations of p38 MAPK phosphorylation and heat shockprotein expression in red blood cells of Sparus aurata.Involvement of p38 MAPK in the induction of hsp70

K. Feidantsis (Aristotle University of Thessaloniki, Greece); T. Markou(NHLI Division, Faculty of Medicine, Imperial College London, RoyalBrompton Campus, London SW3 6LY, UK); H.O. Pörtner (Alfred-egener-nstitut für Polar-nd Meeresforschung, Physiologie marinerTiere, Germany); A. Lazou, B. Michaelidis (Aristotle University ofThessaloniki, Greece)

For ectothermic marine animals including fish, environmentaltemperature has a pervasive impact and influences functions at alllevels of biological organization, from the organismal to themolecular. Understanding of how “environmental signals” such assurface and water temperature are translated into signals at the scaleof the organism or cell is essential for assessing the impact of climatechanges on marine animals. The aim of this project was to investigatethe seasonal stress response in the red blood cells of theMediterranean fish Sparus aurata during one year acclimation periodin the field (at sea surface and at depth of 10–12 m). Molecularresponses were addressed through the expression of hsp70 andhsp90 and the phosphorylation of stress-activated protein kinases








(p38 MAPK). The expression of hsp70 and hsp90 was induced whenthe fish were acclimatized to the lowest seasonal temperatures.Increased phosphorylation of p38MAPK at the same temperaturesindicated the parallel activation of MAPK signaling cascades. Theexpression and the activation of these particular proteins can be usedas indicators of a general cold stress response mechanism known as“winter syndrome”. In order to investigate the potential involvementof p38 MAPK in the induction of the hsps in these cells, red bloodcells were incubated in the presence of a p38 MAPK inhibitor atdifferent temperatures. Inhibition of p38 MAPK resulted in thedownregulation of hsp70 implicating this kinase in hsp70 induction.

doi:10.1016/j.cbpa.2010.06.141

3.Integrated effects of molecular and metabolic stress in Modiolusbarbatus in the field

M. Katsikatsou, A. Anestis (Aristotle University of Thessaloniki,Greece); H.O. Pörtner (Alfred-egener-nstitut für Polar-nd Meeres-forschung, Physiologie mariner Tiere, Germany); B. Michaelidis(Aristotle University of Thessaloniki, Greece)

Following our previous study on the seasonal stress profile of thebivalve Modiolus barbatus, we also investigated the ubiquitin-conjugates and the succinate seasonal changes of the mantle andthe posterior adductor muscle (PAM) in order to assess thephysiological condition in more detail, in view of the climaticchanges. The bivalves were placed in the water column in twodepths, namely in 3 m from the sea-surface and in 17 m deep near thesea-floor, i.e. their natural habitat. The ubiquitin-conjugates displayedinteresting fluctuations. Specifically, for samples placed 3 m deep,one pick was made during winter and another during spring for bothtissues. For the samples in 17 m simultaneous downregulation wasobserved in PAM, while in mantle a winter downregulation wasfollowed by a gradual upregulation during spring. Interestingly, thesamples placed near the sediment demonstrated enhanced levels ofsuccinate, indicating higher levels of hypoxia. In both tissues thesuccinate pattern was biphasic, indicating cellular hypoxia mainlyduring early spring and summer. This pattern was more intense in themantle for the samples in 17 m. The above results imply thatadditional cellular stress occurs during the gametogenesis and thespawning, when ATP turnover demands are elevated. Furthermore,there is a potent correlation between the stress effects and thesediment nearness. Regarding the environmental stress, mortalityevents, have frequently been associated with limited glycogencontent, in eutrophic ecosystems, as was the case of this study.Moreover, the high temperatures which lasted for longer dailyperiods in 3 m samples had adverse effects for the bivalves.

doi:10.1016/j.cbpa.2010.06.142


4.Environmental metabolomics and multibiomarker approaches onbiomonitoring of aquatic habitats

F. Iacono, T. Cappello (Department of Animal Biology and MarineEcology, University of Messina, Italy); C. Corsaro, C. Branca (Depart-ment of Physics, University of Messina, Italy); M. Maisano, G. Gioffrè,E. De Domenico, A. Mauceri, S. Fasulo (Department of Animal Biologyand Marine Ecology, University of Messina, Italy)

Organic compounds, heavy metals and drugs poured into naturalenvironments engender harmful effects on aquatic organisms due totheir biotransformation. The degree of pollution in natural habitatshas given a new urgency in developing of novel technologies to earlyassess the environmental health status. Environmental metabolomicsis an emerging approach for examining metabolic profiles inbiological systems exposed to environmental stress. Recently, it isapplied to value the anthropic impact on aquatic habitats throughbioindicators and its results integrate information obtained byconventional cellular and molecular methods.

In this study, specimens of Serranus scriba, a benthonic andterritorial organism, were collected from Priolo (Siracusa, IT), anindustrial site, and from Vendicari (Siracusa, IT) a natural reserve.Moreover, some cageswithMytilus galloprovincialis individuals, havinga high filtration capacity, were located in both sites. Liver andhepatopancreas metabolic profiles were obtained by high resolutionNMRspectroscopy, providing fingerprints ofmetabolic phenotypes.1HNMR spectra quali-quantitative analysis allowed to characterize ametabolite pool, in which the amount of each metabolite varies inrelation to the physiological responses of organisms. Multivariatestatistical analysis allowed to discriminate amongmetabolic profiles ofspecimens living in both areas. Histochemical analyses revealedmorphological alterations on gills of organisms from Priolo, comparedwith control. Furthermore, in the same samples immunohistochemicaltechniques showed greater immunopositivity for MT and CYP,biomarkers detecting heavymetals and IPApollution. Genic expressionlevels of both biomarkers have been valued by RT-PCR, confirmingimmunohistochemical observations. The applied approaches are thususeful and efficient to assess the environmental health status.

“PRIN 2007” Project Funds.

doi:10.1016/j.cbpa.2010.06.143








Session 12: Cellular reaction and modelling of cell processes in response to pollutants

INVITED LECTURE

1.Autophagy as a protective process in environmentally-inducedoxidative stress

M.N. Moore (Plymouth Marine Laboratory, Prospect Place, The Hoe,Plymouth PL1 3DH, UK)

This conceptual paper addresses the role of lysosomal autophagyin cellular defence against environmentally-induced oxidative stressusing a marine mollusc (the blue mussel) as an experimental model.It is proposed that augmented autophagic removal of oxidativelydamaged organelles and proteins provides a second tier of defenceagainst oxidative stress. Age pigment or lipofuscin is a product ofoxidative attack on proteins and lipids and can accumulate inlysosomes, where it may generate reactive oxygen species (ROS)and inhibit lysosomal function, resulting in autophagic failure. Theprotective role of autophagy induced by nutritional deprivationagainst oxidative stress can be explained by this model, whereautophagy boosts “cellular housekeeping” through enhanced removalof ROS-damaged proteins and organelles minimising formation ofharmful stress/age pigment (lipofuscin). Finally, the probable lowlevel triggering of autophagy in mussels by fluctuating environmentalregimes is considered as a potential protective mechanism that willcontribute to tolerance to environmental oxidative stress. It is furtherconjectured that organisms making up functional ecological assem-blages in fluctuating environments, where up-regulation of autop-hagy should provide a selective advantage, may be pre-selected to betolerant of pollutant-induced oxidative stress. Additional support forthis conjecture has come from system-level modelling studies ofhealth and dysfunction in living cells, based on network and graphtheory. The complexity of the whole system increases when sub-systems, such as detoxication and anti-oxidant protective processes,augmented autophagy, protein degradation and induction of stressproteins, are up-regulated and start to interact significantly as part of

a response to low-level stress, (i.e., biphasic or hormetic response).However, with increasing severity of stress, cell injury and higher-level functional impairment lead to physiological dysfunction andbreakdown of the whole interaction network with consequent loss ofcomplexity.

doi:10.1016/j.cbpa.2010.06.145

ORAL PRESENTATIONS

2.Gene and protein regulation in the marine diatom Thalassiosirapseudonana upon exposure to polycyclic aromatic hydrocarbons

R.N. Carvalho, S.K. Bopp, T. Lettieri (Laboratory of Molecular Ecology,Institute for Environment and Sustainability, European CommissionJoint Research Centre, Italy)

The polycyclic aromatic hydrocarbons (PAHs) are environmentalpollutants ubiquitously distributed. They are generated by incompletecombustion of organicmaterials such aswoodor fossil fuels. Due to theircarcinogenic, mutagenic effects and to their wide distribution in theenvironment, these pollutants pose many concerns to researchers andregulators. In our laboratories we investigated the inhibition growtheffect of either single PAHs or amixture of three compounds e.g. pyrene,fluoranthene and Benzo[a]pyrene (BaP) in marine diatom Thalassiosirapseudonana. In order to investigate themechanismof action of PAHs,weexposed the diatoms for 24 h to 36.45 µg/L of B(a)P which inhibits thegrowth by about 30% and analysed the gene and protein expressionprofile by DNA microarray and itraq labelling, respectively.

The DNA microarray analysis revealed more than 500 regulatedgenes, including lacsA, a gene coding for Long-chain Acyl-CoASynthetase protein, involved in fatty acid metabolism. The regulationat protein level was confirmed by immunostaining.








The proteomics’ profile analysis showed that around 10% of thetotal identified proteins were regulated and one fourth of themconfirmed the gene expression changes.

Particularly interesting was the down regulation of the Silicontransporter 1 (ST1), an enzyme that is responsible for the uptake ofsilica from the media into the cell of diatoms. The gene and proteinwere both down regulated and it is considered a promisingbiomarker of exposure to PAHs. Indeed the changes in the ST1expression were confirmed also when the diatoms were exposed tomarine samples contaminated by PAHs.

doi:10.1016/j.cbpa.2010.06.146

3.Utility of biomarkers in the assessment of marine pollution

M.U. Beg (Institute for Scientific Research, Kuwait)

Aquatic life-based water quality criteria are designed to providea reasonable level of protection to all the taxa present in a waterbody. The criterion serves as a distinct numeric value that can beused to compare concentrations measured in water bodies andused to determine whether water bodies are in compliance withwater quality standards. However, chronic presence of mixtures oftoxic chemicals over a large surface area in sub-optimal concentra-tions is the real problem. Organisms are subjected to a number andvariety of suspected pollutants in the environment, therefore,multiple measures of health are needed to help identify andseparate anthropogenic-induced effects of stress from those effectscaused by natural stressors. A variety of stress responses have beenmeasured in biota collected from the marine area of Kuwait toindicate various types of stresses. Exposure to bioavailable PAHswas assessed by determining metabolites in the bile of fish usingfixed-wavelength fluorescence (FF). Fish liver 7-ethoxy resorufin-o-dethylase (EROD) activity was determined as a biomarker ofexposure to planar hydrocarbons. Heat shock proteins (HSP70)were determined in fish gills, muscle and liver tissue as stressbiomarker of temperature variations and other physical orchemical stimuli. A large variation in biomarker response wasobserved in fish collected from the same place at the same time.The utility of the wide range of biomarker data will be discussed inthe assessment of marine pollution and predicting hazard.

doi:10.1016/j.cbpa.2010.06.147


4.The marine environment I.Q. conceptDeveloping an index of the quality of the marine environmentbased on biomarkers: Integration of pollutant effects on marineorganisms

D.M. Pampanin, E. Ravagnan, S. Apeland, N. Aarab, B.F. Godal,S. Westerlund (IRIS, Stavenger, Norway); D.Ø. Hjermann (Uni-versity of Oslo, Norway); T. Eftestøl (Stavanger University,Norway); M. Budka, B. Gabrys (Bournemouth University, UK); A.Viarengo (University of Piemonte Orientale, DiSAV, Italy); J.Barsiene (Institute of Ecology, Vilnius, Lithuania)

The main goals achieved in the project were:

1. A simple stepwise method for risk assessment analysis for themarine environment based on multiple biological responses(biomarker approach) was constructed. The analytical power ofArtificial Neural Networks (ANN) for water quality classificationbased on biomarker data was assessed.

2. Environmental quality information was extracted from biologi-cal responses of marine organisms. The link between biomarkerresponses and water containing PAHs was studied and theoutput is satisfying. General Additive Models proved to be auseful and robust method in describing relationships betweenbiomarkers and combinations of biomarker responses.

3. Suitable sets of biomarkers for monitoring programs, dependingof the type of pollutants present in the environment, were to bedefined. Data deficiencywas themain challenge for this goal. Setsof biomarkers, focused on PAHs andmetals, were established andused in the water quality classification performed with ANN. Afield validation study was also performed.

4. A mapping system (Geographic Information Systems) to make thetool accessible for public environmental agencies was applied. Thisproject has shown the possibility to extract valuable informationfrom complex data indicating pollution (biomarkers), using sophis-ticated statistical and/ormodelling tools, displaying the results in aneasy and comprehensible way. Water quality classification wasattempted with different "classifiers" other than ANN, to explorenew methods extrapolating maximum information from the actualincomplete dataset, and to test possible future use with differentspecies and more complete datasets.

doi:10.1016/j.cbpa.2010.06.148





Session 13: Biological effects of Nanoparticles mixture toxicity

INVITED LECTURE

1.Nanoparticles in Natural Aquatic Systems and Cellular Toxicity

A.H. Ringwood, M. McCarthy (University NC Charlotte, North Carolina,USA); N. Levi-Polyachenko (Wake Forest University, Health Sciences,Winston-Salem, North Carolina, USA); D.L. Carroll (Wake ForestUniversity, Center for Nanotechnology and Molecular Materials,Winston-Salem, North Carolina, USA)

Nanoparticles will be introduced into aquatic environments duringproduction processes and released as wastes following their use inelectronic and biological applications. Environmental factors such asorganics and salt content will affect their bioavailability and toxicity.The complex milieu of natural environments may facilitate theirdissolution into constituent components or may stabilize them. Thereare also many uncertainties about their cellular interactions, potentialroutes of entry, and target processes. Recent findings from mylaboratory and others are reviewed to describe some of the importantcurrent findings regarding carbon and metal nanoparticles, and theirfundamental cellular interactions. Ourmodel system has been oysters,Crassostrea virginica – a filter-feeding bivalve, and some of the cellularresponses that have been studied are lysosomal integrity, oxidativedamage, antioxidant responses, and protein responses (e.g. metal-lothioneins); studies with embryos are also conducted to considerimpacts on developmental responses. Quantum dot and fullerenestudies have demonstrated that nanoparticles are readily accumulatedinto cells, probably via endosomal pathways, and accumulate inlysosomes. Increases in oxidative stress have been observed, support-ing the concept that oxidative damage will be an important mechan-ism of toxicity. Increases inmetallothionein expressionmay reflect thepotential for silver nanoparticle interactions with metal regulationpathways or may also reflect oxidative stress. Silver nanoparticlestudies suggest that there may be shape-dependent toxicity. Whilenanoparticles represent a new class of contaminant, there are

numerous issues regarding their stability, their reactivity with naturalfactors and other contaminants, and how they interact with biologicalsystems that need careful investigation.

doi:10.1016/j.cbpa.2010.06.150

ORAL PRESENTATIONS

2.Dietary exposure of silver nanoparticles in the endobenthicpolychate Nereis diversicolor

J. García-Alonso (Department of Zoology, Natural History Museum ofLondon, UK); S.K. Misra (Department of Mineralogy, Natural HistoryMuseum of London, UK); F.R. Khan (Department of Zoology, NaturalHistory Museum of London, UK); A. Dybowska (Department of Miner-alogy, Natural History Museum of London, UK); B.D. Smith, P.S. Rainbow(Department of Zoology, Natural History Museum of London, UK);S.N. Luoma (US Geological Survey, Menlo Park, USA); E. Valsami-Jones(Department of Mineralogy, Natural History Museum of London, UK)

Nanoparticles such as nanosilver (Ag-NPs) are widely used inindustries such as those producing textiles, biomedical devices andwashing machines. Therefore, we can expect an impact from theirinevitable release into the environment. Estuarine sediments are sinkfor many pollutants and are also the habitat and food source of thepolychaete Nereis diversicolor. Dietary exposure to NPs from sedi-ments is a potential route of toxicity of these chemicals. We describehow ingested citrate capped Ag-NPs (30±5 nm) behave inside thegut, the metal uptake and its accumulation. We test if uptake is due todirect internalisation of particles or by solubilisation of the particlesand subsequent uptake of dissolved Ag. Nereis diversicolor wereexposed to sediment spiked with Ag-NPs (250 ng g-1) in estuarinewater (salinity 16) for 10 days at 10 °C. Electron dense particles the








size of the Ag-NPs were visualized in the lumen and gut epitheliumby TEM. In some cases Ag-NPs appeared to be associated withendocytotic vesicles. Silver Bioaccumulation of Ag the ragwormtissues was evidenced by concentrations in cytosolic fractionsseparated by homogenization and centrifugation. Alternatively,dissolved Ag could also be generated inside the gut and pass intothe cells through dissolved metal uptake routes. Further methods ofanalysis, including energy dispersive x-ray (EDX) are in progress toconfirm the direct uptake of Ag-NPs by the intestinal epithelial cells.

doi:10.1016/j.cbpa.2010.06.151

3.Integration of conventional biomarkers and proteomic analysis toassess the effects of copper nanoparticles inMytilus galloprovincialis

T. Gomes (University of Algarve, Portugal); I. Cancio (University of theBasque Country, Spain); J.P. Pinheiro, M.J. Bebianno (University ofAlgarve, Portugal)

Soluble forms of copper are highly toxic, being involved in redoxreactions which result in the production of reactive oxygen species,leading to damages in lipids, carbohydrates, proteins and DNA.Nonetheless, scarce information exists on copper behavior as ananoparticle in the aqueous environments and its interaction and

toxicity in aquatic organisms. In order to study the effects of coppernanoparticles in Mytilus galloprovincialis, an integrated approach wascarried out, combining conventional biomarkers with proteomics.The biomarkers chosen were the antioxidant enzymes (catalase andsuperoxide dismutase), metallothionein (MT), lipid peroxidation(LPO), acetylcholinesterase (AChE) and DNA damage. With theobjective of comparing copper effects in the nanoparticle and solubleform, mussels were exposed to copper nanoparticles (Cu NPs) andbulk copper as CuSO4 for a period of 15 days and changes wereassessed in the gills and digestive gland. Copper nanoparticlesmodified biomarker responses in Mytilus galloprovincialis, withinhibition/activation of antioxidant enzymes, MT induction, lipidperoxidation and DNA damage during the whole exposure period.Inhibition of AChE was only detected at the end of the experiment.Significant differences were detected in protein expression profilesbetween Cu NPs and bulk copper, with an overall increase of proteinspots in gills and digestive gland. Specific and common proteinexpression signatures were identified for Cu NPs and CuSO4. Twentysix 2-fold up- and down-regulated proteins were common for Cu NPsand CuSO4 in the gills and 56 in the digestive gland. The resultsobtained provide a first insight of copper nanoparticles effects inMytilus galloprovincialis.

doi:10.1016/j.cbpa.2010.06.152





Session 14: Role of biossays and community studies in the understading of biologicaleffects of pollution

INVITED LECTURE

1.Importance of bioassays to understand population impacts andrisk assessment of emerging contaminants

T.H. Hutchinson (Centre for Environment, Fisheries & AquacultureScience, UK)

The Marine Strategy Framework Directive and Water FrameworkDirective both emphasize the need to address the risks posed toaquatic ecosystems from hazardous chemicals. Understanding theexposure scenarios and biological effects of chemicals on ecologicallyand commercially important organisms is central to environmentalrisk assessment. Bioassays have proven invaluable in detecting andassessing the hazards of chemicals for aquatic organisms over manyyears, and it is important to continue to invest in validated bioassaysand biomarkers to meet the needs of European and other policy stakeholders (e.g. OECD and UNEP). However, the rapid increase in thenumbers of synthetic chemicals being invented each year, alongsideeconomic and ethical factors, underlines the need for efficient(intelligent) testing and monitoring strategies based a subset ofspecies. Basically, bioassays need to be deployed based on threecriteria: (1) exposure relevance; (2) ecological relevance; and (3)toxicological mode-of-action (MOA) relevance. For example, anECETOC Task Force (http://www.ecetoc.org Technical Report number102) has approached this challenge using the Verhaar et al (Chemo-sphere (1992) 25: 471-491) categorization system which separateschemicals into classes that can be assigned a toxic mode ofaction (MOA). These are: MOA1 - inert chemicals (baseline toxicity);MOA2 - less inert chemicals; MOA3 - reactive chemicals; and MOA4 -specifically acting chemicals. MOA4 chemicals may have toxicologicaleffects due to (specific) interactions with certain receptors. It wasconcluded that the protein target MOA approach has significantpotential to help prioritize the selection of MOA-relevant bioassays in

sensitive species. The implications for using this approach for bothfreshwater and marine hazard assessment will be discussed.

doi:10.1016/j.cbpa.2010.06.154

ORAL PRESENTATIONS

2.PLHC-1 fish hepatoma cell line as a tool to assess environmentalquality of coastal sediments

S. Schnell, S. Lacorte, C. Porte (Environmental Chemistry Dept., IDAEA–CSIC–, Barcelona, Spain)

Several studies have shown that fish cell lines are useful screeningtools to evaluate the toxic potential of aquatic pollutants. In this workwe used the fish hepatoma cell line (PLHC-1) in order to characterizethe cytotoxic potential and presence of CPY1A inducing agents insediments collected from ten estuarine and coastal areas alongNorthern Spain, including areas affected by industrial and/or urbaneffluents as well as reference sites. Sediments were extracted withdichloromethane/hexane (1:1, v/v) followed by dichloromethane/acetone (1:1, v/v) and the combined extracts applied on PLHC-1 cellsto evaluate their cytotoxic potential by using Alamar Blue (AB) andcarboxyfluoroscein diacetate acetoxymethyl ester (CFDA-AM). Fiveout of ten extracts showed potential to induce cytotoxicity whentested at 50 to 100 mg eQsed/mL. Exposure of PLHC-1 cells to non-toxic concentrations of the sediment extracts in the range of 0.05 to100 mg eQsed/mL lead to a significant induction of 7-ethoxyresor-ufin-O-deethylase (EROD) activity after 24 hours exposure. Inductionwas evident as soon as 3 hours after exposure. Additionally, theability of sediment extracts to induce ROS was assessed by using 2’,7’-dichlorohydrofluorescein diacetate as a probe; those extracts being






http://www.ecetoc.org














cytotoxic to cells led to a 30 to 70% induction in ROS production.Overall, the study highlighted the usefulness of PLHC-1 to assess thetoxic potential of coastal sediments and the environmental risks foraquatic organisms inhabiting those areas.

doi:10.1016/j.cbpa.2010.06.155

3.Lethal and sublethal effects of polybrominated diphenyl ethers(PBDEs) for turbot (Psetta maxima) early life stage (ELS)

L. Mhadhbi (ECIMAT, University of Vigo, Spain); J. Fumega (InstitutoEspañol de Oceanografía, Spain); M. Boumaiza (Faculty of Sciences,Bizerte, Tunisia)R. Beiras (ECIMAT, University of Vigo, Spain)

A new toxic menace, polybrominated diphenyl ethers (PBDEs), isbeing detected in the aquatic environment all over the world. Theenvironmental presence of PBDEs and its entry into the environmentas BDE-47, 99 and 154 make quality aquatic toxicity data necessary toassess the aquatic hazard risk of PBDs. This study examines the effectsof three PBDE-47, 99 and154 on embryo and larval stages of themarineflatfish turbot (Psetta maxima). The acute toxicity of the three PBDEswas examined and NOEC, LOEC, EC10 and EC50 were calculated. Alltested compounds caused lethal as well as nonlethal malformationsduring embryo development. The effects of PBDEs in the different lifestages of turbot were analysed. PBDEs seemed to be teratogenic atconcentrations higher than 7.5; 20 and 30 µgL-1 for BDE-47, 99 and 154respectively, leading to prolonged delays in embryo development andconsequent death (at 48 h), as well as severe malformations andmortality of larvae. PBDEs showed a higher acute toxicity for embryo-larvae (EC50 for lethal endpoints to embryos 24.07 µg BDE-47 L-1,30.89 µg BDE-99 L-1and 47.6 µg BDE-154 L-1, and 16.1, 24.39 and29 µg L-1 for BDE-47, 99 and 154 respectively for larvae).

Generally, the BDE-47 seemed to cause adverse effects atcomparatively low dose rates, whereas much higher doses wereneeded to cause the same effects with BDE-99 and 154. The results ofthe present study show that the acute toxicity of PBDEs decreases asthe degree of bromination increases, since the order of toxicity wasBDE-47 > BDE-99 > BDE-154. The major isomers also exhibited aclear toxic potential for turbot ELS although at high concentrations,above solubility saturation, pointing at particulate matter as the mainvia of uptake for those hydrophobic molecules.

doi:10.1016/j.cbpa.2010.06.156

4.Microbial ecology methods for assessing the effects of xenobioticsin water and soil ecosystems

A. Barra Caracciolo, P. Grenni (Water Research Institute–NationalResearch Council, Rome, Italy)

Microbial communities are valuable indicators of the occurrenceof disturbances due to exogenous physico-chemical stressors. Theassessment of variations in microbial community structure is of basicimportance to permit to evaluate the impact of an environmentalstressor.

Complex microbial communities may serve as ideal and ecologi-cally relevant toxicity indicators. A number of microbiologicallydriven processes have been proposed to evaluate the effects of xeno-biotics on ecosystems. Proteins, genes, metabolites, or lipids that,

when expressed, present a pattern of molecular change in an or-ganism in response to a specific environmental stressor, can bedefined as environmental biomarkers.

The number of techniques to study microbial communities hasincreased exponentially over the last 20 years and the advent ofculture-independent methods, such as molecular biological techni-ques, has changed the view of microbial diversity. Among thesetechniques epifluorescence microscopy techniques, such as directcount of bacterial abundance (DAPI count) and viability (Live/Deadcell viability assay), and Fluorescence In Situ Hybridization (FISH) arevery useful to study microbial communities directly from theirnatural environment. We report some examples of application ofthese techniques for evaluating the effects of some contaminants(pesticide and pharmaceuticals) to soil and water ecosystems.

doi:10.1016/j.cbpa.2010.06.157

5.Coupling chemical data and pollutant-induced biological effectsincreases reliability in environmental risk assessment: Fromsublethal biomarkers to community studies

A. Dagnino, S. Fenoglio, L. Avidano, S. Sforzini, A. Viarengo (Universityof Piemonte Orientale, DiSAV, Italy); T. Forte, C. Ottavi, M. Peleggi(ISPRA, Rome, Italy)

Ecological risk assessment is world-wide applied in estimatingenvironmental impairments due to contamination. Aiming toobjectively evaluate hazards to biota, chemical, ecotoxicological andecological data are integrated into the Triad approach.

In fact, pollutant concentrations “per-se” are inadequate todescribe real exposure patterns in soils due to the large number oftoxic substances (more than 248000), the lack of reliable estimatorsof bioavailability, and the uncertainty in predicting interactions incontaminant mixtures. Direct measures of pollutant-induced effectsat different levels of biological organization, applying ecotoxicologicaland ecological techniques, can lead to clearly estimate real impacts onbiota and consequently environmental risk and biological vulner-ability.

In the last decade, this exposure-effect approach was applied byour research group in studying different contaminated sites through-out Italy. Obtained results demonstrate that coupling chemical andbiological data consistently improve reliability in environmental riskassessment.

Nowadays, relevant outcomes in terms of biodiversity decline areinferred through investigations on soil community structure (e.g.microarthropods, bacteria) and functions (e.g. feeding activity, soilrespiration), discriminating areas affected by high environmentalrisk. Impairments of high-level ecotoxicological endpoints (i.e.survival and reproduction rate) in model organisms exposed tocontaminated soils suggest potential consequences at population andcommunity level, characterizing sites with potential threats to bio-diversity. Also when no alteration in high-level endpoint is detected,effects on sublethal biomarkers indicate damages of organismalfunctioning and, consequently, a reduction in the ability of theorganism to cope with environmental stressors: these early-warningevidences suggest high biological vulnerability and consequentlypossible future concerns.

(Activity supported by ISPRA)

doi:10.1016/j.cbpa.2010.06.158



6.Macroinvertebrate community structure and sediment bioassayresults from three Italian lotic systems

S. Fenoglio, A. Dagnino, T. Bo, A. Copetta, N.Massa, L. Oliveri, A. Viarengo,(University of PiemonteOrientale, DiSAV, Italy); C.Ottavi, A. Felli (ISPRA,Rome, Italy)

In the last decades, biological monitoring of running watersystems has become increasingly important and, in most countries,it has become an indispensable complement to traditional chemico-physical techniques in the evaluation of human impacts. In thiscontext, the analysis of benthic macroinvertebrate community andthe use of bioassays are among the most utilized tools, due to theirdiffering sensitivity to changes in both chemical characteristics of thewater column and physical properties of habitats, but at the momentfew studies have approached the study of lotic systems utilizing bothmethods. In this study we report the results of the coupledapplication of bioassays and community ecology in three Italian loticenvironments: Bormida river (prime example of contaminated area

of the Northern Apennines), Regi Lagni and Sarno (that are amongthe largest and most contaminated rivers of southern Italy). SomeBiotic Index were used to describe macroinvertebrate taxonomicrichness and diversity, while bioassays were realised utilising abattery of organisms, such as Daphnia magna, Vibrio fischeri,Pseudokirchneriella subcapitata and the innovative and highly sensi-tive Dictyostelium discoideum.

In Bormida, bioassay responses and alterations of macroinverte-brate communities were closely related each other, highlightingbiological problems at different scales (from sublethal to biodiversityloss). Interestingly, in the Regi Lagni and Sarno benthic communitywas severely altered, while bioassays gave results slightly discordant:this difference could be caused by the huge organic load of this river.The resulting sediment hypoxia/anoxia had a different impact ondifferent ecological levels. Our work indicates that a multi-levelapproach is an effective tool in environmental monitoring, allowingdetection and deepen and better understand the conditions of thematrix river.

doi:10.1016/j.cbpa.2010.06.159


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