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Contract n° ECK3-CT2000-00025

F I N A L R E P O R T

BEEP PROJECT

(Biological Effects of Environmental Pollution in marine coastal

ecosystems)

Feb 2001-Jan 2004

Coordinator: Dr Philippe GARRIGUES

Home page:

http://beep.lptc.u-bordeaux.fr

TABLE OF CONTENT

Participants' information

p. 4

Executive Summary of the project

p. 7

Work Package 0: Coordination and Management

Task 1: Web site

p. 9

Task 2: Project workshops

p. 9

Task 3: Preparation of the project reports

p. 9

Task 4: Exploitation and dissemination of the results

p. 9

Task 5: Administrative and financial management

p. 10

Work Package 1: Novel Biomarkers

Task 1: Development of new cellular and molecular biomarkers of stress

p. 11

Task 2: Development of new cellular and molecular biomarkers of exposure

p. 23

Task 3: Development of a molecular biomarker approach by using protein antibodies and
Mrna probes

p. 33

Task 4: Development of biomarkers able to evaluate pollutant effects on the reproductive
performance of mussels

p. 43

Task 5: Development of biomarkers able to evaluate pollutant effects on the reproductive
performance of fish

p. 54

Task 6: Application of the new developed biomarkers in two common, large, lab
experiments. Application of the new biomarkers on field samples. Comparison of the value
(sensitivity, specificity, etc.) of the new biomarkers with “core biomarkers” used in WP 2 – 3
– 4

p. 60

Work Package 2: Biomonitoring in the Baltic Sea

Task 1:Intercalibration exercises on core biomarkers

p. 70

Task 2: Collection of samples and background information on sampling sites

p. 72

Task 3: Measurements of biomarkers (core and research) on the target species

p. 75

Task 4: Measurements of supporting parameters: chemical contaminants

p. 99

Task 5: Data handling

p. 100

Work Package 3: Biomonitoring in the Mediterannean Sea

Task 1:Intercalibration exercises on core biomarkers

p. 110

Task 2: Collection of samples and background information on sampling sites

p. 113

Task 3: Measurements of biomarkers (core and research) on the target species

p. 124

Task 4: Measurements of supporting parameters in fish and bivalves: chemical
contaminants and physiological parameters

p. 131

Task 5: Data handling

p. 137

Work Package 4: Biomonitoring in the North Atlantic Sea

Task 1:Intercalibration exercises on core biomarkers

p. 142

Task 2: site surveys: preparation, collection of samples and background information

p. 143

Task 3: Measurements of biomarkers (core and research) on the target species

p. 146

Task 4: Measurements of supporting parameters in fish: chemical contaminants and
physiological parameters

p. 154

Task 5: Data handling

p. 155

Work Package 5: QA/QC and data management

Task 0:Intercalibration exercises on core biomarkers

p. 158

Task 1: Preparation of an expert system

p. 158

Task 2: Multivariate analyses of biomarker measurements

p. 168

Task 3: Scale classification of pollution level of coastal areas based on multimarker
approach

p. 169

List of publications

p. 182

Multivariate Analysis WP2, WP3, WP4

p. 207

PARTICIPANTS INFORMATION

P1 Dr Philippe GARRIGUES, Coordinator, Dr Hélène BUDZINSKI, Prof. Jean-François NARBONNE

LPTC – Université Bordeaux I, UMR 5472 CNRS, 351 Cours de la Libération, 33405 Talence Cedex

France

Tel: 33 5 40 00 63 05

Fax: 33 5 40 00 22 67

E-mail:

p.garrigues@lptc.u-bordeaux1.fr

P2 Prof. Aldo VIARENGO, Coordinator WP1

Dipartimento di Scienze e Avanzate Tecnologie, Universita ‘Amedeo Avogadro’, Corso Borsalino 54, 15100
Alessandria – Italy
Tel: 39 0131 283804

Fax: 39 0131 254410

E-mail:

viarengo@unipmn.it

P3 Dr Kari LEHTONEN, Coordinator WP2

Finnish Institute of Marine Research, Lyypekinkuja 3A, PO Box 33, 00931 Helsinki – Finland

Tel: 358 9 613941

Fax: 358 9 613944 94

E-mail:

lehtonen@fimr.fi

P4 Dr Gilles BOCQUENE, Coordinator WP3, Dr Thierry BURGEOT

IFREMER DEL/PC, Rue de l’Ile d’Yeu, 44311 Nantes Cedex 3 - France

Tel: 33 2 40 37 41 20

Fax: 33 2 40 37 40 75

E-mail:

gbocquen@ifremer.fr

P5 Dr Odd-Ketil ANDERSEN, Coordinator WP4

RF- Rogaland Research, Prof. Olav. Hanssensvei 15, P.O. Box 8046, 4068 Stavanger – Norway

Tel.: 47 51 87 50 00 / 50 50 Fax: 47 51 87 52 00

E-mail:

odd-ketil.andersen@rf.no

P6 Dr David LÖWE / Pr JONES

NERC – Plymouth Marine Laboratory, Prospect Place – The Hoe, Plymouth, PL1 3DH – United Kingdom

Tel.: 44 1752 633 208

Fax: 44 1752 633 102

E-mail:

d.lowe@pml.ac.uk

P7 Dr Miren P. CAJARAVILLE

Biologia Zelularra cta Histologi Laborategia, Euskal Herrido Unibertsitatea, Universidad del Pais Vasco, 644PKE,
48080 Bilbao – Spain
Tel.: 34 94 6012697

Fax: 34 94 4648500

E-mail:

zopcabem@lg.ehu.es

P8 Dr Vangelis PAPATHANASSIOU

National Center for Marine Research - Institute of Oceanography, PO Box 712 Anavissos 19013 – Greece

Tel.:

302910.76.368

Fax: 302910.76.323

E-mail:

vpapath@ncmr.gr

P9 Dr Vasilis DIMITRIADIS

Aristotle University of Thessaloniki, School of Biology, Dept. of Biology, Dept. of Genetics, Development and
molecular biology, University Campus, 54006 Thessaloniki – Greece

Tel.: 0310-998351

Fax: 0310-998298

E-mail:

vdimitr@bio.auth.gr

P10 Dr Christophe MINIER, Prof. François LEBOULENGER

Université du Havre – Laboratoire d’Ecotoxicologie, 25 Rue Philippe Lebon, BP 540, 76058 Le Havre – France

Tel.: 33 2 32 74 43 03

Fax: 33 2 32 74 43 14

E-mail:

minier@univ-lehavre.fr

P11 Dr Cinta PORTE

Departament de Quemica Ambiental – CSIC, Jordi Girona 18, 08034 Barcelona – Spain

Tel.: 34 93 400 61 75

Fax: 34 93 204 59 04

E-mail:

cpvqam@cid.csic.es

P12 Dr Michel AUFFRET

Université de Bretagne Occidentale, Institut Universitaire Européen de la Mer, Technopole Brest-Iroise

29280 Plouzane – France

Tel.: 33 2 98 49 86 49

Fax: 33 2 98 49 86 45

E-mail:

michel.auffret@univ-brest.fr

P13 Dr James DEVILLERS, Coordinator WP5

CTIS, 3 Chemin de la Gravière, 69140 Rillieux la Pape

Tel.: 04 78 08 49 84

Fax: 04 78 08 56 37

E-mail:

j.devillers@ctis.fr

P14 Prof. Peter D. HANSEN

Institut für Ökologie, Technische Universität Berlin, Keplerstrasse 4-6, 10589 Berlin – Germany

Tel.: 49(0)30 31 42 14 63

Fax: 49(0)30 83 18 113

E-mail:

pd.hansen@tu-berlin.de

P15 Prof. Lars FÖRLIN

Goteborg University, Dept. Zoophysiology, Box 463, 40530 Göteborg – Sweden

Tel.: 46 31 773 3676 Fax: 46 31 773 38 07

E-mail

lars.forlin@zool.gu.se

4 sur 238

P16 Dr Angela KÖHLER

Alfred Wegener Institutes for Polar and Marine Research, Laborgebäude 3, Notkestrasse 85, 22607 Hamburg –
Germany

Tel.: 49(0)471 4831 1407

Fax: 49(0)471 4831 1425 E-mail:

akoehler@awi-bremerhaven.de

P17 Dr Janina BARSIENE

Institute of Ecology, Akademijos 2, 2600 Vilnius – Lithuania

Tel./Fax: 370 2 700666

E-mail:

janbar@takas.lt

P18 Prof. Lennart BALK

Institute of Applied Environmental Research, Laboratory for Aquatic Ecotoxicology, Stockholm University, 10596
Stockholm – Sweden

Tel.: 46 8 674 7251

Fax: 46 8 674 7638

E-mail:

lennart.balk@itm.su.se

P19 Prof. Bjorn Munro JENSSEN

Dept. of Zoology, Norwegian University of Science and Technology, 7491 Trondheim - Norway

Tel.: 47 7359 6267

Fax: 47 7359 1309

E-mail:

bjorn.munro.jenssen@chembio.ntnu.no

P20 Dr Demetris SAVVA

Division of Cell and Molecular Biology, University of Reading, Whiteknights, P.O. Box 228, RG6 6AJ Reading –
United Kingdom

Tel.: 44(0)118 987 5123

Fax: 44 118(0)931 6671 E-mail:

d.savva@reading.ac.uk

P21 Dr Roger RAHMANI

Centre de Recherches INRA, Laboratoire de Pharmaco-Toxicologie Cellulaire et Moléculaire, BP 2078, 06606
Antibes Cedex – France

Tel.: 33 4 93 67 88 60

Fax: 33 4 93 67 30 40

E-mail:

rahmani@antibes.inra.fr

P22 Dr Doris SCHIEDEK, Dr Rolf SCHNEIDER

Institut für Ostseeforschung Warnemünde, Seestrasse 15, 18119 Rostock - Germany

Tel.: 49(0)381 5197205

Fax: 49(0)381 5197440

E-mail:

doris.schiedek@io-warnemuende.de

P23 Dr Pekka VUORINEN

Finnish Game and Fisheries Research Institute, P.O. Box 6, Pukinmaenaukio 4, 00721 Helsinki – Finland

Tel.: 358 205 751277

Fax: 358 205 751201

E-mail:

pekka.vuorinen@rktl.fi

P24 Dr Thomas LANG

Bundesforschungsanstalt für Fischerei, Institut für Fischerökologie, Deichstrasse 12, 27472 Cuxhaven – Germany

Tel.: 49(0)4721 38034

Fax: 49(0)4721 53583

E-mail:

t.lang@t-online.de

P25 Dr Janusz PEMPKOWIAK

Marine Chemistry and Biochemistry Department, Institute of Oceanology, P.O. Box 197, Sopot - Poland

Tel.: 48 58 55 17 281

Fax: 48 58 55 12 130

E-mail:

pempa@iopan.gda.pl

P26 Dr Edward SKORKOWSKI

Biological Station, Gdansk University, 80680 Gdansk-Sobieszewo – Poland

Tel.: 48 58 308 07 14

Fax: 48 58 308 07 03

E-mail:

skorkows@biotech.univ.gda.pl

P27 Dr Aminadav YAWETZ

Tel Aviv University, Faculty for Life Sciences, Institute Nature Conservation Research, P.O. Box 39040, Ramat
Aviv, 69978 Tel Aviv – Israël

Tel.: 972 3 640 80 04

Fax: 972 3 640 73 04

E-mail:

amidav@post.tau.ac.il

P28 Dr Jens GERCKEN

Institute of Applied Ecology, Lindenweg 2, 18184 Neu Broderstorf – Germany

Tel. 49(0)38204 6180

Fax: 49(0)38204 61810

E-mail:

gercken@ifaoe.de

P29 Dr Claudia BOLOGNESI

Toxicological Evaluation Unit, National Cancer Institute, L. go Rosanna Benzi 10, 16132 Genova – Italy

Tel.: 39 010 5600215

E-mail:

blgcld@hp380.ist.unige.it

P30 Prof. Malcolm JONES

Plymouth Environmental Research Center – University of Plymouthv - Drake Circus, Plymouth PL4 8AA, UK.
Tel: +44 1752-232940 - Fax: +44 1752-232970

Email:

mal.jones@plymouth.ac.uk

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List of the workpackages:

WP0 – Management and Coordination (Dr Philippe GARRIGUES)

WP1 – Novel Biomarkers (Prof. Aldo VIARENGO)

WP2 – Biomonitoring in the Baltic Sea (Dr Kari LEHTONEN)

WP3 – Biomonitoring in the Mediterranean Sea (Dr Gilles BOCQUENE)

WP4 – Biomonitoring in the North Atlantic Sea (Dr Odd-Ketil ANDERSEN)

WP5 – QA/QC and Data Management (Dr Philippe GARRIGUES)

6 sur 238

Executive Summary

Biological markers allow the direct determination of pollutant impact on living organisms in aquatic systems.
While new emerging biomarkers are actually under evaluation, some common markers are in a
validation-phase and may be used as assessment tools for the quality of the marine environment. The goal
of the European Research Project BEEP (Biological Effects of Environmental Pollution in Marine
Ecosystems) was to evaluate the use of biological markers determined in marine organisms as a means of
assessment of chemical contamination.

This integrated multi-disciplinary, -site and -marker research project combines special European expertise in
biology, biochemistry, ecotoxicology, environmental chemistry and data handling has enabled a
comprehensive study of selected coastal European environments and their responses when exposed to
varying levels of pollution and numerous chemical contaminants (heavy metals, pesticides, hydrocarbons,
chlorinated compounds).

Different types of coastal European environments (Baltic Sea, North Atlantic Sea, and Mediterranean Sea)
have been investigated by 30 participants who have co-operated on the three selected coastal environments
through a long term study. Further more two joint studies organised in the laboratory of Aquamiljo (RF
Rogaland, Stavanger) have put together during several days several dozens of BEEP researchers who have
performed complementary works on a exeperimentally polluted mesocosms.

The BEEP Project is also part of the EU funded projects supported by the Europen Commission (RTD
programme) within the EESD (Energy, Environment and Sustainable Development) programme of the EC. It
belongs to the Cluster IMPACTS coordinated by C. Eccles at the European Commission

The specific objectives of the project are as follows:

•

To develop new biological markers ranging over different levels of biological organizations.

•

To validate the use of selected biomarkers in specific sites for both routine assessment of chemical

contamination and for the improvement of national and international monitoring programmes

•

To prepare information and advices for user group, policy-makers and fishery institutions about

biological effects of chemical contamination on coastal marine resources,

•

To determine the effects of environmental contamination on end-users (fisheries, marine

aquaculture)

•

To establish a network of biomarker researchers through European countries.

In order to assess these objectives, the research programme has been organised into different
workpackages:

Project Management (WP0): The main coordinator and the 5 workpackage coordinators were part of the co-
ordination committee responsible for the integration of the works of the partners, the communication between
participants, the exploitation of the results and the production of deliverables . The BEEP Web site has been
set up as an essential tool for the project partner (infos, database, presentations, results).

Novel biomarkers (WP1): Development of new biomarkers of stress/exposure at both the cellular levels and
subsequent effects at the population level. This workpackage was in charge of developping new approaches
in biomarker methodology to be transferred to in situ studies.

Biomonitoring Programmes in Baltic Sea (WP2), Mediterranean Sea (WP3) and North Atlantic Sea (WP4):
Selected sites in each coastal environment have been monitored during three years for deploying a set of 5
common biomarkers for all the workpackages. In addition other specific biomarkers have been also studied
for in situ validation.

Data Management (WP5): Various data treatment approaches have been developped to sort and to analyse
the data: expert system, statistical analyses and pollution ranking scale based on biomarkers. A data base
has been built for an easy access to all the BEEP partners.

7 sur 238

The expected achievements of the BEEP project have been obtained:

•

Improvement and developpement of the knowledge on biological markers in marine organisms

exposed to chemical stresses in coastal environments

•

Selection a standardized battery of biological markers for implementation of biomarker techniques in

national/international monitoring programmes (OSPARCOM, HELCOM)

•

Improvement the quality of data realted to biomarker meassurments in view of coming EU directives

for the environment and the consumer protection.

This final report present the results obtained during the three-year project by all the BEEP participants and
has been organised according to the different workpackages. Three worshops (Starting meeting, Plymouth,
2001; Athens Workshop, 2002; Barcelona Workshop, 2003) have put together more than 60 researchers and
students representing the 30 organisms participating to the BEEP Project. More than 110 publications, 130
presentations in international meetings and 35 PhD dissertaions have been produced in the frame work of
the BEEP project. A comprehensive book compiling all the results will be prepared in the two next years.
Finally the BEEP project has been able to create an active European network of researchers involved into
the biomarker approaches into the marine coastal ecosystems, of which results are supporting the
implementation of several EU directives dealing with the aquatic environment.

8 sur 238

Work Package 0

(Management and Coordination)

The main task of WPO was to organise the communication and the dissemination of the information between
the participants of the consortium.

The coordinator of the BEEP project and the project assistant were responsible for the major taks of the
WP0.

The scientific management and the promotion of the project
The administrative and financial management
The control and the respect of the Work Plan
The communication (contact with the WP leader, the partners and the European Commission)
The dissemination and the exploitation of the results

They were assisted by the steering committee composed of the coordinator and the WP leaders. The
steering committee has had several meeting during the duration of the project to discuss update results,
actions and possible reorientations into the BEEP project.

Task 1: Set up the BEEP Web site

http://beeep.lptc.u-bordeaux.fr

The web site contents:

The description of the project (free access)
Inforamtion on the partners (free access)
Description of the Work Packages and the "in the field" monitored sites (free access).
Database containing all the results obtained during sampling cruises, sorted by working sites (login

access).

Meeting reports with all the presentations (oral and poster) as electronic files (login acess).
Project reports (management and scientific reports) (login access)
List of publications and presentations in scientific meetings.

Task2: Project workshops

The coordination team has organized the Project Workshops (one per year) for all the participants in addition
to the Meeting of the coordination Committee during these Workshops . Three general meeting took place
respectively :

June 2001 in Plymouth (starting meeting with 60 participants)
September 2002 in Athens (mid-term meeting with 70 participants)
December 2003 in Barcelona (final meting with 70 participants).

All the presentations made during these meetings can be found on the BEEP web site.

Task 3: Preparation of the reports

The coordinator prepared management, financial (cost statements) and scientific reports that maybe found
partially on the web site. The Technological Implemetation Plan has been also documented by the
coordination team.

Task 4: Exploitation of the results and dissemination

The coordinator was in charge of the publication in international journals, communications in International
Meetings, Meeting with end-users.

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The list of all the publications and presentations in scientific meetings is on the BEEP web site. The
coordinator and the Workpackage leaders have participated to various meetings including also regulatoru
meetings to set up standardized procedures based on results obtained into the BEEP project.

Task5: Administrative and financial management

All the administrative aspects were managed between the institution of the coordinator (University of
Bordeaux 1) and all the partner institutions, including the contact with the European Commission (Scientific
officer, Adminsitrative officer).

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Work Package 1

(Novel biomarkers)

The WP1 program was mainly related to the development of new and more sensitive biomarkers of stress
and exposure in both mussels and fish (Task 1 and 2). Due to the growing importance of genomics and
proteomics a special effort was devoted to the implementation of new “molecular” biomarkers (Task 3).

In addition, due to the importance of linking the biological effects of pollutants to their possible consequences
at the population level, research has been developed aiming to identify biomarkers capable of reflecting the
effects of toxic chemicals on the reproductive performance of studied sentinel organisms (Task 4 and 5).

The newly developed biomarkers were validated in comparison with “core” biomarkers in a series of common
experiments undertaken at Stavenger (Norway). Novel and core biomarkers were also compared using field
samples from animals collected along a well known pollution gradient on the Norwegian coast (Task 6).

TASK 1: Development of new cellular and molecular biomarkers of stress

Participants: P2, P6, P9, P12, P20, P21, P24, P27

This task activity comprised the development of novel and more sensitive biomarkers of stress i.e. able to
integrate the different effects of the numerous pollutants present in the marine coastal water carried out by
Partner 2. In particular, it focused on the study of pollutant induced biological changes of the cell
plasmamembrane, the first cellular component to come in contact with toxic chemicals. Special emphasis
was paid to the possible effects on cell signaling because alterations of this physiological aspect result in
major changes in cell physiology, inducing an alteration in hormonal responses that represents a common
way by which pollutant stressed organisms attempt to re-establish the physiological balance.
In addition, research was carried out to identify simple and sensitive methods capable of demonstrating
mitochondrial damage, an important aspect of cell function that is underestimated in biomonitoring
programmes.

Some effort was also dedicated to render one of the most sensitive biomarkers, lysosomal membrane
stability, of more general use (i.e. adapted for use in molluscs living in low saline marine environments).
Finally, new technologies for the evaluation of DNA damage were developed with the aim of rendering the
methods more sensitive and/or easier to use. Emphasis was also given to the possible development of
biomarkers of apoptosis a well known phenomenon that may be also in part related to the effects of toxic
chemicals.

The study of the effects of chemicals on signal transduction were conducted in an integrated way using the
group Partner 2 and Partner 9. In particular, the studies have concentrated on three of the main aspects of
signal transduction including Ca and tyrosine kinase dependent transduction pathways (P2) and the
signaling cyclic AMP (cAMP) dependent (Partner 9) transduction pathway.

Partner 2 studied the effects of heavy metals on cell calcium. In the research about the alteration of Ca

signalling by heavy metals, as a starting point we have studied in vitro effects of Hg

and Cu

on trout

hepatoma cells (RTH 149), using confocal imaging of fluo 3-loaded cells. Both metals have shown the
potential of inducing a rise in cytosolic Ca

in the range of 5-50 µM. The mechanisms evoked by mercury,

which produced the strongest effect were then explored in more detail. Hg

triggered intracellular Ca

waves, stimulated Ca

-ATPase activity, and promoted InsP

production. Use of various inhibitors has

indicated that Hg

induces Ca

entry through verapamil-sensitive channels, and intracellular Ca

release

via the G protein-PLC-InsP

pathway. However, in cells loaded with heparin and exposed to Hg

the [Ca

]

rise is almost abolished, indicating that the global effect of Hg

is not simply a sum of Ca

entry plus Ca

release, but involves a Ca

-induced Ca

release mechanism.

After having studied the in vitro effects of Cu

and Hg

on cell Ca

, the in vivo effects of these two metals

was investigated using mussels as model organisms. Deregulation of Ca

homeostasis can have serious

effects on cell functioning due to an alteration in Ca

signaling, the variations in plasma membrane Ca

ATPase (PMCA) were therefore evaluated. After in vivo mussel exposure to Cu

(0.3-1.3 µM) or Hg

(0.6-

2.4 µM) for 1-6 days, plasma membrane PMCA activity was cytochemically assayed on cryostat tissue
sections. In the digestive gland, Cu

inhibits PMCA, whereas Hg

induces a rise in PMCA activity. Similar

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results were found using a biochemical assay on gill PMCA. PMCA activity was compared to
immunoprecipitation data. The effect of Cu

treatment does not appear to alter PMCA expression and

reduces PMCA activity, indicating PMCA activity inhibition. Hg

treatment induces a rise in PMCA activity

that is lower than the rise in PMCA expression, suggesting an inhibition of enzyme activity that is more than
compensated by the induction of protein expression. PMCA induction is a newly discovered effect of Hg that
will possibly be further investigated by designing
molecular probes for mussel PMCA gene(s), presently
unidentified, in order to allow the use of quantitative
PCR analysis.

Finally, due to recent evidence that the activation of
Ca

-dependent phospholipase A2 (cPLA2) induces

lysosomal membrane destabilisation, the involvement
of this mechanism in lysosomal membrane
destabilisation induced by Hg

and Cu

in mussel

haemolymph cells was investigated. This is most
relevant for biomarker studies, since lysosome
membrane stability is one of the most sensitive and
widely used stress indexes in environmental
biomonitoring. The effects of heavy metals on free
cytosolic Ca

was studied using Fura2/AM-loaded

cells, and lysosomal membrane destabilisation was
studied using neutral red staining. Both metals induce
Ca

-dependent lysosome destaining and lysosomal

volume increase, indicating destabilisation of lysosomal
membranes. These effects are partially, but
significantly, reduced by a specific Ca

-dependent

PLA2 inhibitor (AACOCF3), but not by a Ca

independent PLA2 inhibitor (BEL), indicating an
involvement of cPLA2 in lysosomal membrane
destabilisation induced by heavy metals. However,
preliminary Western blot analysis also indicates a role
of p38 MAP kinase phosphorylation in this process.

Tyrosine phosphorylation promotes cell growth, differentiation
and apoptosis, due to the activity of receptor and non-
receptor tyrosine kinases. Different stressors are known to
stimulate tyrosine kinase activities and we have initially
studied the effects of heavy metals and pro-oxidants in RTH
149 cells by Western immunoblotting. Proteins from cell
lysates have been separated by SDS-PAGE electrophoresis,
blotted onto filters and probed with phosphotyrosine
antibodies. Filters were developed by chemiluminescence
and analysed by digital imaging. Use of antiphosphotyrosine
showed that Hg

and Cu

in the µM range, and H

in the

mM range, induce a significant rise in phosphotyrosine.
Phosphospecific antibodies against the three types of MAPKs
have shown that ERK is activated by heavy metals only,
while p38 and SAPK/JNK are activated by H

, Hg

, and

plus low H

. ERK activation by H

is prevented by

concomitant activation of p38. Phosphospecific STAT

antibodies have revealed activation by H

only.

Thereafter, in order to assess the suitability of protein tyrosine

phosphorylation levels as a biomarker of stress, we investigated pollutant effects on mussel digestive gland
and gill tissue after in vivo treatments of animals. Short-term in vivo treatments, consisting in the exposure of
animals in the aquarium to 0.6 µM Hg2+ or Cu2+ for 5, 10, 20 and 60 min, showed a marked increase in
phosphotyrosine after stimulation by Hg2+ for 60 min. Longer exposures of 4 and 7 days yielded less clear
results, showing either phosphotyrosine rises or decreases in a limited number of bands.

Fig. 2 Effects of Hg

and Cu

on PMCA activity

(cytochemical analysis, above) and on PMCA expression
(immunoprecipitation, below).

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Another set of analyses was made using samples derived from an experiment performed by the BEEP
Partner 5 research unit (RF-Rogaland Research, Stavanger, Norway). Mussels were exposed in vivo for 1
and 3 weeks to 0.5 ppm North Sea oil, or to a mixture of 0.5 ppm North Sea oil, 0.1 ppm alkylphenol, and 0.1
ppm PAH. The clearest results were obtained with gill tissue, showing a significant rise in phosphotyrosine
after exposure to oil for 3 weeks and to the mixture for 1 and 3 weeks. A similar but weaker effect was found
with oil in the digestive gland.
The comparison between heavy metals and organic xenobiotic compounds suggests that the mercury
induced effect is rapid but tends to disappear over time, whereas organic xenobiotics induce a delayed effect
on phosphotyrosine levels.
In order to test phosphotyrosine as a biomarker
of stress on a vertebrate organism, other
analyses were carried out based on an
experiments conducted again by the Partner 5
unit, in which turbot fish were exposed for 3
weeks to 0.5 ppm North Sea oil, or to
xenoestrogen compounds (30 ppb nonylphenol,
50 ppb dialkyl phthalate, 50 ppb bisphenol A, and
5 ppb tetrabromodiphenylether). We analysed
liver homogenates by Western immunoblotting
using antophosphotyrosine, as described above.
Increases in intensity were found in a few bands
after treatment with oil, dialkyl phthalate, and
tetrabromodiphenylether. However,
xenoestrogens also induced a decrease in
intensity in a band of 35 kDa.

Fig. 4 Results of Western immunoblotting on gill tissue,
after mussel exposure to Cu

and Hg

for short time

periods (top), or to oil and mix for 1 or 3 weeks (middle).
In this latter case, measurements of lane intensity are
shown in the bar chart (bottom).

Fig. 3 Cross-talk effect: H

activates p38

(top) but not ERK (middle), however, in the
presence of p38 inhibitors ERK is activated by
H

(bottom).

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PMCA

100

120

140

160

3 weeks

1 week

3 weeks

1 week

3 weeks

Control

50 ppb Dialkyl phthalate (F)

50 ppb Bisphenol A (B)

5 ppb TBDE

D
e
nsitometric v

a
lue

(
%
)

Catalase

100

120

3 weeks

1 week

3 weeks

1 week

3 weeks

Control

50 ppb Dialkyl phthalate (F)

50 ppb Bisphenol A (B)

5 ppb TBDE

D
e
nsit

et
ric

v
a
lue

(
%
)

Fig. 5 Results of cytochemical detection of PMCA and catalase in mussel digestive gland tissue. Both enzymes show

strong inhibition after in vivo mussel exposure to dialkyl phthalate, bisphenol A, or tetrabromodiphenylether (TBDE).

Mussels from the above in vivo experiment performed by the P5 unit were also tested using a previously-
developed cytochemical procedure for the quantification of plasma membrane Ca

-ATPase and catalase. In

this experiment, mussels were exposed in vivo for 1 and 3 weeks to hydrocarbons (0.5 ppm North Sea oil,
mix of North Sea oil, 0.1 ppm alkylphenol, and 0.1 ppm PAH), or to xenoestrogen compounds (50 ppb dialkyl
phthalate, 50 ppb bisphenol A, 5 ppb tetrabromodiphenylether). Thereafter, the digestive gland was used for
cytochemical detection and digital image quantification of PMCA and catalase activity in cryostat sections of
fresh tissue or semi-thin sections of resin embedded tissue, respectively. Significant inhibition of PMCA was
shown after exposure to oil for 3 weeks and strong inhibition was shown with all xenoestrogen compounds.
Catalase was inhibited with the mixture and oil, the latter only after 3 weeks, and with all xenoestrogens.
We have made an effort in order to develop novel biomarkers, mostly linked to cell signaling mechanisms,
able to detect the stress syndrome deriving from exposure to environmental pollutants. All the cellular targets
that were chosen for our analyses showed responses to the different pollutants, but the complexity of the
data indicates that different techniques should be used in relation to the length of exposure time and to the
kind of pollutant. Direct measurement of cytosolic Ca2+ variations in isolated cells, such as mussel
haemocytes, is indicated for detecting short-term (minutes to hours) deregulation of Ca2+ signaling, while
the cytochemical evaluation of Ca2+-ATPase activity is more suitable for long-term (weeks) effects on Ca2+
homeostasis.

Cellular levels of protein tyrosine phosphorylation can be used to detect the short-term effects of heavy
metals, while this parameter seems much more suitable for the detection of long term effects when
hydrocarbon pollutants are present. The harmful effects of organic xenobiotics, and in particular of
xenoestrogen compunds, on the mussel digestive gland seem also readily detectable using cytochemical
evaluation of Ca

ATPase or catalase.

Research concerning the effects of pollutants on cyclic AMP signal transduction pathways was carried out by
Partner 9 and aimed at standardising a method suitable for quantification of the level of cyclic AMP in various
mussel tissues.

In particular, cAMP content was estimated in three tissues (digestive gland, gills and mantle/gonad complex)
of the mussel Mytilus galloprovincialis. The mussels were collected from different stations along the Gulf of
Thermaikos (North Greece) and Olympiada, being a reference station located in the Gulf of Strymonikos, for
a period of three years (2001-2003).
cAMP was measured using [8-3H] adenosine 3´-5´ cyclic phosphate, radioimmunoassay kit (Amersham,
TRK 432).

The results showed that cAMP content in the digestive gland was generally raised in stations regarded as
being more polluted in relation to the reference station, Olympiada. cAMP values obtained in October were
lower compared to those measured in June during the three year sampling period.
Similarly, cAMP in the gills also increased in most sites along the Gulf of Thermaikos in relation to the
reference station. The latter is more clearly observed in values obtained in October during the three years
sampling period.

14 sur 238

The third tissue that was examined, the mantle/gonad complex, presented a similar profile of cAMP values to
that of gills during the sampling period. It is noteworthy that cAMP content in the mantle/gonad complex
showed significantly higher values than those measured in the other tissues (digestive gland and gills).

Furthermore, it was shown that during the sampling period of 2001 (June and October), cAMP content in the
mantle/gonad complex was significantly negatively correlated with acetylcholinesterase (AChE) activity
measured in the digestive gland of mussels collecting from the same sampling stations (r =-0.74), (Dailianis
et al., 2003). Moreover, when the cAMP and AChE values during the three years sampling were correlated,
a strong negative correlation between AChE activity in the digestive gland and cAMP in the mantle/gonad
complex (r = -0.89) and AChE activity in the gills and cAMP in the mantle/gonad complex (r = -0.97) was
observed. Also, in consistence with our results, when cAMP content in the mantle/gonad complex, during the
three years of sampling were correlated with metallothionein (MT) content in the digestive gland (r = 0.86), a
positive correlation was shown, and when correlated with AChE activity in gills and digestive gland a
negative correlation was shown. Therefore, from the three tissues examined and taking into consideration
the above mentioned correlations, the mantle/gonad tissue probably represents the most suitable tissue for
estimating cAMP. The latter cAMP content could be used as a possible novel biomarker of pollution.

Digestive gland

100

150

200

250

300

June 2001

October 2001

June 2002

October 2002

June 2003

October 2003

sam pling period

ol c

A
M
P
/
g
r
w
e
t
t
i
s
s
u
e

Olim piada

Aggelochori

Peraia

Outlet tube

Halastra

Kim ina

Gills

100

150

200

250

300

350

400

450

500

June 2001

October 2001

June 2002

October 2002

June 2003

October 2003

sam pling period

p
m
o
l
cAM

P
/
g
r
w
et

t
i
ssu

Mantle/gonad com plex

100

200

300

400

500

600

700

800

900

1000

June 2001

October 2001

June 2002

October 2002

June 2003

October 2003

sam pling period

ol c

A
M
P
/
g
r
w
e
t
t
i
s
s
u
e

15 sur 238

Figure 6. cAMP concentrations in the a: digestive gland; b: gills; c: mantle/gonad complex, of mussel Mytilus
galloprovincialis collected from different stations (Aggelochorion, Peraia, Outlet tube, Halastra and Kimina)
along the gulf of Thermaikos and Olympiada, a reference station located in the Gulf of Strymonikos during
samplings from 2001 to 2003. The results are expressed as means (pmol cAMP/gr wet tissue) ± S.D.
Significant differences between pairs of mean values are indicated in the upper triangular matrix by
asterisks. Statistical signification is based on Mann-Whitney U-test (P<0.05).

A second interesting aspect of the research on pollutant effects at the plasma membrane level was
undertaken by Partner 27 who developed a new biomarker related to “displaced haemolymph GST activity
(DH-GST)” associated with membrane stability and function in mollusks”.

The hypothesis that lead us to develop the novel biomarker Displaced Glutathione S Transferase (DGST)
activity in haemolymph is close to that of the biomarker Lysosomal Stability. Instability of the lysosomal
membrane found after exposure to xenobiotic pollutants may very well be exemplified also by cell
membranes in various tissues. Cytosolic enzymes such as reduced glutathione s-transferase may, in
pollutant exposed molluscs , leak into the haemolymph. The presence of displaced GST activity in the
haemolymph may indicate exposure of the mollusc to pollutants, as this enzymatic activity is not normally
found in the haemolymph of unexposed molluscs. The method was tested with heavy metals and chlorinated
hydrocarbons.

Displaced GST activity (DGST) in the haemolymph was very low in untreated Patella (Fig.7) Upon exposure
of Patella to heavy metal ions DGST activity appeared in the haemolymph of treated limpets. The response
was selective and dependent on the metal ion to which the limpet was exposed. Thus the response to Hg

and to Cu

ions appeared at a very low concentrations. These metal ions were also very toxic to the

mollusc. The reaction to Ni

and Pb

was moderate while the response to Cd

was more pronounced and

thus this metal served as a model stressor in this work. There was a linear correlation (Rval=0.881) between
DGST activity and the volume of haemolymph drawn from the limpet that was exposed for three days to 2
ppm Cd

ions. On the other hand, there was no correlation (Rval=0.0232) between protein concentration

and DGST activity in the small aliquot of haemolymph (5µl) drawn from Patella that was exposed three days
to daily addition of 2ppm Cd

. This finding exclude the possibility that the increase in DGST activity in the

haemolymph originates from excessive injury inflicted on the tissues by incorrect insertion of the needle. The
biomarker DGST activity in haemolymph did not compel killing of the test animal. It was possible to draw
haemolymph from each individual twice, at the beginning and at the end of experiment, without causing
death of more then 30% of the experimental animals. The response of the biomarker to chlorinated
hydrocarbons in the form of Aroclor 1254 was very low. No DGST appeared in the haemolymph of Patella
exposed to concentrations of up to 3 times the daily dose of 5 ppm Aroclor 1254. Study of the response of
the DGST biomarker in other mollusc species included, until now, only the limpet Cellana rota. DGST activity
in haemolymph of Cellana exposed for three days to 2 ppm Cd

ions in artificial seawater was about 73% of

the value recorded for exposed Patella. DGST recorded for Cellana exposed for three days to 2 ppm Pb

ions in artificial seawater 3.0±1.5 nmole/min/5µl was about the same as the value recorded for exposed
Patella.

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

TREATMENTS

0.00

2.50

5.00

7.50

10.00

12.50

15.00

D-GST activity (nmole/min/5ul)

Exposure
to Pb

Exposure
to Ni

Exposure
to Cu

Exposure
to Hg

Exposure
to Cd

Fig 7. DGST activity in haemolymph of Patella exposed for three days to indicated dose of metal ions in artificial

seawater. are mean±SD. n= about 6.

16 sur 238

Displaced DGST activity was detected in haemolymph of Patella sampled at different seasons from clean
and polluted sampling sites along the Israeli Mediterranean coastline together with determination of the
gonadal index and lysosomal stability (Fig. 8). The biomarker DGST was found to be independent of the
reproductive stage of Patell</i>a. Results obtained with this biomarker were relevant both in September when
the gonadal index was 1.0 and in September was 1.0 and in November when the gonadal index was 12.
DGST activity in the mollusc haemolymph was normally very low in Patella caerulea sampled from clean
sites at any season of the year. The clean sites were DGST activity in haemolymph of the sampled Patella
was very small were: Shiqmona, Sdot-Yam and Michmoret, all known clean resort sites. Tel Aviv also joined
the clean site criteria by the DGST biomarker, and indeed the pollution in Tel Aviv is composed of organics
from anthropogenic sources and scarcely of toxic industrial waste. In three sampling sites the DGST
biomarker showed moderate response. Of this three sites Akko and Motzqin are located in Haifa bay and
may be exposed to some level of pollution from the industry located 10 Km and 5 Km south to this sites.
These sites may also suffer occasionally from oil spills from tankers. The third sit that exemplified moderate
response of the biomarker DGST activity was Hadera. This site is located very near to coal electric power
plant. The highest response of the DGST biomarker was detected in haemolymph of Patella from the highly
polluted Haifa harbor site and Shemen beach site located very near to the harbor at the estuary of the
Qishon River that carries petrochemical sludge.

The validity of the proposed biomarker DGST activity in haemolymph was verified by comparing it to
known biomarkers in Patella collected from the highly polluted site, Haifa harbor, located near the
outlet of Qishon river that carries petrochemical sludge, and two adjacent sites that were expected to
be realatively clean: Akko, about 10 Km to the north and Shiqmona, about 8 Km to to the south. The
molluscs from the highly polluted site Haifa harbour showed by far a higher DGST activity in
haemolymph (P<0.01) and higher membrane instability compared to that in haemolymph of Patella
sampled from the two adjacent relatively clean sites: Akko and Shiqmona (P<0.01) as well as higher
Cytochrome P450 content (P<0.05). Microsomal Cytochrome b5 content, NADPH-cytochrome c
reductase as well as the cytosolic activity of GST in hepatopancreas of Patella from all the three
sampling sites in Haifa bay did not differ significantly and were practically similar.

Akko

Motzq

i
n

Shemen Bea

c
h

Haifa Harbor

Sikmo

n
a

Sdo

t-Ya

m

Hadera

Michmoret

Tel Aviv

SAMPLING

SITES

0

10

20

30

D-GST

activity (nmol/min/5µl)

September
2002, GI=1

November
2002, GI=12

November
2003, GI=12

Fig. 8. Displaced DGST activity in haemolymph of Patella sampled at different seasons from clean and polluted sampling

sites along the Israeli Mediterranean coastline. Gonadal index value in September was 1.0 and in November 12. Results

are mean±SD. n= about 15.

Concerning the assessment of DGST activity in the haemolymph of Patella cerullea it was confirmed as an
extremely sensitive index of the cellular condition very similar to the lysosomal stability technique. The dose-
response was not linear but rather polinomial, meaning that the exposure to the metal ions triggered low
response at low concentrations and a very high response at high concentrations. This may indicate that cell
membranes structure changed gradually than at a given ion concentration the membrane underwent
massive disruption and the cytosolic enzyme was poured into the haemolymph. The destabilisation of the
cell membranes had a quantitative relationship to the concentration of the metal-ions examined. The impact
of metals ions on the cell membrane (Koizumi et al., 1996) may be mediated by active oxygen species that
either modulate the activities of enzymes and ion transporters which are contained in membranes by

17 sur 238

oxidising sulfhydryl groups of these proteins (Kaenko et al., 1992) or by inducing membrane damage by lipid
peroxidation (Tsppel, 1973). The displaced GST activity leakage from tissue cells to haemolymph may thus
be metal-ions-induced membrane disintegration that develops to enzyme leakage and finally death of cells in
the limpet organs.
A method was also developed to evaluate the metabolic state of mitochondria by Partner 20. To assess the
metabolic state in tissue of living mollusks sampled from clean and polluted sites the activity of mitochondria
in living cells was identified. This was measured by microfluorometry the inherent blue fluorescence
(excitation at 365 nm, emission at 420-450 nm) of reduced nicotinamide adenine dinucleotide (NADH), and
green fluorescence (excitation at 400 nm, emission at 530 nm) of oxidized flavins of respiratory chains. The
use of known uncoupler, dinitrophenol (DNP), and an inhibitor of the respiratory chain, antimycin A, allowed
assessing the redox state of the NAD and flavins.
The results clearly demonstrate that the intensity of inherent NADH-blue fluorescence of Donax mantle was
significantly higher (P<0.01) in specimens from Akko (control site) as compared to that recorded for
specimens from Qiryat Yam and Frutarom (polluted sites). On the other hand, the intensity of green
fluorescence elicited by oxidised flavins was significantly higher (P<0.01) in Donax from the two polluted
sites, Qiryat Yam and Frutatom, as compared to that recorded in the mollusks collected from the clean site,
Akko The use of actinomycin A, a mitochondrial complex III inhibitor demonstrated that in mollusks from
Akko 70% available NAD is normally reduced while only 35-39% of available NAD is reduced in Donax from
the polluted sites, Qiryat Yam and Frutarom. Using the protonophoric uncoupler 2,4-dinitrophenol (DNP) it
was demonstrated that in Donax from the clean site Akko 55% of the available flavins are oxidised while in
the mollusks from the polluted sites, Qiryat Yam and Frutarom, 92% of the available flavins are present in the
oxidised form.

In parallel a set of experiments were established by Partner 6 to adopt the biochemical MTT assay as a
cytochemical method on living cells. The MTT assay was designed as a colorimetric cytotoxicity test and is
based on the reduction of MTT, a yellow soluble dye, by mitochondrial succinate dehydrogenase to form an
insoluble dark blue formazan product. Only viable cells with active mitochondria reduce significant amounts
of MTT to formazan. Much like the original neutral red method for lysosomes, once the reaction has taken
place the dye is extracted and the amount determined colorimetrically. The objective of these studies was
therefore to modify the assay for use with a microscope using the same rationale as for neutral red i.e. if it
can be extracted and read it can be seen down a microscope. Following incubation of attached mussel blood
cells in the MTT there was evidence of mitochondrial staining within 15 minutes. A series of assays were
undertaken using mussels from two populations in the U.K., one from a clean reference site and the other
from a population impacted by shipyard activities. The results of these studies indicated that at thirty minutes
incubation mitochondria in blood cells from the impacted mussels were paler staining, fewer in number and
smaller in size than those from reference mussels. At 180 minutes incubation, and despite evidence of
cytotoxicity in some cells, the mitochondria in blood cells from clean reference mussels were more intensely
stained.

The results of these preliminary investigations indicate that mussels from impacted sites have lower
mitochondrial activity than mussels from clean reference sites and that MTT, especially when combined with
neutral red, may have utility as a biomarker of contaminant effect. However, when the technique was applied
to field mussels from some of the sites from the Norwegian campaigns the animals inter-animal variability
was such that it was not possible to draw any conclusions and it was judged that the test was unreliable as a
biomarker for general use.

A second attempt to realise a simple cytochemical method for evaluation of mitochondrial activity was
realized utilizing Rhodamine 123. Whilst this methodology requires the use of fluorescence microscopy
therefore limiting its utility as a field tool, Rhodamine 123 stains mitochondria with a high degree of specificity
and so was considered as a possible method of choice. The protocol used for these studies was very similar
to that used for the lysosomal neutral red retention assay. Having incubated attached mussel blood cells in
the dye the preparation was then washed in fresh physiological saline to remove the Rh123, coverslipped
and the preparation viewed immediately under a fluorescent microscope using FITC filters.

The results indicated good positive staining of subcellular organelles whose size and distribution indicated
that they were mitichondria and not lysosomes. However, within a short space of time lysosomal staining
became evident which may have been the result of either direct dye uptake or the activity of the lysosomes
in taking up affected mitochondria. Whilst the method was able to identify mitochondria the subsequent
uptake into the lysosomal compartment and the necessity to view slide immediately limits its use as a
monitoring tool and it was not therefore employed in field campaigns.

In the framework of Task 1 activities some studies have also been developed by Partner 6 to improve the
lysosome membrane stability test (one of the more sensitive biomarkers ever discovered) and as a second

18 sur 238

but fundamental part to adapt the neutral red lysosmal membrane stability test methodology to analyse
organisms living in both salty marine water (i.e. the Baltic Sea).

The determination of damage to the lysosomal system of invertebrate blood cells using the dye neutral red
has proven to be very robust when used by experienced researchers, however, the end point can sometimes
be difficult to establish for the untrained eye. Acridine orange (AO), like neutral red, is taken up by
lysosomes. However, unlike neutral red which is a chromogenic dye, AO fluoresces in a range of colours
from green through to red; the colour being dependent on the amount of enzyme within the lysosome. High
levels of enzyme fluoresce red whereas low concentrations fluoresce green. In addition, secondary large
lysosomes should have lower concentration of enzymatic protein and in this case may represent more active
lysosomes. Lysosomal membrane damage results in leakage of the enzyme complement from the lysosome
into the cytosol and it was considered that leaked enzyme, being at low concentration, would fluoresce green
thus proving a simple unequivocal marker of damage. A series of laboratory studies were carried out where
mussels were exposed to a range of compounds, including North Sea crude oil, alkylphenol, nonylphenol
and mixtures thereof, and blood samples collected and incubated in AO at different concentrations and for
different periods of time. In addition, samples of blood from field mussels exposed to a range of
contaminants were also tested using AO. The objective of these studies was to develop and test the utility of
AO as a marker of contaminant exposure and effect. The results of these studies indicated that AO was less
toxic to the cells under test than neutral red. However, within ecotoxicology it is common practice to apply
additional stress (so called stress on stress) to the samples to determine their capacity to withstand further
toxic insult. The fact that AO did not impose additional stress as a result of its low toxicity meant that in all but
extreme cases of toxic insults there was insufficient enzyme leakage to generate a fluorescent signal.
However, incubation in AO did demonstrate the wide variation in the level of enzyme present in the
lysosomes with blood cells from ‘healthy’ mussels showing lysosomes with the full spectrum of colour from
green to red. Perhaps significantly there was less variability in lysosomal fluorescence in mussels from
experimental studies than field mussels and less variability in field mussels from exposed sites than clean
sites.

An adaptation of the methodology was realised during the course of the WP4 field programme in Goteborg.
In fact, it became apparent that the neutral red retention core biomarker did not work satisfactorily with
mussels that were adapted to life at low salinity. The physiological saline used routinely for this assay is 30-
32‰ salinity whereas populations of mussels in the waters around Goteborg are conditioned to living at 10-
15‰ salinity. The osmotic shock associated with immediate transfer of blood from freshly sampled mussels
to the physiological saline resulted in almost immediate dye loss from the lysosomes to the cytosol
regardless of the condition of the site from where the mussels had been sampled. As a consequence, a
sample of seawater was also collected along with mussels from each site sampled and the blood withdrawn
into a100µL of the seawater. In addition the neutral red working solution was also prepared in the seawater
and the seawater was used as a mountant prior to coverslipping the preparation for microscopy.

The studies concerning the effects of pollutants at the nuclear level were focused on two possible new
approaches for evaluating DNA damage in living organisms.
Partner 20 has developed and studied the possible use of an arbitrarily primed PCR fingerprinting method to
monitor the action of genotoxins.

An arbitrarily primed PCR (AP-PCR) fingerprinting method has been developed to examine the genotoxic
effects of certain pollutants on the genome as a whole. The method involves using short arbitrary primers to
amplify sections of the genome and variations in the sequences amplified, result in the fingerprint. It operates
on the same basis as standard PCR but instead of amplifying a particular sequence many sequences are
amplified resulting in many bands being visualised on a gel. This process detects polymorphisms in the
absence of known sequence data and is a relatively rapid way of obtaining a fingerprint. The method relies
on the use of a single primer that binds at arbitrary sites in the genome to amplify multiple products. The
primers are shorter than standard, being just 10 nucleotides long. A series of primers have been examined
for use with this methodology for a number of species. Those primers are chosen that give more than 10
clear bands and also produce fingerprints that show reproducibility for an individual and also show a high
degree of homogeneity between individuals from the reference sites.
To measure the effect of any genotoxic pollutants and to limit the effect of inter-individual heterogeneity the
fingerprints were compared by first comparing the similarity of all fingerprints produced from one site, with
the similarity of all fingerprints produced from a second site. To analyse differences in fingerprints between
sites a measure called the band-sharing index (BSI) is used. This gives a measure of the similarity between
fingerprints. The BSI is calculated by:

BSI = 2s / (a + b)

where a is the number of bands in sample 1, b is the number of bands in sample 2 and s is the number of
bands shared between samples 1 and 2.

19 sur 238

In the case of the shore crab (Carcinus maenas) we have checked a range of different random primers (10-
mers) for the generation of reproducible fingerprints and the results so far are consistent with those obtained
earlier indicating that primers OPA09 (5’-GGGTAACGCC) and OPA20 (5’-GTTGCGATCC) appear to be the
most suitable for use in detecting genotoxicity in this species. Optimistation of the reaction was of upmost
importance. An array of reactions was carried out with the identified primers with differing PCR conditions.
Annealing temperature, Mg

++

concentration, dNTP concentration, primer concentration and DNA

concentration was examined and the optimum conditions for each of these parameters were established.

Analysis has been completed on all the shore crab samples collected from the waters around Stavanger,
Gothenburg (2002) and Mosjoen during the sampling expeditions for WP4. This work has been extended to
other species that have been sampled at this time. The methodology has been developed for application with
eelpout samples and a single 10mer primer (OPA11; 5’-CAATCGCCAG) has been identified. Results so far
demonstrate that the shore crab samples collected from certain polluted areas (Hogevarde in Stavanger
sampling and Gothenburg harbour in the Sweden sampling) are more diverse than those collected from
reference areas.

Along similar lines, further work has also been carried out on the development of fingerprinting assays for
cDNA which will allow us to compare the fingerprints of expressed genes in samples collected from different
sites. Early results have indicated differences in the gene expression patterns in different areas with some
genes suppressed and other genes induced. Work is in progress to characterise these genes, which may
subsequently be used to develop gene specific assays for different pollutants. Data from such studies will
enable comparisons to be made with data that will emerge from studies using proteomic approaches in other
BEEP laboratories.

In addition, a new simple and sensitive biochemical method for the evaluation of DNA damage was
developed by Partner 29. This new method for the evaluation of DNA unwinding using fluorescence dye
PicoGreen have been applied in fish liver and in mussel digestive gland as a new biomarker in a battery of
validated genotoxicity biomarkers.

Pico Green is a fluorophore that selectively binds dsDNA and it appears to exhibit high affinity for DNA and a
large fluorescence enhancement upon DNA binding. Pico Green is very stable and little background occurs
since the unbound dye has virtually no fluorescence. The fluorescence enhancement of PicoGreen dye on
binding to DNA is nearly 2000 fold for dsDNA but is very low upon binding to ssDNA or RNA. All these
characteristics allow to evaluate low levels of DNA fragmentation induced by genotoxic insult.

The method could be applied to detect and quantify small amounts of DNA and may allow development of a
microplate-based assay to evaluate DNA single strand breaks in small amounts of tissues.
The method has been applied in fish liver and in the digestive glands of mussels in laboratory experiments
as well as in field samples revealing a sufficient efficiency and discrimination power in the classification of
the coastal sites along a pollution gradient. The most critical point in the application of the method is DNA
denaturation which is obtained in different conditions in mussels and fish.

The new method for the evaluation of DNA unwinding using the fluorescent dye PicoGreen has been applied
in fish liver and in mussel digestive gland as a new biomarker in a battery of validated genotoxicity
biomarkers. The method was validated in laboratory experiments as well as in the field.
DNA single strand breaks were evaluated, using alkaline elution and DNA unwinding with Picogreen
determination, in the digestive glands of mussels from different samplings carried out along the Ligurian and
the Sardinian coast. The results, expressed as strand scission factor, demonstrated significant increases in
DNA damage in polluted sites with respect to the reference area, along a polluted gradient (ssf 0.004

±

0.009

in the control area with respect to 0.255 for the most polluted sites). A correlation between these results and
data obtained from the alkaline elution test was also found.

The method was also applied to liver from fish treated with known genotoxic compounds or mixtures under
controlled conditions. The results obtained demonstrated efficiency in revealing the increase in single strand
breaks induced by solvents and sea oil mixtures.
DNA damage determinations in liver from the fish Mullus barbatus sampled during the two BEEP cruises
from different stations (Portofino, Voltri, Fos and Cortiou) revealed an increase of strand scission factor along
a pollution gradient. In addition, higher values of DNA damage were detected in Autumn with respect to May
and samples with the highest value were found in Fos (Fig. 9 and 10).
Further data must be collected in order to validate this biomarker, but the results obtained in the framework
of the BEEP program allow us to suggest this test as a primary screen for DNA damage in samples. The

20 sur 238

assay is very simple to perform and not time consuming and its application to the microplate allows the
evaluation of a large number of samples at the same time.

Fig

9

Fig

10

The study of the effects of inorganic and organic chemicals on the biochemical pathways that regulate
apoptosis were carried out on fish liver cells by Partner 21.
Apoptosis is a common form of cell death that is essential for normal biological processes. It represents a
tightly regulated event that is characterised by specific, morphologic and biochemical properties. Interest in
apoptosis has increased significantly during the last decade, due to the fact that its deregulation (inhibition or
activation) may result in various diseases including carcinogenesis and immune system disorders Little
attention has been devoted however to apoptosis in fish cells as well as in ecotoxicology, although this
process has already been described in rainbow trout hepatocytes and epithelial cells. On the other hand,
various lines of evidence indicate that the transcription factor NF-

κ

B is involved in the regulation of many

genes involved in cellular defense, survival or death and directly respond, either in a negative or positive
fashion, to the intracellular «redox state». Therefore, in the framework of Task 1 the possible use of novel
liver diagnostic biomarkers, such as apoptosis, induction of oxidative stress and activation of NF-

κ

B, for

detecting the biological effects of chemical contamination in fish of marine coastal ecosystems (pesticides,
PAHs, metals) were investigated and validated. To that end, these types of biological responses in fish
hepatic cells (primary cultures of trout hepatocytes and RTH hepatoma cells) exposed to these xenobiotics
were thoroughly characterised. These cells were selected because they are broadly used for investigating
hepatic uptake, biotransformation and toxicity of xenobiotics. Hepatocytes are also unique because they
belong to the category of a tissue which keep constant metabolic vigil.
In particular, part of the research was formed on the molecular mechanisms of heavy metals-induced
apoptosis in rainbow trout hepatocytes and on the possible involvement of the mitochondrial pathway and of
oxidative stress.

The purpose of this work was to determine whether heavy metals induced apoptosis in trout hepatocytes and
to examine whether or not reactive oxygen species (ROS) were involved in this process. Only data on
Cadmium (Cd) will be presented in this report. Hepatocytes exposed to increasing Cd concentrations (1-10
µM) showed a molecular hallmark of apoptosis resulting from an activation of endogenous endonucleases
and recognized as a “DNA ladder”. This result was correlated with an increase of the caspase-3 enzyme
activity, which reached more than 42% above the control level at 10 µM (after 24h of exposure), and more
than 71, 88, 113%, at 1 to 10 µM (after 48 h of exposure). The involvement of the mitochondrial pathway in
the initiation of apoptosis and the possible role of oxidative stress in that process was then investigated. The
study demonstrates that hepatocyte exposure to Cd (2, 5 and 10 µM) triggers significant caspase-3, but also
caspase-8 and -9 activation in a dose-dependent manner. Western-blot analysis of hepatocyte mitochondrial
and cytosolic fractions revealed that cytochrome c was released in the cytosol in a dose-dependent manner,
whereas the pro-apoptotic protein Bax was redistributed to mitochondria after 24 and 48 h exposure. We
also found that the expression of anti-apoptotic protein Bcl-xL, known to be regulated under mild oxidative
stress to protect cells from apoptosis, did not change after 3 and 6 h exposure to Cd, then increased after 24
and 48 h exposure to 10 µM Cd.

We further examined the effects of Cd on the intracellular production of reactive oxygen species (ROS): -
either directly by using the 2'-7'-dichlorofluorescein diacetate probe (DCFDA), which is specifically oxidized
to fluorescent dichlorofluorescein (DCF) by H

2

O

2

, or indirectly by measuring lipid peroxidation

0

10

20

30

40

50

60

70

80

Portofino

Voltri

Fos

Cortiou

0

10

20

30

40

50

Portofino

Voltri

Fos

Cortiou

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(Malondialdehyde (MDA) production) and catalase (CAT) activity. Trout hepatocytes were exposed to
increasing Cd concentrations (1-100 µM) for 1 hour. Our results indicated that below 25 µM Cd, ROS
production increased in a dose-dependent (42% above the control cells at 25 µM), while no significant
change was observed above this concentration. Moreover, in hepatocytes treated for 1 hour, an increase of
lipid peroxidation was observed at 2-25 µM Cd: the highest value was found at 2 µM (more than 53% above
the control level). Finally, Cd induced an increase of the CAT activity (more than 57% above the control level
at 10 µM Cd) when the exposure-time was prolonged over 48 hours.

In the second part of this work, two antioxidant agents, TEMPO (100 µM) and N-acetylcysteine (NAC, 100
µM) were used to determine the involvement of reactive oxygen species in Cd-induced apoptosis.
Simultaneously exposing trout hepatocytes to Cd and TEMPO or NAC significantly reduced DNA
fragmentation and caspase-3 activation after 48h It also had a suppressive effect on caspases-8 and -9 also,
mostly after 24 h. Lastly, the presence of either one of these antioxidants in the treatment medium also
attenuated Cd-induced Cyt c release in cytosol and the level of Bax in the mitochondria after 24 and 48 h,
while high Bcl-xL expression was observed. These results lead to a working hypothesis that cadmium-
induced apoptosis in trout hepatocytes are partially triggered by the generation of ROS.
Taken together, these data clearly evidenced the key role of mitochondria in the cascade of events leading
to trout hepatocyte apoptosis in response to Cd and the relationship that exists between oxidative stress and
cell death.

A second part of the study has concerned the possible in vitro application of apoptotic biomarkers for aquatic
ecotoxicology: in particular considering trout hepatocytes in primary culture versus the RTH-149 cell line.
Our aim was to compare the molecular mechanisms of apoptosis in RTH-149 hepatoma cells and normal
trout hepatocytes in primary culture, in order to try to define new possible targets that can be used as
biomarkers in fish. We focused our studies: -first, on the expression of the antiapoptotic protein Bcl-xl, which
can act as a blocker of proapoptotic signals; and second, on the activation of the transcription factor NF-

κ

B

which is a key stress sensor within the cell. We therefore first analysed the apoptotic process in both cell
types when treated for 24 and 48 hours by known proapoptotic inducers such as H

2

O

2

, TGF and

staurosporine. RTH cells seem to be refractory to these compounds, unlike hepatocytes, that became
rounded and detached from the substrate after treatment. This morphological effect was correlated to an
activation of caspases 3, 8 and 9, which demonstrates that apoptotic process is triggered in normal
hepatocytes only. Furthermore, analyzing the molecular basis of this phenomenon shows that in contrast to
trout hepatocytes, resistance of hepatoma cells to apoptosis is related to a constitutive activation of NF-

κ

B

and a high constitutive expression of Bcl-xL-like anti-apoptotic protein, even in the absence of xenobiotic
treatment. Complementary experiments were performed by exposing both cell types to pesticides and
cadmium: these chemicals led to apoptosis in hepatocytes, but not in RTH cells which died by necrosis at
high dose.

On the whole, these results confirm that in contrast to fish hepatocytes primary cultures, RTH cells are less
sensitive to pro-apoptotic stimuli and therefore do not represent a suitable in vitro model for developing
apoptosis biomarkers tests in ecotoxicology. On the other hand, they represent the basis of the activator of
apoptosis as a novel biomarker in fish liver.

Finally, an integrative biomarker of exposure was developed by partner 6 studying the potentiality of
evaluation of the parental exposure by an “early life stage history”.
The use of early life stage bioassays was considered as a means of providing an integrated response to
environmental contaminants in adult mussels. For that purpose mussels were collected from field sites at
Visnes (1), Austvik (2), Håvik (3), Bukkøy (4), and Førlandsfjordens (5), induced to spawn in the usual way,
and larvae obtained from animals from sites 2, 3, 4, and 5. The mussels from Visnes did not spawn. Sub
samples (3) of the stock were inoculated into clean seawater and allowed to grow on. The stock from which
the replicates were prepared was retained and subsequently analysed Mussel larvae were examined using
light microscopy and the numbers recorded that had either reached the ‘D’ stage, or had remained at the
trochophore stage. Unfertilised eggs were also counted. 100 + larvae per sample were examined. The
results of the assay were tested using an ANOVA to determine if there was difference between groups
followed by a multiple range test to identify those values significantly different to the others

There was no significant difference between Austvik, Havik, and Bukkoy (p>0.05, Fisher’s LSD, n=10
replicates per site). There was however a significant difference between the Førlandsfjordens site and the
Austvik and Bukkøy sites (p<0.05, Fisher’s LSD, n=10 replicates per site).
It is therefore possible to conclude that the Førlandsfjordens site shows the greatest number of larvae
developing to the D shell stage which is consistent with the choice of this site as the control.

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The Austvik and Bukkoy sites have significantly less larvae developing to the D shell stage than the
Førlandsfjordens site which may reflect on possibly, bioaccumulation of contaminants in the lipid moiety of
the eggs as a consequence of exposure to contaminants.

Early life stage bioassays provide a simple and inexpensive means of deriving data on the consequences for
larval viability of adult exposure to contaminants. In addition, the utility of the assay can be extended by
spawning unimpacted mussels and then exposing the resultant larvae to contaminated waters as a method
of determining water quality.

TASK 2: Development of new cellular and molecular biomarkers of exposure

Participants: P7, P10, P16, P27

Introduction

The presence of xenobiotics in the environment represents a risk for marine organisms. One of the most
promising and world-wide applied approaches to quantify the impact on organisms is biological-effect
monitoring with the use of biomarkers. Biomarkers can provide information on exposure, toxic effects and the
individual susceptibility to anthropogenic chemical compounds and help to assess and predict the risk of
long-term effects of exposure to xenobiotics such as heavy metals, aromatic hydrocarbons, pesticides and
polychlorinated biphenyls (PCBs). Proteins of MXR-mediating genes play a key role in the first line defence
of aquatic organisms against environmental xenobiotics.

With respect to ‘seafood quality control’, these biomarkers may be used to monitor human health care in
relation to sea food consumption. For example, chemosensitizers may accumulate and/or be generated by
biotransformation of originally inert substances in mussel tissue. Consumption of these substances may lead
to a reduction of protective barrier functions (e.g. blood/brain barrier) in humans and cause an additional,
and until now, less regarded risk by the disruption of inherent resistance against xenobiotics (Schinkel 1997,
Abu-Qare et al. 2003, Eisenblatter et al. 2003). Multixenobiotic resistance (MXR) refers to the ability of cells
to lower the intracellular concentration of many different structurally unrelated toxic compounds below their
toxic level. This phenomenon was first described in mammalian cancer cell lines, where the expression and
activity of a membrane-bound glycoprotein (Pgp) which belongs to the ABC gene family is responsible for
failure of chemotherapy in approx. 50% of treated cancers that have undergone metastasis (Gottesman and
Pastan, 1993). Evidence of xenobiotic transport or export out of the cell has also been identified in tissues of
a wide range of terrestric and aquatic species and is regarded as an important defence mechanism which
prevents the accumulation of toxic xenobiotics or endogenous metabolites (Minier et al., 2002; Bard 2000;
Koehler et al., in press). Yet, it was not clear for many relevant species which other genes of the transporter
family are involved in xenobiotic resistance additionally to the P-gp encoding ones. Inhibition of this
mechanism leads to dramatic effects as shown experimentally (Toomey and Epel 1993; Waldmann et al.
1995) while field data are still missing. Many pollutants, including components of oil and heavy metals can
inhibit directly energy production (Cotran et al., 1989, Bresler and Yanko, 1995). Prolonged action of the
pollutants on cell may, at list in part, be similar with that produced by ischemia and anoxia (Cotran et al.,
1989). Loss of metabolic energy produces changes in cellular glycolysis, pH, membrane structure and
permeability, transport of inorganic ions and water which may seriously affect successful drug elimination.

The contribution of UPV/EHU (partner no. 7) to task 2 has been focussed on peroxisome proliferation as a
possible novel biomarker of exposure to organic pollutants in molluscs and fish (reviewed in Cajaraville et al.,
2003a, b). .

In order to implement the MXR-defence system as a novel biomarker in sentinel species, several goals were
identifies and achieved during this task of the BEEP project:

(1) Identification and characterisation of genes coding for the relevant transporters of the ABC gene family
are involved and have to be characterised by molecular approaches in selected organisms (Partner 10, 16)
(2) Analysis of the influence of natural parameters such as temperature on gene expression and transport
activity for implementation of these assays on a large geographical scale (Partner 16)
(3) Application of gene expression and activity of MXR in situ at differently contaminated sites and laboratory
studies to test the responsiveness of MXR related biomarkers (Partner 10, 16, 27).
(4) New implementation of activity assays in different species by various approaches using whole animals
(partner 27), living gill explants (partner 16) or cell cultures of primary hepatocytes (partner 10).
(5) Analysis of available energy needed for transport processes related to MXR (System of active transport
of organic anions (SATOA), metabolic state of mitochondria, Partner 27)

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(6) Development of to measure peroxisome proliferation, including proteomics, measurement of peroxisomal
enzyme activities (catalase and acyl-CoA oxidase-AOX-)
(7) Implementation of cytochemical and immunochemical techniques using specific antibodies against
peroxisomal proteins and the nuclear receptor PPAR (peroxisome proliferator activated receptor)

These tasks were undertaken in both, fish and molluscs, using various target species such as blue mussel
(Partner 7; 16), oyster and turbot (Partner 10) and truncate donax (Partner 27) that have the potential to be
used as sentinel species. Furthermore, different approaches were chosen for activity studies of the drug
efflux by using whole animals (Partner 27); tissue explants (Partner 16), isolated cultured cells and
homogenates (Partner 10).

Material and Methods

Molecular characterisation in turbot and blue mussel (Partner 10, 16)

Since the genetic study is mainly based on RNA isolation, it was essential to optimise the species-specific
isolation procedures of RNA of mussel tissues. It is known that molluscs contain high amounts of
polysaccharides with RNA-like behaviour in phase separation-based isolation procedures that interfere with
the quantification of RNA concentrations since the amount of polysaccharides may vary greatly between
individuals. Furthermore, polysaccharides can block columns that are used as an alternative for RNA
isolation. This problem was solved by the sequential use of phase separation and column-based RNA
isolation. Together with improvements in purification of RNA from polysaccharides and proteins, columns
were used to remove residual DNA that often is a contaminant in PCR procedures. Quality of the starting
RNA material appeared to be a critical factor in the reproducibility of RT-PCR results. Only the combination
of both methods resulted in sufficient quality and reproducibility. The use of this isolation procedure ensured
a reproducibility of RT-PCR in the range of 20%. MXR genes and biotransformation genes are present and
detectable in M. edulis. Partial cDNA sequences of the selected genes pgp, mrp, mvp, gst-pi, hsp70,
CYP4A, topoII and actin were amplified by degenerate primers which were designed to bind to conserved
regions in the cDNA. Degenerate primers are single-stranded synthetic oligonucleotides designed to
hybridise to DNA encoding a particular protein sequence (for technical reviews see (von Eggeling &
Spielvogel 1995, Mitsuhashi 1996). Degenerate primers are widely used for screening DNA libraries and in
degenerate PCR to identify homologues of genes in animals (Wechselberger 1998, Kobayashi et al. 1999),
plants (Schmidt et al. 1994, Hertzberg & Olsson 1998, van Tegelen et al. 1999), bacteria (Laging et al. 2001)
and fungi (Record et al. 1999). In the experience of partner 16, this method is fast and effective to identify
genes in evolutionary-distinct species. The obtained sequence information can be used in PCR-based
approaches or for probe synthesis. Promising fragments were cloned into a plasmid vector and sequenced.
Further characterisation by Northern blots and PCR with specific primers affirmed the predicted identity of
the genes. MXR genes and biotransformation genes are present and detectable in M. edulis.
Partial cDNA sequences of the selected genes pgp, mrp, mvp, gst-pi, hsp70, CYP4A, topoII and actin were
amplified by degenerate primers which were designed to bind to conserved regions in the cDNA by Partner
16. In our study, we used DNA sequence information to set up a multiplex PCR for semi-quantitative RT-
PCR and investigated tissue-specific expression and regulatory mechanisms. The multiplex system enabled
us to investigate expression levels of genes of interest in a single PCR reaction, saving time and money.

Protein identification has been conducted by partner 10 by western blotting using the C219 monoclonal
antibody in oyster and mussel.

Activity studies in isolated cells (Partner 10)

In order to study the transport activity of live cells, primary cultures of turbot hepatocytes were performed
from juvenile aquarium-reared turbots. Cell viability was studied by measurement of non-specific esterase
activity using fluorescein diacetate. Indeed, fluorescence measurement of cells incubated in 96-well
microplate indicated that cell concentration and FDA activity were closely related up to 100 000 cells/well
(r2=0.96). Thus the cell FDA activity was used to assess viability along the culture. This revealed that turbot
hepatocytes can be cultured for a few days with a viability decreasing to 38 % after 48 h. The 24-h cultured
cells have then been used to study the MXR activity of turbot hepatocytes. Two different approaches were
tested. exclusion activity of the transporter was assessed using the fluorescent test compounds, rhodamine
B (RB) or calcein-AM (CAM) and cell fluorescence was recorded with a microplate reader or a laser confocal
microscope.

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Activity studies in gill explants of Mytilus edulis (Partner 16)

We investigated the optima of transport activity in Mytilidae from different climate zones pre-adapted to
different temperatures (0oC, 5oC, 15oC and 25oC water temperature for 12h).
Mytilus edulis were sampled at Fjord sites of the North Atlantic (Norway), the North Sea (Island of Sylt) and
the White Sea. Mytilus galloprovincialis were obtained from a Mediterranean aquaculture company (La
Spezia, Italy). Sections of living gills were placed into a 24-well microtiter plate with 500

µ

l filtered sea water

(FSW). 1 M Calcein AM (VybrantTM Multidrug Resistance Assay Kit, Molecular Probes), diluted in 0,15 M
PBS buffer, was added to each well, mixed, and gills were incubated at the different temperatures (0°C, 5°C,
15°C, 25°C) for 20 min. A parallel assay was carried out with Calcein AM solution including a pre-incubation
of the tissue with 20 µM of the P-gp transport inhibitor Verapamil (VybrantTM Multidrug Resistance Assay
Kit, Molecular Probes). After incubation gills were rinsed in FSW for 15 s to remove the extra-cellular dye and
transferred to a slide covered with 100 ul FSW. The fluorescence was measured by image analysis
(excitation at 494 nm and emission at 517 nm) and the software KS 300 (Sony CCD Camera HTV Horn
Aalen, Zeiss, Kontron). Camera and image analysis system were adjusted and calibrated according to
Chieco et al.(2001).

Activity studies in intact Donax individuals and energy budget (Partner 27)

System of active transport of organic anions (SATOA):The SATOA (system of active transport of organic
anions) transport activity was visualized and measured by using its fluorescent marker substrate FLU,
specific competitive inhibitors p-aminohippurate (pAH) and probenecid (PRO), and contact microfluorometry
as described previously (Bresler et al., 1975, Bresler and Fishelson, 1994, Bresler and Yanko, 1995). Living
gill and mantle tissues were loaded by FLU with or without inhibitors by using cold preincubation with
corresponding solution or incubation with 1

µ

M solution of esterases’ fluorogenic substrate, fluorescein

diacetate (FDA), then transferred in clean seawater and efflux of FLU was determined microfluorometrically.
In addition, nine living D. trunculus from each collection site were injected in the foot with 10

µ

L of a solution

containing 100

µ

M/L FLU and 5 mM/L PRO or 100

µ

M FLU and 1 mM pAH. The control molluscs were

injected with 100

µ

M FLU only. The concentration of FLU in the kidney was measured within 2 hours after

injection.
The MXR (multixenobiotic resistance transporter) transport system: The MXR (multixenobiotic resistance
transporter) transport activity was visualized and determined by using its fluorescent marker substrates
(acridine orange - AO, ethidium bromide - EBr, or rhodamine B- Rh), specific competitive inhibitor Verapamil
(VER) and contact microfluorometry. Living molluscs were incubated for 1 hr in seawater containing the
marker substrate only, substrate plus inhibitor, or two various markers, accumulation was measured
microfluorometrically, the molluscs were transferred for 1 hr in clean seawater or seawater with inhibitor and
finally, efflux of marker was measured.
Redox state of mitochondria: To assess the metabolic state in tissue of living molluscs sampled from clean
and polluted sites, we diagnosed the activity of mitochondria in living cells. For this we measured by
microfluorometry the inherent blue fluorescence (excitation at 365 nm, emission at 420-450 nm) of reduced
nicotinamide adenine dinucleotide (NADH), and green fluorescence (excitation at 400 nm, emission at 530
nm) of oxidized flavins of respiratory chains. Using known uncoupler, dinitrophenol (DNP), and an inhibitor of
the respiratory chain, antimycin A, allowed assessing the redox state of the NAD and flavins.

Results and Discussions

Characterisation of MXR-related genes (Partner 10, 16)

MXR genes and biotransformation genes are present and detectable in M. edulis. Partial cDNA sequences
of the selected genes pgp, mrp, mvp, gst-pi, hsp70, CYP4A, topoII and actin were amplified by degenerate
primers which were designed to bind to conserved regions in the cDNA. Degenerate primers are single-
stranded synthetic oligonucleotides designed to hybridise to DNA encoding a particular protein sequence (for
technical reviews see (von Eggeling & Spielvogel 1995, Mitsuhashi 1996). Partial sequence homologies of
the identified genes varied between 44% and 96% as compared with the homologue in H. sapiens.
Sequence homologies for the MXR-related genes ranges from 44-70%.
In turbot, a 473 bp cDNA fragment has been cloned by Partner 10 and the deduced amino-acid sequence
showed that this turbot mxr gene shares approximately 80 % homology with class I or class II mammalian
MXR proteins. Using this cDNA as a probe, a major messenger RNA of 5.6 kb has been identified by
northern blot.
In oyster, RT-PCR led to the identification of 2 related genes but only one of them is homologous to the class
I of mxr genes. The second one, although part of the wide super family of ABC transporters, cannot
univocally be classified as mxr gene.

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A single band corresponding to a protein of 83 kDa could be visualised from turbot protein extracts whereas
two bands were observed in oyster tissues preparations. The most predominant one was of 170 kDa and the
second was of 220 kDa. Similar observations were made for the mussel Mytilus edulis. Interestingly, only the
220 kDa band was recognised by the anti-MRP MAB4122 monoclonal antibody suggesting that the
Multidrug-resistant-associated protein system is present in mussel and could be assessed immunologically.

In summary, our experiments showed that it is possible to identify evolutionary highly- conserved genes at
the mRNA level by the degenerate primer approach in Mytilus edulis. All genes showed tissue-specific
expression patterns. Hsp70 was identified as a suitable internal standard whereas actin expression showed
to be regulated in relation to the reproductive cycle. Furthermore, a correlation was found between mitotic
activity and topoII expression.

Environmental factors influencing regulation of MXR related genes (Partner 16)

After implementation of the method, we analysed whether RT-PCR-based analysis of gene expression can
be applied in field studies for implementation in monitoring programmes. However, fluctuations of
parameters such as temperature, salinity and anaerobiosis often occur within sampling sites. Estuaries,
endangered by high loads of xenobiotics, are especially subject to fluctuations of salinity due to the tide.
Fluctuations in temperature and oxygen availability (anaerobiosis) in relation to the tidal rhythm should be
taken into account for biomarker application in costal areas.
In the present work, the effects of environmental parameters on gene expression patterns were analysed in
detail in laboratory experiments. P-gp showed to be affected by different salinities and anaerobiosis.
Expression levels at low salinity were decreased to nearly zero in all individuals while anaerobiosis increases
expression levels. For mvp, an increase in gene expression levels was observed after anaerobiosis.
Additionally, the effects of environmental parameters showed to be tissue-specific. Environmental
parameters affect gene expression of MXR-related genes in M. edulis. To avoid false positive or negative
results in field sampling, the sampling strategy should take these effects into consideration. Ecological
consequences of decreased expression of detoxification genes due to fluctuations in environmental factors
should be further investigated.
Besides fluctuating environmental parameters, different life histories and habitat structure effects have to be
taken into consideration with respect to the general metabolism of xenobiotics in mussels. This has been
shown in comparative studies in mussels collected at rocky shores compared to those collected at boyes.
Differences in protein turnover rate and energy household may affect expression levels of detoxification
genes in response to stress. Consequences of these results for monitoring programs are obvious. Beside
wild catches, caged mussels obtained from aquaculture or a clean site have to be used as internal controls
when different habitats are investigated.

Site-specific gene expression (The Norwegian Fjord sites, Partner 16)

Sampling of point-source contaminated sites in Norway (Stavanger) in comparison with clean reference sites
at a Norway fjord system affirmed the usefulness of MXR-related gene expression analysis for environmental
biomonitoring to detect effects of PAHs and heavy metals. Sampling at two sites, exposed to similar
contamination, gave similar results and proved the reliability of MXR-related gene expression analysis in field
situations. On the basis of gene expression, we were able to identify an until now unknown contaminated
site, formerly used as reference site. Figures 1, 2 and 3. A detailed description of these results is presented
by WP4 (North Atlantic).

MXR actvitiy in a human cell line, isolated cells and cell fragments (Partner 10)

Confocal lasermicroscopy indicated that turbot hepatocytes accumulated the fluorescent dye RB. Different
patterns of accumulation could be observed depending on the presence or absence of verapamil in the
incubation medium. Fluorescence was enhanced by 26% (p<0.001) when the MXR inhibitor was present.
This increased dye accumulation was similar to the results obtained with Mytilus edulis hemocytes (Minier
and Moore, 1996) and was the result of a competition mechanism between verapamil and rhodamine B as
observed in MXR cancer cells. Rhodamine accumulated in the whole cytoplasm with an increased labelling
in part of the cellular membrane and some intracellular structures. These intracellular spots correspond to
the lysosomes which were the only labelled structures when mussel hemocytes were exposed to rhodamine
B (Minier and Moore, 1996).
In oyster, both fragments of adult gill tissues and hemocytes were used to analyse their rhodamine B
retention properties. Fluorescence intensity of lysates from gill fragments previously incubated in RB solution
were recorded using the microplate reader while single cell fluorescence was analysed by confocal

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microscopy and subsequent image analysis. Incubation of gill tissues with either verampamil or cyclosporin A
led to a 50% increase of the cellular RB content (p=0.02) thus giving a good indication of the presence of an
active MXR system. To test whether environmental contaminants could interact with the oyster system, two
concentrations of atrazine or benzo(a)pyrene were added to the incubation medium. These compounds led
to a slight increase in the rhodamine content of gill cells (from 10 to 30%). However, this effect was
significant only for the highest concentration (100 µg/L) of atrazine. Images of oyster hemocytes indicated
that these cells accumulated Rb in a verapamil-sensitive manner and that the lysosomal compartment
retained most of the fluorescent dye that was not excluded from the cell.
As some studies suggest interactions between the P-gp function, the cell regulatory volume decrease (RVD),
the apoptotic process and the chloride channels gating these phenomenon were investigated. A new
approach has been developed, based on patch-clamp recording of ionic currents, to investigate early P-gp
responses to chemical stressors. Two cell types have been compared. The MCF7 cell line, originating from a
human breast cancer, is naturally sensitive to cytotoxins (MDR- phenotype, used as control). P-gp
overexpression can be induced by culturing MCF7 in the presence of 1 µM doxorubicin. This MDR+
phenotype was compared to a native MXR phenotype, or P-gp-like activity, measured in mussel blood cells
(MBC) collected from mussels Mytilus edulis sampled in the seaport of Le Havre (France). The viability of the
cells, cultured in microplates, was estimated using a standard colorimetric MTT assay and the MDR/MXR
activities were assessed by measuring the fluorescence of cells loaded with the calcein-AM P-gp probe.
A first set of experiments was performed to characterize the MDR and MXR phenotypes. In MBC, the P-gp
activity clearly increased with temperature since calcein accumulation diminished gradually from 4°C to
18°C. At 18°C, MBC accumulated less calcein than sensitive MCF7 cells at 37°C. However, MBC can be
cultured in microplates more than 10 days without any marked effect of temperature (4 or 12°C) on cell
viability. In P-gp overexpressing MCF7 cells, verapamil (VRP, 50 µM), a prototypic P-gp antagonist,
provoked a huge blockage of the calcein efflux (2000 % of control). At 12°C, MBC responded to 50 µM VRP
by a less pronounced but significant increased of fluorescence accumulation (150 % of control). At 4°C, VRP
has only a small if any effect on MBC. Finally, after a 5-day exposure to anti-cancer cytotoxics (doxorubicin
or vincristin), MBC displayed a decreased level of calcein fluorescence consistent with an induction of the P-
gp activity. These results indicate that the MXR phenotype in MBC and the MDR phenotype in MCF7 seem
to be similarly regulated. However responses of MBC are of several orders of magnitude lower than
responses of P-gp overexpressing cells.
In a second set of experiments, cross-regulations between the hypo-activated chloride channels, responsible
for the RVD during hypo-osmotic swelling, and the P-gp activity have been investigated. Patch-clamp
recordings of MCF7 in the whole-cell configuration revealed that chloride currents activated by short-term
perfusions of moderate hypotonic solutions (20 % hypotonicity, 15 minutes) elicited outward rectifying
chloride currents. These currents have identical electrophysiological and pharmacological properties (current
density, ATP-activation, voltage-dependence and inhibition by DIDS) in both MDR- and MDR+ cells.
Interestingly, VRP was able to dramatically inhibit hypo-activated currents in MCF7 cells. Conversely, a
moderate hypotonic shock significantly inhibited P-gp activity in resistant MCF7 and in MBC at 12°C. As
observed before, hypotonicity had no effect in MBC cultured at 4°C or in MCF7 that do not express P-gp.
The chloride channel blocker DIDS increased the P-gp activity in both MDR+ and MBC at 12°C. Inversely,
doxorubicin, known to promote the MDR/MXR phenotypes, markedly blocked the hypo-activated chlorides
currents in MDR+ cells.
Taken together, the results demonstrate that regulations of P-gp activities are very similar in a human cell
line overexpressing the P-gp and in blood cells collected from the blue mussel and cultured at a temperature
above 12°C. In addition, hypo-activated chloride channels and P-gp activity seem to be inversely regulated
by several xenobiotics, suggesting a molecular dialogue between both mechanisms.
MXR activity studies in gill explants of Mytilus edulis (Partner 16)

Seasonal temperature fluctuations and changes due to climate change may significantly influence the
successful elimination of hazardous substances from marine invertebrates. Therefore, we investigated the
optima of transport activity in Mytilidae from different climate zones pre-adapted to different temperatures
(0°C, 5°C, 15°C and 25°C water temperature for 12h).
In the frame of the development of novel biomarkers in BEEP it was a relevant aim to test the MXR activity
assay for rapid application in individuals collected at differently polluted sites. MXR activity in gills of Mytilus
edulis sampled in Norwegian fjords showed significant inhibition of drug transport at the sites of high PAH
contamination while at the site of high copper and iron no effects on xenobiotic transport activity was noted in
comparison to the reference sites.
Mytilus edulis from polar and cold regions transported best at lower water temperatures between 0oC and
5oC while Mytilus galloprovincialis from the southern hemisphere showed high transport activity at all
temperatures selected without any temperature preference. Additionally, transport efficiency in Mytilus
galloprovincialis was significantly higher than that of Mytilus edulis from the NA, the North Sea and the White
Sea.

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Fig.2 Mediterranean Sea (yellow box = transport inhibited with Verapamil)

Fig.3 North Atlantic

Fig.4 North Sea

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Fig.4 White Sea

Fig. 5 Norwegian Field sites - No effects on the MXR transport in mussel gills were observed for the three environmental

pollutants (Fig.6)

The assays for MXR transport in living gills evidenced that copper does not seem to impair drug efflux as
fluorescence did not differ from that at the 2 reference sites. Exposure to organic pollution at site 6 led to
slightly reduced drug efflux as indicated by increased fluorescence. Most pronounced impairment of drug
elimination was recorded at the site 5 which is regarded as highly polluted by mixtures of PAHs (Fig5.).

MXR Acticity in intact Donax trunculus (Partner 27)

After incubation with acridine orange (AO), ethidium bromide (Ebr) or rhodamine B (Rh) showed that these
fluorescent substrates were accumulated in the gills epithelium, but their concentration decreased after
washing in clean water (Fig. 3). Accumulation of these markers enhanced markedly in the presence of the
competative inhibitor of the multixenobiotic transporter system - verapamil (VER) while VER in washing
seawater inhibit their efflux (Fig. 1). Loading the cells with AO and Ebr or AO and Rh demonstrated a mutual
inhibition of their efflux. The MXR activity in the gills of D. trunculus from the three sampling sites was
calculated as a difference between AO accumulation with and without verapamil, and expressed as AO
amount (in arbitrary units) pumped out by the MXR in one hour from the gill’s cells. Rate of verapamil-
sensitive efflux of AO increased significantly (P<0.05) in Donax from Qiryat Yam compared to those from
Akko, while the increase in AO efflux in molluscs from Frutarom was not significant.

Min-Max
25%-75%
Median

Relative fluorescence

0

20

40

60

80

100

120

140

160

180

200

Organic

Organic

Copper

Copper

PAH

PAH

Reference

Reference

Reference

Reference

inhibited

inhibited

inh.

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