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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
P2Prof. 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
P3Dr 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
P4Dr Gilles BOCQUENE, Coordinator WP3, Dr Thierry
BURGEOT
IFREMER DEL/PC, Rue de l’Ile d’Yeu, 44311 Nantes Cedex 3 - France
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
P8Dr 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
P9Dr 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
P10Dr 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
P11Dr 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
P12Dr 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
P13Dr 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
P14Prof. 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
P15Prof. 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
P16Dr 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
P17Dr Janina BARSIENE
Institute of Ecology, Akademijos 2, 2600 Vilnius – Lithuania
Tel./Fax: 370 2 700666
E-mail:
janbar@takas.lt
P18Prof. 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
P19Prof. 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
P20Dr 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
P21Dr 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
P22Dr 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
P23Dr 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
P24Dr 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
P25Dr Janusz PEMPKOWIAK
Marine Chemistry and Biochemistry Department, Institute of
Oceanology, P.O. Box 197, Sopot - Poland
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
P28Dr 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
P29Dr 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
P30Prof. 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
5 sur 238
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
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.
9 sur 238
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).
10 sur 238
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
2+
signalling by heavy metals, as a starting point we have studied in
vitro effects of Hg
2+
and Cu
2+
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
2+
in the range of 5-50 µM. The mechanisms evoked by mercury,
which produced the strongest effect were then explored in more
detail. Hg
2+
triggered intracellular Ca
2+
waves, stimulated Ca
2+
-ATPase activity, and promoted InsP
3
production. Use of various inhibitors has
indicated that Hg
2+
induces Ca
2+
entry through verapamil-sensitive channels, and intracellular
Ca
2+
release
via the G protein-PLC-InsP
3
pathway. However, in cells loaded with heparin and exposed to
Hg
2+
the [Ca
2+
]
i
rise is almost abolished, indicating that the global effect of
Hg
2+
is not simply a sum of Ca
2+
entry plus Ca
2+
release, but involves a Ca
2+
-induced Ca
2+
release mechanism.
After having studied the in vitro effects of
Cu
2+
and Hg
2+
on cell Ca
2+
, the in vivo effects of these two metals
was investigated using mussels as model organisms. Deregulation of
Ca
2+
homeostasis can have serious
effects on cell functioning due to an alteration in
Ca
2+
signaling, the variations in plasma membrane Ca
2+
-
ATPase (PMCA) were therefore evaluated. After in vivo mussel exposure
to Cu
2+
(0.3-1.3 µM) or Hg
2+
(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
2+
inhibits PMCA, whereas Hg
2+
induces a rise in PMCA activity. Similar
11 sur 238
results were found using a biochemical assay on gill PMCA. PMCA
activity was compared to immunoprecipitation data. The effect of
Cu
2+
treatment does not appear to alter PMCA expression and
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
2+
-dependent phospholipase A2 (cPLA2) induces
lysosomal membrane destabilisation, the involvement of this
mechanism in lysosomal membrane destabilisation induced by
Hg
2+
and Cu
2+
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
2+
was studied using Fura2/AM-loaded
cells, and lysosomal membrane destabilisation was studied using
neutral red staining. Both metals induce Ca
2+
-dependent lysosome destaining and lysosomal
volume increase, indicating destabilisation of lysosomal
membranes. These effects are partially, but significantly, reduced by a
specific Ca
2+
-dependent
PLA2 inhibitor (AACOCF3), but not by a Ca
2+
-
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
2+
and Cu
2+
in the µM range, and H
2
O
2
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
2
O
2
, Hg
2+
, and
Cu
2+
plus low H
2
O
2
. ERK activation by H
2
O
2
is prevented by
concomitant activation of p38. Phosphospecific STAT
antibodies have revealed activation by H
2
O
2
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
2+
and Cu
2+
on PMCA activity
(cytochemical analysis, above) and on PMCA expression
(immunoprecipitation, below).
12 sur 238
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
2+
and Hg
2+
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
2
O
2
activates p38
(top) but not ERK (middle), however, in the presence of p38
inhibitors ERK is activated by H
2
O
2
(bottom).
13 sur 238
*
*
PMCA
0
20
40
60
80
100
120
140
160
3 weeks
1 week
3 weeks
1 week
3 weeks
3 weeks
Control
50 ppb Dialkyl phthalate (F)
50 ppb Bisphenol A (B)
5 ppb TBDE
D e nsitometric v
a lue
( % )
*
*
*
*
*
Catalase
0
20
40
60
80
100
120
3 weeks
1 week
3 weeks
1 week
3 weeks
3 weeks
Control
50 ppb Dialkyl phthalate (F)
50 ppb Bisphenol A (B)
5 ppb TBDE
D e nsit
om
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
2+
-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
2+
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
0
50
100
150
200
250
300
June 2001
October 2001
June 2002
October 2002
June 2003
October 2003
sam pling period
pm
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
0
50
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
e
Mantle/gonad com plex
0
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
pm
ol c
A M P / g r w e t t i s s u e
a
b
c
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
2+
and to Cu
2+
ions appeared at a very low concentrations. These metal ions were
also very toxic to the
mollusc. The reaction to Ni
2+
and Pb
2+
was moderate while the response to Cd
2+
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
2+
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
2+
. 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
2+
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
2+
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
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Fos
Cortiou
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10
20
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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 proliferationas 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.
25 sur 238
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
26 sur 238
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.
27 sur 238
Fig.2 Mediterranean Sea (yellow box = transport inhibited with
Verapamil)
Fig.3 North Atlantic
Fig.4 North Sea
28 sur 238
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
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