WILL BE POSTED AS SUBMITTED TO BRUSSELS
EXECUTIVE PUBLISHABLE SUMMARIES
FIRST REPORTING PERIOD (12
Contract n° EVK-CT-2001-00057 Reporting period: November, 1st
2001 – October 31st, 2002
Title Genomic tools for bio-monitoring of pollutant coastal impact.
Development of biochemical and molecular tools for the bio monitoring
of the European marine environment
The basic assumptions underlying this project are:
1) Fish inhabiting coastal waters, interacting with the sediment
and water column of their habitat, and fed from local resources,
can serve as indicators for environmental biological impact.
2) Gene products, transcripts and proteins, whose expression in
the indicator fish is affected by environmental factors, are excellent
environmental biomarkers. They underlie most biological events,
therefore, a variety of environmental perturbations are assumed
to affect their level. In addition, gene products can be measured
using common methodologies, simplifying their application as biomarkers.
Two indicator species were selected, enabling the examination
of the two major European water bodies: The flounder (Platichthys
flesus), and the striped sea bream (Lithognathus mormyrus), covering
the North-Eastern Atlantic and the Mediterranean coasts, respectively.
The scientific objectives of the project are:
1) Identification of a variety of environment-affected fish genes,
by screening suitable cDNA microarrays for affected transcripts
and analyzing 2D electrophoretic gels coupled to mass spectrometry
sequencing, revealing influenced proteins.
2) Semi-quantitative evaluation of environmental effects on multiple
gene expression, using cDNA operational microarray.
3) Fully quantitative measurement of specific expression of transcript
and protein biomarkers, in absolute units, using real time PCR
and Enzyme Linked Immuno Sorbent Assay (ELISA), respectively.
Scientific achievements (1.11.01 – 1.11.02:
1) Induction of pollutant-affected gene expression was completed
in both species, resulting in hepatic RNA populations enriched
with environment-affected cDNAs and proteins.
2) Preparation of cDNA plasmid library of several thousands clones
from flounder RNA populations, using the subtractive-suppression
hybridization (SSH) methodology. Similar striped sea bream library
is constructed at present.
3) Analysis of liver samples of both species by 2D gel electrophoresis,
comparing proteins from induced and uninduced individual fish.
The experiments are at the stage of optimizing the gel system
and the protein extraction procedure.
1) A DNA minarray had been printed with fragments of around 100
candidate genes cloned from PCR products obtained with degenerate
primers. This has been used to optimize spotting andfhybridization
conditions and has been tested with RNA samples from flounders
collected from polluted and non-polluted sites.
1) Real time PCR measurement methods were completed for flounder
cytochrome P4501A, Ah receptor and ARNT, all related to the P4501A
expression system. Similar procedures were completed for the sea
bream cytochrome P4501A, metallothionein, vitellogenin and 18S
rRNA, which is used as normalizing agent.
2) Competitive Enzyme Linked Immuno Sorbent Assay (ELISA) was
developed for the measurement of the sea bream cytochrome P4501A
3) The pattern of expression was characterized for the striped
sea bream cytochrome P4501A, metallothionein and vitellogenin,
including maximal induced levels, baseline levels and its natural
fluctuations, not related to pollution and variation of biomarker
levels within fish samples, which may indicate natural fluctuations
among individuals as well as common exposure history of the component
Socio-economic relevance and policy implications:
Part of the developed methods in the framework of objective 3,
are ready for implementation in natural habitats for the evaluation
of pollution biological impacts. Actually, the IOLR partner is
involved in the set up of a pilot bio monitoring system in the
Eastern Mediterranean, to be submitted to the EU-LIFE third countries
The GENIPOL project is in its first year, and generally proceed
according to plan with minor time schedule deviations, demonstrating
also methods ready for implementation.
Bio-monitoring, marine environment, North-eastern Atlantic, Mediterranean,
flounder, striped sea bream, microarray, proteomics, real time
Publications (cumulative list)
Peer Reviewed Articles:
Dixon, T.J., Taggart, J.B., George, S.G. (2002) Application of
real time PCR determination to assess interanimal variabilities
in CYP1A induction in the European Flounder (Platichthys flesus).
Mar. Environ. Res. (54) 267-270
Tom M., Myers C.R., Waterman M.R.( 2002) Competitive ELISA utilizing
membrane-free recombinant fish CYP1A as standard for evaluation
of its microsomal homologue molar concentrations. Aquat. Toxicol.
Tom, M., Shmul, M., Shefer, E., Chen, N., Slor, H., Rinkevich,
B., Herut, B. (2002) Quantitative evaluation of hepatic cytochrome
P4501A transcript, protein and catalytic activity in the striped
sea bream, Lithognathus mormyrus. Environ. Toxicol. Chem. Accepted
Kamer, I., Douek, J., Tom. M., Rinkevich, B. (2002 )Metallothionein
induction in RTH-149 cell line as an indicator for heavy metal
pollution in a brackish environment: assessment by competitive
RT-PCR.Arch.Environ.Contam. Toxicol. Accepted
18 month Partner
meeting held at PRIMO 12, Tampa Florida May 2003.
Overview and Results
presented as Platform and Poster Presentations..see Effects
page for field studies and Publications
page for presentations