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:
Objective 1:
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.
Objective 2:
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.
Objective 3:
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 protein levels.
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 individuals.
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 program.

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 PCR, ELISA.

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. (59)101-114.
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