Final Activity Report Summary - PHOCOENA2004 (Evaluation of the immunotoxicity of mercury, zinc, polychlorobiphenyls and methyl sulfonyl polychlorinated biphenyls on cytokine and proteome expression)
Marine mammals such as the harbour porpoise Phocoena phocoena and the harbour seal Phoca vitulina occupy the top of the North Sea food web and as such, integrate the whole ecosystem. The regulatory role of these predators in trophic chains is of utmost importance, justifying a constant monitoring of the species and their biotopes. Indeed, as long-lived and apex predators, marine mammals become sinks for trace metals (such as mercury Hg) and organic pollutants (polychlorinated biphenyls -PCBs and many other toxic organic compounds). This project aims to better apprehend the effects of Hg and various PCB congeners on the health of harbour porpoise and harbour seal, focusing on their immune system.
- Achievement 1. Blood sampling and pollutant levels in the tissues of harbour porpoises and harbour seals.
Blood of harbour seals from the German coast were be collected on a regular basis in collaboration with Dr U. Siebert (Forschung- und Technologie Zentrum, Kiel University, Germany), from free-ranging seals. These samplings take place at least twice a year in the German North Sea (Schleswig-Holstein). Blood from captive seals was used for proteomic assay (collaboration with the Rehabilitation Center of Friedrichskoog, Germany) that keeps harbour seals on a regular basis for scientific and education purposes. Blood sampling will be continuously collected in 2006 and 2007 to increase sample number. Blood of freshly stranded harbour porpoises were also collected for pollutant analyses.
- Achievement 2. Pollutant levels and stable isotope ratios in harbour seal blood:
Circulating levels of Hg and PCBs (PCBs 28, 52, 101, 118, 138, 153, 180) were measured in the blood of 24 harbour seals caught between 2001 and 2004. As pollutant level may be linked to the trophic position in the food web, carbon and nitrogen isotopic ratios (d13C and d15N) were measured in clotted blood cells. The average concentration of mercury in these living seals did not differ significantly from the high levels observed previously in the blood of stranded harbour seals. Mean blood concentrations of total PCBs were 11 ng/ml (collaboration with Pr JP Thome, CART, ULg). These preliminary results suggest high levels of mercury and PCBs in the blood of wild harbour seals caught in the North Sea.
- Achievement 3. Lymphocyte cell culture and exposure to pollutant.
Blood lymphocytes (Peripheral Blood Mononuclear Cells or PBMCs) from harbour seals were successfully isolated and maintained in cell culture medium before being exposed to methylmercury (collaboration with Dr U Siebert and Dr MC Gillet, Laboratory for Histology and Cytology, ULg).
- Achievement 4. mRNA expression of cytokines in seal lymphocytes.
The immune system is composed of many interdependent cell types that collectively protect the body from bacterial, parasitic, fungal and viral infections. These cells depend on the T helper subset for activation signals in the form of secretions formally known as cytokines. Cytokines such as interleukine-2 (IL-2) and TGF-beta (Transforming Growth Factor- beta) are thus major mediators of the immune system. Our results showed methylmercury immunosuppressive effects even at low concentration (0.2 and 1 microM, often encountered in free-ranging seals from the southern North Sea): IL-2 and TGF-beta mRNA expression were indeed weaker in exposed lymphocytes compared to non-contaminated cells.
- Achievement 5. Protein expression in seal lymphocytes.
Proteome (proteins constituting a cell compartment) was studied in these control and contaminated lymphocytes in order to understand the impact of a contamination by methyl-Hg on the expression of proteins. After mercury exposure, cytosolic proteins were extracted following well established protocols and identified on 2D-gels followed by mass spectrometry. Protein identification is currently under investigation (collaboration with Pr E De Pauw, CART, Ulg).
- Achievement 1. Blood sampling and pollutant levels in the tissues of harbour porpoises and harbour seals.
Blood of harbour seals from the German coast were be collected on a regular basis in collaboration with Dr U. Siebert (Forschung- und Technologie Zentrum, Kiel University, Germany), from free-ranging seals. These samplings take place at least twice a year in the German North Sea (Schleswig-Holstein). Blood from captive seals was used for proteomic assay (collaboration with the Rehabilitation Center of Friedrichskoog, Germany) that keeps harbour seals on a regular basis for scientific and education purposes. Blood sampling will be continuously collected in 2006 and 2007 to increase sample number. Blood of freshly stranded harbour porpoises were also collected for pollutant analyses.
- Achievement 2. Pollutant levels and stable isotope ratios in harbour seal blood:
Circulating levels of Hg and PCBs (PCBs 28, 52, 101, 118, 138, 153, 180) were measured in the blood of 24 harbour seals caught between 2001 and 2004. As pollutant level may be linked to the trophic position in the food web, carbon and nitrogen isotopic ratios (d13C and d15N) were measured in clotted blood cells. The average concentration of mercury in these living seals did not differ significantly from the high levels observed previously in the blood of stranded harbour seals. Mean blood concentrations of total PCBs were 11 ng/ml (collaboration with Pr JP Thome, CART, ULg). These preliminary results suggest high levels of mercury and PCBs in the blood of wild harbour seals caught in the North Sea.
- Achievement 3. Lymphocyte cell culture and exposure to pollutant.
Blood lymphocytes (Peripheral Blood Mononuclear Cells or PBMCs) from harbour seals were successfully isolated and maintained in cell culture medium before being exposed to methylmercury (collaboration with Dr U Siebert and Dr MC Gillet, Laboratory for Histology and Cytology, ULg).
- Achievement 4. mRNA expression of cytokines in seal lymphocytes.
The immune system is composed of many interdependent cell types that collectively protect the body from bacterial, parasitic, fungal and viral infections. These cells depend on the T helper subset for activation signals in the form of secretions formally known as cytokines. Cytokines such as interleukine-2 (IL-2) and TGF-beta (Transforming Growth Factor- beta) are thus major mediators of the immune system. Our results showed methylmercury immunosuppressive effects even at low concentration (0.2 and 1 microM, often encountered in free-ranging seals from the southern North Sea): IL-2 and TGF-beta mRNA expression were indeed weaker in exposed lymphocytes compared to non-contaminated cells.
- Achievement 5. Protein expression in seal lymphocytes.
Proteome (proteins constituting a cell compartment) was studied in these control and contaminated lymphocytes in order to understand the impact of a contamination by methyl-Hg on the expression of proteins. After mercury exposure, cytosolic proteins were extracted following well established protocols and identified on 2D-gels followed by mass spectrometry. Protein identification is currently under investigation (collaboration with Pr E De Pauw, CART, Ulg).