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See detailImmunotoxicology of methylmercury and other contaminants in harbour seals (Phoca vitulina) from the North Sea
Dupont, Aurélie ULg

Doctoral thesis (2013)

Environmental contaminants are suspected to have detrimental effects on marine mammal health. They were notably hypothesised to be involved in the severity and extent of catastrophic viral epidemics that ... [more ▼]

Environmental contaminants are suspected to have detrimental effects on marine mammal health. They were notably hypothesised to be involved in the severity and extent of catastrophic viral epidemics that recently affected the harbour seal (Phoca vitulina) population from the North Sea. High levels were indeed found in their tissues. However, the exact effects of those pollutants on free-ranging harbour seal health are not yet elucidated. In the field of immunotoxicology, in vitro cell culture techniques have been developed and are considered as valuable tools to assess specific toxicity mechanisms. The first objective of this study was to investigate the relationships between environmental contaminant levels found in harbour seal blood and lymphocyte proliferation responses in vitro. Indeed, wild harbour seal cells used in the framework of in vitro culture studies are isolated from the blood of animals that are contaminated, but little is known about the possible relationships between them. They were thus investigated in harbour seals from the North Sea. Peripheral blood leucocytes were isolated and cultured during 72h with Concanavalin A (ConA), a mitogen agent, to evaluate the lymphocyte proliferative responses as a stimulation index. The ConA-induced lymphocyte proliferation assay evaluates their ability to proliferate in response to a polyclonal stimulation, which is considered to reflect the activation of the immune response after an antigenic stimulation in vivo. No statistically significant relationship was found between the lymphocyte stimulation index and blood pollutants or class of pollutants studied (i.e. mercury, lead, persistent organic pollutants, pentachlorophenol, tribromoanisole) in harbour seals sampled while in good body condition and presenting no sign of disease. However, the number of lymphocytes per milliliter of whole blood appeared to be negatively correlated to pentachlorophenol, an organochlorine pesticide. A high interindividual variability of lymphocyte stimulation indexes was observed although cells were isolated in the same way and cultured in identical conditions. This is probably linked to a whole set of parameters such as precise age, sex, life history leading to particular immune status (probably partly related to pollutant loads), physiological parameters and inevitable experimental variability. So, in a general manner, experiments with in vitro immune cell cultures of wild marine mammals should be designed so as to minimise confounding factors in which case they remain a valuable tool to study pollutant effects in vitro. The second objective of this study was to determine underlying mechanisms of methylmercury immunotoxicity. Indeed, among the numerous contaminants found in harbour seal tissues, methylmercury is one of the most hazardous organic compounds known to have numerous detrimental effects on living organisms. It has the property to bioaccumulate and biomagnify in the trophic chain, and can reach very high levels in harbour seal tissues. Peripheral blood leucocytes exposed to relatively low concentrations of methylmercury chloride (MeHg) in vitro revealed a significant decrease of viable cell counts with obvious ultrastructural effects. A higher frequency of apoptosis was observed in lymphocytes, in which mitochondria and nuclei may have been targeted, as demonstrated by ultrastructural analysis. These results were in accordance with the significant decrease of the mitochondrial membrane potential and of lymphocyte proliferation (BrdU assay) observed. The toxicity of this compound was shown to be counteracted by selenium. However, little is known about that interaction in marine mammal blood and the underlying mechanisms still remain unclear. The present study aimed to investigate the in vitro effects of relatively low (0.75 µM) MeHgCl levels on harbour seal ConA-stimulated leucocytes while added concomitantly to Se. Two different forms of Se were studied: selenite and selenomethionine. Their concentrations were within the range of those measured in free-ranging harbour seal blood, and in a Hg:Se ratio of 1:10. Lymphocyte proliferation as well as the mitochondrial membrane potentials were significantly reduced following in vitro exposure to MeHgCl during 72h. No significant difference was found when sodium selenite or selenomethionine was added concomitantly to MeHgCl. Conversely, on average, Se alone did not lead to significant decreased proliferation and mitochondrial membrane potentials. The absence of protection of Se, either in organic or inorganic form, against Hg toxicity in harbour seal blood leucocytes in vitro raises concern as regards the deleterious effects this toxic compound could have on marine mammal immune system. The range of current Hg levels found in free-ranging harbour seals from the North Sea encompasses exposure levels which elicited deleterious effects on lymphocytes in the present study. This highlights the potential for effects on host resistance to disease. [less ▲]

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See detailRelationships between in vitro lymphoproliferative responses and levels of contaminants in blood of free-ranging adult harbour seals (Phoca vitulina) from the North Sea
Dupont, Aurélie ULg; Siebert, Ursula; Covaci, Adrian et al

in Aquatic Toxicology (2013), 142-143

tIn vitro culture of peripheral blood leucocytes (PBLs) is currently used in toxicological studies of marinemammals. However, blood cells of wild individuals are exposed in vivo to environmental ... [more ▼]

tIn vitro culture of peripheral blood leucocytes (PBLs) is currently used in toxicological studies of marinemammals. However, blood cells of wild individuals are exposed in vivo to environmental contaminantsbefore being isolated and exposed to contaminants in vitro. The aim of this study was to highlightpotential relationships between blood contaminant levels and in vitro peripheral blood lymphocyteproliferation in free-ranging adult harbour seals (Phoca vitulina) from the North Sea. Blood samplesof 18 individuals were analyzed for trace elements (Fe, Zn, Se, Cu, Hg, Pb, Cd) and persistent organiccontaminants and metabolites ( PCBs, HO-PCBs, PBDEs, 2-MeO-BDE68 and 6-MeO-BDE47, DDXs,hexachlorobenzene, oxychlordane, trans-nonachlor, pentachlorophenol and tribromoanisole). The samesamples were used to determine the haematology profiles, cell numbers and viability, as well as thein vitro ConA-induced lymphocyte proliferation expressed as a stimulation index (SI). Correlation tests(Bravais-Pearson) and Principal Component Analysis with multiple regression revealed no statisticallysignificant relationship between the lymphocyte SI and the contaminants studied. However, the numberof lymphocytes per millilitre of whole blood appeared to be negatively correlated to pentachlorophenol(r = −0.63, p = 0.005). In adult harbour seals, the interindividual variations of in vitro lymphocyte pro-liferation did not appear to be directly linked to pollutant levels present in the blood, and it is likelythat other factors such as age, life history, or physiological parameters have an influence. In a generalmanner, experiments with in vitro immune cell cultures of wild marine mammals should be designed soas to minimize confounding factors in which case they remain a valuable tool to study pollutant effectsin vitro. [less ▲]

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See detailMercury in blood of free-ranging seals Phoca vitulina from the North Sea: Time-trend and association with environmental factors
Das, Krishna ULg; Brochoire, Charlène ULg; Chambosse, Mélanie et al

Conference (2012, March 27)

The harbour seal (Phoca vitulina) population from the North Sea has experienced various fluctuations these last decades due to habitat loss, prey fluctuation and pollution of the marine environment ... [more ▼]

The harbour seal (Phoca vitulina) population from the North Sea has experienced various fluctuations these last decades due to habitat loss, prey fluctuation and pollution of the marine environment. Recently, development of monitoring programs and non-invasive sampling techniques, including seal catches allowed blood sampling on a regular basis together with measurements of blubber thickness, body mass, sex and body length. Concentrations of total mercury (T-Hg) and other trace elements (Cd, Pb, Zn, Cu, Fe) as well as ∂13C and ∂15N values were determined by mass spectrometry in blood of 75 wild harbour seals caught in the German Wadden Sea between 1997 and 2011. ∂13C and ∂15N mean values (-17.5‰ and 18.1 ‰ respectively) were strongly similar to that measured previously in the muscle of stranded harbour seals from the Wadden Sea. In contrast, ∂15N mean value was strikingly higher than that recorded in grey seals (Halichoerus grypus) from Scotland (14.1 ‰ respectively; Habran et al. submitted.) while ∂13C values remained similar between the two seal species. These values confirmed the high trophic position of the harbour seal in the North Sea. In contrast to Cd and Pb, T-Hg in blood harbour seals reached concentrations as high as 2.1 μg.g-1 dry weight (10 times higher than the 0.21 μg.g-1 dry weight recorded for grey seals from Scotland) but depended on several factors including ∂15N values, age group and the body mass. T-Hg was detected in juveniles confirming maternal transfer to offspring and time-trend revealed no decrease of T-Hg in blood of harbour seals these last 15 years. [less ▲]

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See detailPotential effects of blood contaminants on immune responses in harbour seals (Phoca vitulina)
Dupont, Aurélie ULg; Weijs, Liesbeth; Siebert, Ursula et al

Poster (2011, August 23)

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See detailSTUDY OF SELENITE AND SELENOMETHIONINE EFFECT ON METHYLMERCURY IN VITRO TOXICITY
Dupont, Aurélie ULg; Siebert, Ursula; Rosenberger, Tanja et al

Conference (2011, May 16)

Methylmercury (MeHg) and selenium (Se) can be found at elevated concentrations in blood of marine mammals and both display modulatory effects on the immune system. Whereas mercury (Hg)-Se antagonism in ... [more ▼]

Methylmercury (MeHg) and selenium (Se) can be found at elevated concentrations in blood of marine mammals and both display modulatory effects on the immune system. Whereas mercury (Hg)-Se antagonism in liver of marine mammals is well known, the protective role of Se against Hg immunotoxicity in marine mammals has been poorly described. We propose here an in vitro approach using combined Hg and Se in vitro exposure of peripheral blood mononuclear cells (PBMCs) of the harbor seal (Phoca vitulina). PBMCs were isolated from the blood of 10 harbor seals and exposed to environmental concentrations of MeHg (1µM) and selenite or selenomethionine (5µM), respectively inorganic and organic forms of Se. MeHg leaded to a decrease of lymphocyte proliferation, to an increase of cells with compromised mitochondrial membrane potentials and cell death. Preliminary results evidenced that none of the two Se forms had a protective effect against MeHg toxicity, although cells were slightly stimulated by Se alone. Therefore MeHg expresses its toxicity among blood circulating lymphocytes in presence or absence of selenite or selenomethionine. [less ▲]

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See detailThe harbor seal and the harbor porpoise from the North Sea: review of their ecotoxicological status based on stranded and free-ranging individuals and potential threaths to the population
Das, Krishna ULg; Weijs, Liesbeth; Habran, Sarah ULg et al

Conference (2011, May)

The harbor seal (Phoca vitulina) and the harbor porpoise (Phocoena phocoena) from the North Sea have experienced major fluctuations these last decades due to habitat loss, prey fluctuation and pollution ... [more ▼]

The harbor seal (Phoca vitulina) and the harbor porpoise (Phocoena phocoena) from the North Sea have experienced major fluctuations these last decades due to habitat loss, prey fluctuation and pollution of the marine environment. Recently, development of monitoring programs and non-invasive sampling techniques, including seal catches in Germany allowed blood sampling together with measurements of blubber thickness, body mass, sex and body length. This approach is complementary to the study of stranded and by-caught individuals sampled during necropsies. Essential (Se, Zn, Cu, Fe) and non-essential elements (T-Hg, MeHg, Cd, Pb), perfluorinated organochemicals (PFCs) polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) were measured in blubber and in blood (for selected compounds) of stranded, by-caught and free-ranging individuals from Belgian and German coasts. In contrast to Cd and Pb, hepatic T-Hg can reach concentrations as high as 2.1 μg.g-1 dry weight but depended on several factors including position in the trophic chain (inferred from δ13C and δ15N values), age group and the body mass. This level has been shown to generate in vitro toxicity on harbor seal lymphocytes. Organic compound analysis (PFCs, PCBs, PBDEs) revealed widespread dispersion of contaminants in the marine environment with higher concentrations in seals compared to porpoises in agreement with the higher trophic position of the harbor seal. However, metabolization of these compounds differed between the two species, as revealed by the higher contribution of the persistent BDE-47 and lower chlorinated and non-persistent congeners (e.g. CB 52, CB95) in tissues of harbor porpoises. T-Hg, PFCs, PCBs and PBDEs were detected in calves confirming maternal transfer to offspring. These pollutants are strongly suspected to affect the immune and endocrine systems as well as vitamin A metabolism and this raises concern about exposure-related health effects, especially in younger individuals. [less ▲]

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See detailNew insights in the toxicology and health status of marine marine mammals: Use of free-ranging harbour seals from the Wadden Sea
Das, Krishna ULg; Seibel, Henrike; Hasseilmeier, Ilka et al

Conference (2011, March 20)

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See detailPOTENTIAL EFFECTS OF BLOOD CONTAMINANTS ON IMMUNE RESPONSES IN HARBOUR SEALS (PHOCA VITULINA)
Dupont, Aurélie ULg; Weijs, Liesbeth; Siebert, Ursula et al

in Organohalogen Compounds (2011), 73

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See detailMethylmercury and selenium in vitro effects on harbor seal (Phoca vitulina) lymphocytes : a multidisciplinary approach
Dupont, Aurélie ULg; Bouquegneau, Jean-Marie ULg; Das, Krishna ULg et al

Poster (2010, May 26)

Methylmercury (MeHg) bioaccumulates along the food web, leading to the highest levels in tissues of predatory species. It constitutes the predominant form present in the blood of marine mammals. The blood ... [more ▼]

Methylmercury (MeHg) bioaccumulates along the food web, leading to the highest levels in tissues of predatory species. It constitutes the predominant form present in the blood of marine mammals. The blood cells, including the immune cells, are therefore exposed to the toxic properties of that chemical. Nevertheless, selenium (Se) is an essential element absorbed concomitantly to MeHg which seems to modulate this toxicity. The goal of this study is to evaluate the immunotoxicity of MeHg on the harbor seal (Phoca vitulina) T lymphocytes, highly important in the adaptive immune response, and to investigate the modulating effect of Se on that toxicity. In parallel, the concentrations of MeHg, total mercury (T-Hg) and Se are determined in free-ranging harbour seal blood in order to follow their contamination levels. The T lymphocytes were isolated from the whole blood, exposed to various MeHg and Se concentrations and the exposure effects were estimated by functional tests including the evaluation of viability, proliferation, metabolic activity, DNA and protein synthesis, and by morphological analysis by transmission electron microscopy. The mean T-Hg concentration was 172 ± 143 µg/l of whole blood. The T lymphocytes cultures in vitro displayed a decreasing number of viable cells with increasing concentrations of MeHg, and numerous ultrastructural defects. The cells exposed to MeHg notably displayed distortion of the plasmic membrane, nucleus fragmentations, chromatin compaction, swelling mitochondrias and cytoplasmic vacuolisations. Those results highlighted various immunotoxic effects of MeHg, both at the functional and ultrastructural levels. The antagonistic role of Se on MeHg immunotoxicity is discussed. [less ▲]

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See detailMethylmercury in vitro exposure of harbor seal (Phoca vitulina) lymphocytes: a multidisciplinary approach
Dupont, Aurélie ULg; Siebert, Ursula; Rosenberger, Tanja et al

Poster (2009, June 08)

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See detailMercury immune toxicity in harbour seals: Links to in vitro toxicity
Das, Krishna ULg; Siebert, Ursula; Gillet, Audrey et al

in Environmental Health : A Global Access Science Source (2008), 7

Background Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be ... [more ▼]

Background Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes). Methods Total mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 µM). The expression of cytokines (IL-2; IL-4 and TGF-beta was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 µM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 µM (n = 3). Results The results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 µM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-beta mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 µM). Proteomics showed some variation in the protein expression profile (e.g. vimentin). [less ▲]

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See detailDevelopment of an in vitro model for mercury exposure in marine mammals
Dupont, Aurélie ULg; Bouquegneau, Jean-Marie ULg; Siebert, Ursula et al

Poster (2007, November 01)

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