Immunotoxicology of methylmercury and other contaminants in harbour seals (Phoca vitulina) from the North Sea

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.Immunotoxicology of methylmercury and other contaminants in harbour seals (Phoca vitulina) from the North Se