Héloïse Frouin

Science and policy on endocrine disrupters must not be mixed: a reply to a “common sense” intervention by toxicology journal editors

The “common sense” intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Recent progress on our understanding of the biological effects of mercury in fish and wildlife in the Canadian Arctic

This review summarizes our current state of knowledge regarding the potential biological effects of mercury (Hg) exposure on fish and wildlife in the Canadian Arctic. Although Hg in most freshwater fish from northern Canada was not sufficiently elevated to be of concern, a few lakes in the Northwest Territories and Nunavut contained fish of certain species (e.g. northern pike, Arctic char) whose muscle Hg concentrations exceeded an estimated threshold range (0.5-1.0 μg g(-1) wet weight) within which adverse biological effects begin to occur. Marine fish species generally had substantially lower Hg concentrations than freshwater fish; but the Greenland shark, a long-lived predatory species, had mean muscle Hg concentrations exceeding the threshold range for possible effects on health or reproduction. An examination of recent egg Hg concentrations for marine birds from the Canadian Arctic indicated that mean Hg concentration in ivory gulls from Seymour Island fell within the threshold range associated with adverse effects on reproduction in birds. Mercury concentrations in brain tissue of beluga whales and polar bears were generally lower than levels associated with neurotoxicity in mammals, but were sometimes high enough to cause subtle neurochemical changes that can precede overt neurotoxicity. Harbour seals from western Hudson Bay had elevated mean liver Hg concentrations along with comparatively high muscle Hg concentrations indicating potential health effects from methylmercury (MeHg) exposure on this subpopulation. Because current information is generally insufficient to determine with confidence whether Hg exposure is impacting the health of specific fish or wildlife populations in the Canadian Arctic, biological effects studies should comprise a major focus of future Hg research in the Canadian Arctic. Additionally, studies on cellular interactions between Hg and selenium (Se) are required to better account for potential protective effects of Se on Hg toxicity, especially in large predatory Arctic fish, birds, and mammals.

Effects of individual polybrominated diphenyl ether (PBDE) congeners on harbour seal immune cells in vitro.

Effects of polybrominated diphenyl ethers (PBDEs) on the immune system of marine mammals are poorly understood. One important innate immune function of granulocytes is the respiratory burst which generates reactive oxygen species (ROS) used to kill engulfed microorganisms. The present study investigates in vitro the effects of BDE-47, -99 and -153, on the formation of ROS, on intracellular level of thiols, on activity and efficiency of phagocytosis and on apoptosis in granulocytes of harbour seals. Compounds were tested at four different concentrations ranging from 1.5 to 12 microM. Results showed that ROS levels, thiol levels and phagocytosis were all affected when harbour seal cells were exposed to the highest concentration (12 microM) of PBDE congeners. Apoptosis was not affected by PBDEs. The observed effects were similar in adults, pups and in the 11B7501 cell line of harbour seals.

Toxic effects of tributyltin and its metabolites on harbour seal (Phoca vitulina) immune cells in vitro.

The widespread environmental contamination, bioaccumulation and endocrine disruptor effects of butyltins (BTs) to wildlife are well documented. Although suspected, potential effects of BTs exposure on the immune system of marine mammals have been little investigated. In this study, we assessed the effects of tributyltin (TBT) and its dealkylated metabolites dibutyltin (DBT) and monobutyltin (MBT) on the immune responses of harbour seals. Peripheral blood mononuclear cells isolated from pup and adult harbour seals were exposed in vitro to varying concentrations of BTs. DBT resulted in a significant decrease at 100 and 200 nM of phagocytotic activity and reduced significantly phagocytic efficiency at 200 nM in adult seals. There was no effect in phagocytosis with TBT and MBT. In pups, the highest concentration (200 nM) of DBT inhibited phagocytic efficiency. A reduction of tumor-killing capacity of adult natural killer (NK) cells occurred when leukocytes were incubated in vitro with 50 nM DBT and 200 nM TBT for 24h. In adult seals, T-lymphocyte proliferation was significantly suppressed when the cells were exposed to 200 nM TBT and 100 nM DBT. In pups, the proliferative response increased after an exposure to 100 nM TBT and 50 nM DBT, but decreased with 200 nM TBT and 100 nM DBT. The immune functions were more affected by BTs exposure in adults than in pups, suggesting that other unsuspected mechanisms could trigger immune parameters in pups. The toxic potential of BTs followed the order of DBT>TBT>MBT. BT concentrations of harbour seal pups from the St. Lawrence Estuary (Bic National Park) ranged between 0.1-0.4 ng Sn/g wet weight (ww) and 1.2-13.4 ng Sn/g ww in blood and blubber, respectively. For these animals, DBT concentrations were consistently below the quantification limit of 0.04 ng Sn/g ww in blood and 0.2 ng Sn/g ww in blubber. Results suggest that concentrations measured in pups are considered too low to induce toxic effects to their immune system during first days of life. However, based on our in vitro results, we hypothesize that BTs, and DBT in particular, could pose a serious threat to the immune functions in free-ranging harbour seal adults.

Mercury toxicity in beluga whale lymphocytes: Limited effects of selenium protection

Increasing emissions of anthropogenic mercury represents a growing concern to the health of high trophic level marine mammals. In its organic form, this metal bioaccumulates, and can be toxic to several physiological endpoints, including the immune system. In this study, we (1) evaluated the effects of inorganic mercury (mercuric chloride, HgCl2) and organic mercury (methylmercuric chloride, MeHgCl) on the in vitro function of lymphocytes isolated from the peripheral blood of beluga whales (Delphinapterus leucas); (2) characterized the potential protective effects of sodium selenite (Na2SeO3) on cell proliferation of HgCl2 or MeHgCl-treated beluga whale lymphocytes; and (3) compared these dose-dependent effects to measurements of blood Hg in samples collected from traditionally harvested beluga whales in the western Canadian Arctic. Lymphocyte proliferative responses were reduced following exposure to 1 μM of HgCl2 and 0.33 μM of MeHgCl. Decreased intracellular thiol levels were observed at 10 μM of HgCl2 and 0.33 μM of MeHgCl. Metallothionein induction was noted at 0.33 μM of MeHgCl. Concurrent exposure of Se provided a degree of protection against the highest concentrations of inorganic Hg (3.33 and 10 μM) or organic Hg (10 μM) for T-lymphocytes. This in vitro protection of Se against Hg toxicity to lymphocytes may contribute to the in vivo protection in beluga whales exposed to high Hg concentrations. Current Hg levels in free-ranging beluga whales from the Arctic fall into the range of exposures which elicited effects on lymphocytes in our study, highlighting the potential for effects on host resistance to disease. The implications of a changing Arctic climate on Hg fate in beluga food webs and the consequences for the health of beluga whales remain pressing research needs.

PBDEs in serum and blubber of harbor, grey and harp seal pups from Eastern Canada.

Serum and blubber of pup harbor (Phoca vitulina), grey (Halichoerus grypus) and harp (Phoca groenlandica) seals from the Estuary and the Gulf of St. Lawrence were analyzed for polybrominated diphenyl ethers (PBDEs). Concentrations of ∑PBDEs (on lipid weight basis) in tissues of harbor seal pups inhabiting the St. Lawrence Estuary were about five times higher than in those from a colony located in the northern Gulf. Harp seal pups have the lowest levels of ∑PBDEs among the seal species born in the Gulf of St. Lawrence. Lipid normalized concentrations of ∑PBDEs in serum and blubber were strongly correlated, suggesting that the blood collection as a less invasive method could be used to monitor ∑PBDEs in pups. This study shows that fluvial inputs of PBDEs are important to the St. Lawrence marine ecosystem and that inhabiting harbor seal pups have a substantial exposure to PBDEs at a critical developmental stage. In addition, the observed difference in PBDE levels between harp and grey or harbor seal pups from the Gulf of St. Lawrence is explained by the difference in diets of their mothers which is linked with their residency time in the Gulf and their seasonal migration pattern.

T Lymphocyte-Proliferative Responses of a Grey Seal (Halichoerus grypus) Exposed to Heavy Metals and PCBs in Vitro

This study investigated in vitro the effects of methylmercury chloride (CH3HgCl), zinc chloride (ZnCl2), cadmium chloride (CdCl2), lead acetate (Pb(C2H3O2)2), and PCBs (Aroclor mixtures) on the proliferation of T lymphocytes from the thymus, lymph node, and blood from one female grey seal (Halichoerus grypus) juvenile. After exposure to heavy metals, a dose-response curve was observed with a decrease in proliferation of both T lymphocytes from blood and the lymph node. Exposure to Aroclor mixtures led to a mostly reduced proliferation of thymocytes and T lymphocytes from the lymph node and blood. Lymph node cells seem less sensitive to heavy metals than peripheral blood lymphocytes. Lymph node lymphocytes are more sensitive to PCBs than peripheral blood lymphocytes but less than thymocytes. These results suggest that the sensitivity of T lymphocytes from one grey seal to contaminants may be due to inherent tissue/matrix differences in the sensitivity of these cells to contaminants; however, an individual response cannot be excluded in the present case. That is, samples from a single individual are not extrapolated to the species as a whole in this paper but discussed relative to exposure response by tissues.

Immune status and function in harbor seal pups during the course of rehabilitation.

Routine hematological and serum chemistry parameters are important tools for the evaluation of health and the treatment of marine mammals admitted to rehabilitation centers. The evaluation of phagocytosis, oxidative burst and immunoglobulin G (IgG), as markers of immune system function, and haptoglobin (Hp), as a stress marker, were evaluated alongside routine hematology and chemistry as potentially informative diagnostic tools for marine mammal health assessments. Blood samples from harbor seal pups (Phoca vitulina) admitted to (n=46), and released from (n=28), the Vancouver Aquariums Marine Mammal Rescue Center (VAMMRC) were collected (1) to perform routine and novel functional approaches to evaluate the health of pups at admission; (2) to determine how these parameters changed during the rehabilitation process; and (3) to generate baseline values for reference purposes. Sodium was the only blood parameter which differed between seal pups that survived and those that died, with the surviving pups exhibiting higher levels on admission diagnostics. Positive correlations between total protein concentrations, IgG and Hp levels were observed with globulin concentrations of seal pups. Changes in serum chemistry values between admission and release included a decrease in red blood cells (RBCs), glucose, bicarbonate, total bilirubin, γ-glutamyltransferase (GGT) levels, and an increase in mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), lymphocytes, eosinophils, urea, potassium, anion gap, calcium, phosphorus, total protein, albumin, globulin and osmolality levels. During the rehabilitation process, phagocytosis decreased, while Hp levels increased. Age and improved health appeared to underlie changes in these parameters during the rehabilitation period.

Phagocytosis in pup and adult harbour, grey and harp seals

Knowledge on pinniped immunology is still in its infancy. For instance, age-related and developmental aspects of the immune system in pinnipeds need to be better described. The present study examined the phagocytic activity and efficiency of harbour, grey and harp seal leukocytes. In the first part of the study, peripheral blood was collected from captive female harbour seals of various ages. Data showed an age-related decrease in phagocytosis in female harbour seals from sub-adult to adulthood. In the second part of the study, changes in phagocytosis were quantified during lactation in wild newborn harbour, grey and harp seals and in their mothers (harp and grey seals). In newborns of the same age, leukocytes of harbour and harp seals phagocytosed less than those of grey seal pups. The phagocytic activity and efficiency increased significantly from early to mid-lactation in newborn harbour seals, and from early to late lactation in newborn grey seals, which could suggest that the transfer of phagocytosis-promoting factor(s) in colostrum is an important feature of temporary protection for pups. In contrast, no changes in phagocytic activity and efficiency were observed in lactating females of the two seal species, harp and grey, examined. At late lactation, phagocytic activity in both grey and harp seal pups and phagocytic efficiency in grey seal pups were significantly higher than in their mothers. These results could reflect either the capacity of phagocytes of the newborn harp and grey seals to respond to pathogens. Results from this study suggest that the phagocytosis of the seal species examined is not fully developed at birth as it generally increases in pups during lactation. Thereafter, the phagocytic activity of seals appears to decrease throughout adulthood.