In this thesis, we examined the effects of the exposures to anthropogenic pollutants on the fish, primarily juvenile chinook salmon, immune system using newly and recently developed immune assays. In addition, we developed a new assay for measuring immunocompetence of fish. In the first chapter, the Alamar Blue assay was developed to quantify the proliferation of chinook salmon (Oncorhynchus tshawytscha) leukocytes. Isolated splenic and pronephric leukocytes were stimulated with different concentration of mitogens (LPS, PWM, and ConA) for various incubation times. Optimum cell culture conditions (cell density, mitogen concentration, and incubation time) for the Alamar Blue assay were evaluated by comparison with flow cytometric analysis. The Alamar Blue dye was non-toxic for leukocytes, and the assay proved to be able to quantify the mitogenic responses using LPS, but PWM and ConA. In the second chapter, we determined the effects and mechanisms by which p,p'- DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha). Isolated salmon splenic and pronephric leucocytes were incubated with different concentrations of p,p'-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p'- DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p'-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leucocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p'-DDE. The effect of p,p'-DDE on leucocytes appeared to vary between developmental stages or season. The mitogenic response of leukocytes of chinook salmon exposed to p,p'-DDE in vivo exhibited a biphasic dose-response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p'-DDE had a significantly lower percentage of Ig+ blasting cells than controls. Our results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression. In the third chapter, we evaluated the direct effects of in vitro exposures to tributyltin (TBT), widely used biocide, on the cell mediated immune system of chinook salmon (Oncorhynchus tshawytscha). Splenic and pronephric leukocytes isolated from juvenile chinook salmon were exposed for 6, 24, or 96 hr to a concentration range of 0.03 0.1 mg TBT 1����� in cell cultures. Effects of TBT on cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry. Splenic and pronephric leukocytes in the presence of TBT experienced a concentration-dependent decrease in the viability in cell cultures following the induction of apoptosis. In addition, pronephric lymphocytes exhibited a greater sensitivity to TBT exposure than pronephric granulocytes. The functional ability of splenic B-cells to undergo blastogenesis upon LPS stimulation was also significantly inhibited in the presence of 0.05, 0.07, or 0.10 mg 1����� of TBT in the cell cultures. Flow cytometric assay with the fluorescent conjugated monoclonal antibody against salmon surface immunoglobulin was employed for the conclusive identification of B-cell in the chinook salmon leukocytes. Our findings suggest that adverse effects of TBT on the function or development of fish immune systems could lead to an increase in disease susceptibility and its subsequent ecological implications. / Graduation date: 2004

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Impaired function of the human intestinal epithelial barrier (IEB) might allow for permeability of harmful substances, such as obesogens, which induce obesity and further implications. Tight junction (TJ) proteins are the key component for normal functions of the barrier. In this master thesis, the correlation between increased TJ permeability of the IEB and absorption of obesogens was studied. The effect of obesogens on TJ expression was also investigated. Permeability tests performed on Caco-2 cell monolayers exposed to obesogens showed altered permeability, indicating that obesogens might have an effect on TJ protein expression. Furthermore, impaired monolayers showed increased permeability, which implies that impaired functions of IEB lead to increased absorption of obesogens.

No description available.

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

Un régime alimentaire déséquilibré et/ou la présence de composés contaminantsexogènes peuvent modifier la signalisation endocrine et l’homéostasie des lipides et induirel’obésité. Les travaux réalisés dans le cadre de cette thèse ont permis, dans un premier temps,de développer une méthode simple et rapide, dénommée "zebrafish obesogenic (ZO) test",pour identifier in vivo, par utilisation de la larve de poisson zèbre, des facteurs qui peuventaugmenter ou diminuer la taille de l’adipocyte blanc et ainsi moduler le niveau de l’adiposité(Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). Ce test permetd’identifier des composés et des mélanges de molécules obésogènes et anti-obésogènes etfournit des informations pertinentes pour l'évaluation des risques liés leur présence maiségalement pour élucider les mécanismes impliqués. Les travaux ont, dans un second temps,permis d’apporter des réponses quant aux modalités d’action d’un obésogène puissant, lechlorure de tributylétain, contaminant retrouvé très largement dans notre environnement.Cette molécule agit sur l’adipocyte blanc à une concentration de l’ordre du nano molaire viales récepteurs nucléaires RXR et LXR, et non pas via les isoformes PPARgamma/delta(Ouadah et Babin, manuscrit en préparation). / An unbalanced diet and / or the presence of exogenous compounds contaminants mayalter endocrine signaling and lipid homeostasis and induce obesity. The work done in thisthesis have, at first, developed a simple and rapid method, called "zebrafish obesogenic (ZO)test" to identify in vivo by using the zebrafish larva, the factors that may increase or decreasethe size of the white adipocyte and therefore modulate the level of adiposity (Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). This test helps to identifycompounds and mixtures of obesogenic and anti-obesogenic molecules and providesinformation relevant to the risk assessment of their presence but also to elucidate themechanisms involved. Work in a second time allowed to answer as to how the action oftributyltin chloride, a powerful obesogenic contaminant found widely in the environment.This molecule acts in vivo on white adipocytes in a concentration of the order of nano molarvia nuclear receptors LXR and RXR, and not via the PPARgamma isoforms / delta (Ouadahand Babin, manuscript in preparation).

Le bassin d’Arcachon est une lagune semi-fermée qui concentre de forts enjeux économiques grâce à la pratique de l’ostréiculture. Cette activité est affectée depuis une trentaine d’années par des phénomènes de mortalités estivales, et plus récemment par des surmortalités du naissain. Ces travaux se sont intéressés au rôle des polluants majeurs du bassin d’Arcachon dans ce contexte de crise en étudiant leurs effets sur la biologie de Crassostrea gigas. Une approche in situ a d’abord été adoptée afin d’identifier les contaminants les plus présents dans le milieu naturel. Des opérations de transplantation d’huîtres et des prélèvements de sédiments ont permis de quantifier divers contaminants et d’associer leur présence à des réponses biologiques. Les polluants ainsi identifiés ont ensuite été employés en conditions contrôlées au laboratoire. Trois expérimentations ont été réalisées : la première concerne l’étude des voies de contamination par le tributylétain ; la seconde concerne les effets des pesticides et du cuivre ; la troisième concerne l’effet des HAP sur des huîtres diploïdes et triploïdes. Nos résultats indiquent que les organismes transplantés au coeur de la lagune sont plus exposés aux polluants, en lien avec les caractéristiques hydrodynamiques du système. La plupart des paramètres biologiques étudiés sur le terrain ont par ailleurs montré une saisonnalité liée aux processus de gamétogenèse. Au laboratoire, la plupart des contaminants testés ont induit une réponse adaptative chez les huîtres exposées. Notre étude souligne l’importance de coupler approche de terrain et approche expérimentale pour comprendre le fonctionnement des écosystèmes côtiers. / The Arcachon Bay is a semi-enclosed lagoon and represents the core of strong economic stakes through the practice of oyster-farming. This activity has been affected for around thirty years by summer mortality events, and more recently by abnormally high death rates of juveniles. This work focused on the role of the Arcachon Bay’s main contaminants in this crisis, by studying their effects on the cupped oyster’s biology. Firstly, an in situ approach was adopted in order to identify the major pollutants of the bay: caged oysters were transplanted and sediments were sampled. The presence of contaminants in the samples was associated to biological responses. Secondly, the contaminants identified in situ were used in controlled conditions at the laboratory. Three experimentations were performed; the first dealt with the contamination pathways of tributyltin; the second focused on the biological effects of pesticides and copper; the third concerned the effects of PAH towards diploid and triploid oysters. Our results indicate that the inner stations present higher accumulation of metals and PAH, in accordance with the hydrodynamic features of the bay. The bioindicators used in situ exhibited seasonal trends related to the oysters’ gametogenesis. In the laboratory, most of the contaminants used at environmental levels induced an adaptive response of the exposed oysters. Our study highlights the importance of coupling in situ and laboratory approaches in order to understand the functioning of coastal ecosystems.