| All living organisms have the ability, to some extent, to get rid of toxic molecules due to their biotransformation system. The multixenobiotic resistance (MXR) can expel the unwanted molecules out of the cell before biotransformation (phase 0) or later on (phase III). The MXR transporters act as a first line of defence for the cell. Mixtures of environmental contaminants can compromise this defence mechanism when they interact with MXR proteins and the active transport of molecules. Toxic substances can then be accumulated into cells instead of being expelled. Among marine invertebrates, echinoderms of the cold St. Lawrence play a very important ecological role in ecosystem and few studies are available on their physiology and ecotoxicology. Given the interest and importance of cœlomocytes for the echinoderm body itself (circulatory system responsible for, among other things, the immune system) and for ecotoxicologists (potential tools for assessing a particular population health), we studied the resistance of these cells to xenobiotics. The overall objective of this project was to study the biochemical mechanisms of cellular resistance to xenobiotics in cœlomocytes of Leptasterias polaris, Strongylocentrotus drœbachiensis and Cucumaria frondosa. We also examined MXR activity in connection with environmental stressors such as temperature and contaminants.;In a second step, we confirmed a particular fatty acids (FA) profile in the cœlomocytes of echinoderms L. polaris and S. drœbachiensis; with a high proportion of FA: eicosapentænoic acid (20:5n-3), 20:2 NMI and gadoleic acid (20: 1n-11). We also demonstrated the capacity for the reorganization of membrane phospholipids in both species by reducing the degree of membrane insaturation, especially FA 20:5n-3 when temperature is increasing. However, cholesterol was not involved in the membrane remodeling because its amount remained unchanged with temperature increase. Thus, coelomocytes of L. polaris and S. drœbachiensis are partially capable of an homeoviscous adaptation with no difference between species. In addition, a temperature increase did not seem to contribute to a re-activation of the winter metabolic slowdown (measured on cœlomocytes by the method of the microculture tetrazolium salt assay). Temperature is certainly not the only factor bringing echinoderms out their winter metabolic slowdown, especially when food is not available. Finally, by remodeling their membrane lipids, the physical environment of membrane MXR transporters, and thus their protective function of the cell, should be maintained.;In a third step, working with a toxic stress induced by a single contaminant or a combination of two, we established that the L. polaris cœlomocytes were less sensitive to tributyltin (TBT) than similar cells from invertebrate species or vertebrates, and were not sensitive, to either phenanthrene (Phe) or dibultytin (DBT). In addition, a non-toxic concentration of TBT appeared to increase Phe toxicity in L. polaris cœlomocytes. In first instance, Phe is probably directly involved in the transport by MXR proteins because it led to an increase of calcein-AM incorporation in the cœlomocytes. On the other hand, TBT would seem to have an indirect effect on the MXR mechanism, since no detectable effect on the incorporation of any substrate was recorded, but rather an additional toxic effect was observed when combined with cyclosporin-A.;The study of multiple contamination and its interpretation is a difficult task. We believe that the study of MXR transporters and the potential for chemosensitization of contaminants is a very important topic in environmental biomonitoring (from the descriptive level to the decision-making level). Research on the environmental behavior of chemicals that act as chemosensitizers is still in its infancy.;In a first step, we established the presence of a MXR mechanism in cœlomocytes of L. polaris, S. drœbachiensis and C. frondosa. Results indicated a probable presence of a family of transporters of multidrug resistance-associated proteins (MRPs) and at least one additional MXR protein, may be a P-glycoprotein (Pgp). Proteins were determined by two different methods. The first method using flow cytometry was very successful and led us to the hypothesis that a MRP-like protein was probably present in a membrane inside an unidentified cell organelle. The second method of identification was a direct measurement and should tell us more precisely which MXR protein was involved. The results were less significant, probably due to a lack of specificity of some antibodies (Western blot analysis) and an incomplete purification of samples (mass spectrometry analysis). MXR activity was however confirmed in cœlomocytes and a protein MVP (Major Vault Protein) has been detected in cœlomocytes of the sea urchin, S. droebachiensis..;Mots clés : echinoderms, Leptasterias polaris, Strongylocentrotus drœbachiensis, Cucumaria frondosa, cœlomocytes, tributyltin, dibutyltin, phenanthrene, multixenobiotic resistance, environmental stress, temperature, toxic mixture. |
