Les oeufs d’escargot terrestre de l’espèce petit-gris Helix aspersa (syn. Cantareusaspersus) peuvent être utilisés pour évaluer l’écotoxicité de substances chimiques pures ou enmélange. La mesure des effets embryotoxiques classiquement réalisée est le succès d’éclosionaprès 15 à 20 jours d’exposition (Druart et al., 2012). Cependant, les mécanismes impliquésdans la mise en place des effets toxiques à différents niveaux d’organisation biologique chezl’embryon ne sont pas connus. Des oeufs d’escargots ont été exposés à des solutions decontaminants métallique (Cd) ou organiques (pesticides: le Round Up® flash, le Corail® et laBouillie Bordelaise) selon deux modalités différentes (en continu sur la totalité dudéveloppement embryonnaire ou sur une période de 24 heures) afin de 1/ déterminer denouveaux paramètres de mesure au cours du développement embryonnaire pouvant rendrecompte d’un effet toxique, 2/ détecter des effets génotoxiques de divers contaminants(solution métallique de Cd ou de formulations commerciales de pesticides) par la méthodeRandom Amplified Polymorphic DNA (RAPD) et 3/ d’étudier des systèmes de défense métalspécifiques(métallothionéines).Les paramètres morphologiques et physiologiques suivis au cours d’expositionscontinues au Cd ont montré des effets néfastes sur le rythme cardiaque, la durée del’incubation, la taille et le poids à l’éclosion chez les exposés à la plus forte concentrationtestée. Chez ces derniers des signes de fragmentation de l’ADN ont également été détectés enfin d’exposition. Le couplage de la méthode RAPD avec un système d’électrophorèse hauterésolution (SHR) a permis de détecter des effets génotoxiques suite à des expositionscontinues au Cd, au Round Up® et au Corail®. L’étude par PCR quantitative de l’expressiondes gènes des métallothionéines (MTs) a mis en évidence une expression constitutive des MTsainsi qu’un haut niveau d’expression du gène mixte CdCuMT chez les embryons non exposés.Chez les embryons exposés au Cd durant 24 heures, une surexpression du gène spécifiqueCdMT a été mise en évidence alors qu’aucune augmentation significative des taux detranscrits des 2 autres isogènes étudiés (CuMT et CdCuMT) n’a été démontrée.Les résultats de toxicité du Cd basés sur le taux d’éclosion et l’expression des gènes desMTs ont démontré que des facteurs comme le régime d’exposition (24 heures ou en continu)ou le stade de développement (âge des embryons lors de l’exposition) peuvent modulerl’embryotoxicité des substances chimiques.206Les données obtenues durant cette étude intégrative permettent de proposer un largepanel de paramètres de mesure des effets toxiques des substances chimiques chez l’embryond’escargot terrestre H. aspersa au niveau individuel (rythme cardiaque, taille, durée dedéveloppement et succès d’éclosion) et au niveau moléculaire (expression de gènes dessystèmes de défense, détection des signes de génotoxicité et de la fragmentation de l’ADN)pour l’évaluation de la toxicité des substances chimiques. L’approche RAPD-SHR, bien quenécessitant une certaine expertise pour l’analyse des profils d’amplifications obtenus, apparaîtadaptée pour une détection rapide et efficace du potentiel embryogénotoxiques de substancesvariées (métaux, pesticides. / The land snail species Helix aspersa (syn. Cantareus aspersus) eggs can be used to assess theecotoxicity of chemicals. Measurement of embryotoxic effect is classically based on hatching successafter 15-20 days of exposure (Druart et al., 2012). However, the mechanisms involved in toxic effectsin embryos at different levels of biological organization are not known. Eggs of snails were exposedto solutions metallic contaminants (Cd) or organic (pesticides: Round Up® Flash, Corail® andBordeaux Mixture) in two different regimes (continuous over the entire embryonic development orduring a period of 24 hours), in order to 1 / identify of new endpoints of toxic effect measurementsduring embryonic development, 2 / detect of genotoxic effects of metal solution (Cd) or threepesticides commercial formulations by Random Amplified Polymorphic DNA method (RAPD) and 3 /study metal-specific defense systems (metallothionein).Morphological and physiological parameters monitored during Cd continuous exposures showedadverse effects on heart rate, duration of incubation, size and weight of new hatchlings exposed to thehighest concentration tested. In the latter, signs of DNA fragmentation were detected at the end ofexposure. Coupling the RAPD with a high-resolution electrophoresis system (SHR) has enabled todetect genotoxic effects of Cd, Round Up® and Corail® after continuous exposures. Quantitative PCRstudy of metallothioneins (MTs) gene expression has showed constitutive expression of MTs genesand a high level of mRNA for the mixed gene CdCuMT in unexposed embryos. In embryos exposedto Cd for 24 hours, an overexpression of the specific gene CdMT has been demonstrated whereas thetwo other isogenes (CuMT and CdCuMT) didn’t show significant induction of expression rates.The toxicity results based on the hatching rate and MTs genes expression obtained with Cd haveshowed that factors such as the exposure regime (24 hours or continuous) or the stage of development(age of embryos upon exposure) can modulate embryotoxicity of chemicals. This thesis provides awide range of endpoints usable at the individual level (heart rate, height, hatching monitoring) and atthe molecular level (gene expression of defense systems, detection of genotoxicity signs and DNAladdering) for the assessment of the ecotoxicity of chemical substances. The RAPD-SHR, althoughrequiring some expertise to analyze profiles obtained, appears suitable for rapid and efficientdetection of potential embryogenotoxic effects of various substances (metals, pesticides).

[pt] No presente trabalho foram desenvolvidas duas metodologias analíticas para a determinação de talidomida, um composto de reconhecida importância farmacológica. As metodologias espectrofluorimétrica e voltamétrica desenvolvidas foram comparadas em termos de desempenho analítico.As características fluorescentes e eletroquímicas da talidomida foram estudadas para que se encontrassem condições experimentais que fornecessem máximo sinal fluorescente do analito em solução na temperatura ambiente e possibilidade de pré-concentração do analito no eletrodo de mercúrio.Nas condições experimentais otimizadas para a talidomida, limites de detecção compreendidos entre 10-6 e 10-9 g L-1 foram obtidos para o método espectrofluorimétrico e voltamétrico, respectivamente. Faixas lineares dinâmicas entre 2 e 4 ordens de grandeza foram alcançadas dependendo do método e do interferente presente na amostra. Esses parâmetros de mérito se mostraram adequados para o problema proposto. O possível efeito interferente de substâncias geralmente usadas em associação com o analito foi estudado, e estratégias para minimização das interferências foram desenvolvidas. Enquanto a tetraciclina não interferiu no método espectrofluorimétrico, o uso combinado de meio ácido e irradiação UV da amostra foi necessária para permitir a quantificação do analito em presença de sulfanilamida. Na voltametria, as interferências da tetraciclina e da sulfanilamida puderam ser compensadas pelo uso da quantificação pelo método de adição do analito. Nas determinações dos fluídos biológicos, o uso da extração em coluna de sílica C18 mostrou-se bastante eficiente na separação do analito dos interferentes da matriz para o método espectrofluorimétrico e para o voltamétrico, no caso do soro sangüíneo. Para a urina, apenas a clarificação da amostra com sulfato de amônio foi suficiente no caso da determinação voltamétrica.Os métodos desenvolvidos foram testados na dosagem de talidomida presente em uma formulação comercial e em amostras de urina e soro sangüíneo enriquecidas com o analito. Para tal, utilizaram-se os procedimentos de curva de calibração (espectrofluorimetria) e método da adição do analito (voltametria). Em todos os casos, as recuperações obtidas estiveram compreendidas na faixa de 96,5 a 107,6 %, dentro da faixa de recuperação estabelecida pela Farmacopéia dos Estados Unidos da América. / [en] In the present work two analytical methodologies were developed aiming the determination of thalidomide, an important pharmacological compound. The developed spectrofluorimetric and the voltammetric based analytical methodologies were compared in terms of analytical performance. The thalidomide fluorescent and the electrochemical characteristics were studied in order to find experimental conditions for maximum fluorescence in solution and at room temperature and to allow analyte pre- concentration on the mercury electrode. Using the optimized experimental conditions, limits of detection between 10-6 to 10-9 g L-1 were achieved respectively for the spectrofluorimetric method and for the voltammetric method. Dynamic linear ranges between 2 and 4 orders of magnitude were obtained depending on the method utilized and the interferent substances present in the sample. Those parameters of merit were suitable for this proposed analytical problem. The potential interference effect from substances usually used in association with thalidomide, were studied and strategies for the minimization of such interferences were developed. While no interference in the spectrofluorimetric method was observed for tetracycline, the combined use of acidic medium and UV irradiation of the samples was necessary to allow the analyte determination in the presence of sulfanilamide. For the voltammetric method, interferences from tetracycline and sulfanilamide could be compensated by quantifying thalidomide using the analyte addition method. For the determination in biological fluids, the use a solid-liquid extraction on a C18 column was found to be very effective for the analyte separation and elimination of matrix interferences for the spectrofluorimetric method and for the voltammetric method developed for blood serum. For urine samples, a clean-up step using ammonium sulfate was found to be sufficient for the voltammetric determination of thalidomide. The developed methodologies were tested by determining the thalidomide content in a commercial pharmaceutical formulation and in analyte spiked biological fluids using calibration curves (spectrofluorimetric) and analyte addition method (voltammetric). In all cases, the recoveries were between the 96,5 and 107,6 %, within the recovery range considered adequate according to the United States Pharmacopoeia.