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Les tests écotoxicologiques en conditions écosystémiques, tels que ceux que l’on peut réaliser en mésocosmes,présentent une plus grande pertinence écologique que les tests normalisés en laboratoire. Ilspermettent d’intégrer les interactions complexes entre les espèces, entre le biotope et la biocénose. Cependant,en contrepartie de leur complexité, ces systèmes présentent une variabilité importante qu’il s’agitde réduire, par une standardisation expérimentale poussée, ou d’identifier les facteurs explicatifs pourmieux mettre en lumière des différences entre traitements. Dans le cadre de cette thèse, nous avons enoutre fait le choix d’une espèce sur laquelle focaliser nos efforts, le poisson, ici l’épinoche à trois épines,Gasterosteus aculeatus, en le considérant comme un bon indicateur de l’état de santé du mésocosme. Ilnous est apparu plus judicieux, dans le cadre des études en mésocosmes, de se concentrer sur une espèceintégratrice en parallèle d’une analyse globale d’abondance des espèces.L’objectif de cette thèse était donc de mieux comprendre la dynamique de populations de l’épinoche àtrois épines dans les mésocosmes de l’Ineris afin d’améliorer les protocoles utilisés et l’analyse des donnéesobtenues lors des études écotoxicologiques. Pour y parvenir, des expériences ont été réalisées afinde caractériser les processus et les traits d’histoire de vie de l’espèce dans notre site d’étude ; un protocolea été proposé pour effectuer des expériences en mésocosmes ciblées sur l’étude de la dynamique despoissons ; deux années d’expérience ont permis d’acquérir des données en conditions témoins et de mieuxsaisir les facteurs confondants ; nous avons mis en application nos connaissances en évaluant les effets dubisphénol A (BPA) sur la dynamique de population de poissons exposés à 0, 1, 10 et 100 μg/L de BPA.Nous avons ainsi pu décrire et expliquer la dynamique de la population de l’épinoche dans les mésocosmespar quatre phases distinctes : une augmentation massive des effectifs en raison de la reproduction desfondateurs ; une diminution importante des effectifs en raison d’une saturation des capacités du système ;une faible reprise ; une nouvelle chute à la fin de la période de reproduction. S’agissant du BPA, des effetssignificatifs ont été mis en évidence sur les individus (atrophie des gonades), mais également à l’échellede la population (structure de la population). / Ecotoxicity tests in ecosystemic conditions, such as those that can be performed in mesocosms, providea substantial ecological relevance. They permit to integrate complex inter-species interactions, interactionsbetween the organisms and the environmental conditions, and permit to assess perturbations witha systemic view. However, in return, these systems present a high variability that should be reduced,through experimental standardization or by identifying explicative factors, to better assess the differencebetween the outputs of the different treatments. In this thesis, we also focused on one species, of fish,the three-spined stickleback (Gasterosteus aculeatus), considering that it is good indicator of the healthstatus of the experimental ecosystem. We suggest that is more appropriate to focus on an integrativespecies in parallel to a global analysis in the context of mesocosms studies.The goal of this thesis was thus to better understand sticklebacks population dynamics in INERIS mesocosmsto improve the experimental protocols and the data analysis in ecotoxicity studies. To achieve thisgoal, studies were performed to characterize life cycle traits and processes of the species ; an experimentalprotocol was proposed to perform mesocosms studies with a focus on fish population dynamics; the datafrom two years of mesocosms studies in control conditions permitted to better understand the confoundingfactors ; we applied the methodolgoy to assess the effects of bisphenol A (BPA) on fish populationdynamics with expsoure concentrations 0, 1, 10 and 100 μg/L BPA.We could describe and explain stickleback population dynamics divided by four periods: first a massiveabundance increase due to the first reproduction of the founders in the mesocosms; second an importantdecrease due to the saturation of the system ; third a short increase ; finaly a new drop due to the endof reproduction. For BPA, effects were observed both at individual and at population levels.

L’exposition au bisphénol A (BPA) au cours du développement fœtal est suspectée d’être impliquée dans l’initiation d’effets biologiques. Dans ce contexte, l’objectif de cette thèse est d’évaluer l’exposition humaine fœtale au BPA. Une étude pharmacocinétique (PK) réalisée sur le modèle du fœtus ovin a montré que le BPA glucuronide (BPAG), métabolite majeur du BPA piégé dans le compartiment fœtal, est lentement éliminé via sa réactivation en BPA. A partir de ces données PK, le développement d’un modèle PK humanisé a permis de prédire des concentrations plasmatiques fœtales de BPAG maintenues autour de 40ng/L chez le fœtus humain en fin de grossesse, soit environ 1000 fois supérieures aux concentrations en BPA correspondantes. Une analyse protéomique de tissus fœtaux ovins a montré qu’une même dose molaire de BPAG ou de BPA affecte des voies physiologiques similaires, suggérant que l’hydrolyse du BPAG pourrait exposer les tissus à des concentrations en BPA compatibles avec l’expression d’un effet biologique. L’ensemble de ces résultats indique que les concentrations de BPAG dans le sang de cordon constituent un marqueur pertinent de l’exposition fœtale au BPA et qu’elles doivent être prises en compte dans l’évaluation du risque / Prenatal exposure to bisphenol A (BPA) is suspected to induce adverse effects later in life. In this context, the goal of this thesis was to evaluate the human fetal exposure to BPA. Using a pharmacokinetic (PK) approach, we showed that BPA glucuronide (BPAG), the main metabolite of BPA that remains trapped in the fetal compartment, is slowly eliminated through its back conversion into BPA. A humanized PK model developed from these data predicted steady BPAG fetal plasma concentrations of 40ng/L in late pregnancy, i.e. about 1000 folds higher than corresponding BPA concentrations. A proteomic analysis of fetal tissues has shown that equimolar BPAG or BPA fetal exposures trigger similar physiological shifts in the ovine model, suggesting that BPAG hydrolysis might expose fetal tissues to BPA at levels of functional significance. It is concluded that BPAG levels in human cord blood are relevant indicators of fetal exposure to BPA during late pregnancy and should be taken into account for risk assessment

Billions of pounds of bisphenol A (BPA) are produced annually around the globe for the manufacture of numerous consumer products, including polycarbonate food and water containers, the protective resin linings of food cans, thermal printing paper, and dental fillings. BPA exposure during nervous system development has been associated with learning and behavioral impairments in animal models. The mode of action for these effects is not clearly defined. While BPA is a weak estrogen receptor (ER) agonist, it is also an estrogen-related receptor gamma (ERR��) agonist. ERR�� binds BPA with 100 times greater affinity than ERs. This study was designed to test the hypothesis that exposure to human-relevant BPA concentrations impacts nervous system development and behavior through ERR�� activation. To examine whether BPA behaves more like an ER or ERR�� ligand, two positive control compounds were used throughout the study: 17��-estradiol (E2) and GSK4716, ER and ERR�� agonists, respectively. Initial behavior testing results included the observation that neurodevelopmental exposure to 0.01 or 0.1 ��M BPA led to hyperactivity in larvae, while exposure to 0.1 or 1 ��M BPA led to learning deficits in adult zebrafish. Exposure to 0.1 ��M E2 or GSK4716 also led to larval hyperactivity. To identify early molecular signaling events that lead to the observed neurobehavioral phenotypes, a global gene expression analysis using a 135K zebrafish microarray was conducted. The concentrations of compounds tested were anchored to the common larval hyperactivity phenotype they elicited. Gross abnormalities, in the case of higher concentrations of BPA and E2, were also anchored phenotypes included in the analysis. Functional pathway analysis of the BPA versus E2 results predicted an impact on prothrombin signaling from the two lower concentrations of BPA and E2. Both BPA and GSK4716 were also predicted to impact nervous system development, potentially involving inhibition of the upstream regulator, SIM1. Additionally, GSK4716 exposure was predicted to inhibit neuron migration. There were fewer similarities in transcriptional responses between BPA and E2 when the lower versus higher concentrations were compared, suggesting different mechanisms operated at the higher concentrations. Subsequent experiments were focused on the role of ERR�� in the larval hyperactivity phenotype. Transient ERR�� knockdown by antisense oligonucleotide morpholino during the first 24 hours of development abrogated the hyperactive phenotype induced by 0.1 ��M BPA exposure. Transient ERR�� knockdown during the first 48 hours of development resulted in developmental delays, craniofacial defects, pericardial edema, and severe body axis curvature. This work is the first to identify behavioral effects in a fish from developmental BPA exposure. It is also the first study to confirm a role for ERR�� in mediating BPA's neurobehavioral effects in any animal model. The global gene expression analysis identified similarities between BPA, E2, and GSK4716, suggesting that BPA's mode of action may involve crosstalk between ERR�� and other ERs. These results from human-relevant BPA exposures help explain the widely documented in vivo effects of BPA, despite low binding affinity exhibited by nuclear ERs. ERR�� is an evolutionarily conserved vertebrate receptor and the developmental impacts of BPA in the zebrafish are an indication of hazard potential to vertebrates. They are also an important translational step toward knowing the hazard potential from human developmental exposure to BPA and yet unknown environmental ligands of ERR��. / Graduation date: 2013 / Access restricted to the OSU Community, at author's request, from Dec. 21, 2012 - June 21, 2013

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Die COVID-19-Pandemie machte deutlich, wie wichtig die Verfügbarkeit eines Schnelltests für ein funktionierendes Gesundheitssystem ist. Mit dieser einfachen Technologie kann die Unsicherheit in den unterschiedlichen und teils kontroversen statistischen Kennzahlen signifikant verringert werden. Immunoassays wie LFIA, FPIA und ELISA (Lateral Flow Immunoassay, Fluoreszenzpolarisationsimmunoassay und Enzymimmunoassay) werden in der Diagnostik und der Umweltanalyse eingesetzt. Die Verwendung von LFIA für ein Screening vor Ort bietet eine Alternative zu instrumentellen Methoden sowie komplizierteren Immunoassay-Formaten und liefert innerhalb von 10 bis 15 Minuten Ergebnisse. Verfahren mit Membranen als feste Träger erlauben parallele Assays in derselben Probe. In dieser Arbeit wurden Latex-Mikropartikel-basierte und Gold-Nanopartikel-basierte LFIAs zum Nachweis von Bisphenol A (BPA) entwickelt. Ein Ziel der Arbeit war die Entwicklung von Nitrocellulose-Membranen, Konjugatpads, Membranbehandlungen sowie einer geeigneten LFIA-Vorbehandlung zwecks Optimierung. Die Ergebnisse eines LFIA-Tests können sowohl mit bloßem Auge als auch instrumentell interpretiert werden. Die visuelle Nachweisgrenze (vLOD) wurde bei 10 μg/L gefunden, die berechnete instrumentelle Nachweisgrenze (cLOD) war 0,14 μg/L. Die Synthese der Haptenproteinkonjugate und deren weitere Bewertung in einem ELISA-Aufbau führte zu einem hochempfindlichen Assay mit einer Nachweisgrenze von 0,05 μg/L. Zusätzlich wurde ein Mix-and-Read-FPIA zur Bestimmung von BPA entwickelt. Dafür wurden neue Tracer-Moleküle, welche Fluorophore mit Derivaten des Analyten verbinden, einschließlich eines C6-Spacers (Ahx) synthetisiert. Der Einfluss der Ahx-Tracer-Brückenlänge auf die Assay-Empfindlichkeit wurde abgeschätzt, die Nachweisgrenze lag bei 1,0 μg/L mit einem Arbeitsbereich von 2 bis 155 μg/L. Die Methoden wurden für reale Proben gegen LC-MS/MS als Referenzmethode mit guter Übereinstimmung mit LFIA, FPIA und ELISA validiert. / The COVID-19 pandemic made it obvious how important the availability of a rapid test is for an efficient healthcare system. This simple technology can significantly reduce the uncertainty in the various and sometimes highly controversial statistical figures of a pandemic. Immunoassays, like LFIA, FPIA and ELISA (lateral flow immunoassay, fluorescence polarization immunoassay, enzyme immunoassay) are used in diagnostics and environmental analysis. The use of LFIA for on-site screening is an alternative to instrumental methods and to more sophisticated immunoassay formats. Results from LFIA are obtained within 10 to 15 min. Methods that use membranes as solid supports allow parallel assays to be performed in the same sample. In this work, latex microparticles-based and gold nanoparticles-based LFIAs for a rapid detection of bisphenol A (BPA) were developed. The work focused on the search for suitable nitrocellulose membranes, conjugate pads, membrane treatments, as well as for a proper LFIA pretreatment to optimize the performance. The results of an LFIA test can be interpreted both by naked eye and instrumentally. The visual limit of detection (vLOD) was found to be 10 μg/L, the calculated instrumental limit of detection (cLOD) was 0.14 μg/L. The synthesis of the required hapten-protein conjugates and further evaluation in an ELISA setup resulted in a highly sensitive assay with a limit of detection of 0.05 μg/L. Additionally, a mix-and-read FPIA for determination of BPA was developed. Here, new tracer molecules that link fluorophores to derivatives of the analyte were synthesized, including a C6 spacer (Ahx). The influence of the Ahx tracer bridge length on the assay sensitivity was estimated. The limit of detection was 1.0 μg/L with a working range from 2 to 155 μg/L. The methods were validated for real samples against LC-MS/MS as reference method with good agreement with LFIA, FPIA, and ELISA.

This thesis aims to assess the human health impact of exposure to estrogenic compounds from the diet. A multi-disciplinary approach is taken to address various aspects of this issue. An introduction to xenoestrogens, including international research priorities, wildlife and human health effects, mechanisms of action, structure activity relationships and additivity of estrogenic effects is provided as background information. An assessment of exposure to a range of naturally occurring and synthetic estrogenic compounds found in food is derived in Chapter 2. The assessment combines new and existing data on food concentration, food consumption and serum levels for each xenoestrogen. Exposure is combined with relative estrogenic potency data from published bioassasy data to estimate risk relative to normal circulating levels of estradiol. Assuming additivity of xenoestrogens, for an average New Zealand male and for post-menopausal women, xenoestrogens in the diet contribute an additional 12-90% of estrogenicity above normal circulating levels. For a pre-menopausal female, the contribution from the diet represents in the order of an additional 2%. The level of exposure determined in this thesis would seem to be of pharmacological relevance, especially for men with low levels of estrogen and for post-menopausal women. Bisphenol A (BPA) is an important monomer used in the manufacture of epoxy resins for internal food can linings. A survey of the BPA content of a range of 80 canned foods available to the New Zealand consumer was undertaken and the results used in the exposure and risk assessments. BPA was detected in all foods analysed except soft drinks, at concentrations ranging from <10-29 µg/kg, except for individual samples of tuna, corned beef and coconut cream that were 109, 98 and 191 µg/kg respectively. None, of over 4000 individual exposure scenarios, exceeded the temporary Tolerable Daily Intake (TDI) of 10 µg/kg body weight per day set by the Scientific Committee on Food in 2002. Intestinal microflora influence the bioavailability of the naturally occurring xenoestrogens genistein and daidzein that contribute significantly to total estrogenicity from the diet. The degradation of genistein and daidzein by the faecal microfloral of 5 human subjects was variable and unpredictable between individuals and within an individual. These findings have important implications for the promotion and prescription of soy foods and supplements for disease prevention and health benefits. The "yeast assay" is one of a number of methods available to measure estrogenicity. This assay was established and validated. In utero exposure to estrogenic compounds at critical periods of sexual differentiation and endocrine development may imprint for health effects observed later in life. Placental transfer of estrogenicity, from 17β-estradiol was studied using the human placental perfusion model and the yeast assay. The placenta provides a protective barrier to the transfer of estrogenicity. Experiments with genistein showed that 5-15% placental transfer occurred, suggesting that in utero exposure might be in the order of 10% of maternal exposure. The thesis concludes with consideration of a genomic approach to substantiate, or refute, the mechanistic link between exposure to xenoestrogens and claimed human health effect. Such an approach offers exciting opportunity to clarify the mode of action of the synthetic versus the naturally occurring xenoestrogens, to confirm or dispute additivity of effect that is an important premise of the exposure assessment, to identify key genes involved in the many possible health effects and thence risk to the individual from dietary exposure to xenoestrogens.

The antineoplastic drug doxorubicin is widely used in the treatment of various types of cancers including breast, colon and lung cancer. However, doxorubicin has adverse effects on the heart and prolonged doxorubicin administration results in cardiomyopathy and congestive heart failure. In the present study we have established that treatment of rat cardiac myoblasts (H9c2 cells) for 24 hours with doxorubicin resulted in concentration and time dependent cell death as determined by proliferation assay. Almost 50-55% cell death was attained at 24 hours treatment of H9c2 cells with 5 μM doxorubicin. We have selected about 50% cell injury as an optimum doxorubicin-induced cell injury because once this threshold is reached, cells became irreversibly injured and are unable to respond to protective treatment. We have observed that another potent antineoplastic drug, cyclophosphamide, had no cardiotoxic effects even with exposure at 35 μM concentrations for a treatment time of up to 72 hours. Pretreatment of H9c2 cells for 24 hours with 100 nM 17β-estradiol protects about 30% cell death against subsequent treatment for 24 hours with 5 μM doxorubicin. Interestingly 500 nM quecertin and 20 μM resveratrol pretreatment provide about 30% and 40% protection, respectively, to the H9c2 cells against subsequent doxorubicin treatment. However, diethylstilbestrol (DES), bisphenol A, and estrone exhibit no protective effects. It is concluded that 17β-estradiol, resveratrol and quercetin considerably protect H9c2 cells against doxorubicin-induced cell death.