Cyprodinil and pyraclostrobin are widely used pesticides to protect many plants from attack of microorganisms and fungal diseases. Although cyprodinil and pyraclostrobin exist in the aquatic environment at low concentration, their leftovers in the ecosystems lead to oxidative damage and interfere development in aquatic animals at environmental-relevant concentration. To figure out the combined effect of cyprodinil and pyraclostrobin, mortality and morphology were investigated in zebrafish embryos exposed to 0.1 and 1μM of cyprodinil, pyraclostobin, and their mixture. Also, gene expression profiles were investigated following exposure to both the compounds and their mixture. Mortality and morphology data showed that the mixture is extremely toxic to zebrafish embryos as it increased mortality in concentration-dependent manner and interfered development of zebrafish embryos at 72 hpf. For gene expression analysis, 0.01μM of cyprodinil, pyraclostobin, and their mixture were used. A total of 47 genes were analyzed. Genes involved in apoptosis, cell cycle arrest, DNA damage repair, drug metabolism, stress response, immune response, and lipid metabolism and transport were analyzed. Upregulation of bax for pyraclostrobin suggests that this chemical could induce mitochondrial apoptotic pathway in zebrafish embryos at 144 hpf. Upregulation of cyp1a for cyprodinil indicates that it could produce ROS and cause oxidative damage in zebrafish embryos. Altered expression of several genes for mix suggests that the mixture of cyprodinil and pyraclostrobin have inhibitory effect on gene expression. Overall, the present study suggests that the mixture of cyprodinil and pyraclostrobin could delay of zebrafish embryonic development, increase mortality of zebrafish embryos, and regulate the expression of several genes.

<p>Accumulating evidence is suggesting that exposure to some environmental contaminants may alter adipogenesis, resulting in accumulation of adipocytes, and often significant weight gain. Thus these types of contaminants are often referred to as obesogens. Many of these contaminants act via the activation (i.e. agonism) of the peroxisome proliferator activated receptor &#947; (PPAR&#947;) nuclear receptor. To date, very few chemicals have been identified as possible PPAR&#61543; ligands. In the thesis, our goal was to determine the PPAR&#947; ligand binding potency and activation of several groups of major semi-volatile organic compounds (SVOCs) that are ubiquitously detected in indoor environments, including flame retardants such as polybrominated diphenyl ethers (PBDEs) and Firemaster 550 (FM550), and other SVOCs such as phthalates, organotins, halogenated phenols and bisphenols. Additional attention was also given to the potential activity of the major metabolites of several of these compounds. Since the primary sink for many of these SVOCs is dust, and dust ingestion has been confirmed as an important pathway for SVOCs accumulation in humans, the potential PPAR&#61543; binding and activation in extracts from environmentally relevant dust samples was also investigated. </p><p> Previous studies have also shown that SVOCs sorbed to organic matrices (e.g., soil and sediment), were only partially bioaccessible (bioavailable), but it was unclear how bioaccessible these compounds are from indoor dust matrices. In addition, bioactivation of SVOCs (via metabolism) could exacerbate their PPAR&#61543; potency. Therefore, to adequately assess the potential risk of PPAR&#947; activation from exposure to SVOC mixtures in house dust, it is essential that one also investigates the bioaccessibility and bioactivation of these chemicals following ingestion. </p><p> In the first research aim of this thesis, the bioaccessibility and bioactivation of several important SVOCs in house dust was investigated. To accomplish this, Tenax beads (TA) encapsulated within a stainless steel insert were used as an infinite adsorption sink to estimate the dynamic absorption of a suite of flame retardants (FRs) commonly detected in indoor dust samples, and from a few polyurethane foam samples for comparison. Experimental results demonstrate that the bioaccessibility and stability of FRs following ingestion varies both by chemical and by matrix. Organophosphate flame retardants (OPFRs) had the highest estimated bioaccessibility (~80%) compared to brominated compounds (e.g. PBDEs), and values generally decreased with increasing Log Kow, with <30% bioaccessibility measured for the most hydrophobic compound tested, BDE209. In addition, the stability of the more labile SVOCs that contained ester groups (e.g. OPFRs and 2-ethylhexyl-tetrabromo-benzoate (TBB)) were examined in a simulated digestive fluid matrix. No significant changes in the OPFR concentrations were observed in this fluid; however, TBB was found to readily hydrolyze to tetrabromobenzoic acid (TBBA) in the intestinal fluid in the presence of lipases. </p><p> In research aims 2 and 3, two commercially available high-throughput bioassays, a fluorescence polarization PPAR&#61543; ligand binding assay (PolarScreenTM PPAR&#947;-competitor assay kit, Invitrogen, Aim 2) and a PPAR&#61543; reporter gene assay (GeneBLAzer PPAR&#947; non-DA Assay, Invitrogen, Aim 3) were used to investigate the binding potency and activation of several groups of SVOCs and dust extracts with human PPAR&#947; LBD; respectively. In the PPAR&#61543; binding assay (Aim 2), most of the tested compounds exhibited dose-dependent binding to PPAR&#947;. Mono(2-ethylhexyl) tetrabromophthalate (TB-MEHP), halogenated bisphenol/phenols, triphenyl phosphate and hydroxylated PBDEs were found to be potent or moderate PPAR&#947; ligands, based on the measured ligand binding dissociation constant (Kd). The most potent compound was 3-OH-BDE47, with an IC50 of 0.24 &#956;M. The extent of halogenation and the position of the hydroxyl group strongly affected binding. Of the dust samples tested, 21 of 24 samples showed significant PPAR&#61543; binding potency at a concentration of 3 mg dust equivalents (DEQ)/mL. In the PPAR&#61543; reporter assay (Aim 3), many SVOCs or their metabolites were either confirmed (based on previous reports) or for the first time were found to be potential PPAR&#947; agonists with various potency and efficacy. We also observed that 15 of 25 dust extracts examined showed an activation percentage more than 8% (calculated activation threshold) of the maximal activation induced by rosiglitazone (positive control). In some cases, activation was as high as 50% of the rosiglitazone activation for the dust extracts with the highest efficacy. Furthermore, the correlation between the reporter assay and the ligand binding assay among the house dust extracts was significant and positive (r = 0.7, p < 0.003), suggesting the binding potency was predicting activation. In research aim 2, the effect of bioactivation on the PPAR&#947; binding potency was also investigated. In vitro bioactivation of house dust extracts incubated with rat and human hepatic S9 fractions was used to investigate the role of in vivo biotransformation on PPAR gamma activity. The result showed that metabolism may lead to an increased binding affinity, as a 3-16% increase in PPAR&#947; binding activity was observed following bioactivation of the dust extracts.</p><p> In research aim 4, an effect-directed analysis (EDA) was used to identify compounds likely contributing to the observed PPAR&#61543; activity among the dust extract. Three dust extracts which showed significant PPAR&#61543; activity with approximately 25, 30, and 50% of the maximal response induced by rosiglitazone at the highest efficacy were fractionated using normal phase high-performance liquid chromatography (NP-HPLC) and each fraction was individually tested for PPAR&#61543; activity. Active fractions were then analyzed using gas-chromatography mass spectrometry (GC-MS) and possible compounds identified. Three dust extracts showed a similar PPAR&#61543; activity distribution among the NP-HPLC fractions. In the most active fractions, fatty acids (FAs) were identified as the most active chemicals. The concentrations of four FAs were measured in the house dust extracts, and the concentrations were found to be highly correlated with the observed PPAR&#61543; activity. These four FAs were also tested for PPAR&#61543; activity and found to be partial PPAR&#61543; agonists, particularly oleic and myristic acid. To tentatively identify sources of FAs, FAs in human/animal hair, dead skin cells, and two brands of cooking oil were analyzed. We found the same FAs in those samples and there concentrations were relatively abundant, ranging from 186 to 14,868 µg/g. Therefore, these results suggest that FAs are likely responsible for the observed PPAR&#61543; activity in indoor dust. Also, this is the first study reporting on the level of FAs in dust samples. The source of these FAs in dust may be either from the cooking or accumulation of human/animal cells in indoor dust.</p><p> In conclusion, this research demonstrates that many SVOCs ubiqutiously detected in house dust, and/or their metabolites, can be weak or moderate PPAR&#61543; ligands. In addition, chemical mixtures in house dust can effectively bind to and activate PPAR&#61543;. However, our results suggest FAs are probably responsible for these observations, and likely outcompeting the synthetic environmental contaminants present in the dust extract. Furthermore, bioactivation of contaminants present in house dust can potentially increase their affinity for PPAR&#61543;. And lastly, the bioaccessibility and stability of SVOCs in house dust after ingestion are likely to modulate the PPAR&#61543; activity in the environmental mixtures and should be considered in future risk assessments.</p> / Dissertation

L’objectif principal de cette thèse était de mieux comprendre le comportement photochimique des pesticides pulvérisés dans les serres agricoles à Menyeh-Liban Nord. La première partie du travail a été consacrée à l’analyse des échantillons de poivrons verts, concombres, tomates, des films de plastique et de l’air récupérés dans ces serres agricoles. Nous avons quantifié et suivi par HPLC-HR-MS les résidus de 4 pesticides: bifénazate (BIF), acétamipride, imidaclopride et thiophanate méthyl (TM) dans les échantillons de légumes extraits par la méthode QUECHERS. Les quantités retrouvées durant les 6 jours suivant la pulvérisation étaient bien supérieures aux MRLs imposées pour chaque pesticide. Par ailleurs, des photoproduits de TM, BIF et de l’imidaclopride ont été détectés dans les solutions de rinçage de la surface des tomates, des concombres et des poivrons verts, démontrant ainsi la possibilité de photodégradation des pesticides à l’intérieur d’une serre agricole. L’analyse de la phase gazeuse par GC-MS a révélé la libération de terpènes, d’alcanes et d’aldéhydes comme réponse des plantes au stress de température et de pulvérisation. Dans une deuxième partie, on a détaillé les mécanismes de transformation photochimique du BIF et du TM sous irradiations simulées. La photodégradation de TM avait un caractère autoaccéléré qui n’a jamais été rapporté dans la littérature. Une étude détaillée nous a permis de démontrer que cette auto-accélération est due à la formation d’un photoproduit photosensibilisateur dérivé de la quinoxaline. Quant au BIF, sa vitesse de dégradation et la nature des produits dépendaient du milieu (ACN, eau, cire et poivron verts). Ces travaux ont mis en évidence l’effet de support et de mélange sur le comportement photochimique des pesticides. / The main purpose of this work was to better understand the photochemical behavior of pesticides used in agricultural greenhouses in Menyeh-North Lebanon. The first part was dedicated to the analysis of green peppers, cucumbers, tomatoes, plastic films and air samples from these agricultural greenhouses. HPLC-HR-MS was used to quantify and monitor the residues of 4 pesticides: bifenazate (BIF), acetamiprid, imidacloprid and thiophanate methyl (TM) after QUECHERS extraction of the vegetables samples. The quantities of pesticides found within 6 days after spraying were well above the MRLs imposed for each one. Then, the photoproducts of TM, BIF and imidacloprid were identified after rinsing the surface of tomatoes, cucumbers and green peppers. This demonstrated the possibility of photodegradation of pesticides even inside an agricultural greenhouse. Gas phase analysis by GC-MS revealed the presence of terpenes, alkanes and aldehydes as the response of plants to temperature and spraying stresses. In a second part, we studied the photochemical mechanisms of BIF and TM in the laboratory under simulated irradiation. The auto-accelerated photodegradation of TM was reported for the first time. A detailed study enabled us to demonstrate that this auto-acceleration was due to the formation of a photosensitizing photoproduct derived from quinoxaline. The rate of degradation of BIF and the nature of the products were dependent on the medium (ACN, water, wax and green peppers). These results highlighted the effect of support and mixture on the photochemical behavior of pesticides.

Endocrine disrupting chemicals, or EDCs, are some of the most prevalent toxic chemicals found in the environment because of human activity and they have a variety of adverse effects on both humans and wildlife. A proposed mechanism through which EDCs can negatively affect an organism is via an epigenetic mechanism known as DNA methylation, which can affect the development of the organism with negative outcomes later in life. The aim of this project was to investigate the effect of a prenatal exposure to mixture of EDCs, called Mixture N1, and its adverse effects. Mixture N1 has in a previous study been detected in the first semester of mothers, and linked to language delay in the offspring in the SELMA cohort study. We investigated relationships with DNA methylation pattern changes in key genes with exposure, gene expression and behaviour in the adult brains of the exposed mice.  Our results showed correlative as well as linear relationships between methylation and different behavioural outcomes for target genes. One gene in particular - Nr3c1 - stood out among the results, having links to both stress and sociability. Specifically, DNA methylation of this gene correlates to active stress coping behaviours as well as sociability, but with no mediation component in these relationships. These results are promising for the use of methylation analysis as a biomarker of EDC mixture exposure, but more so as a predictor of negative behavioural outcomes later in life. More research could strengthen this use, and uncover the mechanism through which methylation alone might affect changes in behaviour.