Toxicity testing is moving from animal-based studies to faster, more ethical in vitro approaches that focus on mechanistic toxicology. The use of bisphenol A (BPA) replacement compounds is increasing and there is limited toxicity data available for these compounds in avian species. The overall goals of this thesis were to: a) determine if avian cell lines are suitable alternatives to primary hepatocytes for chemical screening; b) generate toxicity data for five BPA replacement compounds: bis(4-hydroxyphenyl)methane (BPF), bis(3-allyl-4-hydroxyphenyl)sulfone (TGSH), 7-bis(4-hydroxyphenylthio)-3,5-dioxaheptane (DD-70), 2,2-bis(4-hydroxyphenyl)hexafluoropropane (BPAF) and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP) in three in vitro models: primary chicken embryonic hepatocytes (CEH), double-crested cormorant (DCCO) embryonic hepatocytes (DCEH) and chicken LMH cell line; and 3) prioritize two replacements for early-life stage testing (ELS). LMH cells cultured as 3D spheroids, as opposed to 2D monolayer, had enhanced mRNA expression and CYP1A activity and were therefore used for screening. Additionally, an immortalized DCCO hepatic cell line, DCH22, was established, which may be useful for future avian toxicity testing. DD-70 and BPAF were the most cytotoxic across the three in vitro models. TGSH and DD-70 altered expression of genes associated with multiple toxicity pathways, but not estrogen response, and are potential non-estrogenic replacements. BPAF, BPF and BPSIP are potential estrogenic replacements. In general, the replacements were more cytotoxic and/or transcriptionally active than BPA. There was species-specific variability in toxicity; the replacements were more transcriptionally active in CEH compared to DCEH. LMH spheroids were more sensitive to estrogenic endpoints than CEH. DD-70 and BPAF were prioritized for ELS studies based on in vitro results. All of the replacements modulated the expression of genes related to bile acid regulation in vitro and an increase in gallbladder mass was observed in chicken embryos after exposure to DD-70 or BPAF. Overall, this thesis evaluated the utility of LMH cells cultured as spheroids as an animal free alternative for chemical screening, established a DCCO cell line, and generated novel cytotoxicity and gene expression data for five BPA replacement compounds in three in vitro avian models and determined ELS toxicity of two replacement compounds.

Background. Bisphenol A (BPA) and BPS are environmental pollutants that are associated with the development of insulin resistance and type 2 diabetes (T2D). Although skeletal muscle and adipose tissue dysfunction are involved the development of insulin resistance, there are few studies that have investigated the effects of bisphenols on their metabolism. In this study, we investigated the effects of BPA and BPS exposure on skeletal muscle and adipose tissue metabolism to determine how they contribute to the development of T2D. Methods. L6 muscle cells were treated with BPA during the last 24 hours of differentiation, and mitochondrial function and glucose metabolism was measured. Human subcutaneous adipose tissue was incubated for 24 or 72 hours with BPA or BPS, and adipokine gene expression and glucose metabolism was measured in adipose tissue. Results. L6 muscle cells treated with high concentrations of BPA (10⁵ nM) had mitochondrial dysfunction and a compensatory increase in glucose metabolism; however, there were no effects at environmentally-relevant concentrations. Adipose tissue treated with BPA for 24 hours had reduced expression of proinflammatory cytokines and adipokines, and reduced insulin-stimulated glucose uptake. Conclusions. BPA exposure for 24 hours did not alter L6 muscle cell mitochondrial function and glucose metabolism at environmentally-relevant concentrations; however, adipose tissue had altered proinflammatory expression and glucose metabolism at low concentrations. This has important implications in regulatory guidelines in the use of BPA in the manufacturing of consumer products.

Bisphenols are used in the process of polymerization of polycarbonate plastics and epoxy resins. Bisphenols can easily migrate out of plastic products and enter the gastrointestinal system. By increasing colonic inflammation in mice, disrupting the intestinal bacterial community structure and altering the microbial membrane transport system in zebrafish, bisphenols seem to interfere with the gut microbiome. The highly abundant human commensal bacterium Bacteroides thetaiotaomicron was exposed to bisphenols (Bisphenol A (BPA), Bisphenol F (BPF), Bisphenol S (BPS)), to examine the mode of action, in particular of BPF. All chemicals caused a concentration-dependent growth inhibition and the half-maximal effective concentration (EC50) corresponded to their individual logP values, a measure of their hydrophobicity. B. thetaiotaomicron exposed to BPF decreased membrane fluidity with increasing BPF concentrations. Physiological changes including an increase of acetate concentrations were observed. On the proteome level, a higher abundance of several ATP synthase subunits and multidrug efflux pumps suggested an increased energy demand for adaptive mechanisms after BPF exposure. Defense mechanisms were also implicated by a pathway analysis that identified a higher abundance of members of resistance pathways/strategies to cope with xenobiotics (i.e., antibiotics). Here, we present further insights into the mode of action of bisphenols in a human commensal gut bacterium regarding growth inhibition, and the physiological and functional state of the cell. These results, combined with microbiota-directed effects, could lead to a better understanding of host health disturbances and disease development based on xenobiotic uptake.

No description available.

Le bisphénol A (BPA) est un perturbateur endocrinien couramment employé dans l’industrie agroalimentaire, en particulier pour les matériaux en contact des denrées alimentaires. Son utilisation dans la fabrication de polycarbonates et de résines époxy, recouvrant la face interne des boîtes de conserve et des canettes, en fait un contaminant ubiquitaire de l’alimentation humaine. L’augmentation de l’exposition humaine à ce contaminant a été corrélée avec la récurrence de certains troubles comme l’intolérance alimentaire, l’obésité ou le diabète de type 2. Ces dernières années, une attention particulière a été portée sur sa capacité à perturber différentes fonctions physiologiques, dont celle du système immunitaire, après exposition périnatale à des niveaux environnementaux pertinents pour l’Homme. Dans une première étude, nous avons montré qu’une exposition périnatale au BPA à 50 µg/kg de poids corporel/jour induit une diminution de l’activité anti-microbienne corrélée à une chute de l’expression du lysozyme dans l’iléon de la descendance femelle. La perméabilité intestinale de ces individus augmente en association avec le niveau d’IFN- dans les muqueuses du côlon. De plus, nous observons une diminution des plasmocytes à IgA associée à une perte de sécrétion d’IgA dans les fèces, démontrant un défaut de la fonction barrière et des défenses de l’intestin chez la descendance BPA. De manière intéressante, une diminution de la fréquence des ILC3 intestinaux est observée chez ces individus, avec une augmentation du niveau d’IgG sanguin dirigé contre une E.coli commensale. Ces effets sont associés à un défaut de maturation et de capacité migratoire des cellules de la lamina propria (LP) et de la rate. L’exposition périnatale au BPA provoque une augmentation de la sécrétion d’IFN- et d’IL-17 après re-stimulation in vitro CD3/CD28 des cellules de la LP, et une réponse de type Th17 dans la rate. Réunis, ces effets confortent la capacité du BPA, lors d’une exposition périnatale, à induire une intolérance alimentaire chez la descendance femelle. Dans une seconde étude, nous avons mis en évidence que l’exposition périnatale au BPA, à la même dose, induit des perturbations de l’homéostasie du système immunitaire intestinal et systémique chez la descendance mâle au jour 45, via une diminution de la fréquence des Th1 et Th17 dans la LP et une augmentation de la réponse Th1 et Th17 dans la rate. Ces impacts apparaissent en parallèle avec une altération de la sensibilité au glucose, une diminution de la sécrétion d’IgA dans les fèces et un appauvrissement des bifodobacteria dans le microbiote intestinal de ces individus. L’ensemble de ces évènements précède l’infiltration de macrophages M1 pro-inflammatoires dans le tissu adipeux péri-gonadique, en association avec une diminution de la sensibilité à l’insuline et une augmentation du poids corporel apparaissant avec le vieillissement (jour 170) chez la descendance BPA. Cette étude longitudinale a permis de proposer une séquence d’évènements aboutissant à un phénotype obèse et au T2D lors d’une exposition périnatale au BPA, et ainsi de comprendre le rôle du système immunitaire en lien avec le microbiote intestinal dans le développement de ces désordres métaboliques. Enfin, nous émettons l’hypothèse que les bisphénols S et F, deux analogues structuraux du BPA, peuvent disposer de capacités immunomodulatrices équivalentes à celles du BPA, susceptibles de provoquer les mêmes troubles chez la descendance. Nous avons testé cette hypothèse de manière préliminaire chez la descendance femelle après exposition périnatale au BPS et au BPF. Des résultats préliminaires d’études in vitro sur ces deux composés en comparaison au BPA sont également apportés. Ce travail de thèse contribue à accroître les connaissances relatives aux effets immunotoxiques des bisphénols dans le contexte de l’origine développementale des pathologies chroniques de l’adulte (DOHaD). / The endocrine disruptor bisphenol A (BPA) is commonly found in food industry, more precisely in food contact packaging. BPA is used to manufacture polycarbonate plastics and epoxy resins lining food and beverage cans, becoming an ubiquitous contaminant in human food. The extent of human exposure to this chemical is thought to be correlated with the occurrence of disorders like food intolerance, obesity and type-2 diabetes (T2D). During last years, a particular interest have been raised about its ability to disrupt various physiological functions, including immune system, after perinatal exposure at relevant environmental doses for Human. In the first study, we showed that perinatal exposure to BPA (50 µg/kg body weight/day) decreased anti-microbial activity and ileal lysozyme expression in the female mouse offspring. In those mice, we observed an increased gut permeability, in association with an increase of colonic IFN- level. Moreover, we observed a decrease of IgA+ cells with a loss of IgA secretion into faeces, depicting intestinal barrier and defense function defects in BPA female offspring. Interestingly, a decrease of the intestinal ILC3 frequency associated with an increase of IgG against commensal E.coli in sera have been observed in these individuals. These effects were linked to a defect of maturation and migratory ability of dendritic cells from lamina propria (LP) and spleen. Perinatal exposure to BPA also increased IFN- and IL-17 secretions after in vitro stimulation in the gut and elicited Th17 response in the spleen. Altogether, these effects support the ability of a perinatal exposure to BPA to induce oral intolerance with ageing in female offspring. Secondly, we showed that perinatal exposure to BPA at the same dose led to intestinal and systemic immune system homeostasis disturbances in male mouse offspring at day 45, through a decrease of Th1 and Th17 frequencies in the LP and an increase of Th1 and Th17 response in spleen. These effects were associated with an altered glucose sensibility, a decrease of faecal IgA secretion and a fall of bifidobacteria in the microbiota of these individuals. These BPA-mediated events precede infiltration of pro-inflammatory M1 macrophages in gonadal white adipose tissue, together with a decreased insulin sensitivity and an increased weight gain. This longitudinal study allowed us to better understand the sequential events linked to perinatal exposure to BPA that lead to obesity and T2D, and highlighted the role of immune system linked to gut microbiota in the development of these metabolic disorders. Finally, we hypothesized that two structural analogs of BPA –i.e., Bisphenol S and F- can display similar immune-modulatory effects that could lead to similar developmental disturbances than BPA in exposed-offspring. This hypothesis was tested in a preliminary experiment in female mouse offspring perinatally exposed to BPS and BPF. We also provided preliminary results of these two compounds, compared to BPA, from an in vitro study. This thesis contributes to the increase knowledge about the immunotoxic effects of bisphenol compounds in the context of the Developmental Origins of Health and Disease (DOHaD).

Les perturbateurs endocriniens (PE) posent des risques pour la santé Humaine et pour la faune. L’utilisation de tests biologiques basés sur des mécanismes d’action spécifiques permet de caractériser le potentiel PE des substances chimiques dans le cadre de l'évaluation toxicologique, mais les capacités de biotransformation de ces modèles sont rarement prises en compte. Or, le métabolisme conditionne le devenir des xénobiotiques (détoxication vs. bioactivation) et donc in fine l'activité biologique mesurée. Dans ce travail, le devenir de deux contaminants œstrogéniques émergents (benzophénone-2, bisphénol S) a été comparé dans de nouveaux modèles in vitro et in vivo de poisson zèbre (cellules ZELH-zfERs, larve transgénique cyp19a1b-GFP) et des lignées humaines. Nos résultats démontrent que les modèles de poissons zèbres sont métaboliquement compétents et soulignent leur pertinence dans une approche intégrée in vivo/in vitro pour le criblage de l'activité PE des substances chimiques. / Endocrine disruptors (ED) are potentially harmful for Human beings and for wildlife species. Species-specific mechanism-based screening tests nowadays allow characterizing the ED potency of chemicals in the context of their toxicological assessment. However, the biotransformation capabilities of the biological models used are seldom taken into consideration, despite they ultimately condition the fate of the tested xenobiotics (detoxification vs. bioactivation), and, consequently, the biological response. In this work, we examined the fate of two emerging ED (benzophenone-2, bisphenol S) using novel in vitro and in vivo zebrafish models (ZELH-zfERs cells and cyp19a1b-GFP transgenic larvae) as well as human cell lines. Our results demonstrate that the zebrafish models are metabolically competent, and underline their relevance, accuracy and usefulness in an integrated in vivo/in vitro screening approach designed to assess ED's biological activity

Endocrine disrupting chemicals (EDCs) are emerging contaminants that can interfere with the hormone system and may cause cancers, birth defects and reproductive system disorders. Prevalence of endocrine-related dysfunction and disease has increased steadily over the past decades. Although accumulating data suggest that these diseases have fetal origins, associations of EDC exposure during pregnancy and adverse health effects on both mothers and fetuses have not been thoroughly evaluated, particularly at multiple points in time. We firstly developed an analytical method for quantification of 28 EDCs (9 phthalates, 8 bisphenols, 5 parabens, 5 benzophenones and triclosan) in urine samples using ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometer. The method was applied to measure targeted compounds in a total of 5220 urine samples collected from 951 pregnant women at three trimesters and 1501 pregnant women at one or two trimesters in Wuhan, China between 2014 and 2015. Based on the quantification results, exposure patterns and health risks of 28 EDCs on participants were evaluated and discussed in detail below. Among these samples, bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), 4-hydroxybenzophenone (4-OH-BP), 2,4-dihydroxybenzophenone (BP-1), 2-hydroxy-4-methoxybenzophenone (BP-3), triclosan, mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethylhexyl) phthalate (MEHHP), monobenzyl phthalate (MBzP), mono-n-butyl phthalate (MnBP), monoisobutyl phthalate (MiBP) were determined with detection rates exceeding 50%, in which BPA, BP-3, MeP and MnBP were the predominant compounds. We found the U-shaped trends of urinary concentrations of phthalate metabolites over trimesters. Parabens, benzophenones and triclosan displayed a downward trend over three visits. We also found the levels of targeted compounds varied by exposure-related factors, such as sampling seasons, physical activities, computer using time and decoration information. In addition, multiple EDCs were mostly determined at low doses over trimesters, indicating that real-world exposure of pollutants were dominated by low-dose mixtures. We then evaluated the combined health hazards induced by EDC exposure via calculating the estimated daily intakes on the basis of average urinary concentrations at three trimesters. It was found that 24.9% of participants had potential health risks caused by exposure to phthalate mixtures. The most frequency of cumulative risks occurred in women who were exposed to a high dose of one specific phthalate, di-n-butyl phthalate (DnBP) or di(2-ethylhexyl) phthalate (DEHP). We also evaluated the cumulative health risks of BPA and its alternatives and found that about 1.6% of participants were at risks induced by bisphenol exposure. Combined health hazards were mainly driven by one specific bisphenol (BPS or BPA). Our findings suggested that regional interventions of DnBP, DEHP, BPA and BPS in application and production should be tighten and/or taken. Considering the low-dose effects of BPA, we further investigated the associations of BPA and three major natural estrogens, including estrone (E1), estradiol (E2) and estriol (E3), at three trimesters of pregnancy. We observed non-monotonic dose-response relationships of BPA to E1, E2 and E3 over trimesters even when BPA concentrations were below the current safety thresholds. In the gender-stratified models, we found significant negative relationships (β < 0, p < 0.05) between BPA and E2 among mothers with male fetuses in the first trimester. However, we found that no significant relationship between BPA and E2 among mothers with female fetuses over three trimesters. Significant non-monotonic associations (from significant negative to positive associations) between BPA and E3 were observed among mothers with female fetuses in the second trimester. The above mentioned findings suggested the gender-specific and trimester-specific effects of BPA on estrogens. Our findings also indicated that the current tolerance daily intake value maybe not safe enough to evaluate the potential health risks induced by BPA exposure. We next investigated the effects of maternal exposure to phthalates on both mothers and fetuses. Associations of phthalate exposure with the risks of gestational diabetes mellitus (GDM) and plasma glucose levels were evaluated based on a nested case-control study design. It was found that the levels of phthalate metabolites in women with GDM were significantly higher than those without GDM. Meanwhile, positive associations between urinary concentrations of phthalate metabolites and the risks of GDM were obvious, indicating that phthalate exposure may be a risk factor for GDM. In addition, phthalate levels were related to the increased plasma glucose levels after 75 g oral glucose tolerance test. Our findings suggested that phthalates might disturb the glucose homeostasis and increase GDM risks. Furthermore, we assessed the trimester-specific and gender-specific effects of DEHP exposure on fetal growth, birth size and postnatal growth at 6, 12 and 24 months. We found that among male offspring, 1st-trimester DEHP was negatively related to fetal growth (β < 0, p < 0.05), but positively related to 24-month body mass index (BMI). 2nd-trimester DEHP was negatively related to fetal growth, birth weight and birth length, but positively related to the weight gain rates from birth to 12 months old. 3rd-trimester DEHP was positively (β > 0, p < 0.05) associated with birth weight, BMI at 6 and 12 months. However, among females, 1st-trimester DEHP was associated with increased birth length, while 2nd-trimester DEHP was negatively associated with BMI at 6 and 12 months. A negative association between DEHP and weight gain rates at 6 months was noted among females. Our findings indicated the second trimester maybe the sensitive window of DEHP exposure for offspring growth since 2nd-trimester DEHP levels were related to the decreased fetal growth, decreased birth size, but increased weight gain rates in early childhood age among male offspring. To investigate the mechanism underlying the associations of DEHP exposure with glucose and lipid metabolism, we investigated the biotransformation of DEHP and the disturbed metabolisms induced by MEHP, the putative toxic metabolite of DEHP, in human normal liver cell L02 using metabolomics and lipidomics. We found that MEHP was the major metabolite of DEHP. Decreased uptake of glucose and accumulation of glucose in liver cells were obvious after MEHP exposure. Phospholipid remodeling, incomplete fatty acid β-oxidation, inhibition of purine metabolism and glycolysis, and increased oxidative stress were noted in MEHP-exposed L02 cells, which were related to insulin resistance. In this work, we measured 28 EDCs in a total of 5220 urine samples provided by 951 pregnant women (three trimesters) and 1501 pregnant women (one or two trimesters) and then evaluated the exposure levels, exposure patterns (variations, variability and correlations), health risks and health effects of these compounds on pregnant women and fetuses. Our data suggested that participants had potential health risks induced by exposure to phthalates or bisphenols. Phthalate exposure was related with the increased plasma glucose levels and risks of GDM. Prenatal DEHP exposure may induce the intrauterine growth restriction and catch-up growth among males, which supported the evidence of fetal origin. To explore the underlying mechanisms of MEHP on glucose and lipid metabolic disorders, we exposed the human normal hepatic L02 cells with MEHP, and applied metabolomic and lipidomic approaches for finding potential biomarkers and disturbed pathways. We found that MEHP exposure inhibited glucose uptake, caused phospholipid remodeling and increased oxidative stress in L02. These findings suggest that the usage of products containing EDCs, particularly phthalates, in pregnant women should be limited in China, intervention of BPS should be considered, and threshold values of BPA are called for reevaluation.

This study analyzes the reactivity and selectivity of Friedel-Crafts alkylations using benzylic alcohols and phenols in the presence of a Lewis acid, to synthesize methoxylated bisphenols as a benign alternative to BPA. The degree of methoxylation on the electrophile appears to affect the yield of the reaction while the degree of methoxylation on the nucleophile appears to affect the selectivity. A more methoxylated electrophile results in a lower yield whereas a more methoxylated nucleophile results in a change in ratio between the bisphenol isomers and/or causes other isomers to form. Neither the yield nor the selectivity appears to be affected significantly by the temperature.