The organochlorine pesticide endosulfan has been shown to be highly toxic to fish and there is some evidence to support that it may act as an endocrine disrupting chemical. Juvenile rainbow trout (Oncorhynchus mykiss) were caged at 4 sites in Oshawa Creek during the fall and spring of 2008 and 2009 while another group was intra-peritoneal injected in the laboratory with varying concentrations (ppm) of endosulfan. Plasma vitellogenin (VTG) levels, liver ethoxyresorufin-O-deethylase (EROD), citrate synthase (CS), lactate dehydrogenase (LDH), and brain acetylcholine esterase (AChE) (caged fish only) enzymatic activities were measured. Trout injected with endosulfan experienced an increase of the anaerobic (LDH activity) and a decrease of the aerobic (CS activity) metabolic pathways, while male VTG levels increased. Since it was a singular injection, VTG results have to be confirmed. Fall caged trout showed increased EROD activity and inhibited AChE activity while those caged in the spring experienced an unexpected exposure to the lampricide 3-Trifluoro-Methyl-4-Nitro-Phenol (TFM) which disrupted metabolic parameters (inhibited CS and increased LDH activity). Both fall and spring caged trout experienced no induction of VTG activity. Further research is needed since the spring exposure was altered due to the unplanned TFM treatment and thus did not represent a valid temporal replicate.

Octylphenol and genistein are two of the growing list of endocrine disrupting chemicals found in the environment that mimic estrogen in reproductive tissue both in vitro and in vivo. It is well established that endogenous estrogens modulate not only the reproductive system, but also the immune system. However, the effects of many endocrine disrupting chemicals, such as octylphenol and genistein, on the immune system have yet to be determined. Preliminary studies on short-term treatment with genistein (0.6 mg) and octylphenol (10 mg) showed that the thymus of orchiectomized (NZBxNZW)F1 males is sensitive to these agents. Further studies focused on the effects of short-term treatment of octylphenol on the morphology and function of the thymus in adult, reproductively intact non-autoimmune C57BL/6 and pre-autoimmune (NZBxNZW)F1 males. Oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol 3 times a week for 3 weeks did not affect the morphology or function of the thymus as assessed by its weight, thymocyte cellularity, proportion of immature and mature thymocytes, level of apoptosis, apoptotic rates of stimulated thymocytes, and proportion of mature T cells in the spleen. Furthermore, oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol did not result in estrogenic changes in the reproductive tract in our model. Subcutaneous injection of 10 mg of octylphenol resulted in skin lesions that confounded the assessment of its affects on the thymus. Further studies are needed to definitively determine the effects of octylphenol on the immune system of both males and females of various ages and to determine the effect of long-term exposure. / Master of Science

Les substances perturbatrices du système endocrinien sont des substances qui, de par leur capacité à induire des changements hormonaux chez les organismes vivants, génèrent des préoccupations dans le domaine de la qualité des eaux et, par extension, dans le domaine du traitement des effluents aqueux. Particulièrement, ce projet de recherche s’est attardé sur l’élimination des perturbateurs endocriniens phénoliques nonylphénol (NP), bisphénol A (BPA) et triclosan (TCS) en solution aqueuse à l’aide de la laccase (E.C. 1.10.3.2) sécrétée par la souche fongique Coriolopsis polyzona. Cette oxydase est une métalloprotéine pouvant catalyser l’oxydation d’une vaste gamme de substances phénoliques. En premier lieu, l’impact du pH et de la température sur l’élimination de ces composés à l’aide de la laccase libre en utilisant un design factoriel. L’oxydation de ces composés produit des oligomères (dimère à pentamère) via le couplage des radicaux phénoxy produits par l’action de la laccase. Il s’avère que les substances produites suite à l’oxydation du NP et du BPA par la laccase ont perdu leurs similitudes structurales avec l’estrogène. Ainsi, l’élimination de l’activité estrogénique de ces substances est directement liée à la transformation des composés. Finalement, l’utilisation d’ABTS comme médiateur a permis d’augmenter le taux d’oxydation enzymatique de ces composés chimiques. Puis, de façon à augmenter la possibilité d’utilisation de la laccase dans des biotechnologies environnementales, cette enzyme a été immobilisée sur un support siliceux et via la réticulation d’agrégats. L’impact des conditions d’immobilisation sur l’activité enzymatique, la stabilité du catalyseur et les propriété biocatalytiques apparentes a été déterminé pour différentes stratégies d’immobilisation. Globalement, l’immobilisation génère un biocatalyseur stable vis-à-vis les dénaturations chimique, physique et biologique. Particulièrement, l’immobilisation sur un support solide produit un biocatalyseur facile à utiliser ayant une faible activité massique et des propriétés cinétiques moindres que celle de l’enzyme libre. La formation de CLEAs de laccase a permis d’obtenir une activité massique élevée et des propriétés cinétiques supérieures à celle de l’enzyme soluble. Ces biocatalyseurs solides ont étés utilisés pour éliminer en continu le NP, BPA et TCS dans différents types de bioréacteur. Le biocatalyseur sur silice a été utilisé pour éliminer ces substances dans un réacteur garni, tandis que les CLEAs ont été utilisés dans un réacteur à lit fluidisé et un réacteur à perfusion développé au cours de ce projet. Ces différentes configurations de bioréacteur ont permis d’éliminer efficacement ces différents perturbateurs endocriniens. Globalement, les différents résultats obtenus, à l’échelle de laboratoire, au cours de ce projet de recherche démontrent que la laccase et particulièrement les biocatalyseurs formés via les différentes stratégies d’immobilisation testées représentent des approches extrêmement prometteuses pour le développement de biotechnologies environnementales vouées à l’élimination des perturbateurs endocriniens phénoliques.

There is concern regarding exposure of aquatic organisms to chemicals that interfere with the endocrine system. Disruption of the endocrine system can lead to impacts on sexual development, altered hormone levels, intersex, and ultimately reproductive failure. While effects of endocrine disrupting chemicals (EDCs) on standard laboratory species have been subject of intense study, to this day there is a large gap in knowledge and a high degree of uncertainty regarding the sensitivity of wild fish species to these compounds. One of the main concerns with current toxicity testing approaches is that they require the use of a large number of live animals, particularly when working with native species. Therefore, the aim of this study was to develop in vitro tissue explant assays that would enable the assessment of the sensitivity of different wild fish species native to North America to the exposure with EDCs. Specifically, two in vitro assays were developed: 1) A liver explant assay to assess effects of EDCs that can interact with the estrogen receptor (environmental estrogens), and 2) a gonadal explant assay to assess effects of EDCs on sex-steroid production. The test species selected were northern pike (Esox lucius), walleye (Sander vitreus), and white sucker (Catostomus commersoni) that were sampled from Lake Diefenbaker, Saskatchewan, Canada, and white sturgeon (Acipenser transmontanus) that were randomly selected from an in house stock reared from eggs. Liver tissue was excised from male fishes and exposed for 24 h to a synthetic estrogen, 17α- ethinylestradiol (EE2). Transcript abundance of vitellogenin (VTG), estrogen receptor (ER) α and β in liver tissue were quantified using qPCR. Gonad tissue from both male and female were excised and exposed for 24 h to a model inducer (forskolin) and inhibitor (prochloraz) of steroidogenesis. 11-ketotestosterone (11-KT) and estradiol (E2) were quantified in media by use of ELISA. Exposure to EE2 resulted in a concentration dependent increase in VTG in all species, and an increase in ERα in northern pike. Walleye males showed the greatest sensitivity to EE2. Gonad tissues exposed to forskolin showed a concentration dependent increase in 11-KT and E2. Exposure to prochloraz resulted in a decrease of 11-KTand E2. Male and female white sucker showed greatest sensitivity to forskolin, while male and female walleye showed greatest sensitivity to prochloraz. The seasonal time point during which gonad explants were excised and exposed had an impact on the potency and magnitude of response, resulting in a seasonal effect on sensitivity. Also, gonad explants from these species were found to have greater sensitivity than responses previously reported for in vitro explants of other fish species such as the fathead minnow (Pimephales promelas), and stable cell lines currently used as screening applications to detect chemicals that might disrupt the endocrine system. Therefore, current approaches that use stable cell lines or tissue explants from standardized small bodied laboratory species might not be protective of some wild fish species. These tissue explants represent a promising approach to help understand species sensitivity to EDCs, and if appropriately validated, could be a powerful tool for chemical screening.

京都大学 / 新制・論文博士 / 博士(工学) / 乙第13495号 / 論工博第4202号 / 新制||工||1786(附属図書館) / (主査)教授 米田 稔, 教授 高野 裕久, 教授 松井 康人 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No description available.

<p>Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardant chemicals that are added to plastics, electronic components, furniture foam, and textiles to reduce their combustibility. Of the three commercial mixtures historically marketed, only DecaBDE, which is constituted almost entirely (~97%) of the fully brominated congener decabromodiphenyl ether (BDE-209), continues to be used in the U.S. today. While decaBDE is scheduled for phase-out in the U.S. at the end of 2013, exposures to BDE-209 and other PBDEs will continue into the foreseeable future as products that contain them continue to be used, recycled, and discarded. In addition, decaBDE use continues to be largely unrestricted across Asia, although restricted from use in electronic equipment in Europe. </p><p>Despite limits placed on PBDE uses, they are ubiquitous contaminants detected worldwide in humans and wildlife. Major health effect concerns for PBDEs come largely from evidence in laboratory rodents demonstrating neurotoxicity, reproductive and developmental impairments, and thyroid disruption. The potential for PBDEs, particularly BDE-209, to disrupt thyroid regulation and elicit other toxic outcomes in fish is less clear. Thus, the overall objective of this thesis research was to answer questions concerning how fish, as important indicators of overall environmental health, are metabolizing PBDEs and whether and how PBDEs are disrupting thyroid hormone regulation. The central hypothesis was that PBDE metabolism in fish is mediated by iodothyronine deiodinase (dio) enzymes, which are responsible for activating and inactivating thyroid hormones, and that PBDE exposures are causing thyroid system dysfunction across fish life stages. </p><p>Under the first research aim, in vitro experiments conducted in liver tissues isolated from common carp (Cyprinus carpio) suggested a role for dio enzymes in catalyzing the reductive debromination of PBDEs. Carp liver microsomes efficiently debrominated BDE-99 to BDE-47, and enzymes catalyzing this reaction were associated predominantly with the endoplasmic reticulum (i.e., microsomal fraction) where dio enzymes are located. Competitive substrate experiments in carp liver microsomes also demonstrated that rates of BDE-99 debromination to BDE-47 were significantly inhibited upon challenges with 3,3',5'-triiodothyronine (rT3) and thyroxine (T4). This finding supported the hypothesis that enzymes involved in the metabolism of PBDEs may have high affinities for thyroid hormones. Indeed, experiments to determine apparent enzymatic kinetics (apparent Vmax and Km values) of BDE-99 hepatic metabolism suggested that enzymes responsible for the catalytic activity appeared to have a higher affinity for native thyroid hormone than BDE-99. </p><p>The second and third research aims were focused on evaluating BDE-209 accumulation, metabolism, and thyroid toxicity in juvenile and adult life stages of fish using the fathead minnow (Pimephales promelas) as a model. BDE-209 bioaccumulated and was debrominated to several reductive metabolites ranging from penta- to octaBDEs in both juvenile and adult fish exposed to BDE-209. In addition, thyroid hormone regulation in juvenile and adult male fathead minnows was severely disrupted by BDE-209 at low, environmentally relevant exposures. In juvenile minnows, the activity of dio enzymes (T4-outer ring deiodination; T4-ORD and T4-inner ring deiodination; T4-IRD) declined by ~74% upon oral doses of 9.8 ± 0.2 µg/g wet weight (ww) food at 3% body weight (bw)/day for 28 days, compared to controls. Declines in dio activity were accompanied by thyroid follicle hypertrophy indicative of over-stimulation and injury. In addition to thyroid disruption, a distinctive liver phenotype characterized by vacuolated hepatocyte nuclei was measured in ~48% of hepatocytes from treated fish that was not observed in controls. </p><p>Under the third research aim, adult male fathead minnows received dietary treatments of BDE-209 at a low dose (95.3 ± 0.41 ng/g-food at 3% bw/day) and a high dose (10.1 ± 0.10 µg/g-food at 3% bw/day) for 28 days followed by a 14-day depuration period to evaluate recovery. Compared to negative controls, adult male fish exposed orally to BDE-209 at the low dose tested for 28 days experienced a 53% and 46% decline in circulating total T4 and T3, respectively, while fish at the high BDE-209 dose tested had total T4 and T3 deficits of 59% and 62%, respectively. Depressed levels of plasma thyroid hormones were accompanied by a 45-50% decline in the rate of T4-ORD in brains of all treatments by day 14 of the exposure. The decreased T4-ORD continued in the brain at day 28 with a ~65% decline measured at both BDE-209 doses. BDE-209 exposures also caused transient, tissue-specific upregulations of relative mRNA transcripts encoding dio enzymes (dio1, dio2), thyroid hormone receptors (TR&alpha, TR&beta), and thyroid hormone transporters (MCT8, OATP1c1) in the brain and liver in patterns that varied with time and dose, possibly as a compensatory response to hypothyroidism. In addition, thyroid perturbations at the low dose tested generally were equal to those measured at the high dose tested, suggesting non-linear relationships between PBDE exposures and thyroid dysfunction in adult fish. Thus, mechanisms for BDE-209 induced disruption of thyroid regulation can be proposed in adult male minnows that involve altered patterns of thyroid hormone signaling at several important steps in their transport and activation. </p><p>A growing body of evidence describing PBDE toxicity in biota, including data generated here, along with studies showing continued and rising PBDE body burdens, raises concern for human and wildlife health. Long delays in removing PBDEs from the market, their ongoing presence in many products still in use, and their active use outside the U.S. and European Union will leave a lasting legacy of rising contamination unless more concerted regulatory and policy actions are taken to reduce future exposures and harm.</p> / Dissertation

Endocrine Disrupting Chemicals (EDC) are exogenous agents that mimic endogenous hormone activity in the body. EDC exposure during the critical period of neonatal development can potentially cause life-long neurological, behavioural and physiological disease. This thesis focuses on the EDC Bisphenol A (BPA), a synthetic xenoestrogen widely prevalent in everyday materials that has significant environmental relevance given its ubiquitous presence in humans around the world. The central research question of my thesis is: Does perinatal exposure to BPA affect rat pup development? A rodent model was selected to study the effects of BPA on the adrenal component of the hypothalamic-pituitary-adrenal axis (HPA axis) stress pathway, which has not been extensively studied. Rat dams were divided into five groups (vehicle control (VEH), positive control diethylstilbestrol (DES), BPA 5, BPA 50 and BPA 500 μg/kg bw/day) and dosed daily throughout gestation and for four days of lactation. Rat pups were sacrificed at two time-points at the beginning and the end of the stress hyporesponsive period (SHRP), at postnatal day (PND) 5 and PND 15. Changes in three morphometric parameters (bodyweight, crown-rump (CR) length and anogenital distance (AGD) were assessed based on the factors of Treatment and Sex. Adrenal gland glucocorticoid receptor (GR) and 18SrRNA expression was determined by qPCR in male pups at PND 5 and PND 15. At PND 5, compared to the VEH group, the BPA 50 pups were significantly heavier (ANOVA, Dunnett’s post-hoc) and the DES and BPA 50 pups had significantly longer CR lengths (ANOVA, Dunnetts’ post-hoc). At PND 15, xenoestrogen treatment significantly influenced CR length (ANOVA). At both time-points, males had significantly longer AGD than females, as physiologically expected (ANOVA). Adrenal gland GR expression in male pups was not significantly affected by treatment, but there was an effect of treatment in18SrRNA gene expression at PND 5 (Kruskal-Wallis). Using the Ct method to determine GR and 18SrRNA fold changes, we cautiously suggest that our experimental doses resulted in a non-monotonic dose response to BPA in the PND 5 animals and a monotonic dose response to BPA exposure in the PND 15 animals. This study highly values the importance of investigating the effects of environmentally relevant, low dose BPA exposure during the critical window of development, given the little that is known about potentially permanent alterations to the stress pathway due to exposure during this delicate period of development.

Endocrine-disrupting chemicals (EDCs) are known to cause endocrine disruption (ED), developmental neurotoxicity (DNT), infertility and impaired embryo development. EDCs do therefore impose a threat to humans, wildlife and the environment. The present study investigated the effects of the reference compounds dihydrotestosterone, estradiol, benzo(a)pyrene, rosiglitazone, as well as the EDCs bisphenol F and perfluorooctanesulfonic acid at various concentrations on zebrafish embryos. The scientific questions of the study were to investigate how these environmental pollutants impact the development of zebrafish, what their molecular mechanisms are and what the link between ED and DNT is. Zebrafish embryos were exposed for 5 days to the compounds and various parameters on development were collected at different time points. The expression of 41 genes (qPCR) related to ED and DNT, and the levels of 23 steroids (LC-MS/MS) were determined. Gene correlations were determined with Pearson’s correlation test and paired t-tests were used to determine significantly altered gene activities. The significant gene expression changes were further related to the pathways of steroids in order to connect how gene activity impacted steroid levels. Exposure to estradiol, dihydrotestosterone and bisphenol F induced cyp19a1b expression which can affect personality traits. Perfluorooctanesulfonic acid interferes with thyroid hormone transport by binding to TTR causing profound effects on neurodevelopmental processes and cognitive functions. The compounds influenced genes that can disrupt endocrine systems which can cause neurodevelopmental impairments.

Humans experience ubiquitous exposures to estrogenic environmental chemicals from food, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. In our study, pregnant CD-1 mice were orally exposed to BPS or ethinyl estradiol (EE2, a positive control for estrogenicity) from gestational day 9 through postnatal day (PND) 2, the period when many estrogen-sensitive organs are developing. After weaning, the offspring were administered either oil (vehicle) or an estrogen challenge (1 μg EE2/kg/day) for ten days starting at PND21 (prior to puberty), PND80 (early adulthood), or PND260 (later adulthood). Timing of puberty was evaluated in females by noting the date on which vaginal opening occurred. After the 10 day estrogen challenge, we evaluated the response of endocrine sensitive organs through measurements of organ weight, tissue morphology, and gene expression in both males and females. We observed dose- and sex-specific effects of BPS and EE2 treatment, as well as alterations in the responses of males and females to the estrogen challenge. This study sheds light on the effects of low dose xenoestrogen exposures on estrogen-sensitive organs including the reproductive tract and mammary gland. Furthermore, it improves our understanding of the influence of environmental chemicals on secular trends of earlier age of puberty in girls reported over the past few decades.