The oil sands, located in north-eastern Alberta, are one of the largest deposits of oil worldwide. Because the Alberta Environmental Protection and Enhancement Act prohibits the release of oil sands process-affected material into the environment, industry is storing vast quantities of tailings on mine lease sites. The oil sands industry is currently accumulating tailings waste at a rate of >105 m3/day, for which reclamation strategies are being investigated. Naphthenic acids (NAs) have been identified as the most toxic component of oil sands tailings as they are considered acutely toxic to a variety of biota, and are therefore a target contaminant for tailings pond reclamation strategies. Current literature based on Microtox® assays (marine bacteria Vibrio fischeri) suggests that lower molecular weight NAs are more toxic than higher molecular weight NAs. The following thesis involves the utilization of NA fractions and their relative toxicities to determine if NA toxicity is related to NA molecular weight. A previous study generated an oil sands-derived naphthenic acid extract (NAE), which was fractionated by distillation at stepped temperatures, yielding five fractions with increasing median molecular weights (Daltons). In the present study, the same extract and five fractions were utilized. To expand on the earlier characterization which involved a low resolution electrospray ionization mass spectrometry (ESI-MS), the whole extract and five fractions were analysed using electrospray ionization high-resolution mass spectrometry (ESI-HRMS) and synchronous fluorescence spectroscopy (SFS). Mean molecular weights were generated for each fraction, and an increase in molecular weight with increasing fraction number was confirmed. Respective mean Daltons and relative proportions for each fraction are as follows: 237 and 11.9 % (fraction 1), 240 and 32.3% (fraction 2), 257 and 33.4% (fraction 3), 308 and 16.8% (fraction 4), and 355 and 5.6% (fraction 5). When chemical analyses of fractions were compared, it was determined that structures contributing to increased molecular weight included increased cyclic structures (up to 7-ring structures), aromaticity (mono- and diaromatics), nitrogen, sulfur, and oxygen heteroatoms, and dihydroxy/dicarboxy compounds. In addition, characterization data suggested the presence of NAs exhibiting estrogenic structures. Following chemical characterization, NA fractions were subject to embryo/larval bioassays using two fish species: Oryzias latipes (Japanese medaka) and Pimephales promelas (fathead minnow). Endpoints evaluated were mortality, time to hatch, hatch length, and abnormalities. Results suggest that relative NA fraction toxicity is not related to molecular weight, as no trend relating mean Dalton weight to toxicity was observed for any endpoint in both species. Acute toxicity data indicated differences between fractions as high as 2-fold, although results were species-dependent. Fraction 1 displayed the lowest potency (highest LC50) for both Japanese medaka (0.291 mM) and fathead minnow (0.159 mM). Fractions 3 and 2 for Japanese medaka (0.149 and 0.157 mM, respectively), and fractions 5 and 2 for fathead minnow (0.061 and 0.080 mM, respectively) displayed the greatest potencies for mortality (lowest LC50). When fraction LC50s for Japanese medaka were compared to the whole NAE (0.143 mM), the mid molecular weight fractions (fractions 2 and 3) appeared most similar to the whole NA. . In terms of relative toxicity and proportion, constituents in the mid molecular range fractions (2 and 3) likely represent greater risk compared to other fractions, and further chemical and toxicological characterization of constituents within these fractions is warranted particularly for long-chained, monocarboxylic acids, with low aromaticity. Japanese medaka and fathead minnow varied in their sensitivity and their relative response to different fractions. In general, fathead minnow were more sensitive than Japanese medaka based on lower estimates of LC50 and threshold (growth) values in addition to the presence of developmental abnormalities (predominately yolk sac edema) associated with a few of the fractions. Compared to differences in toxicity between fractions for a given species (>2-fold for fathead minnow), there was more variability between species for a given fraction (> 3-fold for fraction 5). Also, the relative toxicity of fractions as indicated in the present study is contrary to the results generated using Vibrio fischeri for the same fractions. Thus, there is a need for multi- endpoint and species toxicity evaluations to assess the efficacy of remediation and reclamation options for reducing toxicity of oil sands tailings.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19178号 / 医博第4020号 / 新制||医||1010(附属図書館) / 32170 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 淳, 教授 渡邉 大, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19765号 / 農博第2161号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4981(農学部図書室) / 32801 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 田川 正朋 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22503号 / 農博第2407号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5283(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Les sédiments agissent comme de véritable puits pour certains polluants organiques persistants et métaux représentant ainsi une source de contamination secondaire pour les milieux aquatiques. Dans ce contexte, un test embryo-larvaire utilisant le Medaka japonais (Oryzias latipes) a été développé afin d’évaluer la toxicité des polluants fixés aux particules sédimentaires. Le bioessai consiste à maintenir les embryons de Medaka en contact direct avec le sédiment durant tout leur développement embryonnaire puis à évaluer les effets létaux et sublétaux induits au niveau phénotypique et moléculaire chez les embryons et larves exposés. Tout d’abord, le bioessai a été appliqué à la caractérisation de la toxicité et des modes d’action de substances organiques et métalliques pures. Le pyrène et son dérivé, le méthylpyrène, se sont avérés fortement tératogènes avec un spectre de malformations rappelant le syndrome de la maladie du sac bleu. L’étude du profil d’expression génique a révélé la perturbation de certains processus cellulaires notamment la voie AhR, le métabolisme mitochondrial et la voie de signalétique et de métabolisation des rétinoïdes. Le cuivre et le cadmium ont induit de nombreuses malformations squelettiques et cardio-vasculaires, des dommages à l’ADN ainsi que l’altération de l’expression de gènes impliqués dans le métabolisme mitochondrial, la régulation du cycle cellulaire et la réparation de l’ADN. Enfin, le bioessai a été appliqué à l’évaluation du potentiel toxique de sédiments naturels du système Lot-Garonne et du Bassin d’Arcachon. L’ensemble des sédiments testés a montré un large spectre d’effets tératogènes et seuls quelques-uns ont conduit à des effets létaux. Par ailleurs, l’expression plusieurs gènes impliqués dans le métabolisme mitochondrial, la voie AhR, le contrôle du cycle cellulaire, la réparation de l’ADN et la détoxication des métaux a été modifiée lors de l’exposition à certains sédiments contaminés de la zone d’étude. / Aquatic sediments act as real sink for numerous anthropogenic chemicals such as persistent organic pollutants and heavy metals as well as a secondary contamination source. In this context, a fish embryo-larval assay was developed using the Japanese Medaka (Oryzias latipes) as a test organism to evaluate de toxicity of particle-bound contaminants. In this aim, fish embryos are kept in direct contact to sediment during their whole embryonic development and induced lethal and sub-lethal effects are measured at the phenotypical and molecular levels. This test was first applied to the toxicity evaluation and modes of action characterization of organic and metallic single compounds. Pyrene and its alkylated derivative, methylpyrene, revealed a high teratogenic potency inducing developmental deformities similar to the blue sac disease syndrome. Gene expressions response pattern using RT-PCR method highlighted the alteration of several cellular process including AhR pathway, mitochondrial metabolism and retinoids signaling and metabolism pathways. Copper and cadmium mainly induced numerous skeletal and cardiovascular injuries, DNA damage as well as modifications of gene expression involved in mitochondrial metabolism, DNA damage repair and cell cycle regulation. Finally, the Medaka embryo-larval assay was applied to analyze the toxic potential of natural sediments form the Lot-Garonne system and the Arcachon bay. All tested sediments showed a wide range of teratogenic effects but only few of them revealed to be acutely toxic. Moreover, the expression of several genes involved in mitochondrial metabolism, AhR pathway, cell cycle arrest, DNA repair and heavy metals detoxication proved to be modified in the presence of some contaminated sediments from the studied area.

<p>The liver is a primary target for toxicants and/or their metabolites. Selected fish species now serve as model organisms for laboratory investigations of toxic responses in the liver. One such model is the Japanese medaka (<italic>Oryzias latipes</italic>), a small freshwater teleost with a robust history of usage in liver and biliary toxicity studies. The structural components of the medaka hepatobiliary system have been well-described by recent studies in two- and three-dimensional contexts, but efforts to characterize the molecular mechanisms underlying critical medaka liver functions during normalcy remain sparse. This dearth of information makes it difficult to definitively characterize toxic responses in this model organism. A crucial transcription factor underlying proper hepatobiliary function in both mammalian and non-mammalian species is the farnesoid X receptor alpha (FXR&alpha;), a member of the nuclear receptor superfamily that plays a key role in bile acid homeostasis. This dissertation describes the function of medaka fxr&alpha; during both normalcy and toxicity.</p><p>To achieve this overall objective, <italic>in vitro</italic> techniques were first employed to study the function of medaka <italic>fxr&alpha;</italic>. Two isoforms of <italic>fxr&alpha;</italic> that differ in the AF1 domain, Fxr&alpha;1 and Fxr&alpha;2, were isolated from liver cDNA and are the result of alternative splicing of one gene locus. Fxr&alpha;2 responded significantly to C24 bile acids and the synthetic FXR&alpha; agonist GW4064. On the other hand, Fxr&alpha;1, despite having an identical ligand-binding domain to that of Fxr&alpha;2, showed no response to any agonists tested by transient transactivation assays. Furthermore, Fxr&alpha;2 interacted with nuclear receptor coactivators PGC-1&alpha; and SRC-1 in mammalian two-hybrid assays while Fxr&alpha;1 did not. These findings point to a significant importance of the AF1 domain to overall receptor structure and function. </p><p>Following <italic>in vitro</italic> functional characterization, <italic>in vivo</italic> experiments using medaka larvae were performed to determine <italic>fxr&alpha;'s</italic> function during normalcy. Quantitative, real-time PCR data demonstrated that Fxr&alpha;1 is highly expressed in adult liver, while Fxr&alpha;2 is expressed predominantly in gut. Fxr&alpha;1's expression was higher than Fxr&alpha;2 in embryos and larvae at all developmental timepoints tested. In vivo exposures of medaka hatchlings to GW4064 at various doses significantly altered expression of defined FXR&alpha; targets, including: bile salt export protein (BSEP), small heterodimer partner (SHP), and cytochrome P450 7A1 (CYP7A1). Surprisingly, numerous sublethal hepatic alterations to hepatocytes and bile preductular epithelial cells (BPDECs) were observed following exposure to GW4064; alterations included: lipid accumulation, glycogen depletion, mitochondrial swelling and rupture of mitochondrial membranes, disruption of endoplasmic reticulum, and apoptosis. Significant lipid accumulation, as revealed by oil red O whole mount staining of larvae, was also noted at lower doses of GW4064. These findings were the first observations of sublethal hepatotoxicity of GW4064; to date, no studies in the mammalian literature reported alterations following its administration.</p><p>Because of studies in the mammalian literature that demonstrated alleviation of cholestatic injury induced via the classic hepatotoxicant &alpha;-naphthylisothiocyanate (ANIT) by GW4064, it was originally hypothesized that a similar finding would be observed in medaka coexposed to these compounds. However, because of GW4064's ability to induce sublethal hepatic alterations in medaka, it was anticipated that its coadministration with ANIT would result in enhanced toxicity rather than alleviation as described in rodent models. However, despite the sublethal alterations induced by 1 uM GW4064, alleviation of toxicity following exposure to 15 uM ANIT was observed. Surprisingly, reduction of GW4064's toxicity was also observed in larvae exposed to both compounds. These investigations of <italic>fxr&alpha;</italic> function are an important and essential component in furthering our understanding of hepatobiliary toxicity in small aquarium fish models of human liver disease. These collective findings have created molecular underpinnings necessary for understanding medaka hepatobiliary function during normalcy and toxicity.</p> / Dissertation

No description available.

<p>Endocrine disrupting chemicals (EDCs) are ubiquitous and often act as xenoestrogens with the ability to disrupt estrogen signaling through differential binding to the various estrogen receptors. Exposure to these xenoestrogens has led to detrimental effects on male reproduction. In fish, observed effects include sex reversal, presence of testicular oocytes, altered courting behavior, vitellogenin synthesis in males, altered fertility and gonadal histopathology. Understanding how xenoestrogens exert their effects is complicated by the existence of multiple estrogen receptors (ESR1, ESR2a, ESR2b, and GPER), coupled with their ability for crosstalk and differential binding capability of selective estrogen receptor modulators (SERMS). Additionally, estrogen can signal through both classic genomic signaling and nongenomic signaling. Furthermore, the importance of estrogen signaling in normal male reproduction is just beginning to be understood. The primary goal of this dissertation was to assess the implications of aberrant estrogen signaling on male reproductive capacity, testicular morphology and gene expression changes in the small aquarium model fish, medaka, by investigating effects of a general estrogen receptor agonist, ethinylestradiol (EE2), and those of a G-protein estrogen receptor (GPER) specific agonist, G-1. This was assessed through breeding experiments, histological assessment of testicular morphology and microarray assessment of testicular gene expression changes following exposure to EE2 and G-1. Finally, a comparison of altered testicular morphology between EE2 and G-1 induced changes was further assessed using a variety of histological techniques. The findings demonstrate that a 14-day exposure to EE2 impaired male reproductive capacity and altered testicular morphology and gene expression in a time- and dose-dependent manner. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. These morphologic changes were highly associated with gene expression changes. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell proliferation, collagen production/extracellular matrix organization, and protein ubiquitination among others. Comparatively, a 14-day exposure to G-1 did not affect male reproductive capacity but did alter testicular morphology and gene expression. The histological analysis found an increased cellularity of the interstitium leading to thickened interstitium but no change in germinal epithelium. The microarray data indicate differential expression in genes most commonly involved in cell cycle, cell proliferation, apoptosis, transcription, translation, and ubiquitination. Finally, an assessment of the testicular histological phenotypes following EE2 and G-1 exposure indicate different morphologic changes led to thickened interstitium observed in the two exposures. In EE2 exposed fish, thickening of interstitium was associated with increased collagen deposition on the periphery of the organ while the interior thickening was primarily due to the collapse of intralobular space associated with decreased germinal epithelium. In the G-1 exposed fish, the thickened interstitium was due to increased cellularity. A modest increase in cell proliferation was observed contributing to the increase in interstitial cells, however, it is also possible that there is a decrease in normal apoptosis and cell turnover as well. These findings highlight the importance of anchoring gene expression changes with morphology and ultimately proper tissue/organ function as well as the potential differences in effects that may occur with EDCs and SERMs.</p> / Dissertation

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第24809号 / 人健博第115号 / 新制||人健||8(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 中尾 恵, 教授 岡 昌吾, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

<p>Cytochrome P450s (CYPs) are enzymes that are found throughout the three domains of life. They function in the metabolism of endogenous and exogenous compounds. CYPs are extensively studied in mammalian systems due to their importance in drug metabolism and are highly expressed in detoxification organs like the liver and intestine. Fish CYP3s are not well understood. CYP3s have diversified in fish and subfamilies A, B, C and D constitute the CYP3 clade in fish. In this study, CYP3C1, CYP3C2, CYP3C3 and CYP3C4 in zebrafish (<em>Danio rerio</em>) and CYP3B4, CYP3B5 and CYP3B6 in medaka (<em>Orzyias latipes</em>) were quantified in hepatic and extrahepatic organs. CYP3C genes were quantified throughout development. All CYP3B and 3C isoforms were detected in all organs except CYP3B4 in male organs and in female brain. CYP3C1-C3 were maternally acquired and expressed in all embryonic stages. Higher expression of some of the isoforms occurred in the liver and intestine of zebrafish and medaka. This is indicative of a possible role in xenobiotic metabolism. Differences in expression between males and females gonad was observed, suggesting a possible role for estrogen in gene regulation. Further research will contribute to characterizing the upstream response elements in order to understand whether estrogens or other compounds are responsible for CYP3 regulation in fish. This knowledge will contribute to understanding the potential function these unique families of CYPs serve for fish.</p> / Master of Science (MSc)