Perfluoroalkyl acids (PFAAs) are a family of synthetic substances used in a wide variety of consumer and industrial applications, including non-stick and stain-resistant products. PFAAs, specifically perfluorinated sulfonates and carboxylates, are chemically stable and virtually non-biodegradable in the environment. In recent years, PFAAs have been detected in tissues and blood of humans and wildlife. Furthermore, PFAAs have a tendency to bioaccumulate and biomagnify in biota. Perfluorooctane sulfonate and perfluorooctanoate are known to be toxic when animals are exposed to environmentally-relevant levels, but scientists and regulators are challenged with determining and predicting their modes of action. There is some evidence to suggest that PFAAs can impact the thyroid hormone (TH) pathway and neurodevelopment. The studies presented in this thesis investigated the developmental effects and potential modes of action of newer PFAAs that are being introduced into the global market place. Egg injection experiments were performed in domestic chicken (Gallus gallus domesticus) embryos to assess the in ovo toxicity of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) during development. Real-time RT-PCR was used to measure the transcription of candidate genes in the liver and cerebral hemisphere of day 21-22 embryos. Candidate genes were selected based on their responsiveness to PFAA exposure in an in vitro screening assay conducted previously. In ovo exposure to PFHxS decreased embryo pipping success and overall growth at 38,000 ng/g; several orders of magnitude higher than concentrations reported in wild bird eggs. The expression of TH-responsive genes, including type II and III 5'-deiodinase, neurogranin, and octamer motif binding factor 1, were induced. In addition, PFHxS diminished free thyroxine (T4) levels in plasma. PFHxA had no affect on pipping success, gene expression or T4 levels in chicken embryos at the doses assessed. The transcriptional profiles in the cerebral hemisphere of chicken embryos exposed to 890 and 38,000 ng/g PFHxS were compared to a solvent control using microarray technology. The expression of 78 different genes were significantly altered (fold change > 1.5, p < 0.001) by PFHxS. Functional analysis showed that PFHxS affected genes involved in tissue development and morphology and cellular assembly and organization. Pathway and interactome analysis suggested that gene expression may be affected through integrin receptors and signaling pathways via TH–dependent and –independent modes of action. It is expected that the findings presented in this thesis will be of general relevance and importance to regulatory agencies and of interest to research scientists and risk assessors.

There is a growing interest in assessing the neurotoxic potential and endocrine disrupting properties of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, pre- and post- natal development and behaviour. PFC exposure altered hormone levels (e.g. thyroid hormone, estrogen, and testosterone) and the expression of hormone-responsive genes in mammalian and aquatic species. Hormone-mediated events are critical in central nervous system development and function, especially those controlled by thyroid hormones (THs). The studies presented in this thesis are the first to assess the effects of PFCs on primary cultures of neuronal cells in two avian species; the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following TH-responsive genes were examined using real-time RT-PCR: type II iodothyronine 5’-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif binding factor (Oct-1), and myelin basic protein (MBP). Several PFCs were shown to alter mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. It was determined that short-chained PFCs (<8 carbons) altered the expression of TH-responsive genes to a greater extent than long-chained PFCs (≥8 carbons). Although several significant changes in mRNA expression were observed in TH-responsive genes following PFC exposure in chicken embryonic neuronal (CEN) cells (Chapter 2), there were fewer changes in herring gull embryonic neuronal (HGEN) cells (Chapter 3). The mRNA levels of D2, D3, TTR, and RC3 were altered following treatment with several short-chained PFCs in CEN cells. Oct-1 and RC3 expression were induced following treatment with several short-chained PFCs in HGEN cells. These studies are the first to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and provide insight into the possible mechanisms of action of PFCs in the avian brain.

Perfluoroalkyl acids (PFAAs) are a family of synthetic substances used in a wide variety of consumer and industrial applications, including non-stick and stain-resistant products. PFAAs, specifically perfluorinated sulfonates and carboxylates, are chemically stable and virtually non-biodegradable in the environment. In recent years, PFAAs have been detected in tissues and blood of humans and wildlife. Furthermore, PFAAs have a tendency to bioaccumulate and biomagnify in biota. Perfluorooctane sulfonate and perfluorooctanoate are known to be toxic when animals are exposed to environmentally-relevant levels, but scientists and regulators are challenged with determining and predicting their modes of action. There is some evidence to suggest that PFAAs can impact the thyroid hormone (TH) pathway and neurodevelopment. The studies presented in this thesis investigated the developmental effects and potential modes of action of newer PFAAs that are being introduced into the global market place. Egg injection experiments were performed in domestic chicken (Gallus gallus domesticus) embryos to assess the in ovo toxicity of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) during development. Real-time RT-PCR was used to measure the transcription of candidate genes in the liver and cerebral hemisphere of day 21-22 embryos. Candidate genes were selected based on their responsiveness to PFAA exposure in an in vitro screening assay conducted previously. In ovo exposure to PFHxS decreased embryo pipping success and overall growth at 38,000 ng/g; several orders of magnitude higher than concentrations reported in wild bird eggs. The expression of TH-responsive genes, including type II and III 5'-deiodinase, neurogranin, and octamer motif binding factor 1, were induced. In addition, PFHxS diminished free thyroxine (T4) levels in plasma. PFHxA had no affect on pipping success, gene expression or T4 levels in chicken embryos at the doses assessed. The transcriptional profiles in the cerebral hemisphere of chicken embryos exposed to 890 and 38,000 ng/g PFHxS were compared to a solvent control using microarray technology. The expression of 78 different genes were significantly altered (fold change > 1.5, p < 0.001) by PFHxS. Functional analysis showed that PFHxS affected genes involved in tissue development and morphology and cellular assembly and organization. Pathway and interactome analysis suggested that gene expression may be affected through integrin receptors and signaling pathways via TH–dependent and –independent modes of action. It is expected that the findings presented in this thesis will be of general relevance and importance to regulatory agencies and of interest to research scientists and risk assessors.

There is a growing interest in assessing the neurotoxic potential and endocrine disrupting properties of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, pre- and post- natal development and behaviour. PFC exposure altered hormone levels (e.g. thyroid hormone, estrogen, and testosterone) and the expression of hormone-responsive genes in mammalian and aquatic species. Hormone-mediated events are critical in central nervous system development and function, especially those controlled by thyroid hormones (THs). The studies presented in this thesis are the first to assess the effects of PFCs on primary cultures of neuronal cells in two avian species; the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following TH-responsive genes were examined using real-time RT-PCR: type II iodothyronine 5’-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif binding factor (Oct-1), and myelin basic protein (MBP). Several PFCs were shown to alter mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. It was determined that short-chained PFCs (<8 carbons) altered the expression of TH-responsive genes to a greater extent than long-chained PFCs (≥8 carbons). Although several significant changes in mRNA expression were observed in TH-responsive genes following PFC exposure in chicken embryonic neuronal (CEN) cells (Chapter 2), there were fewer changes in herring gull embryonic neuronal (HGEN) cells (Chapter 3). The mRNA levels of D2, D3, TTR, and RC3 were altered following treatment with several short-chained PFCs in CEN cells. Oct-1 and RC3 expression were induced following treatment with several short-chained PFCs in HGEN cells. These studies are the first to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and provide insight into the possible mechanisms of action of PFCs in the avian brain.

Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the red drum thyroid precluded their use as a bioassay species, the plasma TSH and T4 response to exogenous TSH was next characterized in goldfish. The T4 response in goldfish was stable and repeatable, with T4 levels peaking at 5 hours and remaining elevated for more than 11 hours after bovine TSH injection. Plasma TSH peaked from 2-5 hours following TSH injection with more than 90 percent cleared by 11 hours. The goldfish bioassay was further utilized to evaluate the effects of structural modifications on TSH biological activity. Substitution of four positively charged amino acids at the n-recombinant human TSH, had the same effect in goldfish. The heterothyrotropic potency of mammalian follicle stimulating hormone in goldfish was also enhanced by the same amino acid substitutions. Finally, the importance of oligosaccharides to TSH bioactivity was also examined in goldfish. Deglycosylation abolished TSH bioactivity, even when immunoreactivity persisted in circulation. Furthermore, recombinant canine TSH was less potent when produced in cell lines generating insect-type glycosylation than when produced in a cell line capable of mammalian-type glycosylation. These studies utilizing recombinant mammalian demonstrated conservation of mammalian TSH hormone-receptor interactions in goldfish, suggesting TSH function might likewise be conserved. Thus, I have established goldfish as a sensitive and stable bioassay which can now be utilized to monitor the biological activity of teleost TSH expressed in vitro as well as to evaluate how structural modifications of the TSH molecule influence its vivo biological activity.

In vitro embryo production (IVP) has been successfully used for infertility treatment, farm animal breeding and preservation of endangered species. Embryo culture media and its composition play an important role in the developmental competency of in vitro produced embryos. To optimize IVP it is important to understand the physiology of the in vivo embryo medium. Previously, we confirmed the presence of thyroid hormones (THs) in the bovine reproductive tract and their beneficial effect on embryo quality in vitro. The aim of this study was to further investigate the developmental effect and mechanisms behind THs function in bovine early embryos by supplementing IVP media with 50ng/ml T3 and T4. In vitro embryo culture (IVC) media fortification with THs significantly improved blastocyst and hatching rates, which was observed in both fast and slow cleaving embryos. THs treatment significantly skewed male/female ratio toward femaleness (43% vs. 57%). Only %60 of supplemented THs was bioavailable in the IVC media. Gene microarray and blastocyst genes expression profile analysis showed that THs supplementation altered mRNA expression profile in blastocysts. 1,234 genes were expressed differentially in the treated embryos and these differences were statistically significant (>1.5 fold at p < 0.05); these findings were confirmed by qPCR. TH-related genes, including THs receptor (TRs) mRNA and deiodinases (DIOs), were expressed in the gene array data of both treated and control groups. TR mRNA was expressed in all stages of early embryo development (EED) and the expression was not inducible by THs. This finding was consistent in both gene array and qPCR. Suppression of embryo gene transcription by α-amanitin resulted in a significantly higher level of TR mRNA from 2-cell to 16-cell treated and control embryos. TR protein was expressed in both treated and control blastocysts but, differentially located and distributed in response to THs in the blastocysts. This study highlights the importance of THs in EED and therefore the need to include these factors in culture media. Furthermore, this study has better characterized THs effects and identified possible regulatory roles of these hormones in EED. Further investigation into embryo competency and pregnancy outcome can provide more details. / Canada Research Chair(CRC), EmbryoGene, Ontario Graduate Scholarship(OGS) and Agriculture and Agrifood Canada.

There is a growing interest in assessing the neurotoxic potential and endocrine disrupting properties of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, pre- and post- natal development and behaviour. PFC exposure altered hormone levels (e.g. thyroid hormone, estrogen, and testosterone) and the expression of hormone-responsive genes in mammalian and aquatic species. Hormone-mediated events are critical in central nervous system development and function, especially those controlled by thyroid hormones (THs). The studies presented in this thesis are the first to assess the effects of PFCs on primary cultures of neuronal cells in two avian species; the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following TH-responsive genes were examined using real-time RT-PCR: type II iodothyronine 5’-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif binding factor (Oct-1), and myelin basic protein (MBP). Several PFCs were shown to alter mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. It was determined that short-chained PFCs (<8 carbons) altered the expression of TH-responsive genes to a greater extent than long-chained PFCs (≥8 carbons). Although several significant changes in mRNA expression were observed in TH-responsive genes following PFC exposure in chicken embryonic neuronal (CEN) cells (Chapter 2), there were fewer changes in herring gull embryonic neuronal (HGEN) cells (Chapter 3). The mRNA levels of D2, D3, TTR, and RC3 were altered following treatment with several short-chained PFCs in CEN cells. Oct-1 and RC3 expression were induced following treatment with several short-chained PFCs in HGEN cells. These studies are the first to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and provide insight into the possible mechanisms of action of PFCs in the avian brain.

Current municipal wastewater treatment plants (MWWTP) technologies are insufficiently removing emerging contaminants of concern. These emerging contaminants are an issue as many are known endocrine disrupting compounds (EDCs). EDCs are contaminants that can have severe and irreversible impacts on highly conserved endocrine systems that are critical during developmental periods in vertebrates as well as during adult life. Many EDCs have non-monotonic dose-response curves yet they are not often tested at low, environmentally relevant concentrations. EDC research to date has focused heavily on xenoestrogenic compounds whereas thyroid hormone (TH) disruption has been largely overlooked. TH is conserved in all vertebrates and plays crucial roles in neural development, basal metabolism, and thermoregulation. TH is comprised of thyroxine (T4), often known as the transport form of TH, and triiodothyronine (T3), the more bioactive form of TH. A TH spike occurs in the perinatal period of humans, and when disrupted, this spike can cause severe developmental defects. An analogous, but perhaps more overt, TH spike occurs in amphibians. TH is the sole hormone that drives amphibian metamorphosis, thus providing an excellent model for TH action. Our lab has previously developed the cultured tailfin (C-fin) assay, which uses biopsies from premetamorphic Rana catesbeiana tadpole tailfins cultured in the presence of an exogenous chemical of concern to assess perturbations to TH- and stress-responsive gene transcript levels by QPCR. This thesis uses the C-fin assay to assess the efficacy of removal of biological TH- and stress-altering activity in conventional municipal wastewater treatment systems. We first assess the successive levels of a full-scale conventional activated sludge (CAS) MWWTP in its ability to reduce perturbations of mRNA transcript levels of the critical TH receptors alpha (thra) and beta (thrb), and stress responsive gene transcripts superoxide dismutase (sod), catalase (cat) and heat shock protein 30 (hsp30). Secondary treatment of wastewater effluents removes cellular stress perturbations when compared to influents, but thr disruptions remain after conventional secondary wastewater treatment. We then assess three pilot-sized conventional secondary MWWTP configurations run at two operational conditions. The C-fin assay results suggest that the current understanding of operational conditions and the efficiency of complex MWWTP configurations is not clear-cut when assessed by biological endpoints such as the transcript abundance perturbations in the C-fin assay. Finally, the C-fin assay is used to investigate transcript profiles of genes of interest when the tissues are treated with the endogenous hormones T3, T4, and estradiol (E2). Our results indicate that T4 acts as more than solely a T3-prohormone and that gene expression levels in response to the two different forms of TH can be T3 or T4 specific. E2 effects, although implicated in altering TH-mediated responses in other contexts, do not affect TH-responsive gene transcripts in the C-fin. The data presented use the novel C-fin assay to challenge and advance the currently accepted views of TH-action, as well as develop necessary yet practical biological knowledge for management of emerging contaminant release from MWWTPs. / Graduate / 0383 / 0768 / 0307 / polaw@uvic.ca

The role of thyroid-stimulating hormone (TSH) in the regulation of peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T₄), the principal hormone released from the thyroid gland in response to TSH stimulation, circulates with a robust daily rhythm in the sciaenid fish, red drum. Previous research has suggested that the red drum T₄ cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T₄ feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with driving this T₄ cycle, I developed quantitative real time RT-PCR (qPCR) techniques to quantify the daily cycle of expression of the pituitary TSH subunits GSU[alpha], and TSH[beta]. I found that pituitary TSH expression cycled inversely to, and 6-12 hours out of phase with, the T₄ cycle, consistent with the hypothesis that TSH secretion drives the T₄ cycle. To examine the potential role of deiodinases in negative feedback regulation of this TSH cycle, I also utilized qPCR to assess the pituitary expression patterns of the TH activating enzyme outer-ring deiodinase (Dio2) and the TH deactivating enzyme inner ring deiodinase (Dio3). Whereas Dio2 was not expressed with an obvious daily cycle, Dio3 was expressed in the pituitary mirroring the TSH cycle. These results are consistent with T₄ negative feedback on TSH and suggest that TH inactivation by pituitary cells is an important component of the negative feedback system. To further examine the TH regulation of this Dio3 cycle, I developed an immersion technique to administer physiological doses of T₃ and T₄ in vivo. Both hormones persist in static tank water for at least 40 hours. Immersion in 200ng/ml T₄ significantly increased both plasma T₄ and T₃ within physiological ranges above control at 4.5 hours. Immersion in 100ng/ml T₃ increased plasma T₃ within physiological ranges over control by 22 hours while significantly decreasing plasma T₄ below control, presumably through inhibition of TSH secretion. T₄ also significantly inhibited the expression of the TSH [alpha] and [beta] subunits at 4.5 and 22 hours of immersion whereas T₃ immersion significantly inhibited the expression of the [alpha] and [beta] subunits of TSH by 22 hours. Both Dio2 and Dio3 expression were significantly diminished by T3 and T₄ at 22 hours. Inhibition of circulating THs with the goitrogen methimazole significantly increased the expression of TSH. These results indicate that both T₄ and T₃ are capable of negative feedback regulation of TSH expression in red drum on a time scale consistent with the T₄ daily cycle, and further support Dio3 destruction of THs in the pituitary, potentially regulated by circulating T₄, as a critical component of negative feedback on TSH. This study supports the importance of central mechanisms acting through pituitary TSH secretion in regulating thyroid function in red drum.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.