Thyroid hormone (TH) signaling is conserved across vertebrates, where it is important for normal growth and development, particularly in the perinatal period. TH has an additional critical role in amphibian metamorphosis as the sole signal that initiates the transition from a larval tadpole to juvenile frog. Premetamorphic tadpoles have a thyroid gland but are functionally athyroid, yet can be induced to undergo precocious metamorphosis by exogenous TH administration. This essential dependence upon TH makes amphibian metamorphosis an excellent model to study TH signaling. Metamorphosis is sensitive to environmental stimuli such as temperature. Low temperature delays or slows metamorphosis, whereas high temperature advances or accelerates it. Whether a temperature is considered low or high varies by species and is related to its natural habitat. In temperate climes the North American bullfrog, Rana catesbeiana, does not undergo natural or precocious metamorphosis at low winter temperatures of 4-5°C. Tadpoles injected with TH at low temperature essentially clear it from their bodies after 60-80 days, but some manner of TH signaling has occurred such that they rapidly execute metamorphosis if returned to 20-25°C. This apparent molecular memory is poorly understood, but there is evidence that components of gene expression programs may be involved. This thesis investigated the role of these factors in the molecular memory of TH formed at low temperature in the liver, brain, lung, back skin, and tail fin of Rana catesbeiana. The results suggested that TH receptor beta (thrb), CCAAT/enhancer binding protein 1 (cebp1), and Krüppel-like factor 9 (klf9) may contribute to the molecular memory to different extents in each tissue, and that TH-induced basic leucine zipper-containing protein (thibz) may have an important role in this process for every tissue examined. Assessment of additional genes was hampered by the limited genetic resources available for this species, so de novo high throughput RNA sequencing (RNA-seq) techniques were explored to alleviate this limitation. Trans-ABySS sequence assembly software produced a high quality Rana catesbeiana liver transcriptome that was annotated by BLAST alignment to established sequence databases and resulted in a more than ten-fold increase in Rana catesbeiana sequence information. This approach was supplemented with a software pipeline that was used to refine replicate Rana catesbeiana back skin assemblies, and by construction of a Bullfrog Annotation Resource for the Transcriptome (BART) that was used to quickly annotate more than 97% of the assembled back skin sequences. In the future, the Rana catesbeiana transcriptome sequence resources can be leveraged to identify additional genes that may be involved in formation of the TH molecular memory, and chromatin immunoprecipitation could help characterize the factors and epigenetic marks in the promoter regions of these genes. Elucidation of the molecular memory mechanism provides a means to uncover key events in TH signaling. / Graduate

Os Hormônios Tiroideanos (HT) representam um fenótipo de cardioproteção e influenciam no estado trófico do tecido cardíaco através de diversos mecanismos, dentre eles a modulação dos componentes do Sistema Renina Angiotensina- a Angiotensina II e seus receptores (AT1 e AT2). Este estudo teve como objetivos avaliar o papel do receptor AT2 na cardioproteção induzida pelo HT e a participação da proteína quinase ativada por AMP (AMPK) nesse processo. O modelo de isquemia e reperfusão (I/R) foi desenvolvido em corações de ratos submetidos ao hipertiroidismo experimental na presença ou ausêcia do antagonista do receptor AT2 (PD123319), utilizando-se o aparto de Langendorff. Os resultados mostraram que o HT exerce efeito cardioprotetor com aumento na expressão protéica do receptor AT2 e da AMPK fosforilada, que foi prevenido com a administração do PD, seguido de piora da função cardíaca. Estes dados sugerem que parte da cardioproteção induzida pelo HT é mediada pelo receptor AT2, e que a AMPK também está envolvida proteção do miocárdio após injúria por I/R. / Thyroid Hormones (TH) is a phenotype of cardioprotection and may influence the trophic state of cardiac tissue through several mechanisms, including the modulation of the components of renin-angiotensin system - angiotensin II and its receptors (AT1 and AT2). The present study aimed to evaluate the role of AT2 receptor in cardioprotection mediated by the TH and the involvement of AMP-activated protein kinase (AMPK) in this context. A model of Ischemia and Reperfusion (I/R) was performed in isolated hearts of rats subjected to experimental hyperthyroidism, in the presence or absence of the AT2 receptor antagonist (PD123319), using the Langendorff system. The results showed that TH exerts cardioprotective effect with increased levels of AT2 and phosphorylated AMP protein expression, which was prevented by the administration of PD, with a loss in cardiac function. These data suggest that part of the TH-induced cardioprotection is mediated by the AT2 receptor with the involvement of AMPK in protection of the myocardium after I/R injury.

O hormônio tireotrófico (TSH) é o principal regulador da síntese e da secreção dos hormônios tireoidianos (HTs), os quais exercem um mecanismo de feedback negativo na hipófise reduzindo a síntese das cadeias <font face=\"Symbol\">&#946 e <font face=\"Symbol\">&#945 (CGA - Glycoprotein hormones Alpha Chain) por meio de mecanismos que envolvem modificações na transcrição de genes que codificam essas proteínas (ações genômicas). Na última década tem aumentado o número de evidências de que, em paralelo as ações genômicas clássicas, algumas ações dos HTs são desencadeadas na presença de inibidores da transcrição gênica e em curto espaço de tempo (segundos a minutos), caracterizando-se assim as ações não genômicas dos HTs. Este trabalho tem como foco avaliar a possibilidade de que os HTs regulem a expressão desses genes não genomicamente. Para tal avaliamos as alterações decorrentes do hipotiroidismo, seguido ou não do tratamento agudo com T3 em dose fisiológica ou saturante, sobre o grau de poliadenilação e a expressão do mRNA das subunidades alfa (CGA) e <font face=\"Symbol\">&#946TSH, bem como sua repercussão sobre a síntese e secreção de <font face=\"Symbol\">&#946TSH. Através da metodologia de PCR em Tempo Real observamos nos animais tireoidectomizados tratados com salina (Tx) um aumento de 10 e 4 vezes no conteúdo de mRNA do <font face=\"Symbol\">&#946TSH e CGA, respectivamente, e na razão <font face=\"Symbol\">&#946TSH/CGA quando comparado ao animal eutireoideo. A administração da dose saturante de T3 em 30 min não alterou o conteúdo do mRNA de <font face=\"Symbol\">&#946TSH e CGA, enquanto a dose fisiológica reduziu 52 e 34%, respectivamente, sem alterar a razão <font face=\"Symbol\">&#946TSH/CGA, comparando com o grupo Tx. Com o ensaio RACE-PAT, observou-se que o grupo Tx apresentou um aumento no comprimento da cauda poli-A do mRNA de <font face=\"Symbol\">&#946TSH, não havendo alterações semelhantes para o mRNA de CGA. A administração aguda de T3, apenas na dose saturante, provocou uma redução de 17% no comprimento da cauda poli-A do mRNA do <font face=\"Symbol\">&#946TSH nos animais hipotiroideos comparados com o grupo Tx. Nenhuma alteração foi observada no comprimento da cauda poli-A do mRNA de CGA, indicando um possível efeito específico do T3 sobre a poliadenilação da subunidade <font face=\"Symbol\">&#946. Através dos ensaios Western Blot / ECL, Imunohistoquímica e Histoquímica foi observado que as duas doses de T3 utilizadas promoveram um aumento de 30% no conteúdo protéico de TSH, uma redução na marcação de <font face=\"Symbol\">&#946TSH na periferia dos tireotrofos e aumento na polimerização de actina na hipófise dos animais hipotiroideos tratados, possivelmente por inibir a secreção deste hormônio. Como estes resultados foram observados em 30 min, e parte deles envolveu alterações em etapas pós-transcricionais da regulação da expressão de genes (poliadenilação), podemos inferir que o T3 esteja agindo por uma via não genômica regulando a síntese e secreção do TSH. / The thyroid-stimulating hormone (TSH) is the main regulator of the synthesis and secretion of thyroid hormones (TH), which exert a negative feedback mechanism in the pituitary by reducing the synthesis of <font face=\"Symbol\">&#946 and <font face=\"Symbol\">&#945 (CGA - Glycoprotein hormones alpha chain) chains through mechanisms that involve changes in the transcription of genes that encode these proteins (known as genomic action). However, in the last decade, an increasing body of evidence has shown that, in parallel with the classical genomic mechanisms, some TH actions might be elicited in a short period time (seconds to minutes), and in the presence of gene transcription inhibitors, which indicates that TH can also act nongenomically. In the present study we evaluate if TH could regulate some steps of the expression of <font face=\"Symbol\">&#946 TSH and CGA in a short period of time, which might provide evidence that they could act by non genomic mechanisms. For this, the expression and polyadenylation of alpha (CGA) and <font face=\"Symbol\">&#946 subunits of TSH mRNA, and TSH content, were evaluated by real time PCR and western blot, respectively, in thyroidectomized (hypothyroid) rats, 30 min after they were subjected or not to physiological or saturating doses of T3. It was observed that hyroidectomyzed animals treated with saline (Tx) presented an increase of 10 and 4 times in the content of <font face=\"Symbol\">&#946TSH and CGA mRNA, respectively, and in the <font face=\"Symbol\">&#946TSH / CGA ratio compared with control group. The saturating dose of T3 did not alter the <font face=\"Symbol\">&#946TSH and CGA mRNAs content, but the physiological dose reduced them at 52 and 34% respectively, without changing the <font face=\"Symbol\">&#946TSH / CGA ratio, compared with Tx group. The RACE-PAT assay showed that the Tx rats presented an increase in the mRNA <font face=\"Symbol\">&#946TSH poly-A tail length, whereas no change was observed to the mRNA of CGA. The acute and saturating dose of T3 caused a 17% reduction in the length of mRNA <font face=\"Symbol\">&#946TSH poly-A tail in hypothyroid animals compared with hypothyroid group. No changes were observed in the length of the poly-A tail of mRNA CGA, suggesting a specific effect of T3 on the <font face=\"Symbol\">&#946 subunit polyadenylation. Through the Western blot/ECL, histochemistry and immunohistochemistry methods we could observe that T3 (in both doses used) promoted a 30% increase in TSH protein content, a decrease in <font face=\"Symbol\">&#946TSH labeling near thyrotrophs plasma membrane and increased the actin polymerization in the pituitary of hypothyroid animals, possibly by inhibiting the secretion of this hormone. Considering that these results were observed in 30 min, and some of them involve changes in post-transcriptional regulation of gene expression (polyadenylation), we can infer that in parallel to its genomic action, T3 acts by non genomic pathways in the regulation of the TSH synthesis and secretion.

O T3 aumenta a poliadenilação e estabilidade do GH mRNA. O citoesqueleto (Cy) participa da estabilidade e tradução de mRNAs, pois fatores, como o EF 1a, ligam alguns transcritos a ele, aumentando sua estabilidade e tradução. Cy também participa dos processos secretores celulares. Observamos que no hipotiroidismo (Tx), há um desarranjo do Cy nos somatotrofos que é revertido pela administração aguda de T3. Neste estudo avaliamos a ligação do EF 1a e do GH mRNA ao Cy e, deste aos polissomos, na hipófise, e a expressão do IGF-I mRNA hepático, em ratos controle e Tx tratados com T3 ou salina, e sacrificados após 30 min. Observamos redução da F-actina, da ligação do EF 1a e GH mRNA a ela, do GH mRNA nos polissomos, e da expressão de IGF-I mRNA hepático, nos ratos Tx, o que indicou redução da síntese e secreção do GH. A administração de T3 estimulou esses processos, aumentando a estabilidade, tradução do GH mRNA e a secreção de GH, o que ocorreu em paralelo ao rearranjo do Cy, indicando uma ação não genômica do T3. / T3 increases GH mRNA poly-A tail and stability. Cytoskeleton (Cy) plays a part on mRNA stability and translation, since factors, like EF 1a, can bind some transcripts to it, improving stability and translation efficiency. Cy is also involved in cellular secretory process. We showed that somatotropes Cy is disrupted in hypothyroidism (Tx), and rearranged by acute T3 treatment. In this study we investigated the binding of EF 1a and GH mRNA to Cy and of GH mRNA to polysomes in pituitary, as well as the liver IGF-I mRNA content, in control and Tx rats treated with T3 or saline, and killed 30 min thereafter. We observed that Tx reduced F-actin content, EF 1a and GH mRNA binding to it, GH mRNA recruitment to polysomes, in pituitary, and IGF-I mRNA expression in liver, which indicates that GH synthesis and secretion are impaired. Acute T3 treatment stimulated all these process, indicating that stability, translation of GH mRNA and GH secretion were restored. These events occurred in parallel to the Cy rearrangement, which strongly indicates a non genomic effect of T3.

Os hormônios tireoidianos estão envolvidos em vários efeitos regulatórios, em órgãos diversos. Suas variações no organismo estão relacionadas a quadros clínicos de grande relevância. A presente dissertação trata do estudo de duas proteínas diretamente relacionadas ao complexo sistema regulatório associado a tais hormônios. A primeira delas é a globulina de ligação à tiroxina (TBG), responsável pelo transporte da grande maioria dos hormônios tireoidianos circulantes, e cujas alterações estão relacionadas a falhas na interpretação de testes de avaliação da função tireoidiana, podendo levar a tratamentos desnecessários. A segunda proteína é o receptor tireoidiano (TR), responsável pela mediação dos efeitos regulatórios do hormônio tireoidiano, tendo sua estimulação de atividade transcricional relacionada à ligação do hormônio em um de seus domínios. A TBG foi estudada através da técnica computacional conhecida como modelagem molecular por homologia, aplicada à proteína em sua forma selvagem e a mutantes observados no Brasil, com o intuito de relacionar a inviabilidade de tais mutantes a aspectos estruturais. Foi proposto que, para dois dos mutantes estudados, a formação das estruturas secundárias como na forma nativa da proteína seria inviável, enquanto que para o terceiro mutante a inviabilidade poderia ser causada por enovelamento incorreto causado por uma possível interação entre um resíduo de cisteína adveniente da mutação e outros resíduos do mesmo aminoácido em posição fisicamente próxima. O estudo do TR teve como base as estruturas cristalográficas das duas isoformas humanas do receptor (hTR &#945; e hTR &#946;) quando ligadas ao tiromimético GC-1. Esse composto tem a propriedade de ligar-se preferencialmente à isoforma &#946;, o que pode ter interessantes aplicações farmacológicas. A análise comparativa da ligação do GC-1 às duas isoformas mostrou que, para tal composto, a seletividade se deve a mudanças consideráveis no modo de ligação ao hTR &#945; e hTR &#946;. Para a isoforma , há dois modos de ligação, envolvendo conformações alternativas de ligante e proteína, onde em uma delas a ligação é mais favorável e semelhante à ligação do composto à isoforma , enquanto no outro modo de ligação há a perda de uma interação direta entre composto e proteína, explicando a mais baixa afinidade do GC-1 à isoforma quando comparado à isoforma . O mecanismo de -seletividade para esse composto está relacionado a um átomo de oxigênio específico que não existe no ligante natural do receptor, o que fornece úteis informações para a criação de novos compostos. / The thyroid hormones are involved in various regulatory effects, on diverse organs. Their fluctuations on the body are related to clinical scenarios of great relevance. This work deals with the study of two proteins which are directly related to the regulatory system associated to these hormones. The first one is the thyroxine-binding globulin (TBG), responsible for the transport of a large part of the thyroid hormones in serum, and whose variations are related to misinterpretation of thyroid function tests. The second protein is the thyroid receptor (TR), responsible for the mediation of the thyroid hormone regulatory effects . the transcriptional activity being related to ligand binding to one of the protein domains. The wild-type TBG and three mutants discovered in Brazil were studied by the computational technique known as homology modeling. The purpose of this investigation was to relate protein unviability to structural aspects. It was proposed that, for two mutants, the unviability was related to the impossibility of secondary structure formation as needed to form the native folding, while the third mutant the cause could be the formation of an incorrect folding due to possible interactions involving a cysteine residue created by the mutation and other nearby cysteine residues. The thyroid receptor was studied in the light of the x-ray structures of the two isoforms of the protein (hTR &#945; and hTR&#946;) bound to GC-1, a synthesized compound which resembles the thyroid hormones. This ligand binds preferably the isoform, a feature that may have interesting pharmacological applications. The comparative analysis of GC-1 binding to the two isoforms allowed the construction of a structural basis of its -selectivity property, which is due to considerable differences in the binding modes for the two isoforms. This involves two different configurations of ligand and protein conformations for the isoform - on one of them, the ligand docks to the molecule the same way it docks to hTR&#946;, while on the second configuration it loses one direct interaction to the protein, explaining its lower affinity to hTR &#945; when compared to hTR&#946;. The -selectivity mechanism for this compound is related to a specific oxygen atom which doesn?t exist on the receptor endogenous ligand, providing useful information for the development of new compounds.

Dados mostram que a estimulação do Sistema Nervoso Simpático (SNS) induz osteopenia via receptor &beta;2-adrenérgico, que é expresso no osso. Porém, um estudo recente demonstrou que camundongos knockouts (KO) para os receptores adrenérgicos &alpha;2A e &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) apresentam alta massa óssea, mesmo com hiperatividade simpática. Além disso, esses camundongos são resistentes à osteopenia induzida pelo excesso de hormônio tireoidiano. Esses achados sugerem que o &alpha;2A e/ou &alpha;2C-AR também possam mediar as ações do SNS no esqueleto e que esses receptores estão envolvidos na interação do HT com o SNS para regular o metabolismo ósseo. Neste estudo, tivemos como objetivo: avaliar se o &alpha;2CAR interfere na fisiologia óssea e se a ação do HT no tecido ósseo depende do &alpha;2CAR, avaliando o efeito do HT na fisiologia óssea dos camundongos &alpha;2CAR-/- tratados com dose suprafisiológica de T3. A microtomografia computadorizada mostrou que o volume de osso trabecular foi menor e maior nos animais &alpha;2CAR-/-, no fêmur e na vértebra respectivamente. Os animais KO também apresentaram diminuição da resistência óssea quando comparados com os selvagens. Além disso, vimos que os animais KOs foram resistentes aos efeitos deletérios da tirotoxicose no osso, tanto no fêmur quanto na vértebra. Esses achados sugerem que o &alpha;2C-AR apresenta um papel na mediação dos efeitos da ativação do SNS no osso e que o HT interage via &alpha;2C-AR, para regular massa e resistência óssea. / Data demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via &beta;2-adrenoceptor signaling. A recent study showed that mice with gene inactivation of the adrenergic receptor &alpha;2A and &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) have a high bone mass phenotype, even presenting SNS hyperactivity. Also, these knockout (KO) mice are resistant to the thyroid hormone-induced osteopenia. These findings suggest that SNS interacts with thyroid hormone (TH) to regulate bone mass and &alpha;2A and/or &alpha;2C adrenoceptors may have an important role in mediating the actions of the SNS in the skeleton. In this study, we had the following objectives: (i) to evaluate whether the isolated inactivation of &alpha;2CAR interferes with the bone metabolism and to evaluate whether the action of HT on bone tissue depends on &alpha;2CAR, treated with 20 times the physiological dose of T3. The microtomography analysis showed that the trabecular bone volume of the femur and of the sixth lumbar vertebra (L6) were, respectively, lower and higher in &alpha;2C-AR-/- mice, when compared with WT animals. Furthermore, we showed a resistance of KO animals on the deleterious effects of TH on bone. These findings suggest: (i) &alpha;2C-AR is involved with bone longitudinal growth; (ii) &alpha;2C-AR may mediates the effects of the SNS in the bone in a skeletal site specific manner, (iii) the actions of thyroid hormone on bone metabolism involves interactions with the SNS via &alpha;2C adrenergic receptors.

Doenças neurodegenerativas vem sendo relacionadas a alterações no metabolismo de glicose e deficiência e/ou resistência à insulina encefálica. Além desta relação, parece existir uma modulação dos hormônios tireoideanos (HT) sobre a sinalização de insulina e a função do sistema nervoso central (SNC). O objetivo desse trabalho foi analisar a relação entre o hipertireoidismo e o SNC, associados a DM. Para isso, ratos Wistar adultos foram injetados com monoidratado de aloxana ou veículo e tratados com T3 durante 30 dias, sendo avaliados em testes comportamentais e posteriormente analises proteicas de regiões do córtex e hipocampo. O tratamento crônico com T3 produziu um efeito ansiolítico e um atraso na aquisição de informações aprendidas, apesar da consolidação não ser modificada. Por outro lado, o T3 promoveu a melhora do processo neurodegenerativo e na via da sinalização da insulina no hipocampo de animais diabéticos. Esses dados sugerem, de modo geral, um efeito benéfico do HT sobre o a função encefálica de animais diabéticos. / Neurodegenerative diseases has been related to changes in glucose metabolism and deficiency and /or resistance to brain insulin. In this respect, there appears to be a modulation of thyroid hormones (TH) on insulin signaling and function of the central nervous system (CNS). The objective of this study was to analyze the relationship between hyperthyroidism and the CNS associated with DM. For this, Wistar adult rats were injected with alloxan monohydrate or treated with vehicle and T3 for 30 days and evaluated in behavioral tests and subsequent analyzes of protein in the cortex and hippocampus regions. Chronic treatment with T3 produced an anxiolytic effect and a delay in the acquisition of information, although consolidation not be modified. Furthermore, T3 allowed improvement of the neurodegenerative process and pathway of insulin signaling in the hippocampus of diabetic rats. These data suggest, in general, a beneficial effect of TH on the brain function of the diabetic animals.

Os receptores tireoidianos (TRs) são proteínas envolvidas em várias funções fisiológicas importantes para os organismos, pois são potentes reguladores do desenvolvimento, divisão e diferenciação celular, metabolismo e homeostase. Eles são responsáveis pela regulação da transcrição de genes-alvo específicos, mediando efeitos pleiotrópicos de hormônios lipofílicos nas células. Na ausência de ligantes essas proteínas estão complexadas a correpressores, impedindo a transcrição de genes por elas regulados. Por outro lado, a presença do ligante induz à transcrição através da ligação a elementos responsivos do DNA e coativadores. Nesse trabalho alguns aspectos do TR foram evidenciados, permintindo-se um melhor conhecimento acerca do funcionamento e estrutura desse receptor. Os experimentos de oligomerização revelaram a presença dos tetrâmeros do TR, os quais estavam restritos ao Receptor X Retinóico, sugerindo mecanismos novos na regulação do receptor. Os ensaios de raios-X a baixos ângulos resultaram nos primeiros modelos estruturais de baixa resolução de construções maiores do TR, demonstrando o correto posicionamento de seus domínios em sua estrutura geral, o que forneceu informações importantes sobre sua estrutura geral. Os experimentos de fluorescência avaliaram a ligação desses receptores a diversos elementos responsivos, em termos de constantes de dissociação e seletividade para cada um deles. E, por fim, os experimentos de troca de hidrogênio por deutério revelaram a movimentação que ocorre no domínio de ligação do ligante do receptor antes e após a adição do ligante T3. Esses resultados ampliam um pouco mais os conhecimentos sobre os mecanismos de ação e sobre a estrutura quaternária dos TR, promovendo um melhor entendimento dos conceitos básicos envolvidos na atuação dessas macromoléculas, as quais estão inseridas em redes complexas de regulação e interação com outras proteínas. / The thyroid receptors (TRs) are proteins, which are involved in diverse and important physiological functions in the organisms, since they are regulators of development, cell divison and differentiation, metabolism and homeostasis. They are responsible by the regulation of specific gene transcription, through pleiotropic effects of lipophilic hormones in the cells. In the absence of the ligand these proteins are complexed to correpressors and block the transcription of genes that are regulated by them On the other hand, in the presence of the ligand transcription is induced through the binding of the receptors to DNA response elements and coactivators. New findings about TR described in this study helped to improve the understanding of the function and structure of the receptor. This was accomplished by: oligomerization experiments which showed the presence of TR tetramers, a quarternary structure described before only for the Retinoid Receptor X, and suggested new regulation mechanisms for the receptors; the small angle X-ray scattering assays which resulted in the first low resolution structural models of bigger constructions of TR, showing the correct position of TR domains and providing important information about the global TR structure; the anisotropy fluorescence experiments which evaluated the binding of these receptors to diverse response elements, in terms of dissociation constants and selectivity for each one of the HREs tested; and finally, the hydrogen/deuterium experiments which revealed the ligand binding domain mobility before and after the ligand addition. In summary, we can say that these results all together extended the knowledge about the TR action mechanisms and its quarternary strucuture, providing better understanding of the basic concepts involved in these macromolecules behavior, which are inserted into a complex network of regulation and interaction with other proteins.

An essential element of the reclamation strategy proposed by the oil sands mining industry in northern Alberta, Canada, includes the creation of wetlands for the bioremediation of mining waste materials. The mining process used to extract oil from these deposits results in the production of large volumes of process-affected water (OSPW) and sediments (OSPS), which must be incorporated into wetlands as a component of the reclaimed landscapes. Wood frogs (<i>Rana sylvatica</i>) are an abundant native species that might be expected to inhabit these reclaimed wetlands. The objective of this study was to determine potential detrimental effects of OSPW and OSPS on the growth and development of wood frogs. Several morphological (weight, length, condition factor) and biochemical (whole body tadpole thyroid hormone and triglyceride concentrations and metamorph hepatic glycogen concentration) endpoints were assessed in conjunction with hatchability and survivability of wood frog eggs and tadpoles exposed to process-affected materials (OSPM) under field and laboratory conditions.<p> As part of this study, assay techniques were optimized to enable simultaneous measurement of whole body 3,5,3-triiodothyronine (T3), thyroxine (T4) and triglyceride (TG) concentrations in wood frog tadpoles. These assays were used to monitor changes in T3, T4 and TG in wood frog tadpoles during development from hatching to metamorphosis (Gosner stages 19-46), to establish baseline levels for subsequent application of the assays to evaluate contaminant effects. The results indicated peak T3 and T4 concentrations occurred during metamorphic climax (Gosner stages 40-46) and prometamorphosis (Gosner stages 31-40), respectively. Maximal TG concentrations were also observed during prometamorphosis. These assays were further employed to assess body condition and development in wood frogs during a field study in 2005, and the following laboratory studies in 2006 and 2007.<p> In summer 2005, 29 reclaimed and five unimpacted wetlands were monitored for use by native amphibians, and tadpoles and newly-metamorphosed wood frogs were collected from a subset of sites as a preliminary assessment of contaminant effects. Endpoints such as metamorph hepatic glycogen and whole body tadpole T3, T4 and triglyceride concentrations were compared among six impacted and three reference wetlands. The surveys indicated 60% of OSPW-impacted wetlands were used by breeding adult amphibians, while wood frog tadpoles and newly-metamorphosed frogs were observed in 37 and 30% of OSPW wetlands, respectively. In general, lower whole body tadpole T3 and triglyceride concentrations were observed in wood frogs from wetlands containing OSPM. In contrast, hepatic glycogen concentrations in newly-metamorphosed frogs and whole body tadpole T4 and T3/T4 concentrations were comparable among the reference and impacted wetlands. In addition, the differences observed in total body weight and length of tadpoles and newly-metamorphosed wood frogs among OSPM and reference sites were likely due to minor differences in developmental stages of the animals collected from the various wetlands, rather than any contaminant effect.<p> In 2006 and 2007, wood frog eggs and tadpoles were exposed to several sources of OSPW and OSPS collected from reclaimed Suncor and Syncrude wetlands under controlled laboratory conditions. Hatchability was reduced in eggs exposed to water from only one of the OSPW sites, compared with the other process-affected ponds and the control water (P<0.05). In contrast, survivability of tadpoles was significantly reduced (P<0.05) in all the impacted sites in both years, with nearly all OSPW sites having <10% survival. The exposure study evaluated the toxicity of five types of OSPS. Results indicated no impact of OSPS exposure on survivability of tadpoles, but showed reduced whole body weight (in three OSPS treatments), length (in two OSPS treatments) and body condition (in one OSPS) of tadpoles exposed to process-affected substrates tested (P<0.05). Whole body T3 and T4 concentrations in tadpoles from OSPS treatments were not different from the control treatment, but tadpole TG concentration was reduced in groups exposed to two impacted substrates (P<0.05). Water quality measurements, including determination of dissolved metals were conducted in an initial attempt to relate any potential toxic effect on wood frog growth and development to specific contaminants.<p> Results of the laboratory studies strongly suggest that exposure to OSPW and OSPS may adversely affect wood frog growth and survival. However, these findings were not entirely consistent with field observations and results of concurrent mesocosm studies. Further research is therefore needed to fully evaluate the suitability of reclaimed oil sands wetlands to support indigenous amphibian population. Future work should focus on the cumulative effects of water and substrates, as well as the effect of OSPM ageing on acute and chronic toxicity.

An essential element of the reclamation strategy proposed by the oil sands mining industry in northern Alberta, Canada, includes the creation of wetlands for the bioremediation of mining waste materials. The mining process used to extract oil from these deposits results in the production of large volumes of process-affected water (OSPW) and sediments (OSPS), which must be incorporated into wetlands as a component of the reclaimed landscapes. Wood frogs (<i>Rana sylvatica</i>) are an abundant native species that might be expected to inhabit these reclaimed wetlands. The objective of this study was to determine potential detrimental effects of OSPW and OSPS on the growth and development of wood frogs. Several morphological (weight, length, condition factor) and biochemical (whole body tadpole thyroid hormone and triglyceride concentrations and metamorph hepatic glycogen concentration) endpoints were assessed in conjunction with hatchability and survivability of wood frog eggs and tadpoles exposed to process-affected materials (OSPM) under field and laboratory conditions.<p> As part of this study, assay techniques were optimized to enable simultaneous measurement of whole body 3,5,3-triiodothyronine (T3), thyroxine (T4) and triglyceride (TG) concentrations in wood frog tadpoles. These assays were used to monitor changes in T3, T4 and TG in wood frog tadpoles during development from hatching to metamorphosis (Gosner stages 19-46), to establish baseline levels for subsequent application of the assays to evaluate contaminant effects. The results indicated peak T3 and T4 concentrations occurred during metamorphic climax (Gosner stages 40-46) and prometamorphosis (Gosner stages 31-40), respectively. Maximal TG concentrations were also observed during prometamorphosis. These assays were further employed to assess body condition and development in wood frogs during a field study in 2005, and the following laboratory studies in 2006 and 2007.<p> In summer 2005, 29 reclaimed and five unimpacted wetlands were monitored for use by native amphibians, and tadpoles and newly-metamorphosed wood frogs were collected from a subset of sites as a preliminary assessment of contaminant effects. Endpoints such as metamorph hepatic glycogen and whole body tadpole T3, T4 and triglyceride concentrations were compared among six impacted and three reference wetlands. The surveys indicated 60% of OSPW-impacted wetlands were used by breeding adult amphibians, while wood frog tadpoles and newly-metamorphosed frogs were observed in 37 and 30% of OSPW wetlands, respectively. In general, lower whole body tadpole T3 and triglyceride concentrations were observed in wood frogs from wetlands containing OSPM. In contrast, hepatic glycogen concentrations in newly-metamorphosed frogs and whole body tadpole T4 and T3/T4 concentrations were comparable among the reference and impacted wetlands. In addition, the differences observed in total body weight and length of tadpoles and newly-metamorphosed wood frogs among OSPM and reference sites were likely due to minor differences in developmental stages of the animals collected from the various wetlands, rather than any contaminant effect.<p> In 2006 and 2007, wood frog eggs and tadpoles were exposed to several sources of OSPW and OSPS collected from reclaimed Suncor and Syncrude wetlands under controlled laboratory conditions. Hatchability was reduced in eggs exposed to water from only one of the OSPW sites, compared with the other process-affected ponds and the control water (P<0.05). In contrast, survivability of tadpoles was significantly reduced (P<0.05) in all the impacted sites in both years, with nearly all OSPW sites having <10% survival. The exposure study evaluated the toxicity of five types of OSPS. Results indicated no impact of OSPS exposure on survivability of tadpoles, but showed reduced whole body weight (in three OSPS treatments), length (in two OSPS treatments) and body condition (in one OSPS) of tadpoles exposed to process-affected substrates tested (P<0.05). Whole body T3 and T4 concentrations in tadpoles from OSPS treatments were not different from the control treatment, but tadpole TG concentration was reduced in groups exposed to two impacted substrates (P<0.05). Water quality measurements, including determination of dissolved metals were conducted in an initial attempt to relate any potential toxic effect on wood frog growth and development to specific contaminants.<p> Results of the laboratory studies strongly suggest that exposure to OSPW and OSPS may adversely affect wood frog growth and survival. However, these findings were not entirely consistent with field observations and results of concurrent mesocosm studies. Further research is therefore needed to fully evaluate the suitability of reclaimed oil sands wetlands to support indigenous amphibian population. Future work should focus on the cumulative effects of water and substrates, as well as the effect of OSPM ageing on acute and chronic toxicity.