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Anuran metamorphosis is characterized by profound morphological changes including remodeling of tissues and organs. This transition is initiated by thyroid hormones (THs). However, the current knowledge of changing levels of THs during metamorphosis relies on pooled samples using methods known for high variability with sparse reporting of measured variation. Moreover, establishing a clear linkage between key gene expression bioindicators and TH levels throughout the metamorphic process is needed. Using state-of-the-art ultra-high performance liquid chromatography isotope-dilution tandem mass spectrometry, we targeted 12 THs and metabolites in the serum of Rana [Lithobates] catesbeiana (n=5-10) across seven distinct postembryonic stages beginning with premetamorphic tadpoles (Gosner stage 31-33) and continuing through metamorphosis to a juvenile frog (Gosner stage 46). TH levels were related to TH-relevant gene transcripts (thra, thrb, and thibz) in back skin of the same individual animals. Significant increases from basal levels were observed for thyroxine (T4) and 3,3’,5-triiodothyronine (T3) at Gosner stage 41, reaching maximal levels at Gosner stage 44 (28 ± 10 and 2.3 ± 0.5 ng/mL, respectively), and decreasing to basal levels in juvenile frogs. In contrast, 3,5-diiodothyronine (T2) increased significantly at Gosner stage 40 and was maintained elevated until stage 44. While thra transcript levels remained constant and then decreased at the end of metamorphic climax, thrb and thibz were induced to maximal levels at Gosner stage 41, followed by a decrease to basal levels in the froglet. This exemplifies the exquisite timing of events during metamorphosis as classic early response genes are transcribed in anticipation of peak TH concentrations. The distinct T2 concentration profile suggests a biological role of this biomolecule in anuran postembryonic development and an additional aspect that may be a target of anthropogenic chemicals that can disrupt anuran metamorphosis and TH signalling. Hence, as a second aim of the study, we set out to find additional bioindicators of metamorphosis, which can aid future investigations of developmental disruption. Using a sensitive nanoLC-Orbitrap system an untargeted analysis workflow was applied. Among 6,062 endogenous metabolites, 421 showed metamorphosis-dependent concentration dynamics. These potential bioindicators included several carnitines, prostaglandins and some steroid hormones. [ABSTRACT FROM AUTHOR]

Aquatic and terrestrial environments are increasingly contaminated by anthropogenic sources that include pharmaceuticals, personal care products, and industrial and agricultural chemicals (i. e., pesticides). Many of these substances have the potential to disrupt endocrine function, yet their effect on thyroid hormone (TH) action has garnered relatively little attention. Anuran postembryonic metamorphosis is strictly dependent on TH and perturbation of this process can serve as a sensitive barometer for the detection and mechanistic elucidation of TH disrupting activities of chemical contaminants and their complex mixtures. The ecological threats posed by these contaminants are further exacerbated by changing environmental conditions such as temperature, photoperiod, pond drying, food restriction, and ultraviolet radiation. We review the current knowledge of several chemical and environmental factors that disrupt TH-dependent metamorphosis in amphibian tadpoles as assessed by morphological, thyroid histology, behavioral, and molecular endpoints. Although the molecular mechanisms for TH disruption have yet to be determined for many chemical and environmental factors, several affect TH synthesis, transport or metabolism with subsequent downstream effects. As molecular dysfunction typically precedes phenotypic or histological pathologies, sensitive assays that detect changes in transcript, protein, or metabolite abundance are indispensable for the timely detection of TH disruption. The emergence and application of 'omics techniques—genomics, transcriptomics, proteomics, metabolomics, and epigenomics—on metamorphosing tadpoles are powerful emerging assets for the rapid, proxy assessment of toxicant or environmental damage for all vertebrates including humans. Moreover, these highly informative 'omics techniques will complement morphological, behavioral, and histological assessments, thereby providing a comprehensive understanding of how TH-dependent signal disruption is propagated by environmental contaminants and factors. [ABSTRACT FROM AUTHOR]

Thyroid hormones (THs), l -thyroxine (T 4 ) and 3,5,3′-triiodothyronine (T 3 ), are essential for vertebrate growth and development. Classically, T 4 is 5′-deiodinated to the active hormone, T 3 , in target tissues which then binds nuclear TH receptors (TRs) and regulates gene transcription. However, it is possible that T 4 acts directly on target tissues. Frog metamorphosis is a powerful TR-dependent model for studying TH action. Premetamorphic Rana ( Lithobates ) catesbeiana tadpoles were injected with 0.1–50 T 3 or 0.5–250 T 4 pmol/g body weight to account for their 5-fold difference in biological activity and the mRNA profiles in six tissues from well-characterized TH-responsive genes were evaluated after 48 h using quantitative real time polymerase chain reaction. 5′-deiodinase-poor tissues should produce superimposable dose–response curves if T 4 does not require conversion to T 3 . This was the case in lung and tail fin; the latter tissue recapitulating these responses in organ culture. 5′-deiodinase-rich tissues should convert T 4 to T 3 . Because T 3 has a higher affinity to TRs, a 5-fold higher T 4 dose compared to T 3 should produce greater transcript induction. This was observed in the brain and for most intestinal transcripts. However, some gene transcripts in the intestine and all transcripts in the back skin produced superimposable response curves suggesting that a direct mode of T 4 action is plausible in these tissues. While the liver showed results consistent with its 5′-deiodinase-poor status, we found evidence of an alternate, non-genomic mechanism for two gene transcripts. Therefore, mechanisms not requiring T 4 conversion to T 3 may play a far greater role than previously thought. [ABSTRACT FROM AUTHOR]

Environmental temperature modulates thyroid hormone (TH)-dependent metamorphosis in some amphibian species. The North American bullfrog - Rana (Lithobates) catesbeiana - tadpole is naturally adapted to a wide range of temperatures over multiple seasons. Cold temperatures delay while warmer temperatures accelerate metamorphosis. Exogenous TH exposure of premetamorphic tadpoles results in a rapid precocious induction of metamorphosis at warm temperatures (20-25°C). The same exposure at cold temperatures (4-5°C) does not elicit an overt metamorphic response. However, a molecular memory of TH exposure is established such that cold, TH-exposed tadpoles returned to permissive warm temperatures will rapidly execute TH-induced genetic programs. Previous mRNA profiling has identified TH-regulated transcription factors encoded by thra, thrb, thibz, klf9, and cebp1 as components of the molecular memory after one week post-exposure. However, a further hierarchy may exist within the initiation phase since many gene transcripts demonstrated tissue-specific patterns. Whether the molecular memory is organ autonomous or requires additional modulating factors is unknown. Herein we examine tail fin and back skin and determine that thibz is the only transcript that is TH-responsive after 2 days post-exposure at low temperature in both tissues in the intact animal. In back skin, cebp1 is also TH-responsive under these conditions. Serum-free tail fin organ culture (C-Fin) reveals that the thibz response is organ autonomous whereas cultured back skin (C-Skin) results suggest that thibz and cebp1 require an additional factor for induction from elsewhere within the intact animal. Subsequent investigations are now possible to identify endogenous factors that modulate the molecular memory in intact animals. [ABSTRACT FROM AUTHOR]

Postembryonic frog development requires a thyroid hormone (TH)-dependent metamorphic transition from an aquatic larva to a terrestrial frog. Such change in environment involves lung maturation in preparation for breathing air. However, little is known regarding the underlying molecular events and the role of THs in this process. Using quantitative real-time polymerase chain reaction, we evaluated Rana (Lithobates) catesbeiana lung mRNA transcripts representing key elements of TH and oxidative stress signaling pathways during natural and TH-induced precocious metamorphosis. TH induction was evaluated in two ways: 1) in vivo through interperitoneal injection of 10 pmol/g body weight of 3,3′, 5-triiodothyronine (T3) into premetamorphic tadpoles and analysis after 48 h, and 2) in serum-free organ culture in the presence of 10 nM T3 after 48 h. Abundance of transcripts encoding the transcriptional regulators TH receptors α and β, TH-induced bZip protein, and CCAAT/enhancer binding protein 1 was increased during postembryonic development and following administration of exogenous THs to premetamorphic tadpoles in vivo and culture. In contrast, mRNA representing Krüppel-like factor 9 and cold-inducible RNA binding protein revealed differential effects between natural and precocious metamorphosis. Elevated levels of catalase and Cu/Zn superoxide dismutase mRNA were observed at the end of metamorphosis with transcript levels displaying minimal TH-dependency. No change in stress-responsive heat shock protein 30 mRNA abundance was noted. The results support a role for TH-dependent reprogramming of the lung transcriptome during frog development and reveal a requirement for increased antioxidant capacity following anuran metamorphosis. [ABSTRACT FROM AUTHOR]

Background After completion of embryogenesis, many organisms experience an additional obligatory developmental transition to attain a substantially different juvenile or adult form. During anuran metamorphosis, the aquatic tadpole undergoes drastic morphological changes and remodelling of tissues and organs to become a froglet. Thyroid hormones are required to initiate the process, but the mechanism whereby the many requisite changes are coordinated between organs and tissues is poorly understood. Metabolites are often highly conserved biomolecules between species and are the closest reflection of phenotype. Due to the extensive distribution of blood throughout the organism, examination of the metabolites contained therein provides a system-wide overview of the coordinated changes experienced during metamorphosis. We performed an untargeted metabolomic analysis on serum samples from naturally-metamorphosing Rana catesbeiana from tadpoles to froglets using ultraperformance liquid chromatography coupled to a mass spectrometer. Total and aqueous metabolite extracts were obtained from each serum sample to select for nonpolar and polar metabolites, respectively, and selected metabolites were validated by running authentic compounds. Results The majority of the detected metabolites (74%) showed statistically significant abundance changes (padj < 0.001) between metamorphic stages. We observed extensive remodelling of five core metabolic pathways: arginine and purine/pyrimidine, cysteine/methionine, sphingolipid, and eicosanoid metabolism and the urea cycle, and found evidence for a major role for lipids during this postembryonic process. Metabolites traditionally linked to human disease states were found to have biological linkages to the system-wide changes occuring during the events leading up to overt morphological change. Conclusions To our knowledge, this is the first wide-scale metabolomic study of vertebrate metamorphosis identifying fundamental pathways involved in the coordination of this important developmental process and paves the way for metabolomic studies on other metamorphic systems including fish and insects. [ABSTRACT FROM AUTHOR]

Abstract: Exposure of critical life stages to harmful chemicals at low, environmentally-relevant concentrations can alter how hormones function, and change metabolic pathways or developmental processes that impact reproduction, behavior, or susceptibility to disease later in life. These alterations can be captured through evaluation of changes to transcriptomes, proteomes, and metabolomes occurring at those critical life stages thereby enabling more effective and earlier identification of mechanism of action, individual susceptibilities and adaptation, and prediction of detrimental sublethal effects. Amphibians are “wet canaries in the coalmine” as indicators for environmental health. There are more than 6000 species living in a variety of ecological niches worldwide yet limited ‘omics resources and approaches exist. To provide for a means of addressing this challenge, frogSCOPE (frog Sentinel species Comparative “Omics” for the Environment) combines transcriptomics, proteomics, and metabolomics together to form the foundation for the identification of biological response indicators of harmful effects on a species of wild frog (Rana catesbeiana) at a sensitive tadpole stage. Various exposure and sampling methodologies are possible including standard in vivo exposures, tail fin biopsies, and the C-fin assay. frogSCOPE establishes methodological and analytical approaches applicable to wildlife by using a uniquely-designed frog cDNA array developed to accommodate cross-species hybridization and quantitative real-time polymerase chain reaction (QPCR) assays on poorly genetically-characterized wildlife species. Combination with proteomics (isobaric tags for relative and absolute protein quantitation; iTRAQ) and metabolomics (mass spectrometry) enable the generation of molecular fingerprints to identify mechanisms of action in a more comprehensive fashion to better define suitable indicators of deleterious biological outcomes to wildlife. [Copyright &y& Elsevier]

There is a need for the development of a rapid method for identifying chemicals that disrupt thyroid hormone (TH) action while maintaining complex tissue structure and biological variation. Moreover, no assay to date allows a simultaneous screen of an individual's response to multiple chemicals. A cultured tail fin biopsy or C-fin assay was developed using Rana catesbeiana tadpoles. Multiple tail fin biopsies were taken per tadpole, cultured in serum-free medium, and then each biopsy was exposed to a different treatment condition. The effects of known disruptors of TH action were evaluated in the C-fin assay. Chemical exposure was performed ± 10 nM 3,3′,5-triiodothyronine and real-time quantitative polymerase chain reaction (QPCR) of two TH-responsive transcripts, TH receptor β (TRβ) and the Rana larval keratin type I (RLKI), was performed. Within 48 h of exposure to Triac (1-100 nM), roscovitine (0.6–60 µM), or genistein (1–100 µM), perturbations in TH signaling were detected. Tetrabromobisphenol A (TBBPA) (10-1,000 nM) showed no effect. Acetochlor (1–100 nM) elicited a modest effect on the TH-dependent induction of TRβ transcript. These data reveal that a direct tissue effect may not be critical for TBBPA and acetochlor to disrupt TH action previously observed in intact tadpoles. Environ. Toxicol. Chem. 2010;29:380–388. © 2009 SETAC [ABSTRACT FROM AUTHOR]

Abstract: Inhibitor of growth 2 (ING2) belongs to a family of tumor suppressors that are important regulators of a wide range of cellular processes including proliferation, apoptosis, and DNA repair. ING family members are found in yeast, plants, invertebrates and many vertebrate species. However, to date, ING has not been characterized in reptiles. Herein we describe the isolation of expressed ING2 sequence in the American alligator, Alligator mississippiensis, and compare this sequence with that isolated in the chicken. We identify features that are unique to these two representatives of the Archosaurs including conservation of specific amino acid residues and the absence of an adenylate residue in the 5′ end of the nucleotide sequence relative to frogs and mammals. The latter feature results in an alteration of the coding potential leading to distinctive N-termini. Injection of juvenile alligators with thyroid stimulating hormone (TSH), which increases endogenous thyroid hormones, results in the modulation of ING2 transcript levels. Quantitative real time polymerase chain reaction analyses revealed a reduction in the steady-state levels of ING2 mRNA in the phallus/cliterophallus, lung, and liver by 48 h after TSH injection. ING2 expression in the thyroid gland, gonad, and heart was unaffected by TSH treatment. These data indicate that control of ING2 expression by the thyroid axis may be conserved among species and is tissue-dependent. [Copyright &y& Elsevier]

Abstract: Despite the extensive use of wildlife species in elucidating important biological processes, very few gene expression tools are available. For example, many frog species with different sensitivities and ecological niches are used as sentinel species for environmental contaminants and as developmental models. However, gene expression analyses have been essentially limited to one laboratory species. In an attempt to extend gene expression analyses to relevant indigenous species, we have developed a frog cDNA array with probes designed against conserved protein-encoding sequences. Changes in gene expression profiles were identified in cultured tail tips of Rana catesbeiana tadpoles during induction of tail regression by exogenous thyroid hormone and are associated with a transition from active cell proliferation to increased apoptotic activity. The expression profiles of selected genes representative of different response patterns were further characterized in tails of tadpoles undergoing natural metamorphosis using de novo designed biomarker probes and quantitative real-time polymerase chain reaction analysis. The results support the cross-species application of cDNA arrays that can direct the development of gene expression biomarkers for indigenous wildlife species. [Copyright &y& Elsevier]

Highlights: [•] Heterologous qPCR primer sets were designed for qPCR. [•] These primers were effective on evolutionarily distant anurans. [•] Rigorous validation ensured integrity of qPCR amplification across species. [•] Tail fin, liver, brain and intestine mRNAs were compared between a Pipid and Ranid. [•] Tissue profiles and response to exogenous hormone provide mechanistic insight. [ABSTRACT FROM AUTHOR]

Thyroid hormones (THs) regulate vertebrate growth, development, and metabolism. Despite their importance, there is a need for effective detection of TH-disruption by endocrine disrupting chemicals (EDCs). The frog olfactory system substantially remodels during TH-dependent metamorphosis and the objective of the present study is to examine olfactory system gene expression for TH biomarkers that can evaluate the biological effects of complex mixtures such as municipal wastewater. We first examine classic TH-response gene transcripts using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) in the olfactory epithelium (OE) and olfactory bulb (OB) of premetamorphic Rana (Lithobates) catesbeiana tadpoles after 48 h exposure to biologically-relevant concentrations of the THs, 3,5,3′-triiodothyronine (T 3 ) and L-thyroxine (T 4 ), or 17-beta estradiol (E 2 ); a hormone that can crosstalk with THs. As the OE was particularly sensitive to THs, further RNA-seq analysis found >30,000 TH-responsive contigs. In contrast, E 2 affected 267 contigs of which only 57 overlapped with THs suggesting that E 2 has limited effect on the OE at this developmental phase. Gene ontology enrichment analyses identified sensory perception and nucleoside diphosphate phosphorylation as the top affected terms for THs and E 2 , respectively. Using classic and additional RNA-seq-derived TH-response gene transcripts, we queried TH-disrupting activity in municipal wastewater effluent from two different treatment systems: anaerobic membrane bioreactor (AnMBR) and membrane enhanced biological phosphorous removal (MEBPR). While we observed physical EDC removal in both systems, some TH disruption activity was retained in the effluents. This work lays an important foundation for linking TH-dependent gene expression with olfactory system function in amphibians. [ABSTRACT FROM AUTHOR]

Anuran metamorphosis involves the transformation of an aquatic tadpole into a juvenile frog. This process is completely dependent upon thyroid hormones (THs). Although much research has been focused on changes in gene expression programs during this postembryonic developmental period, transitions in the metabolic profiles are relatively poorly understood. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) is a technique that generates highly multiplexed mass spectra while retaining spatial location information on a thin tissue section. Reconstructed ion heat maps are correlated with morphology of the tissue section for biological interpretation. The present study is the first to use whole-body MALDI-MSI on tadpoles to gain insights into anuran metamorphosis. Approximately 1000 features were detected in each of five tissues examined (brain, eye, liver, notochord, and tail muscle) from premetamorphic North American bullfrog ( Rana [Lithobates] catesbeiana) tadpoles . Of these detected metabolites, 1700 were unique and 136 were significantly affected by exposure to 50 nM thyroxine for 48 h. Of the significantly-affected metabolites, 64 features were tentatively identified using the MassTRIX annotation tool. All tissues revealed changes in lipophilic compounds including phosphatidylcholines, phosphatidylinositols, phosphatidylglycerols, phosphatidylethanolamines, and phosphatidylserines. These lipophilic compounds made up the largest portion of significantly-affected metabolites indicating that lipid signaling is a major target of TH action in frog tadpoles. [ABSTRACT FROM AUTHOR]

Olfaction is critical for survival, facilitating predator avoidance and food location. The nature of the olfactory system changes during amphibian metamorphosis as the aquatic herbivorous tadpole transitions to a terrestrial, carnivorous frog. Metamorphosis is principally dependent on the action of thyroid hormones (THs), l -thyroxine (T 4 ) and 3,5,3′-triiodothyronine (T 3 ), yet little is known about their influence on olfaction during this phase of postembryonic development. We exposed Taylor Kollros stage I-XIII Rana ( Lithobates ) catesbeiana tadpoles to physiological concentrations of T 4 , T 3 , or 17-beta-estradiol (E 2 ) for 48 h and evaluated a predator cue avoidance response. The avoidance response in T 3 -exposed tadpoles was abolished while T 4 - or E 2 -exposed tadpoles were unaffected compared to control tadpoles. qPCR analyses on classic TH-response gene transcripts ( thra , thrb , and thibz ) in the olfactory epithelium demonstrated that, while both THs produced molecular responses, T 3 elicited greater responses than T 4 . Municipal wastewater feed stock was spiked with a defined pharmaceutical and personal care product (PPCP) cocktail and treated with an anaerobic membrane bioreactor (AnMBR). Despite substantially reduced PPCP levels, exposure to this effluent abolished avoidance behavior relative to AnMBR effluent whose feed stock was spiked with vehicle. Thibz transcript levels increased upon exposure to either effluent indicating TH mimic activity. The present work is the first to demonstrate differential TH responsiveness of the frog tadpole olfactory system with both behavioral and molecular alterations. A systems-based analysis is warranted to further elucidate the mechanism of action on the olfactory epithelium and identify further molecular bioindicators linked to behavioral response disruption. [ABSTRACT FROM AUTHOR]

Nanoparticles (NPs) are engineered in the nanoscale (<100 nm) to have unique physico-chemical properties from their bulk counterparts. Nanosilver particles (AgNPs) are the most prevalent NPs in consumer products due to their strong antimicrobial action. While AgNP toxicity at high concentrations has been thoroughly investigated, the sublethal effects at or below regulatory guidelines are relatively unknown. Amphibian metamorphosis is mediated by thyroid hormone (TH), and initial studies with bullfrogs ( Rana catesbeiana ) indicate that low concentrations of AgNPs disrupt TH-dependent responses in premetamorphic tadpole tailfin tissue. The present study examined the effects of low, non-lethal, environmentally-relevant AgNP concentrations (0.018, 0.18 or 1.8 μg/L Ag; ∼10 nm particle size) on naturally metamorphosing Xenopus laevis tadpoles in two-28 day chronic exposures beginning with either pre- or prometamorphic developmental stages. Asymmetric flow field flow fractionation with online inductively coupled plasma mass spectrometry and nanoparticle tracking analysis indicated a mixture of single AgNPs with homo-agglomerates in the exposure water with a significant portion (∼30–40%) found as dissolved Ag. Tadpoles bioaccumulated AgNPs and displayed transient alterations in snout/vent and hindlimb length with AgNP exposure. Using MAGEX microarray and quantitative real time polymerase chain reaction transcript analyses, AgNP-induced disruption of five TH-responsive targets was observed. The increased mRNA abundance of two peroxidase genes by AgNP exposure suggests the presence of reactive oxygen species even at low, environmentally-relevant concentrations. Furthermore, differential responsiveness to AgNPs was observed at each developmental stage. Therefore, low concentrations of AgNPs had developmental stage-specific endocrine disrupting effects during TH-dependent metamorphosis. [ABSTRACT FROM AUTHOR]

Conventional municipal wastewater treatment plants do not efficiently remove contaminants of emerging concern, and so are primary sources for contaminant release into the aquatic environment. Although these contaminants are present in effluents at ng-μg/L concentrations (i.e. microcontaminants), many compounds can act as endocrine disrupting compounds or stress-inducing agents at these levels. Chemical fate analyses indicate that additional levels of wastewater treatment reduce but do not always completely remove all microcontaminants. The removal of microcontaminants from wastewater does not necessarily correspond to a reduction in biological activity, as contaminant metabolites or byproducts may still be biologically active. To evaluate the efficacy of conventional municipal wastewater treatment plants to remove biological activity, we examined the performance of a full scale conventional activated sludge municipal wastewater treatment plant located in Guelph, Ontario, Canada. We assessed reductions in levels of conventional wastewater parameters and thyroid hormone disrupting and stress-inducing activities in wastewater at three phases along the treatment train using a C-fin assay. Wastewater treatment was effective at reducing total suspended solids, chemical and biochemical oxygen demand, and stress-inducing bioactivity. However, only minimal reduction was observed in thyroid hormone disrupting activities. The present study underscores the importance of examining multiple chemical and biological endpoints in evaluating and monitoring the effectiveness of wastewater treatment for removal of microcontaminants. [ABSTRACT FROM AUTHOR]

Abstract: The orchestration of anuran metamorphosis is initiated and integrated by thyroid hormones, which change dynamically during larval development and which may represent a target of disruption by environmental contaminants. Studies have found that some anurans experience increased rates of development when exposed to the insecticide carbaryl later in larval development, suggesting that this insecticide could affect thyroid hormone-associated biological pathways. However, the time in development when tadpoles are sensitive to insecticide exposure has not been clearly defined nor has the mechanism been tested. In two separate studies, we exposed recently hatched green frog (Lithobates clamitans) tadpoles to a single, three day carbaryl exposure in the laboratory at either 2, 4, 8, or 16 weeks post-hatching. We examined the impact of carbaryl exposure on mRNA abundance patterns in the brains of frogs following metamorphosis months after a single three day exposure (experiment 1) and in tadpole tails three days after exposure (experiment 2) using cDNA microarrays and quantitative real time polymerase chain reaction (QPCR) analyses. For tadpoles reared through metamorphosis, we measured tadpole growth and development, as well as time to, mass at, and survival to metamorphosis. Although carbaryl did not significantly impact tadpole development, metamorphosis, or survival, clear exposure-related alterations in both tail and brain transcript levels were evident when tadpoles were exposed to carbaryl, particularly in tadpoles exposed at weeks 8 and 16 post-hatching, indicating both short-term and long-term alterations in mRNA expression. These results indicate that carbaryl can have long-lasting effects on brain development when exposure occurs at sensitive developmental stages, which may have implications for animal fitness and function later in the life cycle. [Copyright &y& Elsevier]

Abstract: Because thyroid hormones (THs) are conserved modulators of development and physiology, identification of compounds adversely affecting TH signaling is critical to human and wildlife health. Anurans are an established model for studying disruption of TH signaling because metamorphosis is dependent upon the thyroid system. In order to strengthen this model and identify new gene transcript biomarkers for TH disruption, we performed DNA microarray analysis of Xenopus laevis tadpole tail transcriptomes following treatment with triiodothyronine (T3). Comparison of these results with previous studies in frogs and mammals identified 36 gene transcripts that were TH-sensitive across clades. We then tested molecular biomarkers for sensitivity to disruption by exposure to wastewater effluent (WWE). X. laevis tadpoles, exposed to WWE from embryo through metamorphosis, exhibited an increased developmental rate compared to controls. Cultured tadpole tails showed dramatic increases in levels of four TH-sensitive gene transcripts (thyroid hormone receptor β (TRβ), deiodinase type II (DIO2), and corticotropin releasing hormone binding protein (CRHBP), fibroblast activation protein α (FAPα)) when exposed to T3 and WWE extracts. TRβ, DIO2, and CRHBP were identified as TH sensitive in other studies, while FAPα mRNA transcripts were highly TH sensitive in our array. The results validate the array and demonstrate TH-disrupting activity by WWE. Our findings demonstrate the usefulness of cross-clade analysis for identification of gene transcripts that provide sensitivity to endocrine disruption. Further, the results suggest that development is disrupted by exposure to complex mixes of compounds found in WWE possibly through interference with TH signaling. [Copyright &y& Elsevier]

Abstract: The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48h exposure to 1 and 100μg/l of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5ng/l of the estrogenic chemical, 17α-ethynylestradiol (EE2), and 673ng/l of the thyroid hormone, 3,5,3′-triiodothyronine (T3), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48h exposure to EE2 but not T3 while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development. [Copyright &y& Elsevier]

Thyroid hormones (THs) are important regulators of growth, development, and homeostasis of all vertebrates. There are many environmental contaminants that are known to disrupt TH action, yet their mechanisms are only partially understood. While the effects of Endocrine Disrupting Chemicals (EDCs) are mostly studied as "hormone system silos", the present critical review highlights the complexity of EDCs interfering with TH function through their interactions with other hormonal axes involved in reproduction, stress, and energy metabolism. The impact of EDCs on components that are shared between hormone signaling pathways or intersect between pathways can thus extend beyond the molecular ramifications to cellular, physiological, behavioral, and whole-body consequences for exposed organisms. The comparatively more extensive studies conducted in mammalian models provides encouraging support for expanded investigation and highlight the paucity of data generated in other non-mammalian vertebrate classes. As greater genomics-based resources become available across vertebrate classes, better identification and delineation of EDC effects, modes of action, and identification of effective biomarkers suitable for HPT disruption is possible. EDC-derived effects are likely to cascade into a plurality of physiological effects far more complex than the few variables tested within any research studies. The field should move towards understanding a system of hormonal systems' interactions rather than maintaining hormone system silos. [Display omitted] • Thyroid hormones are important cellular process regulators in vertebrates. • Endocrine disruptors (EDCs) affect the hypothalamus-pituitary-thyroid (HPT) axis. • Reproductive, stress, dietary, and metabolic axes crosstalk with the HPT axis. • This crosstalk presents an additional mode of HPT axis disruption by EDCs. • The present review addresses new research questions to identify vulnerable species. [ABSTRACT FROM AUTHOR]

Abstract: We investigated whether exposure to environmentally relevant concentrations of the bactericidal agent, triclosan, induces changes in the thyroid hormone-mediated process of metamorphosis of the North American bullfrog, Rana catesbeiana and alters the expression profile of thyroid hormone receptor (TR) α and β, basic transcription element binding protein (BTEB) and proliferating nuclear cell antigen (PCNA) gene transcripts. Premetamorphic tadpoles were immersed in environmentally relevant concentrations of triclosan and injected with 1×10−11 mol/g body weight 3,5,3′-triiodothyronine (T3) or vehicle control. Morphometric measurements and steady-state mRNA levels obtained by quantitative polymerase chain reaction were determined. mRNA abundance was also examined in Xenopus laevis XTC-2 cells treated with triclosan and/or 10nM T3. Tadpoles pretreated with triclosan concentrations as low as 0.15±0.03μg/L for 4 days showed increased hindlimb development and a decrease in total body weight following T3 administration. Triclosan exposure also resulted in decreased T3-mediated TRβ mRNA expression in the tadpole tail fin and increased levels of PCNA transcript in the brain within 48h of T3 treatment whereas TRα and BTEB were unaffected. Triclosan alone altered thyroid hormone receptor α transcript levels in the brain of premetamorphic tadpoles and induced a transient weight loss. In XTC-2 cells, exposure to T3 plus nominal concentrations of triclosan as low as 0.03μg/L for 24h resulted in altered thyroid hormone receptor mRNA expression. Exposure to low levels of triclosan disrupts thyroid hormone-associated gene expression and can alter the rate of thyroid hormone-mediated postembryonic anuran development. [Copyright &y& Elsevier]

Abstract: Presently in the environment, there exist a number of chemical contaminants which share structural similarity with key naturally occurring regulatory hormones. These hormones play pivotal roles in the normal growth and development of wildlife species and humans. In particular, biphenolic chemical compounds may have the potential to act as agonists or antagonists of thyroid hormone (TH) action. We investigated whether there was any biological effect of exposure to low concentrations of the brominated fire retardant, tetrabromobisphenol-A (TBBPA), on the TH-mediated process of metamorphosis of the Pacific tree frog, Pseudacris regilla. Tadpoles exposed to 10nM (5.4μg/L) TBBPA showed an increase in TH-mediated expression of gelatinase B mRNA within 48h in the tadpole tail which was associated with increased tail resorption by 96h. Treatment with 100nM (54.4μg/L) TBBPA resulted in increased TH-mediated thyroid hormone receptor alpha mRNA expression in the tadpole brain and reduced levels of PCNA transcript in the tail. TBBPA alone was also found to alter the mRNA abundance of thyroid hormone receptor alpha in tail, gelatinase B in brain, and PCNA in both tissues of premetamorphic tadpoles. Interestingly, expression of thyroid hormone receptor beta mRNA was not affected by exposure to TBBPA either alone or in the presence of TH. The results suggest that exposure to low levels of TBBPA may act as an agonist of TH action and potentiate TH-mediated gene expression leading to accelerated anuran metamorphosis. [Copyright &y& Elsevier]

Abstract: Thyroid hormones (TH) are important in growth, development and the maintenance of proper cellular metabolism in vertebrates. Amphibian metamorphosis is completely dependent on TH and forms the basis of a screen for thyroid axis disrupting chemicals that currently relies on external morphological endpoints and changes in thyroid gland histology. The requirement for TH-dependent gene expression makes it possible to augment this screen through the addition of molecular endpoints. In order to do this, gene selection, choice of sampling time, tissue sensitivity, and their relationship to morphological change must all be considered. We exposed stage 54 Xenopus laevis tadpoles to a concentration series of the THs, thyroxine (T4) and 3,5,3′-triiodothyronine (T3), and three known TH antagonists, methimazole, propylthiouracil (PTU), and perchlorate. The agonists significantly accelerated metamorphosis as defined by developmental stage attained after 14 days. In contrast, the TH antagonists significantly delayed metamorphosis at 14 days and caused an increase in thyroid gland size at day 8. We assessed the changes in steady-state mRNA levels of thyroid hormone receptor α- and β-isoforms and the basic transcription element binding (BTEB) protein by quantitative real-time polymerase chain reaction. Three tissues (brain, tail and hindlimb) were analyzed at 24, 48 and 96h and we found that TH receptor, TRβ, and BTEB were the most sensitive gene transcripts for the TH agonists, whereas only TRα displayed significant changes upon antagonist exposure. We detected differences in tissue-specific responses between the two agonists. We matched the concentrations of T3 and T4 that elicited similar biological responses at 14 days and compared the induction of gene expression. At 96h, the TRβ and BTEB expression response to T3 and T4 was similar in the tail. In contrast, T3 elicited no concentration-dependent changes in TRβ and BTEB expression in the brain, whereas T4 elevated their expression. The tail showed the highest correlation between TH concentration and morphological outcome whereas the brain was the most sensitive to antagonist treatment. Only methimazole and perchlorate showed significant changes in TRα gene expression in the brain whereas PTU did not suggesting differences in cellular mechanisms of action. The greatest effect on gene expression occurred within 48h with many of the hormone-dependent changes disappearing by 96h. This study accentuates the need to examine multiple tissues and provides critical information required for optimization of exposure regimens and endpoint assessments that focus on the detection of disruption in TH-regulatory systems. [Copyright &y& Elsevier]