Behavioral and molecular analyses of olfaction-mediated avoidance responses of Rana (Lithobates) catesbeiana tadpoles: Sensitivity to thyroid hormones, estrogen, and treated municipal wastewater effluent.

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 (T4) and 3,5,3'-triiodothyronine (T3), 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 T4, T3, or 17-beta-estradiol (E2) for 48h and evaluated a predator cue avoidance response. The avoidance response in T3-exposed tadpoles was abolished while T4- or E2-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, T3 elicited greater responses than T4. 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.

Influence of temperature on thyroid hormone signaling and endocrine disruptor action in Rana (Lithobates) catesbeiana tadpoles.

Thyroid hormones (THs) are essential for normal growth, development, and metabolic control in vertebrates. Their absolute requirement during amphibian metamorphosis provides a powerful means to detect and assess the impact of environmental contaminants on TH signaling in the field and laboratory. As poikilotherms, frogs can experience considerable temperature fluctuations. Previous work demonstrated that low temperature prevents precocious TH-dependent induction of metamorphosis. However, a shift to a permissive higher temperature allows resumption of the induced metamorphic program regardless of whether or not TH remains. We investigated the impact of temperature on the TH-induced gene expression programs of premetamorphic Rana (Lithobates) catesbeiana tadpoles following a single injection of 10pmol/g body wet weight 3,3',5-triiodothyronine (T3). Abundance profiles of several T3-responsive mRNAs in liver, brain, lung, back skin, and tail fin were characterized under permissive (24°C), nonpermissive (5°C), or temperature shift (5-24°C) conditions. While responsiveness to T3 was retained to varying degrees at nonpermissive temperature, T3 modulation of thibz occurred in all tissues at 5°C suggesting an important role for this transcription factor in initiation of T3-dependent gene expression programs. Low temperature immersion of tadpoles in water containing 10nM T3 and the nonsteroidal anti-inflammatory drug, ibuprofen, or the antimicrobial agent, triclosan, perturbed some aspects of the gene expression programs of tail fin and back skin that was only evident upon temperature shift. Such temporal uncoupling of chemical exposure and resultant biological effects in developing frogs necessitates a careful evaluation of environmental temperature influence in environmental monitoring programs.

PCBs are associated with altered gene transcript profiles in arctic Beluga Whales (Delphinapterus leucas).

High trophic level arctic beluga whales (Delphinapterus leucas) are exposed to persistent organic pollutants (POP) originating primarily from southern latitudes. We collected samples from 43 male beluga harvested by Inuvialuit hunters (2008-2010) in the Beaufort Sea to evaluate the effects of POPs on the levels of 13 health-related gene transcripts using quantitative real-time polymerase chain reaction. Consistent with their role in detoxification, the aryl hydrocarbon receptor (Ahr) (r(2) = 0.18, p = 0.045 for 2008 and 2009) and cytochrome P450 1A1 (Cyp1a1) (r(2) = 0.20, p < 0.001 for 2008 and 2009; r(2) = 0.43, p = 0.049 for 2010) transcripts were positively correlated with polychlorinated biphenyls (PCBs), the dominant POP in beluga. Principal Components Analysis distinguished between these two toxicology genes and 11 other genes primarily involved in growth, metabolism, and development. Factor 1 explained 56% of gene profiles, with these latter 11 gene transcripts displaying greater abundance in years coinciding with periods of low sea ice extent (2008 and 2010). δ(13)C results suggested a shift in feeding ecology and/or change in condition of these ice edge-associated beluga whales during these two years. While this provides insight into the legacy of PCBs in a remote environment, the possible impacts of a changing ice climate on the health of beluga underscores the need for long-term studies.

Effects of acute exposure to the non-steroidal anti-inflammatory drug ibuprofen on the developing North American Bullfrog (Rana catesbeiana) tadpole.

A variety of pharmaceutical chemicals can represent constituents of municipal effluent outflows that are dispersed into aquatic receiving environments worldwide. Increasingly, there is concern as to the potential of such bioactive substances to interact with wildlife species at sensitive life stages and affect their biology. Using a combination of DNA microarray, quantitative real-time polymerase chain reaction, and quantitative nuclease protection assays, we assessed the ability of sub-lethal and environmentally relevant concentrations of ibuprofen (IBF), a non-steroidal anti-inflammatory agent and prevalent environmental contaminant, to function as a disruptor of endocrine-mediated post-embryonic development of the frog. While the LC50 of IBF for pre-metamorphic Rana catesbeiana tadpoles is 41.5 mg/L (95% confidence interval: 32.3-53.5 mg/L), exposure to concentrations in the ppb range elicited molecular responses both in vivo and in organ culture. A nominal concentration of 15 µg/L IBF (actual = 13.7 µg/L) altered the abundance of 26 mRNA transcripts within the liver of exposed pre-metamorphic R. catesbeiana tadpoles within 6 d. IBF-treated animals demonstrated subsequent disruption of thyroid hormone-mediated reprogramming in the liver transcriptome affecting constituents of several metabolic, developmental, and signaling pathways. Cultured tadpole tail fin treated with IBF for 48 h also demonstrated altered mRNA levels at drug concentrations as low as 1.5 µg/L. These observations raise the possibility that IBF may alter the post-embryonic development of anuran species in freshwater environs, where IBF is a persistent or seasonal pollutant.

Distinctive metabolite profiles in in-migrating Sockeye salmon suggest sex-linked endocrine perturbation.

The health of Skeena River Sockeye salmon (Onchorhychus nerka) has been of increasing concern due to declining stock returns over the past decade. In the present work, in-migrating Sockeye from the 2008 run were evaluated using a mass spectrometry-based, targeted metabolomics platform. Our objectives were to (a) investigate natural changes in a subset of the hepatic metabolome arising from migration-associated changes in osmoregulation, locomotion, and gametogenesis, and (b) compare the resultant profiles with animals displaying altered hepatic vitellogenin A (vtg) expression at the spawning grounds, which was previously hypothesized as a marker of xenobiotic exposure. Of 203 metabolites monitored, 95 were consistently observed in Sockeye salmon livers and over half of these changed significantly during in-migration. Among the most dramatic changes in both sexes were a decrease in concentrations of taurine (a major organic osmolyte), carnitine (involved in fatty acid transport), and two major polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid). In females, an increase in amino acids was attributed to protein catabolism associated with vitellogenesis. Animals with atypical vtg mRNA expression demonstrated unusual hepatic amino acid, fatty acid, taurine, and carnitine profiles. The cause of these molecular perturbations remains unclear, but may include xenobiotic exposure, natural senescence, and/or interindividual variability. These data provide a benchmark for further investigation into the long-term health of migrating Skeena Sockeye.

PCB related effects thresholds as derived through gene transcript profiles in locally contaminated ringed seals (Pusa hispida).

Causal evidence linking toxic injury to polychlorinated biphenyl (PCB) exposure is typically confounded by the complexity of real-world contaminant mixtures to which aquatic wildlife are exposed. A local PCB "hotspot" on the Labrador coast provided a rare opportunity to evaluate the effects of PCBs on the health of a marine mammal as this chemical dominated their persistent organic pollutant (POP) burdens. The release of approximately 260 kg of PCBs by a military radar facility over a 30 year period (1970-2000) contaminated some local marine biota, including the ringed seal (Pusa hispida). The abundance profiles of eight health-related gene transcripts were evaluated in liver samples collected from 43 ringed seals in the affected area. The mRNA transcript levels of five gene targets, including aryl hydrocarbon receptor (Ahr), interleukin-1 ß (Il1b), estrogen receptor α (Esr1), insulin like growth factor receptor 1 (Igf1), and glucocorticoid receptor α (Nr3c1) correlated with increasing levels of blubber PCBs. PCB threshold values calculated using best-fit hockey-stick regression models for these five genes averaged 1,680±206 ng/g lw, with the lowest, most conservative, being 1,370 ng/g lw for Il1b. Approximately 14% of the seals in the region exceeded this threshold. The dominance of PCBs in the seals studied enabled an assessment of the effects of this chemical on gene transcripts involved in regulating the health of a highly mobile predator, something that is rarely possible in the world of complex mixtures.

Development of a non-lethal method for evaluating transcriptomic endpoints in Arctic grayling (Thymallus arcticus).

With increases in active mining and continued discharge associated with former mine operations, evaluating the health of watersheds in the Canadian Yukon Territory is warranted. Current environmental assessment approaches often employ guidelines established using sentinel species not relevant to Arctic monitoring programs. The present study focused on the successful development of a quantitative real-time polymerase chain reaction (qPCR) assay directed towards the indigenous Arctic grayling (Thymallus arcticus) and examines the feasibility of using non-lethal sampling from the caudal fin as a means for evaluation of mRNA abundance profiles reflective of environmental conditions. In a proof of concept study performed blind, qPCR results from animals in an area with elevated water concentrations of cadmium (Cd) and zinc (Zn) and higher body burdens of Cd, Zn, and lead (Pb) were compared to a reference location in the Yukon Territory. Lower condition factor and a higher abundance of hepatic and caudal fin gene transcripts encoding the metallothionein isoforms (mta/mtb), in addition to elevated heat shock protein 70 (hsp70) and catalase (cat) mRNAs in liver, were observed in fish from the test site. The strong positive correlation between metal body burden and caudal fin mta/mtb mRNA abundance demonstrates a high potential for use of the Arctic grayling assay in non-lethal environmental monitoring programs.

Phenotypic plasticity in the hepatic transcriptome of the European common frog (Rana temporaria): the interplay between environmental induction and geographical lineage on developmental response.

Phenotypic plasticity might facilitate adaptation to new environmental conditions through the enhancement of initial survival of organisms. Once a population is established, further adaptation and diversification may occur through adaptive trait evolution. While several studies have found evidence for this mechanism using phenotypic traits, much less is known at the level of gene expression. Here, we use an islands system of frog populations that show local adaptation and phenotypic plasticity to pool drying conditions in development time until metamorphoses. We examined gene expression differences in Rana temporaria tadpole livers with respect to pool drying at the source population and in response to simulated pool drying in the laboratory. Using a MAGEX cDNA microarray and quantitative real-time polymerase chain reaction (qPCR), we identified an increase in several gene transcripts in response to artificial pool drying including thyroid hormone receptor alpha and beta, carbamoyl phosphate synthetase 1, ornithine transcarbamylase and catalase. In addition, these gene transcripts also showed greater abundance in island populations that developed faster. Hence, the gene transcripts were related to both constitutive response (higher levels in island populations that developed faster) and plastic response (increased abundance under decreasing water levels). This pattern is in accordance with genetic accommodation, which predicts similarities between plastic gene expression and constitutive expression in locally adapted populations.

Characterization of gene expression endpoints during postembryonic development of the northern green frog (Rana clamitans melanota).

Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.

Thyroid hormone-dependent development in Xenopus laevis: a sensitive screen of thyroid hormone signaling disruption by municipal wastewater treatment plant effluent.

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 (T(3)). 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 ß (TRß), deiodinase type II (DIO2), and corticotropin releasing hormone binding protein (CRHBP), fibroblast activation protein α (FAPα)) when exposed to T(3) and WWE extracts. TRß, 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.

Molecular profiling of marine fauna: integration of omics with environmental assessment of the world's oceans.

Many species that contribute to the commercial and ecological richness of our marine ecosystems are harbingers of environmental change. The ability of organisms to rapidly detect and respond to changes in the surrounding environment represents the foundation for application of molecular profiling technologies towards marine sentinel species in an attempt to identify signature profiles that may reside within the transcriptome, proteome, or metabolome and that are indicative of a particular environmental exposure event. The current review highlights recent examples of the biological information obtained for marine sentinel teleosts, mammals, and invertebrates. While in its infancy, such basal information can provide a systems biology framework in the detection and evaluation of environmental chemical contaminant effects on marine fauna. Repeated evaluation across different seasons and local marine environs will lead to discrimination between signature profiles representing normal variation within the complex milieu of environmental factors that trigger biological response in a given sentinel species and permit a greater understanding of normal versus anthropogenic-associated modulation of biological pathways, which prove detrimental to marine fauna. It is anticipated that incorporation of contaminant-specific molecular signatures into current risk assessment paradigms will lead to enhanced wildlife management strategies that minimize the impacts of our industrialized society on marine ecosystems.

PCB-associated changes in mRNA expression in killer whales (Orcinus orca) from the NE Pacific Ocean.

Killer whales in the NE Pacific Ocean are among the world's most PCB-contaminated marine mammals, raising concerns about implications for their health. Sixteen health-related killer whale mRNA transcripts were analyzed in blubber biopsies collected from 35 free-ranging killer whales in British Columbia using real-time quantitative polymerase chain reaction. We observed PCB-related increases in the expression of five gene targets, including the aryl hydrocarbon receptor (AhR; r(2) = 0.83; p < 0.001), thyroid hormone α receptor (TRα; r(2) = 0.64; p < 0.001), estrogen α receptor (ERα; r(2) = 0.70; p < 0.001), interleukin 10 (IL-10; r(2) = 0.74 and 0.68, males and females, respectively; p < 0.001), and metallothionein 1 (MT1; r(2) = 0.58; p < 0.001). Best-fit models indicated that population (dietary preference), age, and sex were not confounding factors, except for IL-10, where males differed from females. While the population-level consequences are unclear, the PCB-associated alterations in mRNA abundance of such pivotal end points provide compelling evidence of adverse physiological effects of persistent environmental contaminants in these endangered killer whales.

Nanometals induce stress and alter thyroid hormone action in amphibia at or below North American water quality guidelines.

Nanometals are manufactured to particle sizes with diameters in the nanometer range and are included in a variety of consumer and health products. There is a lack of information regarding potential effects of these materials on aquatic organisms. Amphibians are regarded as environmental sentinels and demonstrate an exquisite sensitivity to thyroid hormone action, a hormone that is essential for human health. This present study assessed the effect of exposure to nanometals on stress and thyroid hormone signaling in frog tissue using a cultured tail fin biopsy (C-fin) assay derived from Rana catesbeiana tadpoles. The C-fin assay maintains tissue complexity and biological replication while multiple chemical responses can be assessed from the same individual. We tested the ability of nanosilver (0.06 µg/L-5.5 mg/L), quantum dots (0.25 µg/L-22 mg/L), and nanozinc oxide (0.19-10 mg/L) to alter gene expression in the presence or absence of 3,3',5'-triiodothyronine (T(3)) using quantitative real-time polymerase chain reaction. Results were compared to exposure to micrometer-silver, silver nitrate, and micrometer-cadmium telluride. Nanosilver (≥2.75 mg/L) and quantum dots (≥0.22 mg/L) altered the expression of transcripts linked to T(3)- and stress-mediated pathways, while nanozinc oxide had no effect. Lower concentrations of nanosilver (0.6 to 550 µg/L) perturbed T(3)-mediated signaling while not inducing cell stress. The observed effects were orders of magnitude below acute toxicity levels and occurred at or below the current North American water quality guidelines for metals, underscoring the need for evaluating nanoparticles separately from their constituent chemicals.

Characterization of the histone H2A.Z-1 and H2A.Z-2 isoforms in vertebrates.

BACKGROUND: Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues. RESULTS: We report that H2A.Z-1 and H2A.Z-2 are expressed across a wide range of human tissues, they are both acetylated at lysine residues within the N-terminal region and they exhibit similar, but nonidentical, distributions within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1. The phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution. CONCLUSIONS: Our biochemical, gene expression, and phylogenetic data suggest that the H2A.Z-1 and H2A.Z-2 variants function similarly yet they may have acquired a degree of functional independence.

Transcriptomics investigation of thyroid hormone disruption in the olfactory system of the Rana [Lithobates] catesbeiana tadpole.

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 (T3) and L-thyroxine (T4), or 17-beta estradiol (E2); 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, E2 affected 267 contigs of which only 57 overlapped with THs suggesting that E2 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 E2, 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.

Characterization of Inhibitor of Growth 2 tumor suppressor in Alligator mississippiensis, its conservation in Archosauria, and response to thyroid stimulating hormone.

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.

Identification of gene expression indicators for thyroid axis disruption in a Xenopus laevis metamorphosis screening assay. Part 2. Effects on the tail and hindlimb.

Thyroid hormones (TH), thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)), play crucial roles in regulation of growth, development and metabolism in vertebrates and are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the US Environmental Protection Agency is developing a high throughput screen using TH-dependent metamorphosis of the Xenopus laevis tadpole as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. Using a combination of cDNA array and real time quantitative polymerase chain reaction (QPCR) analyses, this study identifies molecular markers in tissues peripheral to the central thyroid axis. We examine the hindlimb and tail of tadpoles up to 96 h of continuous exposure to T(3), T(4), methimazole, propylthiouracil, or perchlorate. Several novel biomarker candidates are indicated that include transcripts encoding importin, RNA helicase II/Gu, and defender against death protein, DAD1. In combination with previously-identified biomarker candidates, these transcripts will greatly augment the predictive and diagnostic power of the Xenopus metamorphosis assay for perturbation of TH action.

Identification of gene expression indicators for thyroid axis disruption in a Xenopus laevis metamorphosis screening assay. Part 1. Effects on the brain.

Thyroid hormones (TH), thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)), play crucial roles in regulation of growth, development and metabolism in vertebrates and their actions are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the US Environmental Protection Agency is developing a high throughput screen using TH-dependent amphibian metamorphosis as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. This study aims to characterize and identify molecular markers in the tadpole brain. Using a combination of cDNA array analysis and real time quantitative polymerase chain reaction (QPCR), we examine the brain of tadpoles following 96 h of continuous exposure to T(3), T(4), methimazole, propylthiouracil, or perchlorate. This tissue was more sensitive to T(4) rather than T(3), even when differences in biological activity were taken into account. This implies that a simple conversion of T(4) to T(3) cannot fully account for T(4) effects on the brain and suggests distinctive mechanisms of action for the two THs. While the brain shows gene expression alterations for methimazole and propylthiouracil, the environmental contaminant, perchlorate, had the greatest effect on the levels of mRNAs encoding proteins important in neural development and function. Our data identify gene expression profiles that can serve as exposure indicators of these chemicals.

De novo assembly of the ringed seal (Pusa hispida) blubber transcriptome: A tool that enables identification of molecular health indicators associated with PCB exposure.

The ringed seal, Pusa hispida, is a keystone species in the Arctic marine ecosystem, and is proving a useful marine mammal for linking polychlorinated biphenyl (PCB) exposure to toxic injury. We report here the first de novo assembled transcriptome for the ringed seal (342,863 transcripts, of which 53% were annotated), which we then applied to a population of ringed seals exposed to a local PCB source in Arctic Labrador, Canada. We found an indication of energy metabolism imbalance in local ringed seals (n=4), and identified five significant gene transcript targets: plasminogen receptor (Plg-R(KT)), solute carrier family 25 member 43 receptor (Slc25a43), ankyrin repeat domain-containing protein 26-like receptor (Ankrd26), HIS30 (not yet annotated) and HIS16 (not yet annotated) that may represent indicators of PCB exposure and effects in marine mammals. The abundance profiles of these five gene targets were validated in blubber samples collected from 43 ringed seals using a qPCR assay. The mRNA transcript levels for all five gene targets, (Plg-R(KT), r2=0.43), (Slc25a43, r2=0.51), (Ankrd26, r2=0.43), (HIS30, r2=0.39) and (HIS16, r2=0.31) correlated with increasing levels of blubber PCBs. Results from the present study contribute to our understanding of PCB associated effects in marine mammals, and provide new tools for future molecular and toxicology work in pinnipeds.

A multi-omic approach to elucidate low-dose effects of xenobiotics in zebrafish (Danio rerio) larvae.

Regulatory-approved toxicity assays such as the OECD Fish Embryo Toxicity Assay (TG236) allow correlation of chemical exposure to adverse morphological phenotypes. However, these assays are ineffective in assessing sub-lethal (i.e. low-dose) effects, or differentiating between similar phenotypes induced by different chemicals. Inclusion of multi-omic analyses in studies investigating xenobiotic action provides improved characterization of biological response, thereby enhancing prediction of toxicological outcomes in whole animals in the absence of morphological effects. In the current study, we assessed perturbations in both the metabolome and transcriptome of zebrafish (Danio rerio; ZF) larvae exposed from 96 to 120h post fertilization to environmental concentrations of acetaminophen (APAP), diphenhydramine (DH), carbamazepine (CBZ), and fluoxetine (FLX); common pharmaceuticals with known mechanisms of action. Multi-omic responses were evaluated independently and integrated to identify molecular interactions and biological relevance of the responses. Results indicated chemical- and dose-specific changes suggesting differences in the time scale of transcript abundance and metabolite production. Increased impact on the metabolome relative to the transcriptome in FLX-treated animals suggests a stronger post-translational effect of the treatment. In contrast, the transcriptome showed higher sensitivity to perturbation in DH-exposed animals. Integration of 'omic' responses using multivariate approaches provided additional insights not obtained by independent 'omic' analyses and demonstrated that the most distinct overall response profiles were induced following low-dose exposure for all 4 pharmaceuticals. Importantly, changes in transcript abundance corroborated with predictions from metabolomic enrichment analyses and the identified perturbed biological pathways aligned with known xenobiotic mechanisms of action. This work demonstrates that a multi-omic toxicological approach, coupled with a sensitive animal model such as ZF larvae, can help characterize the toxicological relevance of acute low-dose chemical exposures.