Comparative analysis of sucrose-embedding for whole-body zebrafish MSI by IR-MALDESI.

Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI) conventionally utilizes fresh-frozen biological tissues with an ice matrix to improve the detection of analytes. Sucrose-embedding with paraformaldehyde fixation has demonstrated feasibility as an alternative matrix for analysis by IR-MALDESI by preserving tissue features and enhancing ionization of lipids. However, investigating multi-organ systems provides broader context for a biological study and can elucidate more information about a disease state as opposed to a single organ. Danio rerio, or zebrafish, are model organisms for various disease states and can be imaged as a multi-organ sample to analyze morphological and metabolomic preservation as a result of sample preparation. Herein, whole-body zebrafish were imaged to compare sucrose-embedding with paraformaldehyde fixation against conventional fresh-frozen sample preparation. Serial sections were analyzed with and without an ice matrix to evaluate if sucrose functions as an alternative energy-absorbing matrix for IR-MALDESI applications across whole-body tissues. The resulting four conditions were compared in terms of total putative lipid annotations and category diversity, coverage across the entire m/z range, and ion abundance. Ultimately, sucrose-embedded zebrafish had an increase in putative lipid annotations for the combination of putative annotations with and without the application of an ice matrix relative to fresh-frozen tissues which require the application of an ice matrix. Upon the use of an ice matrix, a greater number of high mass putative lipid annotations (e.g., glycerophospholipids, glycerolipids, and sphingolipids) were identified. Conversely, without an ice matrix, sucrose-embedded sections elucidated more putative annotations in lower molecular weight lipids, including fatty acyls and sterol lipids. Similar to the mouse brain model, sucrose-embedding increased putative lipid annotation and abundance for whole-body zebrafish.

Vitamin D deficiency promotes accumulation of bioactive lipids and increased endocannabinoid tone in zebrafish.

Vitamin D is well known for its traditional role in bone mineral homeostasis; however, recent evidence suggests that vitamin D also plays a significant role in metabolic control. This study served to investigate putative linkages between vitamin D deficiency (VDD) and metabolic disruption of bioactive lipids by MS imaging. Our approach employed infrared-matrix-assisted laser desorption electrospray ionization MS imaging for lipid metabolite profiling in 6-month-old zebrafish fed either a VDD or a vitamin D-sufficient (VDS) diet. Using a lipidomics pipeline, we found that VDD zebrafish had a greater abundance of bioactive lipids (N-acyls, endocannabinoids [ECs], diacylglycerols/triacylglycerols, bile acids/bile alcohols, and vitamin D derivatives) suggestive of increased EC tone compared with VDS zebrafish. Tandem MS was performed on several differentially expressed metabolites with sufficient ion abundances to aid in structural elucidation and provide additional support for MS annotations. To confirm activation of the EC pathways, we subsequently examined expression of genes involved in EC biosynthesis, metabolism, and receptor signaling in adipose tissue and liver from VDD and VDS zebrafish. Gene expression changes were congruent with increased EC tone, with VDD zebrafish demonstrating increased synthesis and metabolism of anandamide compared with VDS zebrafish. Taken together, our data suggest that VDD may promote accumulation of bioactive lipids and increased EC tone in zebrafish.

Thyroid Receptor Antagonism of Chemicals Extracted from Personal Silicone Wristbands within a Papillary Thyroid Cancer Pilot Study.

Research suggests that thyroid cancer incidence rates are increasing, and environmental exposures have been postulated to be playing a role. To explore this possibility, we conducted a pilot study to investigate the thyroid disrupting bioactivity of chemical mixtures isolated from personal silicone wristband samplers within a thyroid cancer cohort. Specifically, we evaluated TRß antagonism of chemical mixtures extracted from wristbands (n = 72) worn by adults in central North Carolina participating in a case-control study on papillary thyroid cancer. Sections of wristbands were solvent-extracted and analyzed via mass spectrometry to quantify a suite of semivolatile chemicals. A second extract from each wristband was used in a bioassay to quantify TRß antagonism in human embryonic kidney cells (HEK293/17) at concentrations ranging from 0.1 to 10% of the original extract (by volume). Approximately 70% of the sample extracts tested at a 1% extract concentration exhibited significant TRß antagonism, with a mean of 30% and a range of 0-100%. Inhibited cell viability was noted in >20% of samples that were tested at 5 and 10% concentrations. Antagonism was positively associated with wristband concentrations of several phthalates, organophosphate esters, and brominated flame retardants. These results suggest that personal passive samplers may be useful in evaluating the bioactivities of mixtures that people contact on a daily basis. We also report tentative associations between thyroid receptor antagonism, chemical concentrations, and papillary thyroid cancer case status. Future research utilizing larger sample sizes, prospective data collection, and measurement of serum thyroid hormone levels (which were not possible in this study) should be utilized to more comprehensively evaluate these associations.

Windows of Susceptibility and Consequences of Early Life Exposures to 17ß-estradiol on Medaka (Oryzias latipes) Reproductive Success.

Estrogens and estrogen mimics are commonly found in surface waters and are associated with deleterious effects in fish populations. Impaired fertility and fecundity in fish following chronic exposures to estrogens and estrogen mimics during critical windows in development are well documented. However, information regarding differential reproductive effects of exposure within defined developmental stages remains sparse. In this study, reproductive capacity was assessed in Japanese medaka (Oryzias latipes) after exposure to two concentrations of 17ß-estradiol (E2ß; 2 ng/L and 50 ng/L) during four distinct stages of development: gonad development, gonad differentiation, development of secondary sex characteristics (SSC) and gametogenesis. Exposure to E2ß did not adversely impact survival, hatch success, growth, or genotypic ratios. In contrast, exposure to 50 ng/L E2ß during SSC development altered phenotypic ratios and SSC. Exposure to both E2ß treatments reduced reproductive capacity (fertility, fecundity) by 7.3-57.4% in adult medaka breeding pairs, with hindrance of SSC development resulting in the largest disruption in breeding capacity (51.6-57.4% decrease) in the high concentration. This study documents differential effects among four critical stages of development and provides insight into factors (window of exposure, exposure concentration and duration of exposure period) contributing to reproductive disruption in fish.

Sharing the Roles: An Assessment of Japanese Medaka Estrogen Receptors in Vitellogenin Induction.

Teleost fish express at least three estrogen receptor (ER) subtypes. To date, however, the individual role of these ER subtypes in regulating expression of estrogen responsive genes remains ambiguous. Here, we investigate putative roles of three ER subtypes in Japanese medaka (Oryzias latipes), using vitellogenin (VTG) I and II as model genes. We identify specific ligand/receptor/promoter dynamics, using transient transactivation assays that incorporate luciferase reporters comprising 3kb promoter/enhancer regions of medaka VTGI and VTGII genes. Four steroidal estrogens (17ß-estradiol, estrone, estriol, and 17α-estradiol) were tested in these assays. Results indicate that all three medaka ERs (mERs) are capable of initiating transactivation of both VTG I and II, with ERß2 exhibiting greatest activity. Promoter deletion analysis suggests that ligand-specific receptor transactivation and utilization of regional-specific estrogen response elements may be associated with differential activities of each medaka ER. Further, cluster analysis of in vivo gene expression and in vitro transactivation suggests that all three ER subtypes putatively play a role in up-regulation of VTG. Results illustrate that preferential ligand/receptor/promoter interactions may have direct implications for VTG gene expression and other ER-mediated regulatory functions that are relevant to the risk assessment of estrogenic compounds.

Phenotypic characterization of transgenic Japanese medaka (Oryzias latipes) that express a red fluorescent protein in hepatocytes.

Transgenic organisms that express fluorescent proteins are used frequently for in vivo visualization of proteins and cells. The phenotype of a transgenic medaka (Oryzias latipes) strain that expresses a red fluorescent protein (RFP) in hepatocytes was characterized using light and fluorescence microscopy, immunohistochemistry, and transmission electron microscopy (TEM). Expression of RFP was first detected by confocal fluorescence microscopy in the location of the liver bud of live medaka embryos at 60 hr postfertilization (developmental stage 27). Subsequently, RFP signal was observed exclusively in hepatocytes throughout life using fluorescence microscopy in live fish and immunohistochemistry in formalin-fixed, paraffin-embedded liver sections. As the fish aged, prominent intracytoplasmic eosinophilic inclusions immunoreactive for RFP were observed by light microscopy and were correlated with membrane-bound electron dense inclusions on TEM. These results define the onset and location of RFP expression in the Tg(zf.L-fabp:DsRed) medaka and characterize a histologic phenotype that results from RFP expression in hepatocytes.

Bridging the gap from screening assays to estrogenic effects in fish: potential roles of multiple estrogen receptor subtypes.

This study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hERα. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hERα and hERß, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ERß subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ERß subtypes are critically involved in the teleost estrogenic response, with the ERα:ERß ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures.

Transport of steroid hormones, phytoestrogens, and estrogenic activity across a swine lagoon/sprayfield system.

The inflow, transformation, and attenuation of natural steroid hormones and phytoestrogens and estrogenic activity were assessed across the lagoon/sprayfield system of a prototypical commercial swine sow operation. Free and conjugated steroid hormones (estrogens, androgens, and progesterone) were detected in urine and feces of sows across reproductive stages, with progesterone being the most abundant steroid hormone. Excreta also contained phytoestrogens indicative of a soy-based diet, particularly, daidzein, genistein, and equol. During storage in barn pits and the anaerobic lagoon, conjugated hormones dissipated, and androgens and progesterone were attenuated. Estrone and equol persisted along the waste disposal route. Following application of lagoon slurry to agricultural soils, all analytes exhibited attenuation within 2 days. However, analytes including estrone, androstenedione, progesterone, and equol remained detectable in soil at 2 months postapplication. Estrogenic activity in the yeast estrogen screen and T47D-KBluc in vitro bioassays generally tracked well with analyte concentrations. Estrone was found to be the greatest contributor to estrogenic activity across all sample types. This investigation encompasses the most comprehensive suite of natural hormone and phytoestrogen analytes examined to date across a livestock lagoon/sprayfield and provides global insight into the fate of these analytes in this widely used waste management system.

Developmental exposure to pesticides that disrupt retinoic acid signaling causes persistent retinoid and behavioral dysfunction in zebrafish.

Early developmental exposure to environmental toxicants may play a role in the risk for developing autism. A variety of pesticides have direct effects on retinoic acid (RA) signaling and as RA signaling has important roles in neurodevelopment, such compounds may cause developmental neurotoxicity through an overlapping adverse outcome pathway. It is hypothesized that a pesticide's embryonic effects on retinoid function may correspond with neurobehavioral disruption later in development. In the current studies, we determined the effects of RA-acting pesticides on neurobehavioral development in zebrafish. Buprofezin and imazalil caused generalized hypoactivity in the larval motility test, whereas chlorothalonil and endosulfan I led to selective hypoactivity and hyperactivity, respectively. With buprofezin, chlorothalonil, and imazalil, hypoactivity and/or novel anxiety-like behaviors persisted in adulthood and buprofezin additionally decreased social attraction responses in adulthood. Endosulfan I did not produce significant adult behavioral effects. Using qPCR analyses of adult brain tissue, we observed treatment-induced alterations in RA synthesis or catabolic genes, indicating persistent changes in RA homeostasis. These changes were compound-specific, with respect to expression directionality, and potential patterns of homeostatic disruption. Results suggest the likely persistence of disruptions in RA signaling well into adulthood and may represent compensatory mechanisms following early life stage exposures. This study demonstrates that early developmental exposure to environmental toxicants that interfere with RA signaling causes short as well as long-term behavioral disruption in a well-established zebrafish behavioral model and expand upon the meaning of the RA adverse outcome pathway, indicating that observed effects likely correspond with the nature of underlying homeostatic effects.

Anchoring hepatic gene expression with development of fibrosis and neoplasia in a toxicant-induced fish model of liver injury.

Fish have been used as laboratory models to study hepatic development and carcinogenesis but not for pathogenesis of hepatic fibrosis. In this study, a dimethylnitrosamine-induced fish model of hepatic injury was developed in Japanese medaka (Oryzias latipes) and gene expression was anchored with the development of hepatic fibrosis and neoplasia. Exposed livers exhibited mild hepatocellular degenerative changes 2 weeks' postexposure. Within 6 weeks, hepatic fibrosis/cirrhosis was evident with development of neoplasia by 10 weeks. Stellate cell activation and development of fibrosis was associated with upregulation of transforming growth factor beta 1 (tgfb1), tgfb receptor 2, mothers against decapentaplegic homolog 3 (smad3a), smad3b, beta-catenin (ctnnb1), myc, matrix metalloproteinase (mmp2), mmp14a, mmp14b, tissue inhibitors of metalloproteinase (timp) 2a, timp2b, timp3, collagen type I alpha 1a (col1a1a), and col1a1b and a less pronounced increase in mmp13 and col4a1 expression. Tgfb receptor I expression was unchanged. Immunohistochemistry suggested that biliary epithelial cells and stellate cells were the main producers of TGF-ß1. This study identified a group of candidate genes likely to be involved in the development of hepatic fibrosis and demonstrated that the TGF-ß pathway likely plays a major role in the pathogenesis. These results support the medaka as a viable fish model of hepatic fibrosis.

Comprehensive assessment of hormones, phytoestrogens, and estrogenic activity in an anaerobic swine waste lagoon.

In this study, the distribution of steroid hormones, phytoestrogens, and estrogenic activity was thoroughly characterized within the anaerobic waste lagoon of a typical commercial swine sow operation. Three independent rounds of sampling were conducted in June 2009, April 2010, and February 2011. Thirty-seven analytes in lagoon slurry and sludge were assessed using LC/MS-MS, and yeast estrogen screen was used to determine estrogenic activity. Of the hormone analytes, steroidal estrogens were more abundant than androgens or progesterone, with estrone being the predominant estrogen species. Conjugated hormones were detected only at low levels. The isoflavone metabolite equol was by far the predominant phytoestrogen species, with daidzein, genistein, formononetin, and coumestrol present at lower levels. Phytoestrogens were often more abundant than steroidal estrogens, but contributed minimally toward total estrogenic activity. Analytes were significantly elevated in the solid phases of the lagoon; although low observed log KOC values suggest enhanced solubility in the aqueous phase, perhaps due to dissolved or colloidal organic carbon. The association with the solid phase, as well as recalcitrance of analytes to anaerobic degradation, results in a markedly elevated load of analytes and estrogenic activity within lagoon sludge. Overall, findings emphasize the importance of adsorption and transformation processes in governing the fate of these compounds in lagoon waste, which is ultimately used for broadcast application as a fertilizer.

TCDD alters essential transcriptional regulators of osteogenic differentiation in multipotent mesenchymal stem cells.

Differentiation of multipotent mesenchymal stem cells (MSCs) into bone-forming osteoblasts requires strict coordination of transcriptional pathways. Aryl hydrocarbon receptor ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been shown to alter osteoblast differentiation in vitro and bone formation in multiple developmental in vivo models. The goal of the present study was to establish a global transcriptomic landscape during early, intermediate, and apical stages of osteogenic differentiation in vitro in response to TCDD exposure. Human bone-derived mesenchymal stem cells (hBMSCs) were cultured in growth media (GM), osteogenic differentiation media (ODM), or ODM containing 10 nM TCDD (ODM + TCDD), thus enabling a comparison of the transcriptomic profiles of undifferentiated, differentiated, and differentiated-TCDD-exposed hBMSCs, respectively. In this test system, exposure to TCDD attenuated the differentiation of hBMSCs into osteoblasts as evidenced by reduced alkaline phosphatase activity and mineralization. At various timepoints, we observed altered expression of genes that play a role in the Wnt, fibroblast growth factor, bone morphogenetic protein/transforming growth factor beta developmental pathways, as well as pathways related to extracellular matrix organization and deposition. Reconstruction of gene regulatory networks with the interactive dynamic regulatory event miner (iDREM) analysis revealed modulation of transcription factors (TFs) including POLR3G, NR4A1, RDBP, GTF2B, POU2F2, and ZEB1, which may putatively influence osteoblast differentiation and the requisite deposition and mineralization of bone extracellular matrix. We demonstrate that the combination of RNA-Seq data in conjunction with the iDREM regulatory model captures the transcriptional dynamics underlying MSC differentiation under different conditions in vitro. Model predictions are consistent with existing knowledge and provide a new tool to identify novel pathways and TFs that may facilitate a better understanding of the osteoblast differentiation process, perturbation by exogenous agents, and potential intervention strategies targeting those specific pathways.

Maximized Spatial Information and Minimized Acquisition Time of Top-Hat IR-MALDESI-MSI of Zebrafish Using Nested Regions of Interest (nROIs).

Increasing the spatial resolution of a mass spectrometry imaging (MSI) method results in a more defined heatmap of the spatial distribution of molecules across a sample, but it is also associated with the disadvantage of increased acquisition time. Decreasing the area of the region of interest to achieve shorter durations results in the loss of potentially valuable information in larger specimens. This work presents a novel MSI method to reduce the time of MSI data acquisition with variable step size imaging: nested regions of interest (nROIs). Using nROIs, a small ROI may be imaged at a higher spatial resolution while nested inside a lower-spatial-resolution peripheral ROI. This conserves the maximal spatial and chemical information generated from target regions while also decreasing the necessary acquisition time. In this work, the nROI method was characterized on mouse liver and applied to top-hat MSI of zebrafish using a novel optical train, which resulted in a significant improvement in both acquisition time and spatial detail of the zebrafish. The nROI method can be employed with any step size pairing and adapted to any method in which the acquisition time of larger high-resolution ROIs poses a practical challenge.

Legacy and emerging per- and polyfluoroalkyl substances suppress the neutrophil respiratory burst.

Per- and polyfluoroalkyl substances (PFASs) are used in a multitude of processes and products, including nonstick coatings, food wrappers, and fire-fighting foams. These chemicals are environmentally-persistent, ubiquitous, and can be detected in the serum of 98% of Americans. Despite evidence that PFASs alter adaptive immunity, few studies have investigated their effects on innate immunity. The report here presents results of studies that investigated the impact of nine environmentally-relevant PFASs [e.g. perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid potassium salt (PFOS-K), perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorohexane sulfonic acid (PFHxS), perfluorobutane sulfonic acid (PFBS), ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), 7H-perfluoro-4-methyl-3,6-dioxa-octane sulfonic acid (Nafion byproduct 2), and perfluoromethoxyacetic acid sodium salt (PFMOAA-Na)] on one component of the innate immune response, the neutrophil respiratory burst. The respiratory burst is a key innate immune process by which microbicidal reactive oxygen species (ROS) are rapidly induced by neutrophils in response to pathogens; defects in the respiratory burst can increase susceptibility to infection. The study here utilized larval zebrafish, a human neutrophil-like cell line, and primary human neutrophils to ascertain whether PFAS exposure inhibits ROS production in the respiratory burst. It was observed that exposure to PFHxA and GenX suppresses the respiratory burst in zebrafish larvae and a human neutrophil-like cell line. GenX also suppressed the respiratory burst in primary human neutrophils. This report is the first to demonstrate that these PFASs suppress neutrophil function and support the utility of employing zebrafish larvae and a human cell line as screening tools to identify chemicals that may suppress human immune function.

Confirmation of high-throughput screening data and novel mechanistic insights into FXR-xenobiotic interactions by orthogonal assays.

Toxicology in the 21st Century (Tox21) is a federal collaboration employing a high-throughput robotic screening system to test 10,000 environmental chemicals. One of the primary goals of the program is prioritizing toxicity evaluations through in vitro high-throughput screening (HTS) assays for large numbers of chemicals already in commercial use for which little or no toxicity data is available. Within the Tox21 screening program, disruption in nuclear receptor (NR) signaling represents a particular area of interest. Given the role of NR's in modulating a wide range of biological processes, alterations of their activity can have profound biological impacts. Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that has demonstrated importance in bile acid homeostasis, glucose metabolism, lipid homeostasis and hepatic regeneration. In this study, we re-evaluated 24 FXR agonists and antagonists identified through Tox21 using select orthogonal assays. In transient transactivation assays, 7/8 putative agonists and 4/4 putative inactive compounds were confirmed. Likewise, we confirmed 9/12 antagonists tested. Using a mammalian two hybrid approach we demonstrate that both FXR agonists and antagonists facilitate FXRα-coregulator interactions suggesting that differential coregulator recruitment may mediate activation/repression of FXRα mediated transcription. Additionally, we tested the ability of select FXR agonists and antagonists to facilitate hepatic transcription of FXR gene targets Shp and Bsep in a teleost (Medaka) model. Through application of in vitro cell-based assays, in silico modeling and in vivo gene expressions, we demonstrated the molecular complexity of FXR:ligand interactions and confirmed the ability of diverse ligands to modulate FXRα, facilitate differential coregulator recruitment and activate/repress receptor-mediated transcription. Overall, we suggest a multiplicative approach to assessment of nuclear receptor function may facilitate a greater understanding of the biological and mechanistic complexities of nuclear receptor activities and further our ability to interpret broad HTS outcomes.

Correction: Kassotis et al. Nonylphenol Polyethoxylates Enhance Adipose Deposition in Developmentally Exposed Zebrafish. Toxics 2022, 10, 99.

In the original publication [...].

Nonylphenol Polyethoxylates Enhance Adipose Deposition in Developmentally Exposed Zebrafish.

Alkylphenol polyethoxylates (APEOs), such as nonylphenol ethoxylates (NPEOs), are high-production-volume surfactants used in laundry detergents, hard-surface cleaners, pesticide formulations, textile production, oils, paints, and other products. NPEOs comprise -80% of the total production of APEOs and are widely reported across diverse environmental matrices. Despite a growing push for replacement products, APEOs continue to be released into the environment through wastewater at significant levels. Research into related nonionic surfactants from varying sources has reported metabolic health impacts, and we have previously demonstrated that diverse APEOs and alcohol polyethoxylates promote adipogenesis in the murine 3T3-L1 pre-adipocyte model. These effects appeared to be independent of the base alkylphenol and related to the ethoxylate chain length, though limited research has evaluated NPEO exposures in animal models. The goals of this study were to assess the potential of NPEOs to promote adiposity (Nile red fluorescence quantification) and alter growth and/or development (toxicity, length, weight, and energy expenditure) of developmentally exposed zebrafish (Danio rerio). We also sought to expand our understanding of the ability to promote adiposity through evaluation in human mesenchymal stem cells. Herein, we demonstrated consistent adipogenic effects in two separate human bone-marrow-derived mesenchymal stem cell models, and that nonylphenol and its ethoxylates promoted weight gain and increased adipose deposition in developmentally exposed zebrafish. Notably, across both cell and zebrafish models we report increasing adipogenic/obesogenic activity with increasing ethoxylate chain lengths up to maximums around NPEO-6 and then decreasing activity with the longest ethoxylate chain lengths. This research suggests metabolic health concerns for these common obesogens, suggesting further need to assess molecular mechanisms and better characterize environmental concentrations for human health risk assessments.

The evolution of farnesoid X, vitamin D, and pregnane X receptors: insights from the green-spotted pufferfish (Tetraodon nigriviridis) and other non-mammalian species.

BACKGROUND: The farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR) are three closely related nuclear hormone receptors in the NR1H and 1I subfamilies that share the property of being activated by bile salts. Bile salts vary significantly in structure across vertebrate species, suggesting that receptors binding these molecules may show adaptive evolutionary changes in response. We have previously shown that FXRs from the sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) are activated by planar bile alcohols found in these two species. In this report, we characterize FXR, PXR, and VDR from the green-spotted pufferfish (Tetraodon nigriviridis), an actinopterygian fish that unlike the zebrafish has a bile salt profile similar to humans. We utilize homology modelling, docking, and pharmacophore studies to understand the structural features of the Tetraodon receptors. RESULTS: Tetraodon FXR has a ligand selectivity profile very similar to human FXR, with strong activation by the synthetic ligand GW4064 and by the primary bile acid chenodeoxycholic acid. Homology modelling and docking studies suggest a ligand-binding pocket architecture more similar to human and rat FXRs than to lamprey or zebrafish FXRs. Tetraodon PXR was activated by a variety of bile acids and steroids, although not by the larger synthetic ligands that activate human PXR such as rifampicin. Homology modelling predicts a larger ligand-binding cavity than zebrafish PXR. We also demonstrate that VDRs from the pufferfish and Japanese medaka were activated by small secondary bile acids such as lithocholic acid, whereas the African clawed frog VDR was not. CONCLUSIONS: Our studies provide further evidence of the relationship between both FXR, PXR, and VDR ligand selectivity and cross-species variation in bile salt profiles. Zebrafish and green-spotted pufferfish provide a clear contrast in having markedly different primary bile salt profiles (planar bile alcohols for zebrafish and sterically bent bile acids for the pufferfish) and receptor selectivity that matches these differences in endogenous ligands. Our observations to date present an integrated picture of the co-evolution of bile salt structure and changes in the binding pockets of three nuclear hormone receptors across the species studied.

Exposure to the synthetic FXR agonist GW4064 causes alterations in gene expression and sublethal hepatotoxicity in eleutheroembryo medaka (Oryzias latipes).

The small freshwater teleost, medaka (Oryzias latipes), has a history of usage in studies of chronic toxicity of liver and biliary system. Recent progress with this model has focused on defining the medaka hepatobiliary system. Here we investigate critical liver function and toxicity by examining the in vivo role and function of the farnesoid X receptor alpha (FXRalpha, NR1H4), a member of the nuclear receptor superfamily that plays an essential role in the regulation of bile acid homeostasis. Quantitative mRNA analysis of medaka FXRalpha demonstrates differential expression of two FXRalpha isoforms designated Fxralpha1 and Fxralpha2, in both free swimming medaka embryos with remaining yolk (eleutheroembryos, EEs) and adults. Activation of medaka Fxralpha in vivo with GW4064 (a strong FXRalpha agonist) resulted in modification of gene expression for defined FXRalpha gene targets including the bile salt export protein, small heterodimer partner, and cytochrome P450 7A1. Histological examination of medaka liver subsequent to GW4064 exposure demonstrated significant lipid accumulation, cellular and organelle alterations in both hepatocytes and biliary epithelial cells of the liver. This report of hepatobiliary injury following GW4064 exposure extends previous investigations of the intrahepatic biliary system in medaka, reveals sensitivity to toxicant exposure, and illustrates the need for added resolution in detection and interpretation of toxic responses in this vertebrate.

Developmental exposure of zebrafish to vitamin D receptor acting drugs and environmental toxicants disrupts behavioral function.

Vitamin D receptor (VDR) signaling is important for optimal neurobehavioral development. Disruption of VDR signaling by environmental toxicants during early development might contribute to the etiology of behavioral dysfunction. In the current set of studies, we examined ten compounds known to affect VDR function in vitro for neurobehavioral effects in vivo in zebrafish. Zebrafish embryos were exposed to concentrations of the compounds in their water during the first 5 days post-fertilization. On day 5, the embryos were tested in an alternating light-dark locomotor assay using a computerized video tracking system. We found that most of the compounds produced significant changes in locomotor behavior in exposed zebrafish larvae, although the direction of the effect (i.e., hypo- or hyperactivity) and the sensitivity of the effect to changes in illumination condition varied across the compounds. The nature of the behavioral effects generally corresponded to the effects these compounds have been shown to exert on VDR. These studies lay a foundation for further investigation to determine whether behavioral dysfunction persists into adulthood and if so which behavioral functions are affected. Zebrafish can be useful for screening compounds identified in high throughput in vitro assays to provide an initial test for how those compounds would affect construction and behavioral function of a complex nervous system, helping to bridge the gap between in vitro neurotoxicity assays and mammalian models for risk assessment in humans.