BACKGROUND AND PURPOSE: Functional brain imaging using genetically encoded Ca2+ sensors in larval zebrafish is being developed for studying seizures and epilepsy as a more ethical alternative to rodent models. Despite this, few data have been generated on pharmacological mechanisms of action other than GABAA antagonism. Assessing larval responsiveness across multiple mechanisms is vital to test the translational power of this approach, as well as assessing its validity for detecting unwanted drug-induced seizures and testing antiepileptic drug efficacy. EXPERIMENTAL APPROACH: Using light-sheet imaging, we systematically analysed the responsiveness of 4 days post fertilisation (dpf; which are not considered protected under European animal experiment legislation) transgenic larval zebrafish to treatment with 57 compounds spanning more than 12 drug classes with a link to seizure generation in mammals, alongside eight compounds with no such link. KEY RESULTS: We show 4dpf zebrafish are responsive to a wide range of mechanisms implicated in seizure generation, with cerebellar circuitry activated regardless of the initiating pharmacology. Analysis of functional connectivity revealed compounds targeting cholinergic and monoaminergic reuptake, in particular, showed phenotypic consistency broadly mapping onto what is known about neurotransmitter-specific circuitry in the larval zebrafish brain. Many seizure-associated compounds also exhibited altered whole brain functional connectivity compared with controls. CONCLUSIONS AND IMPLICATIONS: This work represents a significant step forward in understanding the translational power of 4dpf larval zebrafish for use in neuropharmacological studies and for studying the events driving transition from small-scale pharmacological activation of local circuits, to the large network-wide abnormal synchronous activity associated with seizures.

Several pharmaceutical and chemical companies are using the zebrafish embryo as an alternative to animal testing for early detection of developmental toxicants. Unfortunately, the protocol of this zebrafish embryo assay varies between labs, resulting in discordant data for identical compounds. The assay also has some limitations, such as low biotransformation capacity and fewer morphological endpoints in comparison with the in vivo mammalian developmental toxicity studies. Consequently, there is a need to standardize and further optimize the assay for developmental toxicity testing. We developed a Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) that can be extended with a metabolic activation system and/or skeletal staining to increase its sensitivity. As such, the ZEDTA can be used as a modular system depending on the compound of interest. • Our protocol is customized with a metabolic activation system for test compounds, using human liver microsomes. This system ensures exposure of zebrafish embryos to metabolites that are relevant for human risk and safety assessment. As human liver microsomes are toxic for the zebrafish embryo, we developed a preincubation system with an ultracentrifugation and subsequent dilution step. • Additionally, we developed a skeletal staining protocol that can be added to the ZEDTA modular system. Our live Alizarin Red staining method detects several bone structures in 5-day old zebrafish larvae in a consistent manner.

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The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e. benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e. 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and sulfotransferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter, i.e. abcb4, was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e. 120 hpf), although CYP1-except CYP1A-and SULT1ST1 were shown to be already mature in early embryonic development.

Functional neuroimaging, using genetically-encoded Ca2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.

There has been increasing focus on generation and assessment of in vitro developmental toxicology models for assessing teratogenic liability of chemicals. The driver for this focus has been to find reliable in vitro assays that will reduce or replace the use of in vivo tests for assessing teratogenicity. Such efforts may be eventually applied in testing pharmaceutical agents where a developmental toxicology assay or battery of assays may be incorporated into regulatory testing to replace one of the two species currently used in teratogenic assessment. Such assays may be eventually applied in testing a broader spectrum of chemicals, supporting efforts aligned with Tox21 strategies and responding to REACH legislation. This review describes the developmental toxicology assays that are of focus in these assessments: rodent whole embryo culture, zebrafish embryo assays, and embryonic stem cell assays. Progress on assay development as well as future directions of how these assays are envisioned to be applied for broader safety testing of chemicals are discussed. Altogether, the developmental model systems described in this review provide rich biological systems that can be utilized in better understanding teratogenic mechanisms of action of chemotypes and are promising in providing proactive safety assessment related to developmental toxicity. Continual advancements in refining/optimizing these in vitro assays are anticipated to provide a robust data set to provide thoughtful assessment of how whole animal teratogenicity evaluations can be reduced/refined in the future.

INTRODUCTION: Despite effective in vitro preclinical strategies to identify cardiovascular (CV) liabilities, there remains a need for early functional assessment prior to complex in vivo mammalian models. The larval zebrafish (Danio rerio, Zf) has been suggested for this role: previous data suggest that cardiac electrophysiology and vascular ultrastructure are comparable with mammals, and also indicate responsiveness of individual Zf CV system endpoints to some functional modulators. Little information is, however, available regarding integrated functional CV responses to drug treatment. Consequently, we developed a novel larval Zf model capable of simultaneous quantification of chronotropic, inotropic and arrhythmic effects, alongside measures of blood flow and vessel diameter. METHODS: Non-invasive video analysis of the heart and dorsal aorta of anaesthetized and agarose-embedded larval ZF was used to measure multiple cardiovascular endpoints, simultaneously, following treatment with a range of functional modulators of CV physiology. RESULTS: Changes in atrial and ventricular beat frequencies were detected in response to acute treatment with cardio-stimulants (adrenaline and theophylline), and negative chrono/inotropes (cisapride, haloperidol, terfenadine and verapamil). Arrhythmias were also observed including terfenadine-induced 2:1 atrial-ventricular (A-V) block, a previously proposed hERG surrogate measure. Significant increases in blood flow were detected in response to adrenaline and theophylline exposure; and decreases after cisapride, haloperidol, terfenadine, and verapamil treatment. Using dorsal aorta (DA) blood flow and ventricular beat rate, surrogate stoke volumes were also calculated for all compounds. DISCUSSION: These data support the use of this approach for CV function studies. Moreover the throughput and compound requirements (approximately 3 compounds/person effort/week and

Clotrimazole (CTZ) is a persistent imidazole antifungal agent which is frequently detected in the aquatic environment and predicted to bio-concentrate in fish. Common carp (Cyprinus carpio) were exposed to mean measured concentrations of either 1.02 or 14.63μgl(-1) CTZ for 4 and 10 days, followed by a depuration period of 4 days in a further group of animals. Following each exposure regimen, plasma and liver CTZ concentrations were measured. Mean measured plasma concentrations of CTZ in animals exposed to the lower concentration of CTZ were 30 and 44μgl(-1) on days 4 and 10, respectively, and in the higher concentration were 318 and 336μgl(-1). Mean measured liver levels in the same animals were 514, 1725, 2111 and 7017μgl(-1) suggesting progressive hepatic accumulation. Measurement of CTZ in plasma after depuration suggested efficient elimination within 4 days, but appreciable levels of CTZ remained in the liver after depuration suggesting a degree of persistence in this tissue. In addition we measured responses of a number of key hepatic detoxification gene targets in the liver associated with the transcription factor pregnane X receptor (PXR); namely cyp450s 2k and 3a, glutathione-S-transferases a and p (gsta and p), and drug transporters multidrug resistance protein1 (mdr1), and MDR-related protein2 (mrp2). CTZ is a potent ligand of the PXR in humans and there is some evidence of PXR activation following exposure to CTZ in fish. The highest concentration of CTZ was adopted to explore the potential for alterations to detoxification gene expression in fish at a pharmacologically relevant dose level, and the lower concentration is within the range reported in effluents from waste water treatment works (WWTW). The genes for all biotransformation enzymes were up-regulated after exposure to the higher concentration of CTZ for 10 days, and alterations in expression occurred for the drug transporter genes mdr1 and mrp2 following exposure to the lower concentration of 1.02μgl(-1) CTZ (mean measured concentration). These data support the potential for CTZ to induce alterations in biotransformation and drug transporter genes associated with PXR in fish at concentrations measured in some WWTW effluents.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L. Stedman, D. B. Ball, J. Hillegass, J. M. Flood, A. Zhang, C. X. Panzica-Kelly, J. Cao, J. Coburn, A. Enright, B. P. et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100μM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000μM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies. © the Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

Pollution is a significant environmental pressure on fish populations in both freshwater and marine environments. Populations subjected to chronic exposure to pollutants can experience impacts ranging from altered reproductive capacity to changes in population genetic structure. Few studies, however, have examined the reproductive vigor of individuals within populations inhabiting environments characterized by chronic pollution. In this study we undertook an analysis of populations of three-spined sticklebacks (Gasterosteus aculeatus) from polluted sites, to determine levels of genetic diversity, assess for evidence of historic population genetic bottlenecks and determine the reproductive competitiveness of males from these locations. The sites chosen included locations in the River Aire, the River Tees and the River Birket, English rivers that have been impacted by pollution from industrial and/or domestic effluents for over 100 years. Male reproductive competitiveness was determined via competitive breeding experiments with males and females derived from a clean water site, employing DNA microsatellites to determine parentage outcome. Populations of stickleback collected from the three historically polluted sites showed evidence of recent population bottlenecks, although only the River Aire population showed low genetic diversity. In contrast, fish collected from two relatively unpolluted sites within the River Gowy and Houghton Springs showed weak, or no evidence of such bottlenecks. Nevertheless, males derived from polluted sites were able to reproduce successfully in competition with males derived from clean water exposures, indicating that these bottlenecks have not resulted in any substantial loss of reproductive fitness in males. © 2013 Elsevier B.V.

This report provides a progress update of a consortium effort to develop a harmonized zebrafish developmental toxicity assay. Twenty non-proprietary compounds (10 animal teratogens and 10 animal non-teratogens) were evaluated blinded in 4 laboratories. Zebrafish embryos from pond-derived and cultivated strain wild types were exposed to the test compounds for 5 days and subsequently evaluated for lethality and morphological changes. Each of the testing laboratories achieved similar overall concordance to the animal data (60-70%). Subsequent optimization procedures to improve the overall concordance focused on compound formulation and test concentration adjustments, chorion permeation and number of replicates. These optimized procedures were integrated into a revised protocol and all compounds were retested in one lab using embryos from pond-derived zebrafish and achieved 85% total concordance. To further assess assay performance, a study of additional compounds is currently in progress at two laboratories using embryos from pond-derived and cultivated-strain wild type zebrafish.

An established three-spined stickleback (Gasterosteus aculeatus) cDNA array was expanded to 14,496 probes with the addition of hepatic clones derived from subtractive and normalized libraries from control males and males exposed to model toxicants. Microarrays and one-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy, together with individual protein and gene biomarkers were employed to investigate the hepatic responses of the stickleback to ethinyl-estradiol (EE(2)) exposure. Male fish were exposed via the water to EE(2), including environmentally relevant concentrations (0.1-100ng/l) for 4 days, and hepatic transcript and metabolite profiles, kidney spiggin protein and serum vitellogenin concentrations were determined in comparison to controls. EE(2) exposure did not significantly affect spiggin concentration but significantly induced serum vitellogenin protein at the threshold concentration of 32ng/l. (1)H NMR coupled with robust univariate testing revealed only limited changes, but these did support the predicted modulation of the amino acid profile by transcriptomics. Transcriptional induction was found for hepatic vitellogenins and choriogenins as expected, together with a range of other EE(2)-responsive genes. Choriogenins showed the more sensitive responses with statistically significant induction at 10ng/l. Real-time polymerase chain reaction (PCR) confirmed transcriptional induction of these genes. Phosvitinless vitellogenin C transcripts were highly expressed and represent a major form of the egg yolk precursors, and this is in contrast to other fish species where it is a minor component of vitellogenic transcripts. Differences in inducibility between the vitellogenins and choriogenins appear to be in accordance with the sequential formation of chorion and yolk during oogenesis in fish.

Copper (Cu) is a micronutrient essential for the biochemical functioning of numerous processes in vertebrates but is also often present in the aquatic environment at concentrations able to cause adverse health effects in aquatic organisms. This study investigated the signaling pathways mediating the effects of exposure to Cu using a toxicogenomic approach in a fish model, the stickleback ( Gasterosteus aculeatus ). Freshwater-acclimated male fish were exposed via the water to Cu, including at environmentally relevant concentrations (3.2-128 microg of Cu/L for 4 days), and the biological responses explored through analyses of the hepatic transcriptome and metabolome and phenotypic end points, including assessment of DNA damage in blood cells. The Cu exposures resulted in DNA strand breaks in blood cells at all exposure concentrations and alterations in hepatic gene expression and metabolite concentrations in a concentration-dependent manner (from 10 microg of Cu/L). Genes associated with the cholesterol biosynthesis pathway were significantly over-represented and consistently down-regulated (at 128 microg of Cu/L), similar to that occurring in a mouse model for Wilson's disease. Additionally, inductions in metallothionein and catalase were also observed. The concentrations of NAD(+) and lactate increased significantly with the Cu exposure, consistent with a shift toward anaerobic metabolism, and these aligned closely with changes observed in gene expression. The pathways of Cu toxicity identified in our study support the conserved mechanisms of Cu toxicity from lower vertebrates to mammals, provide novel insights into the deleterious effects of Cu in fish, and further demonstrate the utility of fish as environmental sentinels for chemical impacts on both environmental and human health.

A three-spined stickleback (Gasterosteus aculeatus) cDNA array and one-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy-based metabolomics approach, together with individual biomarkers,were employed to investigate the responses of male sticklebacks to polycyclic aromatic hydrocarbon exposure. Fish were exposed to 1,2:5,6-dibenzanthracene (DbA) at concentrations between 0.01 and 50 microg per liter dissolved in the ambient water for four days, and hepatic transcript and metabolite profiles were determined in comparison with those of solvent-exposed controls. Induction of gene expression was apparent for cytochrome P450 1A (CYP1A) and CYP2-family monooxygenases and these responses were strongly correlated with DbA exposure concentrations (for CYP1A r > 0.996). Expression of suites of genes related to bile acid biosynthesis, steroid metabolism, and endocrine function were also affected, as demonstrated by gene ontology analyses. Expression changes in selected genes were confirmed by real-time PCR. Metabolomics highlighted notable changes in concentrations of taurine, malonate, glutamate, and alanine. These statistically significant responses to environmentally relevant concentrations of DbA at the transcriptomic and metabolomic levels provided sensitive markers characteristic of environmentally relevant low-level DbA exposure. Metabolic pathways were identified where both gene expression and metabolite concentrations were altered in response to DbA.