AbstractThe clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.

AbstractThe extrapolation of biological data across species is a key aspect of biomedical research and drug development. In this context, comparative biology considerations are applied with the goal of understanding human disease and guiding the development of effective and safe medicines. However, the widespread occurrence of pharmaceuticals in the environment and the need to assess the risk posed to wildlife have prompted a renewed interest in the extrapolation of pharmacological and toxicological data across the entire tree of life. To address this challenge, a biological “read‐across” approach, based on the use of mammalian data to inform toxicity predictions in wildlife species, has been proposed as an effective way to streamline the environmental safety assessment of pharmaceuticals. Yet, how effective has this approach been, and are we any closer to being able to accurately predict environmental risk based on known human risk? We discuss the main theoretical and experimental advancements achieved in the last 10 years of research in this field. We propose that a better understanding of the functional conservation of drug targets across species and of the quantitative relationship between target modulation and adverse effects should be considered as future research priorities. This pharmacodynamic focus should be complemented with the application of higher‐throughput experimental and computational approaches to accelerate the prediction of internal exposure dynamics. The translation of comparative (eco)toxicology research into real‐world applications, however, relies on the (limited) availability of experts with the skill set needed to navigate the complexity of the problem; hence, we also call for synergistic multistakeholder efforts to support and strengthen comparative toxicology research and education at a global level. Environ Toxicol Chem 2023;00:1–13. © 2023 the Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10-100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40-80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures.

Synthetic glucocorticoids have been widely used for more than 70 years to treat a wide range of diseases and human health issues including asthma, rheumatoid arthritis, and allergies, primarily due to their immunosuppressive properties. Due to the excretion of these compounds by patients, over the past 20 years glucocorticoids have been regularly detected in aquatic environments at low ng/L concentrations. The glucocorticoid system is well conserved across vertebrates, acting through genomic and non-genomic pathways, and is essential to a number of physiological processes including glucose homeostasis, immunomodulation, early development including bone formation and stress-related behaviours. Synthetic glucocorticoids have been shown to be pharmacologically active in non-mammalian vertebrates, including fish, and thus, have the potential to affect fish in the aquatic environment. By using zebrafish (Danio rerio) as a model species, in this thesis, I was able to assess the potential for a wide range of glucocorticoids to affect fish at concentrations ranging from environmentally relevant concentrations to ≥ human therapeutic dose concentrations. In order to complete this, I undertook uptake and bioconcentration studies to understand how much of these compounds were able to accumulate in fish tissues, as well as exploring the effects on early development, teratogenicity, immune function, and bone and cartilage formation in early life exposures to embryo-larval zebrafish.
First, 4-day fish embryo-larval toxicity exposures were completed on Wild Indian Karyotype zebrafish to 4 synthetic glucocorticoids individually (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone). Throughout these exposures, embryo-larvae were assessed for effects on early-development and teratogenicity, including presence of abnormalities, assessment of developing morphological tissues and cartilage development. Exposures to glucocorticoids revealed that concentrations similar to human therapeutic dose concentrations were able to induce developmental abnormalities including pericardial oedema, alter development and tissue morphology including the shortening of a variety of jaw cartilage measures in zebrafish embryo-larvae in a number of the compounds tested, however environmentally relevant concentrations had no effect. In additional 4-day exposures, uptake of these compounds into the whole-body tissue of embryo-larvae was measured and bioconcentration was calculated. Uptake and bioconcentration factors were calculated for 3 out of the 4 compounds. These data were then compared to predicted uptake values calculated using the Fish Plasma Model and the effectiveness of this model to accurately predict uptake of these compounds was assessed. The ability of the Fish Plasma Model to predict uptake varied depending on compound, however, it generally overestimated uptake rates in embryo-larvae.
To build upon the effects seen on cartilage development after 4 days of exposure, 10-day exposures to glucocorticoids were completed in embryo-larvae using a transgenic model with fluorescent markers labelling osteoblasts (bone) and chondrocytes (cartilage). After 10 days of exposure to glucocorticoids, both bone and cartilage development was altered for all compounds tested at ≥ human therapeutic dose concentrations. For exposures to beclomethasone dipropionate, cartilage lengths in the lower jaw (intercranial distance) were reduced after exposure to 0.1 μg/L, a concentration considered environmentally relevant. It was also found that at high concentrations (≥10 μg/L), the stacking of the chondrocyte cells in these cartilaginous structures was altered and the shape of these cells was also affected. Analysis of the osteoblasts showed no change in the organisation or shape of these cells, however the total areas of these osteoblasts were reduced at these high concentrations.
As glucocorticoids are primarily used due to their immunosuppressive properties, it is likely that these compounds may also be able to induce these effects in fish. Using two transgenic zebrafish models with fluorescent markers labelling neutrophil and macrophage cells, the effects of a 4-day glucocorticoid exposure on the ability of these innate immune cells to migrate to damage sites was investigated. By exposing these transgenic zebrafish models to neomycin, which results in damage to the hair cells of the lateral line (neuromasts), it was possible to track and count the numbers of the innate immune cells migrating towards damaged neuromasts. All compounds tested were found to alter the migration of both neutrophils and macrophages, although exposure concentrations at which this occurred were not considered environmentally relevant. It was shown that in general, the motility of these cells was unchanged after exposure to glucocorticoids. Therefore, it is suggested that alterations in the ability of neutrophils and macrophages to sense chemoattractants/chemokines and extravasate from the circulatory system may be possible mechanisms behind the immunosuppressive activity of these compounds.
Collectively, my findings demonstrate that glucocorticoids can induce effects in embryo-larval zebrafish including on development, immune function, and cartilage and bone formation generally at concentrations greater than considered environmentally relevant. Therefore, for the most part, it is suggested that glucocorticoids present a low-level risk to fish populations. However, in this thesis, chronic studies (10-days) were able reveal effects at environmentally relevant concentrations for beclomethasone dipropionate exposures, therefore suggesting that lifetime chronic exposures to these compounds in the environment may pose a greater risk to fish species. Considering the total glucocorticoid concentration found in the environment, and in combination with mixtures of other pollutants, further chronic studies, possibly over multiple generations, should be explored to evaluate the risk of these compounds to fish, thus ensuring that this class of compound does not pose a significant threat to fish in the environment.

The glucocorticosteroid, or glucocorticoid (GC), system is largely conserved across vertebrates and plays a central role in numerous vital physiological processes including bone development, immunomodulation, and modification of glucose metabolism and the induction of stress-related behaviours. As a result of their wide-ranging actions, synthetic GCs are widely prescribed for numerous human and veterinary therapeutic purposes and consequently have been detected extensively within the aquatic environment. Synthetic GCs designed for humans are pharmacologically active in non-mammalian vertebrates, including fish, however they are generally detected in surface waters at low (ng/L) concentrations. In this review, we assess the potential environmental risk of synthetic GCs to fish by comparing available experimental data and effect levels in fish with those in mammals. We found the majority of compounds were predicted to have insignificant risk to fish, however some compounds were predicted to be of moderate and high risk to fish, although the dataset of compounds used for this analysis was small. Given the common mode of action and high level of inter-species target conservation exhibited amongst the GCs, we also give due consideration to the potential for mixture effects, which may be particularly significant when considering the potential for environmental impact from this class of pharmaceuticals. Finally, we also provide recommendations for further research to more fully understand the potential environmental impact of this relatively understudied group of commonly prescribed human and veterinary drugs.

We assessed similarities and differences in the electrographic signatures of local field potentials (LFPs) evoked by different pharmacological agents in zebrafish larvae. We then compared and contrasted these characteristics with what is known from electrophysiological studies of seizures and epilepsy in mammals, including humans. Ultimately, our aim was to phenotype neurophysiological features of drug-induced seizures in larval zebrafish for expanding knowledge on the translational potential of this valuable alternative to mammalian models. LFPs were recorded from the midbrain of 4-d-old zebrafish larvae exposed to a pharmacologically diverse panel of seizurogenic compounds, and the outputs of these recordings were assessed using frequency domain analysis. This included analysis of changes occurring within various spectral frequency bands of relevance to mammalian CNS circuit pathophysiology. From these analyses, there were clear differences in the frequency spectra of drug-exposed LFPs, relative to controls, many of which shared notable similarities with the signatures exhibited by mammalian CNS circuits. These similarities included the presence of specific frequency components comparable to those observed in mammalian studies of seizures and epilepsy. Collectively, the data presented provide important information to support the value of larval zebrafish as an alternative model for the study of seizures and epilepsy. These data also provide further insight into the electrophysiological characteristics of seizures generated in nonmammalian species by the action of neuroactive drugs.

Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

Globally, antidepressant drugs are one of the most commonly prescribed classes of pharmaceuticals used for the treatment of psychiatric conditions, such as anxiety and depression. As a result, antidepressants have been widely detected in the aquatic environment, albeit at relatively low concentrations. Antidepressants act via modulation of brain monoaminergic signalling systems (predominantly serotonergic, adrenergic, dopaminergic), which show a high degree of structural conservation across diverse animal phyla. As a consequence, non-target organisms in the aquatic environment may be at risk from the effects of exposure. In this thesis, using the zebrafish (Danio rerio), l investigated the bioconcentration potential of a range of antidepressant drugs in fish tissues (spanning three major therapeutic classes), and assessed their subsequent effects on features of physiology, behaviour and neuronal activity. A wide range of exposure concentrations were employed, to elucidate the potential risk to fish in the wild, including those of environmental relevance.
In the first instance, acute 5-day exposures of zebrafish embryo-larvae to 9 antidepressant drugs (in isolation) were carried out, before subsequently assessing their effects on locomotor behaviours, including thigmotaxis, which is used as a measure of anxiogenesis, or anxiolysis. As part of this assessment, uptake was measured in whole body tissues and the data were compared with internal concentrations predicted using the Fish Plasma Model (FPM). All compounds tested were found to bioconcentrate in fish tissues to varying degrees. The reliability of the FPM to accurately predict uptake based upon compound lipophilicity was found to vary for the different test compounds. The behavioural assessments revealed that exposure to antidepressants at concentrations several orders of magnitude above those of environmental relevance, induced hypolocomotion, most notably during periods of darkness. There was little evidence of antidepressant-induced anxiolytic behaviours at any of the concentrations tested.
In the next phase of work, a chronic sublethal exposure to the tricyclic antidepressant amitriptyline was undertaken to assess the effects on zebrafish physiology and behaviour at early life stages. The phenotypic effects assessed were those linked to established therapeutic effects in humans i.e. changes in behaviour and specific monoamine pathways. Amitriptyline was found to bioconcentrate in both the whole body and brain tissues of 28-day old zebrafish, but was readily transformed to its major active metabolite nortriptyline. Comparisons of the water and internal tissue concentrations (bioconcentration factors) revealed that in the aquatic environment, amitriptyline is unlikely to reach levels in fish tissues that would be expected to induce therapeutic effects (based upon the effective doses reported in humans). Despite this, at these environmentally relevant concentrations, the relative expression of the serotonin transporter slc6a4a was found to be downregulated, suggesting pharmacological activity. Drug exposure at concentrations above those of environmental relevance were found to accelerate hatch rate and induce hypolocomotive behaviours. Following a period of depuration in clean water, drug-induced behavioural phenotypes were recovered, supporting a drug-specific effect.
Using a transgenic zebrafish with a genetically-encoded pan-neuronal Ca2+ indicator in combination with light sheet microscopy, the effects of aqueous antidepressant exposure on zebrafish larvae brain activity was investigated. All drugs tested were found to alter the neural activity and functional connectivity between distinct anatomical regions in the larval zebrafish brain, further supporting pharmacological activity for acute exposures, albeit at concentrations higher than those detected in the environment. The patterns of activity (i.e. which region exhibited increased or decreased activity versus the control) were distinct for each compound, although some commonalities in the brain regions being (de)activated by drug treatment were evident within therapeutic classes. Furthermore, most of the antidepressants tested were found to modify the neural response of larvae to the introduction of a ‘stressor’ stimulus (abrupt light flashes). By gaining a greater understanding of which neural circuits are influenced by exposure to CNS-active drugs, this may help in directing the development of more targeted behavioural tests.
The findings in this thesis collectively suggest that antidepressant-induced effects on zebrafish physiology, behaviour and neuronal activity were clearly evident only at concentrations above those detected in the aquatic environment supporting the notion that they present a low-level risk to fish populations. Despite this, given factors including their pseudopersistance, the influence of mixtures, food-chain transfer, and the potential for transgenerational inheritance, combined with the continual global rise in prescription rates, the potential risk level may rise in the future.

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.

Antidepressants are one of the most commonly prescribed pharmaceutical classes for the treatment of psychiatric conditions. They act via modulation of brain monoaminergic signaling systems (predominantly serotonergic, adrenergic, dopaminergic) that show a high degree of structural conservation across diverse animal phyla. A reasonable assumption, therefore, is that exposed fish and other aquatic wildlife may be affected by antidepressants released into the natural environment. Indeed, there are substantial data reported for exposure effects in fish, albeit most are reported for exposure concentrations exceeding those occurring in natural environments. From a critical analysis of the available evidence for effects in fish, risk quotients (RQs) were derived from laboratory-based studies for a selection of antidepressants most commonly detected in the aquatic environment. We conclude that the likelihood for effects in fish on standard measured end points used in risk assessment (i.e. excluding effects on behavior) is low for levels of exposure occurring in the natural environment. Nevertheless, some effects on behavior have been reported for environmentally relevant exposures, and antidepressants can bioaccumulate in fish tissues. Limitations in the datasets used to calculate RQs revealed important gaps in which future research should be directed to more accurately assess the risks posed by antidepressants to fish. Developing greater certainty surrounding risk of antidepressants to fish requires more attention directed toward effects on behaviors relating to individual fitness, the employment of environmentally realistic exposure levels, on chronic exposure scenarios, and on mixtures analyses, especially given the wide range of similarly acting compounds released into the environment.

Aim: Aggression is a behavioural trait characterized by the intention to harm others for offensive or defensive purposes. Neurotransmitters such as serotonin and dopamine are important mediators of aggression. However, the physiological role of the histaminergic system during this behaviour is currently unclear. Here, we aimed to better understand histaminergic signalling during aggression by characterizing the involvement of the histamine H3 receptor (Hrh3). Methods: We have generated a novel zebrafish Hrh3 null mutant line using CRISPR-Cas9 genome engineering and investigated behavioural changes and alterations to neural activity using whole brain Ca2+ imaging in zebrafish larvae and ribosomal protein S6 (rpS6) immunohistochemistry in adults. Results: We show that genetic inactivation of the histamine H3 receptor (Hrh3) reduces aggression in zebrafish, an effect that can be reproduced by pharmacological inhibition. In addition, hrh3−/− zebrafish show behavioural impairments consistent with heightened anxiety. Larval in vivo whole brain Ca2+ imaging reveals higher neuronal activity in the forebrain of mutants, but lower activity in specific hindbrain areas and changes in measures of functional connectivity between subregions. Adult hrh3−/− zebrafish display brain region-specific neural activity changes in response to aggression of both key regions of the social decision-making network, and the areas containing histaminergic neurons in the zebrafish brain. Conclusion: These results highlight the importance of zebrafish Hrh3 signalling for aggression and anxiety and uncover the brain areas involved. Targeting this receptor might be a potential novel therapeutic route for human conditions characterized by heightened aggression.

Abstract
Drug development is a highly resource intensive process that uses large numbers of animals for assessing the safety and efficacy of drugs prior to clinical testing. Improving the efficiency of drug development in terms of financial expenditure and number of animals used is therefore of utmost concern, not only to industry, but also to animal welfare organisations such as the NC3Rs. Poor efficiency in drug development largely stems from drug attrition, particularly attrition in the latter stages of the testing due to the large amount of resources expended at the point of failure. It is therefore imperative that deleterious off-target effects are identified as early as possible. However, typically, identification of seizure as a side-effect of drugs is performed in the later stages of development due to the highly intensive and low-throughput nature of seizure assays. At which point, if a compound fails, a large amount of resources have been squandered. There therefore exists a need for high-throughput and relatively inexpensive seizure liability assays that can be used early in drug development to prevent compounds destined for failure undergoing unnecessary resource intensive testing.
In this thesis we propose a refined approach using non-invasive imaging techniques in non-protected life stage zebrafish as a method for the detection of seizurogenic compounds early in drug development. In addition, we highlight its utility for elucidating the pharmacodynamics of compounds. In this study, a transgenic zebrafish line containing a GCaMP6s calcium sensor under the control of the pan-neuronal promoter elavl3 was used for functional profiling of compounds with varied pharmacologies. Light sheet microscopy was used to record fluorescent activity in three spatial dimensions over time (4-dimensions) from the zebrafish brain after exposure to forty-three different compounds with varied pharmacodynamics and seizure liability profiles. Hierarchical clustering was employed in order to assess if compounds with seizurogenic activity or similar pharmacodynamics elicited specific functional brain activity. It was found that compounds with dopaminergic and serotonergic mechanisms of action elicited highly specific and similar brain activity patterns and that non-seizurogenic drugs also clustered separately from seizurogenic ones. Subsequent analyses, focussed on the utilisation of machine learning techniques, developing a model that could be used to discriminate between compounds with and without potentially seizurogenic effects. It is clear, from the analyses presented here, that drugs do in fact elicit specific brain patterns in zebrafish and that these brain patterns are effectively detected using light sheet microscopy. This system is highly applicable for use within the drug industry and even in its relatively preliminary stages provided an accurate method of discriminating between compounds based on their physiological effects in zebrafish.

Background: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. Objectives: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. Methods: to achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. Results: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. Discussion: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.

The zebrafish is rapidly emerging as a promising alternative in vivo model for the detection of drug-induced cardiovascular effects. Despite its increasing popularity, the ability of this model to inform the drug development process is often limited by the uncertainties around the quantitative relevance of zebrafish responses compared with non-clinical mammalian species and ultimately humans. In this test of concept study we provide a comparative quantitative analysis of the in vivo cardiovascular responses of zebrafish, rat, dog, and human to three model compounds (propranolol, losartan, and captopril), which act as modulators of two key systems (beta-Adrenergic and renin-Angiotensin systems) involved in the regulation of cardiovascular functions. We used in vivo imaging techniques to generate novel experimental data of drug-mediated cardiovascular effects in zebrafish larvae. This data was combined with a database of inter-species mammalian responses (i.e. heart rate, blood flow, vessel diameter, stroke volume) extracted from the literature to perform a meta-Analysis of effect size and direction across multiple species. In spite of the high heterogeneity of study design parameters, our analysis highlighted that zebrafish and human responses were largely comparable in 80% of drug/endpoint combinations. However, it also revealed a high intra-species variability which, in some cases, prevented a conclusive interpretation of the drug-induced effect. Despite the shortcomings of our study, the meta-Analysis approach, combined with a suitable data visualization strategy, enabled us to observe of patterns of response that would likely remain undetected with more traditional methods of qualitative comparative analysis. We propose that expanding this approach to larger datasets encompassing multiple drugs and modes-of-Action, would enable a rigorous and systematic assessment of the applicability domain of the zebrafish from both a mechanistic and phenotypic standpoint. This will increase the confidence in its application for the early detection of adverse drug reactions in any major organ system.

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Environmental exposure to Bisphenol a (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 μg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.

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.

In mammals, the pregnane X receptor (PXR) is a transcription factor with a key role in regulating expression of several genes involved in drug biotransformation. PXR is present in fish and some genes known to be under its control can be up-regulated by mammalian PXR ligands. Despite this, direct involvement of PXR in drug biotransformation in fish has yet to be established. Here, the full length PXR sequence was cloned from carp (Cyprinus carpio) and used in a luciferase reporter assay to elucidate its role in xenobiotic metabolism in fish. A reporter assay for human PXR (hPXR) was also established to compare transactivation between human and carp (cPXR) isoforms. Rifampicin activated hPXR as expected, but not cPXR. Conversely, clotrimazole (CTZ) activated both isoforms and was more potent on cPXR, with an EC50 within the range of concentrations of CTZ measured in the aquatic environment. Responses to other azoles tested were similar between both isoforms. A range of pharmaceuticals tested either failed to activate, or were very weakly active, on the cPXR or hPXR. Overall, these results indicate that the cPXR may differ from the hPXR in its responses and/or sensitivity to induction by different environmental chemicals, with implications for risk assessment because of species differences.

AbstractPsychoactive drugs are frequently detected in the aquatic environment. The evolutionary conservation of the molecular targets of these drugs in fish suggests that they may elicit mode of action–mediated effects in fish as they do in humans, and the key open question is at what exposure concentrations these effects might occur. In the present study, the authors investigated the uptake and tissue distribution of the benzodiazepine oxazepam in the fathead minnow (Pimephales promelas) after 28 d of waterborne exposure to 0.8 μg L−1, 4.7 μg L−1, and 30.6 μg L−1. Successively, they explored the relationship between the internal concentrations of oxazepam and the effects on fish exploratory behavior quantified by performing 2 types of behavioral tests, the novel tank diving test and the shelter‐seeking test. The highest internal concentrations of oxazepam were found in brain, followed by plasma and liver, whereas muscle presented the lowest values. Average concentrations measured in the plasma of fish from the 3 exposure groups were, respectively, 8.7 ± 5.7 μg L−1, 30.3 ± 16.1 μg L−1, and 98.8 ± 72.9 μg L−1. Significant correlations between plasma and tissue concentrations of oxazepam were found in all 3 groups. Exposure of fish to 30.6 µg L−1 in water produced plasma concentrations within or just below the human therapeutic plasma concentration (HTPC) range in many individuals. Statistically significant behavioral effects in the novel tank diving test were observed in fish exposed to 4.7 μg L−1. In this group, plasma concentrations of oxazepam were approximately one‐third of the lowest HTPC value. No significant effects were observed in fish exposed to the lowest and highest concentrations. The significance of these results is discussed in the context of the species‐specific behavior of fathead minnow and existing knowledge of oxazepam pharmacology. Environ Toxicol Chem 2016;35:2782–2790. © 2016 the Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

In mammals, the peroxisome proliferator-activated receptor α (PPARα) plays a key role in regulating various genes involved in lipid metabolism, bile acid synthesis and cholesterol homeostasis, and is activated by a diverse group of compounds collectively termed peroxisome proliferators (PPs). Specific PPs have been detected in the aquatic environment; however little is known on their pharmacological activity in fish. We investigated the bioavailability and persistence of the human PPARα ligand clofibric acid (CFA) in carp, together with various relevant endpoints, at a concentration similar to therapeutic levels in humans (20mg/L) and for an environmentally relevant concentration (4μg/L). Exposure to pharmacologically-relevant concentrations of CFA resulted in increased transcript levels of a number of known PPARα target genes together with increased acyl-coA oxidase (Acox1) activity, supporting stimulation of lipid metabolism pathways in carp which are known to be similarly activated in mammals. Although Cu,Zn-superoxide dismutase (Sod1) activity was not affected, mRNA levels of several biotransformation genes were also increased, paralleling previous reports in mammals and indicating a potential role in hepatic detoxification for PPARα in carp. Importantly, transcription of some of these genes (and Acox1 activity) were affected at exposure concentrations comparable with those reported in effluent discharges. Collectively, these data suggest that CFA is pharmacologically active in carp and has the potential to invoke PPARα-related responses in fish exposed in the environment, particularly considering that CFA may represent just one of a number of PPAR-active compounds present to which wild fish may be exposed.

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.

Fish in many surface freshwaters are exposed to a range of pharmaceuticals via wastewater treatment works effluent discharges. In mammals the pregnane X receptor (PXR) plays a key role in the regulation of a suite of genes involved in drug biotransformation, but information on the role of this response pathway in fish is limited. Here we investigated the effects of exposure of carp (Cyprinus carpio) primary hepatocytes to the human PXR agonist rifampicin (RIF) on expression of target genes involved in phase I (cyp2k, cyp3a) and phase II (gstα, gstπ) drug metabolism and drug transporters mdr1 and mrp2. RIF induced expression of all target genes measured and the PXR antagonist ketoconazole (KET) inhibited responses of cyp2k and cyp3a. Exposure of the primary carp hepatocytes to the pharmaceuticals ibuprofen (IBU), clotrimazole (CTZ), clofibric acid (CFA) and propranolol (PRP), found responses to IBU and CFA, but not CTZ or PRP. This is in contrast with mammals, where CTZ is a potent PXR-agonist. Collectively our data indicate potential PXR involvement in regulating selected genes involved in drug metabolism in fish, but suggest some divergence in the regulation pathways with those in mammals. The carp primary hepatocyte model serves as a useful system for screening for responses in these target genes involved in drug metabolism.

The conservation of common physiological systems across vertebrate classes suggests the potential for certain pharmaceuticals, which have been detected in surface waters, to produce biological effects in nontarget vertebrates such as fish. However, previous studies assessing the effects of such compounds in fish have not taken into account the potential for metabolism and elimination. This study aimed to assess if propranolol, a β-adrenergic receptor antagonist or β-blocker, could modulate EROD activity (indicative of CYP1A activity) in rainbow trout (Oncorhynchus mykiss) gills and liver. For this, an in vivo time course exposure with 1 mg/L was conducted. Additionally, using measured in vivo plasma concentrations, an in vitro exposure at human therapeutic levels was undertaken. This allowed comparison of in vitro and in vivo rates of EROD activity, thus investigating the applicability of cell preparations as surrogates for whole animal enzyme activity analysis. In vitro exposure of suspended liver and gill cells at concentrations similar to in vivo levels resulted in EROD activity in both tissues, but with significantly higher rates (up to six times in vivo levels). These results show that propranolol exposure elevated EROD activity in the liver and gill of rainbow trout, and that this is demonstrable both in vivo (albeit nonsignificantly in the liver) and in vitro, thus supporting the use of the latter as a surrogate of the former. These data also provide an insight into the potential role of the gill as a site of metabolism of pharmaceuticals in trout, suggesting that propranolol (and feasibly other pharmaceuticals) may undergo "first pass" metabolism in this organ.

The authors review the current data on the presence and reported biological effects in fish of some of the most commonly detected pharmaceuticals in the aquatic environment; namely nonsteroidal anti-inflammatory drugs (NSAIDs), fibrates, beta-blockers, selective serotonin reuptake inhibitors (SSRIs), azoles, and antibiotics. Reported biological effects in fish in the laboratory have often been shown to be in accordance with known effects of pharmaceuticals in mammals. Water concentrations at which such effects have been reported, however, are generally, between microg L(-1) and mg L(-1), typically at least 1 order of magnitude higher than concentrations normally found in surface waters (ng L(-1)). There are exceptions to this, however, as for the case of synthetic oestrogens, which can induce biological effects in the low ng L(-1) range. Although generally effect levels for pharmaceuticals are higher than those found in the environment, the risks to wild fish populations have not been thoroughly characterised, and there has been a lack of consideration given to the likely chronic nature of the exposures, or the potential for mixture effects. As global consumption of pharmaceuticals rises, an inevitable consequence is an increased level of contamination of surface and ground waters with these biologically active drugs, and thus in turn a greater potential for adverse effects in aquatic wildlife.

Over recent years, human pharmaceuticals have been detected in the aquatic environment. This, combined with the fact that many are (by design) biologically active compounds, has raised concern about potential impacts in wildlife species. This concern was realized with two high-profile cases of unforeseen environmental impact (i.e. estrogens and diclofenac), which have led to a flurry of work addressing how best to predict such effects in the future. One area in which considerable research effort has been made, partially in response to regulatory requirements, has been on the potential use of preclinical and clinical pharmacological and toxicological data (generated during drug development from nonhuman mammals and humans) to predict possible effects in nontarget, environmentally relevant species: so-called read across. This approach is strengthened by the fact that many physiological systems are conserved between mammals and certain environmentally relevant species. Consequently, knowledge of how a pharmaceutical works (the “mode-of-action,” or MoA) in nonclinical species and humans could assist in the selection of appropriate test species, study designs, and endpoints, in an approach referred to as “intelligent testing.” Here we outline the data available from the human drug development process and suggest how this might be used to design a testing strategy best suited to the specific characteristics of the drug in question. In addition, we review published data that support this type of approach, discuss the potential pitfalls associated with read across, and identify knowledge gaps that require filling to ensure accuracy in the extrapolation of data from preclinical and clinical studies, for use in the environmental risk assessment of human pharmaceuticals.

Many factors have been considered in evaluations of the risk-benefit balance of hormone replacement therapy (HRT), used for treating menopausal symptoms in women, but not its potential risks for the environment We investigated the possible environmental health implications of conjugated equine estrogens (CEEs), the most common components of HRT, including their discharge into the environment, their uptake, potency, and ability to induce biological effects in wildlife. Influents and effluents from four U.K. sewage treatment works (STWs), and bile of effluent-exposed fish, were screened for six equine estrogens. In vitro estrogen receptor (ER) activation assays were applied in humans and fish to compare their potencies, followed by in vivo exposures of fish to equine estrogens and evaluation of bioaccumulation, estrogenic responses, and ER gene expression. The equine estrogen equilenin (Eqn), and its metabolite 17beta-dihydroequilenin (17beta-Eqn), were detected by tandem GC-MSMS in all STW influent samples and 83% of STW effluent samples analyzed, respectively, at low concentrations (0.07-2.6 ng/L) and were taken-up into effluent-exposed fish. As occurs in humans, these estrogens bound to and activated the fish ERs, with potencies at ERalpha 2.4-3490% of thatfor 17beta-estradiol. Exposure of fish for 21 days to Eqn and 17beta-Eqn induced estrogenic responses including hepatic growth and vitellogenin production at concentrations as low as 0.6-4.2 ng/L. Associated with these effects were inductions of hepatic ERalpha and ERbeta1 gene expression, suggesting ER-mediated mechanism(s) of action. These data provide evidence for the discharge of equine estrogens from HRT into the aquatic environment and highlight a strong likelihood that these compounds contribute to feminization in exposed wildlife.

The presence of endocrine disrupting chemicals (EDCs) in the environment has driven the development of screening and testing assays to both identify chemicals with hormonal activity and evaluate their potential to cause adverse effects. As the number of animals used for research and regulatory purposes rises, and set against a desire to reduce animal testing, there is increased emphasis on the development and application of in vitro techniques to evaluate chemical risks to the environment. Induction of vitellogenin (VTG) in isolated fish liver cells has been used successfully to identify a wide range of EDCs, including both natural and synthetic oestrogens and a variety of other xenoestrogens. However, the vitellogenic response reported for hepatocytes in culture has been shown to vary widely, making comparisons between studies difficult. The work presented in this paper explored the variability of the vitellogenic response in primary cultures of common carp (Cyprinus carpio) hepatocytes following exposure to the model oestrogenic compound, 17beta-oestradiol (E2). As expected, variability in the vitellogenic response was observed, both in terms of the sensitivity and magnitude of VTG induction, for hepatocytes isolated from different fish. An apparent difference was observed in the response of isolated hepatocytes based on the sex of the donor fish; maximum levels of E2-stimulated VTG synthesis in hepatocytes derived from females appeared higher (1962 ng mL(-1)+/-487 [n=9] compared with 1194 ng mL(-1)+/-223 for hepatocytes from males [n=9]) and EC(50) values lower (1.61+/-0.4 microM E2 for females and 2.12+/-0.2 microM E2 for males). However, these differences were not statistically significant, likely in part due to the variation observed in the vitellogenic response. In particular, hepatocytes derived from female fish showed more variation than their male counterparts (the co-efficient of variation for females was 77% compared to 28% for males). Despite the variation observed in the vitellogenic response between different cultures, data from the different donor fish could be compared by standardising responses relative to the maximum VTG induction in each culture following exposure to E2. Adopting this approach in the future will allow for data from different hepatocyte cultures and from donor fish of different sexes, age and stage of maturity to be compared with greater consistency. Measurement of vtg mRNA expression was relatively more sensitive to the oestrogenic effects of E2 exposure than measurement of VTG protein (the LOEC at the transcriptome level was 10-fold lower [0.01 microM E2] than at the protein level [0.1 microM E2]) and changes in vtg mRNA expression showed less variation between individual hepatocyte isolations. Measurement of vtg mRNA in the hepatocyte culture system therefore may offer the most sensitive and consistent option for the screening of chemicals with oestrogenic activity in fish primary hepatocyte cultures.

Complex physiological traits, such as routine aerobic metabolic rate or exercise performance, are indicators of the functional integrity of fish that can reveal sub-lethal toxicological effects of aquatic pollutants. These traits have proved valuable in laboratory investigations of the sub-lethal effects of heavy metals, ammonia and various xenobiotics. It is not known, however, whether they can also function as biomarkers of the complex potential range of effects upon overall functional integrity caused by exposure to mixtures of chemicals in polluted natural environments. The current study used portable swimming respirometers to compare exercise performance and respiratory metabolism of fish exposed in cages for three weeks to either clean or polluted sites on three urban European river systems: the river Lambro, Milan, Italy; the rivers Blythe, Cole and Tame, Birmingham, UK; and the river Amstel, Amsterdam, the Netherlands. The UK and Italian rivers were variously polluted with high levels of both bioavailable heavy metals and organics, and the Amstel by mixtures of bioavailable organics at high concentrations. In both the UK and Italy, indigenous chub (Leuciscus cephalus) exposed to clean or polluted sites swam equally well in an initial performance test, but the chub from polluted sites could not repeat this performance after a brief recovery interval. These animals were unable to raise the metabolic rate and allocate oxygen towards exercise in the second trial, an effect confirmed in successive campaigns in Italy. Swimming performance was therefore a biomarker indicator of pollutant exposure in chub exposed at these sites. Exposure to polluted sites on the river Amstel did not affect the repeat swimming performance of cultured cloned carp (Cyprinus carpio), indicating either a species-specific tolerance or relative absence of heavy metals. However, measurements of oxygen uptake during swimming revealed increased rates of routine aerobic metabolism in both chub and carp at polluted sites in all of the rivers studied, indicating a sub-lethal metabolic loading effect. Therefore, the physiological traits of exercise performance and metabolic rate have potential as biomarkers of the overall sub-lethal toxic effects of exposure to complex mixtures of pollutants in rivers, and may also provide insight into why fish do not colonize some polluted environments.

the caudal neurosecretory system (CNSS) of fish was first defined over 70 years ago yet despite much investigation, a clear physiological role has yet to be elucidated. Although the CNSS structure is as yet thought to be confined to piscine species, the secreted peptides, urotensins I and II (UI and UII), have been detected in a number of vertebrate species, most recently illustrated by the isolation of UII in humans. The apparent importance of these peptides, suggested by their relative phylogenetic conservation, is further supported by the complex control mechanisms associated with their secretion. The CNSS in teleosts is known to receive extensive and diverse innervation from the higher central nervous system, with evidence for the presence of cholinergic, noradrenergic, serotonergic, and peptidergic descending inputs. Recent observations also suggest the presence of glucocorticoid receptors in the flounder CNSS, supporting previous evidence for a possible role as a pituitary-independent mechanism controlling cortisol secretion. The most convincing evidence as to a physiological role for the CNSS in fish has stemmed from the direct and indirect influence of the urotensins on osmoregulatory function. Recent advances allowing the measurement of circulating levels of UII in the flounder have supported this. In addition, there is evidence to suggest some seasonal variation in peptide levels supporting the notion that the CNSS may have an integrative role in the control of coordinated changes in the reproductive, osmoregulatory and nutritional systems of migratory euryhaline species.