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There is growing evidence that rising global temperatures resulting from climate change may exacerbate the toxic effect of pollutants and heterotherms, including fish, in which homestatic mechanisms are directly influenced by environmental temperature will be most affected. Pharmaceuticals discharged into the environment are potentially harmful to wildlife as many of their drug targets are conserved across divergent phyla. Oxidative stress (OS) is a major mechanism by which many pharmaceutical contaminants can induce toxicity but this has received little consideration in the context of effects in wildlife. Further, these mechanisms are relatively poorly understood, particularly regarding multiple stressor interactions. We used transgenic TG(EpRE:mCherry) zebrafish, developed in our laboratory for detecting OS, as our experimental model. We show that the oxidative effects of high concentrations of pharmaceuticals from three different therapeutic classes (paracetamol, diclofenac and doxorubicin) are increased at temperatures elevated by 2-5 °C above those for zebrafish standard husbandry and relevant to their current natural environment (and predicted under the IPCC 2023 scenarios for intermediate to very high greenhouse gas emissions). These OS responses were primarily seen in the pronephros, liver, and gastrointestinal tract. The increase in OS at the increased water temperature may have resulted from the elevated temperature acting as a direct additive physiological stressor to the OS imposed by the drugs and/or via the temperature increasing the chemicals oxidative effect. For paracetamol, it appeared that the elevated responses at the higher temperature of 33 °C were in part due to an increase in uptake of the drug. Our data illustrate that risk assessments for chemicals inducing OS in fish (and likely other heterotherms) should consider the influence of temperature to ensure environmental protection in future environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Synthetic glucocorticoids (GCs) are used to treat a wide range of human health conditions and as such are frequently detected in the aquatic environment. This, together with the highly conserved nature of the glucocorticoid system across vertebrates means that the potential for biological effects of GCs in fish is relatively high. Here, we found that exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of 4 of the most widely used synthetic GCs (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone), from 0 to 4 days post fertilisation (dpf), resulted in no effects on embryo-larval development or bone and cartilage formation. However, after exposure to equivalents of human therapeutic plasma levels, developmental abnormalities were observed that included pericardial oedema, blood pooling and alterations in jaw cartilage. Furthermore, using a double transgenic zebrafish osteoblast and chondrocyte reporter line, exposure up to 10 dpf resulted in alterations to lower jaw cartilage and bone development for all compounds at, and above, human therapeutic plasma concentrations. In the case of beclomethasone dipropionate, a reduction in lower jaw intercranial distance was observed at the environmentally relevant concentration of 0.1 μg/L. Using further transgenic reporter lines with fluorescently tagged neutrophils and macrophages, we also show exposure of embryo-larvae (0-4 dpf) to the GCs tested resulted in altered immune cell migration, but only at relatively high exposure concentrations. Collectively, our findings show GC exposure impacts embryo-larval zebrafish development, immune function, and skeletal formation, but predominantly at concentrations greater than those currently reported for the aquatic environment. Despite this, however, it is suggested that studies with longer exposure times, and to mixtures of multiple GCs (many GCs act via the same mechanism of action) are warranted before we can confidently assert that these commonly detected contaminants do not pose a risk to fish in the wild., Competing Interests: Declaration of competing interest CH was in receipt of a scholarship (awarded to CRT, MJW and LM-C) co-funded by AstraZeneca. SFO is an employee and share holder of AstraZeneca, a biopharmaceutical company with an interest in the discovery, development and commercialisation of prescription medicines including glucocorticoids., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Zebrafish embryo assays are used by pharmaceutical and chemical companies as new approach methodologies (NAMs) in developmental toxicity screening. Despite an overall high concordance of zebrafish embryo assays with in vivo mammalian studies, false negative and false positive results have been reported. False negative results in risk assessment models are of particular concern for human safety, as developmental anomalies may be missed. Interestingly, for several chemicals and drugs that were reported to be false negative in zebrafish, skeletal findings were noted in the in vivo studies. As the number of skeletal endpoints assessed in zebrafish is very limited compared to the in vivo mammalian studies, the aim of this study was to investigate whether the sensitivity could be increased by including a skeletal staining method. Three staining methods were tested on zebrafish embryos that were exposed to four teratogens that caused skeletal anomalies in rats and/or rabbits and were false negative in zebrafish embryo assays. These methods included a fixed alizarin red-alcian blue staining, a calcein staining, and a live alizarin red staining. The results showed a high variability in staining intensity of larvae exposed to mammalian skeletal teratogens, as well as variability between control larvae originating from the same clutch of zebrafish. Hence, biological variability in (onset of) bone development in zebrafish hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. In conclusion, the used skeletal staining methods did not increase the sensitivity of zebrafish embryo developmental toxicity assays., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Steven Van Cruchten reports financial support was provided by University of Antwerp. Jonathan Ball reports financial support and equipment, drugs, or supplies were provided by University of Exeter. Steven Van Cruchten is Associate Editor for Reproductive Toxicology and Guest Editor for the Special Issue on NAMs for DART testing If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Functional brain imaging using genetically encoded Ca 2+ 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 GABA A 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., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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., (Copyright © 2019. Published by Elsevier Ltd.)

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

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.

Estrogen plays fundamental roles in a range of developmental processes and exposure to estrogen mimicking chemicals has been associated with various adverse health effects in both wildlife and human populations. Estrogenic chemicals are found commonly as mixtures in the environment and can have additive effects, however risk analysis is typically conducted for single-chemicals with little, or no, consideration given for an animal's exposure history. Here we developed a transgenic zebrafish with a photoconvertable fluorophore (Kaede, green to red on UV light exposure) in a skin pigment-free mutant element (ERE)-Kaede-Casper model and applied it to quantify tissue-specific fluorescence biosensor responses for combinations of estrogen exposures during early life using fluorescence microscopy and image analysis. We identify windows of tissue-specific sensitivity to ethinylestradiol (EE2) for exposure during early-life (0-5 dpf) and illustrate that exposure to estrogen (EE2) during 0-48 hpf enhances responsiveness (sensitivity) to different environmental estrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. Our findings illustrate the importance of an organism's stage of development and estrogen exposure history for assessments on, and possible health risks associated with, estrogen exposure.

Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.