Publications by year
2021
Winter MJ, Pinion J, Tochwin A, Takesono A, Ball JS, Grabowski P, Metz J, Trznadel M, Tse K, Redfern WS, et al (2021). Functional brain imaging in larval zebrafish for characterising the effects of seizurogenic compounds acting via a range of pharmacological mechanisms.
Br J Pharmacol,
178(13), 2671-2689.
Abstract:
Functional brain imaging in larval zebrafish for characterising the effects of seizurogenic compounds acting via a range of pharmacological mechanisms.
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.
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2020
Fitzgerald JA, Trznadel M, Katsiadaki I, Santos EM (2020). Hypoxia modifies the response to flutamide and linuron in male three-spined stickleback (Gasterosteus aculeatus).
Environ Pollut,
263(Pt A).
Abstract:
Hypoxia modifies the response to flutamide and linuron in male three-spined stickleback (Gasterosteus aculeatus).
Hypoxia is a major stressor in aquatic environments and it is frequently linked with excess nutrients resulting from sewage effluent discharges and agricultural runoff, which often also contain complex mixtures of chemicals. Despite this, interactions between hypoxia and chemical toxicity are poorly understood. We exposed male three-spined stickleback during the onset of sexual maturation to a model anti-androgen (flutamide; 250 μg/L) and a pesticide with anti-androgenic activity (linuron; 250 μg/L), under either 97% or 56% air saturation (AS). We assessed the effects of each chemical, alone and in combination with reduced oxygen concentration, by measuring the transcription of spiggin in the kidney, as a marker of androgen signalling, and 11 genes in the liver involved in some of the molecular pathways hypothesised to be affected by the exposures. Spiggin transcription was strongly inhibited by flutamide under both AS conditions. In contrast, for linuron, a strong inhibition of spiggin was observed under 97% AS, but this effect was supressed under reduced air saturation, likely due to interactions between the hypoxia inducible factor and the aryl hydrocarbon receptor (AhR) pathways. In the liver, hypoxia inducible factor 1α was induced following exposure to both flutamide and linuron, however this was independent of the level of air saturation. This work illustrates the potential for interactions between hypoxia and pollutants with endocrine or AhR agonist activity to occur, with implications for risk assessment and management.
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2019
Mourabit S, Fitzgerald JA, Ellis RP, Takesono A, Porteus CS, Trznadel M, Metz J, Winter MJ, Kudoh T, Tyler CR, et al (2019). New insights into organ-specific oxidative stress mechanisms using a novel biosensor zebrafish.
Environment International,
133Abstract:
New insights into organ-specific oxidative stress mechanisms using a novel biosensor 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.
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Le Page G, Gunnarsson L, Trznadel M, Wedgwood KCA, Baudrot V, Snape J, Tyler CR (2019). Variability in cyanobacteria sensitivity to antibiotics and implications for environmental risk assessment.
Science of the Total Environment,
695Abstract:
Variability in cyanobacteria sensitivity to antibiotics and implications for environmental risk assessment
Once released into the environment antibiotics can kill or inhibit the growth of bacteria, and in turn potentially have effects on bacterial community structure and ecosystem function. Environmental risk assessment (ERA) seeks to establish protection limits to minimise chemical impacts on the environment, but recent evidence suggests that the current regulatory approaches for ERA for antibiotics may not be adequate for protecting bacteria that have fundamental roles in ecosystem function. In this study we assess the differences in interspecies sensitivity of eight species of cyanobacteria to seven antibiotics (cefazolin, cefotaxime, ampicillin, sufamethazine, sulfadiazine, azithromycin and erythromycin) with three different modes of action. We found that variability in the sensitivity to these antibiotics between species was dependent on the mode of action and varied by up to 70 times for β-lactams. Probabilistic analysis using species sensitivity distributions suggest that the current predicted no effect concentration PNEC for the antibiotics may be either over or under protective of cyanobacteria dependent on the species on which it is based and the mode of action of the antibiotic; the PNECs derived for the macrolide antibiotics were over protective but PNECs for β-lactams were generally under protective. For some geographical locations we identify a significant risk to cyanobacteria populations based upon measured environmental concentrations of selected antibiotics. We conclude that protection limits, as determined according to current regulatory guidance, may not always be protective and might be better derived using SSDs and that including toxicity data for a wider range of (cyano-) bacteria would improve confidence for the ERA of antibiotics.
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2018
Brown AR, Green J, Moreman J, Gunnarsson L, Mourabit S, Ball J, Winter M, Trznadel M, Correia A, Hacker C, et al (2018). Cardiovascular Effects and Molecular Mechanisms of Bisphenol a and its Metabolite MBP in Zebrafish. Environmental Science and Technology
Green JM, Lange A, Scott A, Trznadel M, Wai HA, Takesono A, Brown AR, Owen SF, Kudoh T, Tyler CR, et al (2018). Early life exposure to ethinylestradiol enhances subsequent responses to environmental estrogens measured in a novel transgenic zebrafish.
Sci Rep,
8(1).
Abstract:
Early life exposure to ethinylestradiol enhances subsequent responses to environmental estrogens measured in a novel transgenic zebrafish.
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.
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Moreman J, Takesono A, Trznadel M, Winter MJ, Perry A, Wood ME, Rogers NJ, Kudoh T, Tyler CR (2018). Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol a (BPA) and its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.
Environ Sci Technol,
52(11), 6656-6665.
Abstract:
Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol a (BPA) and its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.
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.
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Langan LM, Owen SF, Trznadel M, Dodd NJF, Jackson SK, Purcell WM, Jha AN (2018). Spheroid Size Does not Impact Metabolism of the β-blocker Propranolol in 3D Intestinal Fish Model.
FRONTIERS IN PHARMACOLOGY,
9 Author URL.
2017
Moreman J, Lee O, Trznadel M, David A, Kudoh T, Tyler CR (2017). Acute Toxicity, Teratogenic, and Estrogenic Effects of Bisphenol a and its Alternative Replacements Bisphenol S, Bisphenol F, and Bisphenol AF in Zebrafish Embryo-Larvae.
Environ Sci Technol,
51(21), 12796-12805.
Abstract:
Acute Toxicity, Teratogenic, and Estrogenic Effects of Bisphenol a and its Alternative Replacements Bisphenol S, Bisphenol F, and Bisphenol AF in Zebrafish Embryo-Larvae.
Bisphenol a (BPA), a chemical incorporated into plastics and resins, has estrogenic activity and is associated with adverse health effects in humans and wildlife. Similarly structured BPA analogues are widely used but far less is known about their potential toxicity or estrogenic activity in vivo. We undertook the first comprehensive analysis on the toxicity and teratogenic effects of the bisphenols BPA, BPS, BPF, and BPAF in zebrafish embryo-larvae and an assessment on their estrogenic mechanisms in an estrogen-responsive transgenic fish Tg(ERE:Gal4ff)(UAS:GFP). The rank order for toxicity was BPAF > BPA > BPF > BPS. Developmental deformities for larval exposures included cardiac edema, spinal malformation, and craniofacial deformities and there were distinct differences in the effects and potencies between the different bisphenol chemicals. These effects, however, occurred only at concentrations between 1.0 and 200 mg/L which exceed those in most environments. All bisphenol compounds induced estrogenic responses in Tg(ERE:Gal4ff)(UAS:GFP) zebrafish that were inhibited by coexposure with ICI 182 780, demonstrating an estrogen receptor dependent mechanism. Target tissues included the heart, liver, somite muscle, fins, and corpuscles of Stannius. The rank order for estrogenicity was BPAF > BPA = BPF > BPS. Bioconcentration factors were 4.5, 17.8, 5.3, and 0.067 for exposure concentrations of 1.0, 1.0, 0.10, and 50 mg/L for BPA, BPF, BPAF, and BPS, respectively. We thus show that these BPA alternatives induce similar toxic and estrogenic effects to BPA and that BPAF is more potent than BPA, further highlighting health concerns regarding the use of BPA alternatives.
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2016
Laing LV, Viana J, Dempster EL, Trznadel M, Trunkfield LA, Uren Webster TM, van Aerle R, Paull GC, Wilson RJ, Mill J, et al (2016). Bisphenol a causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio).
Epigenetics,
11(7), 526-538.
Abstract:
Bisphenol a causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio).
Bisphenol a (BPA) is a commercially important high production chemical widely used in epoxy resins and polycarbonate plastics, and is ubiquitous in the environment. Previous studies demonstrated that BPA activates estrogenic signaling pathways associated with adverse effects on reproduction in vertebrates and that exposure can induce epigenetic changes. We aimed to investigate the reproductive effects of BPA in a fish model and to document its mechanisms of toxicity. We exposed breeding groups of zebrafish (Danio rerio) to 0.01, 0.1, and 1 mg/L BPA for 15 d. We observed a significant increase in egg production, together with a reduced rate of fertilization in fish exposed to 1 mg/L BPA, associated with significant alterations in the transcription of genes involved in reproductive function and epigenetic processes in both liver and gonad tissue at concentrations representing hotspots of environmental contamination (0.1 mg/L) and above. of note, we observed reduced expression of DNA methyltransferase 1 (dnmt1) at environmentally relevant concentrations of BPA, along with a significant reduction in global DNA methylation, in testes and ovaries following exposure to 1 mg/L BPA. Our findings demonstrate that BPA disrupts reproductive processes in zebrafish, likely via estrogenic mechanisms, and that environmentally relevant concentrations of BPA are associated with altered transcription of key enzymes involved in DNA methylation maintenance. These findings provide evidence of the mechanisms of action of BPA in a model vertebrate and advocate for its reduction in the environment.
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Green JM, Metz J, Lee O, Trznadel M, Takesono A, Brown AR, Owen SF, Kudoh T, Tyler CR (2016). High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.
Environ Sci Technol,
50(12), 6536-6545.
Abstract:
High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.
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.
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