Detecting Developmental Neurotoxicants Using Zebrafish Embryos

EPA Science Inventory

As part of EPA’s program on the screening and prioritization of chemicals for developmental neurotoxicity, a rapid, cost-effective in vivo vertebrate screen is being developed using an alternative species approach. Zebrafish (Danio rerio), a small freshwater fish with external f...

Halogenated Bisphenol-A Analogs Act as Obesogens in Zebrafish Larvae (Danio rerio)

PubMed Central

Pinto, Caroline L.; Grimaldi, Marina; Hillenweck, Anne; Perdu, Elisabeth; Zalko, Daniel; Bernard, Laure; Laudet, Vincent; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Ake

2014-01-01

Obesity has increased dramatically over the past decades, reaching epidemic proportions. The reasons are likely multifactorial. One of the suggested causes is the accelerated exposure to obesity-inducing chemicals (obesogens). However, out of the tens of thousands of industrial chemicals humans are exposed to, very few have been tested for their obesogenic potential, mostly due to the limited availability of appropriate in vivo screening models. In this study, we investigated whether two commonly used flame retardants, the halogenated bisphenol-A (BPA) analogs tetrabromobisphenol-A (TBBPA) and tetrachlorobisphenol-A (TCBPA), could act as obesogens using zebrafish larvae as an in vivo animal model. The effect of embryonic exposure to these chemicals on lipid accumulation was analyzed by Oil Red-O staining, and correlated to their capacity to activate human and zebrafish peroxisome proliferator-activated receptor gamma (PPARγ) in zebrafish and in reporter cell lines. Then, the metabolic fate of TBBPA and TCBPA in zebrafish larvae was analyzed by high-performance liquid chromatography (HPLC) . TBBPA and TCBPA were readily taken up by the fish embryo and both compounds were biotransformed to sulfate-conjugated metabolites. Both halogenated-BPAs, as well as TBBPA-sulfate induced lipid accumulation in zebrafish larvae. TBBPA and TCBPA also induced late-onset weight gain in juvenile zebrafish. These effects correlated to their capacity to act as zebrafish PPARγ agonists. Screening of chemicals for inherent obesogenic capacities through the zebrafish lipid accumulation model could facilitate prioritizing chemicals for further investigations in rodents, and ultimately, help protect humans from exposure to environmental obesogens. PMID:24591153

Gene Expression Changes in Developing Zebrafish as Potential Markers for Rapid Developmental Neurotoxicity Screening

EPA Science Inventory

Sparse information exists on many chemicals to guide developmental neurotoxicity (DNT) risk assessments. As DNT testing using rodents is laborious and expensive, alternative species such as zebrafish are being adapted for toxicity screening. Assessing the DNT potential of chem...

Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening

EPA Science Inventory

The U.S. Environmental Protection Agency is developing and evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. Towards this goal, we are exploring methods to detect developmental neurotoxicants in very young larval zebrafish. We have...

ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN.

PubMed

Noyes, Pamela D; Garcia, Gloria R; Tanguay, Robert L

2016-12-21

Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world multi-chemical exposures. The zebrafish is poised to be an important model in bridging these two conventionally separate areas of toxicology and characterizing the biological effects of chemical mixtures that could augment its role in sustainable chemistry.

ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN

PubMed Central

Noyes, Pamela D.; Garcia, Gloria R.; Tanguay, Robert L.

2016-01-01

Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world multi-chemical exposures. The zebrafish is poised to be an important model in bridging these two conventionally separate areas of toxicology and characterizing the biological effects of chemical mixtures that could augment its role in sustainable chemistry. PMID:28461781

Behavorial Screens for Detecting Developmental Neurotoxicity in Larval Zebrafish

EPA Science Inventory

As part of the EPA's effort to develop an in vivo, vertebrate screen for toxic chemicals, we have characterized basic behaviors of 6-day post-fertilization (dpf) zebrafish (Danio rerio) larvae in a microtiter plate format. Our main goal is to develop a convenient, reproducible me...

Assessing Locomotor Activity in Larval Zebrafish: Influence of Extrinsic and Intrinsic Variables

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. We are exploring methods to detect developmentally neurotoxic chemicals using zebrafish behavior at 6 days of age. The behavioral paradig...

Studies of the Variables Affecting Behavior of Larval Zebrafish for Developmental Neurotoxicity Testing

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. We are exploring methods to detect developmentally neurotoxic chemicals using zebrafish behavior at 6 days of age. The behavioral paradig...

Juxtaposition of chemical and mutation-induced developmental defects in zebrafish reveal a copper-chelating activity for kalihinol F.

PubMed

Sandoval, Imelda T; Manos, Elizabeth J; Van Wagoner, Ryan M; Delacruz, Richard Glenn C; Edes, Kornelia; Winge, Dennis R; Ireland, Chris M; Jones, David A

2013-06-20

A major hurdle in using complex systems for drug screening is the difficulty of defining the mechanistic targets of small molecules. The zebrafish provides an excellent model system for juxtaposing developmental phenotypes with mechanism discovery using organism genetics. We carried out a phenotype-based screen of uncharacterized small molecules in zebrafish that produced a variety of chemically induced phenotypes with potential genetic parallels. Specifically, kalihinol F caused an undulated notochord, defects in pigment formation, hematopoiesis, and neural development. These phenotypes were strikingly similar to the zebrafish mutant, calamity, an established model of copper deficiency. Further studies into the mechanism of action of kalihinol F revealed a copper-chelating activity. Our data support this mechanism of action for kalihinol F and the utility of zebrafish as an effective system for identifying therapeutic and target pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

High-Content Screening in Zebrafish Embryos Identifies Butafenacil as a Potent Inducer of Anemia

PubMed Central

Leet, Jessica K.; Lindberg, Casey D.; Bassett, Luke A.; Isales, Gregory M.; Yozzo, Krystle L.; Raftery, Tara D.; Volz, David C.

2014-01-01

Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05–50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO) – an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors – flumioxazin – resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models. PMID:25090246

A MULTIFACETED, MEDIUM-THROUGHPUT APPROACH FOR DETECTING AND CHARACTERIZING DEVELOPMENTAL NEUROTOXICITY USING ZEBRAFISH.

EPA Science Inventory

To address the EPA's need to prioritize hundreds to thousands of chemicals for testing, we are developing a rapid, cost-effective in vivo screen for developmental neurotoxicity using zebrafish (Danio rerio), a small freshwater fish with external fertilization. Zebrafish embryos d...

Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity.

PubMed

Tal, Tamara; Kilty, Claire; Smith, Andrew; LaLone, Carlie; Kennedy, Brendán; Tennant, Alan; McCollum, Catherine W; Bondesson, Maria; Knudsen, Thomas; Padilla, Stephanie; Kleinstreuer, Nicole

2017-06-01

Chemically-induced vascular toxicity during embryonic development may cause a wide range of adverse effects. To identify putative vascular disrupting chemicals (pVDCs), a predictive pVDC signature was constructed from 124 U.S. EPA ToxCast high-throughput screening (HTS) assays and used to rank 1060 chemicals for their potential to disrupt vascular development. Thirty-seven compounds were selected for targeted testing in transgenic Tg(kdrl:EGFP) and Tg(fli1:EGFP) zebrafish embryos to identify chemicals that impair developmental angiogenesis. We hypothesized that zebrafish angiogenesis toxicity data would correlate with human cell-based and cell-free in vitro HTS ToxCast data. Univariate statistical associations used to filter HTS data based on correlations with zebrafish angiogenic inhibition in vivo revealed 132 total significant associations, 33 of which were already captured in the pVDC signature, and 689 non-significant assay associations. Correlated assays were enriched in cytokine and extracellular matrix pathways. Taken together, the findings indicate the utility of zebrafish assays to evaluate an HTS-based predictive toxicity signature and also provide an experimental basis for expansion of the pVDC signature with novel HTS assays. Published by Elsevier Inc.

Targeting the-Dopaminergic Nervous System: Altering Behavior in Larval Zebrafish

EPA Science Inventory

Zebrafish (Dania rerio) are becoming an important model system in studying the effects of environmental chemicals on behavior. In order to develop a rapid in vivo screen to prioritize toxic chemicals, we have begun assessing the acute locomotor effects of drugs that act on the do...

Larval Behavioral Toxicity Screening: Light Intensity and the Order of Stimulus Presentation Affect the Outcome

EPA Science Inventory

The U.S. Environmental Protection Agency is screening large numbers of chemicals using 6 day old zebrafish (Danio rerio). We use a behavioral testing paradigm that simultaneously tests individual zebrafish under both light and dark conditions in a 96-well plate using a video tr...

DRUG EFFECTS ON THE LOCOMOTOR ACTIVITY OF LARVAL ZEBRAFISH.

EPA Science Inventory

As part of an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae and the effects of prototype drugs. Zebrafish larvae (6-7 days post-fertilization) were indiv...

Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

PubMed Central

Virto, Juan M.; Holgado, Olaia; Diez, Maria; Izpisua Belmonte, Juan Carlos; Callol-Massot, Carles

2012-01-01

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism. PMID:22615792

Changing the Scale and Efficiency of Chemical Warfare Countermeasure Discovery Using the Zebrafish

PubMed Central

Peterson, Randall T.; MacRae, Calum A.

2013-01-01

As the scope of potential chemical warfare agents grows rapidly and as the diversity of potential threat scenarios expands with non-state actors, so a need for innovative approaches to countermeasure development has emerged. In the last few years, the utility of the zebrafish as a model organism that is amenable to high-throughput screening has become apparent and this system has been applied to the unbiased discovery of chemical warfare countermeasures. This review summarizes the in vivo screening approach that has been pioneered in the countermeasure discovery arena, and highlights the successes to date as well as the potential challenges in moving the field forward. Importantly, the establishment of a zebrafish platform for countermeasure discovery would offer a rapid response system for the development of antidotes to the continuous stream of new potential chemical warfare agents. PMID:24273586

Automated image-based phenotypic analysis in zebrafish embryos

PubMed Central

Vogt, Andreas; Cholewinski, Andrzej; Shen, Xiaoqiang; Nelson, Scott; Lazo, John S.; Tsang, Michael; Hukriede, Neil A.

2009-01-01

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. PMID:19235725

Automated phenotype pattern recognition of zebrafish for high-throughput screening.

PubMed

Schutera, Mark; Dickmeis, Thomas; Mione, Marina; Peravali, Ravindra; Marcato, Daniel; Reischl, Markus; Mikut, Ralf; Pylatiuk, Christian

2016-07-03

Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of 4 different phenotypes can be classified through pattern recognition at 72 h post fertilization (hpf), allowing the software to classify an embryo into 2 distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented.

TOXICITY SCREENING WITH ZEBRAFISH ASSAY

EPA Science Inventory

The proposed toxicity screening will help EPA to prioritize chemicals for further testing, and it may also alert chemical manufacturers that some of their commercial products may be toxic. The proposed toxicity pathway studies will improve the research community’s abi...

Optimisation of Embryonic and Larval ECG Measurement in Zebrafish for Quantifying the Effect of QT Prolonging Drugs

PubMed Central

Dhillon, Sundeep Singh; Dóró, Éva; Magyary, István; Egginton, Stuart; Sík, Attila; Müller, Ferenc

2013-01-01

Effective chemical compound toxicity screening is of paramount importance for safe cardiac drug development. Using mammals in preliminary screening for detection of cardiac dysfunction by electrocardiography (ECG) is costly and requires a large number of animals. Alternatively, zebrafish embryos can be used as the ECG waveform is similar to mammals, a minimal amount of chemical is necessary for drug testing, while embryos are abundant, inexpensive and represent replacement in animal research with reduced bioethical concerns. We demonstrate here the utility of pre-feeding stage zebrafish larvae in detection of cardiac dysfunction by electrocardiography. We have optimised an ECG recording system by addressing key parameters such as the form of immobilization, recording temperature, electrode positioning and developmental age. Furthermore, analysis of 3 days post fertilization (dpf) zebrafish embryos treated with known QT prolonging drugs such as terfenadine, verapamil and haloperidol led to reproducible detection of QT prolongation as previously shown for adult zebrafish. In addition, calculation of Z-factor scores revealed that the assay was sensitive and specific enough to detect large drug-induced changes in QTc intervals. Thus, the ECG recording system is a useful drug-screening tool to detect alteration to cardiac cycle components and secondary effects such as heart block and arrhythmias in zebrafish larvae before free feeding stage, and thus provides a suitable replacement for mammalian experimentation. PMID:23579446

ARQiv-HTS, a versatile whole-organism screening platform enabling in vivo drug discovery at high-throughput rates

PubMed Central

White, David T; Eroglu, Arife Unal; Wang, Guohua; Zhang, Liyun; Sengupta, Sumitra; Ding, Ding; Rajpurohit, Surendra K; Walker, Steven L; Ji, Hongkai; Qian, Jiang; Mumm, Jeff S

2017-01-01

The zebrafish has emerged as an important model for whole-organism small-molecule screening. However, most zebrafish-based chemical screens have achieved only mid-throughput rates. Here we describe a versatile whole-organism drug discovery platform that can achieve true high-throughput screening (HTS) capacities. This system combines our automated reporter quantification in vivo (ARQiv) system with customized robotics, and is termed ‘ARQiv-HTS’. We detail the process of establishing and implementing ARQiv-HTS: (i) assay design and optimization, (ii) calculation of sample size and hit criteria, (iii) large-scale egg production, (iv) automated compound titration, (v) dispensing of embryos into microtiter plates, and (vi) reporter quantification. We also outline what we see as best practice strategies for leveraging the power of ARQiv-HTS for zebrafish-based drug discovery, and address technical challenges of applying zebrafish to large-scale chemical screens. Finally, we provide a detailed protocol for a recently completed inaugural ARQiv-HTS effort, which involved the identification of compounds that elevate insulin reporter activity. Compounds that increased the number of insulin-producing pancreatic beta cells represent potential new therapeutics for diabetic patients. For this effort, individual screening sessions took 1 week to conclude, and sessions were performed iteratively approximately every other day to increase throughput. At the conclusion of the screen, more than a half million drug-treated larvae had been evaluated. Beyond this initial example, however, the ARQiv-HTS platform is adaptable to almost any reporter-based assay designed to evaluate the effects of chemical compounds in living small-animal models. ARQiv-HTS thus enables large-scale whole-organism drug discovery for a variety of model species and from numerous disease-oriented perspectives. PMID:27831568

Making Waves: New Developments in Toxicology With the Zebrafish.

PubMed

Horzmann, Katharine A; Freeman, Jennifer L

2018-05-01

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

Evaluating alterations in Zebrafish retino-tectal projections as an indication of developmental neurotoxicity

EPA Science Inventory

The U.S. EPA is developing alternative screening methods to identify putative developmental neurotoxicants and prioritize chemicals for additional testing. One method developmentally exposes zebrafish embryos and assesses nervous system structure at 2 days post-fertilization (dpf...

Developmental Effects of the ToxCast™ Phase I and Phase II Chemicals in Caenorhabditis elegans and Corresponding Responses in Zebrafish, Rats, and Rabbits

PubMed Central

Boyd, Windy A.; Smith, Marjolein V.; Co, Caroll A.; Pirone, Jason R.; Rice, Julie R.; Shockley, Keith R.; Freedman, Jonathan H.

2015-01-01

Background: Modern toxicology is shifting from an observational to a mechanistic science. As part of this shift, high-throughput toxicity assays are being developed using alternative, nonmammalian species to prioritize chemicals and develop prediction models of human toxicity. Methods: The nematode Caenorhabditis elegans (C. elegans) was used to screen the U.S. Environmental Protection Agency’s (EPA’s) ToxCast™ Phase I and Phase II libraries, which contain 292 and 676 chemicals, respectively, for chemicals leading to decreased larval development and growth. Chemical toxicity was evaluated using three parameters: a biologically defined effect size threshold, half-maximal activity concentration (AC50), and lowest effective concentration (LEC). Results: Across both the Phase I and Phase II libraries, 62% of the chemicals were classified as active ≤ 200 μM in the C. elegans assay. Chemical activities and potencies in C. elegans were compared with those from two zebrafish embryonic development toxicity studies and developmental toxicity data for rats and rabbits. Concordance of chemical activity was higher between C. elegans and one zebrafish assay across Phase I chemicals (79%) than with a second zebrafish assay (59%). Using C. elegans or zebrafish to predict rat or rabbit developmental toxicity resulted in balanced accuracies (the average value of the sensitivity and specificity for an assay) ranging from 45% to 53%, slightly lower than the concordance between rat and rabbit (58%). Conclusions: Here, we present an assay that quantitatively and reliably describes the effects of chemical toxicants on C. elegans growth and development. We found significant overlap in the activity of chemicals in the ToxCast™ libraries between C. elegans and zebrafish developmental screens. Incorporating C. elegans toxicological assays as part of a battery of in vitro and in vivo assays provides additional information for the development of models to predict a chemical’s potential toxicity to humans. Citation: Boyd WA, Smith MV, Co CA, Pirone JR, Rice JR, Shockley KR, Freedman JH. 2016. Developmental effects of the ToxCast™ Phase I and II chemicals in Caenorhabditis elegans and corresponding responses in zebrafish, rats, and rabbits. Environ Health Perspect 124:586–593; http://dx.doi.org/10.1289/ehp.1409645 PMID:26496690

In silico identification of anthropogenic chemicals as ligands of zebrafish sex hormone binding globulin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thorsteinson, Nels; Ban, Fuqiang; Santos-Filho, Osvaldo

2009-01-01

Anthropogenic compounds with the capacity to interact with the steroid-binding site of sex hormone binding globulin (SHBG) pose health risks to humans and other vertebrates including fish. Building on studies of human SHBG, we have applied in silico drug discovery methods to identify potential binders for SHBG in zebrafish (Danio rerio) as a model aquatic organism. Computational methods, including; homology modeling, molecular dynamics simulations, virtual screening, and 3D QSAR analysis, successfully identified 6 non-steroidal substances from the ZINC chemical database that bind to zebrafish SHBG (zfSHBG) with low-micromolar to nanomolar affinities, as determined by a competitive ligand-binding assay. We alsomore » screened 80,000 commercial substances listed by the European Chemicals Bureau and Environment Canada, and 6 non-steroidal hits from this in silico screen were tested experimentally for zfSHBG binding. All 6 of these compounds displaced the [{sup 3}H]5{alpha}-dihydrotestosterone used as labeled ligand in the zfSHBG screening assay when tested at a 33 {mu}M concentration, and 3 of them (hexestrol, 4-tert-octylcatechol, and dihydrobenzo(a)pyren-7(8H)-one) bind to zfSHBG in the micromolar range. The study demonstrates the feasibility of large-scale in silico screening of anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Such studies could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.« less

Acute Neuroactive Drug Exposures alter Locomotor Activity in Larval Zebrafish

EPA Science Inventory

As part of the development of a rapid in vivo screen for prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae by assessing the acute effects of prototypic drugs that act on the central nervous system. Initially,...

Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity

EPA Science Inventory

Chemically-induced vascular toxicity during embryonic development may cause a wide range of adverse effects. To identify putative vascular disrupting chemicals (pVDCs), a predictive signature was constructed from U.S. EPA ToxCast high-throughput screening (HTS) assays that map to...

Drugs Targeting the Dopaminergic Nervous System Alter Locomotion in Larval Zebrafish

EPA Science Inventory

As part of an effort at the US Environmental Protection Agency to develop a rapid in vivo screen for prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae. This includes assessing the acute effects of drugs that ...

Drugs that Target Dopamine Receptors: Changes in Locomotor Activity in Larval Zebrafish

EPA Science Inventory

As part of an effort at the US Environmental Protection Agency to develop a rapid in vivo screen for prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae. This includes assessing the acute effects of drugs known...

Acute neuroactive drug exposures alter locomotor activity in larval zebrafish

EPA Science Inventory

In an effort to develop a rapid in vivo screen for EPA's prioritization of toxic chemicals, we are characterizing the locomotor activity of zebrafish (Danio rerio) larvae after exposure to prototypic drugs that act on the central nervous system. MPTP (1-methyl-4phenyl- 1 ,2,3,6-...

Developmental Exposure to a Dopaminergic Toxicant Produces Altered Locomotor Activity in Larval Zebrafish

EPA Science Inventory

In an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we are characterizing the locomotor activity of zebrafish (Danio rerio) larvae after developmental exposure to various classes of prototypic drugs that act on the central nervous system. ...

ZebraBeat: a flexible platform for the analysis of the cardiac rate in zebrafish embryos

NASA Astrophysics Data System (ADS)

de Luca, Elisa; Zaccaria, Gian Maria; Hadhoud, Marwa; Rizzo, Giovanna; Ponzini, Raffaele; Morbiducci, Umberto; Santoro, Massimo Mattia

2014-05-01

Heartbeat measurement is important in assesssing cardiac function because variations in heart rhythm can be the cause as well as an effect of hidden pathological heart conditions. Zebrafish (Danio rerio) has emerged as one of the most useful model organisms for cardiac research. Indeed, the zebrafish heart is easily accessible for optical analyses without conducting invasive procedures and shows anatomical similarity to the human heart. In this study, we present a non-invasive, simple, cost-effective process to quantify the heartbeat in embryonic zebrafish. To achieve reproducibility, high throughput and flexibility (i.e., adaptability to any existing confocal microscope system and with a user-friendly interface that can be easily used by researchers), we implemented this method within a software program. We show here that this platform, called ZebraBeat, can successfully detect heart rate variations in embryonic zebrafish at various developmental stages, and it can record cardiac rate fluctuations induced by factors such as temperature and genetic- and chemical-induced alterations. Applications of this methodology may include the screening of chemical libraries affecting heart rhythm and the identification of heart rhythm variations in mutants from large-scale forward genetic screens.

Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure

PubMed Central

Nath, Anjali K.; Roberts, Lee D.; Liu, Yan; Mahon, Sari B.; Kim, Sonia; Ryu, Justine H.; Werdich, Andreas; Januzzi, James L.; Boss, Gerry R.; Rockwood, Gary A.; MacRae, Calum A.; Brenner, Matthew; Gerszten, Robert E.; Peterson, Randall T.

2013-01-01

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.—Nath, A. K., Roberts, L. D., Liu, Y., Mahon, S. B., Kim, S., Ryu, J. H., Werdich, A., Januzzi, J. L., Boss, G. R., Rockwood, G. A., MacRae, C. A., Brenner, M., Gerszten, R. E., Peterson, R. T. Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure. PMID:23345455

Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome1,2,3

PubMed Central

Dinday, Matthew T.

2015-01-01

Abstract Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy. PMID:26465006

The Effects of Acute Exposure to Neuroactive Drugs on the Locomotor Activity of Larval Zebrafish

EPA Science Inventory

In an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae using prototypic drugs that act on the central nervous system. Initially, we chose to define the beh...

Behavioral repertoire of larval zebrafish: Baseline activity and response to drug treatment.

EPA Science Inventory

As part of the EPA’s effort to develop an in vivo, vertebrate screen for toxic chemicals, we have begun to characterize basic behaviors of 6-day post-fertilization (dpf) zebrafish (Danio rerio) larvae in a microtiter plate format. Our main goal is to develop a method for rapidly ...

A forward chemical screen in zebrafish identifies a retinoic acid derivative with receptor specificity.

PubMed

Das, Bhaskar C; McCartin, Kellie; Liu, Ting-Chun; Peterson, Randall T; Evans, Todd

2010-04-02

Retinoids regulate key developmental pathways throughout life, and have potential uses for differentiation therapy. It should be possible to identify novel retinoids by coupling new chemical reactions with screens using the zebrafish embryonic model. We synthesized novel retinoid analogues and derivatives by amide coupling, obtaining 80-92% yields. A small library of these compounds was screened for bioactivity in living zebrafish embryos. We found that several structurally related compounds significantly affect development. Distinct phenotypes are generated depending on time of exposure, and we characterize one compound (BT10) that produces specific cardiovascular defects when added 1 day post fertilization. When compared to retinoic acid (ATRA), BT10 shows similar but not identical changes in the expression pattern of embryonic genes that are known targets of the retinoid pathway. Reporter assays determined that BT10 interacts with all three RAR receptor sub-types, but has no activity for RXR receptors, at all concentrations tested. Our screen has identified a novel retinoid with specificity for retinoid receptors. This lead compound may be useful for manipulating components of retinoid signaling networks, and may be further derivatized for enhanced activity.

Zebrafish: A marvel of high-throughput biology for 21st century toxicology.

PubMed

Bugel, Sean M; Tanguay, Robert L; Planchart, Antonio

2014-09-07

The evolutionary conservation of genomic, biochemical and developmental features between zebrafish and humans is gradually coming into focus with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput (HT) screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing.

Zebrafish: A marvel of high-throughput biology for 21st century toxicology

PubMed Central

Bugel, Sean M.; Tanguay, Robert L.; Planchart, Antonio

2015-01-01

The evolutionary conservation of genomic, biochemical and developmental features between zebrafish and humans is gradually coming into focus with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput (HT) screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing. PMID:25678986

The toxicological application of transcriptomics and epigenomics in zebrafish and other teleosts.

PubMed

Williams, Tim D; Mirbahai, Leda; Chipman, J Kevin

2014-03-01

Zebrafish (Danio rerio) is one of a number of teleost fish species frequently employed in toxicology. Toxico-genomics determines global transcriptomic responses to chemical exposures and can predict their effects. It has been applied successfully within aquatic toxicology to assist in chemical testing, determination of mechanisms and environmental monitoring. Moreover, the related field of toxico-epigenomics, that determines chemical-induced changes in DNA methylation, histone modifications and micro-RNA expression, is emerging as a valuable contribution to understanding mechanisms of both adaptive and adverse responses. Zebrafish has proven a useful and convenient model species for both transcriptomic and epigenetic toxicological studies. Despite zebrafish's dominance in other areas of fish biology, alternative fish species are used extensively in toxico-genomics. The main reason for this is that environmental monitoring generally focuses on species native to the region of interest. We are starting to see advances in the integration of high-throughput screening, omics techniques and bioinformatics together with more traditional indicator endpoints that are relevant to regulators. Integration of such approaches with high-throughput testing of zebrafish embryos, leading to the discovery of adverse outcome pathways, promises to make a major contribution to ensuring the safety of chemicals in the environment.

Miconazole protects blood vessels from MMP9-dependent rupture and hemorrhage

PubMed Central

Yang, Ran; Zhang, Yunpei; Huang, Dandan; Luo, Xiao; Zhang, Liangren; Zhu, Xiaojun; Zhang, Xiaolin; Liu, Zhenming; Han, Jing-Yan

2017-01-01

ABSTRACT Hemorrhagic stroke accounts for 10-15% of all strokes and is strongly associated with mortality and morbidity worldwide, but its prevention and therapeutic interventions remain a major challenge. Here, we report the identification of miconazole as a hemorrhagic suppressor by a small-molecule screen in zebrafish. We found that a hypomorphic mutant fn40a, one of several known β-pix mutant alleles in zebrafish, had the major symptoms of brain hemorrhage, vessel rupture and inflammation as those in hemorrhagic stroke patients. A small-molecule screen with mutant embryos identified the anti-fungal drug miconazole as a potent hemorrhagic suppressor. Miconazole inhibited both brain hemorrhages in zebrafish and mesenteric hemorrhages in rats by decreasing matrix metalloproteinase 9 (MMP9)-dependent vessel rupture. Mechanistically, miconazole downregulated the levels of pErk and Mmp9 to protect vascular integrity in fn40a mutants. Therefore, our findings demonstrate that miconazole protects blood vessels from hemorrhages by downregulating the pERK-MMP9 axis from zebrafish to mammals and shed light on the potential of phenotype-based screens in zebrafish for the discovery of new drug candidates and chemical probes for hemorrhagic stroke. PMID:28153846

Acute effects of ethanol or d-amphetamine on the locomotor activity of larval zebrafish in a microtiter plate format.

EPA Science Inventory

As part of an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae. We are assessing the acute effects of prototypic drugs that are known to act on the central ...

Uncertainty Quantification in High Throughput Screening: Applications to Models of Endocrine Disruption, Cytotoxicity, and Zebrafish Development (GRC Drug Safety)

EPA Science Inventory

Using uncertainty quantification, we aim to improve the quality of modeling data from high throughput screening assays for use in risk assessment. ToxCast is a large-scale screening program that analyzes thousands of chemicals using over 800 assays representing hundreds of bioche...

Transcriptomic and phenotypic profiling in developing zebrafish exposed to thyroid hormone receptor agonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haggard, Derik E.; Noyes, Pamela D.; Waters, Katrina M.

There is a need to develop novel, high-throughput screening and prioritization methods to identify chemicals with adverse estrogen, androgen, and thyroid activity to protect human health and the environment and is of interest to the Endocrine Disruptor Screening Program. The current aim is to explore the utility of zebrafish as a testing paradigm to classify endocrine activity using phenotypically anchored transcriptome profiling. Transcriptome analysis was conducted on embryos exposed to 25 estrogen-, androgen-, or thyroid-active chemicals at a concentration that elicited adverse malformations or mortality at 120 hours post-fertilization in 80% of the animals exposed. Analysis of the top 1000more » significant differentially expressed transcripts across all treatments identified a unique transcriptional and phenotypic profile for thyroid hormone receptor agonists, which can be used as a biomarker screen for potential thyroid hormone agonists.« less

First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass

PubMed Central

Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

2015-01-01

Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001 PMID:26218223

Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening**

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. As such, we are exploring a behavioral testing paradigm, which can assess the effect of sublethal and subteratogenic concentrations of de...

Change in Gene Expression in Zebrafish as an Endpoint for Developmental Neurotoxicity Screening

EPA Science Inventory

Chemicals that adversely affect the developing nervous system may have long-term consequences on human health. Little information exists on a large number of environmental chemicals to guide the risk assessments for developmental neurotoxicity (DNT). As traditional developmental ...

Zebrafish: An Important Tool for Liver Disease Research

PubMed Central

Goessling, Wolfram; Sadler, Kirsten C.

2016-01-01

As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. PMID:26319012

Screening for Developmental Neurotoxicity in Zebrafish Larvae: Assessment of Behavior and Malformations.

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. As part of this approach, it is important to be able to separate overt toxicity (Le., malformed larvae) from the more specific neurotoxic...

Repairing quite swimmingly: advances in regenerative medicine using zebrafish.

PubMed

Goessling, Wolfram; North, Trista E

2014-07-01

Regenerative medicine has the promise to alleviate morbidity and mortality caused by organ dysfunction, longstanding injury and trauma. Although regenerative approaches for a few diseases have been highly successful, some organs either do not regenerate well or have no current treatment approach to harness their intrinsic regenerative potential. In this Review, we describe the modeling of human disease and tissue repair in zebrafish, through the discovery of disease-causing genes using classical forward-genetic screens and by modulating clinically relevant phenotypes through chemical genetic screening approaches. Furthermore, we present an overview of those organ systems that regenerate well in zebrafish in contrast to mammalian tissue, as well as those organs in which the regenerative potential is conserved from fish to mammals, enabling drug discovery in preclinical disease-relevant models. We provide two examples from our own work in which the clinical translation of zebrafish findings is either imminent or has already proven successful. The promising results in multiple organs suggest that further insight into regenerative mechanisms and novel clinically relevant therapeutic approaches will emerge from zebrafish research in the future. © 2014. Published by The Company of Biologists Ltd.

A plasmid library of full-length zebrafish rab proteins for in vivo cell biology.

PubMed

Hall, Thomas E; Martel, Nick; Lo, Harriet P; Xiong, Zherui; Parton, Robert G

2017-01-01

The zebrafish is an emerging model for highly sophisticated medium-throughput experiments such as genetic and chemical screens. However, studies of entire protein families within this context are often hampered by poor genetic resources such as clone libraries. Here we describe a complete collection of 76 full-length open reading frame clones for the zebrafish rab protein family. While the mouse genome contains 60 rab genes and the human genome 63, we find that 18 zebrafish rab genes have 2, and in the case of rab38, 3 paralogues. In contrast, we were unable to identify zebrafish orthologues of the mammalian Rab2b, Rab17 or Rab29. We make this resource available through the Addgene repository to facilitate cell biologic approaches using this model.

Zebrafish in the sea of mineral (iron, zinc, and copper) metabolism

PubMed Central

Zhao, Lu; Xia, Zhidan; Wang, Fudi

2014-01-01

Iron, copper, zinc, and eight other minerals are classified as essential trace elements because they present in minute in vivo quantities and are essential for life. Because either excess or insufficient levels of trace elements can be detrimental to life (causing human diseases such as iron-deficiency anemia, hemochromatosis, Menkes syndrome and Wilson's disease), the endogenous levels of trace minerals must be tightly regulated. Many studies have demonstrated the existence of systems that maintain trace element homeostasis, and these systems are highly conserved in multiple species ranging from yeast to mice. As a model for studying trace mineral metabolism, the zebrafish is indispensable to researchers. Several large-scale mutagenesis screens have been performed in zebrafish, and these screens led to the identification of a series of metal transporters and the generation of several mutagenesis lines, providing an in-depth functional analysis at the system level. Moreover, because of their developmental advantages, zebrafish have also been used in mineral metabolism-related chemical screens and toxicology studies. Here, we systematically review the major findings of trace element homeostasis studies using the zebrafish model, with a focus on iron, zinc, copper, selenium, manganese, and iodine. We also provide a homology analysis of trace mineral transporters in fish, mice and humans. Finally, we discuss the evidence that zebrafish is an ideal experimental tool for uncovering novel mechanisms of trace mineral metabolism and for improving approaches to treat mineral imbalance-related diseases. PMID:24639652

Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos

PubMed Central

2013-01-01

Background In mammals, ABCB1 constitutes a cellular “first line of defense” against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water. Results Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a polycyclic aromatic hydrocarbon) and galaxolide and tonalide (two polycyclic musks). Conclusions We show that zebrafish Abcb4 is a cellular toxicant transporter and provides protection of embryos against toxic chemicals dissolved in the water. Zebrafish Abcb4 thus is functionally similar to mammalian ABCB1, but differs from mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in the liver. Our data have important implications: Abcb4 could affect bioavailability - and thus toxicologic and pharmacologic potency - of chemicals to zebrafish embryos and inhibition of Abcb4 therefore causes chemosensitization, that is, enhanced sensitivity of embryos to toxicants. These aspects should be considered in (eco)toxicologic and pharmacologic chemical screens with the zebrafish embryo, a major vertebrate model. PMID:23773777

Developmental Exposure to Valproate or Ethanol Alters Locomotor Activity and Retino-Tectal Projection Area in Zebrafish Embryos

EPA Science Inventory

Given the minimal developmental neurotoxicity data available for the large number of new and existing chemicals, there is a critical need for alternative methods to identify and prioritize chemicals for further testing. We outline a developmental neurotoxicity screening approach ...

Zebrafish: an important tool for liver disease research.

PubMed

Goessling, Wolfram; Sadler, Kirsten C

2015-11-01

As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

A High-Throughput Fluorescence-Based Assay System for Appetite-Regulating Gene and Drug Screening

PubMed Central

Shimada, Yasuhito; Hirano, Minoru; Nishimura, Yuhei; Tanaka, Toshio

2012-01-01

The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish). This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf), knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1), and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant) revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers. PMID:23300705

Zebrafish Models for Human Acute Organophosphorus Poisoning.

PubMed

Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick Ii, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B Lynn; Zorzano, Antonio; Soares, Amadeu M V M; Raldúa, Demetrio

2015-10-22

Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

Screening Estrogenic Activities of Chemicals or Mixtures In Vivo Using Transgenic (cyp19a1b-GFP) Zebrafish Embryos

PubMed Central

Brion, François; Le Page, Yann; Piccini, Benjamin; Cardoso, Olivier; Tong, Sok-Keng; Chung, Bon-chu; Kah, Olivier

2012-01-01

The tg(cyp19a1b-GFP) transgenic zebrafish expresses GFP (green fluorescent protein) under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i) it is only expressed in radial glial progenitors in the brain of fish and (ii) it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture), including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective. PMID:22586461

Application of Caenorhabditis elegans (nematode) and Danio rerio embryo (zebrafish) as model systems to screen for developmental and reproductive toxicity of Piperazine compounds.

PubMed

Racz, Peter I; Wildwater, Marjolein; Rooseboom, Martijn; Kerkhof, Engelien; Pieters, Raymond; Yebra-Pimentel, Elena Santidrian; Dirks, Ron P; Spaink, Herman P; Smulders, Chantal; Whale, Graham F

2017-10-01

To enable selection of novel chemicals for new processes, there is a recognized need for alternative toxicity screening assays to assess potential risks to man and the environment. For human health hazard assessment these screening assays need to be translational to humans, have high throughput capability, and from an animal welfare perspective be harmonized with the principles of the 3Rs (Reduction, Refinement, Replacement). In the area of toxicology a number of cell culture systems are available but while these have some predictive value, they are not ideally suited for the prediction of developmental and reproductive toxicology (DART). This is because they often lack biotransformation capacity, multicellular or multi- organ complexity, for example, the hypothalamus pituitary gonad (HPG) axis and the complete life cycle of whole organisms. To try to overcome some of these limitations in this study, we have used Caenorhabditis elegans (nematode) and Danio rerio embryos (zebrafish) as alternative assays for DART hazard assessment of some candidate chemicals being considered for a new commercial application. Nematodes exposed to Piperazine and one of the analogs tested showed a slight delay in development compared to untreated animals but only at high concentrations and with Piperazine as the most sensitive compound. Total brood size of the nematodes was also reduced primarily by Piperazine and one of the analogs. In zebrafish Piperazine and analogs showed developmental delays. Malformations and mortality in individual fish were also scored. Significant malformations were most sensitively identified with Piperazine, significant mortality was only observed in Piperazine and only at the higest dose. Thus, Piperazine seemed the most toxic compound for both nematodes and zebrafish. The results of the nematode and zebrafish studies were in alignment with data obtained from conventional mammalian toxicity studies indicating that these have potential as developmental toxicity screening systems. The results of these studies also provided reassurance that none of the Piperazines tested are likely to have any significant developmental and/or reproductive toxicity issues to humans when used in their commercial applications. Copyright © 2017. Published by Elsevier Ltd.

Fetal Origins of Life Stage Disease: A Zebrafish Model for the ...

EPA Pesticide Factsheets

In the U.S., childhood obesity has more than doubled in children and quadrupled in adolescents in the past 30 years, affects 35% of adults, and costs the U.S. healthcare industry >$200 billion annually. The chemical environment in the womb may cause susceptibility to different life-stage and life-long metabolic diseases including obesity. The challenge is to understand if exposures during developmentally sensitive windows impact life-stage disease, such as obesity, by increasing adipose tissue mass. In vitro models lack the integrated systems approach needed to assess adipose development, while mammalian models are impractical in a screen of thousands of chemicals. Therefore, an obesogen screening method was developed to interrogate bioactivity using a full systems approach, in a vertebrate zebrafish model with complete metabolic activity, at a time when the full signaling repertoire is expressed and active, to optimally examine how chemical dose and duration impact life-stage adipose mass. A time-line for adipose depot formation was mapped in zebrafish 6−14 days post fertilization (dpf) using the lipophilic dye, Nile Red, in combination with fluorescent microscopy. Those time points were then used to investigate the impact of embryonic tributyltin chloride (TBT, a known obesogen) exposure (10nM daily renewal, 0−5dpf) on adipose mass. Fluorescent microscopy revealed adipose depots that were larger and appeared 2 days earlier in TBT treated compared to contro

Zebrafish Locomotor Responses Reveal Irritant Effects of Fine Particulate Matter Extracts and a Role for TRPA1.

PubMed

Stevens, Joey S; Padilla, Stephanie; DeMarini, David M; Hunter, Deborah L; Martin, W Kyle; Thompson, Leslie C; Gilmour, M Ian; Hazari, Mehdi S; Farraj, Aimen K

2018-02-01

Exposure to fine particulate matter (PM) air pollution causes adverse cardiopulmonary outcomes. Yet, the limited capacity to readily identify contributing PM sources and associated PM constituents in any given ambient air shed impedes risk assessment efforts. The health effects of PM have been attributed in part to its capacity to elicit irritant responses. A variety of chemicals trigger irritant behavior responses in zebrafish that can be easily measured. The purposes of this study were to examine the utility of zebrafish locomotor responses in the toxicity assessment of fine PM and its chemical fractions and uncover mechanisms of action. Locomotor responses were recorded in 6-day-old zebrafish exposed for 60 min in the dark at 26 °C to the extractable organic matter of a compressor-generated diesel exhaust PM (C-DEP) and 4 of its fractions (F1-F4) containing varying chemical classes of increasing polarity. The role of the transient receptor potential (TRP) cation channel TRPA1, a chemical sensor in mammals and zebrafish, in locomotor responses to C-DEP, was also examined. Acrolein, an environmental irritant and known activator of TRPA1, and all extracts induced concentration-dependent locomotor responses whose potencies ranked as follows: polar F3 > weakly polar F2 > C-DEP > highly polar F4 > nonpolar F1, indicating that polar and weakly polar fractions that included nitro- and oxy-polyaromatic hydrocarbons (PAHs), drove C-DEP responses. Irritant potencies in fish positively correlated with mutagenic potencies of the same extracts in strains of Salmonella sensitive to nitro- and oxy-PAHs, further implicating these chemical classes in the zebrafish responses to C-DEP. Pharmacologic inhibition of TRPA1 blocked locomotor responses to acrolein and the extracts. Taken together, these data indicate that the zebrafish locomotor assay may help expedite toxicity screening of fine PM sources, identify causal chemical classes, and uncover plausible biological mechanisms. Published by Oxford University Press on behalf of the Society of Toxicology 2017. This work is written by US Government employees and is in the public domain in the United States.

Evaluation of 14 Organic Solvents and Carriers for Screening Applications in Zebrafish Embryos and Larvae

PubMed Central

Maes, Jan; Verlooy, Lien; Buenafe, Olivia E.; de Witte, Peter A. M.; Esguerra, Camila V.; Crawford, Alexander D.

2012-01-01

Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2–3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies – e.g. omics analyses –should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish. PMID:23082109

Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration.

PubMed

Ye, Baixin; Xiong, Xiaoxing; Deng, Xu; Gu, Lijuan; Wang, Qiongyu; Zeng, Zhi; Gao, Xiang; Gao, Qingping; Wang, Yueying

2017-12-01

Inflammatory disease is a big threat to human health. Leukocyte chemotactic migration is required for efficient inflammatory response. Inhibition of leukocyte chemotactic migration to the inflammatory site has been shown to provide therapeutic targets for treating inflammatory diseases. Our study was designed to discover effective and safe compounds that can inhibit leukocyte chemotactic migration, thus providing possible novel therapeutic strategy for treating inflammatory diseases. In this study, we used transgenic zebrafish model (Tg:zlyz-EGFP line) to visualize the process of leukocyte chemotactic migration. Then, we used this model to screen the hit compound and evaluate its biological activity on leukocyte chemotactic migration. Furthermore, western blot analysis was performed to evaluate the effect of the hit compound on the AKT or ERK-mediated pathway, which plays an important role in leukocyte chemotactic migration. In this study, using zebrafish-based chemical screening, we identified that the hit compound meisoindigo (25 μM, 50 μM, 75 μM) can significantly inhibit zebrafish leukocyte chemotactic migration in a dose-dependent manner (p = 0.01, p = 0.0006, p < 0.0001). Also, we found that meisoindigo did not affect the process of leukocyte reverse migration (p = 0.43). Furthermore, our results unexpectedly showed that indirubin, the core structure of meisoindigo, had no significant effect on zebrafish leukocyte chemotactic migration (p = 0.6001). Additionally, our results revealed that meisoindigo exerts no effect on the Akt or Erk-mediated signalling pathway. Our results suggest that meisoindigo, but not indirubin, is effective for inhibiting leukocyte chemotactic migration, thus providing a potential therapeutic agent for treating inflammatory diseases.

Development of a quantitative morphological assessment of toxicant-treated zebrafish larvae using brightfield imaging and high-content analysis.

PubMed

Deal, Samantha; Wambaugh, John; Judson, Richard; Mosher, Shad; Radio, Nick; Houck, Keith; Padilla, Stephanie

2016-09-01

One of the rate-limiting procedures in a developmental zebrafish screen is the morphological assessment of each larva. Most researchers opt for a time-consuming, structured visual assessment by trained human observer(s). The present studies were designed to develop a more objective, accurate and rapid method for screening zebrafish for dysmorphology. Instead of the very detailed human assessment, we have developed the computational malformation index, which combines the use of high-content imaging with a very brief human visual assessment. Each larva was quickly assessed by a human observer (basic visual assessment), killed, fixed and assessed for dysmorphology with the Zebratox V4 BioApplication using the Cellomics® ArrayScan® V(TI) high-content image analysis platform. The basic visual assessment adds in-life parameters, and the high-content analysis assesses each individual larva for various features (total area, width, spine length, head-tail length, length-width ratio, perimeter-area ratio). In developing the computational malformation index, a training set of hundreds of embryos treated with hundreds of chemicals were visually assessed using the basic or detailed method. In the second phase, we assessed both the stability of these high-content measurements and its performance using a test set of zebrafish treated with a dose range of two reference chemicals (trans-retinoic acid or cadmium). We found the measures were stable for at least 1 week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our computational malformation index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

The sensitivity and reproducibility of the zebrafish (Danio rerio) embryo test for the screening of waste water quality and for testing the toxicity of chemicals.

PubMed

Lahnsteiner, Franz

2008-07-01

The sensitivity of the zebrafish embryo test, a test proposed for routine waste water control, was compared with the acute fish toxicity test, in the determination of six types of waste water and ten different chemicals. The waste water was sampled from the following industrial processes: paper and cardboard production, hide tanning, metal galvanisation, carcass treatment and utilisation, and sewage treatment. The chemicals tested were: dimethylacetamide, dimethylsulphoxide, cadmium chloride, cyclohexane, hydroquinone, mercuric chloride, nickel chloride, nonylphenol, resmethrin and sodium nitrite. For many of the test substances, the zebrafish embryo test and the acute fish toxicity test results showed high correlations. However, there were certain environmentally-relevant substances for which the results of the zebrafish embryo test and the acute fish toxicity test differed significantly, up to 10,000-fold (Hg(2+) > 150-fold difference; NO(2)(-) > 300-fold; Cd(2+) > 200-fold; resmethrin > 10,000-fold). For the investigated waste water samples and chemicals, the survival rate of the zebrafish embryos showed high variations between different egg samples, within the range of the EC50 concentration. Subsequently, 5-6 parallel assays were deemed to be the appropriate number necessary for the precise evaluation of the toxicity of the test substances. Also, it was found that the sensitivities of different ontogenetic stages to chemical exposure differed greatly. During the first 12 hours after fertilisation (4-cell stage to the 5-somite stage), the embryos reacted most sensitively to test substance exposure, whereas the later ontogenetic stages showed only slight or no response, indicating that the test is most sensitive during the first 24 hours post-fertilisation.

Zebrafish Embryo Toxicity Microscale Model for Ichthyotoxicity Evaluation of Marine Natural Products.

PubMed

Bai, Hong; Kong, Wen-Wen; Shao, Chang-Lun; Li, Yun; Liu, Yun-Zhang; Liu, Min; Guan, Fei-Fei; Wang, Chang-Yun

2016-04-01

Marine organisms often protect themselves against their predators by chemical defensive strategy. The second metabolites isolated from marine organisms and their symbiotic microbes have been proven to play a vital role in marine chemical ecology, such as ichthyotoxicity, allelopathy, and antifouling. It is well known that the microscale models for marine chemoecology assessment are urgently needed for trace quantity of marine natural products. Zebrafish model has been widely used as a microscale model in the fields of environment ecological evaluation and drug safety evaluation, but seldom reported for marine chemoecology assessment. In this work, zebrafish embryo toxicity microscale model was established for ichthyotoxicity evaluation of marine natural products by using 24-well microplate based on zebrafish embryo. Ichthyotoxicity was evaluated by observation of multiple toxicological endpoints, including coagulation egg, death, abnormal heartbeat, no spontaneous movement, delayed hatch, and malformation of the different organs during zebrafish embryogenesis periods at 24, 48, and 72 h post-fertilization (hpf). 3,4-Dichloroaniline was used as the positive control for method validation. Subsequently, the established model was applied to test the ichthyotoxic activity of the compounds isolated from corals and their symbiotic microbes and to isolate the bioactive secondary metabolites from the gorgonian Subergorgia mollis under bioassay guidance. It was suggested that zebrafish embryo toxicity microscale model is suitable for bioassay-guided isolation and preliminary bioactivity screening of marine natural products.

A screening approach using zebrafish for the detection and characterization of developmental neurotoxicity.

EPA Science Inventory

Thousands of chemicals have little or no data to support developmental neurotoxicity risk assessments. Current developmental neurotoxicity guideline studies mandating mammalian model systems are expensive and time consuming. Therefore a rapid, cost-effective method to assess de...

Using the zebrafish to understand tendon development and repair

PubMed Central

Chen, Jessica W.; Galloway, Jenna L.

2017-01-01

Tendons are important components of our musculoskeletal system. Injuries to these tissues are very common, resulting from occupational-related injuries, sports-related trauma, and age-related degeneration. Unfortunately, there are few treatment options, and current therapies rarely restore injured tendons to their original function. An improved understanding of the pathways regulating their development and repair would have significant impact in stimulating the formulation of regenerative-based approaches for tendon injury. The zebrafish provides an ideal system in which to perform genetic and chemical screens to identify new pathways involved in tendon biology. Until recently, there had been few descriptions of tendons and ligaments in the zebrafish and their similarity to mammalian tendon tissues. In this chapter, we describe the development of the zebrafish tendon and ligament tissues in the context of their gene expression, structure, and interactions with neighboring musculoskeletal tissues. We highlight the similarities with tendon development in higher vertebrates, showing that the craniofacial tendons and ligaments in zebrafish morphologically, molecularly, and structurally resemble mammalian tendons and ligaments from embryonic to adult stages. We detail methods for fluorescent in situ hybridization and immunohistochemistry as an assay to examine morphological changes in the zebrafish musculoskeleton. Staining assays such as these could provide the foundation for screen-based approaches to identify new regulators of tendon development, morphogenesis, and repair. These discoveries would provide new targets and pathways to study in the context of regenerative medicine-based approaches to improve tendon healing. PMID:28129848

Immune response induced by major environmental pollutants through altering neutrophils in zebrafish larvae.

PubMed

Xu, Hongyan; Zhang, Xiaoyan; Li, Hankun; Li, Caixia; Huo, Xiao-Jing; Hou, Li-Ping; Gong, Zhiyuan

2018-06-06

Environmental pollutants may cause adverse effects on the immune system of aquatic organisms. However, the cellular effects of pollutants on fish immune system are largely unknown. Here, we exploited the transgenic zebrafish Tg(lysC:DsRed2) larva as a preliminary screening system to evaluate the potential inflammatory effects of environmental pollutants. Tg(lysC:DsRED2) larvae aged 7-day-postfertilization (7 dpf) were treated with selected environmental chemicals for 24 h (24 h) and the number of neutrophils were quantified using both image analysis and fluorescence activated cell sorting (FACS). We found that the numbers of neutrophils in the Tg(lysC:DsRED2) larvae were significantly increased by most of the organic chemicals tested, including E2 (17β-estradiol), BPA (Bisphenol-A), NDEA (N-nitrosodiethylamine), 4-NP (4-Nitrophenol) and Lindane (γ-hexachlorocyclohexane). Neutrophil numbers were also increased by all the metals tested (Na2HAsO4· 7H2O, Pb(NO3)2, HgCl 2 , CdCl2, CuSO 4 ·5H 2 O, ZnSO4, and K2Cr2O7). The only exception was TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which significantly reduced the number of neutrophils after exposure. Additionally, the transcription of genes (lyz, mpo, tnfα and il8) related to fish immune system were significantly modulated upon exposure to some of the selected chemicals such as E2, TCDD, Cu and Cd. This study revealed that representatives of major categories of environmental pollutants could cause an acute inflammatory response in zebrafish larvae as shown by alterations in the neutrophils, which may imply a common immunotoxicity mechanism for most environmental pollutants. This study has also demonstrated that Tg(lyz:DsRed2) transgenic zebrafish is an excellent tool for screening environmental chemicals with potential inflammatory effects through FACS-facilitated neutrophil counting. Copyright © 2018 Elsevier B.V. All rights reserved.

A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development

PubMed Central

Ciarlo, Christie; Kaufman, Charles K; Kinikoglu, Beste; Michael, Jonathan; Yang, Song; D′Amato, Christopher; Blokzijl-Franke, Sasja; den Hertog, Jeroen; Schlaeger, Thorsten M; Zhou, Yi; Liao, Eric

2017-01-01

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation. PMID:28832322

Zebrafish model systems for developmental neurobehavioral toxicology.

PubMed

Bailey, Jordan; Oliveri, Anthony; Levin, Edward D

2013-03-01

Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models. Copyright © 2013 Wiley Periodicals, Inc.

Zebrafish Model Systems for Developmental Neurobehavioral Toxicology

PubMed Central

Bailey, Jordan; Oliveri, Anthony; Levin, Edward D.

2014-01-01

Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models. PMID:23723169

Evaluating the Zebrafish Embryo Toxicity Test for Pesticide Hazard Screening

EPA Science Inventory

Given the numerous chemicals used in society, it is critical to develop tools for accurate and efficient evaluation of potential risks to human and ecological receptors. Fish embryo acute toxicity tests are 1 tool that has been shown to be highly predictive of standard, more reso...

From Omics to Drug Metabolism and High Content Screen of Natural Product in Zebrafish: A New Model for Discovery of Neuroactive Compound

PubMed Central

Hung, Ming Wai; Zhang, Zai Jun; Li, Shang; Lei, Benson; Yuan, Shuai; Cui, Guo Zhen; Man Hoi, Pui; Chan, Kelvin; Lee, Simon Ming Yuen

2012-01-01

The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed. PMID:22919414

Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures

PubMed Central

Baxendale, Sarah; Holdsworth, Celia J.; Meza Santoscoy, Paola L.; Harrison, Michael R. M.; Fox, James; Parkin, Caroline A.; Ingham, Philip W.; Cunliffe, Vincent T.

2012-01-01

SUMMARY The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca2+) flux in muscle cells as well as intense locomotor activity. We then screened a library of ∼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities. PMID:22730455

Fishing for Nature's Hits: Establishment of the Zebrafish as a Model for Screening Antidiabetic Natural Products.

PubMed

Tabassum, Nadia; Tai, Hongmei; Jung, Da-Woon; Williams, Darren R

2015-01-01

Diabetes mellitus affects millions of people worldwide and significantly impacts their quality of life. Moreover, life threatening diseases, such as myocardial infarction, blindness, and renal disorders, increase the morbidity rate associated with diabetes. Various natural products from medicinal plants have shown potential as antidiabetes agents in cell-based screening systems. However, many of these potential "hits" fail in mammalian tests, due to issues such as poor pharmacokinetics and/or toxic side effects. To address this problem, the zebrafish (Danio rerio) model has been developed as a "bridge" to provide an experimentally convenient animal-based screening system to identify drug candidates that are active in vivo. In this review, we discuss the application of zebrafish to drug screening technologies for diabetes research. Specifically, the discovery of natural product-based antidiabetes compounds using zebrafish will be described. For example, it has recently been demonstrated that antidiabetic natural compounds can be identified in zebrafish using activity guided fractionation of crude plant extracts. Moreover, the development of fluorescent-tagged glucose bioprobes has allowed the screening of natural product-based modulators of glucose homeostasis in zebrafish. We hope that the discussion of these advances will illustrate the value and simplicity of establishing zebrafish-based assays for antidiabetic compounds in natural products-based laboratories.

Development and automation of a test of impulse control in zebrafish

PubMed Central

Parker, Matthew O.; Ife, Dennis; Ma, Jun; Pancholi, Mahesh; Smeraldi, Fabrizio; Straw, Chris; Brennan, Caroline H.

2013-01-01

Deficits in impulse control (difficulties in inhibition of a pre-potent response) are fundamental to a number of psychiatric disorders, but the molecular and cellular basis is poorly understood. Zebrafish offer a very useful model for exploring these mechanisms, but there is currently a lack of validated procedures for measuring impulsivity in fish. In mammals, impulsivity can be measured by examining rates of anticipatory responding in the 5-choice serial reaction time task (5-CSRTT), a continuous performance task where the subject is reinforced upon accurate detection of a briefly presented light in one of five distinct spatial locations. This paper describes the development of a fully-integrated automated system for testing impulsivity in adult zebrafish. We outline the development of our image analysis software and its integration with National Instruments drivers and actuators to produce the system. We also describe an initial validation of the system through a one-generation screen of chemically mutagenized zebrafish, where the testing parameters were optimized. PMID:24133417

The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

PubMed Central

Uchiyama, Junzo; Koshimizu, Eriko; Qi, Jie; Nanjappa, Purushothama; Imamura, Shintaro; Islam, Asiful; Neuberg, Donna; Amsterdam, Adam; Roberts, Thomas M.

2008-01-01

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms. PMID:18704191

Fishing anti(lymph)angiogenic drugs with zebrafish.

PubMed

García-Caballero, Melissa; Quesada, Ana R; Medina, Miguel A; Marí-Beffa, Manuel

2018-02-01

Zebrafish, an amenable small teleost fish with a complex mammal-like circulatory system, is being increasingly used for drug screening and toxicity studies. It combines the biological complexity of in vivo models with a higher-throughput screening capability compared with other available animal models. Externally growing, transparent embryos, displaying well-defined blood and lymphatic vessels, allow the inexpensive, rapid, and automatable evaluation of drug candidates that are able to inhibit neovascularisation. Here, we briefly review zebrafish as a model for the screening of anti(lymph)angiogenic drugs, with emphasis on the advantages and limitations of the different zebrafish-based in vivo assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fish in a Dish: Drug Discovery for Hearing Habilitation.

PubMed

Esterberg, Robert; Coffin, Allison B; Ou, Henry; Simon, Julian A; Raible, David W; Rubel, Edwin W

2013-01-01

The majority of hearing loss is caused by the permanent loss of inner ear hair cells. The identification of drugs that modulate the susceptibility to hair cell loss or spur their regeneration is often hampered by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of sensory hair cells, uniquely position it as an ideal model organism for the study of hair cell toxicity, protection, and regeneration. We have used this model to screen for drugs that affect each of these aspects of hair cell biology and have identified compounds that affect each of these processes. The identification of such drugs and drug-like compounds holds promise in the future ability to stem hearing loss in the human population.

Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen

PubMed Central

Robertson, Anne L.; Ogryzko, Nikolay V.; Henry, Katherine M.; Loynes, Catherine A.; Foulkes, Matthew J.; Meloni, Marco M.; Wang, Xingang; Ford, Christopher; Jackson, Malcolm; Ingham, Philip W.; Wilson, Heather L.; Farrow, Stuart N.; Solari, Roberto; Flower, Roderick J.; Jones, Simon; Whyte, Moira K. B.

2016-01-01

ABSTRACT Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available ‘cromones’ are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease. PMID:27079522

Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xiaoxia; University of Chinese Academy of Sciences, Beijing; Chen, Xiaowen

The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were allmore » identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.« less

The HDAC Inhibitor TSA Ameliorates a Zebrafish Model of Duchenne Muscular Dystrophy.

PubMed

Johnson, Nathan M; Farr, Gist H; Maves, Lisa

2013-09-17

Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.

Evaluation of spontaneous propulsive movement as a screening tool to detect rescue of Parkinsonism phenotypes in zebrafish models

PubMed Central

Farrell, Thomas C.; Cario, Clinton L.; Milanese, Chiara; Vogt, Andreas; Jeong, Jong-Hyeon; Burton, Edward A.

2011-01-01

Zebrafish models of human neuropsychiatric diseases offer opportunities to identify novel therapeutic targets and treatments through phenotype-based genetic or chemical modifier screens. In order to develop an assay to detect rescue of zebrafish models of Parkinsonism, we characterized spontaneous zebrafish larval motor behavior from 3 to 9 days post fertilization in a microtiter plate format suitable for screening, and clarified the role of dopaminergic signaling in its regulation. The proportion of time that larvae spent moving increased progressively between 3 and 9 dpf, whereas their active velocity decreased between 5 and 6 dpf as sporadic burst movements gave way to a more mature beat-and-glide pattern. Spontaneous movement varied between larvae and during the course of recordings as a result of intrinsic larval factors including genetic background. Variability decreased with age, such that small differences between groups of larvae exposed to different experimental conditions could be detected robustly by 6 to 7 dpf. Suppression of endogenous dopaminergic signaling by exposure to MPP+, haloperidol or chlorpromazine reduced mean velocity by decreasing the frequency with which spontaneous movements were initiated, but did not alter active velocity. The variability of mean velocity assays could be reduced by analyzing groups of larvae for each data point, yielding acceptable screening window coefficients; the sample size required in each group was determined by the magnitude of the motor phenotype in different models. For chlorpromazine exposure, samples of four larvae allowed robust separation of treated and untreated data points (Z=0.42), whereas the milder impairment provoked by MPP+ necessitated groups of eight larvae in order to provide a useful discovery assay (Z=0.13). Quantification of spontaneous larval movement offers a simple method to determine functional integrity of motor systems, and may be a useful tool to isolate novel molecular modulators of Parkinsonism phenotypes. PMID:21669287

A data-driven weighting scheme for multivariate phenotypic endpoints recapitulates zebrafish developmental cascades

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Guozhu, E-mail: gzhang6@ncsu.edu

Zebrafish have become a key alternative model for studying health effects of environmental stressors, partly due to their genetic similarity to humans, fast generation time, and the efficiency of generating high-dimensional systematic data. Studies aiming to characterize adverse health effects in zebrafish typically include several phenotypic measurements (endpoints). While there is a solid biomedical basis for capturing a comprehensive set of endpoints, making summary judgments regarding health effects requires thoughtful integration across endpoints. Here, we introduce a Bayesian method to quantify the informativeness of 17 distinct zebrafish endpoints as a data-driven weighting scheme for a multi-endpoint summary measure, called weightedmore » Aggregate Entropy (wAggE). We implement wAggE using high-throughput screening (HTS) data from zebrafish exposed to five concentrations of all 1060 ToxCast chemicals. Our results show that our empirical weighting scheme provides better performance in terms of the Receiver Operating Characteristic (ROC) curve for identifying significant morphological effects and improves robustness over traditional curve-fitting approaches. From a biological perspective, our results suggest that developmental cascade effects triggered by chemical exposure can be recapitulated by analyzing the relationships among endpoints. Thus, wAggE offers a powerful approach for analysis of multivariate phenotypes that can reveal underlying etiological processes. - Highlights: • Introduced a data-driven weighting scheme for multiple phenotypic endpoints. • Weighted Aggregate Entropy (wAggE) implies differential importance of endpoints. • Endpoint relationships reveal developmental cascade effects triggered by exposure. • wAggE is generalizable to multi-endpoint data of different shapes and scales.« less

Purinergic and adenosine receptors contribute to hypoxic hyperventilation in zebrafish (Danio rerio).

PubMed

Coe, Alisha J; Picard, Alexina J; Jonz, Michael G

2017-12-01

The chemoreceptors involved in oxygen sensing in teleost fish are neuroepithelial cells (NECs) in the gills, and are analogous to glomus cells in the mammalian carotid body. Purinergic signalling mechanisms involving the neurotransmitters, ATP and adenosine, have been identified in mediating hypoxic signalling in the carotid body, but these pathways are not well understood in the fish gill. The present study used a behavioural assay to screen for the effects of drugs, that target purinergic and adenosine receptors, on the hyperventilatory response to hypoxia in larval zebrafish (Danio rerio) in order to determine if the receptors on which these drugs act may be involved in hypoxic signalling. The purinergic receptor antagonist, PPADS, targets purinergic P2X2/3 receptors and inhibited the hyperventilatory response to hypoxia (IC 50 =18.9μM). The broad-spectrum purinergic agonist, ATPγS, elicited a hyperventilatory response (EC 50 =168μM). The non-specific adenosine receptor antagonist, caffeine, inhibited the hyperventilatory response to hypoxia, as did the specific A2a receptor antagonist, SCH58261 (IC 50 =220nM). These results suggest that P2X2/3 and A2a receptors are candidates for mediating hypoxic hyperventilation in zebrafish. This study highlights the potential of applying chemical screening to ventilatory behaviour in zebrafish to further our understanding of the pathways involved in signalling by gill NECs and oxygen sensing in vertebrates. Copyright © 2017 Elsevier Inc. All rights reserved.

CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens.

PubMed

Shankaran, Sunita S; Dahlem, Timothy J; Bisgrove, Brent W; Yost, H Joseph; Tristani-Firouzi, Martin

2017-07-05

The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F 0 screens in this organism a reality. This unit describes a detailed protocol for performing an F 0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection. Next, two approaches for determining the efficiency of mutation induction by the custom CRISPR/Cas9 reagents that are easily performed using standard molecular biology protocols are detailed. Finally, screening for F 0 induced phenotypes using the zebrafish flh gene as an example is discussed. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

The Texas-Indiana Virtual STAR Center: Zebrafish Models for Developmental Toxicity Screening

EPA Pesticide Factsheets

The Texas-Indiana Virtual STAR Center: Zebrafish Models for Developmental Toxicity Screening (Presented by Maria Bondesson Bolin, Ph.D, University of Houston, Center for Nuclear Receptors and Cell Signaling) (3/22/2012)

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

PubMed

Khan, Kanza M; Collier, Adam D; Meshalkina, Darya A; Kysil, Elana V; Khatsko, Sergey L; Kolesnikova, Tatyana; Morzherin, Yury Yu; Warnick, Jason E; Kalueff, Allan V; Echevarria, David J

2017-07-01

Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets. © 2017 The British Pharmacological Society.

Screening of Toxic Effects of Bisphenol A and Products of Its Degradation: Zebrafish (Danio rerio) Embryo Test and Molecular Docking.

PubMed

Makarova, Katerina; Siudem, Pawel; Zawada, Katarzyna; Kurkowiak, Justyna

2016-10-01

Bisphenol A (BPA) acts as an endocrine-disrupting compound even at a low concentration. Degradation of BPA could lead to the formation of toxic products. In this study, we compare the toxicity of BPA and seven intermediate products of its degradation. The accuracy of three molecular docking programs (Surflex, Autodock, and Autodock Vina) in predicting the binding affinities of selected compounds to human (ERα, ERβ, and ERRγ) and zebrafish (ERα, ERRγA, and ERRγB) estrogen and estrogen-related receptors was evaluated. The docking experiments showed that 4-isopropylphenol could have similar toxicity to that of BPA due to its high affinity to ERRγ and ERRγB and high octanol-water partitioning coefficient. The least toxic compounds were hydroquinone and phenol. Those compounds as well as BPA were screened in the zebrafish (Danio rerio) embryo test. 4-isopropylphenol had the strongest toxic effect on zebrafish embryos and caused 100% lethality shortly after exposure. BPA caused the delay in development, multiple deformations, and low heartbeats (30 bps), whereas hydroquinone had no impact on the development of the zebrafish embryo. Thus, the results of zebrafish screening are in good agreement with our docking experiment. The molecular docking could be used to screen the toxicity of other xenoestrogens and their products of degradation.

An Evaluation of ToxCast Angiogenic Disruptors for Effects on ...

EPA Pesticide Factsheets

Angiogenesis is a critical developmental process and a potential target for chemical teratogenesis. Over one-tenth of the Tox21 library of 10,000 compounds have been shown to disrupt mitochondrial function [Attene-Ramos et al., 2015]. Previous studies utilizing ToxCast chemicals have shown a correlation between vascular disruption in Tg(kdrl:EGFP)mitfab692 zebrafish embryos and mitochondrial disruption reported in literature [McCollum et al., submitted]. To more closely examine this correlation, we culled ToxCast data for mitochondrial translocator protein (TSPO; NovaScreen) and mitochondrial membrane potential (MMP) and biomass (Tox21 and Apredica) for a total of 192 chemicals tested for adverse effects on vascular development in transgenic zebrafish embryos [McCollum et al., submitted; Tal et al., submitted]. This set included 40 compounds that disrupted vascular development in zebrafish embryos (zVDC) and 152 compounds that did not. The zVDC set displayed consistent in vitro bioactivity on mitochondrial membrane potential (with a Pearson Chi-Square value of 16.92, p < 0.0001), but did not have consistent effects on mitochondrial biomass (0.4; p = 0.527) or translocator protein ligand binding (0.05; p = 0.823). The effect on MMP is consistent with the hypothesis that disruption of the mitochondrial respiratory complexes is a potential mode of action of angiogenic disruptors (complex I for pyridaben, fenpyroxymate, tebufenpyrad, and rotenone; complex III for py

Pharmacological profiling of zebrafish behavior using chemical and genetic classification of sleep-wake modifiers.

PubMed

Nishimura, Yuhei; Okabe, Shiko; Sasagawa, Shota; Murakami, Soichiro; Ashikawa, Yoshifumi; Yuge, Mizuki; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

2015-01-01

Sleep-wake states are impaired in various neurological disorders. Impairment of sleep-wake states can be an early condition that exacerbates these disorders. Therefore, treating sleep-wake dysfunction may prevent or slow the development of these diseases. Although many gene products are likely to be involved in the sleep-wake disturbance, hypnotics and psychostimulants clinically used are limited in terms of their mode of action and are not without side effects. Therefore, there is a growing demand for developing new hypnotics and psychostimulants with high efficacy and few side effects. Toward this end, animal models are indispensable for use in genetic and chemical screens to identify sleep-wake modifiers. As a proof-of-concept study, we performed behavioral profiling of zebrafish treated with chemical and genetic sleep-wake modifiers. We were able to demonstrate that behavioral profiling of zebrafish treated with hypnotics or psychostimulants from 9 to 10 days post-fertilization was sufficient to identify drugs with specific modes of action. We were also able to identify behavioral endpoints distinguishing GABA-A modulators and hypocretin (hcrt) receptor antagonists and between sympathomimetic and non-sympathomimetic psychostimulants. This behavioral profiling can serve to identify genes related to sleep-wake disturbance associated with various neuropsychiatric diseases and novel therapeutic compounds for insomnia and excessive daytime sleep with fewer adverse side effects.

In vivo alternative assessment of the chemicals that interfere with anterior pituitary POMC expression and interrenal steroidogenesis in POMC: EGFP transgenic zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Lingli; Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072; Xu Wei

2010-11-01

Adrenocorticotropin (ACTH) has been considered a classic adrenocorticotropic hormone and the key pituitary-derived peptide controlling steroidogenesis in the adult adrenal. ACTH is encoded by the propiomelanocortin (POMC) gene, and its active form is mainly synthesized and processed from the POMC-encoded multihormone precursor in the anterior pituitary. The ACTH level has always been precisely controlled in the signaling cascade of the hypothalamo-pituitary-adrenal (HPA) axis due to its central role. The purpose of this study was to investigate whether the transgenic zebrafish line with EGFP driven by the POMC promoter can be used as a surrogate marker to detect the interference effectsmore » on anterior pituitary POMC expression caused by chemicals in teleost. The Tg (POMC:EGFP) fish treated for 4 days with the known adrenergic agents, dexamethasone (Dex) or aminoglutethimide (AG), exhibited altered levels of EGFP and POMC expression in the anterior domain of pituitary corticotrophs. Whole-mount in situ hybridization revealed impaired patterns of expression of the zebrafish ftz-fl gene (ff1b), a key molecular marker for early interrenal development. Next, several chemicals and six commonly used organophosphorus compounds (OPs) were tested for their effects on anterior pituitary POMC expression and early interrenal development. Our preliminary screening analyses indicated that simazine and 3,3',4,4'5-pentachlorobiphenyl (PCB126) could interfere with anterior pituitary POMC expression and interrenal development in fish. In summary, our results demonstrated that the Tg (POMC:EGFP) zebrafish line might be employed as a specific and reproductive in vivo assessment model for the effects of endocrine disruption on HPA signaling.« less

Integration of Microfractionation, qNMR and Zebrafish Screening for the In Vivo Bioassay-Guided Isolation and Quantitative Bioactivity Analysis of Natural Products

PubMed Central

Maes, Jan; Siverio-Mota, Dany; Marcourt, Laurence; Munck, Sebastian; Kamuhabwa, Appolinary R.; Moshi, Mainen J.; Esguerra, Camila V.; de Witte, Peter A. M.; Crawford, Alexander D.; Wolfender, Jean-Luc

2013-01-01

Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation – generic for NP extracts of diverse origin – that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay-guided fractionation can be performed with only 20 mg of NP extract within a few days. PMID:23700445

Diving deeper into Zebrafish development of social behavior: analyzing high resolution data.

PubMed

Buske, Christine; Gerlai, Robert

2014-08-30

Vertebrate model organisms have been utilized in high throughput screening but only with substantial cost and human capital investment. The zebrafish is a vertebrate model species that is a promising and cost effective candidate for efficient high throughput screening. Larval zebrafish have already been successfully employed in this regard (Lessman, 2011), but adult zebrafish also show great promise. High throughput screening requires the use of a large number of subjects and collection of substantial amount of data. Collection of data is only one of the demanding aspects of screening. However, in most screening approaches that involve behavioral data the main bottleneck that slows throughput is the time consuming aspect of analysis of the collected data. Some automated analytical tools do exist, but often they only work for one subject at a time, eliminating the possibility of fully utilizing zebrafish as a screening tool. This is a particularly important limitation for such complex phenotypes as social behavior. Testing multiple fish at a time can reveal complex social interactions but it may also allow the identification of outliers from a group of mutagenized or pharmacologically treated fish. Here, we describe a novel method using a custom software tool developed within our laboratory, which enables tracking multiple fish, in combination with a sophisticated analytical approach for summarizing and analyzing high resolution behavioral data. This paper focuses on the latter, the analytic tool, which we have developed using the R programming language and environment for statistical computing. We argue that combining sophisticated data collection methods with appropriate analytical tools will propel zebrafish into the future of neurobehavioral genetic research. Copyright © 2014. Published by Elsevier B.V.

Advancing epilepsy treatment through personalized genetic zebrafish models.

PubMed

Griffin, A; Krasniak, C; Baraban, S C

2016-01-01

With an increase in the number of disease causing genetic mutations identified from epilepsy cohorts, zebrafish are proving to be an attractive vertebrate model for functional analysis of these allele variants. Not only do zebrafish have conserved gene functions, but larvae harboring mutations in identified human epileptic genes show spontaneous seizure activity and mimic the convulsive behavioral movements observed in humans. With zebrafish being compatible with medium to high-throughput screening, they are also proving to be a unique and powerful system for early preclinical drug screening, including novel target identification, pharmacology, and toxicology. Additionally, with recent advances in genomic engineering technologies, it is now possible to study the precise pathophysiology of patient-specific gene mutations in zebrafish. The following sections highlight how the unique attributes of zebrafish, in combination with genetic modifications, are continuing to transform our understanding of epilepsy and help identify personalized therapeutics for specific patient cohorts. © 2016 Elsevier B.V. All rights reserved.

Importance of Toxicokinetics to Assess the Utility of Zebrafish Larvae as Model for Psychoactive Drug Screening Using Meta-Chlorophenylpiperazine (mCPP) as Example

PubMed Central

Kirla, Krishna Tulasi; Groh, Ksenia J.; Poetzsch, Michael; Banote, Rakesh Kumar; Stadnicka-Michalak, Julita; Eggen, Rik I. L.; Schirmer, Kristin; Kraemer, Thomas

2018-01-01

The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the ever-increasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish (Danio rerio) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration. PMID:29755353

Importance of Toxicokinetics to Assess the Utility of Zebrafish Larvae as Model for Psychoactive Drug Screening Using Meta-Chlorophenylpiperazine (mCPP) as Example.

PubMed

Kirla, Krishna Tulasi; Groh, Ksenia J; Poetzsch, Michael; Banote, Rakesh Kumar; Stadnicka-Michalak, Julita; Eggen, Rik I L; Schirmer, Kristin; Kraemer, Thomas

2018-01-01

The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the ever-increasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish ( Danio rerio ) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration.

Assessing the Developmental Neurotoxicity of 27 ...

EPA Pesticide Factsheets

Assessing the Developmental Neurotoxicity of 27 Organophosphorus Pesticides Using a Zebrafish Behavioral Assay, Waalkes, M., Hunter, D.L., Jarema, K., Mundy, W., and S. Padilla. The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize organophosphorus pesticides for developmental neurotoxicity. As such, we are exploring a behavioral testing paradigm that can assess the effects of sublethal and subteratogenic concentrations of developmental neurotoxicants on zebrafish (Danio rerio). This in vivo assay quantifies the locomotor response to light stimuli under tandem light and dark conditions in a 96-well plate using a video tracking system on 6 day post fertilization zebrafish larvae. Each of twenty-seven organophosphorus pesticides was tested for their developmental neurotoxic potential by exposing zebrafish embryos/larvae to the pesticide at several concentrations (≤ 100 μM nominal concentration) during the first five days of development, followed by 24 hours of depuration and then behavioral testing. Approximately 22% of the chemicals (Acephate, Dichlorvos, Diazoxon, Bensulide,Tribufos, Tebupirimfos) did not produce any behavioral changes after developmental exposure, while many (Malaoxon Fosthiazate, Dimethoate, Dicrotophos, Ethoprop, Malathion, Naled, Diazinon, Methamidophos, Terbufos, Trichlorfon, Phorate, Pirimiphos-methyl, Profenofos, Z-Tetrachlorvinphos, Chlorpyrifos, Coumaphos, Phosmet, Omethoate) produced changes in swi

Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

PubMed Central

Li, Xiang; Rhee, David K.; Malhotra, Rajeev; Mayeur, Claire; Hurst, Liam A.; Ager, Emily; Shelton, Georgia; Kramer, Yael; McCulloh, David; Keefe, David; Bloch, Kenneth D.; Bloch, Donald B.; Peterson, Randall T.

2015-01-01

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism. PMID:26657863

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish.

PubMed

Gamse, Joshua T; Gorelick, Daniel A

2016-10-01

For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals.

A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

PubMed

Zhu, Xiao-Yu; Wu, Si-Qi; Guo, Sheng-Ya; Yang, Hua; Xia, Bo; Li, Ping; Li, Chun-Qi

2018-03-20

Heart failure is a leading cause of death and the development of effective and safe therapeutic agents for heart failure has been proven challenging. In this study, taking advantage of larval zebrafish, we developed a zebrafish heart failure model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days postfertilization) were treated with verapamil at a concentration of 200 μM for 30 min, which were determined as optimum conditions for model development. Tested drugs were administered into zebrafish either by direct soaking or circulation microinjection. After treatment, zebrafish were randomly selected and subjected to either visual observation and image acquisition or record videos under a Zebralab Blood Flow System. The therapeutic effects of drugs on zebrafish heart failure were quantified by calculating the efficiency of heart dilatation, venous congestion, cardiac output, and blood flow dynamics. All 8 human heart failure therapeutic drugs (LCZ696, digoxin, irbesartan, metoprolol, qiliqiangxin capsule, enalapril, shenmai injection, and hydrochlorothiazide) showed significant preventive and therapeutic effects on zebrafish heart failure (p < 0.05, p < 0.01, and p < 0.001) in the zebrafish model. The larval zebrafish heart failure model developed and validated in this study could be used for in vivo heart failure studies and for rapid screening and efficacy assessment of preventive and therapeutic drugs.

An open-source method to analyze optokinetic reflex responses in larval zebrafish.

PubMed

Scheetz, Seth D; Shao, Enhua; Zhou, Yangzhong; Cario, Clinton L; Bai, Qing; Burton, Edward A

2018-01-01

Optokinetic reflex (OKR) responses provide a convenient means to evaluate oculomotor, integrative and afferent visual function in larval zebrafish models, which are commonly used to elucidate molecular mechanisms underlying development, disease and repair of the vertebrate nervous system. We developed an open-source MATLAB-based solution for automated quantitative analysis of OKR responses in larval zebrafish. The package includes applications to: (i) generate sinusoidally-transformed animated grating patterns suitable for projection onto a cylindrical screen to elicit the OKR; (ii) determine and record the angular orientations of the eyes in each frame of a video recording showing the OKR response; and (iii) analyze angular orientation data from the tracking program to yield a set of parameters that quantify essential elements of the OKR. The method can be employed without modification using the operating manual provided. In addition, annotated source code is included, allowing users to modify or adapt the software for other applications. We validated the algorithms and measured OKR responses in normal larval zebrafish, showing good agreement with published quantitative data, where available. We provide the first open-source method to elicit and analyze the OKR in larval zebrafish. The wide range of parameters that are automatically quantified by our algorithms significantly expands the scope of quantitative analysis previously reported. Our method for quantifying OKR responses will be useful for numerous applications in neuroscience using the genetically- and chemically-tractable zebrafish model. Published by Elsevier B.V.

Nanomaterial Toxicity Screening in Developing Zebrafish Embryos

EPA Science Inventory

To assess nanomaterial vertebrate toxicity, a high-content screening assay was created using developing zebrafish, Danio rerio. This included a diverse group of nanomaterials (n=42 total) ranging from metallic (Ag, Au) and metal oxide (CeO2, CuO, TiO2, ZnO) nanoparticles, to non...

Zebrafish Development: High-throughput Test Systems to Assess Developmental Toxicity

EPA Science Inventory

Abstract Because of its developmental concordance, ease of handling and rapid development, the small teleost, zebrafish (Danio rerio), is frequently promoted as a vertebrate model for medium-throughput developmental screens. This present chapter discusses zebrafish as an altern...

Evaluating the zebrafish embryo toxicity test for pesticide hazard screening.

PubMed

Glaberman, Scott; Padilla, Stephanie; Barron, Mace G

2017-05-01

Given the numerous chemicals used in society, it is critical to develop tools for accurate and efficient evaluation of potential risks to human and ecological receptors. Fish embryo acute toxicity tests are 1 tool that has been shown to be highly predictive of standard, more resource-intensive, juvenile fish acute toxicity tests. However, there is also evidence that fish embryos are less sensitive than juvenile fish for certain types of chemicals, including neurotoxicants. The utility of fish embryos for pesticide hazard assessment was investigated by comparing published zebrafish embryo toxicity data from pesticides with median lethal concentration 50% (LC50) data for juveniles of 3 commonly tested fish species: rainbow trout, bluegill sunfish, and sheepshead minnow. A poor, albeit significant, relationship (r 2  = 0.28; p < 0.05) was found between zebrafish embryo and juvenile fish toxicity when pesticides were considered as a single group, but a much better relationship (r 2  = 0.64; p < 0.05) when pesticide mode of action was factored into an analysis of covariance. This discrepancy is partly explained by the large number of neurotoxic pesticides in the dataset, supporting previous findings that commonly used fish embryo toxicity test endpoints are particularly insensitive to neurotoxicants. These results indicate that it is still premature to replace juvenile fish toxicity tests with embryo-based tests such as the Organisation for Economic Co-operation and Development Fish Embryo Acute Toxicity Test for routine pesticide hazard assessment, although embryo testing could be used with other screening tools for testing prioritization. Environ Toxicol Chem 2017;36:1221-1226. © 2016 SETAC. © 2016 SETAC.

Simple animal models for amyotrophic lateral sclerosis drug discovery.

PubMed

Patten, Shunmoogum A; Parker, J Alex; Wen, Xiao-Yan; Drapeau, Pierre

2016-08-01

Simple animal models have enabled great progress in uncovering the disease mechanisms of amyotrophic lateral sclerosis (ALS) and are helping in the selection of therapeutic compounds through chemical genetic approaches. Within this article, the authors provide a concise overview of simple model organisms, C. elegans, Drosophila and zebrafish, which have been employed to study ALS and discuss their value to ALS drug discovery. In particular, the authors focus on innovative chemical screens that have established simple organisms as important models for ALS drug discovery. There are several advantages of using simple animal model organisms to accelerate drug discovery for ALS. It is the authors' particular belief that the amenability of simple animal models to various genetic manipulations, the availability of a wide range of transgenic strains for labelling motoneurons and other cell types, combined with live imaging and chemical screens should allow for new detailed studies elucidating early pathological processes in ALS and subsequent drug and target discovery.

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish

PubMed Central

Gamse, Joshua T.

2016-01-01

Abstract For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals. PMID:27618129

Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Cheng; Graduate University of Chinese Academy of Sciences, Beijing; Jin, Xia

Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in themore » pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.« less

Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish.

PubMed

Long, Si-Mei; Liang, Feng-Yin; Wu, Qi; Lu, Xi-Lin; Yao, Xiao-Li; Li, Shi-Chang; Li, Jing; Su, Huanxing; Pang, Ji-Yan; Pei, Zhong

2014-05-30

High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1-36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37-42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.

Copper toxicology, oxidative stress and inflammation using zebrafish as experimental model.

PubMed

Pereira, Talita Carneiro Brandão; Campos, Maria Martha; Bogo, Maurício Reis

2016-07-01

Copper is an essential micronutrient and a key catalytic cofactor in a wide range of enzymes. As a trace element, copper levels are tightly regulated and both its deficit and excess are deleterious to the organism. Under inflammatory conditions, serum copper levels are increased and trigger oxidative stress responses that activate inflammatory responses. Interestingly, copper dyshomeostasis, oxidative stress and inflammation are commonly present in several chronic diseases. Copper exposure can be easily modeled in zebrafish; a consolidated model in toxicology with increasing interest in immunity-related research. As a result of developmental, economical and genetic advantages, this freshwater teleost is uniquely suitable for chemical and genetic large-scale screenings, representing a powerful experimental tool for a whole-organism approach, mechanistic studies, disease modeling and beyond. Copper toxicological and more recently pro-inflammatory effects have been investigated in both larval and adult zebrafish with breakthrough findings. Here, we provide an overview of copper metabolism in health and disease and its effects on oxidative stress and inflammation responses in zebrafish models. Copper-induced inflammation is highlighted owing to its potential to easily mimic pro-oxidative and pro-inflammatory features that combined with zebrafish genetic tractability could help further in the understanding of copper metabolism, inflammatory responses and related diseases. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates.

PubMed

Poon, Kar Lai; Wang, Xingang; Ng, Ashley S; Goh, Wei Huang; McGinnis, Claudia; Fowler, Stephen; Carney, Tom J; Wang, Haishan; Ingham, Phillip W

2017-03-01

Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.

The importance of Zebrafish in biomedical research.

PubMed

Tavares, Bárbara; Santos Lopes, Susana

2013-01-01

Zebrafish (Danio rerio) is an ideal model organism for the study of vertebrate development. This is due to the large clutches that each couple produces, with up to 200 embryos every 7 days, and to the fact that the embryos and larvae are small, transparent and undergo rapid external development. Using scientific literature research tools available online and the keywords Zebrafish, biomedical research, human disease, and drug screening, we reviewed original studies and reviews indexed in PubMed. In this review we summarized work conducted with this model for the advancement of our knowledge related to several human diseases. We also focused on the biomedical research being performed in Portugal with the zebrafish model. Powerful live imaging and genetic tools are currently available for zebrafish making it a valuable model in biomedical research. The combination of these properties with the optimization of automated systems for drug screening has transformed the zebrafish into "a top model" in biomedical research, drug discovery and toxicity testing. Furthermore, with the optimization of xenografts technology it will be possible to use zebrafish to aide in the choice of the best therapy for each patient. Zebrafish is an excellent model organism in biomedical research, drug development and in clinical therapy.

The zebrafish world of colors and shapes: preference and discrimination.

PubMed

Oliveira, Jessica; Silveira, Mayara; Chacon, Diana; Luchiari, Ana

2015-04-01

Natural environment imposes many challenges to animals, which have to use cognitive abilities to cope with and exploit it to enhance their fitness. Since zebrafish is a well-established model for cognitive studies and high-throughput screening for drugs and diseases that affect cognition, we tested their ability for ambient color preference and 3D objects discrimination to establish a protocol for memory evaluation. For the color preference test, zebrafish were observed in a multiple-chamber tank with different environmental color options. Zebrafish showed preference for blue and green, and avoided yellow and red. For the 3D objects discrimination, zebrafish were allowed to explore two equal objects and then observed in a one-trial test in which a new color, size, or shape of the object was presented. Zebrafish showed discrimination for color, shape, and color+shape combined, but not size. These results imply that zebrafish seem to use some categorical system to discriminate items, and distracters affect their ability for discrimination. The type of variables available (color and shape) may favor zebrafish objects perception and facilitate discrimination processing. We suggest that this easy and simple memory test could serve as a useful screening tool for cognitive dysfunction and neurotoxicological studies.

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

Associative learning performance is impaired in zebrafish (Danio rerio) by the NMDA-R antagonist MK-801

PubMed Central

Sison, Margarette; Gerlai, Robert

2011-01-01

The zebrafish is gaining popularity in behavioral neuroscience perhaps because of a promise of efficient large scale mutagenesis and drug screens that could identify a substantial number of yet undiscovered molecular players involved in complex traits. Learning and memory are complex functions of the brain and the analysis of their mechanisms may benefit from such large scale zebrafish screens. One bottleneck in this research is the paucity of appropriate behavioral screening paradigms, which may be due to the relatively uncharacterized nature of the behavior of this species. Here we show that zebrafish exhibit good learning performance in a task adapted from the mammalian literature, a plus maze in which zebrafish are required to associate a neutral visual stimulus with the presence of conspecifics, the rewarding unconditioned stimulus. Furthermore, we show that MK-801, a non-competitive NMDA-R antagonist, impairs memory performance in this maze when administered right after training or just before recall but not when given before training at a dose that does not impair motor function, perception or motivation. These results suggest that the plus maze associative learning paradigm has face and construct validity and that zebrafish may become an appropriate and translationally relevant study species for the analysis of the mechanisms of vertebrate, including mammalian, learning and memory. PMID:21596149

Advances in the Study of Heart Development and Disease Using Zebrafish

PubMed Central

Brown, Daniel R.; Samsa, Leigh Ann; Qian, Li; Liu, Jiandong

2016-01-01

Animal models of cardiovascular disease are key players in the translational medicine pipeline used to define the conserved genetic and molecular basis of disease. Congenital heart diseases (CHDs) are the most common type of human birth defect and feature structural abnormalities that arise during cardiac development and maturation. The zebrafish, Danio rerio, is a valuable vertebrate model organism, offering advantages over traditional mammalian models. These advantages include the rapid, stereotyped and external development of transparent embryos produced in large numbers from inexpensively housed adults, vast capacity for genetic manipulation, and amenability to high-throughput screening. With the help of modern genetics and a sequenced genome, zebrafish have led to insights in cardiovascular diseases ranging from CHDs to arrhythmia and cardiomyopathy. Here, we discuss the utility of zebrafish as a model system and summarize zebrafish cardiac morphogenesis with emphasis on parallels to human heart diseases. Additionally, we discuss the specific tools and experimental platforms utilized in the zebrafish model including forward screens, functional characterization of candidate genes, and high throughput applications. PMID:27335817

Phenotype-based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis*

PubMed Central

Reynolds, Alison L.; Alvarez, Yolanda; Sasore, Temitope; Waghorne, Nora; Butler, Clare T.; Kilty, Claire; Smith, Andrew J.; McVicar, Carmel; Wong, Vickie H. Y.; Galvin, Orla; Merrigan, Stephanie; Osman, Janina; Grebnev, Gleb; Sjölander, Anita; Stitt, Alan W.; Kennedy, Breandán N.

2016-01-01

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge DiversetTM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μm in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1–2) at micromolar IC50 values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents. PMID:26846851

An Eco-Safety Assessment of Glyoxal-Containing Cellulose Ether on Freeze-Dried Microbial Strain, Cyanobacteria, Daphnia, and Zebrafish

PubMed Central

Park, Chang-Beom; Song, Min Ju; Choi, Nak Woon; Kim, Sunghoon; Jeon, Hyun Pyo; Kim, Sanghun; Kim, Youngjun

2017-01-01

The objective of this study was to investigate the aquatic-toxic effects of glyoxal-containing cellulose ether with four different glyoxal concentrations (0%, 1.4%, 2.3%, and 6.3%) in response to global chemical regulations, e.g., European Union Classification, Labeling and Packaging (EU CLP). Toxicity tests of glyoxal-containing cellulose ether on 11 different microbial strains, Microcystis aeruginosa, Daphnia magna, and zebrafish embryos were designed as an initial stage of toxicity screening and performed in accordance with standardized toxicity test guidelines. Glyoxal-containing cellulose ether showed no significant toxic effects in the toxicity tests of the 11 freeze-dried microbial strains, Daphnia magna, and zebrafish embryos. Alternatively, 6.3% glyoxal-containing cellulose ether led to a more than 60% reduction in Microcystis aeruginosa growth after 7 days of exposure. Approximately 10% of the developmental abnormalities (e.g., bent spine) in zebrafish embryos were also observed in the group exposed to 6.3% glyoxal-containing cellulose ether after 6 days of exposure. These results show that 6.3% less glyoxal-containing cellulose ether has no acute toxic effects on aquatic organisms. However, 6.3% less glyoxal-containing cellulose ether may affect the health of aquatic organisms with long-term exposure. In order to better evaluate the eco-safety of cellulosic products containing glyoxal, further studies regarding the toxic effects of glyoxal-containing cellulose ether with long-term exposure are required. The results from this study allow us to evaluate the aquatic-toxic effects of glyoxal-containing cellulosic products, under EU chemical regulations, on the health of aquatic organisms. PMID:28335565

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

PubMed Central

Evason, Kimberley J.; Francisco, Macrina T.; Juric, Vladislava; Balakrishnan, Sanjeev; Lopez Pazmino, Maria del Pilar; Gordan, John D.; Kakar, Sanjay; Spitsbergen, Jan; Goga, Andrei; Stainier, Didier Y. R.

2015-01-01

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors. PMID:26134322

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults

PubMed Central

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-01-01

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using 3H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances. PMID:28346357

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults.

PubMed

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-03-25

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances.

Comparison of toxicity values across zebrafish early life stages and mammalian studies: Implications for chemical testing.

PubMed

Ducharme, Nicole A; Reif, David M; Gustafsson, Jan-Ake; Bondesson, Maria

2015-08-01

With the high cost and slow pace of toxicity testing in mammals, the vertebrate zebrafish has become a tractable model organism for high throughput toxicity testing. We present here a meta-analysis of 600 chemicals tested for toxicity in zebrafish embryos and larvae. Nineteen aggregated and 57 individual toxicity endpoints were recorded from published studies yielding 2695 unique data points. These data points were compared to lethality and reproductive toxicology endpoints analyzed in rodents and rabbits and to exposure values for humans. We show that although many zebrafish endpoints did not correlate to rodent or rabbit acute toxicity data, zebrafish could be used to accurately predict relative acute toxicity through the rat inhalation, rabbit dermal, and rat oral exposure routes. Ranking of the chemicals based on toxicity and teratogenicity in zebrafish, as well as human exposure levels, revealed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene, and chlorpyrifos ranked in the top nine of all chemicals for these three categories, and as such should be considered high priority chemicals for testing in higher vertebrates. Copyright © 2014 Elsevier Inc. All rights reserved.

Teratogenic Potential of Antiepileptic Drugs in the Zebrafish Model

PubMed Central

Lee, Sung Hak; Kang, Jung Won; Lin, Tao; Lee, Jae Eun; Jin, Dong Il

2013-01-01

The zebrafish model is an attractive candidate for screening of developmental toxicity during early drug development. Antiepileptic drugs (AEDs) arouse concern for the risk of teratogenicity, but the data are limited. In this study, we evaluated the teratogenic potential of seven AEDs (carbamazepine (CBZ), ethosuximide (ETX), valproic acid (VPN), lamotrigine (LMT), lacosamide (LCM), levetiracetam (LVT), and topiramate (TPM)) in the zebrafish model. Zebrafish embryos were exposed to AEDs from initiation of gastrula (5.25 hours post-fertilization (hpf)) to termination of hatching (72 hpf) which mimic the mammalian teratogenic experimental design. The lethality and teratogenic index (TI) of AEDs were determined and the TI values of each drug were compared with the US FDA human pregnancy categories. Zebrafish model was useful screening model for teratogenic potential of antiepilepsy drugs and was in concordance with in vivo mammalian data and human clinical data. PMID:24324971

Teratogenic potential of antiepileptic drugs in the zebrafish model.

PubMed

Lee, Sung Hak; Kang, Jung Won; Lin, Tao; Lee, Jae Eun; Jin, Dong Il

2013-01-01

The zebrafish model is an attractive candidate for screening of developmental toxicity during early drug development. Antiepileptic drugs (AEDs) arouse concern for the risk of teratogenicity, but the data are limited. In this study, we evaluated the teratogenic potential of seven AEDs (carbamazepine (CBZ), ethosuximide (ETX), valproic acid (VPN), lamotrigine (LMT), lacosamide (LCM), levetiracetam (LVT), and topiramate (TPM)) in the zebrafish model. Zebrafish embryos were exposed to AEDs from initiation of gastrula (5.25 hours post-fertilization (hpf)) to termination of hatching (72 hpf) which mimic the mammalian teratogenic experimental design. The lethality and teratogenic index (TI) of AEDs were determined and the TI values of each drug were compared with the US FDA human pregnancy categories. Zebrafish model was useful screening model for teratogenic potential of antiepilepsy drugs and was in concordance with in vivo mammalian data and human clinical data.

Zebrafish models for functional and toxicological screening of nanoscale drug delivery systems: promoting preclinical applications

PubMed Central

Lee, Keon Yong; Jang, Gun Hyuk; Byun, Cho Hyun; Jeun, Minhong

2017-01-01

Preclinical screening with animal models is an important initial step in clinical translation of new drug delivery systems. However, establishing efficacy, biodistribution, and biotoxicity of complex, multicomponent systems in small animal models can be expensive and time-consuming. Zebrafish models represent an alternative for preclinical studies for nanoscale drug delivery systems. These models allow easy optical imaging, large sample size, and organ-specific studies, and hence an increasing number of preclinical studies are employing zebrafish models. In this review, we introduce various models and discuss recent studies of nanoscale drug delivery systems in zebrafish models. Also in the end, we proposed a guideline for the preclinical trials to accelerate the progress in this field. PMID:28515222

Zebrafish models for functional and toxicological screening of nanoscale drug delivery systems: promoting preclinical applications.

PubMed

Lee, Keon Yong; Jang, Gun Hyuk; Byun, Cho Hyun; Jeun, Minhong; Searson, Peter C; Lee, Kwan Hyi

2017-06-30

Preclinical screening with animal models is an important initial step in clinical translation of new drug delivery systems. However, establishing efficacy, biodistribution, and biotoxicity of complex, multicomponent systems in small animal models can be expensive and time-consuming. Zebrafish models represent an alternative for preclinical studies for nanoscale drug delivery systems. These models allow easy optical imaging, large sample size, and organ-specific studies, and hence an increasing number of preclinical studies are employing zebrafish models. In this review, we introduce various models and discuss recent studies of nanoscale drug delivery systems in zebrafish models. Also in the end, we proposed a guideline for the preclinical trials to accelerate the progress in this field. © 2017 The Author(s).

Zebrafish as tools for drug discovery.

PubMed

MacRae, Calum A; Peterson, Randall T

2015-10-01

The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

Zebrafish Models of Prader-Willi Syndrome: Fast Track to Pharmacotherapeutics

PubMed Central

Spikol, Emma D.; Laverriere, Caroline E.; Robnett, Maya; Carter, Gabriela; Wolfe, Erin; Glasgow, Eric

2016-01-01

Prader-Willi syndrome (PWS) is a rare genetic neurodevelopmental disorder characterized by an insatiable appetite, leading to chronic overeating and obesity. Additional features include short stature, intellectual disability, behavioral problems and incomplete sexual development. Although significant progress has been made in understanding the genetic basis of PWS, the mechanisms underlying the pathogenesis of the disorder remain poorly understood. Treatment for PWS consists mainly of palliative therapies; curative therapies are sorely needed. Zebrafish, Danio rerio, represent a promising way forward for elucidating physiological problems such as obesity and identifying new pharmacotherapeutic options for PWS. Over the last decade, an increased appreciation for the highly conserved biology among vertebrates and the ability to perform high-throughput drug screening has seen an explosion in the use of zebrafish for disease modeling and drug discovery. Here, we review recent advances in developing zebrafish models of human disease. Aspects of zebrafish genetics and physiology that are relevant to PWS will be discussed, and the advantages and disadvantages of zebrafish models will be contrasted with current animal models for this syndrome. Finally, we will present a paradigm for drug screening in zebrafish that is potentially the fastest route for identifying and delivering curative pharmacotherapies to PWS patients. PMID:27857842

Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis

PubMed Central

Jimenez, Laura; Wang, Jindong; Morrison, Monique A.; Whatcott, Clifford; Soh, Katherine K.; Warner, Steven; Bearss, David; Jette, Cicely A.; Stewart, Rodney A.

2016-01-01

ABSTRACT The epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, called Tg(snai1b:GFP), which labels epithelial cells undergoing EMT to produce sox10-positive neural crest (NC) cells. Time-lapse and lineage analysis of Tg(snai1b:GFP) embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. Treating Tg(snai1b:GFP) embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RA in vivo and raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells. PMID:26794130

Persistent behavioral effects following early life exposure to retinoic acid or valproic acid in zebrafish

PubMed Central

Bailey, Jordan M.; Oliveri, Anthony N.; Karbhari, Nishika; Brooks, Roy A.J.; De La Rocha, Amberlene J.; Janardhan, Sheila; Levin, Edward D.

2015-01-01

BACKGROUND Moderate to severe dysregulation in retinoid signaling during early development is associated with a constellation of physical malformations and/or neural tube defects, including spina bifida. It is thought that more subtle dysregulation of this system, which might be achievable via dietary (i.e. hypervitaminosis A) or pharmacological (i.e. valproic acid) exposure in humans, will manifest on behavioral domains including sociability, without overt physical abnormalities. METHODS During early life, zebrafish were exposed to low doses of two chemicals that disrupt retinoid signaling. From 0-5 dpf, larvae were reared in aqueous solutions containing retinoic acid (0, 0.02, 0.2 or 2 nM) or valproic acid (0, 0.5, 5.0 or 50 uM). One cohort of zebrafish was assessed using a locomotor activity screen at 6-dpf; another was reared to adulthood and assessed using a neurobehavioral test battery (startle habituation, novel tank exploration, shoaling, and predator escape/avoidance). RESULTS There was no significant increase in the incidence of physical malformation among exposed fish compared to controls. Both retinoic acid and valproic acid exposures during development disrupted larval activity with persisting behavioral alterations later in life, primarily manifesting as decreased social affiliation. CONCLUSIONS Social behavior and some aspects of motor function were altered in exposed fish; the importance of examining emotional or psychological consequences of early life exposure to retinoid acting chemicals is discussed. PMID:26439099

Development of a whole-organism model to screen new compounds for sun protection.

PubMed

Wang, Yun-Hsin; Wen, Chi-Chung; Yang, Zhi-Shiang; Cheng, Chien-Chung; Tsai, Jen-Ning; Ku, Chia-Chen; Wu, Hsin-Ju; Chen, Yau-Hung

2009-01-01

We used zebrafish as a whole-organism model to screen new compounds for sun protection activity. First of all, we designed a series of UVB exposure experiments and recorded the phenotypic changes of zebrafish embryos. Results showed that 100 mJ/cm(2) of UVB given six times separated by 30 min intervals is the best condition. Fin malformation (reduced and/or absent fin) phenotypes are the most evident consequences after exposure to UVB. Each fin was affected by UVB, including pelvic, ventral, caudal, and dorsal fin, but pelvic fin seemed to be the most sensitive target after UVB exposure. We furthermore carried out "prevention" and "treatment" experiments using green tea extract and/or (-)-epigallocatechin (EGCG) to test this whole-organism model by observing the morphological changes of all fins (especially pelvic fin) after UVB exposure. Effects of UVB, green tea extract and EGCG on fin development were assessed using the Kaplan-Meier analysis, log-rank test and Cox proportional hazards regression. Results showed that a zebrafish pelvic fin in the UVB + green tea (treatment) group is 5.51 (range from 2.39 to 14.90) times, one in the UVB + green tea (prevention) group is 7.04 (range from 3.11 to 18.92) times, and one in the 25 ppm of EGCG (prevention) group is 22.19 (range from 9.40 to 61.50) times more likely to return to normal fin than one in the UVB only group. On the basis of these observations, we believe this model is effective for screening the higher stability and lower toxicity of new compounds, such as small chemicals which are derivative from EGCG or other dietary agents for sun protection.

Zebrafish Get Connected: Investigating Neurotransmission Targets and Alterations in Chemical Toxicity

PubMed Central

Horzmann, Katharine A.; Freeman, Jennifer L.

2016-01-01

Neurotransmission is the basis of neuronal communication and is critical for normal brain development, behavior, learning, and memory. Exposure to drugs and chemicals can alter neurotransmission, often through unknown pathways and mechanisms. The zebrafish (Danio rerio) model system is increasingly being used to study the brain and chemical neurotoxicity. In this review, the major neurotransmitter systems, including glutamate, GABA, dopamine, norepinephrine, serotonin, acetylcholine, histamine, and glutamate are surveyed and pathways of synthesis, transport, metabolism, and action are examined. Differences between human and zebrafish neurochemical pathways are highlighted. We also review techniques for evaluating neurological function, including the measurement of neurotransmitter levels, assessment of gene expression through transcriptomic analysis, and the recording of neurobehavior. Finally examples of chemical toxicity studies evaluating alterations in neurotransmitter systems in the zebrafish model are reviewed. PMID:28730152

Modeling mixtures of thyroid gland function disruptors in a vertebrate alternative model, the zebrafish eleutheroembryo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thienpont, Benedicte; Barata, Carlos; Raldúa, Demetrio, E-mail: drpqam@cid.csic.es

2013-06-01

Maternal thyroxine (T4) plays an essential role in fetal brain development, and even mild and transitory deficits in free-T4 in pregnant women can produce irreversible neurological effects in their offspring. Women of childbearing age are daily exposed to mixtures of chemicals disrupting the thyroid gland function (TGFDs) through the diet, drinking water, air and pharmaceuticals, which has raised the highest concern for the potential additive or synergic effects on the development of mild hypothyroxinemia during early pregnancy. Recently we demonstrated that zebrafish eleutheroembryos provide a suitable alternative model for screening chemicals impairing the thyroid hormone synthesis. The present study usedmore » the intrafollicular T4-content (IT4C) of zebrafish eleutheroembryos as integrative endpoint for testing the hypotheses that the effect of mixtures of TGFDs with a similar mode of action [inhibition of thyroid peroxidase (TPO)] was well predicted by a concentration addition concept (CA) model, whereas the response addition concept (RA) model predicted better the effect of dissimilarly acting binary mixtures of TGFDs [TPO-inhibitors and sodium-iodide symporter (NIS)-inhibitors]. However, CA model provided better prediction of joint effects than RA in five out of the six tested mixtures. The exception being the mixture MMI (TPO-inhibitor)-KClO{sub 4} (NIS-inhibitor) dosed at a fixed ratio of EC{sub 10} that provided similar CA and RA predictions and hence it was difficult to get any conclusive result. There results support the phenomenological similarity criterion stating that the concept of concentration addition could be extended to mixture constituents having common apical endpoints or common adverse outcomes. - Highlights: • Potential synergic or additive effect of mixtures of chemicals on thyroid function. • Zebrafish as alternative model for testing the effect of mixtures of goitrogens. • Concentration addition seems to predict better the effect of mixtures of goitrogens.« less

Generation of Tg(cyp1a:gfp) Transgenic Zebrafish for Development of a Convenient and Sensitive In Vivo Assay for Aryl Hydrocarbon Receptor Activity.

PubMed

Xu, Hongyan; Li, Caixia; Li, Yan; Ng, Grace Hwee Boon; Liu, Chunsheng; Zhang, Xiaoyan; Gong, Zhiyuan

2015-12-01

Both dioxins/dioxin-like compounds and polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants and cause multiple adverse health effects on human and wildlife. Cyp1a is the most commonly used biomarker induced by these pollutants through activation of the aryl hydrocarbon receptor (AhR) pathway. Here we generated Tg(cyp1a:gfp) transgenic zebrafish for establishing a convenient in vivo assay for analysing these xenobiotic compounds. The Tg(cyp1a:gfp) larvae at 4 day post-fertilization were tested with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and GFP induction was observed mainly in the kidney, liver and gut. Similar GFP expression was also induced strongly by two dioxin-like chemicals, co-planar polychlorinated biphenyl (PCB126) and polychlorinated dibenzo-p-furan (PeCDF) and relatively weakly by two PAHs, 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BAP). The lowest observed effective concentration (LOEC) of TCDD was estimated to be ∼1 pM and the EC50 (effective concentration to induce GFP in 50 % of Tg(cyp1a:gfp) larvae) was ∼10 pM. PCB126 and PeCDF had ∼10× lower potencies in GFP induction than TCDD, while the potencies for 3-MC and BAP were at least 1000× lower. The sensitivity of Tg(cyp1a:gfp) larvae to respond TCDD was also favourable compared to that of ethoxyresorufin-O-deethylase (EROD) assay in both zebrafish larvae and adult livers. As GFP-based assay in transgenic zebrafish can be easily accommodated in multi-well dishes, the Tg(cyp1a:gfp) zebrafish should provide not only a valuable biomonitoring tool for aquatic contaminants but also a potential high-throughput chemical screening platform for identification of new AhR agonists.

Investigating How the Microbiome Interacts With Environmental Chemicals in Zebrafish

EPA Pesticide Factsheets

This internship will use an innovative experimental system comprised of colonized and microbe-free zebrafish to learn how microbial colonization status affects the toxicity of environmental chemicals.

Influenza A virus infection in zebrafish recapitulates mammalian infection and sensitivity to anti-influenza drug treatment.

PubMed

Gabor, Kristin A; Goody, Michelle F; Mowel, Walter K; Breitbach, Meghan E; Gratacap, Remi L; Witten, P Eckhard; Kim, Carol H

2014-11-01

Seasonal influenza virus infections cause annual epidemics and sporadic pandemics. These present a global health concern, resulting in substantial morbidity, mortality and economic burdens. Prevention and treatment of influenza illness is difficult due to the high mutation rate of the virus, the emergence of new virus strains and increasing antiviral resistance. Animal models of influenza infection are crucial to our gaining a better understanding of the pathogenesis of and host response to influenza infection, and for screening antiviral compounds. However, the current animal models used for influenza research are not amenable to visualization of host-pathogen interactions or high-throughput drug screening. The zebrafish is widely recognized as a valuable model system for infectious disease research and therapeutic drug testing. Here, we describe a zebrafish model for human influenza A virus (IAV) infection and show that zebrafish embryos are susceptible to challenge with both influenza A strains APR8 and X-31 (Aichi). Influenza-infected zebrafish show an increase in viral burden and mortality over time. The expression of innate antiviral genes, the gross pathology and the histopathology in infected zebrafish recapitulate clinical symptoms of influenza infections in humans. This is the first time that zebrafish embryos have been infected with a fluorescent IAV in order to visualize infection in a live vertebrate host, revealing a pattern of vascular endothelial infection. Treatment of infected zebrafish with a known anti-influenza compound, Zanamivir, reduced mortality and the expression of a fluorescent viral gene product, demonstrating the validity of this model to screen for potential antiviral drugs. The zebrafish model system has provided invaluable insights into host-pathogen interactions for a range of infectious diseases. Here, we demonstrate a novel use of this species for IAV research. This model has great potential to advance our understanding of influenza infection and the associated host innate immune response. © 2014. Published by The Company of Biologists Ltd.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

PubMed

Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella

2017-08-01

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. © The Author 2017. Published by Oxford University Press.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta

PubMed Central

Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A.; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella

2017-01-01

Abstract Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. PMID:28475764

Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression

PubMed Central

Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan

2014-01-01

Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants. PMID:24626481

Short-term memory in zebrafish (Danio rerio).

PubMed

Jia, Jason; Fernandes, Yohaan; Gerlai, Robert

2014-08-15

Learning and memory represent perhaps the most complex behavioral phenomena. Although their underlying mechanisms have been extensively analyzed, only a fraction of the potential molecular components have been identified. The zebrafish has been proposed as a screening tool with which mechanisms of complex brain functions may be systematically uncovered. However, as a relative newcomer in behavioral neuroscience, the zebrafish has not been well characterized for its cognitive and mnemonic features, thus learning and/or memory screens with adults have not been feasible. Here we study short-term memory of adult zebrafish. We show animated images of conspecifics (the stimulus) to the experimental subject during 1 min intervals on ten occasions separated by different (2, 4, 8 or 16 min long) inter-stimulus intervals (ISI), a between subject experimental design. We quantify the distance of the subject from the image presentation screen during each stimulus presentation interval, during each of the 1-min post-stimulus intervals immediately following the stimulus presentations and during each of the 1-min intervals furthest away from the last stimulus presentation interval and just before the next interval (pre-stimulus interval), respectively. Our results demonstrate significant retention of short-term memory even in the longest ISI group but suggest no acquisition of reference memory. Because in the employed paradigm both stimulus presentation and behavioral response quantification is computer automated, we argue that high-throughput screening for drugs or mutations that alter short-term memory performance of adult zebrafish is now becoming feasible. Copyright © 2014 Elsevier B.V. All rights reserved.

Large-scale mapping of mutations affecting zebrafish development.

PubMed

Geisler, Robert; Rauch, Gerd-Jörg; Geiger-Rudolph, Silke; Albrecht, Andrea; van Bebber, Frauke; Berger, Andrea; Busch-Nentwich, Elisabeth; Dahm, Ralf; Dekens, Marcus P S; Dooley, Christopher; Elli, Alexandra F; Gehring, Ines; Geiger, Horst; Geisler, Maria; Glaser, Stefanie; Holley, Scott; Huber, Matthias; Kerr, Andy; Kirn, Anette; Knirsch, Martina; Konantz, Martina; Küchler, Axel M; Maderspacher, Florian; Neuhauss, Stephan C; Nicolson, Teresa; Ober, Elke A; Praeg, Elke; Ray, Russell; Rentzsch, Brit; Rick, Jens M; Rief, Eva; Schauerte, Heike E; Schepp, Carsten P; Schönberger, Ulrike; Schonthaler, Helia B; Seiler, Christoph; Sidi, Samuel; Söllner, Christian; Wehner, Anja; Weiler, Christian; Nüsslein-Volhard, Christiane

2007-01-09

Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.

Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

PubMed

Kalueff, Allan V; Echevarria, David J; Homechaudhuri, Sumit; Stewart, Adam Michael; Collier, Adam D; Kaluyeva, Aleksandra A; Li, Shaomin; Liu, Yingcong; Chen, Peirong; Wang, JiaJia; Yang, Lei; Mitra, Anisa; Pal, Subharthi; Chaudhuri, Adwitiya; Roy, Anwesha; Biswas, Missidona; Roy, Dola; Podder, Anupam; Poudel, Manoj K; Katare, Deepshikha P; Mani, Ruchi J; Kyzar, Evan J; Gaikwad, Siddharth; Nguyen, Michael; Song, Cai

2016-01-01

Zebrafish (Danio rerio) are rapidly emerging as an important model organism for aquatic neuropharmacology and toxicology research. The behavioral/phenotypic complexity of zebrafish allows for thorough dissection of complex human brain disorders and drug-evoked pathological states. As numerous zebrafish models become available with a wide spectrum of behavioral, genetic, and environmental methods to test novel drugs, here we discuss recent zebrafish phenomics methods to facilitate drug discovery, particularly in the field of biological psychiatry. Additionally, behavioral, neurological, and endocrine endpoints are becoming increasingly well-characterized in zebrafish, making them an inexpensive, robust and effective model for toxicology research and pharmacological screening. We also discuss zebrafish behavioral phenotypes, experimental considerations, pharmacological candidates and relevance of zebrafish neurophenomics to other 'omics' (e.g., genomic, proteomic) approaches. Finally, we critically evaluate the limitations of utilizing this model organism, and outline future strategies of research in the field of zebrafish phenomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of Chemical Vascular Disruptors During Development Using An Integrative Predictive Toxicity Model and Zebrafish and in Vitro Functional Angiogenesis Assays.

EPA Science Inventory

Identification of chemical vascular disruptors during development using an integrative predictive toxicity model and zebrafish and in vitro functional angiogenesis assays Chemically-induced vascular toxicity during embryonic development can result in a wide range of adverse pre...

Assessment of the lethal and sublethal effects of 20 environmental chemicals in zebrafish embryos and larvae by using OECD TG 212.

PubMed

Horie, Yoshifumi; Yamagishi, Takahiro; Takahashi, Hiroko; Shintaku, Youko; Iguchi, Taisen; Tatarazako, Norihisa

2017-10-01

Fish embryo toxicity tests are used to assess the lethal and sublethal effects of environmental chemicals in aquatic organisms. Previously, we used a short-term toxicity test published by the Organization for Economic Co-operation and Development (test no. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages [OECD TG 212]) to assess the lethal and sublethal effects of aniline and several chlorinated anilines in zebrafish embryos and larvae. To expand upon this previous study, we used OECD TG 212 in zebrafish embryos and larvae to assess the lethal and sublethal effects of 20 additional environmental chemicals that included active pharmaceutical ingredients, pesticides, metals, aromatic compounds or chlorinated anilines. Zebrafish embryos (Danio rerio) were exposed to the test chemicals until 8 days post-fertilization. A delayed lethal effect was induced by 16 of the 20 test chemicals, and a positive correlation was found between heart rate turbulence and mortality. We also found that exposure to the test chemicals at concentrations lower than the lethal concentration induced the sublethal effects of edema, body curvature and absence of swim-bladder inflation. In conclusion, the environmental chemicals assessed in the present study induced both lethal and sublethal effects in zebrafish embryos and larvae, as assessed by using OECD TG 212. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses.

PubMed

Sullivan, Con; Jurcyzszak, Denise; Goody, Michelle F; Gabor, Kristin A; Longfellow, Jacob R; Millard, Paul J; Kim, Carol H

2017-01-20

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Analysis of Lethality and Malformations During Zebrafish (Danio rerio) Development.

PubMed

Raghunath, Azhwar; Perumal, Ekambaram

2018-01-01

The versatility offered by zebrafish (Danio rerio) makes it a powerful and an attractive vertebrate model in developmental toxicity and teratogenicity assays. Apart from the newly introduced chemicals as drugs, xenobiotics also induce abnormal developmental abnormalities and congenital malformations in living organisms. Over the recent decades, zebrafish embryo/larva has emerged as a potential tool to test teratogenicity potential of these chemicals. Zebrafish responds to compounds as mammals do as they share similarities in their development, metabolism, physiology, and signaling pathways with that of mammals. The methodology used by the different scientists varies enormously in the zebrafish embryotoxicity test. In this chapter, we present methods to assess lethality and malformations during zebrafish development. We propose two major malformations scoring systems: binomial and relative morphological scoring systems to assess the malformations in zebrafish embryos/larvae. Based on the scoring of the malformations, the test compound can be classified as a teratogen or a nonteratogen and its teratogenic potential is evaluated.

DarT: The embryo test with the Zebrafish Danio rerio--a general model in ecotoxicology and toxicology.

PubMed

Nagel, Roland

2002-01-01

The acute fish test is an animal test whose ecotoxicological relevance is worthy of discussion. The primary aim of protection in ecotoxicology is the population and not the individual. Furthermore the concentration of pollutants in the environment is normally not in the lethal range. Therefore the acute fish test covers solely the situation after chemical spills. Nevertheless, acute fish toxicity data still belong to the base set used for the assessment of chemicals. The embryo test with the zebrafish Danio rerio (DarT) is recommended as a substitute for the acute fish test. For validation an international laboratory comparison test was carried out. A summary of the results is presented in this paper. Based on the promising results of testing chemicals and waste water the test design was validated by the DIN-working group "7.6 Fischei-Test". A normed test guideline for testing waste water with fish is available. The test duration is short (48 h) and within the test different toxicological endpoints can be examined. Endpoints from the embryo test are suitable for QSAR-studies. Besides the use in ecotoxicology the introduction as a toxicological model was investigated. Disturbance of pigmentation and effects on the frequency of heart-beat were examined. A further important application is testing of teratogenic chemicals. Based on the results DarT could be a screening test within preclinical studies.

Differences of acute versus chronic ethanol exposure on anxiety-like behavioral responses in zebrafish.

PubMed

Mathur, Priya; Guo, Su

2011-06-01

Zebrafish, a vertebrate model organism amenable to high throughput screening, is an attractive system to model and study the mechanisms underlying human diseases. Alcoholism and alcoholic medical disorders are among the most debilitating diseases, yet the mechanisms by which ethanol inflicts the disease states are not well understood. In recent years zebrafish behavior assays have been used to study learning and memory, fear and anxiety, and social behavior. It is important to characterize the effects of ethanol on zebrafish behavioral repertoires in order to successfully harvest the strength of zebrafish for alcohol research. One prominent effect of alcohol in humans is its effect on anxiety, with acute intermediate doses relieving anxiety and withdrawal from chronic exposure increasing anxiety, both of which have significant contributions to alcohol dependence. In this study, we assess the effects of both acute and chronic ethanol exposure on anxiety-like behaviors in zebrafish, using two behavioral paradigms, the Novel Tank Diving Test and the Light/Dark Choice Assay. Acute ethanol exposure exerted significant dose-dependent anxiolytic effects. However, withdrawal from repeated intermittent ethanol exposure disabled recovery from heightened anxiety. These results demonstrate that zebrafish exhibit different anxiety-like behavioral responses to acute and chronic ethanol exposure, which are remarkably similar to these effects of alcohol in humans. Because of the accessibility of zebrafish to high throughput screening, our results suggest that genes and small molecules identified in zebrafish will be of relevance to understand how acute versus chronic alcohol exposure have opposing effects on the state of anxiety in humans. Copyright © 2011 Elsevier B.V. All rights reserved.

[Ideas and methods on efficient screening of traditional medicines for anti-osteoporosis activity based on M-Act/Tox integrated evaluation using zebrafish].

PubMed

Wang, Mo; Ling, Jie; Chen, Ying; Song, Jie; Sun, E; Shi, Zi-Qi; Feng, Liang; Jia, Xiao-Bin; Wei, Ying-Jie

2017-11-01

The increasingly apparent liver injury problems of bone strengthening Chinese medicines have brought challenges for clinical application, and it is necessary to consider both effectiveness and safety in screening anti-osteoporosis Chinese medicines. Metabolic transformation is closely related to drug efficacy and toxicity, so it is significant to comprehensively consider metabolism-action/toxicity(M-Act/Tox) for screening anti-osteoporosis Chinese medicines. The current evaluation models and the number of compounds(including metabolites) severely restrict efficient screening in vivo. By referring to previous relevant research and domestic and abroad literature, zebrafish M-Act/Tox integrative method was put forward for efficiently screening anti-osteoporosis herb medicines, which has organically integrated zebrafish metabolism model, osteoporosis model and toxicity evaluation method. This method can break through the bottleneck and blind spots that trace compositions can't achieve efficient and integrated in vivo evaluation, and realize both efficient and comprehensive screening on anti-osteoporosis traditional medicines based on in vivo process taking both safety and effectiveness into account, which is significant to accelerate discovery of effective and safe innovative traditional Chinese medicines for osteoporosis. Copyright© by the Chinese Pharmaceutical Association.

Automated analysis of brain activity for seizure detection in zebrafish models of epilepsy.

PubMed

Hunyadi, Borbála; Siekierska, Aleksandra; Sourbron, Jo; Copmans, Daniëlle; de Witte, Peter A M

2017-08-01

Epilepsy is a chronic neurological condition, with over 30% of cases unresponsive to treatment. Zebrafish larvae show great potential to serve as an animal model of epilepsy in drug discovery. Thanks to their high fecundity and relatively low cost, they are amenable to high-throughput screening. However, the assessment of seizure occurrences in zebrafish larvae remains a bottleneck, as visual analysis is subjective and time-consuming. For the first time, we present an automated algorithm to detect epileptic discharges in single-channel local field potential (LFP) recordings in zebrafish. First, candidate seizure segments are selected based on their energy and length. Afterwards, discriminative features are extracted from each segment. Using a labeled dataset, a support vector machine (SVM) classifier is trained to learn an optimal feature mapping. Finally, this SVM classifier is used to detect seizure segments in new signals. We tested the proposed algorithm both in a chemically-induced seizure model and a genetic epilepsy model. In both cases, the algorithm delivered similar results to visual analysis and found a significant difference in number of seizures between the epileptic and control group. Direct comparison with multichannel techniques or methods developed for different animal models is not feasible. Nevertheless, a literature review shows that our algorithm outperforms state-of-the-art techniques in terms of accuracy, precision and specificity, while maintaining a reasonable sensitivity. Our seizure detection system is a generic, time-saving and objective method to analyze zebrafish LPF, which can replace visual analysis and facilitate true high-throughput studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of Common Use Brominated Flame Retardant (BFR) Toxicity Using a Zebrafish Embryo Model.

PubMed

Usenko, Crystal Y; Abel, Erika L; Hopkins, Aaron; Martinez, Gerardo; Tijerina, Jonathan; Kudela, Molly; Norris, Nick; Joudeh, Lana; Bruce, Erica D

2016-09-02

Brominated flame retardants (BFRs) are used to reduce the flammability of plastics, textiles, and electronics. BFRs vary in their chemical properties and structures, and it is expected that these differences alter their biological interactions and toxicity. Zebrafish were used as the model organism for assessing the toxicity of nine structurally-diverse BFRs. In addition to monitoring for overt toxicity, the rate of spontaneous movement, and acetylcholinesterase and glutathione- S -transferase (GST) activities were assessed following exposure. The toxicities of BFRs tested can be ranked by LC50 as tetrabromobisphenol A (TBBPA) < 4,4'-isopropylidenebis[2-(2,6-dibromophenoxyl)ethanol] (TBBPA-OHEE) < Pentabromochlorocyclohexane (PBCH) < 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) < hexabromocyclododecane (HBCD) < hexabromobenzene (HBB) < Tetrabromophthalic anhydride (PHT4). No adverse effect was observed in di(2-ethylhexyl) tetrabromophthalate (TBPH) or dibromoneopentyl glycol (DBNPG)-treated embryos. The rate of spontaneous movement was decreased in a concentration-dependent manner following exposure to four of the nine compounds. GST activity was elevated following treatment with PBCH, TBBPA, HBCD, and HBB. The results indicate that exposure to several BFRs may activate an antioxidant response and alter behavior during early development. Some of the BFRs, such as TBBPA and TBBPA-OHEE, induced adverse effects at concentrations lower than chemicals that are currently banned. These results suggest that zebrafish are sensitive to exposure to BFRs and can be used as a comparative screening model, as well as to determine alterations in behavior following exposure and probe mechanisms of action.

Evaluation of Common Use Brominated Flame Retardant (BFR) Toxicity Using a Zebrafish Embryo Model

PubMed Central

Usenko, Crystal Y.; Abel, Erika L.; Hopkins, Aaron; Martinez, Gerardo; Tijerina, Jonathan; Kudela, Molly; Norris, Nick; Joudeh, Lana; Bruce, Erica D.

2016-01-01

Brominated flame retardants (BFRs) are used to reduce the flammability of plastics, textiles, and electronics. BFRs vary in their chemical properties and structures, and it is expected that these differences alter their biological interactions and toxicity. Zebrafish were used as the model organism for assessing the toxicity of nine structurally-diverse BFRs. In addition to monitoring for overt toxicity, the rate of spontaneous movement, and acetylcholinesterase and glutathione-S-transferase (GST) activities were assessed following exposure. The toxicities of BFRs tested can be ranked by LC50 as tetrabromobisphenol A (TBBPA) < 4,4′-isopropylidenebis[2-(2,6-dibromophenoxyl)ethanol] (TBBPA-OHEE) < Pentabromochlorocyclohexane (PBCH) < 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) < hexabromocyclododecane (HBCD) < hexabromobenzene (HBB) < Tetrabromophthalic anhydride (PHT4). No adverse effect was observed in di(2-ethylhexyl) tetrabromophthalate (TBPH) or dibromoneopentyl glycol (DBNPG)-treated embryos. The rate of spontaneous movement was decreased in a concentration-dependent manner following exposure to four of the nine compounds. GST activity was elevated following treatment with PBCH, TBBPA, HBCD, and HBB. The results indicate that exposure to several BFRs may activate an antioxidant response and alter behavior during early development. Some of the BFRs, such as TBBPA and TBBPA-OHEE, induced adverse effects at concentrations lower than chemicals that are currently banned. These results suggest that zebrafish are sensitive to exposure to BFRs and can be used as a comparative screening model, as well as to determine alterations in behavior following exposure and probe mechanisms of action. PMID:29051424

Predicting developmental neurotoxicity in rodents from larval zebrafish - - and vice versa

EPA Science Inventory

The complexity of standard mammalian developmental neurotoxicity tests limits evaluation of large numbers of chemicals. Less complex, more rapid assays using larval zebrafish are gaining popularity for evaluating the developmental neurotoxicity of chemicals; there remains, howeve...

Developmental defects in zebrafish for classification of EGF pathway inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim

2014-01-15

One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairmentmore » of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors.« less

Quaternary and tertiary aldoxime antidotes for organophosphate exposure in a zebrafish model system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidt, Hayden R.; Radić, Zoran; Taylor, Palmer

The zebrafish is rapidly becoming an important model system for screening of new therapeutics. Here we evaluated the zebrafish as a potential pharmacological model for screening novel oxime antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE). The k{sub i} values determined for chlorpyrifos oxon (CPO) and dichlorvos (DDVP) showed that CPO was a more potent inhibitor of both human and zebrafish AChE, but overall zebrafish AChE was less sensitive to OP inhibition. In contrast, aldoxime antidotes, the quaternary ammonium 2-PAM and tertiary amine RS-194B, showed generally similar overall reactivation kinetics, k{sub r}, in both zebrafish and human AChE. However, differences between themore » K{sub ox} and k{sub 2} constants suggest that zebrafish AChE associates more tightly with oximes, but has a slower maximal reactivation rate than human AChE. Homology modeling suggests that these kinetic differences result from divergences in the amino acids lining the entrance to the active site gorge. Although 2-PAM had the more favorable in vitro reactivation kinetics, RS-194B was more effective antidote in vivo. In intact zebrafish embryos, antidotal treatment with RS-194B rescued embryos from OP toxicity, whereas 2-PAM had no effect. Dechorionation of the embryos prior to antidotal treatment allowed both 2-PAM and RS-194B to rescue zebrafish embryos from OP toxicity. Interestingly, RS-194B and 2-PAM alone increased cholinergic motor activity in dechorionated embryos possibly due to the reversible inhibition kinetics, K{sub i} and αK{sub i}, of the oximes. Together these results demonstrate that the zebrafish at various developmental stages provides an excellent model for investigating membrane penetrant antidotes to OP exposure. - Highlights: • Zebrafish AChE shares significant structural similarities with human AChE. • OP-inhibited zebrafish and human AChE exhibit similar reactivation kinetics. • The zebrafish chorion is permeable to BBB penetrant and not charged aldoximes. • Zebrafish are a good aquatic model for studying centrally acting antidotes.« less

Anesthesia and euthanasia in zebrafish.

PubMed

Matthews, Monte; Varga, Zoltán M

2012-01-01

Because of the relative ease of embryonic manipulation and observation, the ability to produce a great number of genetic mutations, efficient screening methods, and the continued advance of molecular genetic tools, such as the progress in sequencing and mapping of the zebrafish genome, the use of zebrafish (Danio rerio) as a biomedical model organism continues to expand. However, studies involving zebrafish husbandry and veterinary care struggle to keep pace with scientific progress. This article outlines some of the current, acceptable methods for providing anesthesia and euthanasia and provides some examples of how performance-based approaches can be used to advance the relatively limited number of anesthetic and euthanizing techniques available for zebrafish.

Atrogin-1 Deficiency Leads to Myopathy and Heart Failure in Zebrafish.

PubMed

Bühler, Anja; Kustermann, Monika; Bummer, Tiziana; Rottbauer, Wolfgang; Sandri, Marco; Just, Steffen

2016-01-30

Orchestrated protein synthesis and degradation is fundamental for proper cell function. In muscle, impairment of proteostasis often leads to severe cellular defects finally interfering with contractile function. Here, we analyze for the first time the role of Atrogin-1, a muscle-specific E3 ubiquitin ligase known to be involved in the regulation of protein degradation via the ubiquitin proteasome and the autophagy/lysosome systems, in the in vivo model system zebrafish (Danio rerio). We found that targeted inactivation of zebrafish Atrogin-1 leads to progressive impairment of heart and skeletal muscle function and disruption of muscle structure without affecting early cardiogenesis and skeletal muscle development. Autophagy is severely impaired in Atrogin-1-deficient zebrafish embryos resulting in the disturbance of the cytoarchitecture of cardiomyocytes and skeletal muscle cells. These observations are consistent with molecular and ultrastructural findings in an Atrogin-1 knockout mouse and demonstrate that the zebrafish is a suitable vertebrate model to study the molecular mechanisms of Atrogin-1-mediated autophagic muscle pathologies and to screen for novel therapeutically active substances in high-throughput in vivo small compound screens (SCS).

Zebrafish models for translational neuroscience research: from tank to bedside

PubMed Central

Stewart, Adam Michael; Braubach, Oliver; Spitsbergen, Jan; Gerlai, Robert; Kalueff, Allan V.

2014-01-01

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes. PMID:24726051

Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function.

PubMed

Sarmah, Swapnalee; Marrs, James A

2016-12-16

Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed.

Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

PubMed

Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

2008-07-01

The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

Modeling Zebrafish Developmental Toxicity using a Concurrent In vitro Assay Battery (SOT)

EPA Science Inventory

We describe the development of computational models that predict activity in a repeat-dose zebrafish embryo developmental toxicity assay using a combination of physico-chemical parameters and in vitro (human) assay measurements. The data set covered 986 chemicals including pestic...

Physical exercise improves learning in zebrafish, Danio rerio.

PubMed

Luchiari, Ana Carolina; Chacon, Diana Marques Martins

2013-11-01

Zebrafish is an ideal vertebrate model for neuroscience studies focusing on learning and memory. Although genetic manipulation of zebrafish is available, behavioral protocols are often lacking. In this study we tested whether physical activity can facilitate zebrafish's learning process in an associative conditioning task. Learning was inferred by the approach of the feeding area just after the conditioned stimulus (light). Unexercised zebrafish showed conditioning response from the 5th testing day while fish previously submitted to swim against the water current showed learning by the 3rd day of testing. It seems that physical activity may accelerate associative learning response in zebrafish, indicating the benefits of exercise for cognitive processes. We suggest that this preliminary work could be useful for high throughput screening. Copyright © 2013 Elsevier B.V. All rights reserved.

Zebrafish models of cardiovascular diseases and their applications in herbal medicine research.

PubMed

Seto, Sai-Wang; Kiat, Hosen; Lee, Simon M Y; Bensoussan, Alan; Sun, Yu-Ting; Hoi, Maggie P M; Chang, Dennis

2015-12-05

The zebrafish (Danio rerio) has recently become a powerful animal model for cardiovascular research and drug discovery due to its ease of maintenance, genetic manipulability and ability for high-throughput screening. Recent advances in imaging techniques and generation of transgenic zebrafish have greatly facilitated in vivo analysis of cellular events of cardiovascular development and pathogenesis. More importantly, recent studies have demonstrated the functional similarity of drug metabolism systems between zebrafish and humans, highlighting the clinical relevance of employing zebrafish in identifying lead compounds in Chinese herbal medicine with potential beneficial cardiovascular effects. This paper seeks to summarise the scope of zebrafish models employed in cardiovascular studies and the application of these research models in Chinese herbal medicine to date. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Regulatory effect of hydroquinone-tetraethylene glycol conjugates on zebrafish pigmentation.

PubMed

Le, Hoa Thi; Hong, Bin Na; Lee, Yeong Ro; Cheon, Ji Hyun; Kang, Tong Ho; Kim, Tae Woo

2016-01-15

We synthesized two hydroquinone-tetraethylene glycol conjugates (HQ-TGs) and investigated their logP, photophysical stability, and redox chemical stability. HQ-TGs are a little more hydrophilic than hydroquinone (HQ) and show an enhanced photophysical and redox chemical stability compared with HQ. In addition we studied the effect of HQ-TGs on cell viability and on zebrafish pigmentation. MTT assay in HF-16 cells showed HQ-TGs are less cytotoxic than HQ. The phenotype-based image analysis of zebrafish larvae suggests that HQ-TGs suppress the pigmentation of zebrafish in a dose-dependent manner. The comparative experiments on stability, cytotoxicity, and zebrafish pigmentation between HQ and HQ-TGs suggest that mono tetraethylene glycol-functionalization of HQ is an alternative solution to overcome the adverse effect of HQ. Copyright © 2015 Elsevier Ltd. All rights reserved.

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin,more » arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. - Highlights: • Compound induced effects on DNA methylation in zebrafish embryos • Global methylation not an informative biomarker • Minimal genome wide site specific changes as detected with DREAM • Compound/class specific effects suggested by pyrosequence of specific targets • Zebrafish embryo may be a screening model for epigenetic effects.« less

Meta-analysis of toxicity and teratogenicity of 133 chemicals from zebrafish developmental toxicity studies

EPA Science Inventory

Zebrafish developmental toxicity testing is an emerging field, which faces considerable challenges regarding data meta-analysis and the establishment of standardized test protocols. Here, we present an initial correlation study on toxicity of 133 chemicals based on data in the li...

Development of an Automated Imaging Pipeline for the Analysis of the Zebrafish Larval Kidney

PubMed Central

Westhoff, Jens H.; Giselbrecht, Stefan; Schmidts, Miriam; Schindler, Sebastian; Beales, Philip L.; Tönshoff, Burkhard; Liebel, Urban; Gehrig, Jochen

2013-01-01

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems. PMID:24324758

Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney.

PubMed

Westhoff, Jens H; Giselbrecht, Stefan; Schmidts, Miriam; Schindler, Sebastian; Beales, Philip L; Tönshoff, Burkhard; Liebel, Urban; Gehrig, Jochen

2013-01-01

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems.

Altered Adipogenesis in Zebrafish Larvae Following High Fat Diet and Chemical Exposure Is Visualised by Stimulated Raman Scattering Microscopy.

PubMed

den Broeder, Marjo J; Moester, Miriam J B; Kamstra, Jorke H; Cenijn, Peter H; Davidoiu, Valentina; Kamminga, Leonie M; Ariese, Freek; de Boer, Johannes F; Legler, Juliette

2017-04-24

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish ( Danio rerio ). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0-6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda , lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo.

Altered Adipogenesis in Zebrafish Larvae Following High Fat Diet and Chemical Exposure Is Visualised by Stimulated Raman Scattering Microscopy

PubMed Central

den Broeder, Marjo J.; Moester, Miriam J. B.; Kamstra, Jorke H.; Cenijn, Peter H.; Davidoiu, Valentina; Kamminga, Leonie M.; Ariese, Freek; de Boer, Johannes F.; Legler, Juliette

2017-01-01

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish (Danio rerio). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0–6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda, lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo. PMID:28441764

Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model.

PubMed

Drabsch, Yvette; He, Shuning; Zhang, Long; Snaar-Jagalska, B Ewa; ten Dijke, Peter

2013-11-07

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

Zebrafish-Based Discovery of Antiseizure Compounds from the Red Sea: Pseurotin A2 and Azaspirofuran A.

PubMed

Copmans, Daniëlle; Rateb, Mostafa; Tabudravu, Jioji N; Pérez-Bonilla, Mercedes; Dirkx, Nina; Vallorani, Riccardo; Diaz, Caridad; Pérez Del Palacio, José; Smith, Alan J; Ebel, Rainer; Reyes, Fernando; Jaspars, Marcel; de Witte, Peter A M

2018-04-19

In search for novel antiseizure drugs (ASDs), the European FP7-funded PharmaSea project used zebrafish embryos and larvae as a drug discovery platform to screen marine natural products to identify promising antiseizure hits in vivo for further development. Within the framework of this project, seven known heterospirocyclic γ-lactams, namely, pseurotin A, pseurotin A 2 , pseurotin F1, 11- O-methylpseurotin A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated from the bioactive marine fungus Aspergillus fumigatus, and their antiseizure activity was evaluated in the larval zebrafish pentylenetetrazole (PTZ) seizure model. Pseurotin A 2 and azaspirofuran A were identified as antiseizure hits, while their close chemical analogues were inactive. Besides, electrophysiological analysis from the zebrafish midbrain demonstrated that pseurotin A 2 and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges. Next, to determine whether these findings translate to mammalians, both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor seizure model. They lowered the seizure duration dose-dependently, thereby confirming their antiseizure properties and suggesting activity against drug-resistant seizures. Finally, in a thorough ADMET assessment, pseurotin A 2 and azaspirofuran A were found to be drug-like. Based on the prominent antiseizure activity in both species and the drug-likeness, we propose pseurotin A 2 and azaspirofuran A as lead compounds that are worth further investigation for the treatment of epileptic seizures. This study not only provides the first evidence of antiseizure activity of pseurotins and azaspirofurans, but also demonstrates the value of the zebrafish model in (marine) natural product drug discovery in general, and for ASD discovery in particular.

Studying disorders of vertebrate iron and heme metabolism using zebrafish

PubMed Central

van der Vorm, Lisa N.; Paw, Barry H.

2017-01-01

Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be – and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions. PMID:28129844

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning.

PubMed

Alexander, J; Stainier, D Y; Yelon, D

1998-01-01

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart.

Assessing the Developmental Neurotoxicity of 27 Organophosphorus Pesticides Using a Zebrafish Behavioral Assay

EPA Science Inventory

Assessing the Developmental Neurotoxicity of 27 Organophosphorus Pesticides Using a Zebrafish Behavioral Assay, Waalkes, M., Hunter, D.L., Jarema, K., Mundy, W., and S. Padilla. The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize organophosphor...

Optimization of high-throughput nanomaterial developmental toxicity testing in zebrafish embryos

EPA Science Inventory

Nanomaterial (NM) developmental toxicities are largely unknown. With an extensive variety of NMs available, high-throughput screening methods may be of value for initial characterization of potential hazard. We optimized a zebrafish embryo test as an in vivo high-throughput assay...

Perspectives on zebrafish models of hallucinogenic drugs and related psychotropic compounds.

PubMed

Neelkantan, Nikhil; Mikhaylova, Alina; Stewart, Adam Michael; Arnold, Raymond; Gjeloshi, Visar; Kondaveeti, Divya; Poudel, Manoj K; Kalueff, Allan V

2013-08-21

Among different classes of psychotropic drugs, hallucinogenic agents exert one of the most prominent effects on human and animal behaviors, markedly altering sensory, motor, affective, and cognitive responses. The growing clinical and preclinical interest in psychedelic, dissociative, and deliriant hallucinogens necessitates novel translational, sensitive, and high-throughput in vivo models and screens. Primate and rodent models have been traditionally used to study cellular mechanisms and neural circuits of hallucinogenic drugs' action. The utility of zebrafish ( Danio rerio ) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. Possessing a fully characterized genome, both adult and larval zebrafish are currently widely used for in vivo screening of various psychotropic compounds, including hallucinogens and related drugs. Recognizing the growing importance of hallucinogens in biological psychiatry, here we discuss hallucinogenic-induced phenotypes in zebrafish and evaluate their potential as efficient preclinical models of drug-induced states in humans.

Perspectives on Zebrafish Models of Hallucinogenic Drugs and Related Psychotropic Compounds

PubMed Central

2013-01-01

Among different classes of psychotropic drugs, hallucinogenic agents exert one of the most prominent effects on human and animal behaviors, markedly altering sensory, motor, affective, and cognitive responses. The growing clinical and preclinical interest in psychedelic, dissociative, and deliriant hallucinogens necessitates novel translational, sensitive, and high-throughput in vivo models and screens. Primate and rodent models have been traditionally used to study cellular mechanisms and neural circuits of hallucinogenic drugs’ action. The utility of zebrafish (Danio rerio) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. Possessing a fully characterized genome, both adult and larval zebrafish are currently widely used for in vivo screening of various psychotropic compounds, including hallucinogens and related drugs. Recognizing the growing importance of hallucinogens in biological psychiatry, here we discuss hallucinogenic-induced phenotypes in zebrafish and evaluate their potential as efficient preclinical models of drug-induced states in humans. PMID:23883191

Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function

PubMed Central

Sarmah, Swapnalee; Marrs, James A.

2016-01-01

Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed. PMID:27999267

Fishing for causes and cures of motor neuron disorders

PubMed Central

Patten, Shunmoogum A.; Armstrong, Gary A. B.; Lissouba, Alexandra; Kabashi, Edor; Parker, J. Alex; Drapeau, Pierre

2014-01-01

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development. PMID:24973750

Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration.

PubMed

Schmidt, Rebecca; Beil, Tanja; Strähle, Uwe; Rastegar, Sepand

2014-08-04

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury. In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

Uncertainty Quantification in High Throughput Screening ...

EPA Pesticide Factsheets

Using uncertainty quantification, we aim to improve the quality of modeling data from high throughput screening assays for use in risk assessment. ToxCast is a large-scale screening program that analyzes thousands of chemicals using over 800 assays representing hundreds of biochemical and cellular processes, including endocrine disruption, cytotoxicity, and zebrafish development. Over 2.6 million concentration response curves are fit to models to extract parameters related to potency and efficacy. Models built on ToxCast results are being used to rank and prioritize the toxicological risk of tested chemicals and to predict the toxicity of tens of thousands of chemicals not yet tested in vivo. However, the data size also presents challenges. When fitting the data, the choice of models, model selection strategy, and hit call criteria must reflect the need for computational efficiency and robustness, requiring hard and somewhat arbitrary cutoffs. When coupled with unavoidable noise in the experimental concentration response data, these hard cutoffs cause uncertainty in model parameters and the hit call itself. The uncertainty will then propagate through all of the models built on the data. Left unquantified, this uncertainty makes it difficult to fully interpret the data for risk assessment. We used bootstrap resampling methods to quantify the uncertainty in fitting models to the concentration response data. Bootstrap resampling determines confidence intervals for

Real-Time Monitoring and Analysis of Zebrafish Electrocardiogram with Anomaly Detection.

PubMed

Lenning, Michael; Fortunato, Joseph; Le, Tai; Clark, Isaac; Sherpa, Ang; Yi, Soyeon; Hofsteen, Peter; Thamilarasu, Geethapriya; Yang, Jingchun; Xu, Xiaolei; Han, Huy-Dung; Hsiai, Tzung K; Cao, Hung

2017-12-28

Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish ( Danio rerio ) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine.

Real-Time Monitoring and Analysis of Zebrafish Electrocardiogram with Anomaly Detection

PubMed Central

Lenning, Michael; Fortunato, Joseph; Le, Tai; Clark, Isaac; Sherpa, Ang; Yi, Soyeon; Hofsteen, Peter; Thamilarasu, Geethapriya; Yang, Jingchun; Xu, Xiaolei; Hsiai, Tzung K.; Cao, Hung

2017-01-01

Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish (Danio rerio) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine. PMID:29283402

Methods for studying the zebrafish brain: past, present and future.

PubMed

Wyatt, Cameron; Bartoszek, Ewelina M; Yaksi, Emre

2015-07-01

The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Sequential assessment via daphnia and zebrafish for systematic toxicity screening of heterogeneous substances.

PubMed

Jang, Gun Hyuk; Park, Chang-Beom; Kang, Benedict J; Kim, Young Jun; Lee, Kwan Hyi

2016-09-01

Environment and organisms are persistently exposed by a mixture of various substances. However, the current evaluation method is mostly based on an individual substance's toxicity. A systematic toxicity evaluation of heterogeneous substances needs to be established. To demonstrate toxicity assessment of mixture, we chose a group of three typical ingredients in cosmetic sunscreen products that frequently enters ecosystems: benzophenone-3 (BP-3), ethylhexyl methoxycinnamate (EHMC), and titanium dioxide nanoparticle (TiO2 NP). We first determined a range of nominal toxic concentration of each ingredient or substance using Daphnia magna, and then for the subsequent organismal level phenotypic assessment, chose the wild-type zebrafish embryos. Any phenotype change, such as body deformation, led to further examinations on the specific organs of transgenic zebrafish embryos. Based on the systematic toxicity assessments of the heterogeneous substances, we offer a sequential environmental toxicity assessment protocol that starts off by utilizing Daphnia magna to determine a nominal concentration range of each substance and finishes by utilizing the zebrafish embryos to detect defects on the embryos caused by the heterogeneous substances. The protocol showed additive toxic effects of the mixtures. We propose a sequential environmental toxicity assessment protocol for the systematic toxicity screening of heterogeneous substances from Daphnia magna to zebrafish embryo in-vivo models. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fishing for causes and cures of motor neuron disorders.

PubMed

Patten, Shunmoogum A; Armstrong, Gary A B; Lissouba, Alexandra; Kabashi, Edor; Parker, J Alex; Drapeau, Pierre

2014-07-01

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development. © 2014. Published by The Company of Biologists Ltd.

Innate Color Preference of Zebrafish and Its Use in Behavioral Analyses.

PubMed

Park, Jong-Su; Ryu, Jae-Ho; Choi, Tae-Ik; Bae, Young-Ki; Lee, Suman; Kang, Hae Jin; Kim, Cheol-Hee

2016-10-01

Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for high-throughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique.

Innate Color Preference of Zebrafish and Its Use in Behavioral Analyses

PubMed Central

Park, Jong-Su; Ryu, Jae-Ho; Choi, Tae-Ik; Bae, Young-Ki; Lee, Suman; Kang, Hae Jin; Kim, Cheol-Hee

2016-01-01

Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for high-throughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique. PMID:27802373

Macrophage–Microbe Interactions: Lessons from the Zebrafish Model

PubMed Central

Yoshida, Nagisa; Frickel, Eva-Maria; Mostowy, Serge

2017-01-01

Macrophages provide front line defense against infections. The study of macrophage–microbe interplay is thus crucial for understanding pathogenesis and infection control. Zebrafish (Danio rerio) larvae provide a unique platform to study macrophage–microbe interactions in vivo, from the level of the single cell to the whole organism. Studies using zebrafish allow non-invasive, real-time visualization of macrophage recruitment and phagocytosis. Furthermore, the chemical and genetic tractability of zebrafish has been central to decipher the complex role of macrophages during infection. Here, we discuss the latest developments using zebrafish models of bacterial and fungal infection. We also review novel aspects of macrophage biology revealed by zebrafish, which can potentiate development of new therapeutic strategies for humans. PMID:29250076

Mycobacteriosis in zebrafish colonies.

PubMed

Whipps, Christopher M; Lieggi, Christine; Wagner, Robert

2012-01-01

Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward.

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

PubMed

Poon, Kar Lai; Wang, Xingang; Lee, Serene G P; Ng, Ashley S; Goh, Wei Huang; Zhao, Zhonghua; Al-Haddawi, Muthafar; Wang, Haishan; Mathavan, Sinnakaruppan; Ingham, Philip W; McGinnis, Claudia; Carney, Tom J

2017-03-01

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cynodon dactylon and Sida acuta extracts impact on the function of the cardiovascular system in zebrafish embryos.

PubMed

Kannan, Rajaretinam Rajesh; Vincent, Samuel Gnana Prakash

2012-03-01

The aim of the present study was to screen cardioactive herbs from Western Ghats of India. The heart beat rate (HBR) and blood flow during systole and diastole were tested in zebrafish embryos. We found that Cynodon dactylon (C. dactylon) induced increases in the HBR in zebrafish embryos with a HBR of (3.968±0.344) beats/s, which was significantly higher than that caused by betamethosone [(3.770±0.344) beats/s]. The EC50 value of C. dactylon was 3.738 µg/mL. The methanolic extract of Sida acuta (S. acuta) led to decreases in the HBR in zebrafish embryos [(1.877±0.079) beats/s], which was greater than that caused by nebivolol (positive control). The EC50 value of Sida acuta was 1.195 µg/mL. The untreated embryos had a HBR of (2.685±0.160) beats/s at 3 d post fertilization (dpf). The velocities of blood flow during the cardiac cycle were (2,291.667±72.169) µm/s for the control, (4,250±125.000) µm/s for C. dactylon and (1,083.333±72.169) µm/s for S. acuta. The LC50 values were 32.6 µg/mL for C. dactylon and 20.9 µg/mL for S. acuta. In addition, the extracts exhibited no chemical genetic effects in the drug dosage range tested. In conclusion, we developed an assay that can measure changes in cardiac function in response to herbal small molecules and determine the cardiogenic effects by microvideography.

Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs.

PubMed

Yang, Li-Ling; Wang, Guo-Quan; Yang, Li-Mei; Huang, Zhi-Bing; Zhang, Wen-Qing; Yu, Lin-Zhong

2014-02-21

Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1β, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1β, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

Cardiac Arrhythmia: In vivo screening in the zebrafish to overcome complexity in drug discovery.

PubMed

Macrae, Calum A

2010-07-01

IMPORTANCE OF THE FIELD: Cardiac arrhythmias remain a major challenge for modern drug discovery. Clinical events are paroxysmal, often rare and may be asymptomatic until a highly morbid complication. Target selection is often based on limited information and though highly specific agents are identified in screening, the final efficacy is often compromised by unanticipated systemic responses, a narrow therapeutic index and substantial toxicities. AREAS COVERED IN THIS REVIEW: Our understanding of complexity of arrhythmogenesis has grown dramatically over the last two decades, and the range of potential disease mechanisms now includes pathways previously thought only tangentially involved in arrhythmia. This review surveys the literature on arrhythmia mechanisms from 1965 to the present day, outlines the complex biology underlying potentially each and every rhythm disturbance, and highlights the problems for rational target identification. The rationale for in vivo screening is described and the utility of the zebrafish for this approach and for complementary work in functional genomics is discussed. Current limitations of the model in this setting and the need for careful validation in new disease areas are also described. WHAT THE READER WILL GAIN: An overview of the complex mechanisms underlying most clinical arrhythmias, and insight into the limits of ion channel conductances as drug targets. An introduction to the zebrafish as a model organism, in particular for cardiovascular biology. Potential approaches to overcoming the hurdles to drug discovery in the face of complex biology including in vivo screening of zebrafish genetic disease models. TAKE HOME MESSAGE: In vivo screening in faithful disease models allows the effects of drugs on integrative physiology and disease biology to be captured during the screening process, in a manner agnostic to potential drug target or targets. This systematic strategy bypasses current gaps in our understanding of disease biology, but emphasizes the importance of the rigor of the disease model.

A Phenotypic Screen in Zebrafish Identifies a Novel Small-Molecule Inducer of Ectopic Tail Formation Suggestive of Alterations in Non-Canonical Wnt/PCP Signaling

PubMed Central

Gebruers, Evelien; Cordero-Maldonado, María Lorena; Gray, Alexander I.; Clements, Carol; Harvey, Alan L.; Edrada-Ebel, Ruangelie; de Witte, Peter A. M.; Crawford, Alexander D.; Esguerra, Camila V.

2013-01-01

Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen – Jasminum gilgianum, an Oleaceae species native to Papua New Guinea – induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME’s mechanism of action will help determine this compound’s pharmacological utility. PMID:24349481

Behavioral effects of MDMA ('ecstasy') on adult zebrafish.

PubMed

Stewart, Adam; Riehl, Russell; Wong, Keith; Green, Jeremy; Cosgrove, Jessica; Vollmer, Karoly; Kyzar, Evan; Hart, Peter; Allain, Alexander; Cachat, Jonathan; Gaikwad, Siddharth; Hook, Molly; Rhymes, Kate; Newman, Alan; Utterback, Eli; Chang, Katie; Kalueff, Allan V

2011-06-01

3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') is a potent psychedelic drug inducing euphoria and hypersociability in humans, as well as hyperactivity and anxiety in rodents. Adult zebrafish (Danio rerio) have become a widely used species in neurobehavioral research. Here, we explore the effects of a wide range (0.25-120 mg/l) of acute MDMA doses on zebrafish behavior in the novel tank test. Although MDMA was inactive at lower doses (0.25-10 mg/l), higher doses reduced bottom swimming and immobility (40-120 mg/l) and impaired intrasession habituation (10-120 mg/l). MDMA also elevated brain c-fos expression, collectively confirming the usage of zebrafish models for screening of hallucinogenic compounds.

High-throughput behavioral screening method for detecting auditory response defects in zebrafish.

PubMed

Bang, Pascal I; Yelick, Pamela C; Malicki, Jarema J; Sewell, William F

2002-08-30

We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles. Copyright 2002 Elsevier Science B.V.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

PubMed

Thomas, Andrew J; Hailey, Dale W; Stawicki, Tamara M; Wu, Patricia; Coffin, Allison B; Rubel, Edwin W; Raible, David W; Simon, Julian A; Ou, Henry C

2013-03-06

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

Maintenance of Zebrafish Lines at the European Zebrafish Resource Center.

PubMed

Geisler, Robert; Borel, Nadine; Ferg, Marco; Maier, Jana Viktoria; Strähle, Uwe

2016-07-01

We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections.

Zebrafish as an early stage screening tool to study the systemic circulation of nanoparticulate drug delivery systems in vivo.

PubMed

Sieber, Sandro; Grossen, Philip; Detampel, Pascal; Siegfried, Salome; Witzigmann, Dominik; Huwyler, Jörg

2017-10-28

Nanomedicines have gained much attention for the delivery of small molecules or nucleic acids as treatment options for many diseases. However, the transfer from experimental systems to in vivo applications remains a challenge since it is difficult to assess their circulation behavior in the body at an early stage of drug discovery. Thus, innovative and improved concepts are urgently needed to overcome this issue and to close the gap between empiric nanoparticle design, in vitro assessment, and first in vivo experiments using rodent animal models. This study was focused on the zebrafish as a vertebrate screening model to assess the circulation in blood and extravasation behavior of nanoparticulate drug delivery systems in vivo. To validate this novel approach, monodisperse preparations of fluorescently labeled liposomes with similar size and zeta potential were injected into transgenic zebrafish lines expressing green fluorescent protein in their vasculature. Phosphatidylcholine-based lipids differed by fatty acid chain length and saturation. Circulation behavior and vascular distribution pattern were evaluated qualitatively and semi-quantitatively using image analysis. Liposomes composed of lipids with lower transition temperature (<28°C) as well as PEGylated liposomes showed longer circulation times and extravasation. In contrast, liposomes composed of lipids with transition temperatures>28°C bound to venous parts of the vasculature. This circulation patterns in the zebrafish model did correlate with published and experimental pharmacokinetic data from mice and rats. Our findings indicate that the zebrafish model is a useful vertebrate screening tool for nanoparticulate drug delivery systems to predict their in vivo circulation behavior with respect to systemic circulation time and exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis

PubMed Central

Patten, Shunmoogum A.; Aggad, Dina; Martinez, Jose; Tremblay, Elsa; Petrillo, Janet; Armstrong, Gary A.B.; Maios, Claudia; Liao, Meijiang; Ciura, Sorana; Wen, Xiao-Yan; Rafuse, Victor; Ichida, Justin; Zinman, Lorne; Julien, Jean-Pierre; Kabashi, Edor; Robitaille, Richard; Korngut, Lawrence; Parker, J. Alexander

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease. PMID:29202456

Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis.

PubMed

Patten, Shunmoogum A; Aggad, Dina; Martinez, Jose; Tremblay, Elsa; Petrillo, Janet; Armstrong, Gary Ab; La Fontaine, Alexandre; Maios, Claudia; Liao, Meijiang; Ciura, Sorana; Wen, Xiao-Yan; Rafuse, Victor; Ichida, Justin; Zinman, Lorne; Julien, Jean-Pierre; Kabashi, Edor; Robitaille, Richard; Korngut, Lawrence; Parker, J Alexander; Drapeau, Pierre

2017-11-16

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease.

Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish

PubMed Central

Ryan, Sean; Willer, Jason; Marjoram, Lindsay; Bagwell, Jennifer; Mankiewicz, Jamie; Leshchiner, Ignaty; Goessling, Wolfram; Bagnat, Michel; Katsanis, Nicholas

2013-01-01

Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the development of whole genome and RNA sequencing approaches for gene discovery. Here we describe a whole exome sequencing (WES) approach that allows rapid and cost-effective identification of mutations. We used our WES methodology to isolate four mutations that cause kidney cysts; we identified novel alleles in two ciliary genes as well as two novel mutants. The WES approach described here does not require specialized infrastructure or training and is therefore widely accessible. This methodology should thus help facilitate genetic screens and expedite the identification of mutants that can inform basic biological processes and the causality of genetic disorders in humans. PMID:24130329

Lethal and sublethal effects of aniline and chlorinated anilines on zebrafish embryos and larvae.

PubMed

Horie, Yoshifumi; Yamagishi, Takahiro; Koshio, Masaaki; Iguchi, Taisen; Tatarazako, Norihisa

2017-07-01

Environmental risk assessments show increased attention to the sublethal effects of chemicals on aquatic organisms. The Organization for Economic Cooperation and Development (OECD) established the "Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages" (OECD test 212) to predict lethal effects. It is still unclear, however, whether this test can predict sublethal effects. Although their sublethal effects are still unknown, chlorinated anilines are widely used in various fields. The purpose of this study, therefore, is to investigate sublethal effects of chlorinated anilines using OECD test 212 with zebrafish, and to examine the correlation of several sublethal effects between embryo and larval stages. Embryos were exposed to aniline and nine chlorinated anilines until 8 days post-fertilization. A delayed lethal effect was observed from three of the 10 anilines tested. In the control group, the swim bladder inflated after hatching, but there was no swim-bladder inflation after exposure to the chlorinated anilines. Fertilized eggs exposed to lower concentrations of test chemicals showed effects during embryogenesis that did not affect mortality rates, such as changes in body curvature and edema. Our results show that chlorinated anilines induce not only lethal effects but also a variety of sublethal effects. Moreover, a detailed estimate of these effects requires study during both embryonic and larval stages. OECD test 212 may therefore prove useful as a method for screening chemicals for lethal and sublethal effects. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights.

PubMed

Harrison, Nicholas R; Laroche, Fabrice J F; Gutierrez, Alejandro; Feng, Hui

2016-01-01

Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients.

Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights

PubMed Central

Harrison, Nicholas R.; Laroche, Fabrice J.F.; Gutierrez, Alejandro

2016-01-01

Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients. PMID:27165361

Differences in Acute Alcohol-Induced Behavioral Responses Among Zebrafish Populations

PubMed Central

Gerlai, Robert; Ahmad, Fahad; Prajapati, Sonal

2009-01-01

Background With the arsenal of genetic tools available for zebrafish, this species has been successfully used to investigate the genetic aspects of human diseases from developmental disorders to cancer. Interest in the behavior and brain function of zebrafish is also increasing as CNS disorders may be modeled and studied with this species. Alcoholism and alcohol abuse are among the most devastating and costliest diseases. However, the mechanisms of these diseases are not fully understood. Zebrafish has been proposed as a model organism to study such mechanisms. Characterization of alcohol’s effects on zebrafish is a necessary step in this research. Methods Here, we compare the effects of acute alcohol (EtOH) administration on the behavior of zebrafish from 4 distinct laboratory-bred populations using automated as well as observation based behavioral quantification methods. Results Alcohol treatment resulted in significant dose-dependent behavioral changes but the dose–response trajectories differed among zebrafish populations. Conclusions The results demonstrate for the first time a genetic component in alcohol responses in adult zebrafish and also show the feasibility of high throughput behavioral screening. We discuss the exploration and exploitation of the genetic differences found. PMID:18652595

The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

PubMed Central

Motaln, Helena; Turnšek, Tamara Lah

2015-01-01

Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma. PMID:26109632

The zebrafish eye—a paradigm for investigating human ocular genetics

PubMed Central

Richardson, R; Tracey-White, D; Webster, A; Moosajee, M

2017-01-01

Although human epidemiological and genetic studies are essential to elucidate the aetiology of normal and aberrant ocular development, animal models have provided us with an understanding of the pathogenesis of multiple developmental ocular malformations. Zebrafish eye development displays in depth molecular complexity and stringent spatiotemporal regulation that incorporates developmental contributions of the surface ectoderm, neuroectoderm and head mesenchyme, similar to that seen in humans. For this reason, and due to its genetic tractability, external fertilisation, and early optical clarity, the zebrafish has become an invaluable vertebrate system to investigate human ocular development and disease. Recently, zebrafish have been at the leading edge of preclinical therapy development, with their amenability to genetic manipulation facilitating the generation of robust ocular disease models required for large-scale genetic and drug screening programmes. This review presents an overview of human and zebrafish ocular development, genetic methodologies employed for zebrafish mutagenesis, relevant models of ocular disease, and finally therapeutic approaches, which may have translational leads in the future. PMID:27612182

Multifaceted toxicity assessment of catalyst composites in transgenic zebrafish embryos.

PubMed

Jang, Gun Hyuk; Lee, Keon Yong; Choi, Jaewon; Kim, Sang Hoon; Lee, Kwan Hyi

2016-09-01

Recent development in the field of nanomaterials has given rise into the inquiries regarding the toxicological characteristics of the nanomaterials. While many individual nanomaterials have been screened for their toxicological effects, composites that accompany nanomaterials are not common subjects to such screening through toxicological assessment. One of the widely used composites that accompany nanomaterials is catalyst composite used to reduce air pollution, which was selected as a target composite with nanomaterials for the multifaceted toxicological assessment. As existing studies did not possess any significant data regarding such catalyst composites, this study focuses on investigating toxicological characteristics of catalyst composites from various angles in both in-vitro and in-vivo settings. Initial toxicological assessment on catalyst composites was conducted using HUVECs for cell viability assays, and subsequent in-vivo assay regarding their direct influence on living organisms was done. The zebrafish embryo and its transgenic lines were used in the in-vivo assays to obtain multifaceted analytic results. Data obtained from the in-vivo assays include blood vessel formation, mutated heart morphology, and heart functionality change. Our multifaceted toxicological assessment pointed out that chemical composites augmented with nanomaterials can too have toxicological threat as much as individual nanomaterials do and alarms us with their danger. This manuscript provides a multifaceted assessment for composites augmented with nanomaterials, of which their toxicological threats have been overlooked. Copyright © 2016 Elsevier Ltd. All rights reserved.

Time-dependent inhibitory effects of Tris(1, 3-dichloro-2-propyl) phosphate on growth and transcription of genes involved in the GH/IGF axis, but not the HPT axis, in female zebrafish.

PubMed

Zhu, Ya; Su, Guanyong; Yang, Dandong; Zhang, Yongkang; Yu, Liqin; Li, Yufei; Giesy, John P; Letcher, Robert J; Liu, Chunsheng

2017-10-01

Growth curves were used to determine sensitive exposure windows for evaluation of developmental toxicity of chemicals to zebrafish. Dose- and time-dependent effects on body mass, body length and expression of genes involved in the growth hormone/insulin-like growth factor (GH/IGF) axis and the hypothalamic-pituitary-thyroid (HPT) axis were examined after exposure to environmentally relevant concentrations of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP). Based on growth curves, zebrafish grew most rapidly between 60 and 90 days post fertilization (dpf). Exposure to environmentally relevant concentrations of TDCIPP significantly decreased body mass and body length and down-regulated expression of several genes involved in the GH/IGF axis of female zebrafish, but no such effects were observed in male zebrafish. Exposure to TDCIPP did not change concentrations of thyroid hormones or expression of genes along the HPT axis in female and male zebrafish. These results suggest that growth stages of zebrafish between 60 and 90 dpf might be most appropriate for evaluation of developmental toxicity of chemicals, and down-regulation of genes involved in the GH/IGF axis, but not the HPT axis, might be responsible for the observed growth inhibition in females exposed to TDCIPP. Copyright © 2017 Elsevier Ltd. All rights reserved.

A homeostatic, chip-based platform for zebrafish larvae immobilization and long-term imaging

NASA Astrophysics Data System (ADS)

Friedrich, Timo; Zhu, Feng; Wlodkowic, Donald; Kaslin, Jan

2015-12-01

Zebrafish larvae are ideal for toxicology and drug screens due to their transparency, small size and similarity to humans on the genetic level. Using modern imaging techniques, cells and tissues can be dynamically visualised and followed over days in multiple zebrafish. Yet continued imaging experiments require specialized conditions such as: moisture and heat control to maintain specimen homeostasis. Chambers that control the environment are generally very expensive and are not always available for all imaging platforms. A highly customizable mounting configuration with built-in means of controlling temperature and media flow would therefore be a valuable tool for long term imaging experiments. Rapid prototyping using 3D printing is particularly suitable as a production method as it offers high flexibility in design, is widely available and allows a high degree of customizing. We study neural regeneration in zebrafish. Regeneration is limited in humans, but zebrafish recover from neural damage within days. Yet, the underlying regenerative mechanisms remain unclear. We developed an agarose based mounting system that holds the embryos in defined positions along removable strips. Homeostasis and temperature control is ensured by channels circulating buffer and heated water. This allows to image up to 120 larvae simultaneously for more than two days. Its flexibility and the low-volume, high larvae ratio will allow screening of small compound libraries. Taken together, we offer a low cost, highly adaptable solution for long term in-vivo imaging.

Automated processing of zebrafish imaging data: a survey.

PubMed

Mikut, Ralf; Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A; Kausler, Bernhard X; Ledesma-Carbayo, María J; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-09-01

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

Automated Processing of Zebrafish Imaging Data: A Survey

PubMed Central

Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A.; Kausler, Bernhard X.; Ledesma-Carbayo, María J.; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-01-01

Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

Age matters: Developmental stage of Danio rerio larvae influences photomotor response thresholds to diazinion or diphenhydramine

PubMed Central

Kristofco, Lauren A.; Cruz, Luis Colon; Haddad, Samuel P.; Behra, Martine L; Chambliss, C. Kevin; Brooks, Bryan W.

2016-01-01

Because basic toxicological data is unavailable for the majority of industrial compounds, High Throughput Screening (HTS) assays using the embryonic and larval zebrafish provide promising approaches to define bioactivity profiles and identify potential adverse outcome pathways for previously understudied chemicals. Unfortunately, standardized approaches, including HTS experimental designs, for examining fish behavioral responses to contaminants are rarely available. In the present study, we examined movement behavior of larval zebrafish over 7 days (4–10 days post fertilization or dpf) during typical daylight workday hours to determine whether intrinsic activity differed with age and time of day. We then employed an early life stage approach using the Fish Embryo Test (FET) at multiple developmental ages to evaluate whether photomotor response (PMR) behavior differed with zebrafish age following exposure to diazinon (DZN), a well-studied orthophosphate insecticide, and diphenhydramine (DPH), an antihistamine that also targets serotonin reuptake transporters and the acetylcholine receptor. 72 h studies were conducted at 1–4, 4–7 and 7–10 dpf, followed by behavioral observations using a ViewPoint system at 4, 7 and 10 dpf. Distance traveled and swimming speeds were quantified; nominal treatment levels were analytically verified by isotope-dilution LC-MSMS. Larval zebrafish locomotion displayed significantly different (p < 0.05) activity profiles over the course of typical daylight and workday hours, and these time of day PMR activity profiles were similar across ages examined (4–10 dpf). 10 dpf zebrafish larvae were consistently more sensitive to DPH than either the 4 or 7 dpf larvae with an environmentally realistic lowest observed effect concentration of 200 ng/L. Though ELS and FET studies with zebrafish typically focus on mortality or teratogenicity in 0–4 dpf organisms, behavioral responses of slightly older fish were several orders of magnitude more sensitive to DPH. Our observations highlight the importance of understanding the influence of time of day on intrinsic locomotor activity, and the age-specific hazards of aquatic contaminants to fish behavior. PMID:26431593

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

The neurogenetic frontier--lessons from misbehaving zebrafish.

PubMed

Burgess, Harold A; Granato, Michael

2008-11-01

One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish.

The neurogenetic frontier—lessons from misbehaving zebrafish

PubMed Central

Granato, Michael

2008-01-01

One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish. PMID:18836206

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line

PubMed Central

Thomas, Andrew J.; Hailey, Dale W.; Stawicki, Tamara M.; Wu, Patricia; Coffin, Allison B.; Rubel, Edwin W.; Raible, David W.; Simon, Julian A.; Ou, Henry C.

2013-01-01

Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line. PMID:23467357

Two-photon-based photoactivation in live zebrafish embryos.

PubMed

Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

2010-12-24

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish

PubMed Central

Xie, Shao-Lin; Bian, Wan-Ping; Wang, Chao; Junaid, Muhammad; Zou, Ji-Xing; Pei, De-Sheng

2016-01-01

Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system. PMID:27680290

Whole organism high content screening identifies stimulators of pancreatic beta-cell proliferation.

PubMed

Tsuji, Naoki; Ninov, Nikolay; Delawary, Mina; Osman, Sahar; Roh, Alex S; Gut, Philipp; Stainier, Didier Y R

2014-01-01

Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.

Zebrafish-based identification of the antiseizure nucleoside inosine from the marine diatom Skeletonema marinoi

PubMed Central

Khamma, Supitcha; Marcourt, Laurence; Righi, Davide; Romano, Giovanna; Esposito, Francesco; Ianora, Adrianna; Queiroz, Emerson F.; Wolfender, Jean-Luc; Crawford, Alexander D.

2018-01-01

With the goal of identifying neuroactive secondary metabolites from microalgae, a microscale in vivo zebrafish bioassay for antiseizure activity was used to evaluate bioactivities of the diatom Skeletonema marinoi, which was recently revealed as being a promising source of drug-like small molecules. A freeze-dried culture of S. marinoi was extracted by solvents with increasing polarities (hexane, dichloromethane, methanol and water) and these extracts were screened for anticonvulsant activity using a larval zebrafish epilepsy model with seizures induced by the GABAA antagonist pentylenetetrazole. The methanolic extract of S. marinoi exhibited significant anticonvulsant activity and was chosen for bioassay-guided fractionation, which associated the bioactivity with minor constituents. The key anticonvulsant constituent was identified as the nucleoside inosine, a well-known adenosine receptor agonist with previously reported antiseizure activities in mice and rat epilepsy models, but not reported to date as a bioactive constituent of microalgae. In addition, a UHPLC-HRMS metabolite profiling was used for dereplication of the other constituents of S. marinoi. Structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution spectrometry. These results highlight the potential of zebrafish-based screening and bioassay-guided fractionation to identify neuroactive marine natural products. PMID:29689077

A larval zebrafish model of bipolar disorder as a screening platform for neuro-therapeutics.

PubMed

Ellis, Lee David; Soanes, Kelly Howard

2012-08-01

Modelling neurological diseases has proven extraordinarily difficult due to the phenotypic complexity of each disorder. The zebrafish has become a useful model system with which to study abnormal neurological and behavioural activity and holds promise as a model of human disease. While most of the disease modelling using zebrafish has made use of adults, larvae hold tremendous promise for the high-throughput screening of potential therapeutics. The further development of larval disease models will strengthen their ability to contribute to the drug screening process. Here we have used zebrafish larvae to model the symptoms of bipolar disorder by treating larvae with sub-convulsive concentrations of the GABA antagonist pentylenetetrazol (PTZ). A number of therapeutics that act on different targets, in addition to those that have been used to treat bipolar disorder, were tested against this model to assess its predictive value. Carbamazepine, valproic acid, baclofen and honokiol, were found to oppose various aspects of the PTZ-induced changes in activity. Lidocaine and haloperidol exacerbated the PTZ-induced activity changes and sulpiride had no effect. By comparing the degree of phenotypic rescue with the mechanism of action of each therapeutic we have shown that the low-concentration PTZ model can produce a number of intermediate phenotypes that model symptoms of bipolar disorder, may be useful in modelling other disease states, and will help predict the efficacy of novel therapeutics. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Heat-shock protein 60 is required for blastema formation and maintenance during regeneration

PubMed Central

Makino, Shinji; Whitehead, Geoffrey G.; Lien, Ching-Ling; Kim, Soo; Jhawar, Payal; Kono, Akane; Kawata, Yasushi; Keating, Mark T.

2005-01-01

Zebrafish fin regeneration requires the formation and maintenance of blastema cells. Blastema cells are not derived from stem cells but behave as such, because they are slow-cycling and are thought to provide rapidly proliferating daughter cells that drive regenerative outgrowth. The molecular basis of blastema formation is not understood. Here, we show that heat-shock protein 60 (hsp60) is required for blastema formation and maintenance. We used a chemical mutagenesis screen to identify no blastema (nbl), a zebrafish mutant with an early fin regeneration defect. Fin regeneration failed in nbl due to defective blastema formation. nbl also failed to regenerate hearts. Positional cloning and mutational analyses revealed that nbl results from a V324E missense mutation in hsp60. This mutation reduced hsp60 function in binding and refolding denatured proteins. hsp60 expression is increased during formation of blastema cells, and dysfunction leads to mitochondrial defects and apoptosis in these cells. These data indicate that hsp60 is required for the formation and maintenance of regenerating tissue. PMID:16204379

Zebrafish Heart Failure Models for the Evaluation of Chemical Probes and Drugs

PubMed Central

Monte, Aaron; Cook, James M.; Kabir, Mohd Shahjahan; Peterson, Karl P.

2013-01-01

Abstract Heart failure is a complex disease that involves genetic, environmental, and physiological factors. As a result, current medication and treatment for heart failure produces limited efficacy, and better medication is in demand. Although mammalian models exist, simple and low-cost models will be more beneficial for drug discovery and mechanistic studies of heart failure. We previously reported that aristolochic acid (AA) caused cardiac defects in zebrafish embryos that resemble heart failure. Here, we showed that cardiac troponin T and atrial natriuretic peptide were expressed at significantly higher levels in AA-treated embryos, presumably due to cardiac hypertrophy. In addition, several human heart failure drugs could moderately attenuate the AA-induced heart failure by 10%–40%, further verifying the model for drug discovery. We then developed a drug screening assay using the AA-treated zebrafish embryos and identified three compounds. Mitogen-activated protein kinase kinase inhibitor (MEK-I), an inhibitor for the MEK-1/2 known to be involved in cardiac hypertrophy and heart failure, showed nearly 60% heart failure attenuation. C25, a chalcone derivative, and A11, a phenolic compound, showed around 80% and 90% attenuation, respectively. Time course experiments revealed that, to obtain 50% efficacy, these compounds were required within different hours of AA treatment. Furthermore, quantitative polymerase chain reaction showed that C25, not MEK-I or A11, strongly suppressed inflammation. Finally, C25 and MEK-I, but not A11, could also rescue the doxorubicin-induced heart failure in zebrafish embryos. In summary, we have established two tractable heart failure models for drug discovery and three potential drugs have been identified that seem to attenuate heart failure by different mechanisms. PMID:24351044

The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver.

PubMed

Yin, Chunyue; Evason, Kimberley J; Maher, Jacquelyn J; Stainier, Didier Y R

2012-11-01

Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity of in vivo data. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity, and interactions between these two cell types are potentially critical for their development and function. Here, we introduce a transgenic zebrafish line, Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene-expression profile, and increased proliferation and matrix production, in response to an acute hepatic insult. Using the Tg(hand2:EGFP) line, we conducted time-course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells, including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development. Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. The Tg(hand2:EGFP) line represents a unique tool for in vivo analysis and molecular dissection of HSC behavior. Copyright © 2012 American Association for the Study of Liver Diseases.

Antibiotic toxicity and absorption in zebrafish using liquid chromatography-tandem mass spectrometry.

PubMed

Zhang, Fan; Qin, Wei; Zhang, Jing-Pu; Hu, Chang-Qin

2015-01-01

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10-1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD50 values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.

Multidimensional In Vivo Hazard Assessment Using Zebrafish

PubMed Central

Tanguay, Robert L.

2014-01-01

There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies. PMID:24136191

Toxicity of Vascular Disrupting Chemicals to Developing Zebrafish

EPA Science Inventory

Vascular development is integral to proper embryonic development and disruption of that process can have serious developmental consequences. We performed static 48-hr exposures of transgenic TG(kdr:EGFP)s843 zebrafish (Danio rerio) embryos with the known vascular inhibitors Vatal...

Anxiogenic-like effects of chronic nicotine exposure in zebrafish.

PubMed

Stewart, Adam Michael; Grossman, Leah; Collier, Adam D; Echevarria, David J; Kalueff, Allan V

2015-12-01

Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological profile has been extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance of finding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens for CNS drug discovery, necessitate novel efficient experimental models for nicotine research. Zebrafish (Danio rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of acute nicotine treatment in zebrafish are consistently reported in the literature. However, while nicotine abuse is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrafish behavior. In the present study, chronic 4-day exposure to 1-2mg/L nicotine mildly increased adult zebrafish shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes robust anxiogenic behavioral responses in zebrafish tested in the novel tank test paradigm. Generally paralleling clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of zebrafish for nicotine research. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro and in vivo structure and activity relationship analysis of polymethoxylated flavonoids: identifying sinensetin as a novel antiangiogenesis agent.

PubMed

Lam, In Kei; Alex, Deepa; Wang, You-Hua; Liu, Ping; Liu, Ai-Lin; Du, Guan-Hua; Lee, Simon Ming Yuen

2012-06-01

Polymethoxylated flavonoids are present in citrus fruit in a range of chemical structures and abundance. These compounds have potential for anticarcinogenesis, antitumor, and cardiovascular protective activity, but the effect on angiogenesis has not been well studied. Human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish (Danio rerio) in vivo models were used to screen and identify the antiangiogenesis activity of seven polymethoxylated flavonoids; namely, hesperetin, naringin, neohesperidin, nobiletin, scutellarein, scutellarein tetramethylether, and sinensetin. Five, excluding naringin and neohesperidin, showed different degrees of potency of antiangiogenesis activity. Sinensetin, which had the most potent antiangiogenesis activity and the lowest toxicity, inhibited angiogenesis by inducing cell cycle arrest in the G0/G1 phase in HUVEC culture and downregulating the mRNA expressions of angiogenesis genes flt1, kdrl, and hras in zebrafish. The in vivo structure-activity relationship (SAR) analysis indicated that a flavonoid with a methoxylated group at the C3' position offers a stronger antiangiogenesis activity, whereas the absence of a methoxylated group at the C8 position offers lower lethal toxicity in addition to enhancing the antiangiogenesis activity. This study provides new insight into how modification of the chemical structure of polymethoxylated flavonoids affects this newly identified antiangiogenesis activity. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The synthetic substance hypoxanthine 3-N-oxide elicits alarm reactions in zebrafish (Danio rerio)

PubMed Central

Parra, Kevin V.; Adrian, James C.; Gerlai, Robert

2009-01-01

Zebrafish, one of the preferred study species of geneticists, is gaining increasing popularity in behavioral neuroscience. This small and prolific species may be an excellent tool with which the biological mechanisms of vertebrate brain function and behavior are investigated. Zebrafish has been proposed as a model organism in the analysis of fear responses and human anxiety disorders. Species-specific cues signaling the presence of predators have been successfully utilized in such research. Zebrafish has been shown to respond to its natural alarm substance with species-typical fear reactions. However, the extraction of this alarm substance and ascertaining its consistent dosing has been problematic. A synthetic substance with a known chemical identity and molecular weight would allow precise dosing and experimental control. Previously, the chemical component, hypoxanthine 3-N-oxide, common to several fish alarm substances has been identified and has been shown to elicit alarm reactions in fish species belonging to the Osteriophysan superorder. In the current study we investigate the effect of hypoxanthine 3-N-oxide by exposing zebrafish to three different concentrations of this synthetic substance. Our results show that the substance efficaciously induces species-typical fear reactions increasing the number of erratic movement episodes and jumps in zebrafish. We discuss the translational relevance of our findings and conclude that hypoxanthine 3-N-oxide will have utility to elicit fear responses in the laboratory in a precisely controlled manner in zebrafish. PMID:19583985

Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish

PubMed Central

Mokalled, Mayssa H.; Patra, Chinmoy; Dickson, Amy L.; Endo, Toyokazu; Stainier, Didier Y. R.; Poss, Kenneth D.

2016-01-01

Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration, a relatively unexplored process. Here, we performed a genome-wide profiling screen for secreted factors that are upregulated during zebrafish spinal cord regeneration. We find that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupt spinal cord repair, while transgenic ctgfa overexpression and local human CTGF recombinant protein delivery accelerate bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration. PMID:27811277

Teratological Effects of a Panel of Sixty Water-Soluble Toxicants on Zebrafish Development

PubMed Central

Ali, Shaukat; Aalders, Jeffrey

2014-01-01

Abstract The zebrafish larva is a promising whole-animal model for safety pharmacology, environmental risk assessment, and developmental toxicity. This model has been used for the high-throughput toxicity screening of various compounds. Our aim here is to identify possible phenotypic markers of teratogenicity in zebrafish embryos that could be used for the assaying compounds for reproductive toxicity. We have screened a panel of 60 water-soluble toxicants to examine their effects on zebrafish development. A total of 22,080 wild-type zebrafish larvae were raised in 250 μL defined buffer in 96-well plates at a plating density of one embryo per well. They were exposed for a 96-h period starting at 24 h post-fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for developmental toxicity assessment. A total of 9017 survivors were analyzed at 5 days post-fertilization for nine phenotypes, namely, (1) normal, (2) pericardial oedema, (3) yolk sac oedema, (4) melanophores dispersed, (5) bent tail tip, (6) bent body axis, (7) abnormal Meckel's cartilage, (8) abnormal branchial arches, and (9) uninflated swim bladder. For each toxicant, the EC50 (concentration required to produce one or more of these abnormalities in 50% of embryos) was also calculated. For the majority of toxicants (55/60) there was, at the population level, a statistically significant, concentration-dependent increase in the incidence of abnormal phenotypes among survivors. The commonest abnormalities were pericardial oedema, yolk sac oedema, dispersed melanophores, and uninflated swim bladder. It is possible therefore that these could prove to be general indicators of reproductive toxicity in the zebrafish embryo assay. PMID:24650241

Exploratory behaviour in the open field test adapted for larval zebrafish: impact of environmental complexity.

PubMed

Ahmad, Farooq; Richardson, Michael K

2013-01-01

This study aimed to develop and characterize a novel (standard) open field test adapted for larval zebrafish. We also developed and characterized a variant of the same assay consisting of a colour-enriched open field; this was used to assess the impact of environmental complexity on patterns of exploratory behaviours as well to determine natural colour preference/avoidance. We report the following main findings: (1) zebrafish larvae display characteristic patterns of exploratory behaviours in the standard open field, such as thigmotaxis/centre avoidance; (2) environmental complexity (i.e. presence of colours) differentially affects patterns of exploratory behaviours and greatly attenuates natural zone preference; (3) larvae displayed the ability to discriminate colours. As reported previously in adult zebrafish, larvae showed avoidance towards blue and black; however, in contrast to the reported adult behaviour, larvae displayed avoidance towards red. Avoidance towards yellow and preference for green and orange are shown for the first time, (4) compared to standard open field tests, exposure to the colour-enriched open field resulted in an enhanced expression of anxiety-like behaviours. To conclude, we not only developed and adapted a traditional rodent behavioural assay that serves as a gold standard in preclinical drug screening, but we also provide a version of the same test that affords the possibility to investigate the impact of environmental stress on behaviour in larval zebrafish while representing the first test for assessment of natural colour preference/avoidance in larval zebrafish. In the future, these assays will improve preclinical drug screening methodologies towards the goal to uncover novel drugs. This article is part of a Special Issue entitled: insert SI title. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparative Analyses of Zebrafish Anxiety-Like Behavior Using Conflict-Based Novelty Tests.

PubMed

Kysil, Elana V; Meshalkina, Darya A; Frick, Erin E; Echevarria, David J; Rosemberg, Denis B; Maximino, Caio; Lima, Monica Gomes; Abreu, Murilo S; Giacomini, Ana C; Barcellos, Leonardo J G; Song, Cai; Kalueff, Allan V

2017-06-01

Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo, whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.

Startle response memory and hippocampal changes in adult zebrafish pharmacologically-induced to exhibit anxiety/depression-like behaviors.

PubMed

Pittman, Julian T; Lott, Chad S

2014-01-17

Zebrafish (Danio rerio) are rapidly becoming a popular animal model for neurobehavioral and psychopharmacological research. While startle testing is a well-established assay to investigate anxiety-like behaviors in different species, screening of the startle response and its habituation in zebrafish is a new direction of translational biomedical research. This study focuses on a novel behavioral protocol to assess a tapping-induced startle response and its habituation in adult zebrafish that have been pharmacologically-induced to exhibit anxiety/depression-like behaviors. We demonstrated that zebrafish exhibit robust learning performance in a task adapted from the mammalian literature, a modified plus maze, and showed that ethanol and fluoxetine impair memory performance in this maze when administered after training at a dose that does not impair motor function, however, leads to significant upregulation of hippocampal serotoninergic neurons. These results suggest that the maze associative learning paradigm has face and construct validity and that zebrafish may become a translationally relevant study species for the analysis of the mechanisms of learning and memory changes associated with psychopharmacological treatment of anxiety/depression. © 2013.

In vivo cell biology in zebrafish - providing insights into vertebrate development and disease.

PubMed

Vacaru, Ana M; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W; Sadler, Kirsten C

2014-02-01

Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.

Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

PubMed

Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

2014-01-01

Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

Pharmacological analyses of learning and memory in zebrafish (Danio rerio).

PubMed

Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D

2015-12-01

Over the last decade, zebrafish (Danio rerio) have become valuable as a complementary model in behavioral pharmacology, opening a new avenue for understanding the relationships between drug action and behavior. This species offers a useful intermediate approach bridging the gap between in vitro studies and traditional mammalian models. Zebrafish offer great advantages of economy compared to their rodent counterparts, their complex brains and behavioral repertoire offer great translational potential relative to in vitro models. The development and validation of a variety of tests to measure behavior, including cognition, in zebrafish have set the stage for the use of this animal for behavioral pharmacology studies. This has led to research into the basic mechanisms of cognitive function as well as screening for potential cognition-improving drug therapies, among other lines of research. As with all models, zebrafish have limitations, which span pharmacokinetic challenges to difficulties quantifying behavior. The use, efficacy and limitations associated with a zebrafish model of cognitive function are discussed in this review, within the context of behavioral pharmacology. Copyright © 2015 Elsevier Inc. All rights reserved.

Myotonia Congenita-Associated Mutations in Chloride Channel-1 Affect Zebrafish Body Wave Swimming Kinematics

PubMed Central

Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

2014-01-01

Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita. PMID:25083883

Microbiota colonization status influences developmental toxicity of bisphenol A in embryonic zebrafish

EPA Science Inventory

There is growing evidence that microbiota can modify the toxicokinetics and/or toxicodynamics of environmental chemicals. Commonly used mammalian systems have limited ability to link phenotypic effects in exposed animals to colonization status. Here, we used gnotobiotic zebrafish...

Using zebrafish in systems toxicology for developmental toxicity testing.

PubMed

Nishimura, Yuhei; Inoue, Atsuto; Sasagawa, Shota; Koiwa, Junko; Kawaguchi, Koki; Kawase, Reiko; Maruyama, Toru; Kim, Soonih; Tanaka, Toshio

2016-01-01

With the high cost and the long-term assessment of developmental toxicity testing in mammals, the vertebrate zebrafish has become a useful alternative model organism for high-throughput developmental toxicity testing. Zebrafish is also very favorable for the 3R perspective in toxicology; however, the methodologies used by research groups vary greatly, posing considerable challenges to integrative analysis. In this review, we discuss zebrafish developmental toxicity testing, focusing on the methods of chemical exposure, the assessment of morphological abnormalities, housing conditions and their effects on the production of healthy embryos, and future directions. Zebrafish as a systems toxicology model has the potential to elucidate developmental toxicity pathways, and to provide a sound basis for human health risk assessments. © 2015 Japanese Teratology Society.

Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish

EPA Science Inventory

Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish Tamara Tal, Integrated Systems Toxicology Division, U.S. EPA Background: There are tens of thousands of chemicals that have yet to be fully evaluated for their toxicity by validated in vivo testing ...

Understanding the Transformation, Speciation, and Hazard Potential of Copper Particles in a Model Septic Tank System using Zebrafish to Monitor the Effluent

PubMed Central

Lin, Sijie; Taylor, Alicia A.; Zhaoxia, Ji; Chang, Chong Hyun; Kinsinger, Nichola M.; Ueng, William; Walker, Sharon L.; Nel, André E.

2015-01-01

Although copper-containing nanoparticles are used in commercial products such as fungicides and bactericides, we presently do not understand the environmental impact on other organisms that may be inadvertently exposed. In this study, we used the zebrafish embryo as a screening tool to study the potential impact of two nano Cu-based materials, CuPRO and Kocide, in comparison to nano-sized and micron-sized Cu and CuO particles in their pristine form (0 – 10 ppm) as well as following their transformation in an experimental wastewater treatment system. This was accomplished by construction of a modeled domestic septic tank system from which effluents could be retrieved at different stages following particle introduction (10 ppm). The Cu speciation in the effluent was identified as non-dissolvable inorganic Cu(H2PO2)2 and non-diffusible organic Cu by X-ray diffraction, inductively coupled plasma mass spectrometry (ICP-MS), diffusive gradients in thin-films (DGT), and Visual MINTEQ software. While the nanoscale materials, including the commercial particles, were clearly more potent (showing 50% hatching interference above 0.5 ppm) than the micron-scale particulates with no effect on hatching up to 10 ppm, the Cu released from the particles in the septic tank underwent transformation into non-bioavailable species that failed to interfere with the function of the zebrafish embryo hatching enzyme. Moreover, we demonstrate that the addition of humic acid, as an organic carbon component, could lead to a dose-dependent decrease in Cu toxicity in our high content zebrafish embryo screening assay. Thus, the use of zebrafish embryo screening, in combination with the effluents obtained from a modeled exposure environment, enables a bioassay approach to follow the change in the speciation, and hazard potential of Cu particles instead of difficult-to-perform direct particle tracking. PMID:25625504

Understanding the transformation, speciation, and hazard potential of copper particles in a model septic tank system using zebrafish to monitor the effluent.

PubMed

Lin, Sijie; Taylor, Alicia A; Ji, Zhaoxia; Chang, Chong Hyun; Kinsinger, Nichola M; Ueng, William; Walker, Sharon L; Nel, André E

2015-02-24

Although copper-containing nanoparticles are used in commercial products such as fungicides and bactericides, we presently do not understand the environmental impact on other organisms that may be inadvertently exposed. In this study, we used the zebrafish embryo as a screening tool to study the potential impact of two nano Cu-based materials, CuPRO and Kocide, in comparison to nanosized and micron-sized Cu and CuO particles in their pristine form (0-10 ppm) as well as following their transformation in an experimental wastewater treatment system. This was accomplished by construction of a modeled domestic septic tank system from which effluents could be retrieved at different stages following particle introduction (10 ppm). The Cu speciation in the effluent was identified as nondissolvable inorganic Cu(H2PO2)2 and nondiffusible organic Cu by X-ray diffraction, inductively coupled plasma mass spectrometry (ICP-MS), diffusive gradients in thin-films (DGT), and Visual MINTEQ software. While the nanoscale materials, including the commercial particles, were clearly more potent (showing 50% hatching interference above 0.5 ppm) than the micron-scale particulates with no effect on hatching up to 10 ppm, the Cu released from the particles in the septic tank underwent transformation into nonbioavailable species that failed to interfere with the function of the zebrafish embryo hatching enzyme. Moreover, we demonstrate that the addition of humic acid, as an organic carbon component, could lead to a dose-dependent decrease in Cu toxicity in our high content zebrafish embryo screening assay. Thus, the use of zebrafish embryo screening, in combination with the effluents obtained from a modeled exposure environment, enables a bioassay approach to follow the change in the speciation and hazard potential of Cu particles instead of difficult-to-perform direct particle tracking.

Zebrafish models of human eye and inner ear diseases.

PubMed

Blanco-Sánchez, B; Clément, A; Phillips, J B; Westerfield, M

2017-01-01

Eye and inner ear diseases are the most common sensory impairments that greatly impact quality of life. Zebrafish have been intensively employed to understand the fundamental mechanisms underlying eye and inner ear development. The zebrafish visual and vestibulo-acoustic systems are very similar to these in humans, and although not yet mature, they are functional by 5days post-fertilization (dpf). In this chapter, we show how the zebrafish has significantly contributed to the field of biomedical research and how researchers, by establishing disease models and meticulously characterizing their phenotypes, have taken the first steps toward therapies. We review here models for (1) eye diseases, (2) ear diseases, and (3) syndromes affecting eye and/or ear. The use of new genome editing technologies and high-throughput screening systems should increase considerably the speed at which knowledge from zebrafish disease models is acquired, opening avenues for better diagnostics, treatments, and therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of acute ethanol administration on zebrafish tail-beat motion.

PubMed

Bartolini, Tiziana; Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

2015-11-01

Zebrafish is becoming a species of choice in neurobiological and behavioral studies of alcohol-related disorders. In these efforts, the activity of adult zebrafish is typically quantified using indirect activity measures that are either scored manually or identified automatically from the fish trajectory. The analysis of such activity measures has produced important insight into the effect of acute ethanol exposure on individual and social behavior of this vertebrate species. Here, we leverage a recently developed tracking algorithm that reconstructs fish body shape to investigate the effect of acute ethanol administration on zebrafish tail-beat motion in terms of amplitude and frequency. Our results demonstrate a significant effect of ethanol on the tail-beat amplitude as well as the tail-beat frequency, both of which were found to robustly decrease for high ethanol concentrations. Such a direct measurement of zebrafish motor functions is in agreement with evidence based on indirect activity measures, offering a complementary perspective in behavioral screening. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy

PubMed Central

Asimaki, Angeliki; Kapoor, Sudhir; Plovie, Eva; Arndt, Anne Karin; Adams, Edward; Liu, ZhenZhen; James, Cynthia A.; Judge, Daniel P.; Calkins, Hugh; Churko, Jared; Wu, Joseph C.; MacRae, Calum A.; Kléber, André G.; Saffitz, Jeffrey E.

2015-01-01

Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte–specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in INa and IK1 current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities. PMID:24920660

What Are We Putting in Our Food That Is Making Us Fat? Food Additives, Contaminants, and Other Putative Contributors to Obesity

PubMed Central

Simmons, Amber L.; Schlezinger, Jennifer J.; Corkey, Barbara E.

2014-01-01

The “chemical obesogen” hypothesis conjectures that synthetic, environmental contaminants are contributing to the global epidemic of obesity. In fact, intentional food additives (e.g., artificial sweeteners and colors, emulsifiers) and unintentional compounds (e.g., bisphenol A, pesticides) are largely unstudied in regard to their effects on overall metabolic homeostasis. With that said, many of these contaminants have been found to dysregulate endocrine function, insulin signaling, and/or adipocyte function. Although momentum for the chemical obesogen hypothesis is growing, supportive, evidence-based research is lacking. In order to identify noxious synthetic compounds in the environment out of the thousands of chemicals that are currently in use, tools and models from toxicology should be adopted (e.g., functional high throughput screening methods, zebrafish-based assays). Finally, mechanistic insight into obesogen-induced effects will be helpful in elucidating their role in the obesity epidemic as well as preventing and reversing their effects. PMID:25045594

In vivo screening and discovery of novel candidate thalidomide analogs in the zebrafish embryo and chicken embryo model systems

PubMed Central

Beedie, Shaunna L.; Rore, Holly M.; Barnett, Shelby; Chau, Cindy H.; Luo, Weiming; Greig, Nigel H.; Figg, William D.; Vargesson, Neil

2016-01-01

Thalidomide, a drug known for its teratogenic side-effects, is used successfully to treat a variety of clinical conditions including leprosy and multiple myeloma. Intense efforts are underway to synthesize and identify safer, clinically relevant analogs. Here, we conduct a preliminary in vivo screen of a library of new thalidomide analogs to determine which agents demonstrate activity, and describe a cohort of compounds with anti-angiogenic properties, anti-inflammatory properties and some compounds which exhibited both. The combination of the in vivo zebrafish and chicken embryo model systems allows for the accelerated discovery of new, potential therapies for cancerous and inflammatory conditions. PMID:27120781

Zebrafish for the Study of the Biological Effects of Nicotine

PubMed Central

Klee, Eric W.; Schneider, Henning; Hurt, Richard D.; Ekker, Stephen C.

2011-01-01

Introduction: Zebrafish are emerging as a powerful animal model for studying the molecular and physiological effects of nicotine exposure. The zebrafish have many advantageous physical characteristics, including small size, high fecundity rates, and externally developing transparent embryos. When combined with a battery of molecular–genetic tools and behavioral assays, these attributes enable studies to be conducted that are not practical using traditional animal models. Methods: We reviewed the literature on the application of the zebrafish model as a preclinical model to study the biological effects of nicotine exposure. Results: The identified studies used zebrafish to examine the effects of nicotine exposure on early development, addiction, anxiety, and learning. The methods used included green fluorescent protein–labeled proteins to track in vivo nicotine-altered neuron development, nicotine-conditioned place preference, and locomotive sensitization linked with high-throughput molecular and genetic screens and behavioral models of learning and stress response to nicotine. Data are presented on the complete homology of all known human neural nicotinic acetylcholine receptors in zebrafish and on the biological similarity of human and zebrafish dopaminergic signaling. Conclusions: Tobacco dependence remains a major health problem worldwide. Further understanding of the molecular effects of nicotine exposure and genetic contributions to dependence may lead to improvement in patient treatment strategies. While there are limitations to the use of zebrafish as a preclinical model, it should provide a valuable tool to complement existing model systems. The reviewed studies demonstrate the enormous opportunity zebrafish have to advance the science of nicotine and tobacco research. PMID:21385906

Methods to study maternal regulation of germ cell specification in zebrafish

PubMed Central

Kaufman, O.H.; Marlow, F.L.

2016-01-01

The process by which the germ line is specified in the zebrafish embryo is under the control of maternal gene products that were produced during oogenesis. Zebrafish are highly amenable to microscopic observation of the processes governing maternal germ cell specification because early embryos are transparent, and the germ line is specified rapidly (within 4–5 h post fertilization). Advantages of zebrafish over other models used to study vertebrate germ cell formation include their genetic tractability, the large numbers of progeny, and the easily manipulable genome, all of which make zebrafish an ideal system for studying the genetic regulators and cellular basis of germ cell formation and maintenance. Classical molecular biology techniques, including expression analysis through in situ hybridization and forward genetic screens, have laid the foundation for our understanding of germ cell development in zebrafish. In this chapter, we discuss some of these classic techniques, as well as recent cutting-edge methodologies that have improved our ability to visualize the process of germ cell specification and differentiation, and the tracking of specific molecules involved in these processes. Additionally, we discuss traditional and novel technologies for manipulating the zebrafish genome to identify new components through loss-of-function studies of putative germ cell regulators. Together with the numerous aforementioned advantages of zebrafish as a genetic model for studying development, we believe these new techniques will continue to advance zebrafish to the forefront for investigation of the molecular regulators of germ cell specification and germ line biology. PMID:27312489

Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

PubMed

Chen, Yunru; Zeng, Shiyang; Hu, Ruikun; Wang, Xiangxiu; Huang, Weilai; Liu, Jiangfang; Wang, Luying; Liu, Guifen; Cao, Ying; Zhang, Yong

2017-01-01

Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

Strain-Specific Changes in Locomotor Behavior in Larval Zebrafish Elicited by Cholinergic Challenge

EPA Science Inventory

Some studies have compared the baseline behavior of different strains of larval zebrafish (Danio rerio), but there is sparse information on strain-specific responses to chemical challenges. The following study examines both the basal activity and response to a pharmacological cha...

Application of chrysophanol in zebrafish to reduce dietary introduced lipid and its possible mechanism

PubMed Central

Chen, Kan; Wang, Chang-Qian; Fan, Yu-Qi; Xie, Yu-Shui; Yin, Zhao-Fang; Xu, Zuo-Jun; Zhang, Hui-Li; Cao, Jia-Tian; Wang, Yue

2015-01-01

Purpose: To explore the therapeutic potential and mechanism of chrysophanol on lipid-lowering function. Methods: Zebrafish or larvae were employed to evaluate the effect of chrysophanol on lipid-lowering. Zebrafish of 5 day post fertilization (dpf, larva) and 13-week old were fed with high-cholesterol diet or high-fat to investigate the influence of chrysophanol comparing with atorvastain and co-administration of chrysophanol and atorvastain on subsistent blood lipid using the fluorescence microscope and lipid panel screen. Thereafter, we enrolled zebrafish of 7 dpf fed with high-fat diet to explore the lipid-lowering mechanism of chrysophanol basing on the frequency of peristalsis and the residual on the digestive wall. Results: Chrysophanol could significantly lower cholesterol both in zebrafish and larvae (P < 0.05), and the co-administration of chrysophanol and atorvastatin had the best performance in reducing cholesterol (P < 0.05). Chrysophanol and atorvastain could also significantly lower triglyceride. Moreover, we found that chrysophanol attached on the digestive wall for a long time and enhanced the frequency of peristalsis. Conclusions: Chrysophanol has lipid-lowering effect both in zebrafish and larvae which may be attributed to the effect on the frequency of peristalsis and fat absorption, and co-administration with atorvastain performs better lipid-lowering effect in zebrafish. PMID:26379845

LITTLE FISH, BIG DATA: ZEBRAFISH AS A MODEL FOR CARDIOVASCULAR AND METABOLIC DISEASE.

PubMed

Gut, Philipp; Reischauer, Sven; Stainier, Didier Y R; Arnaout, Rima

2017-07-01

The burden of cardiovascular and metabolic diseases worldwide is staggering. The emergence of systems approaches in biology promises new therapies, faster and cheaper diagnostics, and personalized medicine. However, a profound understanding of pathogenic mechanisms at the cellular and molecular levels remains a fundamental requirement for discovery and therapeutics. Animal models of human disease are cornerstones of drug discovery as they allow identification of novel pharmacological targets by linking gene function with pathogenesis. The zebrafish model has been used for decades to study development and pathophysiology. More than ever, the specific strengths of the zebrafish model make it a prime partner in an age of discovery transformed by big-data approaches to genomics and disease. Zebrafish share a largely conserved physiology and anatomy with mammals. They allow a wide range of genetic manipulations, including the latest genome engineering approaches. They can be bred and studied with remarkable speed, enabling a range of large-scale phenotypic screens. Finally, zebrafish demonstrate an impressive regenerative capacity scientists hope to unlock in humans. Here, we provide a comprehensive guide on applications of zebrafish to investigate cardiovascular and metabolic diseases. We delineate advantages and limitations of zebrafish models of human disease and summarize their most significant contributions to understanding disease progression to date. Copyright © 2017 the American Physiological Society.

A reduced transcriptome approach to assess environmental toxicants using zebrafish embryo tests

EPA Science Inventory

This paper reports on the pilot testing of a new bioassay platform that monitors expression of 1600 genes in zebrafish embryos exposed to either single chemicals or complex water samples. The method provides a more cost effective, high throughput means to broadly evaluate the pot...

Automated feature extraction for retinal vascular biometry in zebrafish using OCT angiography

NASA Astrophysics Data System (ADS)

Bozic, Ivan; Rao, Gopikrishna M.; Desai, Vineet; Tao, Yuankai K.

2017-02-01

Zebrafish have been identified as an ideal model for angiogenesis because of anatomical and functional similarities with other vertebrates. The scale and complexity of zebrafish assays are limited by the need to manually treat and serially screen animals, and recent technological advances have focused on automation and improving throughput. Here, we use optical coherence tomography (OCT) and OCT angiography (OCT-A) to perform noninvasive, in vivo imaging of retinal vasculature in zebrafish. OCT-A summed voxel projections were low pass filtered and skeletonized to create an en face vascular map prior to connectivity analysis. Vascular segmentation was referenced to the optic nerve head (ONH), which was identified by automatically segmenting the retinal pigment epithelium boundary on the OCT structural volume. The first vessel branch generation was identified as skeleton segments with branch points closest to the ONH, and subsequent generations were found iteratively by expanding the search space outwards from the ONH. Biometric parameters, including length, curvature, and branch angle of each vessel segment were calculated and grouped by branch generation. Despite manual handling and alignment of each animal over multiple time points, we observe distinct qualitative patterns that enable unique identification of each eye from individual animals. We believe this OCT-based retinal biometry method can be applied for automated animal identification and handling in high-throughput organism-level pharmacological assays and genetic screens. In addition, these extracted features may enable high-resolution quantification of longitudinal vascular changes as a method for studying zebrafish models of retinal neovascularization and vascular remodeling.

In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

PubMed Central

Vacaru, Ana M.; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W.; Sadler, Kirsten C.

2014-01-01

ABSTRACT Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease. PMID:24481493

Translating Discovery in Zebrafish Pancreatic Development to Human Pancreatic Cancer: Biomarkers, Targets, Pathogenesis, and Therapeutics

PubMed Central

Kazi, Abid A.; Yee, Rosemary K.

2013-01-01

Abstract Experimental studies in the zebrafish have greatly facilitated understanding of genetic regulation of the early developmental events in the pancreas. Various approaches using forward and reverse genetics, chemical genetics, and transgenesis in zebrafish have demonstrated generally conserved regulatory roles of mammalian genes and discovered novel genetic pathways in exocrine pancreatic development. Accumulating evidence has supported the use of zebrafish as a model of human malignant diseases, including pancreatic cancer. Studies have shown that the genetic regulators of exocrine pancreatic development in zebrafish can be translated into potential clinical biomarkers and therapeutic targets in human pancreatic adenocarcinoma. Transgenic zebrafish expressing oncogenic K-ras and zebrafish tumor xenograft model have emerged as valuable tools for dissecting the pathogenetic mechanisms of pancreatic cancer and for drug discovery and toxicology. Future analysis of the pancreas in zebrafish will continue to advance understanding of the genetic regulation and biological mechanisms during organogenesis. Results of those studies are expected to provide new insights into how aberrant developmental pathways contribute to formation and growth of pancreatic neoplasia, and hopefully generate valid biomarkers and targets as well as effective and safe therapeutics in pancreatic cancer. PMID:23682805

Translating discovery in zebrafish pancreatic development to human pancreatic cancer: biomarkers, targets, pathogenesis, and therapeutics.

PubMed

Yee, Nelson S; Kazi, Abid A; Yee, Rosemary K

2013-06-01

Abstract Experimental studies in the zebrafish have greatly facilitated understanding of genetic regulation of the early developmental events in the pancreas. Various approaches using forward and reverse genetics, chemical genetics, and transgenesis in zebrafish have demonstrated generally conserved regulatory roles of mammalian genes and discovered novel genetic pathways in exocrine pancreatic development. Accumulating evidence has supported the use of zebrafish as a model of human malignant diseases, including pancreatic cancer. Studies have shown that the genetic regulators of exocrine pancreatic development in zebrafish can be translated into potential clinical biomarkers and therapeutic targets in human pancreatic adenocarcinoma. Transgenic zebrafish expressing oncogenic K-ras and zebrafish tumor xenograft model have emerged as valuable tools for dissecting the pathogenetic mechanisms of pancreatic cancer and for drug discovery and toxicology. Future analysis of the pancreas in zebrafish will continue to advance understanding of the genetic regulation and biological mechanisms during organogenesis. Results of those studies are expected to provide new insights into how aberrant developmental pathways contribute to formation and growth of pancreatic neoplasia, and hopefully generate valid biomarkers and targets as well as effective and safe therapeutics in pancreatic cancer.

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

PubMed

Liu, Tianming; Lu, Jianfeng; Wang, Ye; Campbell, William A; Huang, Ling; Zhu, Jinmin; Xia, Weiming; Wong, Stephen T C

2006-06-15

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

PubMed Central

Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

2018-01-01

Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

Pharmacological evaluation of the mechanisms involved in increased adiposity in zebrafish triggered by the environmental contaminant tributyltin.

PubMed

Ouadah-Boussouf, Nafia; Babin, Patrick J

2016-03-01

One proposed contributing factor to the rise in overweight and obesity is exposure to endocrine disrupting chemicals. Tributyltin chloride (TBT), an organotin, induces adipogenesis in cell culture models and may increases adipose mass in vivo in vertebrate model organisms. It has been hypothesized that TBT acts via the peroxisome proliferator activated receptor (PPAR)γ-dependent pathway. However, the mechanisms involved in the effects of TBT exposure on in vivo adipose tissue metabolism remain unexplored. Semitransparent zebrafish larvae, with their well-developed white adipose tissue, offer a unique opportunity for studying the effects of toxicant chemicals and pharmaceuticals on adipocyte biology and whole-organism adiposity in a vertebrate model. Within hours, zebrafish larvae, treated at environmentally-relevant nanomolar concentrations of TBT, exhibited a remarkable increase in adiposity linked to adipocyte hypertrophy. Under the experimental conditions used, we also demonstrated that zebrafish larvae adipose tissue proved to be highly responsive to selected human nuclear receptor agonists and antagonists. Retinoid X receptor (RXR) homodimers and RXR/liver X receptor heterodimers were suggested to be in vivo effectors of the obesogenic effect of TBT on zebrafish white adipose tissue. RXR/PPARγ heterodimers may be recruited to modulate adiposity in zebrafish but were not a necessary requirement for the short term in vivo TBT obesogenic effect. Together, the present results suggest that TBT may induce the promotion of triacylglycerol storage in adipocytes via RXR-dependent pathways without necessary using PPAR isoforms. Copyright © 2016 Elsevier Inc. All rights reserved.

Essential role for fibrillin-2 in zebrafish notochord and vascular morphogenesis.

PubMed

Gansner, John M; Madsen, Erik C; Mecham, Robert P; Gitlin, Jonathan D

2008-10-01

Recent studies demonstrate that lysyl oxidase cuproenzymes are critical for zebrafish notochord formation, but the molecular mechanisms of copper-dependent notochord morphogenesis are incompletely understood. We, therefore, conducted a forward genetic screen for zebrafish mutants that exhibit notochord sensitivity to lysyl oxidase inhibition, yielding a mutant with defects in notochord and vascular morphogenesis, puff daddygw1 (pfdgw1). Meiotic mapping and cloning reveal that the pfdgw1 phenotype results from disruption of the gene encoding the extracellular matrix protein fibrillin-2, and the spatiotemporal expression of fibrillin-2 is consistent with the pfdgw1 phenotype. Furthermore, each aspect of the pfdgw1 phenotype is recapitulated by morpholino knockdown of fibrillin-2. Taken together, the data reveal a genetic interaction between fibrillin-2 and the lysyl oxidases in notochord formation and demonstrate the importance of fibrillin-2 in specific early developmental processes in zebrafish. Copyright (c) 2008 Wiley-Liss, Inc.

A review of monoaminergic neuropsychopharmacology in zebrafish.

PubMed

Maximino, Caio; Herculano, Anderson Manoel

2010-12-01

Monoamine neurotransmitters are the major regulatory mechanisms in the vertebrate brain, involved in the adjustment of motivation, emotion, and cognition. The chemical anatomy of these systems is thought to be highly conserved in the brain of all vertebrates, including zebrafish. Recently, the development of behavioral assays in zebrafish allowed the neuropsychopharmacological investigation of these circuits and its functions. Here we review neuroanatomical, genetic, neurochemical, and psychopharmacological evidence regarding the roles of histaminergic, dopaminergic, noradrenergic, serotonergic, and melatonergic systems in this species. We conclude that, in spite of species differences, zebrafish are suitable for the investigation of neuropsychopharmacology of drugs that affect theses systems; nonetheless, more thorough validation of behavioral methods is still needed.

Nrf2 and Nrf2-Related Proteins in Development and Developmental Toxicity: Insights from studies in Zebrafish (Danio rerio)

PubMed Central

Hahn, Mark E.; Timme-Laragy, Alicia R.; Karchner, Sibel I.; Stegeman, John J.

2015-01-01

Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap’n’collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects. PMID:26130508

Neoplasia and Neoplasm Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design

PubMed Central

Spitsbergen, Jan M.; Buhler, Donald R.; Peterson, Tracy S.

2014-01-01

During the past decade the zebrafish has emerged as a leading model for mechanistic cancer research due to its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost-effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast to other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type (wt) lines of zebrafish. So far a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues in not available for zebrafish as it is for other intensely studied laboratory animal species. This manuscript confirms that as in other species diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wt lines of zebrafish exhibit elevated neoplasm incidences and neoplasm associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation. PMID:23382343

Toxicological and chemical investigation of untreated municipal wastewater: Fraction- and species-specific toxicity.

PubMed

Hrubik, Jelena; Glisic, Branka; Tubic, Aleksandra; Ivancev-Tumbas, Ivana; Kovacevic, Radmila; Samardzija, Dragana; Andric, Nebojsa; Kaisarevic, Sonja

2016-05-01

Absence of a municipal wastewater (WW) treatment plant results in the untreated WW discharge into the recipient. The present study investigated toxic effects and chemical composition of water extracts and fractions from untreated WW and recipient Danube River (DR). Samples were prepared by solid-phase extraction and silica gel fractionation and screened for EROD activity and cytotoxicity using aquatic models, comprising of fish liver cells (PLHC-1) and a model of the early development of zebrafish embryos, while rat (H4IIE) and human (HepG2) hepatoma cells served as mammalian models. Polar fraction caused cytotoxicity and increased the EROD activity in PLHC-1 cells, and increased mortality and developmental abnormalities in developing zebrafish embryos. In H4IIE, polar fraction induced inhibition of cell growth and increased EROD activity, whereas HepG2 exerted low or no response to the exposure. Non-polar and medium-polar fractions were ineffective. Tentative identification by GC/MS showed that WW is characterized by the hydrocarbons, alkylphenols, plasticizers, and a certain number of benzene derivatives and organic acids. In DR, smaller number of organic compounds was identified and toxicity was less pronounced than in WW treatments. The present study revealed the potent toxic effect of polar fraction of untreated WW, with biological responses varying in sensitivity across organisms. Obtained results confirmed that fraction- and species-specific toxicity should be considered when assessing health risk of environmental pollution. Copyright © 2016 Elsevier Inc. All rights reserved.

Pharmacological evaluation of the mechanisms involved in increased adiposity in zebrafish triggered by the environmental contaminant tributyltin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ouadah-Boussouf, Nafia; Babin, Patrick J., E-mail: p.babin@gpp.u-bordeaux1.fr

One proposed contributing factor to the rise in overweight and obesity is exposure to endocrine disrupting chemicals. Tributyltin chloride (TBT), an organotin, induces adipogenesis in cell culture models and may increases adipose mass in vivo in vertebrate model organisms. It has been hypothesized that TBT acts via the peroxisome proliferator activated receptor (PPAR)γ-dependent pathway. However, the mechanisms involved in the effects of TBT exposure on in vivo adipose tissue metabolism remain unexplored. Semitransparent zebrafish larvae, with their well-developed white adipose tissue, offer a unique opportunity for studying the effects of toxicant chemicals and pharmaceuticals on adipocyte biology and whole-organism adipositymore » in a vertebrate model. Within hours, zebrafish larvae, treated at environmentally-relevant nanomolar concentrations of TBT, exhibited a remarkable increase in adiposity linked to adipocyte hypertrophy. Under the experimental conditions used, we also demonstrated that zebrafish larvae adipose tissue proved to be highly responsive to selected human nuclear receptor agonists and antagonists. Retinoid X receptor (RXR) homodimers and RXR/liver X receptor heterodimers were suggested to be in vivo effectors of the obesogenic effect of TBT on zebrafish white adipose tissue. RXR/PPARγ heterodimers may be recruited to modulate adiposity in zebrafish but were not a necessary requirement for the short term in vivo TBT obesogenic effect. Together, the present results suggest that TBT may induce the promotion of triacylglycerol storage in adipocytes via RXR-dependent pathways without necessary using PPAR isoforms. - Highlights: • The environmental contaminant tributyltin (TBT) may promote obesity development. • TBT may induce adipocyte hypertrophy through a PPARγ independent mechanism. • RXR/RXR and RXR/LXR dimers are potential in vivo effectors of TBT in zebrafish.« less

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

PubMed

Hinfray, N; Tebby, C; Garoche, C; Piccini, B; Bourgine, G; Aït-Aïssa, S; Kah, O; Pakdel, F; Brion, F

2016-09-15

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. Copyright © 2016 Elsevier Inc. All rights reserved.

Optimization and Pharmacological Validation of a Leukocyte Migration Assay in Zebrafish Larvae for the Rapid In Vivo Bioactivity Analysis of Anti-Inflammatory Secondary Metabolites

PubMed Central

Vicet-Muro, Liliana; Wilches-Arizábala, Isabel María; Esguerra, Camila V.; de Witte, Peter A. M.; Crawford, Alexander D.

2013-01-01

Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts. PMID:24124487

Zebrafish: an animal model for research in veterinary medicine.

PubMed

Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

2015-01-01

The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic disorders including fatty liver disease and diabetes. The zebrafish is also a valuable tool as a model in behavioral studies connected with feeding, predator evasion, habituation and memory or lateralized control of behavior. The aim of the present article is to familiarize the reader with the possibilities of Danio rerio as an experimental model for veterinary medicine.

Dimethyl sulfoxide (DMSO) exacerbates cisplatin-induced sensory hair cell death in zebrafish (Danio rerio).

PubMed

Uribe, Phillip M; Mueller, Melissa A; Gleichman, Julia S; Kramer, Matthew D; Wang, Qi; Sibrian-Vazquez, Martha; Strongin, Robert M; Steyger, Peter S; Cotanche, Douglas A; Matsui, Jonathan I

2013-01-01

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

PubMed Central

Gleichman, Julia S.; Kramer, Matthew D.; Wang, Qi; Sibrian-Vazquez, Martha; Strongin, Robert M.; Steyger, Peter S.; Cotanche, Douglas A.; Matsui, Jonathan I.

2013-01-01

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO. PMID:23383324

Netting Novel Regulators of Hematopoiesis and Hematologic Malignancies in Zebrafish.

PubMed

Kwan, Wanda; North, Trista E

2017-01-01

Zebrafish are one of the preeminent model systems for the study of blood development (hematopoiesis), hematopoietic stem and progenitor cell (HSPC) biology, and hematopathology. The zebrafish hematopoietic system shares strong similarities in functional populations, genetic regulators, and niche interactions with its mammalian counterparts. These evolutionarily conserved characteristics, together with emerging technologies in live imaging, compound screening, and genetic manipulation, have been employed to successfully identify and interrogate novel regulatory mechanisms and molecular pathways that guide hematopoiesis. Significantly, perturbations in many of the key developmental signals controlling hematopoiesis are associated with hematological disorders and disease, including anemia, bone marrow failure syndromes, and leukemia. Thus, understanding the regulatory pathways controlling HSPC production and function has important clinical implications. In this review, we describe how the blood system forms and is maintained in zebrafish, with particular focus on new insights into vertebrate hematological regulation gained using this model. The interplay of factors controlling development and disease in the hematopoietic system combined with the unique attributes of the zebrafish make this a powerful platform to discover novel targets for the treatment of hematological disease. © 2017 Elsevier Inc. All rights reserved.

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

PubMed Central

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J.; Salomons, Gajja S.

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease. PMID:29053735

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

PubMed

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development.

PubMed

Verbueken, Evy; Alsop, Derek; Saad, Moayad A; Pype, Casper; Van Peer, Els M; Casteleyn, Christophe R; Van Ginneken, Chris J; Wilson, Joanna; Van Cruchten, Steven J

2017-01-22

At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP)-a group of drug-metabolizing enzymes-in microsomes from whole zebrafish embryos (ZEM) of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf) by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR). The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM) to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA) as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

Random Walk of Single Gold Nanoparticles in Zebrafish Embryos Leading to Stochastic Toxic Effects on Embryonic Developments

PubMed Central

Browning, Lauren M.; Lee, Kerry J.; Huang, Tao; Nallathamby, Prakash D.; Lowman, Jill E.; Xu, Xiao-Hong Nancy

2010-01-01

We have synthesized and characterized stable (non-aggregation, non-photobleaching and non-blinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10-11 to 1.3×10-8 cm2/s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in-vivo assays to screen their biocompatibility. PMID:20644873

High Content Screening in Zebrafish Speeds up Hazard Ranking of Transition Metal Oxide Nanoparticles

PubMed Central

Lin, Sijie; Zhao, Yan; Xia, Tian; Meng, Huan; Zhaoxia, Ji; Liu, Rong; George, Saji; Xiong, Sijing; Wang, Xiang; Zhang, Haiyuan; Pokhrel, Suman; Mädler, Lutz; Damoiseaux, Robert; Lin, Shuo; Nel, Andre E.

2014-01-01

Zebrafish is an aquatic organism that can be used for high content safety screening of engineered nanomaterials (ENMs). We demonstrate, for the first time, the use of high content bright-field and fluorescence-based imaging to compare the toxicological effect of transition metal oxide (CuO, ZnO, NiO and Co3O4) nanoparticles in zebrafish embryos and larvae. High content bright-field imaging demonstrated potent and dose-dependant hatching interference in the embryos, with the exception of Co3O4 which was relatively inert. We propose that the hatching interference was due to the shedding of Cu and Ni ions, compromising the activity of the hatching enzyme, ZHE1, similar to what we previously proposed for Zn2+. This hypothesis is based on the presence of metal–sensitive histidines in the catalytic center of this enzyme. Co-introduction of a metal ion chelator, diethylene triamine pentaacetic acid (DTPA), reversed the hatching interference of Cu, Zn and Ni. While neither the embryos nor larvae demonstrated morphological abnormalities, high content fluorescence-based imaging demonstrated that CuO, ZnO and NiO could induce increased expression of the heat shock protein 70:enhanced green fluorescence protein (hsp70:eGFP) in transgenic zebrafish larvae. Induction of this response by CuO required a higher nanoparticle dose than the amount leading to hatching interference. This response was also DTPA sensitive. In conclusion, we demonstrate that high content imaging of embryo development, morphological abnormalities and HSP70 expression can be used for hazard ranking and determining the dose-response relationships leading to ENM effects on the development of the zebrafish embryo. PMID:21851096

Toxicogenomics to Evaluate Endocrine Disrupting Effects of Environmental Chemicals Using the Zebrafish Model

PubMed Central

Caballero-Gallardo, Karina; Olivero-Verbel, Jesus; Freeman, Jennifer L.

2016-01-01

The extent of our knowledge on the number of chemical compounds related to anthropogenic activities that can cause damage to the environment and to organisms is increasing. Endocrine disrupting chemicals (EDCs) are one group of potentially hazardous substances that include natural and synthetic chemicals and have the ability to mimic endogenous hormones, interfering with their biosynthesis, metabolism, and normal functions. Adverse effects associated with EDC exposure have been documented in aquatic biota and there is widespread interest in the characterization and understanding of their modes of action. Fish are considered one of the primary risk organisms for EDCs. Zebrafish (Danio rerio) are increasingly used as an animal model to study the effects of endocrine disruptors, due to their advantages compared to other model organisms. One approach to assess the toxicity of a compound is to identify those patterns of gene expression found in a tissue or organ exposed to particular classes of chemicals, through new technologies in genomics (toxicogenomics), such as microarrays or whole-genome sequencing. Application of these technologies permit the quantitative analysis of thousands of gene expression changes simultaneously in a single experiment and offer the opportunity to use transcript profiling as a tool to predict toxic outcomes of exposure to particular compounds. The application of toxicogenomic tools for identification of chemicals with endocrine disrupting capacity using the zebrafish model system is reviewed. PMID:28217008

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

PubMed

Busquet, François; Strecker, Ruben; Rawlings, Jane M; Belanger, Scott E; Braunbeck, Thomas; Carr, Gregory J; Cenijn, Peter; Fochtman, Przemyslaw; Gourmelon, Anne; Hübler, Nicole; Kleensang, André; Knöbel, Melanie; Kussatz, Carola; Legler, Juliette; Lillicrap, Adam; Martínez-Jerónimo, Fernando; Polleichtner, Christian; Rzodeczko, Helena; Salinas, Edward; Schneider, Katharina E; Scholz, Stefan; van den Brandhof, Evert-Jan; van der Ven, Leo T M; Walter-Rohde, Susanne; Weigt, Stefan; Witters, Hilda; Halder, Marlies

2014-08-01

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Metabolism of clofibric acid in zebrafish embryos (Danio rerio) as determined by liquid chromatography-high resolution-mass spectrometry.

PubMed

Brox, Stephan; Seiwert, Bettina; Haase, Nora; Küster, Eberhard; Reemtsma, Thorsten

2016-01-01

The zebrafish embryo (ZFE) is increasingly used in ecotoxicology research but detailed knowledge of its metabolic potential is still limited. This study focuses on the xenobiotic metabolism of ZFE at different life-stages using the pharmaceutical compound clofibric acid as study compound. Liquid chromatography with quadrupole-time-of-flight mass spectrometry (LC-QToF-MS) is used to detect and to identify the transformation products (TPs). In screening experiments, a total of 18 TPs was detected and structure proposals were elaborated for 17 TPs, formed by phase I and phase II metabolism. Biotransformation of clofibric acid by the ZFE involves conjugation with sulfate or glucuronic acid, and, reported here for the first time, with carnitine, taurine, and aminomethanesulfonic acid. Further yet unknown cyclization products were identified using non-target screening that may represent a new detoxification pathway. Sulfate containing TPs occurred already after 3h of exposure (7hpf), and from 48h of exposure (52hpf) onwards, all TPs were detected. The detection of these TPs indicates the activity of phase I and phase II enzymes already at early life-stages. Additionally, the excretion of one TP into the exposure medium was observed. The results of this study outline the high metabolic potential of the ZFE with respect to the transformation of xenobiotics. Similarities but also differences to other test systems were observed. Biotransformation of test chemicals in toxicity testing with ZFE may therefore need further consideration. Copyright © 2016 Elsevier Inc. All rights reserved.

Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio).

PubMed

Strykowski, Jennifer L; Schech, Joseph M

2015-01-01

The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge.

Effectiveness of Recommended Euthanasia Methods in Larval Zebrafish (Danio rerio)

PubMed Central

Strykowski, Jennifer L; Schech, Joseph M

2015-01-01

The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge. PMID:25651096

Dechorionation of Zebrafish Embryos on Day 1 Post Fertilization Alters Response to an Acute Chemical Challenge at 6 Days Post Fertilization

EPA Science Inventory

Dechorionation is a method used to enable image acquisition in embryonic and larval zebrafish studies. As it is assumed that dechorionation has no long-term effects on fish embryo development, it is important to determine if that assumption is correct. The present study explored ...

Altered Gene Expression in the Brain and Ovaries of Zebrafish Exposed to the Aromatase Inhibitor Fadrosole: Microarray Analysis for Hypothesis Generation

EPA Science Inventory

A part of an overall program of research aimed at examining system-wide responses of the hypothalamic-pituitary-gonadal axis in fish to endocrine active chemicals acting through a variety of modes of action, we exposed zebrafish (Danio rerio) to the aromatase inhibitor fadrozole ...

A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae.

PubMed

Mills, Margaret G; Gallagher, Evan P

2017-01-01

Chemical-induced oxidative stress and the biochemical pathways that protect against oxidative damage are of particular interest in the field of toxicology. To rapidly identify oxidative stress-responsive gene expression changes in zebrafish, we developed a targeted panel of antioxidant genes using the Affymetrix QuantiGene Plex (QGP) platform. The genes contained in our panel include eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1a, gstp1, gclc, nqo1, prdx1, gpx1a, sod1, sod2), a stress response gene (hsp70), an inducible DNA damage repair gene (gadd45bb), and three reference genes (actb1, gapdh, hprt1). We tested this platform on larval zebrafish exposed to tert-butyl hydroperoxide (tBHP) and cadmium (Cd), two model oxidative stressors with different modes of action, and compared our results with those obtained using the more common quantitative PCR (qPCR) method. Both methods showed that exposure to tBHP and Cd induced expression of prdx1, gstp1, and hmox1a (2- to 12-fold increase via QGP), indicative of an activated Nrf2 response in larval zebrafish. Both compounds also elicited a general stress response as reflected by elevation of hsp70 and gadd45bb, with Cd being the more potent inducer. Transient changes were observed in sod2 and gpx1a expression, whereas nqo1, an Nrf2-responsive gene in mammalian cells, was minimally affected by either tBHP or Cd chemical exposures. Developmental expression analysis of the target genes by QGP revealed marked upregulation of sod2 between 0-96hpf, and to a lesser extent, of sod1 and gstp1. Once optimized, QGP analysis of these experiments was accomplished more rapidly, using far less tissue, and at lower total costs than qPCR analysis. In summary, the QGP platform as applied to higher-throughput zebrafish studies provides a reasonable cost-effective alternative to qPCR or more comprehensive transcriptomics approaches to rapidly assess the potential for chemicals to elicit oxidative stress as a mechanism of chemical toxicity.

simUfish: An Interactive Application to Teach K-12 Students About Zebrafish Behavior.

PubMed

Mwaffo, Violet; Korneyeva, Veronika; Porfiri, Maurizio

2017-10-01

As the zebrafish is rapidly becoming a species of choice in preclinical research, several efforts are being placed toward creating educational programs for K-12 students based on this promising model organism. However, as any other model organisms, the use of zebrafish in classroom settings requires additional experimental resources and poses ethical challenges related to animal use. To mitigate these factors, we have developed an application (app), simUfish, which implements a mathematical model of zebrafish behavior for generating multiple fish trajectories and animating their body undulations. simUfish is developed using a multiplatform game engine and is expected to promote the knowledge of zebrafish behavior to both K-12 students and the general public. Specifically, it demonstrates basic principles of fish individual and social behaviors, including environment interaction; fear response toward a predator; shoaling; and attraction toward a stimulus, which can be a food source or simply a finger placed on the touch screen. The effectiveness of the app as an accessible experimental tool for learning was tested in an outreach activity on middle school students from the New York City school system. The results from this activity show an immediate, tangible improvement of students' satisfaction and willingness to learn about key concepts on zebrafish behavior, accompanied by high level of interest in life sciences.

Nrf2 and Nrf2-related proteins in development and developmental toxicity: Insights from studies in zebrafish (Danio rerio).

PubMed

Hahn, Mark E; Timme-Laragy, Alicia R; Karchner, Sibel I; Stegeman, John J

2015-11-01

Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap'n'collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects. Copyright © 2015. Published by Elsevier Inc.

Zebrafish Whole-Adult-Organism Chemogenomics for Large-Scale Predictive and Discovery Chemical Biology

PubMed Central

Lam, Siew Hong; Mathavan, Sinnakarupan; Tong, Yan; Li, Haixia; Karuturi, R. Krishna Murthy; Wu, Yilian; Vega, Vinsensius B.; Liu, Edison T.; Gong, Zhiyuan

2008-01-01

The ability to perform large-scale, expression-based chemogenomics on whole adult organisms, as in invertebrate models (worm and fly), is highly desirable for a vertebrate model but its feasibility and potential has not been demonstrated. We performed expression-based chemogenomics on the whole adult organism of a vertebrate model, the zebrafish, and demonstrated its potential for large-scale predictive and discovery chemical biology. Focusing on two classes of compounds with wide implications to human health, polycyclic (halogenated) aromatic hydrocarbons [P(H)AHs] and estrogenic compounds (ECs), we generated robust prediction models that can discriminate compounds of the same class from those of different classes in two large independent experiments. The robust expression signatures led to the identification of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. Knowledge-based data mining of human homologs of zebrafish genes revealed highly conserved chemical-induced biological responses/effects, health risks, and novel biological insights associated with AHR and ER that could be inferred to humans. Thus, our study presents an effective, high-throughput strategy of capturing molecular snapshots of chemical-induced biological states of a whole adult vertebrate that provides information on biomarkers of effects, deregulated signaling pathways, and possible affected biological functions, perturbed physiological systems, and increased health risks. These findings place zebrafish in a strategic position to bridge the wide gap between cell-based and rodent models in chemogenomics research and applications, especially in preclinical drug discovery and toxicology. PMID:18618001

In silico molecular comparisons of C. elegans and mammalian pharmacology identify distinct targets that regulate feeding.

PubMed

Lemieux, George A; Keiser, Michael J; Sassano, Maria F; Laggner, Christian; Mayer, Fahima; Bainton, Roland J; Werb, Zena; Roth, Bryan L; Shoichet, Brian K; Ashrafi, Kaveh

2013-11-01

Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.

Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds.

PubMed

Zeng, Wendy R; Beh, Siew-Joo; Bryson-Richardson, Robert J; Doran, Pauline M

2017-09-01

The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts. The cardiospheres contained a mixed population of c-kit + and Mef2c + cells; proliferative peripheral cells of possible mesenchymal lineage were also observed. Cellular outgrowth from cardiac explants and cardiospheres was enhanced significantly using conditioned medium harvested from cultures of a rainbow trout cell line, suggesting that fish-specific trophic factors are required for zebrafish cardiac cell expansion. Three-dimensional culture of zebrafish heart cells in fibrous polyglycolic acid (PGA) scaffolds was carried out under dynamic fluid flow conditions. High levels of cell viability and cardiomyocyte differentiation were maintained within the scaffolds. Expression of cardiac troponin T, a marker of differentiated cardiomyocytes, increased during the first 7 days of scaffold culture; after 15 days, premature disintegration of the biodegradable scaffolds led to cell detachment and a decline in differentiation status. This work expands our technical capabilities for three-dimensional zebrafish cardiac cell culture with potential applications in tissue engineering, drug and toxicology screening, and ontogeny research. Biotechnol. Bioeng. 2017;114: 2142-2148. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Analysis of Chemical Bioactivity through In Vitro Profiling ...

EPA Pesticide Factsheets

Safety assessment of drugs and environmental chemicals relies extensively on animal testing. However, the quantity of chemicals needing assessment and challenges of species extrapolation drive the development of alternative approaches. The EPA’s ToxCast and the multiagency Tox21 programs address this through use of an extensive in vitro screening program to generate data on a large library of important environmental chemicals. These in vitro assays encompass both cell-free, biochemical assays targeting proteins that may be potential molecular initiating events and cellular assays that provide coverage of critical signaling pathways and toxicity phenotypes. Effects on model organisms such as the developing zebrafish, are also part of the testing strategy. A variety of computational approaches are used to analyze the resulting complex data sets to gain insight in to inherent biological activity of chemicals and possible mechanisms of toxicity. Several case studies including identification of modulators of estrogen receptor and aromatic hydrocarbon receptor pathways with effects in primary human cell systems will be described. In addition, existing in vivo data from a subset of the chemicals was used to anchor predictive models using in vitro data for a number of adverse endpoints including reproductive and developmental toxicities. The strengths and weaknesses of this approach will be described. This work does not necessarily reflect official Agency policy. Pres

Neurobeachin is required postsynaptically for electrical and chemical synapse formation

PubMed Central

Miller, Adam C.; Voelker, Lisa H.; Shah, Arish N.; Moens, Cecilia B.

2014-01-01

Summary Background Neural networks and their function are defined by synapses, which are adhesions specialized for intercellular communication that can be either chemical or electrical. At chemical synapses transmission between neurons is mediated by neurotransmitters, while at electrical synapses direct ionic and metabolic coupling occurs via gap junctions between neurons. The molecular pathways required for electrical synaptogenesis are not well understood and whether they share mechanisms of formation with chemical synapses is not clear. Results Here, using a forward genetic screen in zebrafish we find that the autism-associated gene neurobeachin (nbea), which encodes a BEACH-domain containing protein implicated in endomembrane trafficking, is required for both electrical and chemical synapse formation. Additionally, we find that nbea is dispensable for axonal formation and early dendritic outgrowth, but is required to maintain dendritic complexity. These synaptic and morphological defects correlate with deficiencies in behavioral performance. Using chimeric animals in which individually identifiable neurons are either mutant or wildtype we find that Nbea is necessary and sufficient autonomously in the postsynaptic neuron for both synapse formation and dendritic arborization. Conclusions Our data identify a surprising link between electrical and chemical synapse formation and show that Nbea acts as a critical regulator in the postsynaptic neuron for the coordination of dendritic morphology with synaptogenesis. PMID:25484298

The Zebrafish Xenograft Platform: Evolution of a Novel Cancer Model and Preclinical Screening Tool.

PubMed

Wertman, Jaime; Veinotte, Chansey J; Dellaire, Graham; Berman, Jason N

2016-01-01

Animal xenografts of human cancers represent a key preclinical tool in the field of cancer research. While mouse xenografts have long been the gold standard, investigators have begun to use zebrafish (Danio rerio) xenotransplantation as a relatively rapid, robust and cost-effective in vivo model of human cancers. There are several important methodological considerations in the design of an informative and efficient zebrafish xenotransplantation experiment. Various transgenic fish strains have been created that facilitate microscopic observation, ranging from the completely transparent casper fish to the Tg(fli1:eGFP) fish that expresses fluorescent GFP protein in its vascular tissue. While human cancer cell lines have been used extensively in zebrafish xenotransplantation studies, several reports have also used primary patient samples as the donor material. The zebrafish is ideally suited for transplanting primary patient material by virtue of the relatively low number of cells required for each embryo (between 50 and 300 cells), the absence of an adaptive immune system in the early zebrafish embryo, and the short experimental timeframe (5-7 days). Following xenotransplantation into the fish, cells can be tracked using in vivo or ex vivo measures of cell proliferation and migration, facilitated by fluorescence or human-specific protein expression. Importantly, assays have been developed that allow for the reliable detection of in vivo human cancer cell growth or inhibition following administration of drugs of interest. The zebrafish xenotransplantation model is a unique and effective tool for the study of cancer cell biology.

Altered Gene Expression in the Brain and Ovaries of Zebrafish Exposed to the Aromatase Inhibitor Fadrozole: Microarray analysis and Hypothesis Generation

EPA Science Inventory

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine active chemicals (EACs) with different modes of action, we exposed zebrafish (Danio rerio) to 25 or 100 ìg/L of the aromatase inhibitor fadrozole for...

Zebrafish Model of NF1 for Structure-Function Analysis, Mechanisms of Glial Tumorigenesis, and Chemical Biology

DTIC Science & Technology

2014-05-01

identify modulators of this important disease. 15. SUBJECT TERMS Neurofibromatosis ; learning and memory; glioma; MPNST 16. SECURITY CLASSIFICATION...12-13 Page 3 INTRODUCTION Neurofibromatosis type 1...result indicates that sox10 is highly expressed in high-grade gliomas and MPNSTs in our zebrafish model of type I neurofibromatosis . Figure 4. Sox10

Regulation of zebrafish sleep and arousal states: current and prospective approaches

PubMed Central

Chiu, Cindy N.; Prober, David A.

2013-01-01

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish has emerged as a promising model system for sleep and arousal research. Here we review experimental evidence that the zebrafish, a diurnal vertebrate, exhibits fundamental behavioral and neurochemical characteristics of mammalian sleep and arousal. We also propose how specific advantages of the zebrafish can be harnessed to advance the field. These include tractable genetics to identify and manipulate molecular and cellular regulators of behavioral states, optical transparency to facilitate in vivo observation of neural structure and function, and amenability to high-throughput drug screens to discover novel therapies for neurological disorders. PMID:23576957

Behavioural screening of zebrafish using neuroactive traditional Chinese medicine prescriptions and biological targets

NASA Astrophysics Data System (ADS)

Wang, Ya-Nan; Hou, Yuan-Yuan; Sun, Ming-Zhu; Zhang, Chun-Yang; Bai, Gang; Zhao, Xin; Feng, Xi-Zeng

2014-06-01

The mechanism of the therapeutic action of antidepressants remains uncertain in traditional Chinese medicine (TCM). In this study, we selected 7 classical TCM prescriptions and utilised an automatic video-tracking system to monitor the rest/wake behaviour of larval zebrafish at 4 days post-fertilisation (dpf) for 48 hours. We found that the curative effects of the prescriptions were dose-dependent. K-means clustering was performed according to the shared behavioural phenotypes of the zebrafish. The results revealed that the rest/wake behavioural profiles induced by the same class of prescriptions were similar. A correlation analysis was conducted between the TCM prescriptions and the known compounds. The results showed that the TCM prescriptions correlated well with some well-known compounds. Therefore, we predicted that they may share a similar mechanism of action. This paper describes the first study to combine TCM research with zebrafish rest/wake behaviour in vivo and presents a powerful approach for the discovery of the mechanism of action of TCM prescriptions.

OpenSource lab-on-a-chip physiometer for accelerated zebrafish embryo biotests.

PubMed

Akagi, Jin; Hall, Chris J; Crosier, Kathryn E; Cooper, Jonathan M; Crosier, Philip S; Wlodkowic, Donald

2014-01-02

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening. Copyright © 2014 John Wiley & Sons, Inc.

Zebrafish heart failure models: opportunities and challenges.

PubMed

Shi, Xingjuan; Chen, Ru; Zhang, Yu; Yun, Junghwa; Brand-Arzamendi, Koroboshka; Liu, Xiangdong; Wen, Xiao-Yan

2018-05-03

Heart failure is a complex pathophysiological syndrome of pumping functional failure that results from injury, infection or toxin-induced damage on the myocardium, as well as genetic influence. Gene mutations associated with cardiomyopathies can lead to various pathologies of heart failure. In recent years, zebrafish, Danio rerio, has emerged as an excellent model to study human cardiovascular diseases such as congenital heart defects, cardiomyopathy, and preclinical development of drugs targeting these diseases. In this review, we will first summarize zebrafish genetic models of heart failure arose from cardiomyopathy, which is caused by mutations in sarcomere, calcium or mitochondrial-associated genes. Moreover, we outline zebrafish heart failure models triggered by chemical compounds. Elucidation of these models will improve the understanding of the mechanism of pathogenesis and provide potential targets for novel therapies.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

PubMed

O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

2009-05-07

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

PubMed Central

O'Connor, Marie N.; Salles, Isabelle I.; Cvejic, Ana; Watkins, Nicholas A.; Walker, Adam; Garner, Stephen F.; Jones, Chris I.; Macaulay, Iain C.; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L.; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H.; Stemple, Derek L.; Ouwehand, Willem H.

2009-01-01

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. PMID:19109564

In vivo imaging and quantitative analysis of changes in axon length using transgenic zebrafish embryos.

PubMed

Kanungo, Jyotshnabala; Lantz, Susan; Paule, Merle G

2011-01-01

We describe an imaging procedure to measure axon length in zebrafish embryos in vivo. Automated fluorescent image acquisition was performed with the ImageXpress Micro high content screening reader and further analysis of axon lengths was performed on archived images using AcuityXpress software. We utilized the Neurite Outgrowth Application module with a customized protocol (journal) to measure the axons. Since higher doses of ethanol (2-2.5%, v/v) have been shown to deform motor neurons and axons during development, here we used ethanol to treat transgenic [hb9:GFP (green fluorescent protein)] zebrafish embryos at 28 hpf (hours post-fertilization). These embryos express GFP in the motor neurons and their axons. Embryos after ethanol treatment were arrayed in 384-well plates for automated fluorescent image acquisition in vivo. Average axon lengths of high dose ethanol-treated embryos were significantly lower than the control. Another experiment showed that there was no significant difference in the axon lengths between the embryos grown for 24h at 22°C and 28.5°C. These test experiments demonstrate that using axon development as an end-point, compound screening can be performed in a time-efficient manner. Published by Elsevier Inc.

20170312 - Adverse Outcome Pathway (AOP) framework for ...

EPA Pesticide Factsheets

Vascular development commences with de novo assembly of a primary capillary plexus (vasculogenesis) followed by its expansion (angiogenesis) and maturation (angio-adaptation) into a hierarchical system of arteries and veins. These processes are tightly regulated by genetic signals and environmental factors linked to morphogenesis and microphysiology. Gestational exposure to some chemicals disrupts vascular development leading to adverse outcomes. To broadly assess consequences of gestational toxicant exposure on vascular development, an Adverse Outcome Pathway (AOP) framework was constructed that integrates data from ToxCast high-throughput screening (HTS) assays with pathway-level information from the literature and public databases. The AOP-based model resolved the ToxCast library (1065 compounds) into a matrix based on several dozen molecular functions critical for developmental angiogenesis. A sample of 38 ToxCast chemicals selected across the matrix tested model performance. Putative vascular disrupting chemical (pVDC) bioactivity was assessed by multiple laboratories utilizing diverse angiogenesis assays, including: transgenic zebrafish, complex human cell co-cultures, engineered microscale systems, and human-synthetic models. The ToxCast pVDC signature predicted vascular disruption in a manner that was chemical-specific and assay-dependent. An AOP for developmental vascular toxicity was constructed that focuses on inhibition of VEGF receptor (VEGFR2). Thi

Adverse Outcome Pathway (AOP) framework for embryonic ...

EPA Pesticide Factsheets

Vascular development commences with de novo assembly of a primary capillary plexus (vasculogenesis) followed by its expansion (angiogenesis) and maturation (angio-adaptation) into a hierarchical system of arteries and veins. These processes are tightly regulated by genetic signals and environmental factors linked to morphogenesis and microphysiology. Gestational exposure to some chemicals disrupts vascular development leading to adverse outcomes. To broadly assess consequences of gestational toxicant exposure on vascular development, an Adverse Outcome Pathway (AOP) framework was constructed that integrates data from ToxCast high-throughput screening (HTS) assays with pathway-level information from the literature and public databases. The AOP-based model resolved the ToxCast library (1065 compounds) into a matrix based on several dozen molecular functions critical for developmental angiogenesis. A sample of 38 ToxCast chemicals selected across the matrix tested model performance. Putative vascular disrupting chemical (pVDC) bioactivity was assessed by multiple laboratories utilizing diverse angiogenesis assays, including: transgenic zebrafish, complex human cell co-cultures, engineered microscale systems, and human-synthetic models. The ToxCast pVDC signature predicted vascular disruption in a manner that was chemical-specific and assay-dependent. An AOP for developmental vascular toxicity was constructed that focuses on inhibition of VEGF receptor (VEGFR2). Thi

Zebrafish—on the move towards ophthalmological research

PubMed Central

Chhetri, J; Jacobson, G; Gueven, N

2014-01-01

Millions of people are affected by visual impairment and blindness globally, and the prevalence of vision loss is likely to increase as we are living longer. However, many ocular diseases remain poorly controlled due to lack of proper understanding of the pathogenesis and the corresponding lack of effective therapies. Consequently, there is a major need for animal models that closely mirror the human eye pathology and at the same time allow higher-throughput drug screening approaches. In this context, zebrafish as an animal model organism not only address these needs but can in many respects reflect the human situation better than the current rodent models. Over the past decade, zebrafish have become an established model to study a variety of human diseases and are more recently becoming a valuable tool for the study of human ophthalmological disorders. Many human ocular diseases such as cataract, glaucoma, diabetic retinopathy, and age-related macular degeneration have already been modelled in zebrafish. In addition, zebrafish have become an attractive model for pre-clinical drug toxicity testing and are now increasingly used by scientists worldwide for the discovery of novel treatment approaches. This review presents the advantages and uses of zebrafish for ophthalmological research. PMID:24503724

The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

PubMed

Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

2011-02-01

In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12

PubMed Central

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K.; Nielsen, G. Petur; Peterson, Randall T.; Drummond, Iain A.; Haber, Daniel A.

2014-01-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. PMID:24311731

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

PubMed

Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

2017-08-01

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways.

PubMed

Liang, Fang; Han, Yuxiang; Gao, Hao; Xin, Shengchang; Chen, Shaodan; Wang, Nan; Qin, Wei; Zhong, Hanbing; Lin, Shuo; Yao, Xinsheng; Li, Song

2015-10-08

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products.

Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways

PubMed Central

Liang, Fang; Han, Yuxiang; Gao, Hao; Xin, Shengchang; Chen, Shaodan; Wang, Nan; Qin, Wei; Zhong, Hanbing; Lin, Shuo; Yao, Xinsheng; Li, Song

2015-01-01

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products. PMID:26446489

Pathfinder Innovation Projects: Awardees for 2013

EPA Pesticide Factsheets

PIP3 awardees will tackle high-risk, high-reward research ideas with gated funding. Projects include PM toxicity for zebrafish, lab-grown neuron networks, research bias for pharmaceutical chemicals, and innovative chemical life cycle assessments.

Polysaccharides from astragali radix restore chemical-induced blood vessel loss in zebrafish

PubMed Central

2012-01-01

Background Astragali Radix has been used widely for the treatment of cardiovascular and cerebrovascular diseases, and to enhance endurance and stamina in traditional Chinese medicine (TCM) for over 2000 years. The polysaccharide constituents of Astragali Radix (ARP) are considered as one of the major constituents contributing to the multiple pharmacological effects of this medicinal plant. The purpose of the study is to evaluate the vascular regenerative activities of ARPs in a chemically-induced blood vessel loss model in zebrafish. Methods Blood vessel loss was induced in both Tg(fli-1a:EGFP)y1 and Tg(fli-1a:nEGFP)y7 embryos by administration of 300 nM VEGFR tyrosine kinase inhibitor II (VRI) for 3 h at 24 hpf (hour post-fertilization). Then, the blood vessel damaged zebrafish were treated with ARPs for 21 h and 45 h after VRI withdrawal. Morphological changes in intersegmental vessels (ISVs) of zebrafish larvae were observed under the fluorescence microscope and measured quantitatively. The rescue effect of ARPs in the zebrafish models was validated by measuring the relative mRNA expressions of Kdrl, Kdr and Flt-1 using real-time PCR. Results Two polysaccharide fractions, P4 (50000 D < molecular weight & diameter < 0.1 μm) and P5 (molecular diameter > 0.1 μm), isolated from Astragali Radix by ultrafiltration, produced a significant and dose-dependent recovery in VRI-induced blood vessel loss in zebrafish. Furthermore, the down-regulation of Flk-1 and Flt-1 mRNA expression induced by VRI was reversed by treatment with P4. Conclusion The present study demonstrates that P4 isolated from Astragali Radix reduces VRI-induced blood vessel loss in zebrafish. These findings support the hypothesis that polysaccharides are one of the active constituents in Astragali Radix, contributing to its beneficial effect on treatment of diseases associated with a deficiency in angiogenesis. PMID:22357377

In vitro and in vivo estrogenic activity of BPA, BPF and BPS in zebrafish-specific assays.

PubMed

Le Fol, Vincent; Aït-Aïssa, Selim; Sonavane, Manoj; Porcher, Jean-Marc; Balaguer, Patrick; Cravedi, Jean-Pierre; Zalko, Daniel; Brion, François

2017-08-01

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERβ subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1-10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinfray, N., E-mail: nathalie.hinfray@ineris.fr

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase genemore » under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. - Highlights: • Combined effects of EE2 and LNG were assessed on ER-dependent cyp19a1b expression. • EE2 and LNG alone induced brain aromatase in zebrafish specific bioassays. • Experimental ray design allowed complete concentration-response surfaces modeling. • EE2 and LNG exerted additive effects on brain aromatase in radial glial cells.« less

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

PubMed

Bouwmeester, Manon C; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M; van den Brandhof, Evert-Jan; Pennings, Jeroen L A; Kamstra, Jorke H; Jelinek, Jaroslav; Issa, Jean-Pierre J; Legler, Juliette; van der Ven, Leo T M

2016-01-15

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative Developmental Toxicity of Flavonoids Using an Integrative Zebrafish System

PubMed Central

Bugel, Sean M.; Bonventre, Josephine A.; Tanguay, Robert L.

2016-01-01

Flavonoids are a large, structurally diverse class of bioactive naturally occurring chemicals commonly detected in breast milk, soy based infant formulas, amniotic fluid, and fetal cord blood. The potential for pervasive early life stage exposures raises concerns for perturbation of embryogenesis, though developmental toxicity and bioactivity information is limited for many flavonoids. Therefore, we evaluated a suite of 24 flavonoid and flavonoid-like chemicals using a zebrafish embryo-larval toxicity bioassay—an alternative model for investigating developmental toxicity of environmentally relevant chemicals. Embryos were exposed to 1–50 µM of each chemical from 6 to 120 h postfertilization (hpf), and assessed for 26 adverse developmental endpoints at 24, 72, and 120 hpf. Behavioral changes were evaluated in morphologically normal animals at 24 and 72 hpf, at 120 hpf using a larval photomotor response (LPR) assay. Gene expression was comparatively evaluated for all compounds for effects on biomarker transcripts indicative of AHR (cyp1a) and ER (cyp19a1b, esr1, lhb, vtg) pathway bioactivity. Overall, 15 of 24 flavonoids elicited adverse effects on one or more of the developmental or behavioral endpoints. Hierarchical clustering and principle component analyses compared toxicity profiles and identified 3 distinct groups of bioactive flavonoids. Despite robust induction of multiple estrogen-responsive biomarkers, co-exposure with ER and GPER antagonists did not ameliorate toxicity, suggesting ER-independence and alternative modes of action. Taken together, these studies demonstrate that development is sensitive to perturbation by bioactive flavonoids in zebrafish that are not related to traditional estrogen receptor mode of action pathways. This integrative zebrafish platform provides a useful framework for evaluating flavonoid developmental toxicity and hazard prioritization. PMID:27492224

Auditory sensitivity of larval zebrafish (Danio rerio) measured using a behavioral prepulse inhibition assay

PubMed Central

Bhandiwad, Ashwin A.; Zeddies, David G.; Raible, David W.; Rubel, Edwin W.; Sisneros, Joseph A.

2013-01-01

SUMMARY Zebrafish (Danio rerio) have become a valuable model for investigating the molecular genetics and development of the inner ear in vertebrates. In this study, we employed a prepulse inhibition (PPI) paradigm to assess hearing in larval wild-type (AB) zebrafish during early development at 5–6 days post-fertilization (d.p.f.). We measured the PPI of the acoustic startle response in zebrafish using a 1-dimensional shaker that simulated the particle motion component of sound along the fish's dorsoventral axis. The thresholds to startle-inducing stimuli were determined in 5–6 d.p.f. zebrafish, and their hearing sensitivity was then characterized using the thresholds of prepulse tone stimuli (90–1200 Hz) that inhibited the acoustic startle response to a reliable startle stimulus (820 Hz at 20 dB re. 1 m s−2). Hearing thresholds were defined as the minimum prepulse tone level required to significantly reduce the startle response probability compared with the baseline (no-prepulse) condition. Larval zebrafish showed greatest auditory sensitivity from 90 to 310 Hz with corresponding mean thresholds of −19 to −10 dB re. 1 m s−2, respectively. Hearing thresholds of prepulse tones were considerably lower than previously predicted by startle response assays. The PPI assay was also used to investigate the relative contribution of the lateral line to the detection of acoustic stimuli. After aminoglycoside-induced neuromast hair-cell ablation, we found no difference in PPI thresholds between treated and control fish. We propose that this PPI assay can be used to screen for novel zebrafish hearing mutants and to investigate the ontogeny of hearing in zebrafish and other fishes. PMID:23966590

Development of an opioid self-administration assay to study drug seeking in zebrafish.

PubMed

Bossé, Gabriel D; Peterson, Randall T

2017-09-29

The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization

PubMed Central

Greenald, David; Wilson, Garrick K.; Peron, Margherita; Markham, Eleanor; Sinnakaruppan, Mathavan; Matthews, Laura C.; McKeating, Jane A.; Argenton, Francesco; van Eeden, Fredericus J. M.

2017-01-01

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src–mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL. PMID:28851829

Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment.

PubMed

Li, Pulin; Lahvic, Jamie L; Binder, Vera; Pugach, Emily K; Riley, Elizabeth B; Tamplin, Owen J; Panigrahy, Dipak; Bowman, Teresa V; Barrett, Francesca G; Heffner, Garrett C; McKinney-Freeman, Shannon; Schlaeger, Thorsten M; Daley, George Q; Zeldin, Darryl C; Zon, Leonard I

2015-07-23

Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.

Akt3 is a privileged first responder in isozyme-specific electrophile response.

PubMed

Long, Marcus J C; Parvez, Saba; Zhao, Yi; Surya, Sanjna L; Wang, Yiran; Zhang, Sheng; Aye, Yimon

2017-03-01

Isozyme-specific post-translational regulation fine tunes signaling events. However, redundancy in sequence or activity renders links between isozyme-specific modifications and downstream functions uncertain. Methods to study this phenomenon are underdeveloped. Here we use a redox-targeting screen to reveal that Akt3 is a first-responding isozyme sensing native electrophilic lipids. Electrophile modification of Akt3 modulated downstream pathway responses in cells and Danio rerio (zebrafish) and markedly differed from Akt2-specific oxidative regulation. Digest MS sequencing identified Akt3 C119 as the privileged cysteine that senses 4-hydroxynonenal. A C119S Akt3 mutant was hypomorphic for all downstream phenotypes shown by wild-type Akt3. This study documents isozyme-specific and chemical redox signal-personalized physiological responses.

Behavioral barcoding in the cloud: embracing data-intensive digital phenotyping in neuropharmacology.

PubMed

Kokel, David; Rennekamp, Andrew J; Shah, Asmi H; Liebel, Urban; Peterson, Randall T

2012-08-01

For decades, studying the behavioral effects of individual drugs and genetic mutations has been at the heart of efforts to understand and treat nervous system disorders. High-throughput technologies adapted from other disciplines (e.g., high-throughput chemical screening, genomics) are changing the scale of data acquisition in behavioral neuroscience. Massive behavioral datasets are beginning to emerge, particularly from zebrafish labs, where behavioral assays can be performed rapidly and reproducibly in 96-well, high-throughput format. Mining these datasets and making comparisons across different assays are major challenges for the field. Here, we review behavioral barcoding, a process by which complex behavioral assays are reduced to a string of numeric features, facilitating analysis and comparison within and across datasets. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bromodomain and extraterminal (BET) proteins regulate biliary-driven liver regeneration.

PubMed

Ko, Sungjin; Choi, Tae-Young; Russell, Jacquelyn O; So, Juhoon; Monga, Satdarshan P S; Shin, Donghun

2016-02-01

During liver regeneration, hepatocytes are derived from pre-existing hepatocytes. However, if hepatocyte proliferation is compromised, biliary epithelial cells (BECs) become the source of new hepatocytes. We recently reported on a zebrafish liver regeneration model in which BECs extensively contribute to hepatocytes. Using this model, we performed a targeted chemical screen to identify important factors that regulate BEC-driven liver regeneration, the mechanisms of which remain largely unknown. Using Tg(fabp10a:CFP-NTR) zebrafish, we examined the effects of 44 selected compounds on BEC-driven liver regeneration. Liver size was assessed by fabp10a:DsRed expression; liver marker expression was analyzed by immunostaining, in situ hybridization and quantitative PCR. Proliferation and apoptosis were also examined. Moreover, we used a mouse liver injury model, choline-deficient, ethionine-supplemented (CDE) diet. We identified 10 compounds that affected regenerating liver size. Among them, only bromodomain and extraterminal domain (BET) inhibitors, JQ1 and iBET151, blocked both Prox1 and Hnf4a induction in BECs. BET inhibition during hepatocyte ablation blocked BEC dedifferentiation into hepatoblast-like cells (HB-LCs). Intriguingly, after JQ1 washout, liver regeneration resumed, indicating temporal, but not permanent, perturbation of liver regeneration by BET inhibition. BET inhibition after hepatocyte ablation suppressed the proliferation of newly generated hepatocytes and delayed hepatocyte maturation. Importantly, Myca overexpression, in part, rescued the proliferation defect. Furthermore, oval cell numbers in mice fed CDE diet were greatly reduced upon JQ1 administration, supporting the zebrafish findings. BET proteins regulate BEC-driven liver regeneration at multiple steps: BEC dedifferentiation, HB-LC proliferation, the proliferation of newly generated hepatocytes, and hepatocyte maturation. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

(Eco)toxicological effects of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in zebrafish (Danio rerio) and permanent fish cell cultures.

PubMed

Vincze, Krisztina; Gehring, Martin; Braunbeck, Thomas

2014-01-01

2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a high-production volume chemical used in paper, ink, pesticide, and adhesive industries as a wetting and anti-foaming agent. The physicochemical properties and slow biodegradation rate of TMDD indicate a low bioaccumulation potential but a high prevalence in the environment. As a consequence, TMDD has been detected in several European rivers in the nanogram per liter and lower microgram per liter range; however, its environmental risk to aquatic organisms is considered low. Recent studies almost exclusively focused on acute effects by TMDD, little is known about cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity so far. The present study aims to provide more specific baseline information on the ecotoxicological effects of TMDD in fish. For this end, cyto- and genotoxicity assays were carried out in vitro with the permanent fish cell line RTL-W1; in addition, in vivo studies were conducted with the early life stages of zebrafish (Danio rerio) in order to fill the data gaps in developmental toxicity and endocrine disruption. TMDD showed a cytotoxic and slight genotoxic potential in fish cell lines; moreover, various sublethal and lethal effects could be detected in developing zebrafish embryos. There was no evidence of endocrine-disrupting effects by TMDD; however, mortality following prolonged exposure to TMDD during fish sexual development test was clearly higher than mortality in the fish embryo test after 96-h exposure. Our results thus confirmed previous findings of laboratory screening tests, suggesting short-term toxic effects of TMDD in the intermediate, and long-term effects in the lower milligram per liter range.

Knockdown of Zebrafish Lumican Gene (zlum) Causes Scleral Thinning and Increased Size of Scleral Coats*

PubMed Central

Yeh, Lung-Kun; Liu, Chia-Yang; Kao, Winston W.-Y.; Huang, Chang-Jen; Hu, Fung-Rong; Chien, Chung-Liang; Wang, I-Jong

2010-01-01

The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans ∼4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5′-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5′-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia. PMID:20551313

Zebrafish models for the functional genomics of neurogenetic disorders.

PubMed

Kabashi, Edor; Brustein, Edna; Champagne, Nathalie; Drapeau, Pierre

2011-03-01

In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

Characterization of synergistic embryotoxicity of nickel and buprofezin in zebrafish.

PubMed

Ku, Tingting; Yan, Wei; Jia, Wuyao; Yun, Yang; Zhu, Na; Li, Guangke; Sang, Nan

2015-04-07

Multiple pollutants, usually at low levels, coexist and may interact in the environment. It is therefore important to analyze the toxicity of mixtures of coexisting chemicals to evaluate the potential ecological risk. Concern regarding the co-occurrence and combined bioeffects of heavy metals and organic insecticides in aquatic settings has existed for many years, but a clear understanding of the interactions between and potential combined toxicity of these chemicals remains elusive. In the present study, the combined effects of the heavy metal nickel (NiSO4) and insect growth regulator buprofezin on the induction of embryo toxicity in zebrafish were assessed. By applying nonlinear regression to the concentration-response data with each of the chemicals using the Hill and Langmuir functions and computing the predictions using the model of concentration addition (CA), we confirmed that NiSO4 and buprofezin acted together to produce synergistic embryotoxicity in zebrafish. Subsequently, we further found that the combination of NiSO4 and buprofezin formed a complex that facilitated the uptake of nickel (Ni) and buprofezin by the embryos. Following this, we clarified that an oxidative mechanism of the complex might underlie the synergistic embryotoxicity of NiSO4 and buprofezin.

Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish

PubMed Central

Kedikian, Ximena; Faillace, Maria Paula; Bernabeu, Ramón

2013-01-01

Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and β2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine-associated reward in vertebrates. PMID:23894483

Food and conspecific chemical cues modify visual behavior of zebrafish, Danio rerio.

PubMed

Stephenson, Jessica F; Partridge, Julian C; Whitlock, Kathleen E

2012-06-01

Animals use the different qualities of olfactory and visual sensory information to make decisions. Ethological and electrophysiological evidence suggests that there is cross-modal priming between these sensory systems in fish. We present the first experimental study showing that ecologically relevant chemical mixtures alter visual behavior, using adult male and female zebrafish, Danio rerio. Neutral-density filters were used to attenuate the light reaching the tank to an initial light intensity of 2.3×10(16) photons/s/m2. Fish were exposed to food cue and to alarm cue. The light intensity was then increased by the removal of one layer of filter (nominal absorbance 0.3) every minute until, after 10 minutes, the light level was 15.5×10(16) photons/s/m2. Adult male and female zebrafish responded to a moving visual stimulus at lower light levels if they had been first exposed to food cue, or to conspecific alarm cue. These results suggest the need for more integrative studies of sensory biology.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

PubMed

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC(10)) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate "pattern-only" PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals

PubMed Central

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC10) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate “pattern-only” PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress. PMID:22084678

Zebrafish (Danio rerio) as a screen for attenuation of Lancefield group C streptococci and a model for streptococcal pathogenesis.

PubMed

Borst, L B; Patterson, S K; Lanka, S; Suyemoto, M M; Maddox, C W

2013-05-01

Group C streptococci are highly contagious pyogenic bacteria responsible for respiratory tract, lymph node, urogenital tract, and wound infections. Wild-type strains of Streptococcus equi ssp equi (S. equi) and Streptococcus equi ssp zooepidemicus (S. zoo) as well as a commercially available modified live vaccine strain of S. equi were evaluated for virulence in zebrafish. Survival times, histologic lesions, and relative gene expression were compared among groups. Based on the intramuscular route of infection, significantly shorter survival times were observed in fish infected with wild-type strain when compared to modified live vaccine and S. zoo strains. Histologically, S. zoo-infected fish demonstrated a marked increase in inflammatory infiltrates (predominantly macrophages) at the site of infection, as well as increased cellularity in the spleen and renal interstitium. In contrast, minimal cellular immune response was observed in S. equi-injected fish with local tissue necrosis and edema predominating. Based on whole comparative genomic hybridization, increased transcription of positive acute-phase proteins, coagulation factors, and antimicrobial peptides were observed in S. equi-injected fish relative to S. zoo-injected fish, while mediators of cellular inflammation, including CXC chemokines and granulin, were upregulated in S. zoo-injected fish relative to S. equi-injected fish. In a screen of 11 clinical isolates, S. equi strains with a single nucleotide deletion in the upstream region of szp, a known virulence factor of streptococci, were found to be significantly attenuated in zebrafish. These collective findings underscore the value of the zebrafish as a model of streptococcal pathogenesis.

Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation

PubMed Central

Rihel, Jason; Prober, David A.; Arvanites, Anthony; Lam, Kelvin; Zimmerman, Steven; Jang, Sumin; Haggarty, Stephen J.; Kokel, David; Rubin, Lee L.; Peterson, Randall T.; Schier, Alexander F.

2010-01-01

A major obstacle for the discovery of psychoactive drugs is the inability to predict how small molecules will alter complex behaviors. We report the development and application of a high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found that the multi-dimensional nature of observed phenotypes enabled the hierarchical clustering of molecules according to shared behaviors. Behavioral profiling revealed conserved functions of psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds. In addition, behavioral profiling implicated new factors such as ether-a-go-go-related gene (ERG) potassium channels and immunomodulators in the control of rest and locomotor activity. These results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors. PMID:20075256

Identification of vascular disruptor compounds by analysis in zebrafish embryos and mouse embryonic endothelial cells

EPA Science Inventory

The large number of diverse chemicals in production or in the environment has motivated medium to high throughput in vitro or small animal approaches to efficiently profile chemical-biological interactions and to utilize this information to assess risks of chemical exposures on h...

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

PubMed

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K; Nielsen, G Petur; Peterson, Randall T; Drummond, Iain A; Haber, Daniel A

2014-07-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. © 2014. Published by The Company of Biologists Ltd.

A simple automated system for appetitive conditioning of zebrafish in their home tanks.

PubMed

Doyle, Jillian M; Merovitch, Neil; Wyeth, Russell C; Stoyek, Matthew R; Schmidt, Michael; Wilfart, Florentin; Fine, Alan; Croll, Roger P

2017-01-15

We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. Arduino microprocessors were used to control the delivery of auditory or visual stimuli to groups of adult or juvenile zebrafish in their home tanks in a conventional zebrafish facility. An automatic feeder dispensed precise amounts of food immediately after the conditioned stimuli, or at variable delays for controls. Responses were recorded using inexpensive cameras, with the video sequences analysed with ImageJ or Matlab. Fish showed significant conditioned responses in as few as 5 trials, learning that the conditioned stimulus was a predictor of food presentation at the water surface and at the end of the tank where the food was dispensed. Memories of these conditioned associations persisted for at least 2days after training when fish were tested either as groups or as individuals. Control fish, for which the auditory or visual stimuli were specifically unpaired with food, showed no comparable responses. This simple, low-cost, automated system permits scalable conditioning of zebrafish with minimal human intervention, greatly reducing both variability and labour-intensiveness. It will be useful for studies of the neural basis of learning and memory, and for high-throughput screening of compounds modifying those processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Anti-angiogenic activity and phytochemical screening of fruit fractions from Vitex agnus castus.

PubMed

Certo, Giovanna; Costa, Rosaria; D'Angelo, Valeria; Russo, Marina; Albergamo, Ambrogina; Dugo, Giacomo; Germanò, Maria Paola

2017-12-01

Although the antitumour activity of Vitex agnus castus fruits has been already addressed, no work has yet assessed their anti-angiogenic potential. To this purpose, several extractive fractions of such fruits were tested on zebrafish embrios by EAP assay, so that only the bioactive fractions could be subsequently tested on the chick chorioallantoic membrane by CAM assay. Bioactive fractions were also phytochemically screened to identify those bioactive compounds responsible for anti-angiogenic activity. A marked inhibition of vessel formation was detected only in zebrafish embryos treated with chloroform or ethyl acetate fractions. Considering CAM assay, chloroform fraction induced a strong reduction of microvasculature and haemoglobin content; while lower anti-angiogenic effects of the ethyl acetate fraction were determined. Phytochemical analyses confirmed the presence of several bioactive anti-angiogenic compounds. Overall, obtained preliminary results highlighted a potential anti-angiogenic activity of V. agnus castus fruits.

Nanotoxicological and teratogenic effects: A linkage between dendrimer surface charge and zebrafish developmental stages.

PubMed

Calienni, Maria Natalia; Feas, Daniela Agustina; Igartúa, Daniela Edith; Chiaramoni, Nadia Silvia; Alonso, Silvia Del Valle; Prieto, Maria Jimena

2017-12-15

This article reports novel results about nanotoxicological and teratogenic effects of the PAMAM dendrimers DG4 and DG4.5 in zebrafish (Danio rerio). Zebrafish embryos and larvae were used as a rapid, high-throughput, cost-effective whole-animal model. The objective was to provide a more comprehensive and predictive developmental toxicity screening of DG4 and DG4.5 and test the influence of their surface charge. Nanotoxicological and teratogenic effects were assessed at developmental, morphological, cardiac, neurological and hepatic level. The effect of surface charge was determined in both larvae and embryos. DG4 with positive surface charge was more toxic than DG4.5 with negative surface charge. DG4 and DG4.5 induced teratogenic effects in larvae, whereas DG4 also induced lethal effects in both zebrafish embryos and larvae. However, larvae were less sensitive than embryos to the lethal effects of DG4. The platform of assays proposed and data obtained may contribute to the characterization of hazards and differential effects of these nanoparticles. Copyright © 2017 Elsevier Inc. All rights reserved.

The zebrafish embryo model in toxicology and teratology, September 2–3, 2010, Karlsruhe, Germany.

PubMed

Busch, Wibke; Duis, Karen; Fenske, Martina; Maack, Gerd; Legler, Juliette; Padilla, Stephanie; Strähle, Uwe; Witters, Hilda; Scholz, Stefan

2011-05-01

The use of fish embryos is gaining popularity for research in the area of toxicology and teratology. Particularly embryos of the zebrafish offer an array of different applications ranging from regulatory testing to mechanistic research. For this reason a consortium of two research centres and a company with the support of the COST Action EuFishBiomed has organised the Workshop “The zebrafish embryo model in toxicology and teratology”, in Karlsruhe, Germany, 2nd–3rd September 2010. The workshop aimed at bringing together experts from different areas of toxicology using the (zebra)fish embryo and stimulating networking between scientists and representatives from regulatory bodies, research institutions and industry. Recent findings, presented in various platform presentations in the area of regulatory toxicity, high throughput screening, toxicogenomics, as well as environmental and human risk assessment are highlighted in this meeting report. Furthermore, the constraints and possibilities of the model as discussed at the workshop are described. A follow up-meeting was appreciated by the about 120 participants and is planned for 2012.

PFOS, PFNA, and PFOA Sub-Lethal Exposure to Embryonic Zebrafish Have Different Toxicity Profiles in Terms of Morphometrics, Behavior and Gene Expression

PubMed Central

Jantzen, Carrie E.; Annunziato, Kate A.; Bugel, Sean M.; Cooper, Keith R.

2016-01-01

Polyfluorinated compounds (PFC) are a class of anthropogenic, persistent and toxic chemicals. PFCs are detected worldwide and consist of fluorinated carbon chains of varying length, terminal groups, and industrial uses. Previous zebrafish studies in the literature as well as our own studies have shown that exposure to these chemicals at a low range of concentrations (0.02 µM – 2.0 µM; 20–2000 ppb) resulted in chemical specific developmental defects and reduced post hatch survival. It was hypothesized that sub-lethal embryonic exposure to perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), or perfluorooctanoic acid (PFOA) would result in different responses with regard to morphometric, behavior, and gene expression in both yolk sac fry and larval zebrafish. Zebrafish were exposed to PFOS, PFOA, and PFNA (0.02, 0.2, 2.0 µM) for the first five days post fertilization (dpf) and analyzed for morphometrics (5 dpf, 14 dpf), targeted gene expression (5 dpf, 14 dpf), and locomotive behavior (14 dpf). All three PFCs commonly resulted in a decrease in total body length, increased tfc3a (muscle development) expression and decreased ap1s (protein transport) expression at 5dpf, and hyperactive locomotor activity 14 dpf. All other endpoints measured at both life-stage time points varied between each of the PFCs. PFOS, PFNA, and PFOA exposure resulted in significantly altered responses in terms of morphometric, locomotion, and gene expression endpoints, which could be manifested in field exposed teleosts. PMID:27058923

Triclosan Exposure Is Associated with Rapid Restructuring of the Microbiome in Adult Zebrafish

PubMed Central

Barton, Carrie L.; Proffitt, Sarah; Tanguay, Robert L.; Sharpton, Thomas J.

2016-01-01

Growing evidence indicates that disrupting the microbial community that comprises the intestinal tract, known as the gut microbiome, can contribute to the development or severity of disease. As a result, it is important to discern the agents responsible for microbiome disruption. While animals are frequently exposed to a diverse array of environmental chemicals, little is known about their effects on gut microbiome stability and structure. Here, we demonstrate how zebrafish can be used to glean insight into the effects of environmental chemical exposure on the structure and ecological dynamics of the gut microbiome. Specifically, we exposed forty-five adult zebrafish to triclosan-laden food for four or seven days or a control diet, and analyzed their microbial communities using 16S rRNA amplicon sequencing. Triclosan exposure was associated with rapid shifts in microbiome structure and diversity. We find evidence that several operational taxonomic units (OTUs) associated with the family Enterobacteriaceae appear to be susceptible to triclosan exposure, while OTUs associated with the genus Pseudomonas appeared to be more resilient and resistant to exposure. We also found that triclosan exposure is associated with topological alterations to microbial interaction networks and results in an overall increase in the number of negative interactions per microbe in these networks. Together these data indicate that triclosan exposure results in altered composition and ecological dynamics of microbial communities in the gut. Our work demonstrates that because zebrafish afford rapid and inexpensive interrogation of a large number of individuals, it is a useful experimental system for the discovery of the gut microbiome’s interaction with environmental chemicals. PMID:27191725

Existence of Inverted Profile in Chemically Responsive Molecular Pathways in the Zebrafish Liver

PubMed Central

Zhang, Xun; Li, Hu; Ma, Jing; Zhang, Louxin; Li, Baowen; Gong, Zhiyuan

2011-01-01

How a living organism maintains its healthy equilibrium in response to endless exposure of potentially harmful chemicals is an important question in current biology. By transcriptomic analysis of zebrafish livers treated by various chemicals, we defined hubs as molecular pathways that are frequently perturbed by chemicals and have high degree of functional connectivity to other pathways. Our network analysis revealed that these hubs were organized into two groups showing inverted functionality with each other. Intriguingly, the inverted activity profiles in these two groups of hubs were observed to associate only with toxicopathological states but not with physiological changes. Furthermore, these inverted profiles were also present in rat, mouse, and human under certain toxicopathological conditions. Thus, toxicopathological-associated anti-correlated profiles in hubs not only indicate their potential use in diagnosis but also development of systems-based therapeutics to modulate gene expression by chemical approach in order to rewire the deregulated activities of hubs back to normal physiology. PMID:22140468

Developmental and Persistent Toxicities of Maternally Deposited Selenomethionine in Zebrafish (Danio rerio).

PubMed

Thomas, Jith K; Janz, David M

2015-08-18

The objectives of this study were (1) to establish egg selenium (Se) toxicity thresholds for mortality and deformities in early life stages of zebrafish (Danio rerio) after exposure to excess selenomethionine (SeMet, the dominant chemical species of Se in diets) via in ovo maternal transfer and (2) to investigate the persistent effects of developmental exposure to excess SeMet on swim performance and metabolic capacities in F1-generation adult zebrafish. Adult zebrafish were fed either control food (1.3 μg Se/g, dry mass or d.m.) or food spiked with increasing measured concentrations of Se (3.4, 9.8, or 27.5 μg Se/g, d.m.) in the form of SeMet for 90 d. In ovo exposure to SeMet increased mortality and deformities in larval zebrafish in a concentration-dependent fashion with threshold egg Se concentrations (EC10s) of 7.5 and 7.0 μg Se/g d.m., respectively. Impaired swim performance and greater respiration and metabolic rates were observed in F1-generation zebrafish exposed in ovo to 6.8 and 12.7 μg Se/g d.m and raised to adulthood in clean water. A species sensitivity distribution (SSD) based on egg Se developmental toxicity thresholds suggests that zebrafish are the most sensitive fish species studied to date.

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

PubMed

Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

2009-05-15

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

Establishment of a novel experimental protocol for drug-induced seizure liability screening based on a locomotor activity assay in zebrafish.

PubMed

Koseki, Naoteru; Deguchi, Jiro; Yamashita, Akihito; Miyawaki, Izuru; Funabashi, Hitoshi

2014-08-01

As drug-induced seizures have severe impact on drug development, evaluating seizure induction potential of candidate drugs at the early stages of drug discovery is important. A novel assay system using zebrafish has attracted interest as a high throughput toxicological in vivo assay system, and we tried to establish an experimental method for drug-induced seizure liability on the basis of locomotor activity in zebrafish. We monitored locomotor activity at high-speed movement (> 20 mm/sec) for 60 min immediately after exposure, and assessed seizure liability potential in some drugs using locomotor activity. However this experimental procedure was not sufficient for predicting seizures because the potential of several drugs with demonstrated seizure potential in mammals was not detected. We, therefore, added other parameters for locomotor activity such as extending exposure time or conducting flashlight stimulation (10 Hz) which is a known seizure induction stimulus, and these additional parameters improved seizure potential detection in some drugs. The validation study using the improved methodology was used to assess 52 commercially available drugs, and the prediction rate was approximately 70%. The experimental protocol established in this present study is considered useful for seizure potential screening during early stages of drug discovery.

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development

PubMed Central

Covassin, L. D.; Siekmann, A. F.; Kacergis, M. C.; Laver, E.; Moore, J. C.; Villefranc, J. A.; Weinstein, B. M.; Lawson, N. D.

2009-01-01

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development. PMID:19269286

Zebrafish as a Model for Systems Medicine R&D: Rethinking the Metabolic Effects of Carrier Solvents and Culture Buffers Determined by (1)H NMR Metabolomics.

PubMed

Akhtar, Muhammad T; Mushtaq, Mian Y; Verpoorte, Robert; Richardson, Michael K; Choi, Young H

2016-01-01

Zebrafish is a frequently employed model organism in systems medicine and biomarker discovery. A crosscutting fundamental question, and one that has been overlooked in the field, is the "system-wide" (omics) effects induced in zebrafish by metabolic solvents and culture buffers. Indeed, any bioactivity or toxicity test requires that the target compounds are dissolved in an appropriate nonpolar solvent or aqueous media. It is important to know whether the solvent or the buffer itself has an effect on the zebrafish model organism. We evaluated the effects of two organic carrier solvents used in research with zebrafish, as well as in drug screening: dimethyl sulfoxide (DMSO) and ethanol, and two commonly used aqueous buffers (egg water and Hank's balanced salt solution). The effects of three concentrations (0.01, 0.1, and 1%) of DMSO and ethanol were tested in the 5-day-old zebrafish embryo using proton nuclear magnetic resonance ((1)H NMR) based metabolomics. DMSO (1% and 0.1%, but not 0.01%) exposure significantly decreased the levels of adenosine triphosphate (ATP), betaine, alanine, histidine, lactate, acetate, and creatine (p < 0.05). By contrast, ethanol exposure did not alter the embryos' metabolome at any concentration tested. The two different aqueous media noted above impacted the zebrafish embryo metabolome as evidenced by changes in valine, alanine, lactate, acetate, betaine, glycine, glutamate, adenosine triphosphate, and histidine. These results show that DMSO has greater effects on the embryo metabolome than ethanol, and thus is used with caution as a carrier solvent in zebrafish biomarker research and oral medicine. Moreover, the DMSO concentration should not be higher than 0.01%. Careful attention is also warranted for the use of the buffers egg water and Hank's balanced salt solution in zebrafish. In conclusion, as zebrafish is widely used as a model organism in life sciences, metabolome changes induced by solvents and culture buffers warrant further attention for robust systems science, and precision biomarkers that will stand the test of time.

High-throughput assessment of oxidative respiration in fish embryos: Advancing adverse outcome pathways for mitochondrial dysfunction.

PubMed

Souders, Christopher L; Liang, Xuefang; Wang, Xiaohong; Ector, Naomi; Zhao, Yuan H; Martyniuk, Christopher J

2018-06-01

Mitochondrial dysfunction is a prevalent molecular event that can result in multiple adverse outcomes. Recently, a novel high throughput method to assess metabolic capacity in fish embryos following exposure to chemicals has been adapted for environmental toxicology. Assessments of oxygen consumption rates using the Seahorse XF(e) 24/96 Extracellular Flux Analyzer (Agilent Technologies) can be used to garner insight into toxicant effects at early stages of development. Here we synthesize the current state of the science using high throughput metabolic profiling in zebrafish embryos, and present considerations for those wishing to adopt high throughput methods for mitochondrial bioenergetics into their research. Chemicals that have been investigated in zebrafish using this metabolic platform include herbicides (e.g. paraquat, diquat), industrial compounds (e.g. benzo-[a]-pyrene, tributyltin), natural products (e.g. quercetin), and anti-bacterial chemicals (i.e. triclosan). Some of these chemicals inhibit mitochondrial endpoints in the μM-mM range, and reduce basal respiration, maximum respiration, and spare capacity. We present a theoretical framework for how one can use mitochondrial performance data in zebrafish to categorize chemicals of concern and prioritize mitochondrial toxicants. Noteworthy is that our studies demonstrate that there can be considerable variation in basal respiration of untreated zebrafish embryos due to clutch-specific effects as well as individual variability, and basal oxygen consumption rates (OCR) can vary on average between 100 and 300 pmol/min/embryo. We also compare OCR between chorionated and dechorionated embryos, as both models are employed to test chemicals. After 24 h, dechorionated embryos remain responsive to mitochondrial toxicants, although they show a blunted response to the uncoupling agent carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP); dechorionated embryos are therefore a viable option for investigations into mitochondrial bioenergetics. We present an adverse outcome pathway framework that incorporates endpoints related to mitochondrial bioenergetics. High throughput bioenergetics assays conducted using whole embryos are expected to support adverse outcome pathways for mitochondrial dysfunction. Copyright © 2018 Elsevier B.V. All rights reserved.

Patient-derived xenograft in zebrafish embryos: a new platform for translational research in neuroendocrine tumors.

PubMed

Gaudenzi, Germano; Albertelli, Manuela; Dicitore, Alessandra; Würth, Roberto; Gatto, Federico; Barbieri, Federica; Cotelli, Franco; Florio, Tullio; Ferone, Diego; Persani, Luca; Vitale, Giovanni

2017-08-01

Preclinical research on neuroendocrine tumors usually involves immortalized cell lines and few animal models. In the present study we described an in vivo model based on patient-derived xenografts of neuroendocrine tumor cells in zebrafish (Danio rerio) embryos, allowing a rapid analysis of the angiogenic and invasive potential. Patient-derived neuroendocrine tumor cells were transplanted in 48 hours post-fertilization Tg(fli1a:EGFP) y1 zebrafish embryos that express enhanced green fluorescent protein in the entire vasculature. Neuroendocrine tumor cells, stained with CM-Dil, were injected into the subperidermal (perivitelline) space, close to the developing subintestinal venous plexus. A proper control group, represented by zebrafish injected with only D-PBS, was included in this study. Angiogenic and invasive potentials of each patient-derived xenograft were evaluated by both epifluorescence and confocal microscopes. Six out of eight neuroendocrine tumor samples were successfully transplanted in zebrafish embryos. Although the implanted tumor mass had a limited size (about 100 cells for embryos), patient-derived xenografts showed pro-angiogenic (5 cases) and invasive (6 cases) behaviors within 48 hours post injection. Patient-derived xenograft in zebrafish embryos appears to be a reliable in vivo preclinical model for neuroendocrine tumors, tumors with often limited cell availability. The rapidity of this procedure makes our model a promising platform to perform preclinical drug screening and opens a new scenario for personalized treatment in patients with neuroendocrine tumors.

An automatic method to calculate heart rate from zebrafish larval cardiac videos.

PubMed

Kang, Chia-Pin; Tu, Hung-Chi; Fu, Tzu-Fun; Wu, Jhe-Ming; Chu, Po-Hsun; Chang, Darby Tien-Hao

2018-05-09

Zebrafish is a widely used model organism for studying heart development and cardiac-related pathogenesis. With the ability of surviving without a functional circulation at larval stages, strong genetic similarity between zebrafish and mammals, prolific reproduction and optically transparent embryos, zebrafish is powerful in modeling mammalian cardiac physiology and pathology as well as in large-scale high throughput screening. However, an economical and convenient tool for rapid evaluation of fish cardiac function is still in need. There have been several image analysis methods to assess cardiac functions in zebrafish embryos/larvae, but they are still improvable to reduce manual intervention in the entire process. This work developed a fully automatic method to calculate heart rate, an important parameter to analyze cardiac function, from videos. It contains several filters to identify the heart region, to reduce video noise and to calculate heart rates. The proposed method was evaluated with 32 zebrafish larval cardiac videos that were recording at three-day post-fertilization. The heart rate measured by the proposed method was comparable to that determined by manual counting. The experimental results show that the proposed method does not lose accuracy while largely reducing the labor cost and uncertainty of manual counting. With the proposed method, researchers do not have to manually select a region of interest before analyzing videos. Moreover, filters designed to reduce video noise can alleviate background fluctuations during the video recording stage (e.g. shifting), which makes recorders generate usable videos easily and therefore reduce manual efforts while recording.

Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio).

PubMed

Collymore, Chereen; Banks, E Kate; Turner, Patricia V

2016-11-01

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish.

TBBPA induces developmental toxicity, oxidative stress, and apoptosis in embryos and zebrafish larvae (Danio rerio).

PubMed

Wu, Shengmin; Ji, Guixiang; Liu, Jining; Zhang, Shenghu; Gong, Yang; Shi, Lili

2016-10-01

Tetrabromobisphenol A (TBBPA) is currently one of the most frequently used brominated flame retardants and can be considered as a high production volume chemical. In this study, zebrafish embryos and larvae served as a biological model to evaluate TBBPA-induced developmental toxicity, oxidative stress, oxidant-associated gene expression, and cell apoptosis. Abnormalities, including hyperemia and pericardial edema, were induced in zebrafish larvae. The results showed that toxicity endpoints such as hatching rate, survival rate, malformation rate, and growth rate had a significant dose-response relationship with TBBPA. Further studies revealed that TBBPA did not alter the enzyme activities of Copper/Zinc Superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathioneperoxidase (GPx) at 0.10 mg/L, but decreased activities following exposure to 0.40, 0.70, and 1.00 mg/L. Despite the significantly decreased gene expression of Cu/Zn-SOD, CAT, and GPx1a in the 1.00 mg/L treatment group, other treatments (0.10, 0.40, 0.70 mg/L) did not alter gene expression. Moreover, Acridine orange staining results showed that apoptotic cells mainly accumulated in the brain, heart, and tail, indicating possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. Histological analysis also showed evidence of obvious heart impairment in TBBPA-treated groups. This study provides new evidence on the developmental toxicity, oxidative stress, and apoptosis of embryos and zebrafish larvae, which is important for the evaluation of environmental toxicity and chemical risk. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1241-1249, 2016. © 2015 Wiley Periodicals, Inc.

Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio)

PubMed Central

Collymore, Chereen; Banks, E Kate; Turner, Patricia V

2016-01-01

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish. PMID:27931323

Behavioral, morphometric, and gene expression effects in adult zebrafish (Danio rerio) embryonically exposed to PFOA, PFOS, and PFNA.

PubMed

Jantzen, Carrie E; Annunziato, Kate M; Cooper, Keith R

2016-11-01

Perfluoroalkylated substances (PFAS) are a class of persistent anthropogenic chemicals that have been detected worldwide. PFASs consist of fluorinated carbon chains of varying length, terminal groups, and have a number of industrial uses. A previous zebrafish study from our laboratory showed that acute (3-120h post fertilization, 0.02-2.0μM), waterborne embryonic exposure to these chemicals resulted in chemical specific alterations at 5days post fertilization (dpf), and some effects persisted up to 14 dpf. Using a gene battery consisting of 100 transcripts identified several genes that were up or down regulated. This current study looks at the long-term impacts of PFASs in adult zebrafish using the same exposure regimen. It was hypothesized that sub-lethal exposure of perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), or perfluorooctane sulfonate (PFOA) in embryonic zebrafish (3-120 hpf) would result in permanent morphometric, gene expression, and behavioral changes in adult fish similar to those observed at 5 and 14 dpf. Zebrafish were exposed to PFOS, PFOA, and PFNA (Control 0μM, 2.0μM) for the first five days post fertilization. At six months post fertilization, no PFAS treatment resulted in a significant change in total body length or weight. In terms of behavior, PFNA males showed a reduction in total distance traveled and time of immobility, and an increase in thigmotaxis behavior, aggressive attacks, and preference for the bright section of the tank. PFOS treated males had a reduced aggression behavior, and PFOA females preferred the dark section of the tank. Gene expression of slco2b1, slco1d1, and tgfb1a were analyzed because these transcripts were previously found to be affected by PFAS exposure in 5dpf and 14 dpf zebrafish and resulted in: significant decrease in expression of slco2b1 for both sexes in PFNA and PFOS treated groups, significant decrease of slco1d1 in all treatment groups for females and PFOS and PFOA exposed males, significant increase of tgfb1a in males treated with PFOS and PFNA, and a significant increase of bdnf in all PFAS male groups. This study demonstrates that acute, embryonic exposure (5days) to individual PFASs result in significant biochemical and behavioral changes in young adult zebrafish 6 months after exposure. These three PFASs have long term and persistent impacts following short term embryonic exposure that persists into adulthood. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic Analysis of Digestive Physiology Using Fluorescent Phospholipid Reporters

NASA Astrophysics Data System (ADS)

Farber, Steven A.; Pack, Michael; Ho, Shiu-Ying; Johnson, Iain D.; Wagner, Daniel S.; Dosch, Roland; Mullins, Mary C.; Hendrickson, H. Stewart; Hendrickson, Elizabeth K.; Halpern, Marnie E.

2001-05-01

Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae

PubMed Central

Pelkowski, Sean D.; Kapoor, Mrinal; Richendrfer, Holly A.; Wang, Xingyue; Colwill, Ruth M.; Creton, Robbert

2011-01-01

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image twelve multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red ‘bouncing ball’ stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders. PMID:21549762

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.

PubMed

Pelkowski, Sean D; Kapoor, Mrinal; Richendrfer, Holly A; Wang, Xingyue; Colwill, Ruth M; Creton, Robbert

2011-09-30

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image 12 multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red 'bouncing ball' stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

PubMed Central

Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

2012-01-01

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

Developmental exposure to triclosan alters microbiota community structure and locomotor activity in larval zebrafish

EPA Science Inventory

Growing evidence indicates that host-associated microbiota modify the toxicokinetics and/or toxicodynamics of environmental chemicals; however, current risk assessment methods do not consider interactions between microbiota and chemical toxicity. We previously reported that micro...

Host-associated microbiota modifies the toxicokinetics of environmental chemicals

EPA Science Inventory

Host-associated microbiota are known to biotransform drugs and some environmental chemicals like arsenic and polycyclic aromatic hydrocarbons. However, the metabolic capacity of microbiota treated with anti-microbial agents has not been assessed. Here, we exposed zebrafish with a...

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lemaire, Benjamin; Kubota, Akira; O'Meara, Conor M.

Cytochrome P450 (CYP) enzymes for which there is no functional information are considered “orphan” CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences inmore » the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to “deorphanization”, that is, identifying CYP20A1 functions and its roles in health and disease. - Highlights: • The “orphan” CYP20A1 was cloned from zebrafish and its sequence analyzed. • Knockdown of CYP20A1 reduced an optomotor response and elicited bursts of activity. • Effects of knockdown resemble some features of a microdeletion of CYP20A1 in human. • Expression of CYP20A1 was downregulated by the neurotoxicant methylmercury. • CYP20A1 may be involved in neurobehavioral processes and effects of some chemicals.« less

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Danio rerio: Small Fish Making a Big Splash in Leukemia.

PubMed

Squiban, Barbara; Frazer, J Kimble

2014-06-01

Zebrafish ( Danio rerio ) are widely used for developmental biology studies. In the past decade, D. rerio have become an important oncology model as well. Leukemia is one type of cancer where zebrafish are particularly valuable. As vertebrates, fish have great anatomic and biologic similarity to humans, including their hematopoietic and immune systems. As an experimental platform, D. rerio offer many advantages that mammalian models lack. These include their ease of genetic manipulation, capacity for imaging, and suitability for large-scale phenotypic and drug screens. In this review, we present examples of these strategies and others to illustrate how zebrafish have been and can be used to study leukemia. Besides appraising the techniques researchers apply and introducing the leukemia models they have created, we also highlight recent and exciting discoveries made using D. rerio with an eye to where the field is likely headed.

Early zebrafish development: It’s in the maternal genes

PubMed Central

Abrams, Elliott W.; Mullins, Mary C.

2009-01-01

Summary The earliest stages of embryonic development in all animals examined rely on maternal gene products that are generated during oogenesis and supplied to the egg. The period of maternal control of embryonic development varies among animals according to the onset of zygotic transcription and the persistence of maternal gene products. This maternal regulation has been little studied in vertebrates, due to the difficulty in manipulating maternal gene function and lack of basic molecular information. However, recent maternal-effect screens in the zebrafish have generated more than 40 unique mutants that are providing new molecular entry points to the maternal control of early vertebrate development. Here we discuss recent studies of 12 zebrafish mutant genes that illuminate the maternal molecular controls on embryonic development, including advances in the regulation of animal-vegetal polarity, egg activation, cleavage development, body plan formation, tissue morphogenesis, microRNA function and germ cell development. PMID:19608405

Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies

PubMed Central

Dang, Michelle; Henderson, Rachel E.; Garraway, Levi A.

2016-01-01

ABSTRACT Zebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAFV600E inhibitor, Vemurafenib, in adult zebrafish harboring BRAFV600E melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases. PMID:27482819

A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information.

PubMed

Knowlton, Michelle N; Li, Tongbin; Ren, Yongliang; Bill, Brent R; Ellis, Lynda Bm; Ekker, Stephen C

2008-01-07

The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies. The MOrpholino DataBase is a MySQL relational database with an online, PHP interface. Multiple quality control levels allow differential access to data in raw and finished formats. MODBv1 includes sequence information relating to almost 800 morpholinos and their targets and phenotypic data regarding the dose effect of each morpholino (mortality, toxicity and defects). To improve the searchability of this database, we have incorporated a fixed-vocabulary defect ontology that allows for the organization of morpholino affects based on anatomical structure affected and defect produced. This also allows comparison between species utilizing Phenotypic Attribute Trait Ontology (PATO) designated terminology. MODB is also cross-linked with ZFIN, allowing full searches between the two databases. MODB offers users the ability to retrieve morpholino data by sequence of morpholino or target, name of target, anatomical structure affected and defect produced. MODB data can be used for functional genomic analysis of morpholino design to maximize efficacy and minimize toxicity. MODB also serves as a template for future sequence-based functional genetic screen databases, and it is currently being used as a model for the creation of a mutagenic insertional transposon database.

Overview of ToxCast™ | Science Inventory | US EPA

EPA Pesticide Factsheets

In 2007, EPA launched ToxCast™ in order to develop a cost-effective approach for prioritizing the toxicity testing of large numbers of chemicals in a short period of time. Using data from state-of-the-art high throughput screening (HTS) bioassays developed in the pharmaceutical industry, ToxCast™ is building computational models to forecast the potential human toxicity of chemicals. These hazard predictions will provide EPA regulatory programs with science-based information helpful in prioritizing chemicals for more detailed toxicological evaluations, and lead to more efficient use of animal testing. In its first phase, ToxCast™ is profiling over 300 well-characterized chemicals (primarily pesticides) in over 400 HTS endpoints. These endpoints include biochemical assays of protein function, cell-based transcriptional reporter assays, multi-cell interaction assays, transcriptomics on primary cell cultures, and developmental assays in zebrafish embryos. Almost all of the compounds being examined in Phase 1 of ToxCast™ have been tested in traditional toxicology tests, including developmental toxicity, multi-generation studies, and sub-chronic and chronic rodent bioassays. ToxRefDB, a relational database being created to house this information, will contain nearly $1B worth of toxicity studies in animals when completed. ToxRefDB is integrated into a more comprehensive data management system developed by NCCT called ACToR (Aggregated Computational Toxicology

Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species -- an update.

PubMed

Braunbeck, Thomas; Boettcher, Melanie; Hollert, Henner; Kosmehl, Thomas; Lammer, Eva; Leist, Erik; Rudolf, Mark; Seitz, Nadja

2005-01-01

After its standardisation at the national level in Germany (DIN 38415-6, 2001, 2001), the 48 h sewage testing assay with zebrafish (Danio rerio) embryos has been submitted for standardisation to ISO. As an alternative to the conventional acute (96 h) fish test, a modified fish embryo test will be submitted to the OECD for chemical testing in late 2005. For this, a protocol originally designed for zebrafish was adapted to fit also the requirements of other OECD species, namely medaka (Oryzias latipes) and fathead minnow (Pimephales promelas). Results document that the transfer of the protocol is possible with only minor modifications. Data obtained from embryo tests with the three species are comparable. Statistical analysis of existing zebrafish embryo toxicity data resulted in the conclusions (1) that there is a reliable correlation between the fish embryo test and the acute fish test, (2) that the confidence belt of the regression line was relatively small, but that the prediction range was relatively wide. The regression thus seems appropriate to describe the relationship between acute fish and embryo LC(50) with good confidence, but is less appropriate as a prediction model. Investigations into oxygen requirements of zebrafish embryos reveal that they adapt to a broad range of oxygen levels and survive at concentrations of 2 mg/l without malformations. Zebrafish embryos can thus be exposed in very small toxicant volumes (100 microl), which is of particular interest for the testing of metabolites. Dechorionation studies with 48 h old zebrafish embryos indicate that the barrier function of the chorion increases with the lipophilicity of the test compound. Finally, examples are given as to how additional endpoints can be incorporated into the fish embryo test protocol to extend its scope, e.g. to sediment toxicity assessment or genotoxicity and mutagenicity testing.

HCV IRES-Mediated Core Expression in Zebrafish

PubMed Central

Zhang, Jing-Pu; Hu, Zhan-Ying; Tong, Jun-Wei; Ding, Cun-Bao; Peng, Zong-Gen; Zhao, Li-Xun; Song, Dan-Qing; Jiang, Jian-Dong

2013-01-01

The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism. PMID:23469178

The Transcriptome of the Zebrafish Embryo After Chemical Exposure: A Meta-Analysis.

PubMed

Schüttler, Andreas; Reiche, Kristin; Altenburger, Rolf; Busch, Wibke

2017-06-01

Numerous studies have been published in the past years investigating the transcriptome of the zebrafish embryo (ZFE) upon being subjected to chemical stress. Aiming at a more mechanistic understanding of the results of such studies, knowledge about commonalities of transcript regulation in response to chemical stress is needed. Thus, our goal in this study was to identify and interpret genes and gene sets constituting a general response to chemical exposure. Therefore, we aggregated and reanalyzed published toxicogenomics data obtained with the ZFE. We found that overlap of differentially transcribed genes in response to chemical stress across independent studies is generally low and the most commonly differentially transcribed genes appear in less than 50% of all treatments across studies. However, effect size analysis revealed several genes showing a common trend of differential expression, among which genes related to calcium homeostasis emerged as key, especially in exposure settings up to 24 h post-fertilization. Additionally, we found that these and other downregulated genes are often linked to anatomical regions developing during the respective exposure period. Genes showing a trend of increased expression were, among others, linked to signaling pathways (e.g., Wnt, Fgf) as well as lysosomal structures and apoptosis. The findings of this study help to increase the understanding of chemical stress responses in the developing zebrafish embryo and provide a starting point to improve experimental designs for this model system. In future, improved time- and concentration-resolved experiments should offer better understanding of stress response patterns and access to mechanistic information. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro.

PubMed

Kroehne, Volker; Tsata, Vasiliki; Marrone, Lara; Froeb, Claudia; Reinhardt, Susanne; Gompf, Anne; Dahl, Andreas; Sterneckert, Jared; Reimer, Michell M

2017-01-01

Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neurons. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. In zebrafish, OPCs remyelinate axons of the optic tract after lysophosphatidylcholine (LPC)-induced demyelination back to full thickness myelin sheaths. In contrast to zebrafish, mammalian OPCs are highly vulnerable to excitotoxic stress, a cause of secondary injury, and remyelination remains insufficient. Generally, injury induced remyelination leads to shorter internodes and thinner myelin sheaths in mammals. In this study, we show that myelin sheaths are lost early after a complete spinal transection injury, but are re-established within 14 days after lesion. We introduce a novel, easy-to-use, inexpensive and highly reproducible OPC culture system based on dormant spinal OPCs from adult zebrafish that enables in vitro analysis. Zebrafish OPCs are robust, can easily be purified with high viability and taken into cell culture. This method enables to examine why zebrafish OPCs remyelinate better than their mammalian counterparts, identify cell intrinsic responses, which could lead to pro-proliferating or pro-differentiating strategies, and to test small molecule approaches. In this methodology paper, we show efficient isolation of OPCs from adult zebrafish spinal cord and describe culture conditions that enable analysis up to 10 days in vitro . Finally, we demonstrate that zebrafish OPCs differentiate into Myelin Basic Protein (MBP)-expressing OLs when co-cultured with human motor neurons differentiated from induced pluripotent stem cells (iPSCs). This shows that the basic mechanisms of oligodendrocyte differentiation are conserved across species and that understanding the regulation of zebrafish OPCs can contribute to the development of new treatments to human diseases.

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo.

PubMed

Kim, Dong-Jae; Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Na, Yi-Rang; Park, Sung-Hoon; Lee, Hyun-Kyoung; Dutta, Noton Kumar; Kawakami, Koichi; Park, Jae-Hak

2009-05-01

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up-regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 x 10(-6), 40 x 10(-6) and 80 x 10(-6) M of benomyl-treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase-based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 x 10(-6) M concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds.

Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

PubMed Central

Rajendran, R. Samuel; Shen, Chia-Ning; Chen, Te-Hao; Yen, Chueh-Chuan; Chuang, Chih-Kuang; Lin, Dar-Shong; Hsiao, Chung-Der

2012-01-01

Background Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. Methodology/Principal Findings Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1)cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. Conclusion/Significance Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity. PMID:22623957

Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

PubMed

Wen, Dingsheng; Liu, Aiming; Chen, Feng; Yang, Julin; Dai, Renke

2012-10-01

Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates. Copyright © 2012 John Wiley & Sons, Ltd.

Longitudinal visualization of vascular occlusion, reperfusion, and remodeling in a zebrafish model of retinal vascular leakage using OCT angiography

NASA Astrophysics Data System (ADS)

Spitz, Kathleen; Bozic, Ivan; Desai, Vineet; Rao, Gopikrishna M.; Pollock, Lana M.; Anand-Apte, Bela; Tao, Yuankai K.

2017-02-01

Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are two of the leading causes of blindness and visual impairment in the world. Neovascularization results in severe vision loss in DR and AMD and, thus, there is an unmet need to identify mechanisms of pathogenesis and novel anti-angiogenic therapies. Zebrafish is a leading model organism for studying human disease pathogenesis, and the highly conserved drug activity between zebrafish and humans and their ability to readily absorb small molecules dissolved in water has benefited pharmaceutical discovery. Here, we use optical coherence tomography (OCT) and OCT angiography (OCT-A) to perform noninvasive, in vivo retinal imaging in a zebrafish model of vascular leakage. Zebrafish were treated with diethylaminobenzaldehyde (DEAB) to induce vascular leakage and imaged with OCT and OCT-A at six time points over two weeks: baseline one day before treatment and one, three, six, eight, and ten days post treatment. Longitudinal functional imaging showed significant vascular response immediately after DEAB treatment. Observed vascular changes included partial or complete vascular occlusion immediately after treatment and reperfusion during a two-week period. Increased vascular tortuosity several days post treatment indicated remodeling, and bifurcations and collateral vessel formation were also observed. In addition, significant treatment response variabilities were observed in the contralateral eye of the same animal. Anatomical and functional normalization was observed in most animals by ten days post treatment. These preliminary results motivate potential applications of OCT-A as a tool for studying pathogenesis and therapeutic screening in zebrafish models of retinal vascular disease.

A rapid throughput approach identifies cognitive deficits in adult zebrafish from developmental exposure to polybrominated flame retardants.

PubMed

Truong, Lisa; Mandrell, David; Mandrell, Rick; Simonich, Michael; Tanguay, Robert L

2014-07-01

A substantial body of evidence has correlated the human body burdens of some polybrominated diphenyl ether (PBDE) flame retardants with cognitive and other behavioral deficits. Adult zebrafish exhibit testable learning and memory, making them an increasingly attractive model for neurotoxicology. Our goal was to develop a rapid throughput means of identifying the cognitive impact of developmental exposure to flame retardants in the zebrafish model. We exposed embryos from 6h post fertilization to 5 days post fertilization to either PBDE 47 (0.1μM), PBDE 99 (0.1μM) or PBDE 153 (0.1μM), vehicle (0.1% DMSO), or embryo medium (EM). The larvae were grown to adulthood and evaluated for the rate at which they learned an active-avoidance response in an automated shuttle box array. Zebrafish developmentally exposed to PBDE 47 learned the active avoidance paradigm significantly faster than the 0.1% DMSO control fish (P<0.0001), but exhibited significantly poorer performance when retested suggestive of impaired memory retention or altered neuromotor activity. Learning in the PBDE 153 group was not significantly different from the DMSO group. Developmental exposure to 0.1% DMSO impaired adult active avoidance learning relative to the sham group (n=39; P<0.0001). PBDE 99 prevented the DMSO effect, yielding a learning rate not significantly different from the sham group (n=36; P>0.9). Our results underscore the importance of vehicle choice in accurately assessing chemical effects on behavior. Active avoidance response in zebrafish is an effective model of learning that, combined with automated shuttle box testing, will provide a highly efficient platform for evaluating persistent neurotoxic hazard from many chemicals. Copyright © 2014 Elsevier Inc. All rights reserved.

Immediate and long-term consequences of vascular toxicity during zebrafish development

EPA Science Inventory

Proper formation of the vascular system is necessary for embryogenesis, and chemical disruption of vascular development may be a key event driving developmental toxicity. In order to test the effect of environmental chemicals on this critical process, we developed a quantitative ...

Epigenetic Mechanism Mediating the Regulation of the Estrogen Exposure Biomarker Vitellogenin in Zebrafish

EPA Science Inventory

An increasing number of pharmaceuticals and industrial chemicals are being classified as endocrine disrupting compounds (EDCs). These chemicals can interfere with hormonal homeostasis and lead to developmental disorders, cancer and other pathologies. One such EDC is 17α-et...

Differential effects of exposure to bisphenol analogues and estradiol on zebrafish host-associated microbiota and behavior

EPA Science Inventory

Host-associated microbiota can biotransform xenobiotics, mediate health effects of chemical exposure, and play important roles in early development. Bisphenol A (BPA) is a widespread environmental chemical that has been associated with adverse endocrine and neurodevelopmental eff...

Differential effects of exposure to bisphenol analogues and estradiol on zebrafish host-associated microbiota and neurobehavior

EPA Science Inventory

Host-associated microbiota can biotransform xenobiotics, mediate health effects of chemical exposure, and play important roles in early development. Bisphenol A (BPA) is a widespread environmental chemical that has been associated with adverse endocrine and neurodevelopmental eff...

Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban

PubMed Central

Torres, Tiago; Cunha, Isabel; Martins, Rosário; Santos, Miguel M.

2016-01-01

Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent need to gather ecotoxicological data on PCPs as a proxy to improve risk assessment. Here, the toxicity of three different PCPs (4-Methylbenzylidene Camphor (4-MBC), propylparaben and triclocarban) was tested using embryo bioassays with Danio rerio (zebrafish) and Paracentrotus lividus (sea urchin). The No Observed Effect Concentration (NOEC) for triclocarban was 0.256 µg/L for sea urchin and 100 µg/L for zebrafish, whereas NOEC for 4-MBC was 0.32 µg/L for sea urchin and 50 µg/L for zebrafish. Both PCPs impacted embryo development at environmentally relevant concentrations. In comparison with triclocarban and 4-MBC, propylparaben was less toxic for both sea urchin (NOEC = 160 µg/L) and zebrafish (NOEC = 1000 µg/L). Overall, this study further demonstrates the sensitivity of embryo bioassays as a high-throughput approach for testing the toxicity of emerging pollutants. PMID:27775672

Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban.

PubMed

Torres, Tiago; Cunha, Isabel; Martins, Rosário; Santos, Miguel M

2016-10-21

Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent need to gather ecotoxicological data on PCPs as a proxy to improve risk assessment. Here, the toxicity of three different PCPs (4-Methylbenzylidene Camphor (4-MBC), propylparaben and triclocarban) was tested using embryo bioassays with Danio rerio (zebrafish) and Paracentrotus lividus (sea urchin). The No Observed Effect Concentration (NOEC) for triclocarban was 0.256 µg/L for sea urchin and 100 µg/L for zebrafish, whereas NOEC for 4-MBC was 0.32 µg/L for sea urchin and 50 µg/L for zebrafish. Both PCPs impacted embryo development at environmentally relevant concentrations. In comparison with triclocarban and 4-MBC, propylparaben was less toxic for both sea urchin (NOEC = 160 µg/L) and zebrafish (NOEC = 1000 µg/L). Overall, this study further demonstrates the sensitivity of embryo bioassays as a high-throughput approach for testing the toxicity of emerging pollutants.

Sleep deprivation effects on object discrimination task in zebrafish (Danio rerio).

PubMed

Pinheiro-da-Silva, Jaquelinne; Silva, Priscila Fernandes; Nogueira, Marcelo Borges; Luchiari, Ana Carolina

2017-03-01

The zebrafish is an ideal vertebrate model for neurobehavioral studies with translational relevance to humans. Many aspects of sleep have been studied, but we still do not understand how and why sleep deprivation alters behavioral and physiological processes. A number of hypotheses suggest its role in memory consolidation. In this respect, the aim of this study was to analyze the effects of sleep deprivation on memory in zebrafish (Danio rerio), using an object discrimination paradigm. Four treatments were tested: control, partial sleep deprivation, total sleep deprivation by light pulses, and total sleep deprivation by extended light. The control group explored the new object more than the known object, indicating clear discrimination. The partially sleep-deprived group explored the new object more than the other object in the discrimination phase, suggesting a certain degree of discriminative performance. By contrast, both total sleep deprivation groups equally explored all objects, regardless of their novelty. It seems that only one night of sleep deprivation is enough to affect discriminative response in zebrafish, indicating its negative impact on cognitive processes. We suggest that this study could be a useful screening tool for cognitive dysfunction and a better understanding of the effect of sleep-wake cycles on cognition.

Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity.

PubMed

Kruger, Matthew; Boney, Robert; Ordoobadi, Alexander J; Sommers, Thomas F; Trapani, Josef G; Coffin, Allison B

2016-01-01

Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity

PubMed Central

Kruger, Matthew; Boney, Robert; Ordoobadi, Alexander J.; Sommers, Thomas F.; Trapani, Josef G.; Coffin, Allison B.

2016-01-01

Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20–30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment. PMID:27065807

Conditioned place avoidance of zebrafish (Danio rerio) to three chemicals used for euthanasia and anaesthesia.

PubMed

Wong, Devina; von Keyserlingk, Marina A G; Richards, Jeffrey G; Weary, Daniel M

2014-01-01

Zebrafish are becoming one of the most used vertebrates in developmental and biomedical research. Fish are commonly killed at the end of an experiment with an overdose of tricaine methanesulfonate (TMS, also known as MS-222), but to date little research has assessed if exposure to this or other agents qualifies as euthanasia (i.e. a "good death"). Alternative agents include metomidate hydrochloride and clove oil. We use a conditioned place avoidance paradigm to compare aversion to TMS, clove oil, and metomidate hydrochloride. Zebrafish (n = 51) were exposed to the different anaesthetics in the initially preferred side of a light/dark box. After exposure to TMS zebrafish spent less time in their previously preferred side; aversion was less pronounced following exposure to metomidate hydrochloride and clove oil. Nine of 17 fish exposed to TMS chose not to re-enter the previously preferred side, versus 2 of 18 and 3 of 16 refusals for metomidate hydrochloride and clove oil, respectively. We conclude that metomidate hydrochloride and clove oil are less aversive than TMS and that these agents be used as humane alternatives to TMS for killing zebrafish.

Using visual lateralization to model learning and memory in zebrafish larvae

PubMed Central

Andersson, Madelene Åberg; Ek, Fredrik; Olsson, Roger

2015-01-01

Impaired learning and memory are common symptoms of neurodegenerative and neuropsychiatric diseases. Present, there are several behavioural test employed to assess cognitive functions in animal models, including the frequently used novel object recognition (NOR) test. However, although atypical functional brain lateralization has been associated with neuropsychiatric conditions, spanning from schizophrenia to autism, few animal models are available to study this phenomenon in learning and memory deficits. Here we present a visual lateralization NOR model (VLNOR) in zebrafish larvae as an assay that combines brain lateralization and NOR. In zebrafish larvae, learning and memory are generally assessed by habituation, sensitization, or conditioning paradigms, which are all representatives of nondeclarative memory. The VLNOR is the first model for zebrafish larvae that studies a memory similar to the declarative memory described for mammals. We demonstrate that VLNOR can be used to study memory formation, storage, and recall of novel objects, both short and long term, in 10-day-old zebrafish. Furthermore we show that the VLNOR model can be used to study chemical modulation of memory formation and maintenance using dizocilpine (MK-801), a frequently used non-competitive antagonist of the NMDA receptor, used to test putative antipsychotics in animal models. PMID:25727677

Drug Discovery in Fish, Flies, and Worms

PubMed Central

Strange, Kevin

2016-01-01

Abstract Nonmammalian model organisms such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the zebrafish Danio rerio provide numerous experimental advantages for drug discovery including genetic and molecular tractability, amenability to high-throughput screening methods and reduced experimental costs and increased experimental throughput compared to traditional mammalian models. An interdisciplinary approach that strategically combines the study of nonmammalian and mammalian animal models with diverse experimental tools has and will continue to provide deep molecular and genetic understanding of human disease and will significantly enhance the discovery and application of new therapies to treat those diseases. This review will provide an overview of C. elegans, Drosophila, and zebrafish biology and husbandry and will discuss how these models are being used for phenotype-based drug screening and for identification of drug targets and mechanisms of action. The review will also describe how these and other nonmammalian model organisms are uniquely suited for the discovery of drug-based regenerative medicine therapies. PMID:28053067

Adenosine signaling promotes regeneration of pancreatic β-cells in vivo

PubMed Central

Andersson, Olov; Adams, Bruce A.; Yoo, Daniel; Ellis, Gregory C.; Gut, Philipp; Anderson, Ryan M.; German, Michael S.; Stainier, Didier Y. R.

2012-01-01

Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β-cells is still needed. Using a zebrafish model of diabetes, we screened ~7000 small molecules to identify enhancers of β-cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β-cell regeneration was the adenosine agonist 5′-N-Ethylcarboxamidoadenosine (NECA), which acting through the adenosine receptor A2aa increased β-cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β-cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β-cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes. PMID:22608007

Changes in CpG-methylation of Vitellogenin 1 in Adult Male Zebrafish After Exposure to 17α-ethynylestradiol (EE2)

EPA Science Inventory

An increasing number of pharmaceutical and industrial chemicals are being classified as endocrine disrupting compounds (EDCs). These chemicals can interfere with hormonal homeostasis and lead to developmental disorders, cancer and other pathologies. One such EDC is 17α-ethy...

MEETING IN PORTUGAL: LINKAGE OF EXPOSURE AND EFFECTS USING GENOMICS, PROTEOMICS AND METABOLOMICS IN SMALL FISH MODELS

EPA Science Inventory

With an interdisciplinary team of scientists from U.S. Government Agencies and Universities, we are utilizing zebrafish and fathead minnow to develop techniques for extrapolation of chemical stressor impacts across species, chemicals and endpoints. The linkage of responses acros...

Assessment of Toxicological Perturbations and Variants of Pancreatic Islet Development in the Zebrafish Model

PubMed Central

Sant, Karilyn E.; Jacobs, Haydee M.; Xu, Jiali; Borofski, Katrina A.; Moss, Larry G.; Moss, Jennifer B.; Timme-Laragy, Alicia R.

2016-01-01

The pancreatic islets, largely comprised of insulin-producing beta cells, play a critical role in endocrine signaling and glucose homeostasis. Because they have low levels of antioxidant defenses and a high perfusion rate, the endocrine islets may be a highly susceptible target tissue of chemical exposures. However, this endpoint, as well as the integrity of the surrounding exocrine pancreas, is often overlooked in studies of developmental toxicology. Disruption of development by toxicants can alter cell fate and migration, resulting in structural alterations that are difficult to detect in mammalian embryo systems, but that are easily observed in the zebrafish embryo model (Danio rerio). Using endogenously expressed fluorescent protein markers for developing zebrafish beta cells and exocrine pancreas tissue, we documented differences in islet area and incidence rates of islet morphological variants in zebrafish embryos between 48 and 96 h post fertilization (hpf), raised under control conditions commonly used in embryotoxicity assays. We identified critical windows for chemical exposures during which increased incidences of endocrine pancreas abnormalities were observed following exposure to cyclopamine (2–12 hpf), Mono-2-ethylhexyl phthalate (MEHP) (3–48 hpf), and Perfluorooctanesulfonic acid (PFOS) (3–48 hpf). Both islet area and length of the exocrine pancreas were sensitive to oxidative stress from exposure to the oxidant tert-butyl hydroperoxide during a highly proliferative critical window (72 hpf). Finally, pancreatic dysmorphogenesis following developmental exposures is discussed with respect to human disease. PMID:28393070

Method for quantifying NSAIDs and clofibric acid in aqueous samples, lumpfish (Cyclopterus lumpus) roe, and zebrafish (Danio rerio) eleutheroembryos and evaluation of their bioconcentration in zebrafish eleutheroembryos.

PubMed

Molina-Fernandez, N; Perez-Conde, C; Rainieri, S; Sanz-Landaluze, J

2017-04-01

Pharmaceuticals such as nonsteroidal anti-inflammatory drugs (NSAIDs) and lipid regulators are being repeatedly detected at low concentrations (pg · mL -1 -ng · mL -1 ) in the environment. A large fraction of these compounds are ionizable. Ionized compounds show different physico-chemical properties and environmental behavior in comparison to their neutral analogs; as a consequence, the quantification methods currently available, based on the neutral molecules, might not be suitable to detect the corresponding ionized compounds. To overcome this problem, we developed a specific analytical method to quantify NSAIDs and lipid regulators (i.e., ibuprofen, diclofenac, naproxen, and clofibric acid) and their ionized compounds. This method is based on three steps: (1) the extraction of the organic compounds with an organic solvent assisted with an ultrasonic probe, (2) the cleaning of the extracts with a dispersive SPE with C 18 , and (3) the determination of the chemical compounds by GC-MS (prior derivatization of the analytes). We demonstrated that the proposed method can successfully quantify the pharmaceuticals and their ionized compounds in aqueous samples, lumpfish eggs, and zebrafish eleutheroembryos. Additionally, it allows the extraction and the cleanup of extracts from small samples (0.010 g of wet weight in pools of 20 larvae) and complex matrixes (due to high lipid content) and can be used as a basis for bioaccumulation assays performed with zebrafish eleutheroembryos in alternative to OECD test 305.

Generation and Characterization of Neurogeninl-GFP Transgenic Medaka for High Throughput Developmental Neurotoxicity Screening

EPA Science Inventory

Fish models such as zebrafish and medaka are increasingly used as alternatives to rodents in developmental and toxicological studies. These developmental and toxicological studies can be facilitated by the use of transgenic reporters that permit the real-time, noninvasive observa...

A Simplified and Rapid Screening Assay using Zebrafish to Assess Cardiac Effects of Air Pollution-derived Particulate Matter

EPA Science Inventory

Comparative toxicity assessment of particulate matter (PM) from different sources will potentially inform the understanding of regional differences in PM-induced cardiac health effects by identifying PM sources linked to highest potency components. Conventional low-throughput in...

Pituitary adenylate cyclase-activating polypeptide (PACAP) in zebrafish models of nephrotic syndrome

PubMed Central

van den Heuvel, Lambertus P.; Khodaparast, Laleh; Khodaparast, Ladan; van Geet, Chris; Freson, Kathleen

2017-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish. PMID:28759637

Chronic Unpredictable Stress (CUS)-Induced Anxiety and Related Mood Disorders in a Zebrafish Model: Altered Brain Proteome Profile Implicates Mitochondrial Dysfunction

PubMed Central

Chakravarty, Sumana; Reddy, Bommana R.; Sudhakar, Sreesha R.; Saxena, Sandeep; Das, Tapatee; Meghah, Vuppalapaty; Brahmendra Swamy, Cherukuvada V.; Kumar, Arvind; Idris, Mohammed M.

2013-01-01

Anxiety and depression are major chronic mood disorders, and the etiopathology for each appears to be repeated exposure to diverse unpredictable stress factors. Most of the studies on anxiety and related mood disorders are performed in rodents, and a good model is chronic unpredictable stress (CUS). In this study, we have attempted to understand the molecular basis of the neuroglial and behavioral changes underlying CUS-induced mood disorders in the simplest vertebrate model, the zebrafish, Danio rerio. Zebrafish were subjected to a CUS paradigm in which two different stressors were used daily for 15 days, and thorough behavioral analyses were performed to assess anxiety and related mood disorder phenotypes using the novel tank test, shoal cohesion and scototaxis. Fifteen days of exposure to chronic stressors appears to induce an anxiety and related mood disorder phenotype. Decreased neurogenesis, another hallmark of anxiety and related disorders in rodents, was also observed in this zebrafish model. The common molecular markers of rodent anxiety and related disorders, corticotropin-releasing factor (CRF), calcineurin (ppp3r1a) and phospho cyclic AMP response element binding protein (pCREB), were also replicated in the fish model. Finally, using 2DE FTMS/ITMSMS proteomics analyses, 18 proteins were found to be deregulated in zebrafish anxiety and related disorders. The most affected process was mitochondrial function, 4 of the 18 differentially regulated proteins were mitochondrial proteins: PHB2, SLC25A5, VDAC3 and IDH2, as reported in rodent and clinical samples. Thus, the zebrafish CUS model and proteomics can facilitate not only uncovering new molecular targets of anxiety and related mood disorders but also the routine screening of compounds for drug development. PMID:23691016

Phenotypic and biomarker evaluation of zebrafish larvae as an alternative model to predict mammalian hepatotoxicity.

PubMed

Verstraelen, Sandra; Peers, Bernard; Maho, Walid; Hollanders, Karen; Remy, Sylvie; Berckmans, Pascale; Covaci, Adrian; Witters, Hilda

2016-09-01

Zebrafish phenotypic assays have shown promise to assess human hepatotoxicity, though scoring of liver morphology remains subjective and difficult to standardize. Liver toxicity in zebrafish larvae at 5 days was assessed using gene expression as the biomarker approach, complementary to phenotypic analysis and analytical data on compound uptake. This approach aimed to contribute to improved hepatotoxicity prediction, with the goal of identifying biomarker(s) as a step towards the development of transgenic models for prioritization. Morphological effects of hepatotoxic compounds (acetaminophen, amiodarone, coumarin, methapyrilene and myclobutanil) and saccharin as the negative control were assessed after exposure in zebrafish larvae. The hepatotoxic compounds induced the expected zebrafish liver degeneration or changes in size, whereas saccharin did not have any phenotypic adverse effect. Analytical methods based on liquid chromatography-mass spectrometry were optimized to measure stability of selected compounds in exposure medium and internal concentration in larvae. All compounds were stable, except amiodarone for which precipitation was observed. There was a wide variation between the levels of compound in the zebrafish larvae with a higher uptake of amiodarone, methapyrilene and myclobutanil. Detection of hepatocyte markers (CP, CYP3A65, GC and TF) was accomplished by in situ hybridization of larvae to coumarin and myclobutanil and confirmed by real-time reverse transcription-quantitative polymerase chain reaction. Experiments showed decreased expression of all markers. Next, other liver-specific biomarkers (i.e. FABP10a and NR1H4) and apoptosis (i.e. CASP-3 A and TP53) or cytochrome P450-related (CYP2K19) and oxidoreductase activity-related (ZGC163022) genes, were screened. Links between basic mechanisms of liver injury and results of biomarker responses are described. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

The role of Fanconi anemia/BRCA genes in zebrafish sex determination.

PubMed

Rodríguez-Marí, Adriana; Postlethwait, John H

2011-01-01

Fanconi anemia (FA) is a human disease of bone marrow failure, leukemia, squamous cell carcinoma, and developmental anomalies, including hypogonadism and infertility. Bone marrow transplants improve hematopoietic phenotypes but do not prevent other cancers. FA arises from mutation in any of the 15 FANC genes that cooperate to repair double stranded DNA breaks by homologous recombination. Zebrafish has a single ortholog of each human FANC gene and unexpectedly, mutations in at least two of them (fancl and fancd1(brca2)) lead to female-to-male sex reversal. Investigations show that, as in human, zebrafish fanc genes are required for genome stability and for suppressing apoptosis in tissue culture cells, in embryos treated with DNA damaging agents, and in meiotic germ cells. The sex reversal phenotype requires the action of Tp53 (p53), an activator of apoptosis. These results suggest that in normal sex determination, zebrafish oocytes passing through meiosis signal the gonadal soma to maintain expression of aromatase, an enzyme that converts androgen to estrogen, thereby feminizing the gonad and the individual. According to this model, normal male and female zebrafish differ in genetic factors that control the strength of the late meiotic oocyte-derived signal, probably by regulating the number of meiotic oocytes, which environmental factors can also alter. Transcripts from fancd1(brca2) localize at the animal pole of the zebrafish oocyte cytoplasm and are required for normal oocyte nuclear architecture, for normal embryonic development, and for preventing ovarian tumors. Embryonic DNA repair and sex reversal phenotypes provide assays for the screening of small molecule libraries for therapeutic substances for FA. Copyright © 2011 Elsevier Inc. All rights reserved.

Extraction Protocols for Individual Zebrafish's Ventricle Myosin and Skeletal Muscle Actin for In vitro Motility Assays

PubMed Central

Scheid, Lisa-Mareike; Weber, Cornelia; Bopp, Nasrin; Mosqueira, Matias; Fink, Rainer H. A.

2017-01-01

The in vitro motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish—or other origin—and ATP, sliding movement can be visualized using a fluorescence microscope and an intensified CCD camera. Taken together, we introduce a method for functional analysis in zebrafish cardiac and skeletal muscle research to study mutations at the molecular level of thick or thin filament proteins. Additionally, preliminary data indicate the usefulness of the presented method to perform the IVMA with myosin extracted from muscles of other animal models. PMID:28620318

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

PubMed

Spagnoli, Sean; Xue, Lan; Kent, Michael L

2015-09-15

The zebrafish's potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host's brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10 min along with taps at 18 and 60 min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 min, whereas exposed negative fish retained partial habituation at 18 min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the improvement of the zebrafish as a translational model for human behavior. Copyright © 2015 Elsevier B.V. All rights reserved.

Unique and potent effects of acute ibogaine on zebrafish: the developing utility of novel aquatic models for hallucinogenic drug research.

PubMed

Cachat, Jonathan; Kyzar, Evan J; Collins, Christopher; Gaikwad, Siddharth; Green, Jeremy; Roth, Andrew; El-Ounsi, Mohamed; Davis, Ari; Pham, Mimi; Landsman, Samuel; Stewart, Adam Michael; Kalueff, Allan V

2013-01-01

An indole alkaloid, ibogaine is the principal psychoactive component of the iboga plant, used by indigenous peoples in West Africa for centuries. Modulating multiple neurotransmitter systems, the drug is a potent hallucinogen in humans, although its psychotropic effects remain poorly understood. Expanding the range of model species is an important strategy for translational neuroscience research. Here we exposed adult zebrafish (Danio rerio) to 10 and 20mg/L of ibogaine, testing them in the novel tank, light-dark box, open field, mirror stimulation, social preference and shoaling tests. In the novel tank test, the zebrafish natural diving response (geotaxis) was reversed by ibogaine, inducing initial top swimming followed by bottom dwelling. Ibogaine also attenuated the innate preference for dark environments (scototaxis) in the light-dark box test. While it did not exert overt locomotor or thigmotaxic responses in the open field test, the drug altered spatiotemporal exploration of novel environment, inducing clear preference of some areas over others. Ibogaine also promoted 'mirror' exploration in the mirror stimulation test, disrupted group cohesion in the shoaling test, and evoked strong coloration responses due to melanophore aggregation, but did not alter brain c-fos expression or whole-body cortisol levels. Overall, our results support the complex pharmacological profile of ibogaine and its high sensitivity in zebrafish models, dose-dependently affecting multiple behavioral domains. While future investigations in zebrafish may help elucidate the mechanisms underlying these unique behavioral effects, our study strongly supports the developing utility of aquatic models in hallucinogenic drug research. High sensitivity of three-dimensional phenotyping approaches applied here to behavioral effects of ibogaine in zebrafish provides further evidence of how 3D reconstructions of zebrafish swimming paths may be useful for high-throughput pharmacological screening. Copyright © 2012 Elsevier B.V. All rights reserved.

Novel degenerative and developmental defects in a zebrafish model of mucolipidosis type IV

PubMed Central

Li, Huiqing; Pei, Wuhong; Vergarajauregui, Sivia; Zerfas, Patricia M.; Raben, Nina; Burgess, Shawn M.; Puertollano, Rosa

2017-01-01

Abstract Mucolipidosis type IV (MLIV) is a lysosomal storage disease characterized by neurologic and ophthalmologic abnormalities. There is currently no effective treatment. MLIV is caused by mutations in MCOLN1, a lysosomal cation channel from the transient receptor potential (TRP) family. In this study, we used genome editing to knockout the two mcoln1 genes present in Danio rerio (zebrafish). Our model successfully reproduced the retinal and neuromuscular defects observed in MLIV patients, indicating that this model is suitable for studying the disease pathogenesis. Importantly, our model revealed novel insights into the origins and progression of the MLIV pathology, including the contribution of autophagosome accumulation to muscle dystrophy and the role of mcoln1 in embryonic development, hair cell viability and cellular maintenance. The generation of a MLIV model in zebrafish is particularly relevant given the suitability of this organism for large-scale in vivo drug screening, thus providing unprecedented opportunities for therapeutic discovery. PMID:28449103

Analysis of the dynamic co-expression network of heart regeneration in the zebrafish

PubMed Central

Rodius, Sophie; Androsova, Ganna; Götz, Lou; Liechti, Robin; Crespo, Isaac; Merz, Susanne; Nazarov, Petr V.; de Klein, Niek; Jeanty, Céline; González-Rosa, Juan M.; Muller, Arnaud; Bernardin, Francois; Niclou, Simone P.; Vallar, Laurent; Mercader, Nadia; Ibberson, Mark; Xenarios, Ioannis; Azuaje, Francisco

2016-01-01

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration. PMID:27241320

The zebrafish as a model system to study cardiovascular development.

PubMed

Stainier, D Y; Fishman, M C

1994-01-01

The zebrafish, Brachydanio rerio, is rapidly becoming a system of choice for vertebrate developmental biologists. It presents unique embryological attributes and is amenable to saturation style mutagenesis, a powerful approach that, in invertebrates, has already led to the identification of a large number of key developmental genes. Since fertilization is external, the zebrafish embryo develops in the dish and is thus accessible for continued observation and manipulation at all stages of development. Furthermore, because the embryo is transparent, the developing heart and vessels can be resolved at the single-cell level. A large number of mutations that affect the development of cardiovascular form and function have recently been isolated from large-scale genetic screens for zygotic embryonic lethals. Our further understanding of the development of the cardiovascular system is important not only because of the high incidence, and familial inheritance, of congenital abnormalities, but also because it should lead to novel, differentiation-based strategies for the analysis and therapy of the diseased state. Copyright © 1994. Published by Elsevier Inc.

A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

PubMed

Tiefenbach, Jens; Moll, Pamela R; Nelson, Meryl R; Hu, Chun; Baev, Lilia; Kislinger, Thomas; Krause, Henry M

2010-03-22

Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

Analysis of the dynamic co-expression network of heart regeneration in the zebrafish

NASA Astrophysics Data System (ADS)

Rodius, Sophie; Androsova, Ganna; Götz, Lou; Liechti, Robin; Crespo, Isaac; Merz, Susanne; Nazarov, Petr V.; de Klein, Niek; Jeanty, Céline; González-Rosa, Juan M.; Muller, Arnaud; Bernardin, Francois; Niclou, Simone P.; Vallar, Laurent; Mercader, Nadia; Ibberson, Mark; Xenarios, Ioannis; Azuaje, Francisco

2016-05-01

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration.

Identification of specific metabolic pathways as druggable targets regulating the sensitivity to cyanide poisoning.

PubMed

Sips, Patrick Y; Shi, Xu; Musso, Gabriel; Nath, Anjali K; Zhao, Yanbin; Nielson, Jason; Morningstar, Jordan; Kelly, Amy E; Mikell, Brittney; Buys, Eva; Bebarta, Vikhyat; Rutter, Jared; Davisson, V Jo; Mahon, Sari; Brenner, Matthew; Boss, Gerry R; Peterson, Randall T; Gerszten, Robert E; MacRae, Calum A

2018-01-01

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning.

Effect of Gabapentin/Memantine on the Infantile Nystagmus Syndrome in the Zebrafish Model: Implications for the Therapy of Ocular Motor Diseases.

PubMed

Bögli, Stefan Yu; Afthinos, Maresa; Huang, Melody Ying-Yu

2017-06-01

Infantile nystagmus syndrome (INS) is a disorder characterized by typical horizontal eye oscillations. Due to the uncertain etiology of INS, developing specific treatments remains difficult. Single reports demonstrated, on limited measures, alleviating effects of gabapentin and memantine. In the current study, we employed the zebrafish INS model belladonna (bel) to conduct an in-depth study of how gabapentin and memantine interventions alleviate INS signs, which may further restore visual conditions in affected subjects. Moreover, we described the influence of both medications on ocular motor functions in healthy zebrafish, evaluating possible iatrogenic effects. Ocular motor function and INS characteristics were assessed by eliciting optokinetic response, spontaneous nystagmus, and spontaneous saccades in light and in dark, in 5- to 6-day postfertilization bel larvae and heterozygous siblings. Single larvae were recorded before and after a 1-hour drug treatment (200 mM gabapentin/0.2 mM memantine). Both interventions significantly reduced nystagmus intensity (gabapentin: 59.98%, memantine: 39.59%). However, while the application of gabapentin affected all tested ocular motor functions, memantine specifically reduced nystagmus amplitude and intensity, and thus left controls completely unaffected. Finally, both drug treatments resulted in specific changes in nystagmus waveform and velocity. Our study provides deeper insight into gabapentin and memantine treatment effect in the zebrafish INS model. Moreover, this study should establish zebrafish as a pharmacologic animal model for treating nystagmus and ocular motor disease, serving as a basis for future large-scale drug screenings.

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

PubMed

Knöbel, Melanie; Busser, Frans J M; Rico-Rico, Angeles; Kramer, Nynke I; Hermens, Joop L M; Hafner, Christoph; Tanneberger, Katrin; Schirmer, Kristin; Scholz, Stefan

2012-09-04

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

Impacts of chemical modification on the toxicity of diverse nanocellulose materials to developing zebrafish

PubMed Central

Harper, Bryan J.; Clendaniel, Alicea; Sinche, Federico; Way, Daniel; Hughes, Michael; Schardt, Jenna; Simonsen, John; Stefaniak, Aleksandr B.

2016-01-01

Cellulose is an abundant and renewable resource currently being investigated for utility in nanomaterial form for various promising applications ranging from medical and pharmaceutical uses to mechanical reinforcement and biofuels. The utility of nanocellulose and wide implementation ensures increasing exposure to humans and the environment as nanocellulose-based technologies advance. Here, we investigate how differences in aspect ratio and changes to surface chemistry, as well as synthesis methods, influence the biocompatibility of nanocellulose materials using the embryonic zebrafish. Investigations into the toxicity of neutral, cationic and anionic surface functionalities revealed that surface chemistry had a minimal influence on the overall toxicity of nanocellulose materials. Higher aspect ratio cellulose nanofibers produced by mechanical homogenization were, in some cases, more toxic than other cellulose-based nanofibers or nanocrystals produced by chemical synthesis methods. Using fluorescently labeled nanocellulose we were able to show that nanocellulose uptake did occur in embryonic zebrafish during development. We conclude that the benign nature of nanocellulose materials makes them an ideal platform to systematically investigate the inherent surface features driving nanomaterial toxicity in order to create safer design principles for engineered nanoparticles. PMID:27468180

Embryonic senescence and laminopathies in a progeroid zebrafish model.

PubMed

Koshimizu, Eriko; Imamura, Shintaro; Qi, Jie; Toure, Jamal; Valdez, Delgado M; Carr, Christopher E; Hanai, Jun-ichi; Kishi, Shuji

2011-03-30

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape. We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.

Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Zebrafish (Danio rerio) early developmental stage.

PubMed

Zhu, Xiaoshan; Zhu, Lin; Duan, Zhenghua; Qi, Ruiqi; Li, Yan; Lang, Yupeng

2008-02-15

With the emergence of manufactured nanomaterials, it is urgent to carry out researches on their potential environmental impacts and biological effects. To better understand the potential ecotoxicological impacts of metal oxide nanoparticles released to aquatic environments, the zebrafish 96-h embryo-larval bioassay was used to assess and compare the developmental toxicities of nanoscale zinc oxide (nZnO), titanium dioxide (nTiO(2)) and alumina (nAl(2)O(3)) aqueous suspensions. Toxicological endpoints such as zebrafish embryos or larvae survival, hatching rate and malformation were noted and described within 96 h of exposure. Meanwhile, a comparative experiment with their bulk counterparts (i.e., ZnO/bulk, TiO(2)/bulk and Al(2)O(3)/bulk) was conducted to understand the effect of particle size on their toxicities. The results showed that: (i) both nZnO and ZnO/bulk aqueous suspensions delayed zebrafish embryo and larva development, decreased their survival and hatching rates, and caused tissue damage. The 96-h LC(50) of nZnO and ZnO/bulk aqueous suspensions on the zebrafish survival are 1.793 mg/L and 1.550 mg/L respectively; and the 84-h EC(50) on the zebrafish embryo hatching rate are 2.065 mg/L and 2.066 mg/L respectively. Serious tissue ulceration was found on zebrafish larvae exposed to nZnO and ZnO/bulk aqueous suspensions. (ii) In contrast, neither nTiO(2) and TiO(2)/bulk nor nAl(2)O(3) and Al(2)O(3)/bulk showed any toxicity to zebrafish embryos and larvae under the same experimental condition. It revealed that the metal oxide nanoparticles with different chemical composition have different zebrafish developmental toxicities. (iii) Exposures of nTiO(2), nZnO and nAl(2)O(3) produced toxic effects on zebrafish embryos and larvae, which was not different from the effects caused by exposing to their bulk counterparts. This is the first study about the developmental toxicity of metal oxide nanoparticles, and the results demonstrate that nZnO is very toxic to zebrafish embryos and larvae, which highlights the need to evaluate the potential eco-toxicity of these manufactured nanomaterials (MNMs).

Comparison of the Developmental and Acute Neurotoxicity of a Library of Organophosphorus Pesticides Using a Vertebrate Behavioral Assay

EPA Science Inventory

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize organophosphorus pesticides for neurotoxicity using behavioral tests in an in vivo, vertebrate, medium-throughput model (zebrafish; Danio rerio). Our behavioral testing paradigm assesses the e...

An Automated Heart Rate Detection Platform in Wild-Type Zebrafish for Cardiotoxicity Screening of Fine Particulate Matter Air Pollution

EPA Science Inventory

Exposure to air pollution-derived particulate matter (PM) causes adverse cardiovascular health outcomes, with increasing evidence implicating soluble components of PM; however, the enormous number of unique PM samples from different air sheds far exceeds the capacity of conventio...

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

NASA Astrophysics Data System (ADS)

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-02-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle.

Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase

PubMed Central

Mathias, Jonathan R.; Zhang, Zhanying; Saxena, Meera T.

2014-01-01

Abstract Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology. PMID:24428354

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

PubMed

Mathias, Jonathan R; Zhang, Zhanying; Saxena, Meera T; Mumm, Jeff S

2014-04-01

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.

Zebrafish in Toxicology and Environmental Health

PubMed Central

Bambino, Kathryn; Chu, Jaime

2018-01-01

As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. PMID:28335863

Zebrafish in Toxicology and Environmental Health.

PubMed

Bambino, Kathryn; Chu, Jaime

2017-01-01

As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. © 2017 Elsevier Inc. All rights reserved.

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

PubMed Central

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-01-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle. PMID:26898711

Clemizole and modulators of serotonin signalling suppress seizures in Dravet syndrome.

PubMed

Griffin, Aliesha; Hamling, Kyla R; Knupp, Kelly; Hong, SoonGweon; Lee, Luke P; Baraban, Scott C

2017-03-01

Dravet syndrome is a catastrophic childhood epilepsy with early-onset seizures, delayed language and motor development, sleep disturbances, anxiety-like behaviour, severe cognitive deficit and an increased risk of fatality. It is primarily caused by de novo mutations of the SCN1A gene encoding a neuronal voltage-activated sodium channel. Zebrafish with a mutation in the SCN1A homologue recapitulate spontaneous seizure activity and mimic the convulsive behavioural movements observed in Dravet syndrome. Here, we show that phenotypic screening of drug libraries in zebrafish scn1 mutants rapidly and successfully identifies new therapeutics. We demonstrate that clemizole binds to serotonin receptors and its antiepileptic activity can be mimicked by drugs acting on serotonin signalling pathways e.g. trazodone and lorcaserin. Coincident with these zebrafish findings, we treated five medically intractable Dravet syndrome patients with a clinically-approved serotonin receptor agonist (lorcaserin, Belviq®) and observed some promising results in terms of reductions in seizure frequency and/or severity. Our findings demonstrate a rapid path from preclinical discovery in zebrafish, through target identification, to potential clinical treatments for Dravet syndrome. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis.

PubMed

Thambyrajah, Roshana; Ucanok, Deniz; Jalali, Maryam; Hough, Yasmin; Wilkinson, Robert Neil; McMahon, Kathryn; Moore, Chris; Gering, Martin

2016-09-01

A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and up-regulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of Population Bottlenecks and Colonization Factors during Assembly of Bacterial Communities within the Zebrafish Intestine

PubMed Central

Wiles, Travis J.; Martinez, Emily S.; Jemielita, Matthew; Burns, Adam R.; Parthasarathy, Raghuveer; Bohannan, Brendan J. M.

2015-01-01

ABSTRACT The zebrafish, Danio rerio, is a powerful model for studying bacterial colonization of the vertebrate intestine, but the genes required by commensal bacteria to colonize the zebrafish gut have not yet been interrogated on a genome-wide level. Here we apply a high-throughput transposon mutagenesis screen to Aeromonas veronii Hm21 and Vibrio sp. strain ZWU0020 during their colonization of the zebrafish intestine alone and in competition with each other, as well as in different colonization orders. We use these transposon-tagged libraries to track bacterial population sizes in different colonization regimes and to identify gene functions required during these processes. We show that intraspecific, but not interspecific, competition with a previously established bacterial population greatly reduces the ability of these two bacterial species to colonize. Further, using a simple binomial sampling model, we show that under conditions of interspecific competition, genes required for colonization cannot be identified because of the population bottleneck experienced by the second colonizer. When bacteria colonize the intestine alone or at the same time as the other species, we find shared suites of functional requirements for colonization by the two species, including a prominent role for chemotaxis and motility, regardless of the presence of another species. PMID:26507229

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

PubMed

Liverani, Chiara; La Manna, Federico; Groenewoud, Arwin; Mercatali, Laura; Van Der Pluijm, Gabri; Pieri, Federica; Cavaliere, Davide; De Vita, Alessandro; Spadazzi, Chiara; Miserocchi, Giacomo; Bongiovanni, Alberto; Recine, Federica; Riva, Nada; Amadori, Dino; Tasciotti, Ennio; Snaar-Jagalska, Ewa; Ibrahim, Toni

2017-02-15

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays. © 2016. Published by The Company of Biologists Ltd.

Muscle dysfunction in a zebrafish model of Duchenne muscular dystrophy.

PubMed

Widrick, Jeffrey J; Alexander, Matthew S; Sanchez, Benjamin; Gibbs, Devin E; Kawahara, Genri; Beggs, Alan H; Kunkel, Louis M

2016-11-01

Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that sapje mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic sapje larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days postfertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were three- to fivefold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the sapje mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of sapje larvae represent novel physiological endpoints for therapeutic drug screening. Copyright © 2016 the American Physiological Society.

Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish.

PubMed

Carroll, Kelli J; North, Trista E

2014-08-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Oceans of Opportunity: Exploring Vertebrate Hematopoiesis in Zebrafish

PubMed Central

Carroll, Kelli J.; North, Trista E.

2015-01-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell (HSPC) biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor and effector cell emergence, expansion and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell-types can be identified and characterized. Further, a myriad of transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of HSPC biology during development, in response to infection or injury, or in the setting of hematological malignancy, continues to deepen, zebrafish will remain essential for exploring the spatio-temporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. PMID:24816275

Perturbation of metabonome of embryo/larvae zebrafish after exposure to fipronil.

PubMed

Yan, Lu; Gong, Chenxue; Zhang, Xiaofeng; Zhang, Quan; Zhao, Meirong; Wang, Cui

2016-12-01

The escalating demand for fipronil by the increasing insects' resistance to synthetic pyrethroids placed a burden on aquatic vertebrates. Although awareness regarding the toxicity of fipronil to fish is arising, the integral alteration caused by fipronil remains unexplored. Here, we investigated on the development toxicity of fipronil and the metabolic physiology perturbation at 120h post fertilization through GC-MS metabolomics on zebrafish embryo. We observed that fipronil dose-dependently induced malformations including uninflated swim bladder and bent spine. Further, the "omic" technique hit 26 differential metabolites after exposure to fipronil and five significant signaling pathways. We speculated that changes in primary bile acid synthesis pathway and the content of saturated fatty acid in the chemical-related group indicated the liver toxicity. Pathway of Aminoacyl-tRNA biosynthesis changed by fipronil may relate to the macromolecular synthesis. Concurrently, methane metabolism pathway was also identified while the role in zebrafish needs further determination. Overall, this study revealed several new signaling pathways in fipronil-treated zebrafish embryo/larval. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo assessment of impact of titanium oxide nanoparticle on zebrafish embryo

NASA Astrophysics Data System (ADS)

Verma, Suresh K.; Mishra, Anurag K.; Suar, M.; Parashar, S. K. S.

2017-05-01

Technologies and innovations have attended a new height with recent development in nanotechnology in last few decades. With these developments there has a great raise in demand of metal oxides like TiO2, ZnO having versatile physical, chemical and biological application. However the great rise has raised concern over the effect of these nanoparticles in biological system. In this study, we have assessed the impact of titanium oxide nanoparticles synthesized by high energy ball milling (HEBM) by milling bulk TiO2 particles for 15h. The synthesized particles were characterized with XRD, UV-Visible spectroscopy and DLS for their physiochemical properties. Biological impact of these nanoparticles was then studied on zebrafish embryo as invivo model. Mortality and hatching rate were calculated for 48hpf and 96hpf treatment. To determine the mechanism of mortality effect, Reactive oxygen species (ROS) was determined with the help of flow cytometry. 15h nanoparticles were found to have a LC50 of ( ) for zebrafish embryo. However TiO2 nanoparticles were found to be a ROS scavenger for the treated Zebrafish cells.

Zebrafish (Danio rerio) androgen receptor: sequence homology and up-regulation by the fungicide vinclozolin.

PubMed

Smolinsky, Amanda N; Doughman, Jennifer M; Kratzke, Liên-Thành C; Lassiter, Christopher S

2010-03-01

Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available.

Zebrafish as a model for acetylcholinesterase-inhibiting organophosphorus agent exposure and oxime reactivation

PubMed Central

Koenig, Jeffrey A.; Dao, Thuy L.; Kan, Robert K.; Shih, Tsung-Ming

2016-01-01

The current research progression efforts for investigating novel treatments for exposure to organophosphorus (OP) compounds that inhibit acetylcholinesterase (AChE), including pesticides and chemical warfare nerve agents (CWNAs), rely solely on in vitro cell assays and in vivo rodent models. The zebrafish (Danio rerio) is a popular, well-established vertebrate model in biomedical research that offers high-throughput capabilities and genetic manipulation not readily available with rodents. A number of research studies have investigated the effects of subacute developmental exposure to OP pesticides in zebrafish, observing detrimental effects on gross morphology, neuronal development, and behavior. Few studies, however, have utilized this model to evaluate treatments, such as oxime reactivators, anticholinergics, or anticonvulsants, following acute exposure. Preliminary work has investigated the effects of CWNA exposure. The results clearly demonstrated relative toxicity and oxime efficacy similar to that reported for the rodent model. This review surveys the current literature utilizing zebrafish as a model for OP exposure and highlights its potential use as a high-throughput system for evaluating AChE reactivator antidotal treatments to acute pesticide and CWNA exposure. PMID:27123828

Zebrafish as a systems toxicology model for developmental neurotoxicity testing.

PubMed

Nishimura, Yuhei; Murakami, Soichiro; Ashikawa, Yoshifumi; Sasagawa, Shota; Umemoto, Noriko; Shimada, Yasuhito; Tanaka, Toshio

2015-02-01

The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorder, attention deficit hyperactive disorder, schizophrenia, Parkinson's disease, and Alzheimer's disease. Although rodents have been widely used for developmental neurotoxicity testing, experiments using large numbers of rodents are time-consuming, expensive, and raise ethical concerns. Using alternative non-mammalian animal models may relieve some of these pressures by allowing testing of large numbers of subjects while reducing expenses and minimizing the use of mammalian subjects. In this review, we discuss some of the advantages of using zebrafish in developmental neurotoxicity testing, focusing on central nervous system development, neurobehavior, toxicokinetics, and toxicodynamics in this species. We also describe some important examples of developmental neurotoxicity testing using zebrafish combined with gene expression profiling, neuroimaging, or neurobehavioral assessment. Zebrafish may be a systems toxicology model that has the potential to reveal the pathways of developmental neurotoxicity and to provide a sound basis for human risk assessments. © 2014 Japanese Teratology Society.

Functional behavior and reproduction in androgenic sex reversed zebrafish (Danio rerio).

PubMed

Larsen, Mia G; Baatrup, Erik

2010-08-01

Endocrine-disrupting chemicals released into natural watercourses may cause biased sex ratios by sex reversal in fish populations. The present study investigated the androgenic sex reversal of zebrafish (Danio rerio) exposed to the androgenic compound 17beta-trenbolone (TB) and whether sex-changed females would revert to the female phenotype after cessation of TB exposure. 17beta-Trenbolone is a metabolite of trenbolone acetate, an anabolic steroid used as a growth promoter in beef cattle. 17beta-Trenbolone in runoff from cattle feedlots may reach concentrations that affect fish sexual development. Zebrafish were exposed to a concentration of 20 ng/L TB in a flow-through system for five months from egg until sexual maturity. This resulted in an all-male population. It was further found that all these phenotypic males displayed normal male courtship behavior and were able to reproduce successfully, implying that the sex reversal was complete and functional. None of the phenotypic males developed into females after six months in clean water, demonstrating that androgenic sex reversal of zebrafish is irreversible. Copyright 2010 SETAC

Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boxtel, Antonius L. van, E-mail: thijs.van.boxtel@ivm.vu.n; Kamstra, Jorke H.; Fluitsma, Donna M.

2010-04-15

Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effectsmore » observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.« less

A Morpholino-based screen to identify novel genes involved in craniofacial morphogenesis

PubMed Central

Melvin, Vida Senkus; Feng, Weiguo; Hernandez-Lagunas, Laura; Artinger, Kristin Bruk; Williams, Trevor

2014-01-01

BACKGROUND The regulatory mechanisms underpinning facial development are conserved between diverse species. Therefore, results from model systems provide insight into the genetic causes of human craniofacial defects. Previously, we generated a comprehensive dataset examining gene expression during development and fusion of the mouse facial prominences. Here, we used this resource to identify genes that have dynamic expression patterns in the facial prominences, but for which only limited information exists concerning developmental function. RESULTS This set of ~80 genes was used for a high throughput functional analysis in the zebrafish system using Morpholino gene knockdown technology. This screen revealed three classes of cranial cartilage phenotypes depending upon whether knockdown of the gene affected the neurocranium, viscerocranium, or both. The targeted genes that produced consistent phenotypes encoded proteins linked to transcription (meis1, meis2a, tshz2, vgll4l), signaling (pkdcc, vlk, macc1, wu:fb16h09), and extracellular matrix function (smoc2). The majority of these phenotypes were not altered by reduction of p53 levels, demonstrating that both p53 dependent and independent mechanisms were involved in the craniofacial abnormalities. CONCLUSIONS This Morpholino-based screen highlights new genes involved in development of the zebrafish craniofacial skeleton with wider relevance to formation of the face in other species, particularly mouse and human. PMID:23559552

Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures.

PubMed

Luzio, Ana; Matos, Manuela; Santos, Dércia; Fontaínhas-Fernandes, António A; Monteiro, Sandra M; Coimbra, Ana M

2016-08-01

Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE2, 4ng/L) and fadrozole (Fad, 50μg/L) from 2h to 35days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and -6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the "juvenile ovary" development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved in zebrafish spermatogenesis. Both EDCs, EE2 and Fad, increased the apoptosis stimulus in zebrafish gonad. It was noticed that the few females that were resistant to Fad-induced sex reversal had increased anti-apoptotic factor levels, while males exposed to EE2 showed increased pro-apoptotic genes/proteins and were more advanced in gonad differentiation. Overall, our findings show that apoptosis pathways are involved in zebrafish gonad differentiation and that EDCs can disrupt this process. Copyright © 2016 Elsevier B.V. All rights reserved.

Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish.

PubMed

Godfrey, Amy; Hooser, Blair; Abdelmoneim, Ahmed; Horzmann, Katharine A; Freemanc, Jennifer L; Sepúlveda, Maria S

2017-12-01

Endocrine disrupting chemicals (EDCs) can alter thyroid function and adversely affect growth and development. Halogenated compounds, such as perfluorinated chemicals commonly used in food packaging, and brominated flame retardants used in a broad range of products from clothing to electronics, can act as thyroid disruptors. Due to the adverse effects of these compounds, there is a need for the development of safer next generation chemicals. The objective of this study was to test the thyroid disruption potential of old use and next generation halogenated chemicals. Zebrafish embryos were exposed to three old use compounds, perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) and two next generation chemicals, 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxdie (DOPO) and perfluorobutyric acid (PFBA). Sub-chronic (0-6days post fertilization (dpf)) and chronic (0-28dpf) exposures were conducted at 1% of the concentration known to kill 50% (LC 50 ) of the population. Changes in the surface area of the swim bladder as well as in expression levels of genes involved in the thyroid control of swim bladder inflation were measured. At 6dpf, zebrafish exposed to all halogenated chemicals, both old use and next generation, had smaller posterior swim bladder and increased expression in the gene encoding thyroid peroxidase, tpo and the genes encoding two swim bladder surfactant proteins, sp-a and sp-c. These results mirrored the effects of thyroid hormone-exposed positive controls. Fish exposed to a TPO inhibitor (methimazole, MMI) had a decrease in tpo expression levels at 28dpf. Effects on the anterior swim bladder at 28dpf, after exposure to MMI as well as both old and new halogenated chemicals, were the same, i.e., absence of SB in ∼50% of fish, which were also of smaller body size. Overall, our results suggest thyroid disruption by the halogenated compounds tested via the swim bladder surfactant system. However, with the exception of TBBPA and TDCPP, the concentrations tested (∼5-137ppm) are not likely to be found in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

PubMed

Lammer, E; Kamp, H G; Hisgen, V; Koch, M; Reinhard, D; Salinas, E R; Wendler, K; Zok, S; Braunbeck, Th

2009-10-01

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH - Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.

Chemical Form Matters: Differential Accumulation of Mercury Following Inorganic and Organic Mercury Exposures in Zebrafish Larvae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korbas, Malgorzata; MacDonald, Tracy C.; Pickering, Ingrid J.

2013-04-08

Mercury, one of the most toxic elements, exists in various chemical forms each with different toxicities and health implications. Some methylated mercury forms, one of which exists in fish and other seafood products, pose a potential threat, especially during embryonic and early postnatal development. Despite global concerns, little is known about the mechanisms underlying transport and toxicity of different mercury species. To investigate the impact of different mercury chemical forms on vertebrate development, we have successfully combined the zebrafish, a well-established developmental biology model system, with synchrotron-based X-ray fluorescence imaging. Our work revealed substantial differences in tissue-specific accumulation patterns ofmore » mercury in zebrafish larvae exposed to four different mercury formulations in water. Methylmercury species not only resulted in overall higher mercury burdens but also targeted different cells and tissues than their inorganic counterparts, thus revealing a significant role of speciation in cellular and molecular targeting and mercury sequestration. For methylmercury species, the highest mercury concentrations were in the eye lens epithelial cells, independent of the formulation ligand (chloride versus L-cysteine). For inorganic mercury species, in absence of L-cysteine, the olfactory epithelium and kidney accumulated the greatest amounts of mercury. However, with L-cysteine present in the treatment solution, mercuric bis-L-cysteineate species dominated the treatment, significantly decreasing uptake. Our results clearly demonstrate that the common differentiation between organic and inorganic mercury is not sufficient to determine the toxicity of various mercury species.« less

Ecotoxicological bioassays of sediment leachates in a river bed flanked by decommissioned pesticide plants in Nantong City, East China.

PubMed

Zhou, Yan; Wang, Fenghe; Wan, Jinzhong; He, Jian; Li, Qun; Qiang Chen; Gao, Jay; Lin, Yusuo; Zhang, Shengtian

2017-03-01

Traditionally, the toxicity of river contaminants is analyzed chemically or physically through river bed sediments. The biotoxicity of polluted sediment leachates has not caught our attention. This study aims to overcome this deficiency through a battery of biotests which were conducted to monitor comprehensive toxicity of sediment leachates for the Yaogang River in East Jiangsu Province of China, which is in close proximity to former pesticide plants. The general physical and chemical parameters of major pollutants were analyzed from river bed sediments collected at five strategic locations. The ecotoxicity analyses undertaken include overall fish (adult zebrafish) acute toxicity, luminescent bacteria (Vibrio fischeri) bioassay, and zebrafish embryo toxicity assay. Compared with the control group, sediment leachates increased the lethality, inhibited the embryos hatching and induced development abnormalities of zebrafish embryos, and inhibited the luminescence of V. fischeri. The results show that sediment leachates may assume various toxic effects, depending on the test organism. This diverse toxicity to aquatic organisms reflects their different sensitivity to sediment leachates. It is found clearly that V. fischeri was the organism which was characterized by the highest sensitivity to the sediment leachates. The complicated toxicity of leachates was not caused by one single factor but by multiple pollutants together. This indicates the need of estimations of sediment leachate not only taking into account chemical detection but also of applying the biotests to the problem. Thus, multigroup bioassays are necessary to realistically evaluate river ecological risks imposed by leachates.

Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis.

PubMed

Zimmermann, Fernanda Francine; Gaspary, Karina Vidarte; Leite, Carlos Eduardo; De Paula Cognato, Giana; Bonan, Carla Denise

2015-01-01

Changes in social behavior are associated with brain disorders, including mood disorders, stress, schizophrenia, Alzheimer's disease, and autism spectrum disorders (ASD). Autism is a complex neurodevelopmental disorder characterized by deficits in social interaction, impaired communication, anxiety, hyperactivity, and the presence of restricted interests. Zebrafish is one of the most social vertebrates used as a model in biomedical research, contributing to an understanding of the mechanisms that underlie social behavior. Valproic acid (VPA) is used as an anti-epileptic drug and mood stabilizer; however, prenatal VPA exposure in humans has been associated with an increased incidence of autism and it can also affect fetal brain development. Therefore, we conducted a behavioral screening at different periods of zebrafish development at 6, 30, 70, and 120dpf (days postfertilization) after VPA exposure in the early development stage to investigate social behavior, locomotion, aggression, and anxiety. VPA (48μM) exposure during the first 48hpf (hours postfertilization) did not promote changes on survival, morphology, and hatching rate at 24hpf, 48hpf, and 72hpf. The behavioral patterns suggest that VPA exposure induces changes in locomotor activity and anxiety at different developmental periods in zebrafish. Furthermore, a social interaction deficit is present at 70dpf and 120dpf. VPA exposure did not affect aggression in the adult stage at 70dpf and 120dpf. This is the first study that demonstrated zebrafish exposed to VPA during the first 48h of development exhibit deficits in social interaction, anxiety, and hyperactivity at different developmental periods. Copyright © 2015 Elsevier Inc. All rights reserved.

A Forward Genetic Screen in Zebrafish Identifies the G-Protein-Coupled Receptor CaSR as a Modulator of Sensorimotor Decision Making.

PubMed

Jain, Roshan A; Wolman, Marc A; Marsden, Kurt C; Nelson, Jessica C; Shoenhard, Hannah; Echeverry, Fabio A; Szi, Christina; Bell, Hannah; Skinner, Julianne; Cobbs, Emilia N; Sawada, Keisuke; Zamora, Amy D; Pereda, Alberto E; Granato, Michael

2018-05-07

Animals continuously integrate sensory information and select contextually appropriate responses. Here, we show that zebrafish larvae select a behavioral response to acoustic stimuli from a pre-existing choice repertoire in a context-dependent manner. We demonstrate that this sensorimotor choice is modulated by stimulus quality and history, as well as by neuromodulatory systems-all hallmarks of more complex decision making. Moreover, from a genetic screen coupled with whole-genome sequencing, we identified eight mutants with deficits in this sensorimotor choice, including mutants of the vertebrate-specific G-protein-coupled extracellular calcium-sensing receptor (CaSR), whose function in the nervous system is not well understood. We demonstrate that CaSR promotes sensorimotor decision making acutely through Gα i/o and Gα q/11 signaling, modulated by clathrin-mediated endocytosis. Combined, our results identify the first set of genes critical for behavioral choice modulation in a vertebrate and reveal an unexpected critical role for CaSR in sensorimotor decision making. Copyright © 2018 Elsevier Ltd. All rights reserved.

Zebrafish embryotoxicity test for developmental (neuro)toxicity: Demo case of an integrated screening approach system using anti-epileptic drugs.

PubMed

Beker van Woudenberg, Anna; Snel, Cor; Rijkmans, Eke; de Groot, Didima; Bouma, Marga; Hermsen, Sanne; Piersma, Aldert; Menke, Aswin; Wolterbeek, André

2014-11-01

To improve the predictability of the zebrafish embryotoxicity test (ZET) for developmental (neuro)toxicity screening, we used a multiple-endpoints strategy, including morphology, motor activity (MA), histopathology and kinetics. The model compounds used were antiepileptic drugs (AEDs): valproic acid (VPA), carbamazepine (CBZ), ethosuximide (ETH) and levetiracetam (LEV). For VPA, histopathology was the most sensitive parameter, showing effects already at 60μM. For CBZ, morphology and MA were the most sensitive parameters, showing effects at 180μM. For ETH, all endpoints showed similar sensitivity (6.6mM), whereas MA was the most sensitive parameter for LEV (40mM). Inclusion of kinetics did not alter the absolute ranking of the compounds, but the relative potency was changed considerably. Taking all together, this demo-case study showed that inclusion of multiple-endpoints in ZET may increase the sensitivity of the assay, contribute to the elucidation of the mode of toxic action and to a better definition of the applicability domain of ZET. Copyright © 2014 Elsevier Inc. All rights reserved.

Sensitivities of Two Zebrafish TRPA1 Paralogs to Chemical and Thermal Stimuli Analyzed in Heterologous Expression Systems.

PubMed

Oda, Mai; Kurogi, Mako; Kubo, Yoshihiro; Saitoh, Osamu

2016-03-01

Transient receptor potential A1 (TRPA1) is the only member of the mouse, chick, and frog TRPA family, whereas 2 paralogs (zTRPA1a and zTRPA1b) are present in zebrafish. We herein investigated functional differences in the 2 zebrafish TRPA1s. HEK293T cells were used as heterologous expression systems, and the sensitivities of these cells to 4 chemical irritants (allyl isothiocyanate [AITC], caffeine, auto-oxidized epigallocatechin gallate [EGCG], and hydrogen peroxide [H2O2]) were compared with Ca(2+) imaging techniques. Sensitivities to the activators for AITC, oxidized EGCG, and H2O2 were higher in cells expressing zTRPA1a than in those expressing zTRPA1b, whereas caffeine appeared to activate both cells equally. We also characterized the thermal sensitivity of Xenopus oocytes expressing each TRPA1 electrophysiologically using a 2-electrode voltage clamp. Although endogenous currents induced by a cold stimulation were observed in control oocytes in some batches, oocytes expressing zTRPA1b showed significantly stronger cold- and heat-induced responses. However, significant thermal activation was not observed in oocytes expressing zTRPA1a. The results obtained using in vitro expression systems suggest that zTRPA1a is specialized for chemical sensing, whereas zTRPA1b responds to thermal stimuli. Furthermore, characterization of the chimeric molecule of TRPA1a and 1b revealed the importance of the N-terminal region in chemical and thermal sensing by zTRPA1s. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The classification of motor neuron defects in the zebrafish embryo toxicity test (ZFET) as an animal alternative approach to assess developmental neurotoxicity.

PubMed

Muth-Köhne, Elke; Wichmann, Arne; Delov, Vera; Fenske, Martina

2012-07-01

Rodents are widely used to test the developmental neurotoxicity potential of chemical substances. The regulatory test procedures are elaborate and the requirement of numerous animals is ethically disputable. Therefore, non-animal alternatives are highly desirable, but appropriate test systems that meet regulatory demands are not yet available. Hence, we have developed a new developmental neurotoxicity assay based on specific whole-mount immunostainings of primary and secondary motor neurons (using the monoclonal antibodies znp1 and zn8) in zebrafish embryos. By classifying the motor neuron defects, we evaluated the severity of the neurotoxic damage to individual primary and secondary motor neurons caused by chemical exposure and determined the corresponding effect concentration values (EC₅₀). In a proof-of-principle study, we investigated the effects of three model compounds thiocyclam, cartap and disulfiram, which show some neurotoxicity-indicating effects in vertebrates, and the positive controls ethanol and nicotine and the negative controls 3,4-dichloroaniline (3,4-DCA) and triclosan. As a quantitative measure of the neurotoxic potential of the test compounds, we calculated the ratios of the EC₅₀ values for motor neuron defects and the cumulative malformations, as determined in a zebrafish embryo toxicity test (zFET). Based on this index, disulfiram was classified as the most potent and thiocyclam as the least potent developmental neurotoxin. The index also confirmed the control compounds as positive and negative neurotoxicants. Our findings demonstrate that this index can be used to reliably distinguish between neurotoxic and non-neurotoxic chemicals and provide a sound estimate for the neurodevelopmental hazard potential of a chemical. The demonstrated method can be a feasible approach to reduce the number of animals used in developmental neurotoxicity evaluation procedures. Copyright © 2012 Elsevier Inc. All rights reserved.

Early life exposure to PCB126 results in delayed mortality and growth impairment in the zebrafish larvae.

PubMed

Di Paolo, Carolina; Groh, Ksenia J; Zennegg, Markus; Vermeirssen, Etiënne L M; Murk, Albertinka J; Eggen, Rik I L; Hollert, Henner; Werner, Inge; Schirmer, Kristin

2015-12-01

The occurrence of chronic or delayed toxicity resulting from the exposure to sublethal chemical concentrations is an increasing concern in environmental risk assessment. The Fish Embryo Toxicity (FET) test with zebrafish provides a reliable prediction of acute toxicity in adult fish, but it cannot yet be applied to predict the occurrence of chronic or delayed toxicity. Identification of sublethal FET endpoints that can assist in predicting the occurrence of chronic or delayed toxicity would be advantageous. The present study characterized the occurrence of delayed toxicity in zebrafish larvae following early exposure to PCB126, previously described to cause delayed effects in the common sole. The first aim was to investigate the occurrence and temporal profiles of delayed toxicity during zebrafish larval development and compare them to those previously described for sole to evaluate the suitability of zebrafish as a model fish species for delayed toxicity assessment. The second aim was to examine the correlation between the sublethal endpoints assessed during embryonal and early larval development and the delayed effects observed during later larval development. After exposure to PCB126 (3-3000ng/L) until 5 days post fertilization (dpf), larvae were reared in clean water until 14 or 28 dpf. Mortality and sublethal morphological and behavioural endpoints were recorded daily, and growth was assessed at 28 dpf. Early life exposure to PCB126 caused delayed mortality (300 ng/L and 3000 ng/L) as well as growth impairment and delayed development (100 ng/L) during the clean water period. Effects on swim bladder inflation and cartilaginous tissues within 5 dpf were the most promising for predicting delayed mortality and sublethal effects, such as decreased standard length, delayed metamorphosis, reduced inflation of swim bladder and column malformations. The EC50 value for swim bladder inflation at 5 dpf (169 ng/L) was similar to the LC50 value at 8 dpf (188 and 202 ng/L in two experiments). Interestingly, the patterns of delayed mortality and delayed effects on growth and development were similar between sole and zebrafish. This indicates the comparability of critical developmental stages across divergent fish species such as a cold water marine flatfish and a tropical freshwater cyprinid. Additionally, sublethal effects in early embryo-larval stages were found promising for predicting delayed lethal and sublethal effects of PCB126. Therefore, the proposed method with zebrafish is expected to provide valuable information on delayed mortality and delayed sublethal effects of chemicals and environmental samples that may be extrapolated to other species. Copyright © 2015 Elsevier B.V. All rights reserved.

From fish bowl to bedside: The power of zebrafish to unravel melanoma pathogenesis and discover new therapeutics.

PubMed

van Rooijen, Ellen; Fazio, Maurizio; Zon, Leonard I

2017-07-01

Melanoma is the most aggressive and deadliest form of skin cancer. A detailed knowledge of the cellular, molecular, and genetic events underlying melanoma progression is highly relevant to diagnosis, prognosis and risk stratification, and the development of new therapies. In the last decade, zebrafish have emerged as a valuable model system for the study of melanoma. Pathway conservation, coupled with the availability of robust genetic, transgenic, and chemical tools, has made the zebrafish a powerful model for identifying novel disease genes, visualizing cancer initiation, interrogating tumor-microenvironment interactions, and discovering new therapeutics that regulate melanocyte and melanoma development. In this review, we will give an overview of these studies, and highlight recent advancements that will help unravel melanoma pathogenesis and impact human disease. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish

PubMed Central

Kumar, Sunil; Lockwood, Nicola; Ramel, Marie-Christine; Correia, Teresa; Ellis, Matthew; Alexandrov, Yuriy; Andrews, Natalie; Patel, Rachel; Bugeon, Laurence; Dallman, Margaret J.; Brandner, Sebastian; Arridge, Simon; Katan, Matilda; McGinty, James; Frankel, Paul; French, Paul M.W.

2016-01-01

We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models. PMID:27259259

Zebrafish Locomotor Responses Predict Irritant Potential of ...

EPA Pesticide Factsheets

Over the past few decades, the drying and warming trends of global climate change have increased wildland fire (WF) season length, as well as geographic area impacted. Consequently, exposures to WF fine particulate matter (PM2.5; aerodynamic diameter <2.5 µm) are likely to increase in frequency and duration, contributing to a growing public health burden. Given the influence of fuel type and combustion conditions on WFPM2.5 composition, there is pressing need to identify the biomass fuel sources and emission constituents that drive toxicity. Previously, we reported the utility of 6-day post-fertilization (dpf) zebrafish larvae in evaluating diesel exhaust PM-induced irritation, demonstrating responses analogous to those in mammals. In the present study, combustions, separated by smoldering or flaming conditions, of pine needles, red oak, pine, eucalyptus, and peat were achieved using an automated tube furnace paired with a cryo-trapping apparatus to collect condensates of emissions. The condensates were extracted and prepared for use in zebrafish assays. We hypothesized that 1) the extractable organic fractions of biomass smoke PM will elicit dose-dependent irritant responses in 6-dpf zebrafish larvae, and 2) the relative potencies will vary across biomass emissions, potentially driven by varying chemical composition of fuel sources. Six-dpf zebrafish (n= 28-32/group) were exposed acutely to PM extracts (5 concentrations; 0.3-30 µg/ml; half-log intervals) and

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal)--experimental assessment using a zebrafish embryo test.

PubMed

Madureira, Tânia Vieira; Cruzeiro, Catarina; Rocha, Maria João; Rocha, Eduardo

2011-09-01

Fish embryos are a particularly vulnerable stage of development, so they represent optimal targets for screening toxicological effects of waterborne xenobiotics. Herein, the toxicity potential of two mixtures of pharmaceuticals was evaluated using a zebrafish embryo test. One of the mixtures corresponds to an environmentally realistic scenario and both have carbamazepine, fenofibric acid, propranolol, trimethoprim and sulfamethoxazole. The results evidenced morphological alterations, such as spinal deformities and yolk-sac oedemas. Moreover, heart rates decreased after both mixture exposures, e.g., at 48hpf, highest mixture versus blank control (47.8±4.9 and 55.8±3.7 beats/30s, respectively). The tail lengths also diminished significantly from 3208±145μm in blank control to 3130±126μm in highest mixture. The toxicological effects were concentration dependent. Mortality, hatching rate and the number of spontaneous movements were not affected. However, the low levels of pharmaceuticals did interfere with the normal development of zebrafish, which indicates risks for wild organisms. Copyright © 2011 Elsevier B.V. All rights reserved.

Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish

PubMed Central

Irion, Uwe; Frohnhöfer, Hans Georg; Krauss, Jana; Çolak Champollion, Tuǧba; Maischein, Hans-Martin; Geiger-Rudolph, Silke; Weiler, Christian; Nüsslein-Volhard, Christiane

2014-01-01

Interactions between all three pigment cell types are required to form the stripe pattern of adult zebrafish (Danio rerio), but their molecular nature is poorly understood. Mutations in leopard (leo), encoding Connexin 41.8 (Cx41.8), a gap junction subunit, cause a phenotypic series of spotted patterns. A new dominant allele, leotK3, leads to a complete loss of the pattern, suggesting a dominant negative impact on another component of gap junctions. In a genetic screen, we identified this component as Cx39.4 (luchs). Loss-of-function alleles demonstrate that luchs is required for stripe formation in zebrafish; however, the fins are almost not affected. Double mutants and chimeras, which show that leo and luchs are only required in xanthophores and melanophores, but not in iridophores, suggest that both connexins form heteromeric gap junctions. The phenotypes indicate that these promote homotypic interactions between melanophores and xanthophores, respectively, and those cells instruct the patterning of the iridophores. DOI: http://dx.doi.org/10.7554/eLife.05125.001 PMID:25535837

Isolation of Novel CreERT2-Driver Lines in Zebrafish Using an Unbiased Gene Trap Approach

PubMed Central

Jungke, Peggy; Hammer, Juliane; Hans, Stefan; Brand, Michael

2015-01-01

Gene manipulation using the Cre/loxP-recombinase system has been successfully employed in zebrafish to study gene functions and lineage relationships. Recently, gene trapping approaches have been applied to produce large collections of transgenic fish expressing conditional alleles in various tissues. However, the limited number of available cell- and tissue-specific Cre/CreERT2-driver lines still constrains widespread application in this model organism. To enlarge the pool of existing CreERT2-driver lines, we performed a genome-wide gene trap screen using a Tol2-based mCherry-T2a-CreERT2 (mCT2aC) gene trap vector. This cassette consists of a splice acceptor and a mCherry-tagged variant of CreERT2 which enables simultaneous labeling of the trapping event, as well as CreERT2 expression from the endogenous promoter. Using this strategy, we generated 27 novel functional CreERT2-driver lines expressing in a cell- and tissue-specific manner during development and adulthood. This study summarizes the analysis of the generated CreERT2-driver lines with respect to functionality, expression, integration, as well as associated phenotypes. Our results significantly enlarge the existing pool of CreERT2-driver lines in zebrafish and combined with Cre–dependent effector lines, the new CreERT2-driver lines will be important tools to manipulate the zebrafish genome. PMID:26083735

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

PubMed Central

Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

2016-01-01

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

Behavioral, endocrine, and neuronal alterations in zebrafish (Danio rerio) following sub-chronic coadministration of fluoxetine and ketamine.

PubMed

Pittman, Julian; Hylton, Andrew

2015-12-01

Most existing pharmacological treatments have focused on the "monoamine hypothesis" for targeted drug design for major depressive disorder (MDD). Many of these medications have a delayed onset-of-action and limited efficacy. Antidepressants with principal targets outside the monoamine system may offer the potential for more rapid activity with improved therapeutic benefit. Growing evidence suggests that the glutamatergic system is uniquely central to the neurobiology and treatment of MDD. Ketamine (Ketalar®) is a non-competitive glutamatergic antagonist classically used to induce sedation. However, preliminary clinical evidence has been promising with regard to its rapidly acting antidepressant profile. Zebrafish (Danio rerio) have emerged as a promising new animal model to screen the effects of numerous psychotropic compounds. This study aimed to determine if a sub-chronic low (sub-anesthetic) dose of ketamine could be used to augment the antidepressant effects of the widely used antidepressant fluoxetine (Prozac®) in adult zebrafish, employing an ethanol withdrawal model. Sub-chronic exposure to dosages of 100μg/L fluoxetine and 20mg/L of ketamine reduced anxiety/depression-like behaviors, leads to upregulation of serotonin synthesis and elevated whole-body cortisol levels. These results demonstrate the utility of zebrafish as a model for neuropharmacological research, and the possible efficacy of fluoxetine and ketamine coadministration. Copyright © 2015 Elsevier Inc. All rights reserved.

Repositioning drugs for inflammatory disease – fishing for new anti-inflammatory agents

PubMed Central

Hall, Christopher J.; Wicker, Sophie M.; Chien, An-Tzu; Tromp, Alisha; Lawrence, Lisa M.; Sun, Xueying; Krissansen, Geoffrey W.; Crosier, Kathryn E.; Crosier, Philip S.

2014-01-01

Inflammation is an important and appropriate host response to infection or injury. However, dysregulation of this response, with resulting persistent or inappropriate inflammation, underlies a broad range of pathological processes, from inflammatory dermatoses to type 2 diabetes and cancer. As such, identifying new drugs to suppress inflammation is an area of intense interest. Despite notable successes, there still exists an unmet need for new effective therapeutic approaches to treat inflammation. Traditional drug discovery, including structure-based drug design, have largely fallen short of satisfying this unmet need. With faster development times and reduced safety and pharmacokinetic uncertainty, drug repositioning – the process of finding new uses for existing drugs – is emerging as an alternative strategy to traditional drug design that promises an improved risk-reward trade-off. Using a zebrafish in vivo neutrophil migration assay, we undertook a drug repositioning screen to identify unknown anti-inflammatory activities for known drugs. By interrogating a library of 1280 approved drugs for their ability to suppress the recruitment of neutrophils to tail fin injury, we identified a number of drugs with significant anti-inflammatory activity that have not previously been characterized as general anti-inflammatories. Importantly, we reveal that the ten most potent repositioned drugs from our zebrafish screen displayed conserved anti-inflammatory activity in a mouse model of skin inflammation (atopic dermatitis). This study provides compelling evidence that exploiting the zebrafish as an in vivo drug repositioning platform holds promise as a strategy to reveal new anti-inflammatory activities for existing drugs. PMID:25038060

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism

PubMed Central

Spagnoli, Sean; Xue, Lan; Kent, Michael L.

2015-01-01

The zebrafish’s potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host’s brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over ten minutes along with taps at 18 and 60 minutes to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 minutes, whereas exposed negative fish retained partial habituation at 18 minutes. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the improvement of the zebrafish as a translational model for human behavior. PMID:26028515

A microfluidic device to study neuronal and motor responses to acute chemical stimuli in zebrafish.

PubMed

Candelier, Raphaël; Murmu, Meena Sriti; Romano, Sebastián Alejo; Jouary, Adrien; Debrégeas, Georges; Sumbre, Germán

2015-07-21

Zebrafish larva is a unique model for whole-brain functional imaging and to study sensory-motor integration in the vertebrate brain. To take full advantage of this system, one needs to design sensory environments that can mimic the complex spatiotemporal stimulus patterns experienced by the animal in natural conditions. We report on a novel open-ended microfluidic device that delivers pulses of chemical stimuli to agarose-restrained larvae with near-millisecond switching rate and unprecedented spatial and concentration accuracy and reproducibility. In combination with two-photon calcium imaging and recordings of tail movements, we found that stimuli of opposite hedonic values induced different circuit activity patterns. Moreover, by precisely controlling the duration of the stimulus (50-500 ms), we found that the probability of generating a gustatory-induced behavior is encoded by the number of neurons activated. This device may open new ways to dissect the neural-circuit principles underlying chemosensory perception.

Effects of chronic perfluorooctanoic acid (PFOA) at low concentration on morphometrics, gene expression, and fecundity in zebrafish (Danio rerio).

PubMed

Jantzen, Carrie E; Toor, Fatima; Annunziato, Kate A; Cooper, Keith R

2017-04-01

Perfluorooctanoic acid (PFOA) is a persistent, toxic, anthropogenic chemical recalcitrant to biodegradation. Based on previous studies in lower and higher vertebrates, it was hypothesized that chronic, sub-lethal, embryonic exposure to PFOA in zebrafish (Danio rerio) would adversely impact fish development, survival, and fecundity. Zebrafish embryo/sac-fry were water exposed to 2.0 or 0nM PFOA from 3 to 120hpf, and juvenile to adult cohorts were fed spiked food (8 pM) until 6 months. After chronic exposure, PFOA exposed fish were significantly smaller in total weight and length. Gene expression analysis found a significant decrease of transporters slco2b1, slco4a1, slco3a1 and tgfb1a, and a significant increase of slco1d1 expression. PFOA exposed fish produced significantly fewer eggs with reduced viability and developmental stage delay in F 1 . Chronic, low-dose exposure of zebrafish to PFOA significantly altered normal development, survival and fecundity and would likely impact wild fish population fitness in watersheds chronically exposed to PFOA. Copyright © 2017 Elsevier Inc. All rights reserved.

Embryotoxicity of poorly soluble nanoparticles at various stages of Zebrafish development

NASA Astrophysics Data System (ADS)

Morgaleva, Tamara; Morgalev, Yuri; Gosteva, Irina; Morgalev, Sergey; Nesterenya, Daria

2017-11-01

The biological effects of the poorly soluble nanoparticles (NPs) of different chemical nature and structural characteristics were evaluated. It was established that the Zebrafish test response to contamination of aqueous medium with nickel NPs (nNi), platinum (nPt), zinc oxide (nZnO) and cerium oxide (nCeO2) depends on the physicochemical properties of the NPs and embryo development stage. The concentrations of NPs not causing disruptions in embryonic development of Zebrafish were determined. The smallest impact on embryogenesis was exerted by nCeO2: coagulation of a small number of embryos was observed only at C = 20.0 mg/L. The same effect was observed when exposed to lower concentrations of nPt (C = 5.0 mg/L) and nNi (C = 0.1 mg/L). The greatest number of coagulated embryos was observed when grown in the DS nZnO: 37.5% of embryos died at the DS concentration of C = 0.1 mg/L. Zebrafish cultivation in the DS with low concentrations (C ≤ LC10) of nNi and nZnO caused distortions in the development of embryos: development of scoliosis, malformation of somites, inhibited mobility.

A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos.

PubMed

Schiwy, Sabrina; Bräunig, Jennifer; Alert, Henriette; Hollert, Henner; Keiter, Steffen H

2015-11-01

The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario.

Molecular and Chemical Genetic Approaches to Developmental Origins of Aging and Disease in Zebrafish

PubMed Central

Sasaki, Tomoyuki; Kishi, Shuji

2013-01-01

The incidence of diseases increases rapidly with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but mostly inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states in response to different environmental or genetic perturbations. On the one hand, we hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds of adaptive plasticity by chemical genetic approaches, we have been investigating whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during embryogenesis (“embryonic senescence”), subsequently showing shortened lifespan in adulthoods. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes and small molecular compounds that can be linked to the senescence phenotype, and thereby facilitates searching for the evolutionary and developmental origins of aging in vertebrates. PMID:23660559

Cardio-respirometry disruption in zebrafish (Danio rerio) embryos exposed to hydraulic fracturing flowback and produced water.

PubMed

Folkerts, Erik J; Blewett, Tamzin A; He, Yuhe; Goss, Greg G

2017-12-01

Hydraulic fracturing to extract oil and natural gas reserves is an increasing practice in many international energy sectors. Hydraulic fracturing flowback and produced water (FPW) is a hyper saline wastewater returned to the surface from a fractured well containing chemical species present in the initial fracturing fluid, geogenic contaminants, and potentially newly synthesized chemicals formed in the fracturing well environment. However, information on FPW toxicological mechanisms of action remain largely unknown. Both cardiotoxic and respirometric responses were explored in zebrafish (Danio rerio) embryos after either an acute sediment-free (FPW-SF) or raw/sediment containing (FPW-S) fraction exposure of 24 and 48 h at 2.5% and 5% dilutions. A 48 h exposure to either FPW fraction in 24-72 h post fertilization zebrafish embryos significantly increased occurrences of pericardial edema, yolk-sac edema, and tail/spine curvature. In contrast, larval heart rates significantly decreased after FPW fraction exposures. FPW-S, but not FPW-SF, at 2.5% doses significantly reduced embryonic respiration/metabolic rates (MO 2 ), while for 5% FPW, both fractions reduced MO 2 . Expression of select cardiac genes were also significantly altered in each FPW exposure group, implicating a cardiovascular system compromise as the potential cause for reduced embryonic MO 2 . Collectively, these results support our hypothesis that organics are major contributors to cardiac and respiratory responses to FPW exposure in zebrafish embryos. Our study is the first to investigate cardiac and respiratory sub-lethal effects of FPW exposure, demonstrating that FPW effects extend beyond initial osmotic stressors and verifies the use of respirometry as a potential marker for FPW exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

A comparison of fate and toxicity of selenite, biogenically, and chemically synthesized selenium nanoparticles to zebrafish (Danio rerio) embryogenesis.

PubMed

Mal, Joyabrata; Veneman, Wouter J; Nancharaiah, Y V; van Hullebusch, Eric D; Peijnenburg, Willie J G M; Vijver, Martina G; Lens, Piet N L

2017-02-01

Microbial reduction of selenium (Se) oxyanions to elemental Se is a promising technology for bioremediation and treatment of Se wastewaters. But a fraction of biogenic nano-Selenium (nano-Se b ) formed in bioreactors remains suspended in the treated waters, thus entering the aquatic environment. The present study investigated the toxicity of nano-Se b formed by anaerobic granular sludge biofilms on zebrafish embryos in comparison with selenite and chemogenic nano-Se (nano-Se c ). The nano-Se b formed by granular sludge biofilms showed a LC 50 value of 1.77 mg/L, which was 3.2-fold less toxic to zebrafish embryos than selenite (LC 50  =   0.55 mg/L) and 10-fold less toxic than bovine serum albumin stabilized nano-Se c (LC 50  =   0.16 mg/L). Smaller (nano-Se cs ; particle diameter range: 25-80 nm) and larger (nano-Se cl ; particle diameter range: 50-250 nm) sized chemically synthesized nano-Se c particles showed comparable toxicity on zebrafish embryos. The lower toxicity of nano-Se b in comparison with nano-Se c was analyzed in terms of the stabilizing organic layer. The results confirmed that the organic layer extracted from the nano-Se b consisted of components of the extracellular polymeric substances (EPS) matrix, which govern the physiochemical stability and surface properties like ζ-potential of nano-Se b . Based on the data, it is contented that the presence of humic acid like substances of EPS on the surface of nano-Se b plays a major role in lowering the bioavailability (uptake) and toxicity of nano-Se b by decreasing the interactions between nanoparticles and embryos.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

PubMed Central

Moreno, Rosa L.; Josey, Megan; Ribera, Angeles B.

2017-01-01

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model. For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin12345. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6789. PMID:28448016

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons.

PubMed

Moreno, Rosa L; Josey, Megan; Ribera, Angeles B

2017-04-18

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model. For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin 1 , 2 , 3 , 4 , 5 . A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up 4 . The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages 6 , 7 , 8 , 9 .

Decreased Dissolution of ZnO by Iron Doping Yields Nanoparticles with Reduced Toxicity in the Rodent Lung and Zebrafish Embryos

PubMed Central

Xia, Tian; Zhao, Yan; Sager, Tina; George, Saji; Pokhrel, Suman; Li, Ning; Schoenfeld, David; Meng, Huan; Lin, Sijie; Wang, Xiang; Wang, Meiying; Ji, Zhaoxia; Zink, Jeffrey I.; Mädler, Lutz; Castranova, Vincent; Lin, Shuo; Nel, Andre E.

2014-01-01

We have recently shown that the dissolution of ZnO nanoparticles and Zn2+ shedding leads to a series of sub-lethal and lethal toxicological responses at cellular level that can be alleviated by iron-doping. Iron-doping changes the particle matrix and slows the rate of particle dissolution. To determine whether iron doping of ZnO also leads to lesser toxic effects in vivo, toxicity studies were performed in rodent and zebrafish models. First, we synthesized a fresh batch of ZnO nanoparticles doped with 1–10 wt % of Fe. These particles were extensively characterized to confirm their doping status, reduced rate of dissolution in an exposure medium and reduced toxicity in a cellular screen. Subsequent studies compared the effects of undoped to doped particles in the rat lung, mouse lung and the zebrafish embryo. The zebrafish studies looked at embryo hatching and mortality rates as well as the generation of morphological defects, while the endpoints in the rodent lung included an assessment of inflammatory cell infiltrates, LDH release and cytokine levels in the bronchoalveolar lavage fluid. Iron doping, similar to the effect of the metal chelator, DTPA, interfered in the inhibitory effects of Zn2+ on zebrafish hatching. In the oropharyngeal aspiration model in the mouse, iron doping was associated with decreased polymorphonuclear cell counts and IL-6 mRNA production. Doped particles also elicited decreased heme oxygenase 1 expression in the murine lung. In the intratracheal instillation studies in the rat, Fe-doping was associated with decreased polymorphonuclear cell counts, LDH and albumin levels. All considered, the above data show that Fe-doping is a possible safe design strategy for preventing ZnO toxicity in animals and the environment. PMID:21250651

Construction and use of a zebrafish heart voltage and calcium optical mapping system, with integrated electrocardiogram and programmable electrical stimulation

PubMed Central

Lin, Eric; Craig, Calvin; Lamothe, Marcel; Sarunic, Marinko V.; Beg, Mirza Faisal

2015-01-01

Zebrafish are increasingly being used as a model of vertebrate cardiology due to mammalian-like cardiac properties in many respects. The size and fecundity of zebrafish make them suitable for large-scale genetic and pharmacological screening. In larger mammalian hearts, optical mapping is often used to investigate the interplay between voltage and calcium dynamics and to investigate their respective roles in arrhythmogenesis. This report outlines the construction of an optical mapping system for use with zebrafish hearts, using the voltage-sensitive dye RH 237 and the calcium indicator dye Rhod-2 using two industrial-level CCD cameras. With the use of economical cameras and a common 532-nm diode laser for excitation, the rate dependence of voltage and calcium dynamics within the atrial and ventricular compartments can be simultaneously determined. At 140 beats/min, the atrial action potential duration was 36 ms and the transient duration was 53 ms. With the use of a programmable electrical stimulator, a shallow rate dependence of 3 and 4 ms per 100 beats/min was observed, respectively. In the ventricle the action potential duration was 109 ms and the transient duration was 124 ms, with a steeper rate dependence of 12 and 16 ms per 100 beats/min. Synchronous electrocardiograms and optical mapping recordings were recorded, in which the P-wave aligns with the atrial voltage peak and R-wave aligns with the ventricular peak. A simple optical pathway and imaging chamber are detailed along with schematics for the in-house construction of the electrocardiogram amplifier and electrical stimulator. Laboratory procedures necessary for zebrafish heart isolation, cannulation, and loading are also presented. PMID:25740339

Transcriptome alterations in zebrafish embryos after exposure to environmental estrogens and anti-androgens can reveal endocrine disruption.

PubMed

Schiller, Viktoria; Wichmann, Arne; Kriehuber, Ralf; Schäfers, Christoph; Fischer, Rainer; Fenske, Martina

2013-12-01

Exposure to environmental chemicals known as endocrine disruptors (EDs) is in many cases associated with an unpredictable hazard for wildlife and human health. The identification of endocrine disruptive properties of chemicals certain to enter the aquatic environment relies on toxicity tests with fish, assessing adverse effects on reproduction and sexual development. The demand for quick, reliable ED assays favored the use of fish embryos as alternative test organisms. We investigated the application of a transcriptomics-based assay for estrogenic and anti-androgenic chemicals with zebrafish embryos. Two reference compounds, 17α-ethinylestradiol and flutamide, were tested to evaluate the effects on development and the transcriptome after 48h-exposures. Comparison of the transcriptome response with other estrogenic and anti-androgenic compounds (genistein, bisphenol A, methylparaben, linuron, prochloraz, propanil) showed commonalities and differences in regulated pathways, enabling us to classify the estrogenic and anti-androgenic potencies. This demonstrates that different mechanism of ED can be assessed already in fish embryos. Copyright © 2013 Elsevier Inc. All rights reserved.