In vivo zebrafish assays for analyzing drug toxicity.

Review

In vivo zebrafish assays for analyzing drug toxicity

Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by RMIT University on 06/19/14 For personal use only.

Demetrio Rald ua & Benjamin Pinã† IDAEA-CSIC, Environmental Chemistry, Barcelona, Spain

1.

Introduction

2.

Cardiovascular system

3.

Gastrointestinal system

4.

Neurotoxicity assessment

5.

Neuromuscular system

6.

Assessment of the effect on motor behavior

7.

Thyroid and sex hormones disrupting activity

8.

High-throughput approaches

9.

Conclusion

10.

Expert opinion

Introduction: Off-target effects represent one of the major concerns in the development of new pharmaceuticals, requiring large-scale animal toxicity testing. Faster, cheaper and more reliable assays based on zebrafish embryos (ZE) are being developed as major tools for assessing toxicity of chemicals during the drug-discovery process. Areas covered: This paper reviews techniques aimed to the analysis of in vivo sublethal toxic effects of drugs on major physiological functions, including the cardiovascular, nervous, neuromuscular, gastrointestinal and thyroid systems among others. Particular emphasis is placed on high-throughput screening techniques (HTS), including robotics, imaging technologies and image-analysis software. Expert opinion: The analysis of off-target effects of candidate drugs requires systemic analyses, as they often involve the complete organism rather than specific, tissue- or cell-specific targets. The unique physical and physiological characteristics of ZE make this system an essential tool for drug discovery and toxicity assessment. Different HTS methodologies applicable to ZE allow the screening of large numbers of different chemicals for many diverse and relevant toxic endpoints. Keywords: cardiotoxicity, drug discovery, endocrine disruption, neurotoxicity, pharmaceuticals, replacement methods Expert Opin. Drug Metab. Toxicol. (2014) 10(5):685-697

1.

Introduction

Zebrafish is a vertebrate model organism extensively used in developmental biology, drug discovery, safety pharmacology and ecotoxicology [1]. Of small size and easy to maintain, a single pair of adults will breed once a week, generating 100 -- 200 offspring per brood, and their husbandry costs are 100 and 1000 times lower than for mice or other mammals [2]. Zebrafish embryos (ZE) grow rapidly, with the basic vertebrate body plan laid out within 24 hours post-fertilization (hpf) [3]. Hatching occurs normally at 48 -- 72 hpf, and the majority of organs, including the nervous system, cardiovascular system, intestines, liver and kidneys, are differentiated and functional at 5 days post-fertilization (dpf). This stage marks the transition from free-swimming embryo (also called eleutheroembryo) to a self-feeding, autonomous larva. Zebrafish’s organs and tissues have proved similar to their mammalian counterparts on the anatomical, physiological and molecular levels [4]. The zebrafish ex utero development and optical clarity during embryogenesis and early larval stages facilitate visual in vivo observation of early developmental processes and organogenesis. These functional and morphological changes may be observed both in vivo or in whole-mount fixed specimens by using transgenic lines, vital dyes, fluorescent tracers, antibodies, riboprobes and fluorescent markers [4]. As eleutheroembryos can live in as little as 50 l water, and they can easily be accommodated into 96-well plate formats, only micrograms of compounds are required for assays, representing a major cost saving in screening entire molecule libraries [2]. Whereas the main focus of zebrafish research has traditionally been on 10.1517/17425255.2014.896339 © 2014 Informa UK, Ltd. ISSN 1742-5255, e-ISSN 1744-7607 All rights reserved: reproduction in whole or in part not permitted

685

a & B. Pinã D. Raldu

2.

Article highlights. . . . .


.

Off-target effects represent one of the major concerns in the development of new pharmaceuticals. Large-scale, mammal-based toxicological screens are expensive and require elevated numbers of test animals. Zebrafish embryos (ZE) represent a viable alternative due to their specific biological and physiological characteristics. ZE are considered a replacement toxicological model up to 120 h post-fertilization. Circulatory, digestive, neuromuscular and endocrine systems can be tested in ZE using high-throughput screening methodologies.

This box summarizes key points contained in the article.

developmental biology, observations from large-scale genetic screening allowed the identification of mutant strains that constitute phenocopies of diseases and developmental pathologies described in humans. These studies demonstrated the suitability of using zebrafish as a model of human disease, drug discovery and safety pharmacology [5,6]. In this context, zebrafish has been proposed as an intermediate step between single cell-based evaluation and mammalian (and ultimately human) testing. Safety pharmacology tries to identify potential adverse effects in drug candidates as early as possible in the drug development pipeline, in order to reduce the final cost of the drug-discovery process [7]. Among toxic effects induced by drugs, the ones caused by the interaction of a molecular target different from the intended one (off-target effects) are particularly worrisome. Toxicity testing in mammalian models, such as mice, rats, rabbits and dogs, is a time-consuming and expensive process that requires the handling of many animals, with the consequent application of legal constrains from animal welfare legislations. For this reason, many pharmaceutical companies already use zebrafish embryo assays as first-tier screening for hazard identification, thus reducing the number and cost of mammalian studies. This is consistent with the 3Rs rules (replacement, reduction and refinement of animal experiments [8]). ZE and eleutheroembryos (0 -- 5 dpf) are considered unlikely to experience pain, suffering, distress or lasting harm [9]. Therefore, zebrafish assays performed during this initial endotrophic nutritional period may be considered as replacement or refinement methods, and they are excluded from the normative on animal testing by the Directive 2010/63/EU. There are a number of available biotechnological tools for assessing toxicity organotoxicity induced by drugs in zebrafish [2,6]. In this review, we describe the potential of the zebrafish toolbox for the development of in vivo assays to identify detrimental effects of drugs on different organs. Finally, we will assess the capability of new high-throughput method to scale up these assays to meet the ever-growing demands of the drug-discovery research. 686

Cardiovascular system

The circulatory system is a known major target for toxic effects of candidate drugs, and many secondary effects discovered for already marketed drugs are related with heart toxicity [10]. It is therefore essential to identify these risks as early as possible during the drug development process. Zebrafish has a prototypic vertebrate heart, with a single atrium and a single ventricle, and the developmental program involved in its formation is essentially identical to all other vertebrates, including humans [11-13]. Zebrafish heart develops very rapidly, as it starts beating 26 hpf, showing the complete repertoire of ion channels and the typical vertebrate heart metabolic setup [14]. Many of the heart features associated to circulation are similar in zebrafish and in mammals, including blood-flow-directing valves, endocardium musculature and pacemaker activity associated with heart beat [14,15]. Cardiotoxicants are known to induce cardiac enlargement, edema and several dysfunctions in ZE, including reductions in cardiomyocyte numbers, decreased heart size, altered vascular remodeling, pericardial edema and decreased ventricular contraction culminating in ventricular standstill [16,17]. Heart morphology and the relation between atrium and ventricle can be easily evaluated in vivo by using transgenic lines or in fixed embryos by immunofluorescence (Table 1). Heart beating rate, as well as red blood cells perfusion rate, can be analyzed by video recording and further image analysis (Table 1). In addition, cardiac function can be studied by electrocardiograms, given the similarity between zebrafish and human heart electric patterns [18]. As a distinct feature, and because of their small size, ZE are not completely dependent on a functional cardiovascular system, carrying on a relatively normal development for several days in the absence of cardiovascular function [12]. Therefore, ZE can be analyzed in conditions (genetic, environmental, toxic) leading to serious cardiovascular defects that will be lethal in other systems, facilitating the discovery and characterization of toxic compounds severely impairing cardiovascular development [19,20]. The general pattern of formation of new blood vessels, or angiogenesis, during zebrafish development resembles the human one, with similar key genes expressed at the corresponding developmental stages [21,22]. Angiogenesis can be easily followed in zebrafish, either by monitoring specific blood vessel markers, like the endogenous alkaline phosphatase activity, or by visualizing the whole circulatory system with appropriated staining (microangiography), fluorescence transgenic lines or whole-mount in situ hybridization (Table 1). Angiogenesis is a major target for anticancer research, but it also lies on the origin of different degenerating diseases, like diabetic retinopathy or macular degeneration [23]. Therefore, the possibility to use zebrafish to assess the effects of new molecules on it is relevant both in terms of new anticancer drug discovery and of prevention of most relevant off-target effects of candidate drugs.

Expert Opin. Drug Metab. Toxicol. (2014) 10(5)


Table 1. Zebrafish toolbox for drug toxicity assessment. System Cardiovascular system



Parameter Heart: morphology and function Blood circulation

Tg (cmlc2:dsRed2nuc) Tg (cmlc2:gfp) Tg (gata1:gfp)

Angiogenesis

Tg (vegfr2:GRCFP) Tg (fli1:EGFP)y1 Tg (gut:GFP)

Intestinal peristaltism Liver: morphology and function

Nervous system

Transgenic lines

Gastrointestinal tract function Dopaminergic

LF2.8-EGFP

ETvmat2:GFP Tg (dat:EGFP)

Lateral line neuromasts

Tg (Pou4F3:GFP)

Myelination

Tg (mbp:EGFP-CAAX) Tg (mbp:EGFP)

Motor neurons

Tg (mnx1:GFP) ml2/+ AB

Neuromuscular junction

Muscle system

Endocrine system

Tg (smyhc1:GFP)

Estrogenicity

Tg (cyp19a1b: cyp19a1b-GFP)

Alternative method

Ref.

MF20 and S46 antibodies staining Video recording blood circulation in wild type fli1, flk4, flt4 in situ hybridization Video recording and image analysis Streptavidin-CY3 (CY3-SA)/ anti-MDR1 antibody for labeling bile canaliculi PED6, EnzChek

[97-99]

TH and slc6a3 (dat) and slc18a2 (vmat2) in situ hybridization/TH antibody Fluorescent dyes (DiAsp, SASPEI, YO-PRO1, FM1-43) for in vivo staining/acetylated alpha-tubulin antibody for labeling kinocillia Anti-MBP antibody/mbp and P0 in situ hybridization/Luxol blue staining Znp1 or SV2 antibodies for PMNs and zn8 for SMNs/ alpha-tubulin antibody for axonal tracts of spinal nerves AChE staining as presynaptic marker/bungarotoxin linked to a fluorochrome as post-synaptic marker F59 and F310 antibodies for labeling slow and fast muscle fibers, respectively Expression of aromatase gene in brain (quantitative Real-Time PCR, in situ)

[39,41,42]

[100] [101-103] [104,105] [34,106]

[35]

[39,41,107]

[49,57,60,108,109]

[57,68,69,109,110]

[60,65-67]

[73-76]

[94]

EGFP: Enhanced green fluorescent protein; mbp: Myelin basic protein; P0: Protein zero; PMN: Primary motor neuron; SMN: Secondary motor neuron.

Mitochondrial toxicants have profound effects on zebrafish development and cardiovascular functions. Mitochondria are a drug target for some dysfunction diseases and in antiparasitic chemotherapy. Cardiotoxicity-related effects (decreased heart size, altered vascular remodeling, pericardial edema) are induced by mitochondrial toxicants, as well as by substances that exacerbate the oxidative metabolism, for example, through the ectopic activation of the aryl hydrocarbon receptor, among other mechanisms (Figure 1A -- C) [20,24,25]. 3.


Although some components of the zebrafish gastrointestinal tract present important differences with the human gut, its simplified intestine presents most of the cell types observed in the human small intestine, like enterocytes, endocrine, goblet and interstitial cells of Cajal, all of them already present at 5 dpf [26]. Anterograde and retrograde peristaltic contractions,

as well as local rectal contractions, are present in zebrafish intestine between 4 and 7 dpf. Intestinal peristalsis is under the control of the enteric nervous systems, which respond to different pharmaceuticals in similar way as the mammalian counterpart. For example, ZE can be used to predict emetic response to pharmaceuticals, one of the most commonly reported clinical adverse effects to be considered in the development of new drugs [27]. Because of the transparency of the larvae, peristaltic contractions can be observed under the microscope and can be quantified in vivo [6,28]. Hepatotoxicity has been recognized by the pharmaceutical industry as a major toxicological problem. Compounds inducing steatosis in human liver, as amiodarone, simvastatin, tetracycline or valproic acid, have similar effects in ZE [29,4]. Hepatomegaly has been evaluated in vivo by using fluorescent reporters (Table 1) [30]. Thus, the induction of hepatomegaly in zebrafish larvae by the local anesthetic bupivacaine has been detected by using streptavidin-CY3 (Figure 1D -- G).


687


D.

E.

F.

G.

A.

e B.

d

b

c


a

H. 3

C.

e

ys

h

I.

e d b

c

e h

ys

a

Figure 1. Evaluation of the toxic effects of drugs on gross morphology in zebrafish eleutheroembryos, including effects on specific organs. (A -- C) Analysis of the gross morphology defects induced by chemicals. Analysis of the gross morphology in 96 hpf zebrafish non-exposed (A) or exposed to benzo[a]pyrene (B) or benzo[k]fluoranthene (C) indicates strong cardiotoxic effect for these two dioxin-like compounds. Abbreviations: a: pericardial edema; b: malformation of the lower jaw; c: malformation of the tail; d: color of the yolk; e: coagulation. (D -- G) Analysis of hepatomegaly induced by drugs in zebrafish larvae. CY3-SA staining (D,E) and bright-field (F,G) images of 7 dpf zebrafish larvae control (D,F) or treated for 3 days with 0.3 µM of bupivacaine (E,G). The liver is outlined in white. Lateral view, with the anterior to the left, dorsal to the top (H -- I): analysis of malnutrition in zebrafish embryos. Oil red O (ORO) staining of a control (H) and a treated (I) embryos shows that after treatment with clofibrate, there is no transfer of neutral lipids between the yolk sac and the embryo, with the induction of an embryonic malabsorption syndrome characterized by embryos with an small size and a big yolk sac. a et al. [60]. Modified from Raldu dpf: Days post-fertilization; e: Eye; h: Heart; hpf: Hours post-fertilization; ys: Yolk sac.

Other zebrafish tools available for assessing hepatotoxicity include selective labeling of bile canaliculi [30], liver histopathology, induction of hepatotoxicity-associated gene expression [29], and in vivo imaging of hepatic (and other digestive-related) functions using multiple quenched fluorescent reporters (Table 1) [31]. 4.

Neurotoxicity assessment

ZE and eleutheroembryos are exceptionally well suited for developmental neurotoxicity studies integrating cellular, molecular and genetic approaches. Since ZE and eleutheroembryos are transparent, specific neurons and axon tracts, as well as glial cells, can be visualized in vivo using transgenic lines or by injecting reporter dyes [4]. Specific types of neurons and glial cells can be also visualized in fixed intact zebrafish by whole-mount immunohistochemistry or in situ hybridization [32,33]. In addition, the small size of early-stage zebrafish permits performance of quantitative whole animal assays in a 96-well microplate format for neurotoxicity screening. The zebrafish model has been already used for assessing 688

the toxic effect of different xenobiotics on specific cell types in the nervous systems, like dopaminergic neurons or the mechanosensory system [32,33]. Drug effects on dopaminergic neurons in ZE can be assessed in fixed specimens by whole-mount immunochemistry or in situ hybridization against markers for dopaminergic neuron, like tyrosine hydroxylase or the dopamine transporter [32-34]. By using this approach, it has been demonstrated that both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and sodium benzoate induce a strong down-regulation in the expression of tyrosine hydroxylase mRNA and in the tyrosine hydroxylase-positive cells in the ventral diencephalon. These drugs also decreased the expression of slc6a3, a membrane transport protein involved in dopamine reuptake that is a specific marker of dopaminergic neurons [34]. A recent chemical screening of over 5000 compounds in zebrafish eleutheroembryos revealed the impairment of dopaminergic neuronal survival by some cardiac glycosides by using a similar approach [35]. The recent development of transgenic zebrafish lines in which structural or functional markers of dopaminergic neurons are labeled with the green




fluorescent protein (GFP) would facilitate further screening of drug candidates in regard to their neurotoxic effect (Table 1) [36-38]. The mechanosensory systems of fish, including the lateral line, are closely related to the hearing system of higher vertebrates [39]. Besides possessing the typical vertebrate inner ear, fish possess the lateral line organs that contain sensory hair cells. These analogies make the assessment of the zebrafish mechanosensory system as a relevant tool in safety pharmacology. The fish lateral line is composed of rosette-like structures called neuromasts, composed of hair cells and supporting cells. Hair cells are innervated by sensory neurons that are localized in the anterior and posterior lateral line ganglia [40]. Neuromasts are located on the fish surface, with their peripheral sensory neurons in direct contact with the surrounding water containing pollutants. Hair cells of the neuromast can be easily stained by various fluorescent dyes in vivo or in fixed larvae by using whole-mount immunofluorescence or wholemount in situ hybridization (Table 1) [39,41]. The intensity of hair cell staining can be imaged and quantified using image-analysis systems, and the effects can be assessed for the screening of potential ototoxic pollutants [39,42]. Gentamicin and streptomycin are also known to induce hair cell loss [43,44], as well as different heavy metals as cadmium, copper, zinc, iron and silver [39,41]. In a massive study, NINDS Custom Collection II library of 1040 Food and Drug Administration-approved drugs and known bioactives was screened for ototoxic effects in 5 dpf zebrafish eleutheroembryos by using the vital dye YO-PRO1, and 21 compounds were identified as ototoxic, including aminoglycosides, antiprotozoal agents, an anti-neoplastic agent, an anticholinergic agent and an antihyperlipidemic agent [42]. Moreover, a hair cell toxicity screen performed recently in zebrafish on a library of 88 anticancer drugs (National Cancer Institute Approved Oncology Drugs Set) identified successfully an 80% of known ototoxins [45]. Some pharmaceuticals, as amiodarone, suramin, tacrolimus or cis-platin, seem to present primary adverse effects on myelin sheaths [46,47]. Disruption of myelin sheaths or myelinated axons is extremely detrimental, inducing severe neurological disorders. Zebrafish myelination is homologous to the mammalian system, as orthologous genes for all the major mammalian myelin-associated genes have been already identified [48]. The major biochemical difference between zebrafish and mammalian myelin is the presence of protein zero as a major CNS myelin protein in zebrafish, rather than proteolipid protein in mammals. Nevertheless, different studies have revealed fundamental similarities in glial cell development and myelination between teleost and mammals [49], suggesting that the zebrafish model may contribute to the understanding of human myelinopathies [50,51]. Numerous tools, including in situ hybridization probes, primary antibodies and some transgenic lines, are already available to study potential adverse effects of drugs on myelinated axons in zebrafish (Table 1) [51]. Luxol blue staining on

histological sections has been used to demonstrate the adverse effects of acrylamide on the integrity of the myelin sheaths in the zebrafish brain [52]. The potential adverse effect of drugs on zebrafish eleutheroembryos myelin sheaths can be easily been evaluated by analyzing the myelin basic protein (mbp) expression in oligodendrocytes and Schwann cells using whole-mount in situ hybridization and immunofluorescence, a methodology initially developed for genetic screenings of genes involved in myelination in Schwann cells and oligodendrocytes [50,51,53]. The recent generation of different transgenic lines expressing enhanced GFP (EGFP) under the control of the mbp promoter (Table 1) opens the possibility to evaluate in vivo the time-course of the neurotoxic effects of candidate drugs on myelinating oligodendrocytes and Schwann cells [54,55].

5.

Neuromuscular system

Zebrafish axial muscles are innervated by primary and secondary spinal motor neurons. There are three primary motor neurons (PMNs) in each spinal hemisegment, one of which innervates the dorsal trunk musculature, whereas the other two innervate mid and ventral regions of the trunk. Secondary motor neurons (SMNs) follow similar paths to those taken by PMNs, although they do not branch as extensively and are more numerous. The stereotypic pattern of the axonal projections and specific neurotoxic effects on motor neurons can be assessed by whole-mount immunofluorescence with axon-specific antibodies as well as by using different transgenic lines (Table 1). Figure 2A -- D shows an evaluation of the potential adverse effects of caffeine on motor systems [32,56]. It has been demonstrated that exposition to some drugs, like vincristine, bortezomib, nicotine, thiocyclam and disulfiram, induces adverse effects on the motor neuron development and a classification of motor neuron defects has been proposed [57,58]. An important point to take in consideration during the assessment of potential adverse effects of drugs on spinal motor neurons is that during the first days of development axogenesis is an extremely dynamic process, and embryos with small differences in development (a few hours) exhibit strong morphological differences. Thus, it is essential to be familiar with the time-course of spinal motor neurons axogenesis to be able to discriminate between a specific pathological effect and a delay in development. The effect of drugs on the neuromuscular junctions (NMJs), an essential part of the neuromuscular system, may also be easily analyzed by using pre- and post-synaptic markers (Table 1). On one hand, whole-mount acetylcholinesterase staining allows the analysis of the effect of the drug on the morphology and the number of NMJs [59]. On the other hand, fluorophore-conjugated a-bungarotoxin, binding specifically acetylcholine receptors, is commonly used as post-synaptic marker of the NMJ in the evaluation of adverse effects of drugs at the NMJ level (Figure 2G). Using the latter


689


A.

B.

C.

D.


E.

Figure 2. Morphological markers used in the assessment of potential adverse effects of the motor systems. A -- D: adverse effects of caffeine on spinal motor neurons. Immunolabeling with znp-1 (A -- B) shows that the stereotyped pathways followed by the axons of the rostral (RoP, yellow arrow), middle (MiP, white arrow) and caudal (CaP, red arrow) PMNs in 57 hpf zebrafish embryos (A) are clearly disrupted in embryos treated with 0.77 mM caffeine (B), where RoP and MiP axons are missing, and CaP main axons present abnormal morphology and reduced branching. Immunolabeling with zn8 (C -- D) shows that stereotyped pathways followed by the axons of the secondary motor neurons following a rostral-like (yellow arrow) and a caudal-like (red arrow) PMN pathways in 57 hpf zebrafish eleutheroembryos (C) are strongly disrupted in embryos treated with 0.77 mM caffeine (D). E: The highly organized muscle fibers of a control 12 dpf zebrafish larva are able to rotate polarized light inducing birefringency. Asterisks are labeling the choice point of the spinal motor neurons. dpf: Days post-fertilization; hpf: Hours post-fertilization; PMN: Primary motor neuron.

approach, different defects in the NMJs have been found in zebrafish exposed to sodium benzoate and caffeine [60,61]. Zebrafish eleutheroembryos possess two types of skeletal muscle fibers. Slow (red) muscle fibers, a superficial monolayer on the surface of the myotome, are equipped for oxidative phosphorylation, can generate relatively large stores of energy and are most resistant to fatigue. Fast (white) muscle fibers, in the deep portion of the myotome, are least resistant to fatigue because they rely on anaerobic glycolysis for ATP generation. The two types of muscle fibers perform different functions, where fast muscle fibers are inactive during slow swimming episodes and fast and slow muscle fibers are recruited during fast swimming. Fast muscle fibers are innervated by both PMNs and SMNs, while slow muscle fibers are likely only innervated by SMNs. Adverse effects of drugs on muscle fibers organization can be easily evaluated by using birefringence (Figure 2E), a phenomenon in which the highly ordered somitic muscle has the unique property of being able to rotate polarized light. This assay can be easily performed in vivo by placing the eleutheroembryo or larval in lateral view, between two polarizing filters and aligning the filters, until only the rotated light is visible. Using this assay, a decrease in the amount of rotated light could be indicative of the loss of the sarcomeric structure within muscle, whereas dark patches in the muscle could be indicative of muscle tearing or muscle fiber disorganization [62]. Labeling of slow and fast muscle fibers with specific antibodies (Table 1) allows detection of subtle changes in the muscle fiber alignments. Evaluated endpoints included the length, width and number of the muscle fibers as well as 690

disorganized muscle fiber alignment. This disorganization can be reflected by a lack of segment division, presence of fibers extending over two segments rather than one, altered angles between dorsal and ventral hemi-segments, and smaller muscle fibers. Different drugs, including diclofenac, vincristine, bortezomib, lovastatin, nifurtimox and benznidazole, exhibit a myotoxic effect for slow muscle fibers of zebrafish [58,63,64], whereas ethanol has also been proved to be myotoxic for fast muscle fibers [65]. The availability of different GFP transgenic lines labeling the slow-muscle fibers opens the possibility to perform high-throughput screening (HTS) for myotoxicity in small chemical libraries [66,67].

Assessment of the effect on motor behavior

6.

The behavioral repertoire of the zebrafish eleutheroembryos and the behavioral screening assays for neuroactive drugs currently available have been recently reviewed [68,69]. Different assays have been developed for the analysis of the different behaviors, and some of these assays, as locomotor activity and photomotor response, are suitable for HTS. Thus, two largescale small-molecule screens have examined the effects of thousands of drugs on larval zebrafish sleep/wake and photomotor response behaviors. Both screens identified hundreds of molecules that altered zebrafish behavior in distinct ways, and the behavioral profiles induced by these active molecules enabled the clustering of compounds according to shared phenotypes [70,71]. Moreover, it is possible to analyze the effect of drugs on locomotor activity in zebrafish eleutheroembryo



Control A.

C. 25.00

MMI B.

Average pixel intensity


20.00

15.00 *

* *

10.00

‡

5.00 ‡

0.00 DMSO

Atrazine

2,4-D

DDT Fenoxycarb 4-NP

MeHg 60H-DOPA MMI

Figure 3. Analysis of the disrupting effect of the chemicals on the thyroid gland function by T4-immunofluorescence coupled to a further image analysis in zebrafish eleutheroembryos. While the thyroid follicles in 5 dpf untreated zebrafish are already filled by T4 (A), treatment for 3 days with the antithyroid drug methimazole (B) is able to block the thyroid function resulting in a total abolition of the intrafollicular T4-content. T4 immunofluorescence quantitative disruption test (TIQDT) on zebrafish eleutheroembryos allows screen molecules for its potential to disrupt the thyroid gland function (C). Ventral view of the head, with the anterior to the bottom. dpf: Days post-fertilization.

or early larval, under both light and dark conditions, in a 96-well plate, by using commercially available video-tracking systems [72]. Locomotor activity assessment has been also extensively used with drugs inducing tonic--clonic convulsions, as pentylenetetrazole, a seizure-inducing drug, and the results suggest that this assay is suitable for monitoring seizure-like activity as a neurotoxicity marker [73]. All above-mentioned behavioral assays are devoted to quantify changes in the ‘quantity’ of movement, but without considering potential changes in the ‘quality’ of the movement. Escape response evoked by a variety of stimulus in eleutheroembryos or early larvae is a stereotyped complex behavior built from a repertoire of simpler behavioral modules. Neuronal circuits involved in these responses include reticulospinal inputs to the spinal cord, specific spinal interneurons and the spinal motor neurons [68]. The kinematic of this response can be disrupted by exposition to some drugs. For instance, kinematic analysis of the touch-evoked escape response has showed that exposition to the chemotherapy drug vincristine [58] causes uncoordinated swimming behavior, which is coupled to the adverse effects on PMNs and slow-muscle fibers. In contrast, bortezomib increases the

duration and amplitude of the escape response, which was coupled with a decrease in the axonal branching of PMNs and a mild effect on muscle fibers architecture [58]. The kinematic profile of the exposed zebrafish will give not only information about the adverse effects on behavior, but also can provide a valuable information about the components of the neural circuit potentially involved in the anomalous behavior.

Thyroid and sex hormones disrupting activity

7.

Impairment of the thyroid function is an important side effect of many drugs. Thus, many drugs including some sulfonamides and salycilamides, amphenone and ketoconazole exhibit as undesirable side effect the inhibition of synthesis of thyroid hormone [74]. Well-known drugs can impair the thyroid function at different levels of the hypothalamus--pituitary--thyroid axis, including synthesis, transport and metabolism of the thyroid hormone [75-77]. This plurality of possible targets makes it challenging to determine from among a very long list of potential thyroid toxicants, primarily


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tested in rodent and in vitro models, which are relevant to human thyroid function [75]. Zebrafish thyroid system development has been well characterized [78]. Moreover, the essential role played by the thyroid hormone during the transition larva--juvenile (sometimes described as metamorphosis), a complex developmental process including resorption of the larval fin fold and development of adult unpaired fins, changes in the gut, peripheral nervous system and sensory systems (including eyes), alterations in physiology and behavior, substitution of adult hemoglobin for embryonic forms, development of scales and formation of an adult pigment pattern, has been demonstrated [79,80]. Distortions on the timing of the onset of thyroid hormone may have deleterious effects on the developing embryo. Some of these changes can be detected in the zebrafish eleutheroembryos, as several functions, like ossification, visual processes and the hematopoietic system alter their natural developmental pattern upon the presence of heterologous thyroid hormones [81]. Moreover, the development and function of the thyroid follicles can be visualized in the early zebrafish embryo, allowing the screening of individual drugs and mixtures impairing thyroid gland development and/or function (Figure 3) [77,82,83]. Qualitative (nature of phenotypes) and quantitative (effective concentrations) concordance between this kind of zebrafish assays and published mammalian and other vertebrate data has been found to be very high [83], illustrating the convenience of zebrafish assays to monitor the capacity of new drugs to disrupt the human thyroid gland function. As reproductive systems are not fully developed at the early developmental stages, ZE are not well suited for the study of fertility or reproductive effects. However, recent developments suggest that the estrogenic response is fully operational in brain, pancreas and liver, at least at 3 -- 4 dpf, if not earlier [84], and that this may be also the case for the androgen response [85]. This opens the possibility, not fully developed yet, to use zebrafish embryo assays also for testing potential endocrine disruptors based on their ability to activate (or to suppress) hormonal responses through the corresponding hormone receptors.

8.

High-throughput approaches

Zebrafish is presently the only vertebrate system for which comprehensive high-throughput approaches have been developed and tested. Adult mating, embryo sorting and arraying, drug/substance administration, and data acquisition and analysis can be devised in an efficient work flow-trough that allows analysis of hundreds if not thousands of substances in parallel [86]. The high fertility of zebrafish and the small size of the embryo up to the 120 hpf facilitate the multiplate array configuration of the experiments, which in turn allow automatic delivery of substances by different robotized methods [86,87]. In this section, we will focus on the detection of both 692

unspecific and specific toxic endpoints at high-throughput scale. ZE and eleutheroembryos are relatively transparent, making bioimaging approaches possible at levels that are unknown to other whole animal systems. Even the slight pigmentation naturally present in zebrafish can be circumvented, if necessary, either by treatment with the tyrosinase inhibitor 1-phenyl-2-thiourea or by using the transparent zebrafish lines, as casper, which has no pigmentation [88]. Whereas analyzing images used to be tedious and time consuming [1], recent advances in microscope technology and image processing allow processing and analyzing phenotypes from hundreds or thousands of individual embryos, thus enabling the use of complex image-based read-outs for high throughput [89]. There are at least four levels of image processing that can be applied to high-throughput toxicity analyses. In the first place, advanced white-field microscopy coupled to appropriate image-analysis protocols may detect gross defects on embryo development, including egg coagulation and embryo deformities [63,90-92]. Finer analyses of structural changes implicating specific tissues can be performed using transgenic zebrafish lines harboring fluorescent derivatives of specific proteins that act as markers of a given tissue (Table 1). For example, the fli-1: EGFP line allows visualization of the process of neovascularization in ZE, thus enabling the identification of effects affecting angiogenesis [19]. Similar strategies have been applied to different tissues, including cardiac, nervous and muscular [93], and are getting implemented for the estrogenic response as well [94]. A third level of image-base analyses is brought about by the use of fluorescent reporter assays that monitor the expression of specific markers. In zebrafish there are multiple transgenic lines in which the expression of a fluorescent protein is directed by a well-characterized promoter, either natural or artificial. In this case, the output reflects global changes on the fluorescence intensity, giving results comparable to a cell-based reporter assay or a quantitative real-time PCR (qRT-PCR) assay, with the advantage that the fluorescent signal can be followed in vivo, during the normal development of the individual. This strategy allows the direct monitoring of primary transcriptional effects even at levels that would not elicit macroscopic phenotypes, which is useful to determine toxic modes of action for a given substance or to predict its overall toxicity even at low concentrations. This is especially useful to analyze the potential endocrine disruption capacity of a substance, even for endpoints for which embryonic systems are not well suited, like estrogenic effects [94]. It may also give important information on the activation of general stress genes or on the onset of genes involved in tissue formation or differentiation [95]. Finally, the use of cameras, rather than static images, combined with appropriated algorithms, allows the recording of movement or reaction-base phenotypes, particularly important when dealing with neuroactive compounds. Similar



strategies can be used to detect and monitor heartbeat and blood flow.


9.

Conclusion

Widely used as a toxicological model, ZE and eleutheroembryos represent a valuable biotechnological tool to identify and characterize toxic effects of new candidate drugs at much higher level of specificity than standard toxicological analyses. Whereas information on acute toxicity would be always the first relevant data on any screening scheme, the zebrafish system allows dissection of the toxic effect and mode of action in different tissues and functions at the whole organism level. The zebrafish embryo model benefits from the important advances on genomics and different in situ methodologies that have been applied to zebrafish in last years. Future developments, like in situ proteomic analyses [96], are likely to facilitate the analysis of complex toxicological effects of new substances in the next future. The use of molecular techniques allows the monitoring of the initial step(s) of the toxicological mode of action, which in principle should result in a higher predictive capacity and sensitivity of the assay. Perhaps the most relevant characteristic of zebrafish embryo assays is their integrative capacity, as it allows the determination of a wide range of effects in a single system. Finally, the developmental characteristics and transparency of the ZE allow the implementation of image-based high-throughput methodologies, thus allowing the screening of the high number of new products required for the modern pharmaceutical industries. 10.

Expert opinion

Off-target effects of candidate drugs represent a major issue in the development of new remedies or in the reassessment of already existing ones. In many cases, off-target effects occur in tissue and body functions completely unexpected and utterly independent from the intended targets the pharmacological efficacy depends upon. For this reason, in toto approaches involving complete organisms are preferred to partial, single-cell approaches. However, the use of complex organisms (mammals, adult vertebrates) is expensive and time consuming, and it is in direct conflict with the current requirements for minimizing animal testing. Zebrafish offers the unique possibility of following the complete embryo development in a matter of few days, in a system for which abundant and very powerful genetic tools have been

developed and with relatively few requirements in terms of installations and husbandry. Major anatomic systems (digestive, circulatory, nervous, muscular, endocrine) relevant on assessing drug toxicity can be analyzed in early ZE, and all available data indicate that their response to external insults, including toxic effects, are comparable to that observed in humans. ZE are particularly amenable to high-throughput approaches, and several of the existing configurations allow the monitoring of adverse effects in vivo, using nondestructive analytical tools. Nevertheless, as in any other model, using ZE presents some intrinsic disadvantages. ‘Adult’ functions related to reproduction and fertility cannot be adequately assessed in embryos, as well as effects on tissues and cell types that develop during or after larval stages, like the fat tissue, for example. A second level of difficulties on evaluating toxic effects arises from the needed to discriminate between developmental toxicity and specific organ toxicity. During early development, zebrafish embryo is an extremely dynamic system, with huge changes in the morphology of some systems in only a few hours, and is well known that some drugs are able to induce a global delay in development in different degrees. As a result, the morphology and function of specific organs in embryos treated with those drugs inducing a global delay in development, even if this delay is small, can show big differences with the controls. Finally, adverse effects on development, like developmental neurotoxicity, produced by drugs inducing embryonic malabsorption syndrome are not necessarily relevant for placentary vertebrates. Despite the unavoidable drawbacks, ZE are bound to represent an essential tool for drug toxicology in general. Their unique characteristic among vertebrates of allowing the application high-throughput optical imaging methodologies opens the possibility to perform toxicological analyses at high scale and therefore the screening of large numbers of different chemicals for many diverse and relevant toxic endpoints.

Declaration of interest This work was supported by the Spanish Ministry of Economy and Competitiveness (CGL2011-29621 and CTM2011-30471-C02-01) and the Generalitat de Catalunya (grant 2009 SGR 924). The authors do not have any conflict of interest to declare.


693


Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers.

11.

Weinstein BM, Schier AF, Abdelilah S, et al. Hematopoietic mutations in the zebrafish. Development 1996;123:303-9

1.

12.

Stainier DYR. Zebrafish genetics and vertebrate heart formation. Nat Rev Genet 2001;2:39-48


..

Love DR, Pichler FB, Dodd A, et al. Technology for high-throughput screens: the present and future using zebrafish. Curr Opin Biotechnol 2004;15:564-71 Discussion on the applications of HTS methodologies on zebrafish.

2.

Goldsmith P. Zebrafish as a pharmacological tool: the how, why and when. Curr Opin Pharmacol 2004;4:504-12

3.

Zon LI, Peterson RT. In vivo drug discovery in the zebrafish. Nat Rev Drug Discov 2005;4:35-44

4.

McGrath P, Li CQ. Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov Today 2008;13:394-401

5.

Zon L, Peterson R. In vivo drug discovery in the zebrafish. Nat Rev Drug Discov 2005;4:35-44

6.

Berghmans S, Butler P, Goldsmith P, et al. Zebrafish based assays for the assessment of cardiac, visual and gut function -- potential safety screens for early drug discovery. J Pharmacol Toxicol Methods 2008;58:59-68 Monitoring of drug effects on systemic physiological functions.

.

13.

Lambrechts D, Carmeliet P. Genetics in zebrafish, mice, and humans to dissect congenital heart disease: insights in the role of vegf. In: Schatten GP, editor, Developmental vascular biology. Volume 62; 2004. p. 189-224

14.

Baker K, Warren KS, Yellen G, et al. Defective “pacemaker” current (ih) in a zebrafish mutant with a slow heart rate. Proc Natl Acad Sci USA 1997;94:4554-9

15.

Thisse C, Zon LI. Organogenesis -- heart and blood formation from the zebrafish point of view. Science 2002;295:457-62

16.

Bello SM, Heideman W, Peterson RE. 2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal vein in developing zebrafish. Toxicol Sci 2004;78:258-66

17.

Antkiewicz DS, Burns CG, Carney SA, et al. Heart malformation is an early response to tcdd in embryonic zebrafish. Toxicol Sci 2005;84:368-77

18.

Hu N, Yost HJ, Clark EB. Cardiac morphology and blood pressure in the adult zebrafish. Anat Rec 2001;264:1-12

7.

Scholz S. Zebrafish embryos as an alternative model for screening of drug-induced organ toxicity. Arch Toxicol 2013;87:767-9

19.

Chen T, Zhang R-H, He S-C, et al. Synthesis and antiangiogenic activity of novel gambogic acid derivatives. Molecules 2012;17:6249-68

8.

Russell W, Burch R. The principles of humane experimental techniques. Methuen, London, UK; 1959

20.

9.

Broom D, Sherwin C, Gregory N, et al. EFSA: opinion of the scientific panel on animal health and welfare on a request from the commission related to the aspects of the biology and welfare of animals used for experimental and other scientific purposes (efsa-q-2004-105). EFSA J 2005;292:1-46 Theoretical basis of the implementation of zebrafish embryos (and other systems) as replacement models for animal testing.

Pinho BR, Santos MM, Fonseca-Silva A, et al. How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs. Br J Pharmacol 2013;169:1072-90

21.

Fouquet B, Weinstein BM, Serluca FC, et al. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Dev Biol 1997;183:37-48

22.

Liang D, Xu X, Chin AJ, et al. Cloning and characterization of vascular endothelial growth factor (vegf) from zebrafish, danio rerio. Biochim Biophys Acta 1998;1397:14-20

.

10.

694

Ma BL, Ma YM. Pharmacokinetic properties, potential herb-drug interactions and acute toxicity of oral rhizoma coptidis alkaloids. Expert Opin Drug Metab Toxicol 2013;9:51-61

23.

Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57

24.

Incardona JP, Collier TK, Scholz NL. Defects in cardiac function precede


morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol Appl Pharmacol 2004;196:191-205 25.

Scott JA, Incardona JP, Pelkki K, et al. Ahr2-mediated, cyp1a-independent cardiovascular toxicity in zebrafish (danio rerio) embryos exposed to retene. Aquat Toxicol 2011;101:165-74

26.

Wallace KN, Akhter S, Smith EM, et al. Intestinal growth and differentiation in zebrafish. Mech Dev 2005;122:157-73

27.

Barros TP, Alderton WK, Reynolds HM, et al. Zebrafish: an emerging technology for in vivo pharmacological assessment to identify potential safety liabilities in early drug discovery. Br J Pharmacol 2008;154:1400-13

28.

Roach G, Wallace RH, Cameron A, et al. Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility. Dev Biol 2013;376:171-86

29.

Driessen M, Kienhuis AS, Pennings JL, et al. Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling. Arch Toxicol 2013;87:807-23

30.

Sadler KC, Amsterdam A, Soroka C, et al. A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease. Development 2005;132:3561-72

31.

Hama K, Provost E, Baranowski TC, et al. In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters. Am J Physiol Gastrointest Liver Physiol 2009;296:G445-G53

32.

Ton C, Lin Y, Willett C. Zebrafish as a model for developmental neurotoxicity testing. Birth Defects Res A Clin Mol Teratol 2006;76:553-67 Study of developmental neurotoxicity in zebrafish embryos.

.

33.

Parng C, Roy NM, Ton C, et al. Neurotoxicity assessment using zebrafish. J Pharmacol Toxicol Methods 2007;55:103-12

34.

Chen Q, Huang NN, Huang JT, et al. Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neurons in developing


47.

Stillman M, Cata JP. Management of chemotherapy-induced peripheral neuropathy. Curr Pain Headache Rep 2006;10:279-87

48.

Buckley CE, Goldsmith P, Franklin RJM. Zebrafish myelination: a transparent model for remyelination? Dis Model Mech 2008;1:221-8

49.

Brosamle C, Halpern ME. Characterization of myelination in the developing zebrafish. Glia 2002;39:47-57

Wen L, Wei W, Gu W, et al. Visualization of monoaminergic neurons and neurotoxicity of mptp in live transgenic zebrafish. Dev Biol 2008;314:84-92

50.

Pogoda H-M, Sternheim N, Lyons DA, et al. A genetic screen identifies genes essential for development of myelinated axons in zebrafish. Dev Biol 2006;298:118-31

Xi Y, Yu M, Godoy R, et al. Transgenic zebrafish expressing green fluorescent protein in dopaminergic neurons of the ventral diencephalon. Dev Dyn 2011;240:2539-47

51.

zebrafish. Birth Defects Res B Dev Reprod Toxicol 2009;86:85-91 35.

36.


37.

38.

39.

40.

41.

42.

43.

Sun F, Kanthasamy AG, Kanthasamy A. Measurement of proteasomal dysfunction in cell models of dopaminergic degeneration. Methods Mol Biol 2011;758:293-305 Ren CG, Wang L, Jia XE, et al. Activated n-ras signaling regulates arterial-venous specification in zebrafish. J Hematol Oncol 2013;6:34

Froehlicher M, Liedtke A, Groh KJ, et al. Zebrafish (danio rerio) neuromast: promising biological endpoint linking developmental and toxicological studies. Aquat Toxicol 2009;95:307-19 Bricaud O, Chaar V, Dambly-Chaudiere C, et al. Early efferent innervation of the zebrafish lateral line. J Comp Neurol 2001;434:253-61

53.

Yang L, Kemadjou J, Zinsmeister C, et al. Transcriptional profiling reveals barcode-like toxicogenomic responses in the zebrafish embryo. Genome Biol 2007;8:R227

54.

Chiu LL, Cunningham LL, Raible DW, et al. Using the zebrafish lateral line to screen for ototoxicity. J Assoc Res Otolaryngol 2008;9:178-90 Seiler C, Nicolson T. Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants. J Neurobiol 1999;41:424-34

44.

Ton C, Parng C. The use of zebrafish for assessing ototoxic and otoprotective agents. Hear Res 2005;208:79-88

45.

Hirose Y, Simon JA, Ou HC. Hair cell toxicity in anti-cancer drugs: evaluating an anti-cancer drug library for independent and synergistic toxic effects on hair cells using the zebrafish lateral line. J Assoc Res Otolaryngol 2011;12:719-28

46.

52.

Peltier AC, Russell JW. Recent advances in drug-induced neuropathies. Curr Opin Neurol 2002;15:633-8

59.

Raldua D, Andre M, Babin PJ. Clofibrate and gemfibrozil induce an embryonic malabsorption syndrome in zebrafish. Toxicol Appl Pharmacol 2008;228:301-14

60.

Chen YH, Huang YH, Wen CC, et al. Movement disorder and neuromuscular change in zebrafish embryos after exposure to caffeine. Neurotoxicol Teratol 2008;30:440-7

61.

Tsay HJ, Wang YH, Chen WL, et al. Treatment with sodium benzoate leads to malformation of zebrafish larvae. Neurotoxicol Teratol 2007;29:562-9

62.

Guyon JR, Steffen LS, Howell MH, et al. Modeling human muscle disease in zebrafish. Biochim Biophys Acta 2007;1772:205-15

63.

Parng C, Roy NM, Ton C, et al. Neurotoxicity assessment using zebrafish. J Pharmacol Toxicol Methods 2007;55:103-12

Chen S, Zhu Y, Xia W, et al. Automated analysis of zebrafish images for phenotypic changes in drug discovery. J Neurosci Methods 2011;200:229-36

64.

Kazakova N, Li HL, Mora A, et al. A screen for mutations in zebrafish that affect myelin gene expression in schwann cells and oligodendrocytes. Dev Biol 2006;297:1-13

Cao P, Hanai J, Tanksale P, et al. Statin-induced muscle damage and atrogin-1 induction is the result of a geranylgeranylation defect. FASEB J 2009;23:2844-54

65.

Sylvain NJ, Brewster DL, Ali DW. Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers. Neurotoxicol Teratol 2010;32:472-80

66.

Elworthy S, Hargrave M, Knight R, et al. Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for hedgehog and prdm1 activity. Development 2008;135:2115-26

67.

Nguyen-Chi ME, Bryson-Richardson R, Sonntag C, et al. Morphogenesis and cell fate determination within the adaxial cell equivalence group of the zebrafish myotome. PLoS Genet 2012;8:e1003014

68.

Fero KE, Jalota L, Hornuss C, et al. Pharmacologic management of postoperative nausea and vomiting. Expert Opin Pharmacother 2011;12:2283-96

69.

Rihel J, Schier AF. Behavioral screening for neuroactive drugs in zebrafish. Dev Neurobiol 2012;72:373-85

70.

Rihel J, Prober DA, Arvanites A, et al. Zebrafish behavioral profiling links drugs

Czopka T, Lyons DA. Dissecting mechanisms of myelinated axon formation using zebrafish. In: Detrich HW, Westerfield M, Zon LI, editors, Zebrafish: disease models and chemical screens. Methods in Cell Biology. Volume 105. 3rd edition. 2011. p. 25-62

Jung S-H, Kim S, Chung A-Y, et al. Visualization of myelination in gfptransgenic zebrafish. Dev Dyn 2010;239:592-7

55.

Almeida RG, Czopka T, Ffrench-Constant C, et al. Individual axons regulate the myelinating potential of single oligodendrocytes in vivo. Development 2011;138:4443-50

56.

de Soysa TY, Ulrich A, Friedrich T, et al. Macondo crude oil from the deepwater horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis. BMC Biol 2012;10:40

57.

Muth-Kohne E, Wichmann A, Delov V, et al. The classification of motor neuron defects in the zebrafish embryo toxicity test (zfet) as an animal alternative approach to assess developmental neurotoxicity. Neurotoxicol Teratol 2012;34:413-24

58.

outgrowth and behavioral defects in larval zebrafish. J Peripher Nerv Syst 2012;17:76-89

Khan TM, Benaich N, Malone CF, et al. Vincristine and bortezomib cause axon


695


Toxicol Appl Pharmacol 2013;269:169-75

to biological targets and rest/wake regulation. Science 2010;327:348-51 71.

Kokel D, Bryan J, Laggner C, et al. Rapid behavior-based identification of neuroactive small molecules in the zebrafish. Nat Chem Biol 2010;6:231-7

72.

Padilla S, Hunter DL, Padnos B, et al. Assessing locomotor activity in larval zebrafish: influence of extrinsic and intrinsic variables. Neurotoxicol Teratol 2011;33:624-30


73.

74.

75.

76.

Hortopan GA, Dinday MT, Baraban SC. Zebrafish as a model for studying genetic aspects of epilepsy. Dis Model Mech 2010;3:144-8

86.

Crofton KM. Thyroid disrupting chemicals: mechanisms and mixtures. Int J Androl 2008;31:209-23

Raldua D, Thienpont B, Babin PJ. Zebrafish eleutheroembryos as an alternative system for screening chemicals disrupting the mammalian thyroid gland morphogenesis and function. Reprod Toxicol 2012;33:188-97

82.

696

.

Brent GA, Braverman LE, Zoeller RT. Thyroid health and the environment. Thyroid 2007;17:807-9

78.

81.

84.

85.

Rald ua D, Babin PJ. Simple, rapid zebrafish larva bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. Environ Sci Technol 2009;43:6844-50

80.

.

Sarne D. Effects of the environment, chemicals and drugs on thyroid function. In: deGroot LJ, Hennemann G, editors, The thyroid and its diseases. Endocrine Education, Inc., Massachusetts; 2010

77.

79.

83.

Brown DD. The role of thyroid hormone in zebrafish and axolot development. Proc Natl Acad Sci USA 1997;94:13011-16 Parichy DM, Tumer JM. Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis. Dev Biol 2003;256:242-57 Pelayo S, Oliveira E, Thienpont B, et al. Triiodothyronine-induced changes in the zebrafish transcriptome during the eleutheroembryonic stage: implications for bisphenol a developmental toxicity. Aquat Toxicol 2012;110:114-22 Thienpont B, Barata C, Raldua D. Modeling mixtures of thyroid gland function disruptors in a vertebrate alternative model, the zebrafish eleutheroembryo.

..

Thienpont B, Tingaud-Sequeira A, Prats E, et al. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environ Sci Technol 2011;45:7525-32 Screening of potential thyroid disruptors in zebrafish. Hao RX, Bondesson M, Singh AV, et al. Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis. PLoS One 2013;8:18 Development of tools for detecting estrogens in zebrafish embryos. Gorelick DA, Watson W, Halpern ME. Androgen receptor gene expression in the developing and adult zebrafish brain. Dev Dyn 2008;237:2987-95 Delvecchio C, Tiefenbach J, Krause HM. The zebrafish: a powerful platform for in vivo, hts drug discovery. Assay Drug Dev Technol 2011;9:354-61 Description of zebrafish as a platform for drug discovery.

zebrafish line that expresses gfp in radial glial cells. Genesis 2009;47:67-73 95.

Walker SL, Ariga J, Mathias JR, et al. Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish. PLoS One 2012;7:14

96.

Nelson KA, Daniels GJ, Fournie JW, et al. Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging. J Biomol Tech 2013;24:119-27

97.

Hofsteen P, Plavicki J, Johnson SD, et al. Sox9b is required for epicardium formation and plays a role in tcdd-induced heart malformation in zebrafish. Mol Pharmacol 2013;84:353-60

98.

Nevis K, Obregon P, Walsh C, et al. Tbx1 is required for second heart field proliferation in zebrafish. Dev Dyn 2013;242:550-9

99.

Wu TS, Yang JJ, Yu FY, et al. Cardiotoxicity of mycotoxin citrinin and involvement of microrna-138 in zebrafish embryos. Toxicol Sci 2013;136:402-12

100. Heideman W, Antkiewicz DS, Carney SA, et al. Zebrafish and cardiac toxicology. Cardiovasc Toxicol 2005;5:203-14

87.

Wang KIK, Salcic Z, Yeh J, et al. Toward embedded laboratory automation for smart lab-on-a-chip embryo arrays. Biosens Bioelectron 2013;48:188-96

88.

White RM, Sessa A, Burke C, et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2008;2:183-9

101. Tran TC, Sneed B, Haider J, et al. Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish. Cancer Res 2007;67:11386-92

89.

Mikut R, Dickmeis T, Driever W, et al. Automated processing of zebrafish imaging data: a survey. Zebrafish 2013;10:401-21

102. Evensen L, Odlo K, Micklem DR, et al. Contextual compound screening for improved therapeutic discovery. ChemBioChem 2013;14(18):2512-18

90.

Xu X, Xu X, Huang X, et al. A high-throughput analysis method to detect regions of interest and quantify zebrafish embryo images. J Biomol Screen 2010;15:1152-9

91.

Vogt A, Cholewinski A, Shen X, et al. Automated image-based phenotypic analysis in zebrafish embryos. Dev Dyn 2009;238:656-63

103. Lam KH, Alex D, Lam IK, et al. Nobiletin, a polymethoxylated flavonoid from citrus, shows anti-angiogenic activity in a zebrafish in vivo model and huvec in vitro model. J Cell Biochem 2011;112:3313-21

92.

Jeanray N, Maree R, Pruvot B, et al. Phenotype classification of zebrafish embryos by supervised learning. Toxicol Lett 2012;211:S152

93.

Rubinstein AL. Zebrafish assays for drug toxicity screening. Expert Opin Drug Metab Toxicol 2006;2:231-40

94.

Tong SK, Mouriec K, Kuo MW, et al. A cyp19a1b-gfp (aromatase b) transgenic


104. Thakur PC, Davison JM, Stuckenholz C, et al. Dysregulated phosphatidylinositol signaling promotes endoplasmic reticulum stress-mediated intestinal mucosal injury and inflammation in zebrafish. Dis Model Mech 2014;7(1):93-106 105. Ng ANY, de Jong-Curtain TA, Mawdsley DJ, et al. Formation of the digestive system in zebrafish: iii. Intestinal epithelium morphogenesis. Dev Biol 2005;286:114-35


106.

107.

Moon IS, So J-H, Jung Y-M, et al. Fucoidan promotes mechanosensory hair cell regeneration following amino glycoside-induced cell death. Hear Res 2011;282:236-42 Pogoda HM, Sternheim N, Lyons DA, et al. A genetic screen identifies genes

essential for development of myelinated axons in zebrafish. Dev Biol 2006;298:118-31 109.

Khan TM, Benaich N, Malone CF, et al. Vincristine and bortezomib cause axon outgrowth and behavioral defects in larval zebrafish. J Peripher Nerv Syst 2012;17:76-89

110.

Kanungo J, Cuevas E, Ali SF, et al. Ketamine induces motor neuron toxicity and alters neurogenic and proneural gene

expression in zebrafish. J Appl Toxicol 2013;33:410-17

Affiliation

Demetrio Rald ua & Benjamin Pinã† † Author for correspondence IDAEA-CSIC, Environmental Chemistry, Jordi Girona 18, 08034 Barcelona, Spain Tel: +34 93400 6157; Fax: +34 93204 5904; E-mail: [email protected]


108.

Her GM, Chiang CC, Chen WY, et al. In vivo studies of liver-type fatty acid binding protein (l-fabp) gene expression in liver of transgenic zebrafish (danio rerio). FEBS Lett 2003;538:125-33


697

Interpreting human genetic variation with in vivo zebrafish assays.

Analyzing the role of heparan sulfate proteoglycans in axon guidance in vivo in zebrafish.

Strategies for analyzing cardiac phenotypes in the zebrafish embryo.

Chromatin immunoprecipitation assays: analyzing transcription factor binding and histone modifications in vivo.

Zebrafish Assays as Developmental Toxicity Indicators in The Design of TAML Oxidation Catalysts.

In vivo assays for drug resistance in Babesia divergens using the Mongolian gerbil, Meriones unguiculatus.

Evaluation of toxicity of Calophyllum brasiliense stem bark extract by in vivo and in vitro assays.

Analyzing craniofacial morphogenesis in zebrafish using 4D confocal microscopy.

Sodium cholate-templated blue light-emitting Ag subnanoclusters: in vivo toxicity and imaging in zebrafish embryos.

Solid-phase biological assays for drug discovery.

Comparison of effects of anti-angiogenic agents in the zebrafish efficacy-toxicity model for translational anti-angiogenic drug discovery.

Fish in chips: an automated microfluidic device to study drug dynamics in vivo using zebrafish embryos.

Exposure-based validation list for developmental toxicity screening assays.

Analyzing search behavior of healthcare professionals for drug safety surveillance.

ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish.

Zebrafish as an in vivo high-throughput model for genotoxicity.

Topographical and drug specific sensitivity of hair cells of the zebrafish larvae to aminoglycoside-induced toxicity.

A crystal-clear zebrafish for in vivo imaging.

Zebrafish earns its stripes for in vivo ASC speck dynamics.

Assays of drug sensitivity for cells from human tumours: in vitro and in vivo tests on a xenografted tumour.

Developmental toxicity assays using the Drosophila model.

Toxicity of chlorine to zebrafish embryos.

Utilizing Zebrafish Visual Behaviors in Drug Screening for Retinal Degeneration.

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.