Send Orders for Reprints to reprints@benthamscience.net

5416 Current Pharmaceutical Design, 2014, 20, 5416-5429

Zebrafish Model in Drug Safety Assessment

Jyotshna Kanungo*, Elvis Cuevas, Syed F. Ali and Merle G. Paule

Division of Neurotoxicology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR road, Jefferson, AR 72079

Abstract: Over the past decade, zebrafish are being increasingly used in assessing the effects of chemical compounds. Especially, the embryos and larvae, due to their microscopically small size and optical transparency, are compatible with multi-well microtiter plates for high throughput screening. Being transparent, they allow for non-invasive visualization of internal organs during early development. The organization of the genome, the genetic pathways controlling signal transduction and the developmental pattern appear to be significantly conserved between zebrafish and humans. Major organ systems including the nervous, cardiovascular, digestive and visual systems of zebrafish are also similar to their mammalian counterparts at the anatomical, physiological and molecular levels. Therefore, zebrafish as-says are ideal for evaluating multiple organ toxicities simultaneously that contrast in vitro assays performed on cultured cells or tissue explants and organ slices. Although research on zebrafish as a model system began a few decades ago, later studies on zebrafish devel-opmental biology and developmental genetics resulted in the characterization of a large number of genes involved in vertebrate develop-ment and biological pathways thus establishing zebrafish as a relevant human disease model for research. Recently, zebrafish have be-come an attractive vertebrate model for pharmaceutical and toxicological studies. We have outlined in this review some of the toxicologi-cal screens and tools that used zebrafish early life stages, and the efforts made to validate zebrafish assays against mammalian drug screens.

Keywords: Zebrafish, drug, toxicity screening, high throughput assay, neurotoxicity, hepatotoxicity, cardiotoxicity.

INTRODUCTION An average success rate for all therapeutic areas approximately at 11% implies that only one in nine compounds passes the drug development process and the major causes of attrition in the clinic are safety issues (reviewed in [1]). Between 1975 and 2007, 47 drugs were withdrawn from the market due to their significant adverse effects. For this reason, ongoing efforts have focused on increasing the success rate of drug development while minimizing the risk to patients by developing reliable prediction methods for drug safety assessment. Based on available data, a high rate of failure of drug candidates appears to be mainly due to three com-mon toxicities encountered as clinical adverse events: neurotoxicity (22%), cardiotoxicity (16%), and hepatotoxicity (14%). Most of the drugs withdrawn between 1975 and 2007 were linked to hepatotoxicity (15/47) and cardiotoxicity (21/47). Hence, during the drug discovery process, special emphasis is given to eliminate compounds exhibiting these specific toxicities. In order to deter-mine safety, in pre-clinical tests, cell-based assays are commonly used and these assays do not offer an effect on the organismic level but only display cell-autonomous effects. Therefore, in vivo models for early-stage toxicity screening that would offer quick and non-cumbersome cost-effective platforms have been sought after. In the context of animal use in research, the three R’s, (reduc-tion, refinement, and replacement) have become a major focus of discussion among scientists for the humane use of animals. The Three R's originated in a proposal made in 1954 in the publication of the book The Principles of Humane Experimental Technique in 1959. Reduction alternatives describe methods for obtaining com-parable levels of information from the use of fewer animals in sci-entific research. There is a direct correlation between larger number of animals used to higher overall costs and animal suffering. There-fore, the number of animals used should be kept to a minimum that would be consistent with the research objectives. Refinement alter-natives include methods that alleviate or minimize potential

*Address correspondence to this author at the Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Admini-stration, 3900 NCTR Road, Jefferson, AR 72079, USA; Tel: 870-543-7591; Fax: 870-543-7143; E-mail: jyotshnabala.kanungo@fda.hhs.gov

pain and distress thus emphasizing on animal well-being. Replace-ment alternatives advocate methods to be used in achieving scien-tific results without conducting experiments on animals. To this end, the European Union established the European Cen-ter for the Validation of Alternative Methods (ECVAM) in 1993. In 1997, the United States government established the Interagency Coordinating Center for the Validation of Alternative Methods (ICCVAM). These entities focus on the reduction of the number of animals used for safety testing to promote the development and validation of alternative tests [2]. In addition, the International Con-ference on Harmonisation (ICH) S7A guideline requires that any clinical candidate is evaluated before first exposure to man on basic vital (cardiac, central nervous and respiratory system) functions. These safety pharmacology investigations as well as toxicology studies often provide data that require further clarification and therefore, can be expensive and time consuming. However, studies in vertebrates are indispensable to obtain a comprehensive toxicity profile of chemicals. Almost a decade ago, the German Federal Institute for Risk Assessment estimated that 45 million laboratory animals would be required to meet the European Community legis-lative framework for the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) legislation [3]. In 2007, as a successful initiative in the direction of using alternative animal models in evaluation of chemicals, the zebrafish embryo test was introduced in Germany as a standardized ISO (International Orga-nization for Standardization) assay for water testing, replacing tra-ditional toxicological tests [ISO 15088:2007, Determination of the acute toxicity of waste water to zebrafish (Danio rerio) eggs] for testing environmental contaminants. While ethical issues and cost may preclude the excessive use of higher vertebrates in research, there has been a steady increase in the use of zebrafish embryos and larvae as an alternative model to assess the effects of drugs and chemicals both in toxicity/safety screens and mechanistic studies.

ADVANTAGES OF ZEBRAFISH EMBRYOS/LARVAE IN TOXICITY SCREENING With its emergence as an alternative model in biomedical re-search due to its comparable genetic and physiologic make-up with mammals, zebrafish have several advantages for screening large

1873-4286/14 $58.00+.00 © 2014 Bentham Science Publishers

Zebrafish Model in Drug Safety Assessment Current Pharmaceutical Design, 2014, Vol. 20, No. 34 5417

numbers of compounds, both for drug discovery and for toxicologi-cal studies. The toxicities of drugs and environmental pollutants have been shown to be well conserved between humans and zebraf-ish [4-12]. Furthermore, zebrafish embryos and larvae are not con-sidered to be test animals in the same sense as rodents as far as animal care rules and regulations are concerned, thereby reducing the cost and administrative labor for large-scale safety testing. In the research community, zebrafish embryo up to 120 hpf is consid-ered as a non-animal model for research since it is only after 120 hpf that the embryos begin to feed independently, at which time they are subjected to animal care regulations [13]. Several inherent advantages of zebrafish for drug screening are: they are small, inexpensive to maintain and easily bred in large numbers year-round, a single spawning producing ~200 eggs. Adult zebrafish are ~ 3 cm long and embryos/larvae are only 1- 4 mm long. The latter can live up to a few days after fertilization without external feeding. Drug treatment is easy since zebrafish em-bryos/larvae absorb small molecules diluted in the surrounding water through their skin and gills. Drugs can also enter the body orally when zebrafish begin to swallow at 72 hours post fertilization (hpf). Highly hydrophobic compounds, large molecules and pro-teins can be microinjected into the yolk sac, the sinus venosus or the circulation. In addition, the transparency of zebrafish for several days post fertilization enables in vivo observation. Besides, in whole mount fixed specimens, visualization of organs and tissues using specific dyes, fluorescent tracers, antibodies and riboprobes can also be accomplished. By 120 hpf, zebrafish develop discrete organs and tissues, including brain, heart, liver, pancreas, kidney, intestines, bone, muscles, nerve systems and sensory organs. These organs and tissues have been shown to be similar to their mammal-ian counterparts at the anatomical, physiological and molecular levels. With these unique features, zebrafish embryo is emerging as a promising alternative animal model for developmental toxicity screening [5, 9, 14-17]. Optimum standards for adult zebrafish maintenance and breeding have been well established and the em-bryos develop rapidly ex utero with most organs becoming fully functional at 2-5 days post fertilization (dpf) [18]. Zebrafish can be raised and maintained in aquaria needing less space and cost com-pared to rodent facilities. The zebrafish genome is already se-quenced (http://www.sanger.ac.uk/Projects/D_rerio/) [19, 20] and multiple genetic markers and gene chips are available commercially for experiments at the molecular level. The organization of the ge-nome and the genetic pathways controlling signal transduction and development are highly conserved between zebrafish and humans [21, 22]. Protein homologies among research animal models show high conservation between mammals and zebrafish. For example, compared with the human, the G-protein-coupled receptor (GPCR) protein homologies are: Monkey 98%, Rat 97%, Zebrafish 91%, Drosophila 54%, and C. elegans 47%. The conservation of most cell signaling pathways is also another appealing aspect to using this vertebrate model [23-25]. Another very attractive feature of zebrafish embryo assays for toxicity screening is its amenability to medium-to high- throughput experiments. Experimental manipulation and direct observation of organ development and function can be easily performed as the embryos are transparent. Although both larval and adult zebrafish have been used for pharmacological and toxicological research, its unique advantage remains in the early life stages as the latter can be used for multi-well plate screening paradigms. From the fertilized egg stage, zebrafish embryos can survive for a few days individu-ally in multi-well plates and therefore can be assessed for drug ef-fects in high throughput [26]. The zebrafish larvae are not consid-ered ‘fetal’ like rodents but are closer to the juvenile state. In these early life stages, the nervous system is mature, vital organs are functional and tissue complexity is fully established. Furthermore, very little test compounds are needed for screening in multi-well plates as the larvae can live in as little as 50 �l of water. Zebrafish

are tolerant to low concentrations of dimethyl sulfoxide (DMSO) generally used as a vehicle for many test compounds [27]. For many years, zebrafish have been used for evaluating the toxicity of environmental and agrochemical agents [28]. Moreover, toxicity screening platforms also provide high content analyses of the em-bryos offering an in vivo assay for fast and inexpensive alternative in the drug discovery process [12, 27, 29-31]. Although conventional in vitro assays using cultured cells can be used to evaluate potential drug toxicity effects, results are fre-quently not predictive of results in vivo which involve drug absorp-tion, distribution, metabolism and excretion (ADME). To stream-line the drug development time-line, prioritize drug candidates for animal testing and reduce unnecessary costs for mammalian studies, drug-screening assays using zebrafish are becoming increasingly popular since this convenient, predictive animal model can serve as an intermediate step between cell-based evaluation and conven-tional animal testing.

ZEBRAFISH EMBRYO BEHAVIORAL ASSAYS Zebrafish larvae are capable of free swimming from 4 dpf and therefore, have been used in assays for locomotor activity [32] thus making them well suited for behavioral testing, and functionality of the motor, sensory, and environmental stress-modulating systems [32-41]. These features are also successfully utilized in drug dis-covery related to psychiatric disorders [42]. Zebrafish larval beha-vioral/locomotion assays have been performed using multi-well plates [39, 40, 43-47] that include endpoints like acoustic startle response [33, 43, 48], and visual motor response [35, 38, 47, 49, 50]. While some drugs, such as clozapine, fluoxetine , melatonin , diazepam and pentobarbital have been found to induce hypomotility in zebrafish larvae [51-53], ethanol causes hyperactivity at interme-diate concentrations (1–2%), and depress spontaneous activity and causes toxicity at higher concentrations (4%) [7, 41]. Thus zebraf-ish model can predict hypo/hyperactivity-inducing potential of drugs and chemicals. Another attractive feature of the zebrafish model for neurobe-havioral studies is based on the reports that they display learning, sleep, drug addiction and neurobehavioral phenotypes that are com-parable to those of humans [36, 53-57]. Zebrafish larvae at 7 dpf respond to pentylenetetrazol (PTZ) with a series of movements [58] that begins with increased swimming activity to rapid ‘whirlpool-like’ behavior and then display clonic seizures. The patterns of this three-stage phenomenon depend on the dose of the drug. Data ob-tained by a video-tracking system provided evidence of reduction and suppression of PTZ-induced seizure caused by13 known antie-pileptic drugs [43]. In another blinded study using 17 compounds known to produce seizure and 8 negative controls, data showed 72% predictability [48]. Zebrafish larvae exposed to domoic acid demonstrated reduced latency time to first PTZ-induced seizure [59]. Using 2 dpf zebrafish embryos, another study developed and validated an in vivo, high-throughput assay based on the expression of fos, which is unregulated in response to chemically induced sei-zures [60]. Using this assay 2000 bioactive small molecules were assayed and 46 compounds with anticonvulsant activity were iden-tified. Further analysis of a subset of these compounds, which in-cluded both known and newly identified anticonvulsants, showed that they exhibited dose-dependent inhibition of convulsant-induced locomotor activity and fos transcription. This robust, scalable ap-proach for in vivo screening of compounds with anticonvulsant activity in a vertebrate model organism provides a relatively low-cost, high throughput platform [60]. Zebrafish have been used to study learning and memory thus enabling identification of potential toxicities of CNS-active drugs during early drug discovery. Non-associative learning has been studied in larval zebrafish by measuring the reduction/alteration in startle response to various stimuli [34, 44]. Donepezil and meman-tine, the therapeutics for improving cognitive ability in Alzheimer’s

5418 Current Pharmaceutical Design, 2014, Vol. 20, No. 34 Kanungo et al.

disease patients, and the phosphodiesterase-4 inhibitor rolipram that has been shown to increase learning and memory in rodent studies, modulated habituation to the acoustic stimuli in zebrafish larvae [34]. Ketamine, a pediatric anesthetic and an NMDA receptor an-tagonist, altered the startle response behavior of the larvae [44]. Zebrafish treated with ethanol or lead for the first 24 hpf of devel-opment showed significant learning and memory deficits during adulthood [61]. These reports signify the reliability of the zebrafish model in neurobehavioral research pertaining to drug effects.

MORPHOLOGY-BASED TERATOLOGIC ASSAYS USING ZEBRAFISH MODEL Morphology-based toxicological assessments at an organismic level have focused on zebrafish as a model organism [12, 62, 63] for in vivo toxicity and teratogenicity screening [64-75]. Routine endpoints used for assessing the effects of test compounds include embryo mortality, yolk sac edema, tail malformation, changes in hatching rates and deficiencies in gastrulation, somite formation, head formation, heart rate and pigmentation [76, 77]. Zebrafish embryo toxicity test presents a 75% success rate in identifying non-teratogenic compounds and a 100% success rate in identifying tera-togenic compounds [78]. Screens of chemical libraries that already contain FDA-approved drugs, based simply on the morphology of the embryos, have also been performed to assist in the rapid transla-tion of hits from zebrafish chemical screens to clinical trials [79]. The National Institute of Environmental Health Sciences (NIEHS) in the United States and the Institute for Environment and Sustain-ability (IES) in Europe have supported the use of zebrafish as a model organism for the assessment of environmental toxicity. The US Food and Drug Administration (FDA) established protocols to be used for assessing drug effects on reproduction and development before approval for human use after the devastating teratogenic effects of thalidomide were realized in 1966. The Environmental Protection Agency (EPA), USA issued similar guidelines for pesti-cides in 1982 and for industrial chemicals in 1985 to protect public health. Based on the current international guidelines, developmental toxicity testing involves exposing pregnant animals, usually rats or rabbits, to compounds and subsequently assessing toxic effects on fetuses. Since the early 1980s, there has been a lot of focus on al-ternative methods for assessing developmental toxicity that include in vitro cell differentiation assays using either primary cell cultures or cell lines. In addition, in vitro rodent whole embryo culture test [80] and in vivo frog embryo teratogenesis assay-Xenopus (FE-TAX) [81] have also been in practice for compound testing [82-84]. However, in vitro tests are inefficient in predicting drug effects on human embryonic and fetal development [85]. Furthermore, one of the drawbacks of the in vivo FETAX assay that uses Xenopus em-bryos is due to the distinct chemical metabolic pathways that are different from mammals; and therefore, the embryos do not respond to halogenated aromatic hydrocarbons [86]. With these obvious limitations in the Xenopus embryos, developmental toxicity studies in zebrafish embryos, however, pose some selective advantages, since zebrafish have been shown to be sensitive to compounds that are teratogenic in mammals. In contrast to rodent embryos in cul-ture, zebrafish embryos can be reared until advanced organogene-sis. In EPA’s five-year US$100M ToxCast Program (http:/www.epa.gov/comptox/toxcast), an initiative to prioritize chemicals based on rigorous toxicological testing, zebrafish was included as the sole animal model for assessing the effects envi-ronmental contaminants for their potential as developmental toxi-cants [87] . In another study aimed at validating zebrafish as a predictive model for assessing developmental toxicity, 12 reference com-pounds provided by Bristol-Myers Squibb Pharmaceutical Research Institute (New Brunswick, NJ, USA) were evaluated for develop-mental toxicity in zebrafish embryos using a visual assessment protocol. Seng and colleagues treated the larvae with test com-pounds from 6 hpf to 5 dpf displayed a range of developmental

abnormalities in the body, heart, liver and gastrointestinal tract (GI) were observed (reviewed in [88]). After comparing the resulting data with teratogenicity properties observed in vivo in mammals, assessment of assay predictivity ranked it well (>70% <80%) for specificity and excellent (>80%) for sensitivity. Furthermore, this assay format presented a 75% success rate in identifying nonterato-gens and a 100% success rate in identifying teratogens [78]. This study demonstrated that for simple and direct assessment of devel-opmental toxicity, zebrafish embryo can be an ideal model. Several other morphology-based developmental toxicological studies have been successfully performed using the zebrafish em-bryo model [89-91]. Six compounds including known teratogens (all-trans-retinoic acid, valproic acid, lithium chloride, and caffeine) as well as non-teratogens (glucose and saccharin) were tested using zebrafish embryos at 2 dpf and evaluation of phenotypes at 24, 48, 72, or 144 hpf correctly identified teratogenic and non-teratogenic compounds [17]. In another study, embryos were treated at 4-6 hpf for five days and several morphological parameters, such as sur-vival, motility, cardiovascular function, pigmentation, brain mor-phology, facial structure, jaw, pharyngeal arch, somite, notochord, tail, fins, and heart were assessed in vivo [90]. Of the 31 known developmental toxicants tested, 13 compounds were non-teratogens, and 18 were teratogens. Using their prediction model, the authors correctly classified 87% (27/31) of the compounds with 15% false positives and 11% false negatives. Another novel quanti-tative evaluation method called the zebrafish embryotoxicity test (ZET) was used by treating embryos at 4- to 32-cell stages for three days with eight glycol ethers and six 1,2,4-triazole anti-fungals. Assessment of morphological parameters, such as locomotion, heart rate, blood circulation, tail detachment, somite formation, eye de-velopment, pigmentation development, mouth/jaw development, pectoral fin development, and hatching rate detected embryotoxic properties of this class of compounds [92] suggesting that the method can also identify toxicities of other classes of compounds. In another test where zebrafish embryos were treated with drugs beginning at the sphere stage until 96 hpf, 28 morphological end-points were evaluated and the results showed an overall predictivity of 75% although the false positive and false negative rates were both at 40% [77]. Several reports from the EPA described zebrafish developmen-tal toxicity assays and one of these reports indicated only 55% of the chemicals tested were concordant with in vivo mammalian data [93]. This conclusion was derived from the assessment of a total of 271 chemicals, mostly environmental chemicals, tested as part of EPA’s ToxCast initiative. EPA also tested a number of compounds (ToxCast 309 Phase I chemical library) on 6 dpf larvae using a ‘terata score’ [57]. Terata scores were categorized by target organs, such as spine, fins, eyes, jaws, etc., that could be morphologically identified. Combined together the terata scores provided an overall teratogenicity index. Based on five separate studies, when com-pared with mammalian data, zebrafish data showed 55-100% con-cordance [17, 57, 90, 94-97]. Altogether, the zebrafish embryotox-icity testing presents several advantages over other in vivo toxicity testing methods.

SYSTEM-SPECIFIC TOXICITY ASSAYS USING ZEBRAF-ISH MODEL Zebrafish embryos are suitable as an alternative model for screening of drug-induced organ toxicity [98]. They have been shown to be an excellent model for assessing drug-induced car-diotoxicity [30, 99-102]. Although zebrafish lack a pulmonary sys-tem, they exhibit similar functional characteristics of the heart to humans [103, 104] and comparative transcriptome analyses indicate the relatedness of the zebrafish swim bladder and mammalian lung [105]. Due to the conservation of most physiological functions between zebrafish and mammals, zebrafish have been successfully used for organ- or system-specific toxicological studies.

Zebrafish Model in Drug Safety Assessment Current Pharmaceutical Design, 2014, Vol. 20, No. 34 5419

Cardiotoxicity While cardiac functions such as heart rate, contractility, and gross morphology can be visually assessed in live embryos, micro-electrodes can record compound action potentials [106]. Heart rate can be recorded manually from live embryos or from live observa-tion [107, 108] and/or videotape recording [109]. Larvae can be immobilized using tricaine during these recordings [110]. Electro-cardiogram recordings can be made noninvasively from 5 dpf em-bryonic zebrafish [111]. For the analysis of cardiac conduction, a transgenic zebrafish line (Tg(cmlc2:gCaMP)s878) has been used [112] allowing the mapping of the spread of calcium excitation in the heart during each cardiac cycle [113]. Several reports have shown that zebrafish pharmacologic responses to compounds that are capable of affecting cardiac function in humans are strikingly similar to that in humans [30, 102, 103, 109, 114-117]. Validation of zebrafish larvae as a reliable model to test compounds for their ability to induce QT prolongation have also been reported [103, 117, 118] . A screen of one hundred small molecules showed that 22 of 23 drugs that cause QT prolongation in humans consistently caused bradycardia and blocked AV conduction in zebrafish [118]. A study from the Abbott Laboratories further validated zebraf-ish embryos as an in vivo model for determining the inhibition of hERG, showing that almost all of the drugs known to cause QT prolongation induced a dissociation between atrial and ventricular rates in zebrafish embryos upon acute exposure to these compounds [108]. A number of the above studies of cardiotoxicity (mostly categorized as developmental toxicity) also showed high predictiv-ity according to the guidelines of ECVAM that classifies alternative toxicity assays based on their capability to predict adverse effects of chemicals known to be toxic in humans.

Hepatotoxicity and Endocrine Disruption Hepatotoxicity assessment in zebrafish has been performed successfully since studies show that in zebrafish, mechanisms of enzyme induction and oxidative stress are similar to those in mam-mals [119, 120]. Many zebrafish homologs of mammalian lipid metabolizing enzymes are present in the zebrafish liver, including 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, HMG-CoA lyase and members of the peroxisome proliferator-activated receptors (PPARs) [121]. Primary liver morphogenesis in zebrafish is completed by 48 hpf and liver is fully formed and functional at 72 hpf. Merbarone and carbamate, two mammalian liver toxicants, caused similar toxicities in zebrafish [122]. Initial evaluation of the predictive utility of zebrafish embryo assay of a series of com-pounds that are known hepatotoxicants in humans (amiodarone, clofibrate, diclofenac, paracetamol, tetracycline and troglitazone) also showed predictive potential of the assay [122]. The assay used biotin, ketorolac and rosiglitazone as negative controls. While changes in liver size and liver-specific marker expression were observed in embryos treated with these hepatotoxicants, negative controls showed negative results at the highest concentrations tested. In another assay, hepatotoxicity of 6 known mammalian hepatotoxic drugs [acetaminophen (APAP)], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantita-tively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% as compared with mammalian results, suggesting that hepato-toxic drugs in mammals also caused hepatotoxicity in zebrafish [123]. Cytochrome p450 (CYP) enzymes are a diverse superfamily of the heme-containing monooxygenase enzymes [124, 125] involved in the metabolism and bioactivation of both endogenous and exogenous chemicals [125, 126]. Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals

[127]. CYP19 aromatase is a crucial steroidogenic enzyme that catalyzes the final, rate-limiting step in the conversion of androgens into estrogens [128]. In mammals, with the exception of pigs, there is a single CYP19 gene which is expressed in a variety of tissues [129]. However, owing to genome duplication during evolution, teleosts including zebrafish contain a pair of aromatase genes: cyp19a1a and cyp19a1b present on two separate chromosomes [130]. Using human CYP-specific substrates, CYP3A4 and CYP2D6 functional activity assays have been performed in zebraf-ish after drug treatment and CYP3A4 was up regulated in zebrafish treated with dexamethasone [131], similar to the CYP3A4 response in humans [132]. Brefeldin A induced liver necrosis in zebrafish embryos [122] while valproic acid and simvastatin treatment re-sulted in liver phenotypes similar to those observed in mammalian fatty liver disease (FLD) [88]. In addition, using cyp19a1a and cyp19a1b as biomarkers of endocrine disruption zebarfish embryo has been successfully utilized in detecting endocrine disruptors [98, 133, 134]. Several biomarkers of endocrine disruption have also been described in small fish models including zebrafish larvae that support the development of simple molecular-based screening as-says (reviewed in [135]).

Neurotoxicity The overall organization of the zebrafish brain is similar to other vertebrates with comparable areas such the hypothalamus, olfactory bulb, and the lateral pallium, which is considered ho-mologous to the mammalian hippocampus [136]. Additionally, neurotransmitter systems such as the cholinergic, 5 hydroxytryp-taminergic, dopaminergic and noradrenergic pathways are also functional [137-139]. The blood–brain barrier (BBB) of zebrafish is structurally and functionally similar to those of mammals [140, 141]. Zebrafish also display a functional BBB early during devel-opment [142]. Reports on neurotoxicity assessment in zebrafish have de-scribed this organism to be an ideal model system. Neurotoxicity, according to NRC (National Research Council) and EPA, is defined as adverse functional or structural changes in the nervous system caused by exposure to chemical, biological or physical agents. Neu-rotoxicity profiles of many approved drugs are still incomplete and they cause neurotoxic side effects [55, 143]. Routine methods for assessing neurotoxicity in mammals are based on behavioral, neu-rohistopathological, and biochemical assays. The former two pro-cedures are labor-intensive. Therefore, research on biochemical markers for neurotoxicity testing is ongoing in federal agencies as well as other research entities, since biochemical assays that corre-late brain functions with metabolism have not been rigorously tested [143]. In an effort toward developing rapid assays for neurotoxicity testing using alternative animal models, zebrafish embryos and larvae have been used by numerous investigators focusing on cellu-lar, molecular and genetic analyses [144]. A unique advantage of the zebrafish embryo is, for several days of early development, specific neurons and axons can be visualized in vivo using differen-tial interference contrast microscopy or by using vital dyes [145]. Neuron-types can also be identified in intact zebrafish by whole mount immunohistochemistry or in situ hybridization [146]. The function of specific neurons can also be elucidated by behavioral assays, the latter gaining more popularity among researchers as a non-invasive procedure. A number of neurotoxicants, both drugs and industrial chemicals, have been tested in zebrafish embryos [7, 143, 147, 148]. Additionally, studies to identify potential mecha-nisms and neuroprotectants are also being pursued [149, 150]. Neu-rotoxins tested in zebrafish include dopaminergic neurotoxins, non-NMDA (N-methyl-D-aspartate) type glutamate receptor (AMPA) agonists or antagonists, nicotinic acetylcholine receptors (nAChRs) antagonists or acetylcholinesterase (AChE) inhibitors, TCDD (2,3,7,8-tetrachlorodibenzodioxin) and NMDA receptor antagonist

5420 Current Pharmaceutical Design, 2014, Vol. 20, No. 34 Kanungo et al.

[88, 151-156]. In zebrafish, taxol treatment caused extensive neu-ronal apoptosis in the brain and spinal cord [157] as observed in mammals [158]. Nicotine’s ability to act as an endocrine disruptor and a neurotoxicant (affecting motor neurons and axons) was also shown in zebrafish embryos [153, 159]. Ketamine, a pediatric anes-thetic that has been shown to be neurotoxic to rodents as well as non-human primates [160-163] was also neurotoxic to zebrafish embryos [149, 152]. Thus, neurotoxicity data from zebrafish studies show concordance with those from mammalian studies. Ethanol has been studied extensively in the zebrafish model both in adults and embryso/larvae with the endpoints being behav-ior and startle responses [164, 165], predator avoidance and anx-iolytic behavior [166]. Zebrafish larvae have been used as a model vertebrate organism in large-scale genetic analyses to study bio-logical effects of alcohol [41]. Using this method, many signaling molecules regulating acute locomotor behavior in response to etha-nol have been identified [167]. Zebrafish embryos have also been used in several studies of ethanol teratogenesis [168], and the phe-notypic outcomes include developmental retardation, pericardial and yolk sac edema [169-171], reduction in body length [172], branchial skeleton defects [61], abnormal eye development [173-177] as well as cognitive defects [8, 61, 178] and higher mortality [179]. These phenotypes are manifested in human fetal alcohol syndrome (FAS). Most studies of ethanol toxicity in zebrafish use chronic exposure, often over several hours or days [168, 172, 175, 176, 180-182]. However, a recent study on brief exposure of staged embryos to an ethanol pulse identified that prim-6 and prim-16 stages are particularly susceptible for the induction of pharyngeal defects by ethanol [183]. It is also revealed that high doses of etha-nol are required to induce defects that are similar to mammalian models [177, 184, 185]. This may be due to the difference in ab-sorption and real exposure. In zebrafish embryos, ethanol appears to alter neuromuscular junction development [186]. Zebrafish em-bryos exposed to ethanol for 16 h, between 8 and 24 hpf, develop defective motor neurons, motor axons and muscle fibers [187] and embryos exposed at 24 hpf for one day develop defective axono-genesis [188]. Using conventional immunostaining with axon-specific antibodies, ethanol treatment has been shown to cause pri-mary motor neuron loss in spinal region of zebrafish [7] and similar effects have been reported in mammals [189]. In vivo studies on hb9:GFP transgenic embryos using image-based high content analysis also showed ethanol-induced reduction in motor axon length [188]. These studies indicate that zebrafish embryos can be successfully utilized to test drugs especially those with potential neurotoxic effects.

Ocular Toxicity Toxicity assays based on visual function using the zebrafish model have been reported. Eye development is rapid in zebrafish embryos and the larvae exhibit visually-triggered behaviors that can be used in the assessment of ocular toxicity [190]. To assess visual function in zebrafish, assays such as the optokinetic response and optomotor response (OMR) have previously been developed [191]. In a preliminary validation study of the zebrafish OMR assay, six compounds (chloroquine, chlorpromazine, diazepam, nicotine, oua-bain and phenytoin) out of eight were correctly predicted to have effects on visual function, with aspirin correctly identified as a negative control. A more detailed study of the effects of 27 com-pounds using the OMR assay revealed a good correlation between the effects of compounds in 8 dpf larval zebrafish with the data available from other in vivo and in vitro models. Thirteen out of 19 positive compounds produced the expected phenotype, whereas 6 of the 8 negative compounds were also correctly predicted. The over-all predictability was 70% for adverse effects on visual function [50]. Although not commonly used as a toxicological screening assay, the visual function assay can be helpful in developing an “assay for purpose” when drugs are suspected to have adverse ef-fects on the visual system.

Ototoxicity Ototoxicity assays in zebrafish embryos/larvae are relatively common compared to many other organ-specific assays (Table 1).There are clear differences in the general anatomy of the ears of zebrafish and mammals. However, the structure and function of the hair cells are highly conserved between fish and mammals. Zebraf-ish do not have outer or middle ears, but have a typical vertebrate inner ear with biomineralized structures called otoliths with sensory hair bundles underneath them [192]. Hair cells are also found in the sensory lateral line systems in zebrafish [193]. In contrast to hu-mans, hair cells of zebrafish larvae can regenerate after injury. In these larvae, hair cells are also present in neuromasts (the lateral line organs that are parts of the peripheral sensory nervous system) and can serve as endpoints for developmental toxicological studies relating to hearing loss [194]. These cells are innervated by sensory neurons projecting to the CNS [195, 196]. Several genes involved in normal hair cell development and function are conserved be-tween zebrafish and mammals [197]. A simple approach to screen for auditory defects in zebrafish is to monitor alterations in patterns. An indirect approach to assess hearing in zebrafish is to assess acoustically-triggered startle response. The startle response is a fast contraction of body muscles caused by a sudden acoustic, tactile or visual stimulus and zebrafish larvae display a characteristic startle response to auditory and visual stimuli that is maintained through adulthood [198, 199]. In one study, 7 dpf larvae were exposed to an acoustic stimulus that resulted in an increase in locomotor activity [34]. Hair cell integrity can also be assessed through staining with vital dyes like DASPEI (2-(4-(dimethylamino)styryl)-N-ethylpyri-diniumiodide) [200]. Zebrafish larvae have been used to demon-strate lateral line hair cell loss upon treatment with a number of agents, including gentamicin, neomycin, and cisplatin, and otopro-tection was reported with glutathione and other antioxidants in cis-platin-treated fish [200-203]. Drug screening using the zebrafish lateral line for FDA approved libraries of compounds that could prevent or cause hearing loss has been reported [204, 205]. DMSO has been reported to exacerbate cisplatin-induced sensory hair cell death in zebrafish larvae [206]. These studies support the use of zebrafish larvae to assess drugs that cause hearing impairment in humans.

Nephrotoxicity In teleost fish like zebrafish, the kidney is called pronephros, which is a simple structure comprising of a pair of nephrons with two glomeruli fused at the midline with two bilateral pronephric ducts directing the blood filtrate for excretion. By 40 hpf the pronephros begins blood filtration [207]. The glomerulus has vari-ous cell types similar to those in the kidneys of higher vertebrates, including the capillary endothelial cells, podocytes and polarized tubular epithelial cells [207]. Despite such a simple organization, zebrafish pronephros functionally phenocopies the mammalian kidney when exposed to gentamicin. Gentamicin, an antibiotic, is nephrotoxic in mammals. Injection of gentamicin into the cardiac venous sinus of at 50–54 hpf larvae caused several morphological anomalies including pericardial and endocranial edema, and renal system malfunction [208, 209] that is similar to acute renal failure in mammals [210]. In zebrafish larvae, treatment with cisplatin and puromycin, both nephrotoxic agents, also caused responses similar to renal injury in higher vertebrates [211]. These results suggest that zebrafish early life stages can serve as an in vivo model to evaluate nephrotoxicity.

Other Organ-Specific Toxicities Other internal organ toxicities induced by certain drugs have also been reported in the zebrafish embryo model since zebrafish intestine, liver and pancreas develop within the first 4 dpf [212]. Intestinal contractions start early in development with erratic and spontaneous contraction waves reported as early as 3 dpf, before

Zebrafish Model in Drug Safety Assessment Current Pharmaceutical Design, 2014, Vol. 20, No. 34 5421

independent feeding starts [212, 213]. Effects of 10 compounds on gut motility in 7 dpf zebrafish larvae showed 8 of the 10 tested expectedly decreased the number of contractions. In addition, two smooth muscle relaxants, isoprenaline and chlorpromazine, caused the intestinal lumen to expand [99]. Skeletal defects as endpoints of drug toxicity assays have also been reported in the zebrafish model. Structurally, zebrafish and mammalian bones are similar and both have osteoblasts and osteo-clasts [214, 215]. A high-throughput in vivo screening assay for compounds that alter bone density has been reported in zebrafish [216]. The larvae were exposed to the compounds from 5 to 10 dpf in a 96-well plate. Alizarin red staining of the bones of the whole embryos showed 50% reduction in mineralization [217]. These assays are performed on fixed larvae and therefore are in situ as-says, which can be used to evaluate drugs with potentially adverse effects on the human skeletal system.

ZEBRAFISH AS HUMAN DISEASE MODELS The National Institutes of Health (NIH) recognizes the zebrafish as an alternative model for exploring human disease, development, and physiology [30, 218]. Although research on ze-brafish as a model system goes back to the early 1970s when ze-brafish larvae were used for forward genetic screening [219, 220], later on zebrafish were predominantly used in developmental biol-ogy and developmental genetics studies. These later studies resulted in the functional characterization of a large number of genes in-volved in vertebrate development and biological pathways thus establishing zebrafish as a relevant human disease model for re-search [221, 222]. Several human disease models for drug efficacy testing have also been developed in zebrafish for cardiovascular disease, infection/immunity, cancer, inflammation, vitamin defi-ciency, endocrine diseases (e.g., hypothyroidism, growth hormone disorder), metabolic diseases [12, 15, 26, 31, 221-225] and tubercu-losis [226]. Interestingly, disease phenotypes of zebrafish models,

such as muscular dystrophy and myopathies, closely phenocopy the severity of human clinical presentation than corresponding mouse models [29, 227-229].

MICROINJECTION OF INSOLUBLE DRUGS INTO ZEBRAFISH EMBRYOS FOR TOXICITY ASSAYS Although microinjection of the zebrafish early embryos with a single glass micropipette is most commonly used for delivering macromolecules, automated systems are being developed in order to enable fast and robust screening in high throughput [29]. Molecular and genetic analyses of zebrafish development depend on the injection of foreign molecules into early zebrafish embryos [230, 231]. Few years ago a technique for robotically micro-injecting compounds into the embryonic tissue was developed [232]. Since injection of thousands of fertilized eggs needs to be conducted within 1.5 hpf for effective results, efforts in automating the microinjection process have emerged [233-235]. While engineered DNA injection is used to generate transgenic zebrafish lines, mRNA injection is used to overexpress specific proteins in zebrafish embryos and compounds that are insoluble in water need to be injected into the eggs for toxicological studies. Additionally, reverse genetics or loss-of-function studies are based on micro-injection of antisense morpholino oligonucleotides (morpholinos) to specifically inhibit RNA splicing and/or translation in the embryos [231]. One way to overcome the impermeability provided by the chorion is to digest it away with the enzyme, Pronase (www.zfin.org). However, for manual or automated injections, the chorion barely poses any problem. Microinjection that involves delivering the drug through the chorion into the underlying perivitelline space (the gap between the early embryo/yolk and the chorion) has also been described [236]. Although compared to cultured cells, zebrafish fertilized eggs are relatively larger (~600 �m or ~1.2 mm including chorion), zebrafish embryos inside the protective chorion are still a mass of

Table 1. Common types of toxicity screening using zebrafish early life stages.

Types of Toxicity Screening Representative References

Morphology-based Developmental Toxicity:

Embryotoxicity & Teratogenicity Parng et al. 2005 [30]; Eimon and Rubinstein 2009 [100]; Knudesen et al. 2011 [14]; McCollum et al.2011 [96]; Sipes et al. 2011 [93]; Tamplin et al. 2012 [79]

Organ-specific toxicity:

Cardiotoxicity Berghmans et al. 2008 [99]; Eimon and Rubinstein 2009 [100] ; MacRae 2013 [101]

Neurotoxicity Parng et al. 2007 [7]; Peterson et al. 2008 [260]; Tierney 2011 [261]; de Esch et al. 2012 [144]; Kanungo et al. 2011 2013 a; 2013b [149, 152, 188]

Ton and Parng 2006 [78]

Ocular Toxicity Dlugos and Rabin 2007 [174]; Richards et al. 2008 [50]; Fleisch and Neuhauss 2006 [190]; Berghmans et al. 2008 [99]

Vascular toxicity Teraoka et al. 2003 [10]; Tran et al. 2007 [242]

Hepatotoxicity He et al. 2013 [123] ; Driessen et al. 2013 [262]; Hill et al. 2012 [263]

Ototoxicity Eimon and Rubinstein 2009 [100]; Froehlicher 2009 [194]; Ou et al. 2009 2010 2012 [204, 205, 264]; Coffin et al. 2010 [265]; Hirose et al. 2011 [266]

Endocrine disruption Scolz et al. 2008 [133]; Scholz and Mayer 2008 [135] ; Segner 2009 [134]; Trickler et al. 2013 [258]; Kanungo et al. 2012 [153]

Behavioral Toxicity:

Locomotion, Sedation, Hyperactivity Sipes et al. 2011 [93]; Ali et al. 2012 [267]; Padilla et al. 2012 [57]; Bailey et al. 2013 [268]

5422 Current Pharmaceutical Design, 2014, Vol. 20, No. 34 Kanungo et al.

cells that are very delicate and can be easily damaged, especially during microinjections. Thus high-throughput microinjection of these early stage embryos can be a considerably challenging task. Quickly immobilizing a large number of zebrafish fertilized eggs with proper orientation for rapid microinjection using microrobots poses an additional challenge. In spite of such difficult challenges, fortunately, there are instruments available for zebrafish embryo high-throughput injections featuring full automation, high-speed sample immobilization, with high (~ 98.5%) survival rate [232]. Latest developments in instrumentation with automation can facilitate large-scale molecule screening using zebrafish embryos, especially while dealing with insoluble drugs and biologics.

IMAGE-BASED HIGH THROUGHPUT ANALYSIS OF ZEBRAFISH EMBRYOS FOR TOXICITY SCREENING One of the simplest zebrafish toxicity screening assays is based on optical imaging and evaluating the general morphology and developmental status of the embryos and larvae, which is facilitated by automated imaging-based toxicity assays (Fig. 1). Considerable advances have been made in screening technologies including image-based high throughput assays that use zebrafish embryos [237, 238]. In order to isolate and quantify the image based data, the majority of the published studies on zebrafish high content screening have resorted primarily to fluorescence-based microscopy using specifically developed transgenic zebrafish lines [188, 239-244]. Through the use of fluorescence intensity and distribution, an automated high-throughput mapping of promoter-enhancer inter-actions in zebrafish embryos was recently developed [241]. The reporter gene expression in the embryos was registered (i.e., categorized) to eight domains (yolk sac, eye, skin, brain, midbrain-hindbrain boundary, heart, spinal cord, and notochord) via an image-based method exhibiting an average registration accuracy of

Fig. (1). Schematic of image-based high content analysis of zebrafish em-bryos exposed to chemical compounds. An example of analyzing neurotox-icity (axonogenesis) is presented [188] although other transgenic zebrafish embryos that express tissue-specific fluorescence can similarly be used for rapid organ-specific toxicity assays. 86%. Another recent study also adopted fluorescence-based micro-scopy and employed cognition network technology (an object-oriented image analysis method that emulates cognitive processes in the human mind) to quantify intersegmental blood vessel development from images of zebrafish embryos with an error rate of 4.5% [243, 244]. For non-fluorescence based high throughput screening HTS, a bright-field (grayscale) zebrafish image analysis

algorithm, based on a heuristic approach, was proposed that detects and segments a region enclosing an area surrounding the pigments [245]. The approach was tested using 18 images of zebrafish embryos treated with DMSO and gamma secretase inhibitor (GSI-18). The false positive and false negative identification rates were 28.6% and 37.5%, respectively [245]. Fully automated mani-pulation of animals, including automated orienting and positioning the zebrafish for imaging regions of interest has also been developed [246]. High content analyses of zebrafish embryos is fast evolving and would provide a platform for rapid assessment of drug effects in vivo [237].

DRAWBACKS OF ZEBRAFISH MODEL IN TOXICO-LOGICAL RESEARCH As discussed above, in the last decade, studies with zebrafish toxicity assays have been reported including developmental toxic-ity, neurotoxicity, hepatotoxicity, and ototoxicity among others. Although these results are promising, systematic evaluations of zebrafish embryo-based toxicity assays using a large number of drugs are still lacking. Large-scale screens will help establish the predictive power of zebrafish embryo models in toxicity screens. Assessment of a large number of compounds from diverse drug classes is necessary to further validate the model. In terms of assays on the absorption, distribution, metabolism and excretion (ADME) properties of compounds, zebrafish embryo screens may face addi-tional challenges. Although it is unclear how ADME after drug delivery in fish water compares to ADME after delivery by other routes of administration, studies in adult zebrafish following con-ventional drug delivery methods such as injection and intubation show promising outcomes supporting the use of this model for comparative animal studies [30, 247]. Reported results show that inter- and intra-laboratory standards vary widely, making it difficult to interpret the outcomes of drug-induced effects. Such variations can hinder the acceptance of this animal model by the regulatory scientists. Additionally, mechanistic studies will add to fully under-standing the potential of the zebrafish embryo model for drug screening improving the efficiency and reliability of the assays. Safety assessment using zebrafish in early non-good laboratory practice (GLP) studies can adequately serve in the selection process of safe lead candidates very early in the drug discovery process. With adequate validation, high-throughput zebrafish assays would be helpful in rapidly testing tens of thousands of compounds in a matter of hours or days. This ability should enable better decision-making in critical stages of the drug discovery process. Combined with mechanistic studies, the efforts can also provide insights into how a drug works in all its complexity on a whole organism com-pared to its simplistic effects on cultured cells and may warrant for its acceptance in to regulatory purposes. However, given the amount of data generated, the use of zebrafish early during the drug discovery phase, appears to be very promising. Another drawback of zebrafish assays is that unless uptake of compounds into the zebrafish embryos and larvae are measured, accurate interpretation of results may be hindered. In an effort to eliminate false negatives and to rank compounds within a chemical library, such bioanalysis will be required. While bioanalysis can only be performed on whole embryos/larvae, specific tissue concen-trations are difficult to assess in these specimens and therefore can-not be compared with mammalian tissue concentrations. In a liquid chromatography tandem mass spectrometry (LC-MS/MS) study, uptake of compounds by zebrafish larvae showed a correlation be-tween CLogP and the amount of compound accumulating in the larvae, whereas less polar compounds with CLogP values under 3.8 showed lowest accumulation [99]. Rather more disconcerting is limited uptake of modestly soluble compounds by the zebrafish embryos that may result in a lack of sensitivity of the model and consequently produce false negatives. This issue of varying uptake into the zebrafish complicates the process of validating a particular

Zebrafish Model in Drug Safety Assessment Current Pharmaceutical Design, 2014, Vol. 20, No. 34 5423

biological system with a diverse set of compounds in determining the predictability and sensitivity of each assay. Although zebrafish larvae are reared at 28o C, which is different from mammalian physiological temperature of 37o C, recently, drug metabolism systems were shown to have a high degree of functional similarity to that of mammals (reviewed in [248]). In zebrafish larvae, key enzymes involved in phase I drug metabolism including the cytochrome P450 family, flavin-containing monooxygenases, and epoxide hydrolases have been identified and presence of transcripts of several key phase II drug metabolism enzymes including UDP-glucuronosyltransferase, sulfotransferases, catechol-O-methyltransferase, and glutathione-S-transferases have been reported (reviewed in [248]. More research in this area will lend strong support for the use of zebarfish model in risk assessment of drugs and chemicals.

CONCLUSION The evolutionary divergence of zebrafish and mammals oc-curred approximately 445 million years ago [22, 249]. However, there is a high degree of conservation at both the developmental processes and genetic pathways. While 25% of the zebrafish genes are known to be essential for early development, 99% of these genes are homologous to human genes, suggesting that results obtained in zebrafish may be comparable and translatable to humans [250, 251]. Furthermore, zebrafish embryos can activate proteratogenic/procarcinogenic substances without any addi-tion of exogenous metabolic activation systems as they present phase I enzyme activities at very early stages of development [252]. Reports show that drugs with specific effects in humans can cause similar effects in the zebrafish supporting the predictive ca-pability of the zebrafish model. For example, a comparison of the toxicity of 18 compounds between zebrafish and rodents showed a good correlation [102]. Data from the zebrafish embryo toxicologi-cal screen has also been compared with those of the aquatic crusta-cean (Daphnia magna) [253], validated rat whole embryo culture (WEC) and mouse embryonic stem cells [95, 254] in order to de-termine inter-species similarities and variations (reviewed in [255]). Such comparative toxicity studies can help develop databases like the well-known ‘‘Registry of Cytotoxicity’’, which examines the predictive power of cell-based assays [256]. A rapidly growing rapporteur of research on zebrafish-based toxicity studies, both in biomedical research and drug discovery entities, underscores the emerging popularity of this vertebrate model organism among re-searchers. Additionally, the zebrafish embryos can be used for ki-netic measurements [257, 258]. More importantly, zebrafish offer advantages in certain areas of potential safety assessment that can-not be easily achieved using rodent models, especially in mechanis-tic studies that rely on in vivo anatomical endpoints that can be recorded live in a non-invasive manner. For example, manifesta-tions of drug–drug interactions arising from the use of combination drugs could be monitored in vivo relatively easily in zebrafish [59, 206, 259]. With the availability of thousands of transgenic lines (www.zfin.org) with single and multiple organs specifically ex-pressing fluorescent reporters, live monitoring of multiple or-gan/tissue toxicities in a single embryo is an advantage no other vertebrate animal model can offer. Although full validation will be necessary for its acceptance by the regulatory science community, zebrafish embryos are and can be used to eliminate potentially hazardous compounds during early stages of drug development before prioritizing compounds for fur-ther preclinical and clinical studies. In this respect, existing conven-tional instrumentation combined with new high throughput tech-nologies will facilitate and diversify its use in drug-related research. Although, more efforts are needed to establish and accept the pre-dictive power of the zebrafish model, zebrafish embryo toxicity assays have proved to be a versatile in vivo assessment method in various studies. Therefore, it can be successfully utilized to reduce

the current, high degree of attrition in drug development. Given the species-specific variations in responses to drugs even among mammals, drugs that are effective in both zebrafish and rodent models will have the greatest probability of being effective in the human patients. Furthermore, following discovery of lead com-pounds using the zebrafish model during risk assessment, subse-quent testing in rodents, could lead to much faster, cheaper and more efficient drug development.

DISCLAIMER This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents neces-sarily reflect the position or opinions of the FDA, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The findings and conclusions in this re-port are those of the authors and do not necessarily represent the views of the FDA.

ACKNOWLEDGEMENTS This work was supported by the National Center for Toxico-logical Research (NCTR)/U.S. Food and Drug Administration (FDA).

REFERENCES[1] Stevens JL, Baker TK. The future of drug safety testing: expanding

the view and narrowing the focus. Drug Discov Today 20 2009; 14: 162-7.

[2] Spielmann H, Grune B, Liebsch M, Seiler A, Vogel R. Successful validation of in vitro methods in toxicology by ZEBET, the Na-tional Centre for Alternatives in Germany at the BfR (Federal Insti-tute for Risk Assessment). Exp Toxicol Pathol 2008; 60: 225-33.

[3] Hofer T, Gerner I, Gundert-Remy U, et al. Animal testing and alternative approaches for the human health risk assessment under the proposed new European chemicals regulation. Arch Toxicol 2004; 78: 549-64.

[4] Brittijn SA, Duivesteijn SJ, Belmamoune M, et al. Zebrafish de-velopment and regeneration: new tools for biomedical research. Int J Dev Biol 2009; 53: 835-50.

[5] Hill AJ, Teraoka H, Heideman W, Peterson RE. Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol Sci 2005; 86: 6-19.

[6] Kari G, Rodeck U, Dicker AP. Zebrafish: an emerging model sys-tem for human disease and drug discovery. Clin Pharmacol Ther 2007; 82: 70-80.

[7] Parng C, Roy NM, Ton C, Lin Y, McGrath P. Neurotoxicity as-sessment using zebrafish. J Pharmacol Toxicol Methods 2007; 55: 103-12.

[8] Reimers MJ, La Du JK, Periera CB, Giovanini J, Tanguay RL. Ethanol-dependent toxicity in zebrafish is partially attenuated by antioxidants. Neurotoxicol Teratol 2006; 28: 497-508.

[9] Spitsbergen JM, Kent ML. The state of the art of the zebrafish model for toxicology and toxicologic pathology research--advantages and current limitations. Toxicol Pathol 2003; 31 Suppl: 62-87.

[10] Teraoka H, Dong W, Tsujimoto Y, et al. Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish. Biochem Biophys Res Commun 2003; 304: 223-8.

[11] Van den Belt K, Van Puymbroeck S, Witters H. Toxicity of cad-mium-contaminated clay to the zebrafish Danio rerio. Arch Envi-ron Contam Toxicol 2000; 38: 191-6.

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

[13] Strahle U, Scholz S, Geisler R, et al. Zebrafish embryos as an al-ternative to animal experiments--a commentary on the definition of the onset of protected life stages in animal welfare regulations. Re-prod Toxicol 2012; 33: 128-32.

[14] Knudsen T, Martin M, Chandler K, Kleinstreuer N, Judson R, Sipes N. Predictive models and computational toxicology. Methods Mol Biol 2013; 947: 343-74.

5424 Current Pharmaceutical Design, 2014, Vol. 20, No. 34 Kanungo et al.

[15] Lieschke GJ, Currie PD. Animal models of human disease: zebraf-ish swim into view. Nat Rev Genet 2007; 8: 353-67.

[16] Peterson RT, Macrae CA. Systematic approaches to toxicology in the zebrafish. Annu Rev Pharmacol Toxicol 2012; 52: 433-53.

[17] Selderslaghs IW, Van Rompay AR, De Coen W, Witters HE. De-velopment of a screening assay to identify teratogenic and embryo-toxic chemicals using the zebrafish embryo. Reprod Toxicol 2009; 28: 308-20.

[18] Stainier DY, Fishman MC. The zebrafish as a model system to study cardiovascular development. Trends Cardiovasc Med 1994; 4: 207-12.

[19] Howe K, Clark MD, Torroja CF, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 2013; 496: 498-503.

[20] Kettleborough RN, Busch-Nentwich EM, et al. A systematic ge-nome-wide analysis of zebrafish protein-coding gene function. Na-ture 2013; 496: 494-7.

[21] Granato M, Nusslein-Volhard C. Fishing for genes controlling development. Curr Opin Genet Dev 1996; 6: 461-8.

[22] Postlethwait JH, Woods IG, Ngo-Hazelett P, et al. Zebrafish com-parative genomics and the origins of vertebrate chromosomes. Ge-nome Res 2000; 10: 1890-902.

[23] Lam KH, Alex D, Lam IK, Tsui SK, Yang ZF, Lee SM. 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.

[24] Stein C, Caccamo M, Laird G, Leptin M. Conservation and diver-gence of gene families encoding components of innate immune re-sponse systems in zebrafish. Genome Biol 2007; 8: R251.

[25] Zimmerman AM, Romanowski KE, Maddox BJ. Targeted annota-tion of immunoglobulin light chain (IgL) genes in zebrafish from BAC clones reveals kappa-like recombining/deleting elements within IgL constant regions. Fish Shellfish Immunol 2011; 31: 697-703.

[26] MacRae CA, Peterson RT. Zebrafish-based small molecule discov-ery. Chem Biol 2003; 10: 901-8.

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

[28] Bretaud S, Lee S, Guo S. Sensitivity of zebrafish to environmental toxins implicated in Parkinson's disease. Neurotoxicol Teratol 2004; 26: 857-64.

[29] Gibbs EM, Horstick EJ, Dowling JJ. Swimming into prominence: the zebrafish as a valuable tool for studying human myopathies and muscular dystrophies. FEBS J 2013.

[30] Parng C. In vivo zebrafish assays for toxicity testing. Curr Opin Drug Discov Devel 2005; 8: 100-6.

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

[32] Drapeau P, Saint-Amant L, Buss RR, Chong M, McDearmid JR, Brustein E. Development of the locomotor network in zebrafish. Prog Neurobiol 2002; 68: 85-111.

[33] Best JD, Alderton WK. Zebrafish: An in vivo model for the study of neurological diseases. Neuropsychiatr Dis Treat 2008; 4: 567-76.

[34] Best JD, Berghmans S, Hunt JJ, Clarke SC, Fleming A, Goldsmith P, Roach AG. Non-associative learning in larval zebrafish. Neuro-psychopharmacology 2008; 33: 1206-15.

[35] Emran F, Rihel J, Dowling JE. A behavioral assay to measure responsiveness of zebrafish to changes in light intensities. J Vis Exp 2008.

[36] Guo S. Linking genes to brain, behavior and neurological diseases: what can we learn from zebrafish? Genes Brain Behav 2004; 3: 63-74.

[37] Guo S. Using zebrafish to assess the impact of drugs on neural development and function. Expert Opin Drug Discov 2009; 4: 715-726.

[38] Irons TD, MacPhail RC, Hunter DL, Padilla S. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish. Neuro-toxicol Teratol 2010; 32: 84-90.

[39] Kokel D, Bryan J, Laggner C, et al. Rapid behavior-based identifi-cation of neuroactive small molecules in the zebrafish. Nat Chem Biol 2010; 6: 231-237.

[40] Kokel D, Peterson RT. Chemobehavioural phenomics and behav-iour-based psychiatric drug discovery in the zebrafish. Brief Funct Genomic Proteomic 2008; 7: 483-90.

[41] Lockwood B, Bjerke S, Kobayashi K, Guo S. Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screen-ing. Pharmacol Biochem Behav 2004; 77: 647-54.

[42] Alderton W, Karran E, Ward S. Current and Future Perspectives in Psychiatric Drug Discovery. Drug News Perspect 2009; 22: 360-4.

[43] Berghmans S, Hunt J, Roach A, Goldsmith P. Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants. Epilepsy Res 2007; 75: 18-28.

[44] Burgess HA, Granato M. Sensorimotor gating in larval zebrafish. J Neurosci 2007; 27: 4984-94.

[45] Gerlai R. High-throughput behavioral screens: the first step to-wards finding genes involved in vertebrate brain function using ze-brafish. Molecules 2010; 15: 2609-22.

[46] Muto A, Orger MB, Wehman AM, et al. Forward genetic analysis of visual behavior in zebrafish. PLoS Genet 2005; 1: e66.

[47] Rihel J, Prober DA, Arvanites A, et al. Zebrafish behavioral profil-ing links drugs to biological targets and rest/wake regulation. Sci-ence 2010; 327: 348-51.

[48] Winter MJ, Redfern WS, Hayfield AJ, Owen SF, Valentin JP, Hutchinson TH. Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J Pharmacol Toxicol Methods 2008; 57: 176-87.

[49] MacPhail RC, Brooks J, Hunter DL, Padnos B, Irons TD, Padilla S. Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol. Neurotoxicology 2009; 30: 52-8.

[50] Richards FM, Alderton WK, Kimber GM, Liu Z, Strang I, Redfern WS, Valentin JP, Winter MJ, Hutchinson TH. Validation of the use of zebrafish larvae in visual safety assessment. J Pharmacol Toxi-col Methods 2008; 58: 50-8.

[51] Airhart MJ, Lee DH, Wilson TD, Miller BE, Miller MN, Skalko RG. Movement disorders and neurochemical changes in zebrafish larvae after bath exposure to fluoxetine (PROZAC). Neurotoxicol Teratol 2007; 29: 652-64.

[52] Boehmler W, Petko J, Woll M, et al. Identification of zebrafish A2 adenosine receptors and expression in developing embryos. Gene Expr Patterns 2009; 9: 144-51.

[53] Zhdanova IV, Wang SY, Leclair OU, Danilova NP. Melatonin promotes sleep-like state in zebrafish. Brain Res 2001; 903: 263-8.

[54] Cahill GM. Clock mechanisms in zebrafish. Cell Tissue Res 2002; 309: 27-34.

[55] Ninkovic J, Bally-Cuif L. The zebrafish as a model system for assessing the reinforcing properties of drugs of abuse. Methods 2006; 39: 262-74.

[56] Orger MB, Gahtan E, Muto A, Page-McCaw P, Smear MC, Baier H. Behavioral screening assays in zebrafish. Methods Cell Biol 2004; 77: 53-68.

[57] Padilla S, Corum D, Padnos B, et al. Zebrafish developmental screening of the ToxCast Phase I chemical library. Reprod Toxicol 2012; 33: 174-87.

[58] Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 2005; 131: 759-68.

[59] Tiedeken JA, Ramsdell JS. Embryonic exposure to domoic Acid increases the susceptibility of zebrafish larvae to the chemical con-vulsant pentylenetetrazole. Environ Health Perspect 2007; 115: 1547-52.

[60] Baxendale S, Holdsworth CJ, Meza Santoscoy PL, et al. Identifica-tion of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures. Dis Model Mech 2012; 5: 773-84.

[61] Carvan MJ, 3rd, Loucks E, Weber DN, Williams FE. Ethanol ef-fects on the developing zebrafish: neurobehavior and skeletal morphogenesis. Neurotoxicol Teratol 2004; 26: 757-68.

[62] 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.

[63] Xia T, Zhao Y, Sager T, et al. Decreased dissolution of ZnO by iron doping yields nanoparticles with reduced toxicity in the rodent lung and zebrafish embryos. ACS Nano 2011; 5: 1223-35.

[64] Bai W, Tian W, Zhang Z, et al. Effects of copper nanoparticles on the development of zebrafish embryos. J Nanosci Nanotechnol 2010; 10: 8670-6.

[65] Chen YH, Tsai IT, Wen CC, Wang YH, Cheng CC, Hu SC. Fin reduction is a novel and unexpected teratogenic effect of amikacin-treated zebrafish embryos. Toxicol Mech Methods 2012; 22: 151-8.

[66] Cheng J, Cheng SH. Influence of carbon nanotube length on toxic-ity to zebrafish embryos. Int J Nanomedicine 2012; 7: 3731-9.

Zebrafish Model in Drug Safety Assessment Current Pharmaceutical Design, 2014, Vol. 20, No. 34 5425

[67] King Heiden TC, Spitsbergen J, Heideman W, Peterson RE. Persis-tent adverse effects on health and reproduction caused by exposure of zebrafish to 2,3,7,8-tetrachlorodibenzo-p-dioxin during early de-velopment and gonad differentiation. Toxicol Sci 2009; 109: 75-87.

[68] King-Heiden TC, Wiecinski PN, Mangham AN, et al. Quantum dot nanotoxicity assessment using the zebrafish embryo. Environ Sci Technol 2009; 43: 1605-11.

[69] Lee KJ, Browning LM, Nallathamby PD, Desai T, Cherukuri PK, Xu XH. In vivo quantitative study of sized-dependent transport and toxicity of single silver nanoparticles using zebrafish embryos. Chem Res Toxicol 2012; 25: 1029-46.

[70] Truong L, Harper SL, Tanguay RL. Evaluation of embryotoxicity using the zebrafish model. Methods Mol Biol 2011; 691: 271-9.

[71] Usenko CY, Hopkins DC, Trumble SJ, Bruce ED. Hydroxylated PBDEs induce developmental arrest in zebrafish. Toxicol Appl Pharmacol 2012; 262: 43-51.

[72] Usenko CY, Robinson EM, Usenko S, Brooks BW, Bruce ED. PBDE developmental effects on embryonic zebrafish. Environ Toxicol Chem 2011; 30: 1865-72.

[73] Zhu X, Tian S, Cai Z. Toxicity assessment of iron oxide nanoparti-cles in zebrafish (Danio rerio) early life stages. PLoS One 2012; 7: e46286.

[74] Zhu X, Zhu L, Duan Z, Qi R, Li Y, Lang Y. Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Zebraf-ish (Danio rerio) early developmental stage. J Environ Sci Health A Tox Hazard Subst Environ Eng 2008; 43: 278-84.

[75] Zhu X, Zhu L, Li Y, Duan Z, Chen W, Alvarez PJ. Developmental toxicity in zebrafish (Danio rerio) embryos after exposure to manu-factured nanomaterials: buckminsterfullerene aggregates (nC60) and fullerol. Environ Toxicol Chem 2007; 26: 976-9.

[76] Oliveira R, Domingues I, Koppe Grisolia C, Soares AM. Effects of triclosan on zebrafish early-life stages and adults. Environ Sci Pol-lut Res Int 2009; 16: 679-88.

[77] Van den Bulck K, Hill A, Mesens N, Diekman H, De Schaepdrijver L, Lammens L. Zebrafish developmental toxicity assay: A fishy so-lution to reproductive toxicity screening, or just a red herring? Re-prod Toxicol 2011; 32: 213-9.

[78] 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.

[79] Tamplin OJ, White RM, Jing L, et al. Small molecule screening in zebrafish: swimming in potential drug therapies. Wiley Interdiscip Rev Dev Biol 2012; 1: 459-68.

[80] Peters PW, Piersma AH. In vitro embryotoxicity and teratogenicity studies. Toxicol In vitro 1990; 4: 570-6.

[81] Dawson DA, Fort DJ, Smith GJ, Newell DL, Bantle JA. Evaluation of the developmental toxicity of nicotine and cotinine with frog embryo teratogenesis assay: Xenopus. Teratog Carcinog Mutagen 1988; 8: 329-38.

[82] Bantle JA, Fort DJ, Rayburn JR, DeYoung DJ, Bush SJ. Further validation of FETAX: evaluation of the developmental toxicity of five known mammalian teratogens and non-teratogens. Drug Chem Toxicol 1990; 13: 267-82.

[83] Fort DJ, Bantle JA. Use of Frog Embryo Teratogenesis Assay-Xenopus and an exogenous metabolic activation system to evaluate the developmental toxicity of diphenylhydantoin. Fundam Appl Toxicol 1990; 14: 720-33.

[84] Fort DJ, Bantle JA. Analysis of the mechanism of isoniazid-induced developmental toxicity with frog embryo teratogenesis as-say: Xenopus (FETAX). Teratog Carcinog Mutagen 1990; 10: 463-76.

[85] Bernauer U, Oberemm A, Madle S, Gundert-Remy U. The use of in vitro data in risk assessment. Basic Clin Pharmacol Toxicol 2005; 96: 176-81.

[86] Fort DJ, Dawson DA, Bantle JA. Development of a metabolic activation system for the frog embryo teratogenesis assay: Xenopus (FETAX). Teratog Carcinog Mutagen 1988; 8: 251-63.

[87] Martin MT, Judson RS, Reif DM, Kavlock RJ, Dix DJ. Profiling chemicals based on chronic toxicity results from the U.S. EPA ToxRef Database. Environ Health Perspect 2009; 117: 392-9.

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

[89] Brannen KC, Charlap JH, Lewis EM. Zebrafish teratogenicity testing. Methods Mol Biol 2013; 947: 383-401.

[90] Brannen KC, Panzica-Kelly JM, Danberry TL, Augustine-Rauch KA. Development of a zebrafish embryo teratogenicity assay and

quantitative prediction model. Birth Defects Res B Dev Reprod Toxicol 2010; 89: 66-77.

[91] Panzica-Kelly JM, Zhang CX, Augustine-Rauch K. Zebrafish em-bryo developmental toxicology assay. Methods Mol Biol 2012; 889: 25-50.

[92] Hermsen SA, van den Brandhof EJ, van der Ven LT, Piersma AH. Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo po-tencies. Toxicol In vitro 2011; 25: 745-53.

[93] Sipes NS, Padilla S, Knudsen TB. Zebrafish: as an integrative model for twenty-first century toxicity testing. Birth Defects Res C Embryo Today 2011; 93: 256-67.

[94] Gustafson AL, Stedman DB, Ball J, et al. Inter-laboratory assess-ment of a harmonized zebrafish developmental toxicology assay - progress report on phase I. Reprod Toxicol 2012; 33: 155-64.

[95] Hermsen SA, Pronk TE, van den Brandhof EJ, van der Ven LT, Piersma AH. Chemical class-specific gene expression changes in the zebrafish embryo after exposure to glycol ether alkoxy acids and 1,2,4-triazole antifungals. Reprod Toxicol 2011; 32: 245-52.

[96] McCollum CW, Ducharme NA, Bondesson M, Gustafsson JA. Developmental toxicity screening in zebrafish. Birth Defects Res C Embryo Today 2011; 93: 67-114.

[97] Selderslaghs IW, Blust R, Witters HE. Feasibility study of the zebrafish assay as an alternative method to screen for developmen-tal toxicity and embryotoxicity using a training set of 27 com-pounds. Reprod Toxicol 2012; 33: 142-54.

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

[99] 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 Meth-ods 2008; 58: 59-68.

[100] Eimon PM, Rubinstein AL. The use of in vivo zebrafish assays in drug toxicity screening. Expert Opin Drug Metab Toxicol 2009; 5: 393-401.

[101] MacRae CA. Recent advances in in vivo screening for antiarrhyth-mic drugs. Expert Opin Drug Discov 2013; 8: 131-41.

[102] Parng C, Seng WL, Semino C, McGrath P. Zebrafish: a preclinical model for drug screening. Assay Drug Dev Technol 2002; 1: 41-8.

[103] Langheinrich U, Vacun G, Wagner T. Zebrafish embryos express an orthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia. Toxicol Appl Phar-macol 2003; 193: 370-82.

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

[105] Zheng W, Wang Z, Collins JE, Andrews RM, Stemple D, Gong Z. Comparative transcriptome analyses indicate molecular homology of zebrafish swimbladder and mammalian lung. PLoS One 2011; 6: e24019.

[106] Hassel D, Scholz EP, Trano N, et al. Deficient zebrafish ether-a-go-go-related gene channel gating causes short-QT syndrome in zebrafish reggae mutants. Circulation 2008; 117: 866-75.

[107] George S, Xia T, Rallo R, et al. Use of a high-throughput screening approach coupled with in vivo zebrafish embryo screening to de-velop hazard ranking for engineered nanomaterials. ACS Nano 2011; 5: 1805-17.

[108] Mittelstadt SW, Hemenway CL, Craig MP, Hove JR. Evaluation of zebrafish embryos as a model for assessing inhibition of hERG. J Pharmacol Toxicol Methods 2008; 57: 100-5.

[109] Langheinrich U. Zebrafish: a new model on the pharmaceutical catwalk. Bioessays 2003; 25: 904-12.

[110] Craig MP, Gilday SD, Hove JR. Dose-dependent effects of chemi-cal immobilization on the heart rate of embryonic zebrafish. Lab Anim (NY) 2006; 35: 41-7.

[111] Forouhar AS, Hove JR, Calvert C, Flores J, Jadvar H, Gharib M. Electrocardiographic characterization of embryonic zebrafish. Conf Proc IEEE Eng Med Biol Soc 2004; 5: 3615-7.

[112] Chi NC, Shaw RM, Jungblut B, et al. Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS Biol 2008; 6: e109.

[113] Chi NC, Bussen M, Brand-Arzamendi K, et al. Cardiac conduction is required to preserve cardiac chamber morphology. Proc Natl Acad Sci USA 2010; 107: 14662-7.

[114] Kanungo J, Cuevas E, Ali SF, Paule MG. l-Carnitine rescues keta-mine-induced attenuated heart rate and MAPK (ERK) activity in zebrafish embryos. Reprod Toxicol 2012; 33: 205-12.

5426 Current Pharmaceutical Design, 2014, Vol. 20, No. 34 Kanungo et al.

[115] Milan DJ, Jones IL, Ellinor PT, MacRae CA. In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation. Am J Physiol Heart Circ Physiol 2006; 291: H269-73.

[116] Milan DJ, Macrae CA. Zebrafish genetic models for arrhythmia. Prog Biophys Mol Biol 2008; 98: 301-8.

[117] Milan DJ, Peterson TA, Ruskin JN, Peterson RT, MacRae CA. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation 2003; 107: 1355-8.

[118] Sedmera D, Reckova M, deAlmeida A, et al. Functional and mor-phological evidence for a ventricular conduction system in zebraf-ish and Xenopus hearts. Am J Physiol Heart Circ Physiol 2003; 284: H1152-60.

[119] Carney SA, Peterson RE, Heideman W. 2,3,7,8-Tetrachlorodibenzo-p-dioxin activation of the aryl hydrocarbon re-ceptor/aryl hydrocarbon receptor nuclear translocator pathway causes developmental toxicity through a CYP1A-independent mechanism in zebrafish. Mol Pharmacol 2004; 66: 512-21.

[120] Wiegand C, Pflugmacher S, Giese M, Frank H, Steinberg C. Up-take, toxicity, and effects on detoxication enzymes of atrazine and trifluoroacetate in embryos of zebrafish. Ecotoxicol Environ Saf 2000; 45: 122-31.

[121] Ibabe A, Grabenbauer M, Baumgart E, Fahimi HD, Cajaraville MP. Expression of peroxisome proliferator-activated receptors in ze-brafish (Danio rerio). Histochem Cell Biol 2002; 118: 231-9.

[122] Zhang C, Willett C, Fremgen T. Zebrafish: an animal model for toxicological studies. Curr Protoc Toxicol 2003; Chapter 1: Unit1 7.

[123] He JH, Guo SY, Zhu F, et al. A zebrafish phenotypic assay for assessing drug-induced hepatotoxicity. J Pharmacol Toxicol Meth-ods 2013; 67: 25-32.

[124] Nebert DW, Dalton TP. The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis. Nat Rev Cancer 2006; 6: 947-60.

[125] Nebert DW, Nelson DR, Coon MJ, et al. The P450 superfamily: update on new sequences, gene mapping, and recommended no-menclature. DNA Cell Biol 1991; 10: 1-14.

[126] Zhang YY, Yang L. Interactions between human cytochrome P450 enzymes and steroids: physiological and pharmacological implica-tions. Expert Opin Drug Metab Toxicol 2009; 5: 621-9.

[127] Goldstone JV, McArthur AG, Kubota A, et al. Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish. BMC Genomics 2010; 11: 643.

[128] Simpson ER, Mahendroo MS, Means GD, et al. Aromatase cyto-chrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 1994; 15: 342-55.

[129] Simpson ER, Clyne C, Rubin G, et al. Aromatase--a brief over-view. Annu Rev Physiol 2002; 64: 93-127.

[130] Cheshenko K, Brion F, Le Page Y, et al. Expression of zebra fish aromatase cyp19a and cyp19b genes in response to the ligands of estrogen receptor and aryl hydrocarbon receptor. Toxicol Sci 2007; 96: 255-67.

[131] Tseng HP, Hseu TH, Buhler DR, Wang WD, Hu CH. Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva. Toxicol Appl Pharmacol 2005; 205: 247-58.

[132] Pascussi JM, Drocourt L, Fabre JM, Maurel P, Vilarem MJ. Dex-amethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor activators. Mol Phar-macol 2000; 58: 361-72.

[133] Scholz S, Fischer S, Gundel U, Kuster E, Luckenbach T, Voelker D. The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing. Environ Sci Pollut Res Int 2008; 15: 394-404.

[134] Segner H. Zebrafish (Danio rerio) as a model organism for investi-gating endocrine disruption. Comp Biochem Physiol C Toxicol Pharmacol 2009; 149: 187-95.

[135] Scholz S, Mayer I. Molecular biomarkers of endocrine disruption in small model fish. Mol Cell Endocrinol 2008; 293: 57-70.

[136] Tropepe V, Sive HL. Can zebrafish be used as a model to study the neurodevelopmental causes of autism? Genes Brain Behav 2003; 2: 268-81.

[137] Mueller T, Vernier P, Wullimann MF. The adult central nervous cholinergic system of a neurogenetic model animal, the zebrafish Danio rerio. Brain Res 2004; 1011: 156-69.

[138] Rink E, Wullimann MF. Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio). Brain Res 2004; 1011: 206-20.

[139] Wullimann MF, Mueller T. Identification and morphogenesis of the eminentia thalami in the zebrafish. J Comp Neurol 2004; 471: 37-48.

[140] Jeong JY, Kwon HB, Ahn JC, et al. Functional and developmental analysis of the blood-brain barrier in zebrafish. Brain Res Bull 2008; 75: 619-28.

[141] Xie J, Farage E, Sugimoto M, Anand-Apte B. A novel transgenic zebrafish model for blood-brain and blood-retinal barrier develop-ment. BMC Dev Biol 2010; 10: 76.

[142] Watanabe K, Nishimura Y, Nomoto T, et al. In vivo assessment of the permeability of the blood-brain barrier and blood-retinal barrier to fluorescent indoline derivatives in zebrafish. BMC Neurosci 2012; 13: 101.

[143] O'Callaghan JP, Sriram K. Glial fibrillary acidic protein and related glial proteins as biomarkers of neurotoxicity. Expert Opin Drug Saf 2005; 4: 433-42.

[144] de Esch C, Slieker R, Wolterbeek A, Woutersen R, de Groot D. Zebrafish as potential model for developmental neurotoxicity test-ing: a mini review. Neurotoxicol Teratol 2012; 34: 545-53.

[145] Appel B, Chitnis A. Neurogenesis and specification of neuronal identity. Results Probl Cell Differ 2002; 40: 237-51.

[146] Mueller T, Wullimann MF. Anatomy of neurogenesis in the early zebrafish brain. Brain Res Dev Brain Res 2003; 140: 137-55.

[147] Hill A, Howard CV, Strahle U, Cossins A. Neurodevelopmental defects in zebrafish (Danio rerio) at environmentally relevant di-oxin (TCDD) concentrations. Toxicol Sci 2003; 76: 392-9.

[148] Kanungo J, Cuevas E, Ali SF, Paule MG. Ketamine induces motor neuron toxicity and alters neurogenic and proneural gene expres-sion in zebrafish. J Appl Toxicol 2011.

[149] Cuevas E, Trickler WJ, Guo X, Ali SF, Paule MG, Kanungo J. Acetyl l-carnitine protects motor neurons and Rohon-Beard sensory neurons against ketamine-induced neurotoxicity in zebrafish em-bryos. Neurotoxicology and Teratology.

[150] Parng C, Ton C, Lin YX, Roy NM, McGrath P. A zebrafish assay for identifying neuroprotectants in vivo. Neurotoxicol Teratol 2006; 28: 509-16.

[151] Ali DW, Buss RR, Drapeau P. Properties of miniature glutamater-gic EPSCs in neurons of the locomotor regions of the developing zebrafish. J Neurophysiol 2000; 83: 181-91.

[152] Kanungo J, Cuevas E, Ali SF, Paule MG. Ketamine induces motor neuron toxicity and alters neurogenic and proneural gene expres-sion in zebrafish. J Appl Toxicol 2013; 33: 410-7.

[153] Kanungo J, Cuevas E, Guo X, et al. Nicotine alters the expression of molecular markers of endocrine disruption in zebrafish. Neuro-sci Lett 2012; 526: 133-7.

[154] Kanungo J, Paule MG. Disruption of blastomeric F-actin: a poten-tial early biomarker of developmental toxicity in zebrafish. Mol Cell Biochem 2011; 353: 283-90.

[155] Legendre P. Pharmacological evidence for two types of postsynap-tic glycinergic receptors on the Mauthner cell of 52-h-old zebrafish larvae. J Neurophysiol 1997; 77: 2400-15.

[156] Rigo JM, Badiu CI, Legendre P. Heterogeneity of postsynaptic receptor occupancy fluctuations among glycinergic inhibitory syn-apses in the zebrafish hindbrain. J Physiol 2003; 553: 819-32.

[157] Parng C, Anderson N, Ton C, McGrath P. Zebrafish apoptosis assays for drug discovery. Methods Cell Biol 2004; 76: 75-85.

[158] Figueroa-Masot XA, Hetman M, Higgins MJ, Kokot N, Xia Z. Taxol induces apoptosis in cortical neurons by a mechanism inde-pendent of Bcl-2 phosphorylation. J Neurosci 2001; 21: 4657-67.

[159] Svoboda KR, Vijayaraghavan S, Tanguay RL. Nicotinic receptors mediate changes in spinal motoneuron development and axonal pathfinding in embryonic zebrafish exposed to nicotine. J Neurosci 2002; 22: 10731-41.

[160] Paule MG, Li M, Allen RR, et al. Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol 2011; 33: 220-30.

[161] Slikker W, Jr., Zou X, Hotchkiss CE, et al. Ketamine-induced neuronal cell death in the perinatal rhesus monkey. Toxicol Sci 2007; 98: 145-58.

[162] Wang C, Sadovova N, Fu X, et al. The role of the N-methyl-D-aspartate receptor in ketamine-induced apoptosis in rat forebrain culture. Neuroscience 2005; 132: 967-77.

Zebrafish Model in Drug Safety Assessment Current Pharmaceutical Design, 2014, Vol. 20, No. 34 5427

[163] Zou X, Patterson TA, Divine RL, et al. Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. Int J Dev Neurosci 2009; 27: 727-31.

[164] Dlugos CA, Rabin RA. Ethanol effects on three strains of zebraf-ish: model system for genetic investigations. Pharmacol Biochem Behav 2003; 74: 471-80.

[165] Gerlai R, Lee V, Blaser R. Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio). Pharma-col Biochem Behav 2006; 85: 752-61.

[166] Mathur P, Berberoglu MA, Guo S. Preference for ethanol in zebraf-ish following a single exposure. Behav Brain Res 2011; 217: 128-33.

[167] Peng J, Wagle M, Mueller T, et al. Ethanol-modulated camouflage response screen in zebrafish uncovers a novel role for cAMP and extracellular signal-regulated kinase signaling in behavioral sensi-tivity to ethanol. J Neurosci 2009; 29: 8408-18.

[168] Loucks E, Ahlgren S. Assessing teratogenic changes in a zebrafish model of fetal alcohol exposure. J Vis Exp 2012.

[169] Chromcova L, Stepanova S, Plhalova L, Praskova E, Svobodova Z. Effect of four selected carrier solvents on embryonal stages of Danio rerio. Neuro Endocrinol Lett 2012; 33 Suppl 3: 60-5.

[170] Hallare A, Nagel K, Kohler HR, Triebskorn R. Comparative em-bryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos. Ecotoxicol Environ Saf 2006; 63: 378-88.

[171] Reimers MJ, Flockton AR, Tanguay RL. Ethanol- and acetalde-hyde-mediated developmental toxicity in zebrafish. Neurotoxicol Teratol 2004; 26: 769-81.

[172] Loucks EJ, Ahlgren SC. Deciphering the role of Shh signaling in axial defects produced by ethanol exposure. Birth Defects Res A Clin Mol Teratol 2009; 85: 556-67.

[173] Bilotta J, Barnett JA, Hancock L, Saszik S. Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome. Neurotoxicol Teratol 2004; 26: 737-43.

[174] Dlugos CA, Rabin RA. Ocular deficits associated with alcohol exposure during zebrafish development. J Comp Neurol 2007; 502: 497-506.

[175] Kashyap B, Frederickson LC, Stenkamp DL. Mechanisms for persistent microphthalmia following ethanol exposure during reti-nal neurogenesis in zebrafish embryos. Vis Neurosci 2007; 24: 409-21.

[176] Kashyap B, Frey RA, Stenkamp DL. Ethanol-induced mi-crophthalmia is not mediated by changes in retinoic acid or sonic hedgehog signaling during retinal neurogenesis. Alcohol Clin Exp Res 2011; 35: 1644-61.

[177] Matsui JI, Egana AL, Sponholtz TR, Adolph AR, Dowling JE. Effects of ethanol on photoreceptors and visual function in devel-oping zebrafish. Invest Ophthalmol Vis Sci 2006; 47: 4589-97.

[178] de Esch C, van der Linde H, Slieker R, et al. Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol. Neu-rotoxicol Teratol 2012; 34: 425-33.

[179] Loucks E, Carvan MJ, 3rd. Strain-dependent effects of develop-mental ethanol exposure in zebrafish. Neurotoxicol Teratol 2004; 26: 745-55.

[180] Arenzana FJ, Carvan MJ, 3rd, Aijon J, Sanchez-Gonzalez R, Are-valo R, Porteros A. Teratogenic effects of ethanol exposure on ze-brafish visual system development. Neurotoxicol Teratol 2006; 28: 342-8.

[181] Bilotta J, Saszik S, Givin CM, Hardesty HR, Sutherland SE. Ef-fects of embryonic exposure to ethanol on zebrafish visual func-tion. Neurotoxicol Teratol 2002; 24: 759-66.

[182] Blader P, Strahle U. Ethanol impairs migration of the prechordal plate in the zebrafish embryo. Dev Biol 1998; 201: 185-201.

[183] Ali S, Champagne DL, Alia A, Richardson MK. Large-scale analy-sis of acute ethanol exposure in zebrafish development: a critical time window and resilience. PLoS One 2011; 6: e20037.

[184] Driscoll CD, Streissguth AP, Riley EP. Prenatal alcohol exposure: comparability of effects in humans and animal models. Neurotoxi-col Teratol 1990; 12: 231-7.

[185] Cudd TA. Animal model systems for the study of alcohol teratol-ogy. Exp Biol Med (Maywood) 2005; 230: 389-93.

[186] Sylvain NJ, Brewster DL, Ali DW. Embryonic ethanol exposure alters synaptic properties at zebrafish neuromuscular junctions. Neurotoxicol Teratol 2011; 33: 313-21.

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

[188] Kanungo J, Lantz S, Paule MG. In vivo imaging and quantitative analysis of changes in axon length using transgenic zebrafish em-bryos. Neurotoxicol Teratol 2011; 33: 618-23.

[189] Jacobs JS, Miller MW. Proliferation and death of cultured fetal neocortical neurons: effects of ethanol on the dynamics of cell growth. J Neurocytol 2001; 30: 391-401.

[190] Fleisch VC, Neuhauss SC. Visual behavior in zebrafish. Zebrafish 2006; 3: 191-201.

[191] Neuhauss SC, Biehlmaier O, Seeliger MW, et al. Genetic disorders of vision revealed by a behavioral screen of 400 essential loci in zebrafish. J Neurosci 1999; 19: 8603-15.

[192] Fekete DM. Developmental biology. Rocks that roll zebrafish. Science 2003; 302: 241-2.

[193] Whitfield TT. Zebrafish as a model for hearing and deafness. J Neurobiol 2002; 53: 157-71.

[194] Froehlicher M, Liedtke A, Groh KJ, Neuhauss SC, Segner H, Eg-gen RI. Zebrafish (Danio rerio) neuromast: promising biological endpoint linking developmental and toxicological studies. Aquat Toxicol 2009; 95: 307-19.

[195] Metcalfe WK. Sensory neuron growth cones comigrate with poste-rior lateral line primordial cells in zebrafish. J Comp Neurol 1985; 238: 218-24.

[196] Lopez-Schier H, Starr CJ, Kappler JA, Kollmar R, Hudspeth AJ. Directional cell migration establishes the axes of planar polarity in the posterior lateral-line organ of the zebrafish. Dev Cell 2004; 7: 401-12.

[197] Froehlicher M, Liedtke A, Groh K, et al. Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio re-rio) larvae. Dev Biol 2009; 330: 32-43.

[198] Easter SS, Jr., Nicola GN. The development of vision in the zebraf-ish (Danio rerio). Dev Biol 1996; 180: 646-63.

[199] Zeddies DG, Fay RR. Development of the acoustically evoked behavioral response in zebrafish to pure tones. J Exp Biol 2005; 208: 1363-72.

[200] Harris JA, Cheng AG, Cunningham LL, MacDonald G, Raible DW, Rubel EW. Neomycin-induced hair cell death and rapid re-generation in the lateral line of zebrafish (Danio rerio). J Assoc Res Otolaryngol 2003; 4: 219-34.

[201] Owens KN, Santos F, Roberts B, et al. Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death. PLoS Genet 2008; 4: e1000020.

[202] Santos F, MacDonald G, Rubel EW, Raible DW. Lateral line hair cell maturation is a determinant of aminoglycoside susceptibility in zebrafish (Danio rerio). Hear Res 2006; 213: 25-33.

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

[204] Ou H, Simon JA, Rubel EW, Raible DW. Screening for chemicals that affect hair cell death and survival in the zebrafish lateral line. Hear Res 2012; 288: 58-66.

[205] Ou HC, Cunningham LL, Francis SP, et al. Identification of FDA-approved drugs and bioactives that protect hair cells in the zebraf-ish (Danio rerio) lateral line and mouse (Mus musculus) utricle. J Assoc Res Otolaryngol 2009; 10: 191-203.

[206] Uribe PM, Mueller MA, Gleichman JS, et al. Dimethyl sulfoxide (DMSO) exacerbates cisplatin-induced sensory hair cell death in zebrafish (Danio rerio). PLoS One 2013; 8: e55359.

[207] Drummond IA, Majumdar A, Hentschel H, et al. Early develop-ment of the zebrafish pronephros and analysis of mutations affect-ing pronephric function. Development 1998; 125: 4655-67.

[208] Hentschel DM, Bonventre JV. Novel non-rodent models of kidney disease. Curr Mol Med 2005; 5: 537-46.

[209] Hentschel DM, Park KM, Cilenti L, Zervos AS, Drummond I, Bonventre JV. Acute renal failure in zebrafish: a novel system to study a complex disease. Am J Physiol Renal Physiol 2005; 288: F923-9.

[210] Kelly M. Chronic renal failure. Am J Nurs 1996; 96: 36-7. [211] Hentschel DM, Mengel M, Boehme L, et al. Rapid screening of

glomerular slit diaphragm integrity in larval zebrafish. Am J Physiol Renal Physiol 2007; 293: F1746-50.

[212] Pack M, Solnica-Krezel L, Malicki J, et al. Mutations affecting development of zebrafish digestive organs. Development 1996; 123: 321-8.

[213] Holmberg A, Schwerte T, Pelster B, Holmgren S. Ontogeny of the gut motility control system in zebrafish Danio rerio embryos and larvae. J Exp Biol 2004; 207: 4085-94.

5428 Current Pharmaceutical Design, 2014, Vol. 20, No. 34 Kanungo et al.

[214] Fleming A, Keynes R, Tannahill D. A central role for the noto-chord in vertebral patterning. Development 2004; 131: 873-80.

[215] Inohaya K, Takano Y, Kudo A. The teleost intervertebral region acts as a growth center of the centrum: in vivo visualization of os-teoblasts and their progenitors in transgenic fish. Dev Dyn 2007; 236: 3031-46.

[216] Fleming A, Sato M, Goldsmith P. High-throughput in vivo screen-ing for bone anabolic compounds with zebrafish. J Biomol Screen 2005; 10: 823-31.

[217] Barrett R, Chappell C, Quick M, Fleming A. A rapid, high content, in vivo model of glucocorticoid-induced osteoporosis. Biotechnol J 2006; 1: 651-5.

[218] Bar-Ilan O, Albrecht RM, Fako VE, Furgeson DY. Toxicity as-sessments of multisized gold and silver nanoparticles in zebrafish embryos. Small 2009; 5: 1897-910.

[219] Grunwald DJ, Streisinger G. Induction of recessive lethal and spe-cific locus mutations in the zebrafish with ethyl nitrosourea. Genet Res 1992; 59: 103-16.

[220] Streisinger G, Singer F, Walker C, Knauber D, Dower N. Segrega-tion analyses and gene-centromere distances in zebrafish. Genetics 1986; 112: 311-9.

[221] Alestrom P, Holter JL, Nourizadeh-Lillabadi R. Zebrafish in func-tional genomics and aquatic biomedicine. Trends Biotechnol 2006; 24: 15-21.

[222] Driever W, Fishman MC. The zebrafish: heritable disorders in transparent embryos. J Clin Invest 1996; 97: 1788-94.

[223] Cui C, Benard EL, Kanwal Z, et al. Infectious disease modeling and innate immune function in zebrafish embryos. Methods Cell Biol 2011; 105: 273-308.

[224] Murphey RD, Stern HM, Straub CT, Zon LI. A chemical genetic screen for cell cycle inhibitors in zebrafish embryos. Chem Biol Drug Des 2006; 68: 213-9.

[225] Zon LI, Le X. Potential of zebrafish for anticancer drug screening. Expert Opin Drug Discov 2008; 3: 1451-60.

[226] Carvalho R, de Sonneville J, Stockhammer OW, et al. A high-throughput screen for tuberculosis progression. PLoS One 2011; 6: e16779.

[227] Berger J, Berger S, Hall TE, Lieschke GJ, Currie PD. Dystrophin-deficient zebrafish feature aspects of the Duchenne muscular dys-trophy pathology. Neuromuscul Disord 2010; 20: 826-32.

[228] Berger J, Currie PD. Zebrafish models flex their muscles to shed light on muscular dystrophies. Dis Model Mech 2012; 5: 726-32.

[229] Telfer WR, Busta AS, Bonnemann CG, Feldman EL, Dowling JJ. Zebrafish models of collagen VI-related myopathies. Hum Mol Genet 2010; 19: 2433-44.

[230] Langenau DM, Keefe MD, Storer NY, et al. Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes Dev 2007; 21: 1382-95.

[231] Nasevicius A, Ekker SC. Effective targeted gene 'knockdown' in zebrafish. Nat Genet 2000; 26: 216-20.

[232] Wang W, Liu X, Gelinas D, Ciruna B, Sun Y. A fully automated robotic system for microinjection of zebrafish embryos. PLoS One 2007; 2: e862.

[233] Hogg RC, Bandelier F, Benoit A, Dosch R, Bertrand D. An auto-mated system for intracellular and intranuclear injection. J Neuro-sci Methods 2008; 169: 65-75.

[234] Veneman WJ, Stockhammer OW, de Boer L, Zaat SA, Meijer AH, Spaink HP. A zebrafish high throughput screening system used for Staphylococcus epidermidis infection marker discovery. BMC Ge-nomics 2013; 14: 255.

[235] Noori A, Selvaganapathy PR, Wilson J. Microinjection in a micro-fluidic format using flexible and compliant channels and elec-troosmotic dosage control. Lab Chip 2009; 9: 3202-11.

[236] Mizell M, Romig ES. The aquatic vertebrate embryo as a sentinel for toxins: zebrafish embryo dechorionation and perivitelline space microinjection. Int J Dev Biol 1997; 41: 411-23.

[237] Mathias JR, Saxena MT, Mumm JS. Advances in zebrafish chemi-cal screening technologies. Future Med Chem 2012; 4: 1811-22.

[238] Walker SL, Ariga J, Mathias JR, et al. Automated reporter quanti-fication in vivo: high-throughput screening method for reporter-based assays in zebrafish. PLoS One 2012; 7: e29916.

[239] Blackburn JS, Liu S, Raimondi AR, Ignatius MS, Salthouse CD, Langenau DM. High-throughput imaging of adult fluorescent ze-brafish with an LED fluorescence macroscope. Nat Protoc 2011; 6: 229-41.

[240] Burns CG, Milan DJ, Grande EJ, Rottbauer W, MacRae CA, Fish-man MC. High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nat Chem Biol 2005; 1: 263-4.

[241] Gehrig J, Reischl M, Kalmar E, et al. Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos. Nat Methods 2009; 6: 911-6.

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

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

[244] Vogt A, McPherson PA, Shen X, et al. High-content analysis of cancer-cell-specific apoptosis and inhibition of in vivo angiogenesis by synthetic (-)-pironetin and analogs. Chem Biol Drug Des 2009; 74: 358-68.

[245] Xu X, Huang X, Xia W, Xia S. A high-throughput analysis method to detect regions of interest and quantify zebrafish embryo images. J Biomol Screen 2010; 15: 1152-9.

[246] Chang TY, Pardo-Martin C, Allalou A, Wahlby C, Yanik MF. Fully automated cellular-resolution vertebrate screening platform with parallel animal processing. Lab Chip 2012; 12: 711-6.

[247] Radi M, Evensen L, Dreassi E, et al. A combined tar-geted/phenotypic approach for the identification of new antiangio-genics agents active on a zebrafish model: from in silico screening to cyclodextrin formulation. Bioorg Med Chem Lett 2012; 22: 5579-83.

[248] Hung MW, Zhang ZJ, Li S, et al. From omics to drug metabolism and high content screen of natural product in zebrafish: a new model for discovery of neuroactive compound. Evid Based Com-plement Alternat Med 2012; 2012: 605303.

[249] Postlethwait J, Amores A, Cresko W, Singer A, Yan YL. Subfunc-tion partitioning, the teleost radiation and the annotation of the hu-man genome. Trends Genet 2004; 20: 481-90.

[250] Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N. Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci USA 2004; 101: 12792-7.

[251] Amsterdam A, Sadler KC, Lai K, et al. Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biol 2004; 2: E139.

[252] Weigt S, Huebler N, Strecker R, Braunbeck T, Broschard TH. Zebrafish (Danio rerio) embryos as a model for testing proterato-gens. Toxicology 2011; 281: 25-36.

[253] Martins J, Oliva Teles L, Vasconcelos V. Assays with Daphnia magna and Danio rerio as alert systems in aquatic toxicology. Envi-ron Int 2007; 33: 414-25.

[254] de Jong E, Barenys M, Hermsen SA, et al. Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles. Toxicol Appl Pharmacol 2011; 253: 103-11.

[255] Lee HY, Inselman AL, Kanungo J, Hansen DK. Alternative models in developmental toxicology. Syst Biol Reprod Med 2012; 58: 10-22.

[256] Halle W. The Registry of Cytotoxicity: toxicity testing in cell cul-tures to predict acute toxicity (LD50) and to reduce testing in ani-mals. Altern Lab Anim 2003; 31: 89-198.

[257] Schreiber R, Altenburger R, Paschke A, Schuurmann G, Kuster E. A novel in vitro system for the determination of bioconcentration factors and the internal dose in zebrafish (Danio rerio) eggs. Che-mosphere 2009; 77: 928-33.

[258] Trickler WJ, Guo X, Cuevas E, Ali SF, Paule MG, Kanungo J. Ketamine attenuates cytochrome p450 aromatase gene expression and estradiol-17beta levels in zebrafish early life stages. J Appl Toxicol 2013.

[259] Kim S, Ji K, Lee S, Lee J, Kim J, Kho Y, Choi K. Perfluorooctane sulfonic acid exposure increases cadmium toxicity in early life stage of zebrafish, Danio rerio. Environ Toxicol Chem 2011; 30: 870-7.

[260] Peterson RT, Nass R, Boyd WA, Freedman JH, Dong K, Narahashi T. Use of non-mammalian alternative models for neurotoxicologi-cal study. Neurotoxicology 2008; 29: 546-55.

[261] Tierney KB. Behavioural assessments of neurotoxic effects and neurodegeneration in zebrafish. Biochim Biophys Acta 2011; 1812: 381-9.

[262] Driessen M, Kienhuis AS, Pennings JL, et al. Exploring the zebraf-ish embryo as an alternative model for the evaluation of liver toxic-

Zebrafish Model in Drug Safety Assessment Current Pharmaceutical Design, 2014, Vol. 20, No. 34 5429

ity by histopathology and expression profiling. Arch Toxicol 2013; 87: 807-23.

[263] Hill A, Mesens N, Steemans M, Xu JJ, Aleo MD. Comparisons between in vitro whole cell imaging and in vivo zebrafish-based approaches for identifying potential human hepatotoxicants earlier in pharmaceutical development. Drug Metab Rev 2012; 44: 127-40.

[264] Ou HC, Santos F, Raible DW, Simon JA, Rubel EW. Drug screen-ing for hearing loss: using the zebrafish lateral line to screen for drugs that prevent and cause hearing loss. Drug Discov Today 2010; 15: 265-71.

[265] Coffin AB, Ou H, Owens KN, et al. Chemical screening for hair cell loss and protection in the zebrafish lateral line. Zebrafish 2010; 7: 3-11.

[266] 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.

[267] Ali S, Champagne DL, Richardson MK. Behavioral profiling of zebrafish embryos exposed to a panel of 60 water-soluble com-pounds. Behav Brain Res 2012; 228: 272-83.

[268] Bailey J, Oliveri A, Levin ED. Zebrafish model systems for devel-opmental neurobehavioral toxicology. Birth Defects Res C Embryo Today 2013; 99: 14-23.

Received: December 11, 2013 Accepted: February 4, 2014