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116 Current Drug Metabolism, 2009, 10, 116-124 Zebrafish: A Complete Animal Model for In Vivo Drug Discovery and Development Chiranjib Chakraborty1,*, Chi Hsin Hsu1, Zhi Hong Wen1, Chang Shing Lin1 and Govindasamy Agoramoorthy2 1 Department of Marine Biotechnology and Resources, College of Marine Science and Division of Marine Biotechnology, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohisung, Taiwan; 2Department of Pharmacy, Tajen University, Yanpu, Pingtung 907, Taiwan Abstract: In last few years, the use of zebrafish (Danio rerio) in scientific research is growing very rapidly. Initially, it was a popular as a model of vertebrate development because zebrafish embryos are transparent and also develop rapidly. Presently, the research using zebrafish is expanding into other areas such as pharmacology, clinical research as a diseases model and interestingly in drug discovery. The use of zebrafish in pharmaceutical research and discovery and drug development is mainly screening of lead compounds, target identification, target validation, morpholino oligonucleotide screens, assay development for drug discovery, physiology based drug discovery, quantitative structure-activity relationship (QSAR) and structure –activity relationships (SAR) study and drug toxicity study. In this paper, we have described properly all the areas of dug discovery where zebrafish is used as a tool. We are hopeful that the use of these techniques or methods will make the zebrafish a prominent model in drug discovery and development research in the forthcoming years. Keywords: Zebrafish, animal model, in vivo drug discovery, physiology based drug discovery, screening of compounds, target identification, target validation, drug toxicity study. INTRODUCTION Fundamentally, drug discovery follows two different routes— physiology based drug discovery and target-based drug discovery. Physiology-based drug discovery follows physiological readouts. One example is the amelioration of a disease phenotype in an animal model or cell-based assay. Compounds are screened first and profiling can be done based on this readout. A purely physiologybased approach would initially forgo target identification/validation and instead jump right into screening. Identification of the drug target and the mechanism of action would follow in later stages of the process by deduction based on the specific pharmacological properties of lead compounds [1]. On the other hand, the path of target-based drug discovery commences with identifying the function of a possible therapeutic target and its role in disease. This path or route relies on target identification and validation. Screening small molecule compounds for specific phenotypes and identification of their targets [2] may provide a short-cut for target identification and validation by combining several steps of the process into one step. The strategy for screening of large groups of chemical compounds for their effects on specific cellular character in cell cultures is well-established. On the other hand, many biological processes cannot be reproduced in cultured cells and often the three-dimensional environment of cells determines their function. Furthermore, metabolism of the compounds may be profoundly different in whole organisms. Therefore, one would like to screen large numbers of small molecule compounds with high throughput for biological activity in whole organisms as early in the screening process as possible [3-5]. Presently, these two routes of drug discovery have become feasible using zebrafish embryos [6] or zebrafish adults. In this review, we highlight the importance of zebrafish for background for dug discovery and development; physiology based drug discovery, target-based drug discovery, drug toxicity study using zebrafish model. ZEBRAFISH A COMPLETE TOOL FOR DUG DISCOVERY AND DEVELOPMENT: A BACKGROUND Due to transparent embryos and rapid organogenesis, zebrafish were recognized as a tool for developmental biologists in the 1970s. In the 1990s, zebrafish were used for the first vertebrate large scale *Address correspondence to this author at the Department Biotechnology, College of Engineering and Technology, IILM Academy, Greater Noida, India; Fax: +91-120-2320058; E-mail: [email protected] 1389-2002/09 $55.00+.00 mutagenesis screen, yielding thousands of mutations, some of which recapitulated human diseases. Several characteristics make zebrafish a convincing tool for drug discovery. First, large numbers of embryos are produced due to the high fecundity of zebrafish. Each mature female can produce 200-250 eggs per mating. Mating is not seasonal. In addition, maintenance costs are considerably lower than those for mammals. Second, embryogenesis is rapid. The entire body plan established by 24 hours post fertilization (hpf) and most of the internal organs like heart, liver kidney and intestine totally developed by 96 hpf [7]. Third, zebrafish larvae are transparent which means organs, cells and tissues are visualized in vivo and investigated in realtime [8,9]. Fourth, the zebrafish is amenable to molecular and genetic analysis through rapid determination of temporal and spatial gene expression, examination of specific gene function by transgenic development, antisense gene knockdown and through large-scale mutagenesis [10,11]. Fifth, zebrafish embryos can be used to screen compounds in 50 microliter volumes. Compounds are added to the water, absorbed and ingested once embryos are able to swallow. Test compounds have been shown to have reproducible effects zebrafish assays. Sixth, Zebrafish has cardiovascular, nervous and digestive systems that are similar to those of mammals. Seventh, automation of fluorescent zebrafish assays will allow medium-throughput, high content screening of compounds (large number of compounds like 1,000 compounds a day). Eight, a high degree of conservation exists between the human and zebrafish genomes (approximately 75% similarity). The Sanger Institute is in the process of sequencing the zebra fish genome. Raw sequence totalling approximately 7.8 times the size of the genome is now publicly available. Updated assemblies are generated twice a year. A complete assembled sequence has already published [12]. Due to above advantages, zebrafish is the most important research tool today (Fig. 1). As a nearby for the human system, zebrafish is the only vertebrate system currently used for rapid in vivo compound screening. Since zebrafish are amenable to large-scale mutagenesis, a forward genetics approach can be used to identify new disease-relevant targets. Once novel targets are identified, the zebrafish has the added benefit of providing a system for their validation through the rapid analysis of gene function. Thus, the zebrafish affords a model to examine not only gene function but also high volume compound screening, target identification, target validation, assay development and drug toxicity study. © 2009 Bentham Science Publishers Ltd. Zebrafish: A Complete Animal Model for In Vivo Drug Discovery and Development Fig. (1). The increase in the use of zebrafish research reported in Pubmed references from the year 1980 to 2008. The term ‘zebrafish’ and the year like “ 2007” were used for searching in Pubmed . ZEBRAFISH AND PRECLINICAL DRUG DISCOVERY The modern drug discovery process can be divided into four major components: screening of lead compounds, target identification, target validation and assay development [1, 13]. Target identification describes to the process of identifying gene or protein (target gene product) that, when modulated by a drug, can have a positive impact on disease state progression. Once a possible target is identified which is very promising, the target validation process begins by determination of protein function and assessment of the 'druggability' of the target [1,14,15]. Furthermore, validated targets for their ability can be tested along with the small molecule compounds to modulate the function of the protein. Compound screening can also be useful in disease models when the target is unknown. The zebrafish has the importance in each of these areas of drug discovery (Fig. 1). Fig. (2). A flowchart that describes the zebrafish has the potentiality in each of the areas of drug discovery. Current Drug Metabolism, 2009, Vol. 10, No. 2 117 Screening of Lead Compounds Screening of drug in zebrafish that have been carried out so far have demonstrated that it is feasible to screen libraries of compounds with specific biological activities. The zebrafish is an outstanding vertebrate system for developing in vivo disease-related assays that can be useful for high volume compound screening. Embryos and larvae are amenable to compound screening applications because they can be assayed in multiwell plates where compounds can be taken up directly from the media or injected as necessary [16]. Moon et al. [17] used zebrafish embryos to screen a small library of triazine compounds for their ability to inhibit tubulin polymerization in vivo which was a first step toward identification of new anticancer drugs. A medium-scale screen with 1100 randomly selected small molecules was performed on morphological defects. This experiment was focusing on morphological defects mainly after 1, 2 or 3 days and the research was leading to the identification of compounds that are involved in development of the central nervous system, the cardiovascular system, the neural crest and the ear [5,18]. Some of the compounds (~ 2%) were lethal at very early stages and some compounds (~1% ) induced specific defects. The molecular targets of these compounds were unknown; however the phenotypes and/or chemical structure suggest drug candidate targets in some cases. For example, 31B4, a compound identified in the screen, affects general pigment production. 31B4’s chemical structure resembles phenylthiourea. It is also an effective tyrosinase inhibitor like phenylthiourea [5]. To screen for novel hypo-pigmenting agents, many methodologies such as cell culture and enzymatic assays are usually used. In a study, Choi et al. [19] validated zebrafish as an animal model for phenotype-based screening of melanogenic inhibitors or stimulators. The results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Schiffer et al. [20] established the zebrafish, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins. Murphey et al. [21] examined a broad range of known cell cycle compounds like the mitotic marker phospho-histone H3 using zebrafish embryos. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery. Target Identification Identification of novel drug targets is a bottleneck in drug discovery [14]. So far, only crude morphological defects [5] and behavioral changes [22] have been considered to evaluate the effects of the compounds, but it is also feasible to observe the protein localization, gene expression and metabolic changes into the specific organ where the morphological changes occur. However, one target identification focused assay was performed where whole-mount in situ hybridization was used to analyze catecholaminergic neurons in mutated fish [23]. For neurodegenerative and psychiatric disorders, class genes that interrupt the formation of specific neuronal classes may be important targets. Another experiment was performed that directly examined the behavior of zebrafish that was exposed to cocaine. In this experiment, identification of mutants was performed with the reduce sensitivity to cocaine [22]. Cocaine-treatment of normal fish increased 118 Current Drug Metabolism, 2009, Vol. 10, No. 2 Chakraborty et al. their aggressive behavior, and changed their conditioned place preference, an indication of the rewarding effect of cocaine. Analysis of mutations that affect these cocaine-induced behaviors may help identify targets for modulation of addictive behavior. Addiction is a complex maladaptive behavior involving alterations in several neurotransmitter networks also. In mammals, psychostimulants trigger elevated extracellular levels of dopamine, which can be modulated by central cholinergic transmission. The elements of the cholinergic system might be targeted for drug addiction therapies remains unknown. Ninkovic et al. (2006) further show that this behavior is dramatically reduced upon genetic impairment of acetylcholinesterase (AChE) function in ache/+ mutants, without involvement of concomitant defects in exploratory activity, learning, and visual performance. These observations demonstrate that the cholinergic system modulates drug-induced reward in zebrafish, and identify genetically AChE as a promising target for systemic therapies against addiction to psychostimulants. More generally, this experiment validate the zebrafish model to study the effect of developmental mutations on the molecular neurobiology of addiction in vertebrates [24]. In another screen, zebrafish mutations with defects in blood clotting were identified [25]. This assay can Analysis of mutations with defects in blood clotting that may help identify drug targets. A fluorescent lipid assay was used to identify genes that may hinder with normal lipid processing [26]. Zebrafish larvae ingested the quenched fluorescent phospholipid substrate N- ((6-(2,4dinitrophenyl)amino)hexanoyl)-1-palmitoyl-2-BODIPYFL- pentanoyl-sn-glycero-3-phosphoethanolamine (PED6) that fluoresces after cleavage by phospholipases in the intestine. This assay can used to identify genes that may hinder with normal lipid processing. To identify novel pigmentation modulators and their cellular targets, chemical genetic screenings were performed with triazinebased combinatorial libraries that include various linkers as intrinsic components of the small molecules in the library. Twelve compounds were identified as novel pigmentation modulators from various screenings performed in normal and albino murine melanocytes and zebrafish. Target identification by affinity chromatography revealed unexpected roles for prohibitin and mitochondrial F1F0-adenotriphosphatase in the regulation of mammalian pigmentation. Results from this screenings provide potential active agents and targets for the medical and aesthetic treatment of disorders of pigmentation [27]. A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a draTable 1. matic increase in the pigmentation of early stage zebrafish embryos. A compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells [28]. Therefore zebrafish can be used to identify new diseaserelevant targets. Target Validation Target validation as well as lead compound optimization can be done through a zebrafish diseases model. Disease model can be created through transgenic line or knockdown line that can help to validate the target [29]. Therefore, creation of transgenic line or knockdown line can validate the target as well as lead optimization. Once novel targets are identified, the zebrafish has the added benefit of providing a system for their validation through the rapid analysis of gene function. Antisense morpholinos can be injected into embryos shortly after fertilization to affect the function of genes for several days of early development that can cause knockdown [30]. To maximize the potential of this approach to target validation, appropriate assays must be available to determine whether knockdown of the gene in question affects a disease-related process. As an example, one can scan the effect of depleting the function of the gene on newly forming blood vessels in the zebrafish embryo to determine whether a blood vessel-specific gene is a legitimate target for anti-angiogenesis drugs or not. Morpholino knockdown of the vascular endothelial cell growth factor, part of a pathway targeted by current angiogenesis drug development, prevents angiogenic blood vessel formation in zebrafish embryos [31]. Transgenic line is also used for target validation (Table 1). Other strategies should be developed and considered to facilitate target validation. Morpholino Oligonucleotide Screens Morpholinos are chemically modified antisense oligonucleotides. It is designed to hybridize to the translation-initiation or splicing acceptor/donor sites of specific mRNAs [30,45]. Morpholino oligonucleotides are nonionic DNA analogs available from Gene Tools LLC [46,47]. They possess altered backbone linkages compared with DNA or RNA (Fig. 2). Morpholinos cause a vigorous knockdown of gene function when injected into zebrafish embryos [45,48-52]. Injection of morpholinos can find out the developmental roles of many individual Different Transgenic Line for Diseases Model Creation and Drug Target Validation Genes for target validation Related diseases References Insulin IA-2 autoantigen Diabetes Milewski et al. 1999 [32]; Cai et al. 2001[33] runx1 cbfb Leukemia Blake et al. 2002 [34] Factor VII COX-1 COX-2 Thrombosis Sheehan et al. 2001[35]; Grosser et al. 2002 [36] Presenilin-1 presenilin-2 acetylcholinesterase amyloid precursor protein apoE Alzheimer's disease Leimer et al. 1999[37]; Groth et al. 2002[38]; Behra et al., 2002[39]; Musa et al. 2001[40]; Monnot et al., 1999[41] Myc myc-induced T cell leukemia Cancer/tumor Langenau et al. 2003[42]; Langenau et al. 2005[43] Huntingtin Huntington's disease Karlovich et al. 1998[44] Zebrafish: A Complete Animal Model for In Vivo Drug Discovery and Development Current Drug Metabolism, 2009, Vol. 10, No. 2 119 Fig. (3). Diagramatic representation that describes morpholino oligonucleotides, nonionic DNA analogs. It can be prepared form phosphodiester DNA. Morpholinos injection cause vigorous knockdown of gene. genes in zebrafish. Other than zebrafish, small-scale morpholino screens have been reported in Xenopus tropicalis [53], Ciona intestinalis [54] and sea urchin [55]. Genetic and morpholino oligonucleotide screens are an efficient means of systematically assessing the roles of individual genes in disease processes in the zebrafish. By itself, this process represents a potential route to the identification and validation of novel drug targets. In traditional forward genetic zebrafish screens, phenotypes have been identified that resemble human diseases [56-58]. The novel genes that underlie the zebrafish disease phenotypes might lead directly to the identification of novel drug targets. Alternatively, the disease phenotypes might form the basis of further screens to find genes or chemicals that can correct the phenotype [59]. Assay Development for Drug Discovery The disease relevant assay development is very necessary to completely understand the potential of zebrafish for both target validation and drug screening. The assays which can do automation, should more fascinating. An Assay was developed for a new gastrointestinal motility using zebrafish. Gastrointestinal (GI) motility disorders such as constipation and dyspepsia are common, but current treatment options are largely ineffective, primarily due to a limited understanding of GI motility. Validation of the assay was performed using two prokinetic agents [60]. Since development of blood vessels in early zebrafish embryos of which was characterized and observed, so, it is suitable for identification of angiogenesis inhibitors [61]. For example, two group of scientist have shown that tyrosine kinase inhibitors targeting VEGF receptors can prevent angiogenesis in the early embryo [62, 63]. Using either endogenous alkaline phosphatase staining of blood vessels or microangiography, angiogenesis was measured in the zebrafish. For that, transgenic lines of zebrafish with fluorescent blood vessels have been developed, which simplifies the process by which blood vessels are visualized [64]. The ability to perform angiogenesis assays in fluorescent zebrafish should facilitate automation. Assays can also be designed by exploiting the typical zebrafish behavioral repertoire. For instance, specific assays have been designed to examine drug and alcohol abuse using the zebrafish [65]. Other assays have been designed to test for hearing defects, by examining either defects in normal swimming behavior, or the response of zebrafish to loud sounds [66, 67]. The visual system is especially amenable to study in the zebrafish, due to the large size of the eyes and the ease with which visual assays can be established [68, 69]. Thus, diseases such as retinal pigmentosa and macular degeneration can be modeled and drug can be developed through that assay. Physiology Based Drug Discovery Physiology based drug discovery is one of the route for drug discovery and development. Physiology-based drug discovery follows physiological diseases models (Table 2) which are created, for example, the disease phenotype is crated in an animal model or cell-based assay and then compounds are screened through this readout. On the other hand, the process of target-based drug discovery starts with identification the therapeutic target and the function of the target for the creation of disease. A physiology-based strategy first start with the screening of compound in an animal model and target identification or validation would follow in later stages [70]. A new hypothesis was also proposed for drug discovery and development that is based on pathophysiology and using animal models [71]. Zebrafish has become a widely used disease model. Mature zebrafish thrombocytes is the same as mammalian platelets [72]. Therefore, a zebrafish thrombocyte-specific cDNA library may provide a rich source of novel genes involved in platelet aggregation and blood clotting. Zygogen (USA), a drug discovery company using zebrafish model, has recently concluded that transgenic zebrafish with fluorescent thrombocytes would make the recovery of these cells and the synthesis of a thrombocyte-specific library straightforward [73]. Transgenic zebrafish with fluorescent blood vessels have been developed, where blood vessels are visualized very easily [74]. Compounds effect on blood vessel of this model can be seen very easily. The gridlock mutation was developed in zebrafish which causes a vascular defect, and crb (crash and burn) and it is a cell-cycle mutation. It was used to screen through diverse small-molecule libraries. In both screens, small molecules that can reverse the phenotypic effects of the mutations were identified [75,76]. Gridlock mutants have a hypomorphic mutation in the hey2 gene, a hairy/enhancer of split-related transcriptional repressor believed to function downstream of Notch in regulating vascular cell-fate decisions [77,78]. An excellent experiment was performed with mutant zebrafish embryos which were developed with dysmorphogenesis of the dorsal aorta that prevents circulation to the trunk and tail. However, in that case, perfusion of the head was normal. Mutant embryos were placed into 96-well plates. The rate of distribution was three em- 120 Current Drug Metabolism, 2009, Vol. 10, No. 2 bryos per well and compounds from a structurally diverse smallmolecule compound were transferred into the water surrounding the zebrafish embryos where the distribution rate of compound was one compound per well. The embryos were permitted to develop. Then image- scored for the gridlock phenotype was observed under a dissecting microscope. With the experiment, two structurally related compounds were identified from 5,000 small molecules that two molecules completely suppress the gridlock mutant phenotype, causing mutant embryos that are exposed to the compounds to develop a normal vasculature [75]. The gridlock-suppressing compounds, represent a novel class of compounds that were not previously known to influence vasculogenesis or angiogenesis. Table 2. List of Different Diseases Model that has Developed so far and can be Used Physiology Based Drug Discovery Different diseases model References ‘Cancer’ as Diseases Model Feitsma et al.[79]; Moore et al.[80] ‘Cardiovascular Disorders’ as Diseases Model Rottbauer et al.[81]; Rottbauer et al.[82] ‘Kidney Diseases’ as Diseases Model Drummond [83]; Solnica-Krezel et al.[84] Hemostasis Jagadeeswaran et al. [85]; Jagadeeswaran and Liu [94] Caveolin-Associated Muscle Disease Nixon et al. [86] Angiogenesis Nicoli et al. [87]; Fouquet et al.[88]; Liao et al.[89]; Thompson et al. [90]; Kidd and Weinstein [119] ‘Neurological Diseases’ as Diseases Model Wilson et al. [91]; McKinley et al. [92] ‘Liver Diseases’ as Diseases Model Sadler et al. [93] ‘Hemophilia’ as Disease Model Jagadeeswaran and Liu [94]; Chakroborty et al.[120] Alzheimer's Disease Liu et al. [95] Osteoporosis Barrett et al.[96]; Campbell et al. [97] Quantitative Structure-Activity Relationship (QSAR) and Structure –Activity Relationships (SAR) Study of a Drug using Zebrafish Quantitative structure-activity relationship (QSAR) is the process by which chemical structure is quantitatively correlated with a well defined process, such as biological activity or chemical reactivity. For example, biological activity can be expressed quantitatively as in the concentration of a substance required to give a certain biological response. The mathematical expression can then be used to predict the biological response of other chemical structures. The basic assumption for all molecule based hypotheses is that similar molecules have similar activities. This principle is also called structure-activity relationship (SAR) [98, 99]. The zebrafish is highly open to the study of structure–activity relationships (SARs). During the above screen for compounds that alter pH3, several compounds were identified. SARs have been established for several of these compounds. Several derivatives of parent compound L were generated and tested for their ability to alter pH3 levels in intact zebrafish. Three compounds induce the zebrafish phenotype at concentrations similar to those of the parent compound, four compounds induce the phenotype only at a fivefold higher concentration, and three compounds have no activity [59]. We hope that in future, with this SAR studies in whole zebrafish, structures can be identified. Chakraborty et al. ZEBRAFISH AND DRUG DEVELOPMENT Drug Toxicity Study Toxicity plays a major role in drug development. The several new molecular entities submitted to the FDA for the approval of new dugs. But FDA has declined about half of the molecules due to their toxicity problem. In a statement, the FDA point out to technological difficulties in toxicology as one of the principal causes of this ‘pipeline problem’. However, new animal models are needed to test the safety of novel drug candidates. The FDA reports that an estimated 10% improvement in predicting failures before clinical trials would save US $100 million per drug in development costs [100-102]. However, much more progress is needed to develop better animal models for toxicological assessment and to involve toxicology earlier in the drug discovery process. To evaluate the toxicity of a drug, it is essential to identify the endpoints of toxicity and the dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxicodynamics of the drug. In addition to detailed toxicological investigations of a drug, there also is a need for high-throughput large scale screening for toxicity of several drugs at a time. In both cases, the zebrafish has numerous attributes. The zebrafish is rapidly gaining acceptance as a promising animal model for drug and chemical toxicology [103, 104]. The ability to efficiently assess the toxicity of a large number of compounds enables whole libraries to be prescreened for potential toxicity. To examine significant toxicities to drug development, some zebrafish assays have been developed specifically. For instance, a susceptible zebrafish assay was performed for detecting smallmolecule-induced mutagenesis which was developed by Amanuma et al. [105]. Zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure [106]. Toxicity of diclofenac, anti-rheumatic drug, was evaluated by using zebrafish model [107]. Until now, zebrafish toxicity studies have paying attention on environmental contaminants like pesticides. The zebrafish is now a well established model for drug toxicology study. However, questions continue about relevant of fish and human toxicity their similarities. Yet, drug toxicology should be focused and considered to facilitate in this unique animal model. CONCLUSION The pharmaceutical screens in zebrafish that have been carried out so far have demonstrated that it is feasible to screen libraries for compounds with specific biological activities. Until now, only crude morphological defects have been considered, but it is feasible to use molecular markers and in situ hybridization or immunohistochemistry to evaluate more subtle effects of the compounds at a large scale. Candidate molecular targets can be rapidly validated, using antisense morpholino mediated knock down. The most potential use of drug screens in the zebrafish is to screen genetically modified zebrafish embryos which is the prominent substitute of wild type embryos. Mutants from the phenotypedriven forward mutagenesis screen can also be used. It can assume that all mutants in disease genes that exhibit an embryonic phenotype, as well as the transgenics with an embryonic phenotype may be used for these drug screens. The targets of the chemical compounds will include factors downstream of the mutant gene and may include the expressed transgene as well. These types of screens present a direct means to screen for chemical compounds that work on disease pathway [6]. The zebrafish has already provided a wealth of fundamental information about embryonic development and disease [57,120]. With the completion of the zebrafish genome project and the establishment of a robust infrastructure for genetic and physiological studies, the zebrafish system sits poised to take on a larger role in Zebrafish: A Complete Animal Model for In Vivo Drug Discovery and Development Table 3. Current Drug Metabolism, 2009, Vol. 10, No. 2 121 Some Drugs and Chemicals Used and Assessed Toxicity in Zebrafish Drugs Drug types Type of toxicity study References Retinoic acid Acidified form of Vitamin A Abnormal pectoral fin bud morphology Abnormal development of the caudal midbrain and anterior hindbrain RA-mediated gene expression in transgenic reporter zebrafish Akimenko and Ekker [108] Hill et al. [109] Perz-Edwards et al. [110] Cyclopamine Treatment agent in basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma Elimination of primary motoneurons Role of shh in the induction and patterning of the pituitary Inhibition of fin outgrowth Role of hedgehog signaling in eye development Chen et al. [111] Sbrogna et al. [112] Quint et al. [113] Stenkamp and Frey [114] 17-beta estradiol, Attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease Effects on mortality and hatching, consequences for CNS Vitellogenin as an estrogenic biomarker Kishida et al. [115] Van den Belt et al. [116] 17alphaethinylestradiol Synthetic steroid Effects on sex ratio and breeding success Hill and Janz [117] Neomycin An aminoglycoside antibiotic Bioassays for assessing toxicity Parng et al. [118] Fig. (4). Example of different strategies for drug discovery and development using zebrafish. A. Screening of lead compounds; B. Physiology based drug discovery; C. Target identification using fluorescent technology D. Target validation & gene expression analysis using microarry. the field of drug development. By contributing to target identification and validation, drug lead discovery and toxicology, the zebrafish might provide a shorter route to developing novel therapies for human disease. However, development of disease relevant assays and disease models in the zebrafish is still in infancy and should be more. With the improvement of modern technology, the zebrafish might be able to important substitute of other mammalian models for the pharmaceutical discovery in next 5 years. ACKNOWLEDGEMENT This work is particularly supported by "Aim for the Top University Plan" of the National Sun Yat-sen University and Ministry of Education, Taiwan. 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