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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.
ABBREVIATIONS
AchE
= Acetylcholinesterase
ATPase = ATP synthase
GI
= Gastro-intestinal
FDA
= Food and Drug Administration
HD
= Huntington Disease
SAR
= Structure Activity Relationship
QSAR = Quantitative Structure Activity Relationship
VEGF = Vascular Endothelial Growth Factor
122 Current Drug Metabolism, 2009, Vol. 10, No. 2
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