Download Zebrafish models for assessing developmental and reproductive

Neurotoxicology and Teratology 42 (2014) 35–42
Contents lists available at ScienceDirect
Neurotoxicology and Teratology
journal homepage: www.elsevier.com/locate/neutera
Review article
Zebrafish models for assessing developmental and reproductive toxicity
Jian-Hui He a,b,c, Ji-Min Gao a, Chang-Jiang Huang a,b, Chun-Qi Li a,b,c,⁎
a
b
c
Zhejiang Provincial Key Lab for Technology and Application of Model Organisms, Wenzhou Medical University, Wenzhou, Zhejiang Province 325035, PR China
Institute of Watershed Science and Environmental Ecology, Wenzhou Medical University, Wenzhou, Zhejiang Province 325035, PR China
Hunter Biotechnology, Inc., Transfarland, Hangzhou, Zhejiang Province 311231, PR China
a r t i c l e
i n f o
Article history:
Received 20 June 2013
Received in revised form 22 January 2014
Accepted 26 January 2014
Available online 3 February 2014
Keywords:
Zebrafish
Developmental toxicity
Embryo toxicity
Reproductive toxicity
Chemical toxicity
a b s t r a c t
The zebrafish is increasingly used as a vertebrate animal model for in vivo drug discovery and for assessing chemical toxicity and safety. Numerous studies have confirmed that zebrafish and mammals are similar in their physiology, development, metabolism and pathways, and that zebrafish responses to toxic substances are highly
predictive of mammalian responses. Developmental and reproductive toxicity assessments are an important
part of new drug safety profiling. A significant number of drug candidates have failed in preclinical tests due to
their adverse effect on development and reproductivity. Compared to conventional mammal testing, zebrafish
testing for assessing developmental and reproductive toxicity offers several compelling experimental advantages, including transparency of embryo and larva, higher throughput, shorter test period, lower cost, smaller
amount of compound required, easier manipulation and direct compound delivery. Toxicity and safety assessments using zebrafish have also been accepted by the FDA and EMEA for investigative new drug (IND) approval.
© 2014 Elsevier Inc. All rights reserved.
Contents
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.
Zebrafish embryo toxicity assay . . . . . . . . . . . . . . . . . .
3.
Developmental toxicity and teratogenicity assessments in the zebrafish
4.
Reproductive toxicity assessment in the zebrafish . . . . . . . . . .
5.
Behavioral toxicity assessment in the zebrafish . . . . . . . . . . .
6.
Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transparency document . . . . . . . . . . . . . . . . . . . . . . . .
Transparency document . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Developmental and reproductive toxicity assessments are important
for both new drugs and environmental chemicals. In drug development,
a significant number of drug candidates have failed in preclinical
tests due to their adverse effect on development and reproductivity
(Boverhof and Zacharewski, 2006; Kola and Landis, 2004; Lühe
⁎ Corresponding author at: Zhejiang Provincial Key Lab for Technology and Application
of Model Organisms, Wenzhou Medical University, Wenzhou, Zhejiang Province 325035,
PR China. Tel.: +86 571 8378 2170; fax: +86 571 8378 2135.
E-mail address: [email protected] (C.-Q. Li).
0892-0362/$ – see front matter © 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ntt.2014.01.006
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et al., 2005; (http://www.technologyreview.com/featuredstory/402459/
can-pfizer-deliver/). On the environmental front, the global production
of chemicals has increased threefold in the past decades (EC, 2001) and
many of these chemicals may have potential toxic effect on humans, including developmental and reproductive toxicity. Although conventional
in vitro assays using cultured cells can be used to evaluate potential drug
and chemical toxicity, results are frequently not predictive of results
in vivo which involve drug absorption, distribution, metabolism and excretion (ADME). Conventional mammalian animal tests for assessing developmental and reproductive toxicity are laborious, costly and timeconsuming (McGrath and Li, 2008; Rubinstein, 2006).
The zebrafish is a vertebrate animal model that is increasingly used
for in vivo drug toxicity and efficacy screening and for assessing
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J.-H. He et al. / Neurotoxicology and Teratology 42 (2014) 35–42
chemical toxicity and safety (Ali et al., 2011; He et al., 2013; McGrath
and Li, 2008; Rubinstein, 2006; Zhu et al., 2013). In contrast to other vertebrate models, the zebrafish completes embryogenesis in the first 72 h
(Kimmel et al., 1995). By 120 h post-fertilization (hpf), the zebrafish
develops discrete organs and tissues (Fig. 1). Although the zebrafish
lack some of the mammalian organs such as the lung, prostate, and
mammary glands, they share many organs and tissues, including the
brain and central nervous system (CNS), heart and vascular system,
kidney, liver for metabolism, pancreas with insulin production,
adipocytes for fat storage, intestines, bone, muscles, and immune and
reproductive systems with ovary and testis regulated by endocrine
and paracrine signals (Lewis and Eisen, 2003; Moens and Prince,
2002; Wilson et al., 2002). Zebrafish organs and tissues have been
shown to be similar to their mammalian counterparts at the anatomical,
physiological, cellular and molecular levels, while their metabolism,
signaling processes, cognitive behavior and sensory systems are comparable to those of mammals (de Esch et al., 2012; Lewis and Eisen, 2003;
Moens and Prince, 2002; Wilson et al., 2002). The zebrafish genome is
fully sequenced and shown to share an approximately 85% homology
with their human counterparts (McCollum et al., 2011; Renier et al.,
2007). More importantly, the amino acid sequences of functionally
relevant protein domains have been proven to be even more evolutionary conserved (de Esch et al., 2012; Reimers et al., 2004; Renier et al.,
2007). Numerous studies have confirmed that zebrafish toxic responses
are predictive of mammalian ones (Haldi et al., 2006; He et al., 2013;
McGrath and Li, 2008; Parng et al., 2002, 2004, 2005; Ton and Parng,
2005; Zhu et al., 2013). Zebrafish is one of the current perspectives on
the use of alternative species in human health and ecological hazard
assessments (Perkins et al., 2013).
Zebrafish toxicity assays offer several compelling experimental advantages, including the transparency of embryo and larva, high throughput, short test period, low cost, small amount of compound required,
easy manipulation and direct compound delivery (Bull and Levin,
2000; Lieschke and Currie, 2007; McGrath and Li, 2008; Rubinstein,
2006). In 2003, the National Institutes of Health (NIH) ranked the
zebrafish as the third most important experimental organism after
rats and mice (http://www.fda.gov/forconsumers/consumerupdates/
ucm343940.htm). The Food and Drug Administration (FDA) and
European Agency for the Evaluation of Medicinal Products (EMEA)
have also accepted zebrafish toxicity and safety assessment data for
investigative new drug (IND) approval, and more than a dozen of new
drugs discovered primarily based on zebrafish models are now in clinical
trials (Chakraborty et al., 2009; Delvecchio et al., 2011; Hill et al., 2012;
Paquet et al., 2009; Zon and Peterson, 2005). Two important recent
developments will likely further spread the use of the zebrafish. First,
the OECD is developing standards for using the zebrafish to assess
environmental risk of chemicals. Second, the European Union has
enacted the Registration, Evaluation, Authorization and Restriction of
Chemicals (REACH), which requires toxicity assessment using animal
testing (including zebrafish) to systematically evaluate the risk to
human health and the environment of chemical substances produced,
used or imported (Combes et al., 2003; Lilienblum et al., 2008;
McCollum et al., 2011; Yang et al., 2009). The use of the zebrafish as an
alternative animal model for in vivo drug and chemical toxicity assays
can greatly increase the speed and decrease the cost of the assessment
process and provide more accurate results than cell-based assays. This
convenient and predictive animal model can serve as an intermediate
step between cell-based evaluation and mammalian animal testing
(McGrath and Li, 2008).
2. Zebrafish embryo toxicity assay
The early stages of life are particularly susceptible to the adverse
effects of drugs and chemicals (Makri et al., 2004). Unfortunately,
these stages are the most difficult to assess in the traditional mammalian models of toxicology. The transparency of zebrafish embryos and
their development outside of the mother allow scoring of teratological
and embryo toxic effects easily.
Fish embryo testing of chemicals has matured to the point that
international standardization, method validation, and broadening of
chemical coverage are rapidly occurring. Current OECD fish testing
guidelines acknowledge the importance of fish embryo testing that
includes fish acute toxicity test (OECD 203 (OECD, 1992a)), early lifestage toxicity test (OECD 210 (OECD, 1992b)), short term toxicity test
Fig. 1. Zebrafish developmental stages. Zebrafish at 6, 24 and 120 h post-fertilization (hpf) are shown. By 120 hpf, the zebrafish develops discrete organs and tissues, including the brain,
heart, liver, intestine, eye, ear, somite and swim bladder.
J.-H. He et al. / Neurotoxicology and Teratology 42 (2014) 35–42
on embryo and sac-fry stages (OECD 212 (OECD, 1998)), and juvenile
growth test (OECD 215 (OECD, 2000). At present, the most promising
alternative approach to classical acute fish toxicity testing is zebrafish
embryo toxicity assay. The whole effluent test with zebrafish embryos
has been standardized at an international level (ISO, 2007), and a
modified version has been submitted by the German Federal Environment Agency as a draft guideline for an alternative to chemical testing
with intact fish (Braunbeck et al., 2005).
The zebrafish embryo toxicity assays in the United States include the
fish acute toxicity test (OPPTS 850.1075) and the fish early-life stage
toxicity test (OPPTS 850.1400) (EPA, 1996a,b). The fish acute toxicity
test assesses the impact of chemicals shortly after fertilization through
96 hpf, with the aim to establish two concentration values: one that
results in 50% fish death (LC50) and one with no observable effect.
The early-life test begins shortly after fertilization and ends at either
the free-feeding stage or the first 30 days of life. The Environmental
Protection Agency (EPA) testing guidelines specify hatching and survival and abnormalities in morphology, behavior and size for testing during
early life stages (McCollum et al., 2011). The zebrafish embryo toxicity
assays developed in our and other laboratories use newly fertilized
zebrafish eggs exposed to potential toxicants for 24 and 48 h to assess
for 4 toxic endpoints: the coagulation of eggs and embryos, failure to
develop somites, lack of heartbeat, as well as non-detachment of the
37
tail from the yolk (Fig. 2) (Gustafson et al., 2012; Lammer et al., 2009;
Selderslaghs et al., 2009; Weigt et al., 2011).
Embryo toxicity assays of various drugs, endogenous signaling
molecules and hormones have also been evaluated in zebrafish and
have been suggested to be valuable in predicting drug safety in humans
(Berghmans et al., 2008; Redfern et al., 2008; Spitsbergen and Kent,
2003). Meanwhile, most classes of environmental contaminants have
been evaluated for early life stage toxicity in zebrafish, including various
metals, pesticides, and organochlorines (Chen et al., 2011, 2012a,b; He
et al., 2011; Huang et al., 2010; Jin et al., 2009). Various industrial
chemicals and pollutants show adverse effects on the developing
zebrafish. Physical stresses such as magnetic fields may perturb
zebrafish development. Skauli et al. (2000) showed an additive interaction of magnetic field stress with the hormone progesterone in
causing delayed hatching. The algal toxin microcystin suppresses
zebrafish growth following early life stage exposure (Spitsbergen and
Kent, 2003). A few molecular mechanisms of toxicity have been
investigated in embryonic and larval zebrafish. Willey and Krone
(2001) used the vasa gene as a marker of primordial germ cells to
track alterations in their homing to gonad caused by endosulfan or
nonylphenol. Dong et al. (2001) applied in situ terminal transferasemediated nick-end-labeling staining (TUNEL) to demonstrate increased
cell death in the dorsal midbrain of TCDD-treated zebrafish embryos.
Fig. 2. Representative phenotypes of zebrafish embryo toxicity, including coagulation, abnormal somite and non-detachment of the tail.
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J.-H. He et al. / Neurotoxicology and Teratology 42 (2014) 35–42
Akimenko and Ekker (1995) showed that zebrafish fin malformations
induced by exogenous all-trans retinoic acid are associated with the
anterior duplication of expression domains of shh (Sonic hedgehog).
3. Developmental toxicity and teratogenicity assessments in
the zebrafish
The current international guidelines for developmental toxicity
testing involve exposing pregnant animals (usually rats or rabbits) to
compounds and subsequently assessing toxic effects on fetuses.
Alternative methods for assessing developmental toxicity have been
developed in the recent decades, including in vitro cell differentiation
assays using either primary cell cultures or immortalized cell lines, the
in vitro rodent whole embryo culture test and the in vivo frog embryo
teratogenesis assay (FETAX) (Marathe and Thomas, 1990). Unfortunately, these in vitro and in vivo tests are not sensitive and have been
of limited value in predicting the effect of drugs and chemicals on
human embryonic and fetal development (Fort et al., 1988; Oberemm,
2000).
Although mammalian models remain the gold standard for assessing
developmental toxicity, acceptance of the zebrafish as a predictive
model is increasing in the USA (Parng, 2005; Spitsbergen and Kent,
2003) EPA has included the zebrafish as the alternative animal model
for assessing environmental contaminants and selected developmental
toxicity as an initial screen (EPA, 1996a,b).
There are strong rationales for performing developmental toxicity
assessment using zebrafish embryos. First, the zebrafish is a distinct
species and has been shown to be sensitive to compounds that exhibit
teratogenicity in vivo in mammals. Second, the developmental processes in the zebrafish are highly conserved. Third, in contrast to rodent
embryo culture, which is limited to early organogenesis, zebrafish
embryos can be cultured until advanced organogenesis. Finally, the
zebrafish genome is well characterized. Abnormal phenotypes linked
to genomic targets can potentially enable rapid evaluation of mechanisms of action for compound-induced teratogenicity (Bailey et al.,
2013; Haldi et al., 2011; McCollum et al., 2011; Sipes et al., 2011).
The zebrafish literature has included a large number of nonregulatory studies in which mortality and common morphological
defects of developmental toxicity and teratogenicity in zebrafish are
assessed through bright-field microscopy. These defects include altered
hatching and a variety of morphological abnormalities such as altered
body size, eye size, head size and formation, body curvature, tail formation, pigmentation, swim bladder inflation, edema, and malformation in
the pericardial sac and yolk sac (Fig. 3) (Gustafson et al., 2012; Weigt
et al., 2011). A common scoring endpoint is the hatching time. Most
compounds that affect hatching either delay or shorten zebrafish hatching. For example, shortened hatching time has been reported for the
insecticide methoxychlor, acetone and 1% dimethylsulfoxide (DMSO),
the latter being one of the most common solvents for chemicals when
testing in zebrafish (Hallare et al., 2006; Versonnen et al., 2004). Two
other common endpoints for developmental toxicity testing are body
size and curvature. Changes in body curvature include axial curvature,
dorsal curvature, altered tail formation, and lordosis. Toxic effects
on skeletal or muscle development might cause some of these
malformations. There are presently commercially available automated
programs that can measure body size and curvature, thus allowing for
high-throughput screening (http://www.thermo.fr/eThermo/CMA/).
Defects in swim bladder inflation are also a commonly measured
endpoint. Several benzene ring-containing compounds, such as
dibenzothiophene, chrysene, and naphthalene, cause swim bladder
defects (Incardona et al., 2004). Yolk-sac edema can also be induced
by a large number of chemicals, including ethanol, petroleum products,
benzene-like chemicals, drugs, plasticizers, pesticides, flame retardants,
and dioxins (Ducharme et al., 2013).
In our laboratory, we assess developmental toxicity and teratogenicity of drugs and chemicals in embryonic and larval zebrafish using 12
major endpoints and 8 minor endpoints. The 12 major endpoints
are the heart, brain, jaw, eye, liver, intestine, trunk/tail/notocord,
muscle/somite, body pigmentation, circulation, body edema and
hemorrhage. The 8 minor endpoints are the fin, ear, swim bladder, red
blood cell formation, kidney cysts, pancreas, motility and body length
(He et al., 2013; Zhu et al., 2013). Similar phenotype endpoints were
used in a recent study with 12 blinded reference compounds aiming
Fig. 3. The representative phenotypes of zebrafish developmental toxicity and teratogenicity. Red arrow in the bottom pictures from left to right showed developmental eye absence, pericardial edema and brain degeneration, respectively, in zebrafish treated with mammalian teratogenic drugs. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
J.-H. He et al. / Neurotoxicology and Teratology 42 (2014) 35–42
to validate zebrafish as a predictive model for assessing developmental
toxicity (Haldi et al., 2011). The latter study found that zebrafish developmental toxicity assay presented a 75% success rate in identifying
nonteratogenic compounds and a 100% success rate in identifying
teratogens. The assay was ranked as good (N70% b 80%) for specificity
and excellent (N 80%) for sensitivity (Genschow et al., 2002; Ton et al.,
2006).
Many of the gross morphological phenotypes seem to be correlated,
as the same compound can cause many different effects. For instance,
tetrachlorodibenzo-p-dioxin (TCDD), perfluorooctanesulfonate (PFOS),
arsenite, and ethanol affect hatching time, body size and curvature, yolk
sac, and swim bladder inflation. This indicates that one compound
might work through a specific mechanism, such as inducing apoptosis
or causing oxidative stress, which then affects many different endpoints.
Chemicals that induce apoptosis include TCDD, PFOS, arsenite and so
on. The insecticide cypermethrin changes the expression of several
apoptosis-related gene products (Jin et al., 2011). Another signaling pathway that is often affected by chemical exposure is the heat shock protein
70 (Hsp70) pathway. Ethanol, acetone, diazinon and chlorpyrifos, among
other chemicals, increase Hsp70 protein levels (Hallare et al., 2006; Scheil
et al., 2010). Linking phenotypes to molecular mechanisms of action may
increase our understanding and ability to predict phenotype(s) for different compounds. Though a substantial body of work exists that examines
the gross morphological effects easily observable in embryonic and larval
zebrafish by light microscopy, more detailed analyses for assessing additional effects at cellular and molecular levels are needed in the future
(McCollum et al., 2011).
4. Reproductive toxicity assessment in the zebrafish
Traditionally, mammals such as rats or mice are used as models for
the reproductive toxicity assessment of drugs and chemicals. The measurement of toxic effects has focused on the histopathology of the testis
or ovary, sperm quality and the early development of offspring derived
from the treated animals (Kuriyama et al., 2005; Lilienthal et al., 2006;
Stoker et al., 2005; Tseng et al., 2006). Unfortunately, reproductive
toxicity assessment using mammals is complex, expensive and timeconsuming, with little possibility for high throughput or large-scale
analysis in experiments. In addition, the oral exposure and high dosage
needed for mammal testing make them unsuitable for predicting
the reproductive toxicity of environmental chemicals because the
concentration of pollutants is usual low and water-soluble pollutants
are diffused in the aquatic system.
In vitro fertilization and embryogenesis make the zebrafish a simpler
and more attractive animal model for investigating reproductive toxicity and teratogenicity (Deng et al., 2010; Du et al., 2009; He et al.,
2011; Heiden et al., 2005; Van den Belt et al., 2001). Using the zebrafish
to assess reproductive toxicity and teratogenicity of drugs and
chemicals can shorten test period, reduce cost and increase throughput.
Most structural classes of toxicants have been evaluated for reproductive toxicity in zebrafish, including metals, organochlorines, and
pesticides, halogenated aromatic hydrocarbons substituted anilines,
synthetic and natural estrogens and other industrial chemicals. The
potential for disrupting the endocrine system has been extensively
investigated in the zebrafish with a growing list of agents (Spitsbergen
and Kent, 2003). The index suggested by OECD 229 for assessing
reproductive toxicity using zebrafish is focused on: (1) the gonad
growth index (gonad weight, GSI) (Brion et al., 2004; Deng et al.,
2010; He et al., 2011); (2) reproduction ability: the egg-laying amount
(Brion et al., 2004; Deng et al., 2010) and sperm quality (He et al.,
2011; Jing et al., 2009; Wang et al., 2010); (3) the gonad histology
(Koc and Muslu, 2007; Leal et al., 2009); (4) the vitellogenin (Vtg)
expression assay (Brion et al., 2002; Fenske et al., 2005); and (5) the
hypothalamus–pituitary–gonad (HPG) axis gene expression analysis.
A few studies found that EDCs (extrogen disruptor compounds)
changed the expression of the HPG axis genes (Liu et al., 2009, 2011;
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Wang et al., 2011). Such gene expression change analysis has been an
important development in exploring toxicity-induced mechanisms
(Deng et al., 2010; Shi et al., 2009). The HPG axis gene expression has
been a key method in assessing endocrine system functions, which
lays the foundation for characterizing both signal pathways and gene
functions.
5. Behavioral toxicity assessment in the zebrafish
Developmental and reproductive toxicity could lead to abnormal
or dysfunctional behaviors. Behavioral neurotoxicology has been
instrumental in identifying and characterizing the functional
consequences of neurotoxicants on the function of both experimental
animals and humans (Levin et al., 2009; Selderslaghs et al., 2010).
Currently, neurotoxicity testing as defined by the OECD and FDA is
based solely on in vivo experiments using large numbers of mammals,
which is expensive and unsuitable for large-scale screening of
chemicals. Due to the inherent advantages of the zebrafish discussed
above, the zebrafish has been used as a relatively high-throughput
and predictive animal model for assessing behavioral neurotoxicity.
Locomotor activity is used extensively as a quantitative endpoint for
measuring behavioral toxicity in the zebrafish. Zebrafish embryos
start to show spontaneous contractions around 17 hpf, followed by
responsiveness to touch as of 21 hpf. Zebrafish embryos start to show
swimming movements in response to a touch to the tail or head after
27 hpf, and the rate of swimming is increased to a rate comparable to
that of an adult zebrafish after 36 hpf (Buss and Drapeau, 2001;
Roberts, 2000; Saint-Amant and Drapeau, 1998). Zebrafish locomotor
behavior can be visually assessed by performing touch response or
escape response (Granato et al., 1996; Li and Dowling, 1997; Samson
et al., 2001). Quantitative analysis can be performed by continuous
image acquisition using an infrared camera to measure the number
and duration of movements and the distance traveled in a given time
period. For example, using this quantitative method, a convulsioninducing agent pentylenetetrazole (PTZ) has been shown to cause
seizures in the zebrafish that can be suppressed by the anti-seizure
drug phenytoin sodium (Fig. 4). The behavioral, electrophysiological
and molecular changes in PTZ-treated zebrafish are comparable to
effects observed in a rodent seizure model (Baraban et al., 2005).
Dose-dependent locomotor responses to ethanol and other neuroactive
or neurodepressive drugs such as amphetamine, cocaine and melatonin
have been well described in the zebrafish and the results are similar to
the behavioral responses observed in mammals (Airhart et al., 2007;
Boehmler et al., 2007; de Esch et al., 2012; Gerlai et al., 2008; Irons
et al., 2009; Levin et al., 2009). The pesticide chlorpyrifos, which elicits
presynaptic serotonergic hyperactivity in juvenile and adolescent rats
(Slotkin and Seidler, 2007), could also cause significant hyperactivity
in zebrafish and this behavioral impairment has been related to
alterations in zebrafish neurochemical indices of dopamine and
serotonin neurotransmitter systems (Levin et al., 2004, 2009).
Zebrafish are social animals and their social behavior heavily
depends on visual color (pigment) patterns. It has been shown that
social behavior can be assessed in zebrafish through an assay using
visual stimulus (Saverino and Gerlai, 2008). In addition, learning and
anxiety can also be reliably measured in zebrafish larvae (Schnorr
et al., 2012; Sison and Gerlai, 2010; Valente et al., 2012). More and
more behavioral tests are becoming available for zebrafish, which
need to be carefully validated in future studies.
6. Outlook
The zebrafish has been extensively used in the study of vertebrate
genetics and developmental biology. Numerous studies have clearly
established their high degree of genetic and physiological similarity to
mammals. Due to the limitations of both traditional mammalian models
and in vitro approaches, researchers are showing increasing interest in
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J.-H. He et al. / Neurotoxicology and Teratology 42 (2014) 35–42
Fig. 4. Graphical representation of movement of a single zebrafish at the stage of 144 hpf. Graph (a) shows movement of an untreated zebrafish, graph (b) shows zebrafish treated with
10 mM pentylenetetrazole (PTZ) and graph (c) shows that zebrafish epilepsy induced by PTZ was recovered by phenytoin sodium treatment. The tracks represent a distinct movement
during a 1-hour period. Black tracks represent inactive state at a speed of b4 mm/s, green tracks represent medium speed between 4 and 20 mm/s, and red color tracks represent fast
movement (N20 mm/s). The increasing amount of red color shown for PTZ-treated zebrafish indicates increased locomotor activity, whereas phenytoin reduced locomotion activity of
PTZ-treated zebrafish. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
zebrafish-based assays to assess toxicity and safety of drugs and
chemicals, including developmental toxicity, teratogenicity, reproductive toxicity and behavioral toxicity (Borski and Hodson, 2003;
Fleming, 2007; Pugsley et al., 2008). In response, an array of zebrafish
toxicity assay standards have been established by OECD and EPA, and
zebrafish toxicity and safety assays on chemicals have been recognized
and accepted in the United States and the European Union. In addition,
there is also increasing cooperation among academic and industry laboratories to develop standard operating procedures for performing drug
and chemical assessments in zebrafish. A European zebrafish biotechnology company Biobide was certificated in 2009 to realize studies of
toxicity and efficacy with zebrafish as animal models under the compliance of GLP principles and the results could be used to apply for the
register of compounds in regulatory organizations, including EMEA,
FDA and EASMP (Environmental and Social Management Plans).
(http://www.biobide.es/news-related-to-biobide/166-biobide-hasobtained-the-certificate-of-good-laboratory-practices-glp.html).
In the future, transgenic zebrafish with fluorescent tissues and
organs could be highly valuable in zebrafish toxicity assays, especially
for developmental and reproductive toxicity analyses. A comprehensive
database of the drug- and chemical-induced zebrafish organ toxicity
and toxic phenotypes would also be very useful in speeding up the
development of further applications for the zebrafish. Finally, the
adaption of conventional technology and instrumentation as well as
rapidly developing microassay technologies offer increasing promise
for unlocking the full potential of zebrafish toxicity and efficacy assays.
Conflict of interest
There are no conflicts of interest.
Transparency document
The Transparency document associated with this article can be
found, in the online version.
Acknowledgments
This work was supported in part by the National Innovation Fund of
China (12C26213302918), the National Torch Plan of China
(2012GH020813), the National Major Specific Project for Innovation of
New Pharmaceuticals (2009ZX09103-649), the Zhejiang Provincial
Major Research Program (2008C14082 and 2010C13007), the
Zhejiang Key Science and Technology Innovation Program for the Cultivation of High-level Innovative Health Talents, and the Wenzhou Municipal Research Program (G20090142). We thank Yuanjian Carla Li at
Massachusetts Institute of Technology for her excellent editorial
assistance.
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