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[II. Zebrafish]: Model Vertebrate Organism, Toxicologically Relevant (and homologous), Endpoints (physical,
behavioral, genetic), Genomic, HTS
(RT: main thrust of chapter)
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Model Vertebrate Organism:
Zebrafish (Danio rerio), native to the Indian subcontinent, are a small teleost freshwater tropical fish. Zebrafish
reach sexual maturity within 3 months, reproduce year round, and produce clutch sizes that range between 100200 embryos per female per week. Spawning occurs within one hour after the onset of light and no followup
parental care occurs. Embryos develop externally with an initial chorion size of 1 mm diameter, weight less than
1 mg, and are optically transparent. At 7 days they are well developed and small (4 mm x 0.5 mm x 1 mm). In
captivity zebrafish breed well for 2 years, survive for 2-4 years and in adulthood are approximately 15-40 mm
long. Zebrafish are also easily maintained in vivariums at high densities and temperatures of 25 - 33 C
(Westerfield, 2000), and the infrastructure and maintenance costs required for housing large numbers of animals
for comprehensive studies is relatively low. From a modest size colony, thousands of embryos can be produced
daily. Zebrafish exhibit a circadian pattern of daytime activity and nighttime rest, similar to other mammals
(Zhdanova, 2005). They also possess all the senses including; sight, hearing, taste, smell, touch, and balance
(Moorman, 2001). The vision system includes both rods and cones cells that respond to the ultraviolet and
visible spectrum (Bilotta, 2000).
Testing can occur in isolated environments (1 well in multiwell plate) or tanks with large numbers of other fish
(e.g., diet). Zebrafish are suitable for many types of endpoint analysis starting from embryos (0 to 3 days), larval
(3 to 30 day), juvenile (30 to 120 day) and adult (> 90 day) to end of life. Zebrafish develop quickly through
morphogenesis with a full backbone and beating heart in 1 day, natural hatching in 2-3 days, and omnivorous
feeding after 5 days (Westerfield, 1995). Embryos and larval up to approximately 5-6 dpf are negatively buoyant.
Around this time it is thought, larval inflate their gas bladder by swimming up and gulping air at the surface. At
this point they are neutrally buoyant and capable of swimming and maintaining position in the entire water
column.
Zebrafish have complex organs and systems and are a robust model for nearly all types of animal testing.
External exposure pathways include contact (skin, eyes), ingestion (gut), inhalation (gills) and testing is
conducted in a water medium. The zebrafish is thought to be a good model to bridge the gap between
human/mouse and fly/worm for understanding development and disease. Currently the ZFIN (ZFIN, 2010)
website lists 655 laboratories using zebrafish.
Toxicologically Relevant:
Much of the anatomy, physiology, and genomics of fish is highly homologous to humans and these similarities
are just beginning to be exploited by the biomedical research community (reviewed in (Zon, 1999; Dodd et al.,
2000; Wixon, 2000; Ackermann and Paw, 2003; Rubinstein, 2003). The National Institutes of Health (NIH) is an
advocate for the use of zebrafish as a health research animal model (NIH, 2010). NIH supports a broad array of
zebrafish research resources including ZIRC and NNN funded research grants. PubMed reports more than
14,000 scientific publications using zebrafish as the model organism. Zebrafish share a high genomic, proteomic,
and metabolomic homology with other vertebrates and a good species correlation to mammals (80% homology
with human genome). Researchers regard it as a good predictor for human toxicity (Carroll et al., 2003).
Zebrafish are a vertebrate organism homologous to humans that can live in 96-well plate format and absorb
chemicals from the water. They have become a model organism for researchers studying human health issues
and provide opportunity to assess the mechanism of action and pathways of drugs and agents in a whole
organism.
Fish have been used since the 1960s for carcinogenicity research. Models indicate that they respond to
carcinogens similar to mammals (Metcalfe, 1989) and they can be complementary to rodent models (Di Giulio,
2008).
Endpoints:
Since there are significant similarities between zebrafish and mammals, toxicity responses can be evaluated at
multiple levels (atomic, molecular, cellular, anatomical, physiological, and behavioral). Early developmental life
stages are often uniquely sensitive to agent insult, due in part to the enormous changes in cellular differentiation,
proliferation and migration required to form multiple cell types, tissues and organs. These endpoints measure an
effect and can be used to predict the effect of hazardous substances on humans based on the animal effects.
Zebrafish embryos and adults are small in size and weight. Assessments require much smaller quantities of
compounds than other tests using mammals. This is especially important when testing costly drugs, chemicals,
and nanomaterials.
Atomic assays: Dose; Molecular assays: ?; Cellular assays: Apoptosis; Anatomical assays: Morphology;
Physiological assays: Cardiovascular, Neurology; Behavioral assays: Motion;
Recently the zebrafish has become an important model for cancer research because its genome contains all the
orthologs of human oncogenes and forms tumors with similar histologic and genetic features as human tumors.
Genetics:
Comparative genomics has revealed remarkable similarity at the genome level between zebrafish and other
vertebrates including humans. The human, mouse, and zebrafish genomes compare as follows (human: 3 billion
bases, 23 chromosomes; mouse: 3 billion bases, 20 chromosomes; zebrafish: 1.5 billion bases, 25
chromosomes). Each of these genomes are fully sequenced and maintained by the Genome Reference
Consortium (GRC, 2010). The mission of the GRC is to ensure that the human, mouse, and zebrafish genomes
are biologically relevant by closing gaps and fixing errors in their assemblies. This data is supported by
informatic tools at the three major genome browser sites (Ensembl, NCBI, UCSC). These tools are useful for
comparative research. For example, a zebrafish ortholog to a human gene can be identified using BLAST with
the human. Over the past 10 years, the path from mutation to function in zebrafish has become more developed
with transgenic fish. Zebrafish mutants provide additional screens that answer specific questions.
High Throughput Screening:
NIH initiative.
Chemical libraries (Univ of Pitt, Univ of Penn).
Due to their small size (4 mm x 0.5 mm x 1 mm for 7-day old zebrafish), zebrafish can be accommodated in
multi-well testing plates. The small size of zebrafish embryos (0 to 3 day) and larval (3 to 30 day) make them
compatible with the standard 96 well microplate. Normal zebrafish length is less than 7 mm by 21 days (ZFIN).
The small size and durability of zebrafish embryos allow the use of microtiter plates and robotic handling.
Models that employ embryogenesis have been proposed as testing screens due to the complicated and multiphase
process during development. More traditional whole animal models such as rat or mouse would be employed
after zebrafish testing screens deem them worthwhile.
Zebrafish swimbladder (SB) consists of the anterior chamber (AC) and posterior chamber (PC).
The bladder initially starts as one chamber at about 1-3 day post-hatching (3-6 dpf) but forms into two chambers
during the Juvenile stage. The swimbladders are homologous to lungs. Zebrafish larvae tend to remain at the
bottom of their water environment for the first 4-5 dpf. At 5-7 dpf the yolk sac is depleted, the first chamber of
the swimbladder inflates, and free swimming behavior begins (G.N. Robertson et al, 2007).
The optical transparency of the zebrafish embryo enables assessment of internal features without the surgery
that is often required in studies using mammals.
CONCLUSION
Zebrafish is a model vertebrate organism with many advantages for health research. This is why it has become
the most preferred model species after rodents by the NIH and its most preferred vertebrate model in lower
species.
Zebrafish testing good for (Biotech and Safety Assessment, Thomas/Myers):
Screening (embryos / larval), Microsurgery, Exposure/Timing, Controlled/Reproducible, Morphogenesis.
Zebrafish lifestage testing opportunities include developmental, acute, and behavioral (embryonic, juvenile),
chronic, cancer, and behavioral (adult) ...
21st century hazard testing goals include testing the broadest coverage of chemicals, end points, life stages, and
mechanistic and dose-response information for human health risk assessment. The NRC Committee on toxicity
testing and assessment of environmental agents recommends an in vitro and in vivo testing strategy that is
primarily focused on human biology, a broad range of doses, medium and high throughput testing, lest cost and
time, fewer animal, perturbs toxic pathways, and allows in silico screens when possible. The 21st testing strategy
includes chemical (agent) characterization, chemical (agent) testing that includes pathways and targets, and doseresponse for a much larger quantity of chemicals and exposures. These tests will aid in evaluation and
interpretation of of human exposures and population effects. Screening using lower vertebrates such as zebrafish
have been used for years. Now scientists have the ability to generate transgenic animals and to screen chemicals
(agents) in medium and high throughput systems to evaluate different phenotypes. As the amount of data and
information increases, challenges will include integration and interpretation.
US EPA continues to manage the ToxCast program for testing chemicals and maintains the ToxRefDB database
on animal toxicology data.
Physiology of different life stages need to be considered. In adult fish, gill and gut epithelia are major routes of
uptake. Larval fish utilize skin as a respiratory interface and thus may uptake more compound than adult fish that
utilize gills for respiration. Although, compounds that are attracted to the gill lamellae of adults could have a
greater dose than larval who lack the large gill surface area of the adults (Reference ???).
Fish are ideal subjects for behavioral assays of various chemicals and stressors due to their: direct contact with
the exposure medium over their entire body; ecological relevance; reproductive readiness, and history of
behavioral testing.
REFERENCES
Detrich, H III, Westerfield M, and Zon L. Overview of the zebrafish system. Methods Cell Biol 59: 3–10, 1999.
Dodd, A., Curtis, P. M., Williams, L. C., and Love, D. R. (2000). Zebrafish: bridging the gap between
development and disease. Hum Mol Genet 9, 2443-2449.
Wixon, J. (2000). Featured organism: Danio rerio, the zebrafish. Yeast 17, 225-231.
Zon, L. I. (1999). Zebrafish: A New Model for Human Disease. Genome Res. 9, 99-100.
NIH (2010). NIH Zebrafish Initiative web site. http://www.nih.gov/science/models/zebrafish/
GRC (2010). Genome Reference Consortium web site.
http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/
Di Giulio, R. T., & Hinton, D. E. (2008). The toxicology of fishes. Taylor & Francis.
Metcalfe CD, (1989) Tests for predicting carcinogenicity in fish. CRC Critical Reviews in Aquatic Sciences
1:111-112
(Zhdanova, 2005)
(Moorman, 2001)
(Bilotta, 2000)
(Haffter et al., 1996; Driever et al., 1996)