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Using twenty chemicals commercially available or provided by NCI, we demonstrated the feasibility of

using zebrafish embryos to study toxicity response by assessing Median Lethal Concentration (LC50),
developmental teratogenicity, and specific liver and kidney toxicity. For the twenty compounds used to
study LC50 and liver and kidney toxicity in zebrafish embryos, we found good correlation with LC50
values in other mammalian models. This is strong preliminary validation for use of zebrafish embryos for
drug toxicity testing. In addition, 90% (18 of 20) of the compounds produced specific tissue, organ and
behavioral toxicity, as expected. The liver toxicity data was obtained using two different assays: a
staining method and a colorimetric endpoint assay. This last method allowed us to analyze livergastrointestinal toxicity (dose-response effect) for all the 20 compounds used during this work.
Figure 1: Schematic representation of a zebrafish embryo at 72 hours of development showing the
location of the liver (brown arrow), the kidney (green arrow) and the heart (red arrow).

Advantages of Zebrafish Embryos for Toxicity Testing


Visual Assessment In contrast to other vertebrate models, the zebrafish embryo is completely transparent,
facilitating visual multi-parameter observation and analysis. Its entire body plan is established by 24
hours and all the precursor cells and tissues of the brain, eyes, heart and musculature can be easily
visualized using light microscopy. The zebrafish completes embryogenesis in the first 72 hours (Figure
1), and most of the internal organs, including the cardiovascular system, gut, liver and kidney, develop
rapidly in the first 24-48 hours (Stainier and Fishman, 1994). An important advantage of this animal
model is that the morphological and molecular basis of tissue and organ development are, in general,
either identical or similar to other vertebrates including man (Chen and Fishman, 1996; Granato and
Nusslein-Volhard, 1996).

Automated Whole Animal Analysis In addition to rapid development, there are several other
advantages of using zebrafish for assay development, including: zebrafish are small, inexpensive
to maintain, and easily bred in large numbers; a single mating produces 100-200 eggs.
Furthermore, since single embryos can be maintained in fluid volumes as small as 100 l for the
first five to six days of development, they can be kept in individual microtiter wells. Chemicals
can then be added directly to the solution in which the embryos develop, simplifying drug
dispensing and facilitating high throughput screening (Westerfield, 1993).

Organ Formation is Easily Visualized in the Zebrafish Embryo: Liver Staining

T
G
E
T
G

Figure 2: Zebrafish treated for five days were used to determine the effects of dexamethasone on liver development
and function. The embryos were fixed with paraformaldehyde, and incubated with streptavidin-peroxidase to
specifically detect the liver after incubating with a chromogenic dye. The arrows indicate the position of the liver.
(Top), six day old untreated embryo (control); (Bottom), six day old embryo treated with 100 ? M of dexamethasone
for five days indicates a dramatic reduction in liver size compared with the control embryo. Scale bar = 1 mm; Eye
(E); Gut (G); Tail (T).

ELISA Assays Can Be Performed on Zebrafish Embryos


Dexamethasone dose response
120

% of Control

100
80
60
40
20
0
0

10

100

Dexamethasone (uM)

Figure 3: Liver toxicity after Dexamethasone treatment: A dose response. Embryos were exposed to different
concentrations of dexamethasone (from 1 ? M to 100 ? M) for five days as described in the Figure 1 legend. After
treatment, they were stained with a soluble dye to specifically detect liver defects. After staining, the product
formed by peroxidase was detected by absorbance at 405 nm. The values were expressed as a percentage of control
(% Control), where the control (untreated embryos) is 100%. The standard deviation was also calculated and added
to the data.
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Table I - Zebrafish Toxicity Testing Compared with Mammalian Modelsa


Compound tested

Ref.

Zebrafish

Mammalian models

LC50 /Log LC50


(mg/l)

Specific toxicity
observed

LC50/Log LC50
(mg/kg)

Specific toxicity
(from the literature*)

Geldanamycin

(1)

3.13/0.49b

Liver

1.0/0.4 (mice, ip.c)

Liver

Phenylurea
dithiocarbamate

(2)

1.5**

Potent teratogenic,
liver

Unknown

Liver

Didemnin B

(3)

6.2/0.79

Developmental delay

4.0/0.6(mice, i.v.d)

Kidney

Merbarone

(4)

4.7/0.67

Teratogen, very toxic,


enlarge liver, kidney
microcephalia

55/1.74 (mice, i.v.)

Liver,
Kidney

Fujisawa peptide

(5)

30/1.48

Teratogen, heart kidney

5.1/0.7 (rats, i.v.)

Lung,heart

Ecteinascidin

(6)

0.42**

Very toxic, teratogen,


kidney

Unknown

Potent
toxin

Trithiophene

(7)

2.7/0.43

Liver, muscle

110/2.04 (rats, i.p.)


e

Liver

4-Ipomeanol

(8)

94/1.97

No apparent specific
toxicity

60/1.77 (mam. , i.p.)

Liver,
kidney, lungs

Ethanol

(9)

11,180/4.0

Teratogen,
neurodevelopment,
craniofacial

7,000/3.8 (rats, or.f)

Liver,
teratogen,
neurodevelopment,
craniofacial

Doxorubicin

(10)

30.3/1.51

Teratogen, liver,
cardiovascular, kidney

21/1.35 (mice, i.v.)

Liver, cardiovascular

Cyclosporin A

(11)

69/1.83

Teratogen, kidney
cardiovascular,
liver

170/2.23 (mice, i.v.)

Kidney,ureter,
bladder

Naproxen

(12)

13.2/1.12

Liver, gastrointestinal

435/2.63 (mice, i.v.)

Gastrointestinal

Ibuprofen

(13)

5.56/0.74

Liver, kidney,
gastrointestinal

495/2.69 (mice, i.p.)

kidney,
gastrointestinal

Aspirin

(14)

100.9/2.0

keratogen, kidney,
muscle contraction,
erratic movements

167/2.22 (mice, i.p.)

kidney, ureter,
cardiovascular,
musculoeskeletal

Dexamethasone

(15)

324/2.51

Liver, gastrointestinal,
kidney

410/2.61 (mice, i.p.)

Liver, heart, kidney


gastrointestinal

Acetaminophen

(16)

252/2.4

Liver

500/2.69 (mice, i.p.)

Liver, kidney,
gastrointestinal

Caffeine

(17)

108.4/2.03

Behavioral: 1. muscle
contraction or spasticity

127/2.1 (mice, i.p.)

Behavioral: 1. muscle
contraction or
spasticity (child)
2. Change in motor activity
activity (mice).

2. Change in motor
Tacrine

(18)

11.13/1.04

Teratogen, liver

20/1.3 (mice, i.p.)

Liver

Dichloroacetic acid

(19)

72/1.85

Teratogen, liver,
kidney

1500/3.17 (rats, or.)

Liver, kidney,
cardiovascular

Polychlorinated
biphenyls (PCBs)

(20)

10/1

Liver, gastrointestinal

8000/3.9 (rats, i.v.)

Liver

Notes: a. Includes: rats, mice, rabbits and other mammals, b. Bold: values of Log LC50 that appear in Graphic I, c. i.p.= intraperitoneal, d. i.v.=
intravenous, e. mam.= mammals, in general, f. or.= orally.* Data obtained from the NIH TOXNET database, NCI and others. ** Two
compounds: Phenylurea dithiocarbamate and Ecteinascidin, were not used to compare toxicity in mammals because LC50 values are unknown in
these systems. References: 1, (Scheibel et al., 1998); 2, (Frank et al., 1987); 3, (Montgomery, 1985); 4, (Fortune & Osheroff, 1998); 5, (Chan et
al., 1997); 6, (Izbicka et al., 1998); 7, (Hudson et al., 1993); 8, (Boyd, 1980); 9, (Baumann & Sander, 1984); 10, (Muller et al., 1997); 11, (Singer
& McCune, 1998); 12, 13, (Cryer & Feldman, 1998); 14, (Bosman, 1994); 15, (Iida et al., 1998); 16, (McDanell et al., 1992); 17, (Marret et al.,
1997); 18, (monteih & Theiss, 1996); 19, (McHugh et al., 1998); 20, (Dubois et al., 1995).

Mammalian-LC 50 (Log mg/Kg)

*For twenty compounds used to study LC50 and liver and kidney toxicity in the zebrafish, we found good
correlation with previous studies using mammalian models (Table I and Figure 3). These data provide
strong preliminary validation of zebrafish as a model for toxicity testing.

*Figure 4: Comparative Drug


Toxicity. LC50 values from Table
1, calculated in mg/L and expressed
in logarithmic scale, are compared
with the corresponding LC50 values
(mg/Kg) obtained with mammals. A
graphical representation of the data
was developed; the diagonal line
represents the perfect regression
between the two sets of values. LC50
values for mammals were obtained
from the NIH TOXNET database,
NCI and others.

5
4
3
2
1
0
-1
-2
-3
-3

-2

-1

Zebrafish-LC 50 (Log mg/L)

*Comparison of the effect of compounds on specific organ toxicity correlated extremely well with results
in mammals. As shown in Table I, 90% (18 of 20) of the compounds produced specific tissue, organ and
behavioral toxicity, as expected.
Although LC50 values are expressed in mg/Liter in the zebrafish model versus mg/Kg in mammalian
models, the lethal concentrations were remarkably similar in both systems. Comparison of the effect of
compounds on specific organ toxicity, another important parameter, correlated extremely well with results
in mammals. 90% (18 of 20) of the compounds produced specific tissue, organ and behavioral toxicity,
as expected. The liver toxicity data was obtained using two different assays, a staining method and a
colorimetric endpoint assay (see Liver staining). This last method allowed us to analyze livergastrointestinal toxicity (dose-response effect) for all the compounds presented. Results show that
compounds affect the same target tissues and organs in both systems, reinforcing use of zebrafish as a
model organism for drug toxicity testing with a high predictive value for humans.

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