Академический Документы
Профессиональный Документы
Культура Документы
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).
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).
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).
% 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.
2
Ref.
Zebrafish
Mammalian models
Specific toxicity
observed
LC50/Log LC50
(mg/kg)
Specific toxicity
(from the literature*)
Geldanamycin
(1)
3.13/0.49b
Liver
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
Liver,
Kidney
Fujisawa peptide
(5)
30/1.48
Lung,heart
Ecteinascidin
(6)
0.42**
Unknown
Potent
toxin
Trithiophene
(7)
2.7/0.43
Liver, muscle
Liver
4-Ipomeanol
(8)
94/1.97
No apparent specific
toxicity
Liver,
kidney, lungs
Ethanol
(9)
11,180/4.0
Teratogen,
neurodevelopment,
craniofacial
Liver,
teratogen,
neurodevelopment,
craniofacial
Doxorubicin
(10)
30.3/1.51
Teratogen, liver,
cardiovascular, kidney
Liver, cardiovascular
Cyclosporin A
(11)
69/1.83
Teratogen, kidney
cardiovascular,
liver
Kidney,ureter,
bladder
Naproxen
(12)
13.2/1.12
Liver, gastrointestinal
Gastrointestinal
Ibuprofen
(13)
5.56/0.74
Liver, kidney,
gastrointestinal
kidney,
gastrointestinal
Aspirin
(14)
100.9/2.0
keratogen, kidney,
muscle contraction,
erratic movements
kidney, ureter,
cardiovascular,
musculoeskeletal
Dexamethasone
(15)
324/2.51
Liver, gastrointestinal,
kidney
Acetaminophen
(16)
252/2.4
Liver
Liver, kidney,
gastrointestinal
Caffeine
(17)
108.4/2.03
Behavioral: 1. muscle
contraction or spasticity
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
Liver
Dichloroacetic acid
(19)
72/1.85
Teratogen, liver,
kidney
Liver, kidney,
cardiovascular
Polychlorinated
biphenyls (PCBs)
(20)
10/1
Liver, gastrointestinal
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).
*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.
5
4
3
2
1
0
-1
-2
-3
-3
-2
-1
*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.
PHYLONIX
2/02