Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 134

11.3 RESULTS

In initial studies, we determined the lethal concentration of each compound. Next we
assessed acute zebrafish organ toxicity of five cancer drug candidates: 17-DMAG,
HDAC inhibitor X, paclitaxel, SMA-838, and zebularine. Zebrafish were treated for
24 h with six drug concentrations

LC 10 and effects on heart, central nervous system,
liver, and kidney morphology, function, and organ-specific cell death were assessed.
Based on these results, we estimated the no observable effect concentration. Next, we
assessed reversibility of toxic effects. After treatment for 24 h, drugs were removed by
washing zebrafish in fresh fish water and incubating for an additional 48 h. Organ
morphology and function were then reassessed. Finally, we compared results in
zebrafish with available results in mammalian models and humans.

11.3.1 LC 10 and LC 50 Determination

Two and four dpf zebrafish were initially treated for 24 h with compound concentra-
tions ranging from 0.01 to 400 M. Triplicate experiments were performed using 10

zebrafish per condition. No lethality was observed for any compound at any
concentration at either stage. However, paclitaxel precipitated from the fish water
and 2

m

M was estimated as the highest soluble concentration. During incubation in
aqueous solution, SMA-838 changed color to dark orange from light yellow,
indicating possible disintegration. Additional 17-DMAG, SMA-838, and zebularine
concentrations, including 800, 1200, 1600, 2000, 3000, 4000, 5000, and 10,000 mM,
were then assessed in single experiments, due to limited compound availability. For
higher drug concentrations, final DMSO concentration was increased to 1.0%.
Percent lethality was calculated for each condition and LC 10 and LC 50 for 17-DMAG,
SMA-838, and zebularinewere then estimated using logistic regression analysis (JMP
Software, v7.0). Results showed that for 17-DMAG, LC 10 and LC 50 at 2dpf were 1664
and 3141

m

M, respectively. For
HDAC inhibitor X, no lethality was detected at concentrations up to 400

m M, respectively, and at 4dpf were 2000 and 2080 m

m M. For

paclitaxel, no lethalitywas detected up to the highest soluble concentration, 2 m M. For

SMA-838, at 2dpf, no lethality was detected up to 5000 m M, and at 4dpf, LC 10 and

LC 50 were 2000 and 2528 m M, respectively. For zebularine, no lethality was detected

up to 10,000 M at either 2 or 4dpf stage.

To assess toxicity, six concentrations

m

LC 10 were selected for each compound
(Table 11.3). Since zebularine was not lethal, and no abnormalities were observed at
concentrations up to 10,000

m M, per Study Sponsor's instructions, organ-specific

toxicity was not assessed.

11.3.2 Morphology/Function Organ Toxicity After
Compound Treatment for 24h

Organmorphology and function were assessed after compound treatment for 24 h at 2
or 4dpf stage. For each experiment, in addition to compound-treated zebrafish,