Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 132

Table 11.2 Final Compound Concentrations for Master Stock Solutions

Compound MW Concentration (mM) Solvent

17-DMAG 653 10.8 Fish water

HDAC inhibitor X 540.7 46.2 DMSO

Paclitaxel 853.9 50.0 DMSO

SMA-838 375.8 55.6 Fish water

Zebularine 228 500.0 Fish water

dissolved in DMSO, 3 L of compound substock solution was added directly to fish

water and final DMSO concentration was 0.4%. For stock solutions prepared in fish
water, 12

m

L DMSO was added and final concentration was 0.4%. During early
embryogenesis, a protective chorion membrane, which may interfere with compound
uptake, is present. Therefore, to facilitate drug delivery, 2dpf zebrafish were dechor-
ionated using amild protease solution (0.5mg/mL for

m

1.5 min at room temperature).
Two or four dpf zebrafish were then incubated with compounds for 24 h.
For each experiment, untreated zebrafish were used as assay control and 0.4%
DMSO-treated zebrafish were used as carrier control. If 10% of embryos in the

untreated control group were dead or malformed, the experiment was considered
invalid and aborted.

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11.2.4 Lethality Assessment

To assess lethality, 10 concentrations were initially tested: 0.01, 0.5, 0.1, 0.5, 1, 5, 10,
50, 100, and 400 M. To establish lethality curves, after treatment for 24 h, surviving

zebrafish were counted to determine number of dead zebrafish; dead zebrafish can
disintegrate, impeding counting. Experiments were performed three times. LC 50 and
LC 10 were calculated using logistic regression analysis (JMP software, v7.0, The SAS
Institute, Cary, NC). If no lethality was observed up to 400

m

m M, additional concen-

trations up to 10,000 m M were assessed.

11.2.5 Assessment of Organ Morphology/Function
Toxicity After Treatment for 24h

Six concentrations LC 10 , determined in lethality studies, were used to assess organ-

specific toxicity. Compound treatment was performed as described above. Thirty
micromolar celecoxib (Celebrex) was used as a positive control to assess cardio-
toxicity; 7.5 and 10

M brefeldin Awere used as positive controls to assess CNS and
liver toxicity, respectively, and 3% ethanol was used as a positive control to assess
kidney toxicity. Controls were assessed concurrentlywith test compounds. After heart
rate and circulation were assessed zebrafish were anesthetized withMESAB (0.5 mM
3-aminobenzoic acid ethyl ester, 2mMNa 2 HPO 4 ) and visually assessed using a Zeiss

m