Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 131

11.2 MATERIALS AND METHODS

11.2.1 Zebrafish Breeding and Handling

Embryos were generated by natural pairwise mating as described in The Zebrafish
Book (Westerfield, 1993). Embryos were maintained at 28 C in fish water (200mg
Instant Ocean Salt (Aquarium Systems, Mentor, OH) per liter of deionized water; pH
6.6-7.0, maintained with 2.5 mg/L Jungle pH Stabilizer (Jungle Laboratories Cor-
poration, Cibolo, TX); conductivity 670-760

S). Embryos were cleaned (dead and
unfertilized embryos removed) and staged at 4 h post fertilization (hpf). Because
embryos receive nourishment from an attached yolk sac, no feeding is required for
7 days post fertilization (dpf).

m

11.2.2 Compounds

Five test compounds (Table 11.1) were supplied blinded by the Developmental
Therapeutics Program, National Cancer Institute (SAIC-Frederick, Frederick, MD).
Compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) or
fish water following Study Sponsor's instructions. Master stock solutions were
aliquoted and stored at 20 C until the day of experiments when substock solutions

were prepared at designated concentrations. Table 11.2 shows compound concentra-
tions and solvents used for master stock solutions.

11.2.3 Compound Treatment

Zebrafish organs form at different stages. To ensure comparability for assessing drug-
induced lethality and acute toxicity, central nervous system and heart were assessed
after treating 2dpf zebrafish for 24 h and liver and kidney were assessed after treating
4dpf zebrafish for 24 h. For each compound concentration, 10 zebrafish were
distributed into six-well plates containing 3mL sterile fish water. For compounds

Table 11.1 Mechanism of Action and Potential Therapeutic Indication for Five Test
Compounds

Compound Mechanism of action Indication

17-DMAG Binds HSP90 and destabilizes

HSP90-oncoprotein complex

Lymphoma

HDAC inhibitor X HDAC inhibitor Lymphoma

Paclitaxel Promotes assembly and prevents depolymerization

of microtubules

Breast cancer

SMA-838 Transcriptional inhibitor—upregulates p53 and

downregulates p21 and survivin

Leukemia

Zebularine DNA methyltransferase inhibitor Leukemia