Radiation-Induced Toxicity and Radiation Response Modifiers in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 288

are imaged with QImaging camera and iVision software; the images are analyzed
using ImageJ software.

21.2.8 Drug Treatments

Stock solutions of the agents described here are typically prepared in DMSO followed
by further dilution in embryo medium. DMSO concentrations in final solutions are
typically

0.1% and nontoxic. In all experiments, embryo medium containing no
more than 0.1% DMSO is used as a vehicle control. Amifostine (MedImmune
Oncology, Inc., Gaithersburg, MD) serves as a positive control for radiation protectors
and is used at 4 mmol as described by us previously. DF-1 was provided by C-Sixty
Inc. and CDDO-TFEA by Reata Pharmaceuticals. The IKK inhibitor 2 (wedelolac-
tone), IKK inhibitor 3 (BMS-345541), IKK-2 inhibitor 4, and IKK-2 inhibitor 5
(IMD-0354) are from Calbiochem as are azakenpaullone, SB361540, and SB361549
(Calbiochem). LiCl is obtained from Sigma and SB216763 from Selleck Chemicals
LLC. All compounds are used at nontoxic concentrations arrived at by toxicity testing
using log dilutions. When possible, target inhibition is assessed by measuring effects
of drug in vivo in zebrafish. Current efforts focus on accomplishing this by monitoring
the activity of reporter plasmids that contain specific promoters responsive to the
molecular pathways targeted by the agents under investigation and injected into
zebrafish embryos at the two- to four-cell stage (Kari et al., in preparation). To observe
drug effects in the presence and absence of radiation, zebrafish embryos are main-
tained at 28.5 C for up to 7 dpf and the time-dependent effects of treatments on
survival and morphology are scored.

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21.3 VALIDATION OF ZEBRAFISH EMBRYOS
AS A MODEL SYSTEM FOR RADIATION
PROTECTORS/SENSITIZERS

Early work focused on proof-of-principle experiments to validate the potential of
zebrafish to identify modulators of the radiation response (Traver et al., 2004;
McAleer et al., 2005). While hematopoietic stem cell transplantation was shown to
protect adult fish against hematopoietic radiation syndrome (Traver et al., 2004),
much of the subsequent work has focused on embryos. In this work, viable zebrafish
embryos were exposed to 0-10 Gy single-fraction 250 kVp X-rays at defined devel-
opmental stages at 1-24 hpf and scored for survival. These experiments revealed
increasing radiation resistance at later stages of development and radiation protection
of 4-8 hpf embryos by the FDA-approved amifostine. In contrast, pharmacological
inhibition of the epidermal growth factor receptor radiosensitized zebrafish embryos
consistent with earlier results in vitro and in mice (for review see Brizel, 2007).
Subsequent studies were done by irradiating at 24 hpf for the following reasons:
(i) at this developmental stage most major organs have started to form, (ii) the effects
of morpholino oligonucleotides (MOs) to downregulate target gene expression are
well established, and (iii) pharmacological agents can be easily administered at