Radiation-Induced Toxicity and Radiation Response Modifiers in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 289

defined time points before and after radiation to assess radiation protection and
mitigation, respectively. For practical reasons, the LD 50 of ionizing radiation (IR;
20 Gy single-dose whole body exposure) was defined by determining survival at 7 dpf
of embryos irradiated at 1 dpf. Principally, this was due to minimal husbandry
requirements in the first 6-7 days of development. However, when embryos irradiated
at 24 hpf were scored for survival at 3 months, the LD 50 decreased to 6 Gy and thus
was close to the LD 50 of humans exposed to whole body radiation (4 Gy).
21.4 GROSS MORPHOLOGICAL ALTERATIONS
ASSOCIATED WITH RADIATION EXPOSURE
Irradiation of zebrafish embryos at or before 4 hpf produces multiple morphological
defects, including microcephaly, microophthalmia, micrognathia, distal notochord
and segmental abnormalities, pericardial edema, and inhibition of yolk resorption. As
outlined in the preceding paragraph, organogenesis has commenced by 24 hpf
rendering this the preferred developmental stage to study radiation-induced effects
on major organ systems. When compared to younger embryos, the range of known
organ-specific functional impairments in embryos irradiated at 24 hpf is more limited
(see below). However, embryos irradiated at 24 hpf with 10-40 Gy demonstrate a
characteristic morphological phenotype readily apparent within 1-2 days following
ionizing radiation exposure and observable without visual aids. This phenotype
consists of dorsal curvature of the body axis previously described as “curly-up” or
CUP and ascribed to defects inmidline development of zebrafish embryos (Fig. 21.1a)
(Brand et al., 1996). The incidence of the CUP phenotype is approximately 60% after
20 Gy at 24 hpf. Although the mechanism causing CUP after IR is presently unknown,
the easewithwhich this phenotype can be observedmakes it an attractive parameter to
score radiation toxicity in zebrafish embryos (Daroczi et al., 2006).
21.5 RADIATION-ASSOCIATED APOPTOSIS
INCIDENCE
Radiation induces apoptosis in zebrafish, which can be easily visualized and scored
using acridine orange (AO) staining of live fish. An example of increased AO staining
after 20 Gy and assayed 6 h after radiation exposure is shown in Fig. 21.1b. Although
several tissues reveal increased AO staining after radiation exposure, the central
nervous system, the eyes, and cells in the spinal cord/notochord region along the body
axis preferentially show marked increases in punctate AO staining and lend them-
selves to quantification of this phenomenon by measuring fluorescence in a defined
area of the developing embryo using ImageJ software. In addition to AO staining,
terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL) and determination of caspase activity by use of fluorescent substrates
have been used to determine radiation-associated apoptosis in zebrafish embryos.
Compared to AO staining, these methods are presently more cumbersome (TUNEL)
or have low signal-to-noise ratios (our unpublished results).