Radiation Research

Published by: Radiation Research Society

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Radiation Research 168(2):149-157. 2007
doi: 10.1667/RR0803.1

Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo

Catherine L. Bladen1a, Melody A. Flowersa, Katsuya Miyakea, Robert H. Podolskyb, John T. Barrettc, David J. Kozlowskiad, and William S. Dynan2a

aInstitute of Molecular Medicine and Genetics,

bCenter for Biotechnology and Genomic Medicine,

cDepartment of Radiology,

dDepartment of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912

1Present address: School of Biochemistry and Molecular Biology, Institute of Medical and Biological Engineering, Division of Microbiology, University of Leeds, Leeds LS2 9JT UK

2Address for correspondence: Institute of Molecular Medicine and Genetics, CB-2803, 1120 15th Street, Medical College of Georgia, Augusta, GA 30912;

Abstract

Bladen, C. L., Flowers, M., Miyake, K., Podolsky, R. H., Barrett, J., Kozlowski, D. J. and Dynan, W. S. Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo. Radiat. Res. 168, 149–157 (2007).

Quantitative studies of radiation cytotoxicity have been performed mostly in cells in culture. For a variety of reasons, however, the response of cells in culture may not reflect the response for cells in situ in a whole organism. We describe here an approach for quantification of radiation-induced cell death in vivo using the transparent embryo of the zebrafish, Danio rerio, as a model vertebrate system. Using this system, we show that the number of TUNEL-positive cells within a defined region increases approximately linearly with radiation dose up to 1 Gy. The results are consistent with predictions of a linear-quadratic model. The use of alternative models, accommodating a response threshold or low-dose hypersensitivity, did not significantly improve the fit to the observed data. Attenuation of the expression of the 80-kDa subunit of Ku, an essential protein for the nonhomologous end-joining pathway of repair, led to a dose reduction of 30- to 34-fold, possibly approaching the limit where each double-strand break causes a lethal hit. In both the Ku80-attenuated and the control embryos, apoptotic cells were distributed uniformly, consistent with a cell-autonomous mechanism of cell death. Together, these results illustrate the potential of the zebrafish for quantitative studies of radiation-induced cell death during embryogenesis and in vivo.

Received: August 15, 2006; Accepted: March 26, 2007



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FIG. 1. Quantification of TUNEL-positive cells within an intact embryo. Panel A: Representative optical sections from a single embryo. Embryos were collected and treated as described in the Materials and Methods. The embryo shown here was microinjected with buffer only at the one-cell stage and received an acute exposure to 800 mGy of 137Cs γ radiation. The 24 h after fertilization embryo shown here is curled around the yolk with the front of the head at left. The developing left eye (50 μM depth) and right eye (250 μM depth) are clearly visible. The approximate location of developing inner ear is also marked. Note that benzoyl aminobenzoate treatment, needed to reduce light scattering, reduces contrast in the bright-field image, making morphological structures more difficult to see. Scale bar = 50 μM. The line drawing below the images is a dorsal view showing the location of the optical sections relative to the embryo as a whole. Panel B: Three different views of the data set obtained from the embryo in panel A. Using a rendered volume representing the data, individual apoptotic cells were classified using Volocity software. Note that the scale varies in these perspective views. Panel C: Representative optical sections of embryos from two different treatment groups. Sagittal sections are shown representing the midline of the embryo. Top row, buffer-microinjected (control) embryos. Bottom row, Ku80 MO-microinjected embryos. Doses of 137Cs γ radiation are noted

FIG. 2. Panel A: Dose–response curves. All embryos are from the same clutch and were treated in parallel. Embryos were irradiated with 137Cs γ rays (Gammacell Exactor, MDS Nordion, Ontario, Canada). Experimental groups generally contained an average of 7.6 embryos. Solid symbols, buffer-microinjected embryos. Solid line reflects linear regression. Open symbols, Ku80 MO-microinjected embryos. Solid curve reflects second-order polynomial fit. Panel B: Residuals from linear regression for buffer-microinjected embryos

FIG. 3. Dose–response curves in the low-dose range. Embryos were treated as in Fig. 2 except that irradiation was performed using a 6 MeV Varian linear accelerator beam, which was attenuated and calibrated to deliver an accurate dose in the 1- to 50-mGy range shown. Solid symbols, buffer-microinjected embryos. Open symbols, Ku80 MO-microinjected embryos. Solid lines reflect linear regression

FIG. 4. High-magnification view of TUNEL-stained embryo counterstained with DAPI. Embryos were collected and treated as in Fig. 2. Figure shows a posterior oblique optical section through developing brain. Panel A: TUNEL-positive cells. Panel B: Differential interference contrast showing anatomical structures. Hindbrain (hb), midbrain (mb) and eye are marked. Ventricle is at the center and yolk is at the bottom. Panel C: DAPI staining. Panel D: Merged image. Scale bar = 100 μM. Embryo was from an experimental group that received Ku80 MO and a 50-mGy radiation dose

table

TABLE 1 Number of Embryos Evaluateda

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