Quantification of ionizing radiation-induced cell death in situ in a vertebrate embryo

Abstract

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.