Evidence for pronounced bystander effects caused by nonuniform distributions of radioactivity using a novel three-dimensional tissue culture model

A new in vitro multicellular cluster model has been developed to assess the impact of nonuniform distributions of radioactivity on the biological response of mammalian cells, and the relative importance of bystander effects compared to conventional radiation effects. Chinese hamster V79 cells are labeled with tritiated thymidine ([³H]dThd), mixed with unlabeled V79 cells, and centrifuged gently to form multicellular clusters about 1.6 mm in diameter. The short range of the ³H β particles effectively allows only self-irradiation of labeled cells and no cross-irradiation of unlabeled cells. The percentage of labeled cells is controlled precisely by varying the number of labeled cells mixed with unlabeled cells prior to assembling the cluster. The clusters are assembled in the absence or presence of 100 μM lindane, a chemical that is known to inhibit formation of gap junctions. After the clusters are maintained at 10.5°C for 72 h, the cells are dispersed and plated for colony formation. In the case of 100% labeling, the survival of cells in the cluster depends exponentially (SF = e^−A/1.8) on the cluster activity A (in kBq), and lindane has no effect on the response. A two-component exponential response is obtained for 50% labeling in the absence of lindane (SF = 0.33 e^−A/0.81 + 0.67 e^−A/11.8), and lindane has a marked effect on the response (SF = 0.33 e^−A/1.6 + 0.67 e^−A/41.6). These data suggest that bystander effects play an important role in the biological response of V79 cells when the ³H is localized in the cell nucleus and distributed nonuniformly among the cells. In contrast, bystander effects cannot be detected above traditional radiation effects (i.e. direct + indirect) when the ³H is localized in the cell nucleus and distributed uniformly among the cells. These results indicate that this multicellular cluster model is well suited for studying the effects of nonuniform distributions of radioactivity, including bystander and “hot-particle” effects. Furthermore, these results suggest that by-stander effects may play an important role in the prediction of the biological effects of radiopharmaceuticals used in medical diagnosis and treatment.