Free Radical-Initiated and Gap Junction-Mediated Bystander Effect due to Nonuniform Distribution of Incorporated Radioactivity in a Three-Dimensional Tissue Culture Model

Abstract

To investigate the biological effects of nonuniform distribution of radioactivity in mammalian cells, we have developed a novel three-dimensional tissue culture model. Chinese hamster V79 cells were labeled with tritiated thymidine and mixed with unlabeled cells, and multicellular clusters (approximately 1.6 mm in diameter) were formed by gentle centrifugation. The short-range beta particles emitted by (3)H impart only self-irradiation of labeled cells without significant cross-irradiation of unlabeled bystander cells. The clusters were assembled in the absence or presence of 10% dimethyl sulfoxide (DMSO) and/or 100 microM lindane. DMSO is a hydroxyl radical scavenger, whereas lindane is an inhibitor of gap junctional intercellular communication. The clusters were maintained at 10.5 degrees C for 72 h to allow (3)H decays to accumulate and then dismantled, and the cells were plated for colony formation. When 100% of the cells were labeled, the surviving fraction was exponentially dependent on the mean level of radioactivity per labeled cell. A two-component exponential response was observed when either 50 or 10% of the cells were labeled. Though both DMSO and lindane significantly protected the unlabeled or bystander cells when 50 or 10% of the cells were labeled, the effect of lindane was greater than that of DMSO. In both cases, the combined treatment (DMSO + lindane) elicited maximum protection of the bystander cells. These results suggest that the bystander effects caused by nonuniform distributions of radioactivity are affected by the fraction of cells that are labeled. Furthermore, at least a part of these bystander effects are initiated by free radicals and are likely to be mediated by gap junctional intercellular communication.

Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect

“…In these experiments, it was argued that due to the very short range (about 1 mm) of the low-energy bparticles emitted by tritium, no crossirradiation of bystander cells could occur. Interestingly, maximal protection of the bystander cells was observed by growth in the presence of both DMSO and lindane, an inhibitor of GJIC (Bishayee et al, 2001). Oxidative metabolism has also been implicated in toxic bystander effects observed in media transfer experiments involving g-radiation (Lyng et al, 2000(Lyng et al, , 2001Mothersill et al, 2000).…”

“…In studies utilizing other types of ionizing radiation, the antioxidant DMSO reduced the lethal effects in bystander cells produced by growing them with 3 H-thymidinelabeled cells in a three-dimensional architecture (Bishayee et al, 2001). In these experiments, it was argued that due to the very short range (about 1 mm) of the low-energy bparticles emitted by tritium, no crossirradiation of bystander cells could occur.…”

Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts

“…Oxidative stress has also been implicated in toxic effects observed in bystander cells in which g-or b-particle radiation was used (Lyng et al, 2000;Bishayee et al, 2001;Lorimore et al, 2001). To investigate whether oxidative stress was involved in the bystander effects in our system, 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a probe with fluorescence microscopy to examine the generation of ROS in AGO1522 cells by X-radiation.…”

Changes in the ornithine cycle following ionising radiation cause a cytotoxic conditioning of the culture medium of H35 hepatoma cells

“…Cytotoxic and clastogenic effects were detected on nonirradiated cells in contact with irradiated cells or exchanged via the culture medium (Hallahan et al, 1989;Mothersill and Seymour, 1997;Bishayee et al, 2001; Lewis et al, 2001;Sawant et al, 2002). The contribution of such bystander effects to radiation-induced cell death can be significant.…”