Assessment of low linear energy transfer radiation-induced bystander mutagenesis in a three-dimensional culture model.

A three-dimensional cell culture model composed of human-hamster hybrid (A(L)) and Chinese hamster ovary (CHO) cells in multicellular clusters was used to investigate low linear energy transfer (LET) radiation-induced bystander genotoxicity. CHO cells were mixed with A(L) cells in a 1:5 ratio and briefly centrifuged to produce a spheroid of 4 x 10(6) cells. CHO cells were labeled with tritiated thymidine ([3H]dTTP) for 12 hours and subsequently incubated with A(L) cells for 24 hours at 11 degrees C. The short-range beta-particles emitted by [3H]dTTP result in self-irradiation of labeled CHO cells; thus, biological effects on neighboring A(L) cells can be attributed to the bystander response. Nonlabeled bystander A(L) cells were isolated from among labeled CHO cells by using a magnetic separation technique. Treatment of CHO cells with 100 microCi [3H]dTTP resulted in a 14-fold increase in bystander mutation incidence among neighboring A(L) cells compared with controls. Multiplex PCR analysis revealed the types of mutants to be significantly different from those of spontaneous origin. The free radical scavenger DMSO or the gap junction inhibitor Lindane within the clusters significantly reduced the mutation incidence. The use of A(L) cells that are dominant negative for connexin 43 and lack gap junction formation produced a complete attenuation of the bystander mutagenic response. These data provide evidence that low LET radiation can induce bystander mutagenesis in a three-dimensional model and that reactive oxygen species and intercellular communication may have a modulating role. The results of this study will address the relevant issues of actual target size and radiation quality and are likely to have a significant effect on our current understanding of radiation risk assessment.