Introduction. Although radiation-induced bystander effects have been defined in a variety of in vitro models using a range of endpoints, the mechanism(s) as well as the presence of such an effect in vivo are not well described. In this study, our aims are to examine radiation-induced bystander mutagenesis in vivo and to determine the mechanism involved using the gpt delta transgenic mouse system. We hypothesize that transforming growth factor beta (TGF-β) initiates mutagenesis via the induction of cyclooxygenase 2 (COX-2) in bystander, non-irradiated lung tissues.

Methods. In these studies, we irradiated a 1 cm ² (1 cm x 1 cm) unshielded area in the lower region of the abdomen of the mice with 5 Gy of X-rays. First, we examined Spi^- mutant frequency (MF) in non-targeted lung tissues and analyzed the mutation spectrum of selected mutant clones. The expression and location of COX-2, the production of prostaglandin and the induction of oxidative DNA damage were determined in non-targeted lung tissues at a series of time points after partial body irradiation. Investigation of various responses to the damage was followed. Next we examined the possible signaling pathways for induction of COX-2 and mutagenesis in bystander lung tissues. We focused on the changes of cytokines especially TGF-β and tumor necrosis factor alpha (TNF-α) in plasma and lung tissues after partial body irradiation. Multiple TGF-β signaling pathways were determined as well. Finally, we tested the role of Nimesulide, a COX-2 inhibitor in suppression of mutagenesis in non-targeted lung tissues after lower extremity irradiation.

Results. COX-2 was induced in the lung of the gpt mice that were not directly irradiated. Interestingly, induction of COX-2 was detected within 1 h after partial, out of field, irradiation whereas liver showed no induction of COX-2 at all. Immunohistochemical staining showed that COX-2 was highly expressed in bronchial epithelial cells of non-targeted lung tissues relative to other stromal cells of the region. Prostaglandin in the bystander lung tissues was higher than in the non-treatment group and the observation was coupled with an increase in 8-oxo-2'-deoxyguanosine (8-oxo-dG) expression levels in bystander lung tissues. Furthermore, the bystander lung tissues showed a 3-4 fold increase in Spi^- mutations 24 h after partial body irradiation relative to non-irradiated controls but only 1.7 fold within 72 h after irradiation. As the responses to damages, apoptosis was induced in bystander lung tissues at 24 h after irradiation. Apart from these observations, lower abdomen irradiation also induced inflammation in bystander lung tissues including neutrophil infiltration, an increase in TGF-β and TGF-β receptor I (TGFBRI) and activation of mitogen-activated protein kinase (MAPK), nuclear factor kappa-B (NFκB) and AKT pathways. Different expression of TGFBRI was consistent with tissue-specific bystander response such as induction of COX-2 and mutagenesis between lung and livers. Finally pre-treatment with Nimesulide, a COX-2 inhibitor, reduced the induction of COX-2 and oxidative DNA damages in non-targeted lung tissues by lower extremity irradiation.

Conclusion. These data suggest that out of field irradiation induces bystander effects in lung tissues especially the bronchial epithelial cells and TGF-β induced COX-2 may be involved in mutagenesis in the bystander tissues. As such, the data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose radiation exposure.