The in vivo mouse transgenic pKZ1 chromosomal inversion assay is a sensitive assay that responds to very low doses of DNA-damaging agents. pKZ1 inversions are measured as the frequency of cells expressing E. coli β-galactosidase protein, which can only be produced from an inverted pKZ1 transgene. In previous studies we reported that a single whole-body low dose of 0.01 mGy X rays alone caused an increase in pKZ1 chromosomal inversions in spleen when analyzed 3 days postirradiation, and yet this same dose could protect from high-dose-induced inversions when delivered as a conditioning dose 4 h before or after a 1 Gy challenge dose. In an attempt to explain these results, we performed temporal studies over a wide radiation dose range to determine if the inversion response was temporally different at different doses. pKZ1 mice were irradiated with a single whole-body X-ray dose of 0.01 mGy, 1 mGy or 1 Gy, and spleen sections were then analyzed for pKZ1 inversions at 7 h, 1 day or 7 days after exposure. No change in inversion frequency was observed at the 7 h time point at any dose. At day 1, an increase in inversions was observed in response to the 0.01 mGy dose, whereas a decrease in inversions below sham-treated frequency was observed for the 1 mGy dose. Inversion frequency for both doses returned to sham-treated inversion frequency by day 7. To our knowledge, this is the first reported study to examine the temporal nature of a radiation response spanning a wide dose range, including doses relevant to occupational exposure, and the results are dynamic and dose specific. The results suggest that inversions induced after low-dose irradiation are removed by homeostatic mechanisms within a short time frame, and underscore the importance of studying responses over a period of time when interpreting radiation effects.

To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed. No significant changes in the apoptosis frequency at any time after low-dose irradiation were detected. Apoptosis was induced above endogenous levels by five- to sevenfold 7 h after 1000 mGy. Using these data, the expected increases in apoptosis 7 h after a dose of 1 mGy or 0.01 mGy were calculated based on the assumption that induction of apoptosis would decrease linearly with dose. The magnitude of potential bystander effects for apoptosis that could be detected above homeostatic levels after these low doses of radiation was determined. A substantial bystander effect for apoptosis (>50-fold above direct effects) would be required before such proposed effects would be identified using 10 animals/treatment group as studied here. These data demonstrate that amplification of apoptosis even due to a substantial bystander effect would fall within the homeostatic range.

The potential for irradiated cells to induce biological effects in their unirradiated neighbors (known as the bystander effect) has been observed repeatedly in vitro . However, whether bystander effects occur in vivo under the specific conditions relevant to low-dose radiation protection is still unclear. To test this, the fate of bystander cells in the mouse spleen was examined using an adoptive transfer method designed to replicate the rare, irradiated cells in an organ that might be expected after a low-dose-rate, low-LET radiation exposure. Splenic lymphocytes radiolabeled with low activities of 3 H-thymidine were introduced into the spleens of unirradiated recipient mice. In this study, the apoptotic and proliferative response of the neighboring bystander spleen cells was compared to the response of spleen cells in parallel control recipients that received sham-irradiated cells. Neither the local area surrounding lodged radiolabeled cells nor the spleen as a whole showed a change in apoptosis or proliferation either 1 or 3 days after adoptive transfer. Increasing the irradiated cell numbers, increasing the mean 3 H-thymidine activity per cell, or exposing cells ex vivo to an acute X-ray dose also had no effect. Possible reasons for the absence of a bystander effect in the spleen under these conditions are discussed.