Radiobiological effects of single helical chest CT scans on cutaneous fibroblasts
Human fibroblasts derived from 12 patients showing different levels of individual radiosensitivity/susceptibility were submitted to one single helical (14.4 mGy) chest CT scan session. This set-up provided an average volumetric CT dose index (CTDIvol) of 8.1 ± 0.6 mGy. The average dose-length product (DLP) was 136.1 ± 11.9 mGy cm. The average absorbed dose at the surface of the phantom was 14.4 ± 1.91 mGy. Concerning the topogram, the CTDIvol was 0.13 ± 0.01 mGy. The average DLP was 6.7 ± 0.6 mGy cm. The average absorbed dose at the surface of the phantom was 1.1 ± 0.06 mGy.
Without exposure to IR, the radioresistant 200CLB control fibroblasts showed 0.31 ± 0.05 spontaneous γH2AX foci per cell on average. Among the other tested fibroblasts, 6 cell lines (GM03399, 85MA, 01HNG, 13HNG, 201CLB, 203CLB) showed significantly more spontaneous γH2AX foci (p < 0.001), suggesting a higher genomic instability. It is however noteworthy that the numbers of γH2AX foci never exceeded 1 focus per cell (Fig. 3a).
Ten minutes after a single helical CT scan exposure, the average number of γH2AX foci was 0.93 ± 0.04 γH2AX foci per cell in the radioresistant controls, corresponding to 0.62 ± 0.05 by omitting the background described above. This value is in agreement with the theoretical rate of DSB induced per Gy per cell currently reported in human diploid fibroblast [25]: a linearly dose-dependent induction of 37 ± 4 γH2AX foci per Gy per cell, corresponds to 0.57 γH2AX foci at 14.4 mGy) (Fig. 3a). Similar conclusions were reached by using the percentage of cells with more than two γH2AX foci as an endpoint (Fig. 3b).
The average numbers of γH2AX and pATM foci per cell assessed at 10 min after post-irradiation were found significantly lower in RACKHAM01, RACKHAM12, RACKHAM39, 02HNA, and 13HNG cells when compared with data obtained from radioresistant controls (p < 0.001) (Fig. 3a and 4). This suggests a less efficient DSB recognition for these cell lines (Supplemental Fig. S1a). In the frame of the RIANS model, these data do not mean that less DSB are induced by IR, but rather that less DSB are recognised by fewer ATM monomers that diffuse to the nucleus and trigger H2AX phosphorylation (Fig. S1a). In the other cell lines, the early DSB recognition rate was found similar to that of radioresistant controls.
In the radioresistant controls, the number of γH2AX foci significantly decreased with repair time and reached a number of residual γH2AX foci comparable to that assessed in non-irradiated cells. All the other fibroblast cell lines showed a different shape of γH2AX foci kinetic (Fig. 3a and S1a). Particularly, there was a difference in both the maximal number of γH2AX foci and the post-irradiation time at which it was reached. The RACKHAM01, 02HNA, and 85MA fibroblasts reached a maximal number of γH2AX foci at 1 h and the 201CLB fibroblasts at 4 h. For 203CLB, 202CLB, 01HNG, and 13HNG cell lines, the number of γH2AX foci remained constant from 10 min to 24 h post-irradiation, suggesting an impairment in both DSB recognition and repair. All the other cell lines reached their maximal γH2AX value at 10 min post-irradiation and decreased thereafter.
At 24 h post-irradiation, the number of γH2AX foci remaining suggested a complete DSB repair in the radioresistant controls. The 85MA and 01HNG cell lines showed a statistically significant higher number of residual γH2AX foci when compared with non-irradiated conditions (p = 0.008 and p = 0.001, respectively), suggesting an impairment of DSB repair. All the other cell lines showed a number of residual γH2AX foci similar to that of radioresistant controls, suggesting a normal DSB repair. Again, similar conclusions were reached with the pATM data and with the percentage of cells with more than 2 γH2AX foci (Figs. 3b and 4).
Radiobiological effects of topogram on cutaneous fibroblasts
The standard protocol of chest CT scan exams generally involves a low-dose topogram, to get a “scout view” of the volume to be imaged. In our conditions, the topogram resulted in a 1.1-mGy dose applied 1 min before the single helical chest CT exposure itself. If the DSB induction rate obeyed a linearly dose-dependent law, a dose of 1 mGy would induce 0.04 DSB per cell, on average, which may be considered negligible. Surprisingly, while this pre-irradiation appeared to have no significant effect in cells up to 4 h post-irradiation, the number of γH2AX foci assessed 24 h post-irradiation was found to be higher than that of non-irradiated cells in the BRCA1-mutated cell lines, 203CLB and 202CLB (3.24 ± 0.53 versus 0.82 ± 0.29 for 202CLB, respectively; p < 0.001 and 2.46 ± 0.77 versus 1.05 ± 0.29 for 203CLB, respectively; p = 0.040) (Fig. 5a).
When data were expressed as a number of γH2AX foci in excess (Fig. S2a) and as a number of cells with more than two foci γH2AX per cell (Fig. 5b), the same conclusions were reached (Fig. 5b). The pATM data also consolidated our conclusions (Fig. 6 and S2b).
Radiobiological effects of chest CT on mammary epithelial cells
The same experimental protocol with the same physical features described above was applied to the four mammary epithelial cell lines provided from the 200CLB, 201CLB, 202CLB, and 203CLB donors. In order to avoid any confusion, “epi” labels were added at the end of the name of each mammary epithelial cell line.
By considering the spontaneous number of γH2AX foci, no difference was found between the radioresistant 200CLB fibroblast cell line and its corresponding mammary epithelial counterpart, 200CLBepi (0.315 ± 0.053 versus 0.6 ± 0.3 γH2AX foci for fibroblasts and mammary epithelial cells, respectively) (Fig. 7). Conversely, for the radiosensitive/susceptible 201CLB, 202CLB, and 203CLB donors, a significant difference was found between the two fibroblastic and mammary epithelial cell types (p < 0.001). For example, for the 201CLB cells, there was an 8-fold difference in the number of γH2AX foci between the two cell types (0.57 ± 0.13 versus 4.48 ± 0.43 foci for fibroblast and epithelial cells, respectively) (Fig. 7). These findings suggest a strong genomic instability that may be specific to the mammary epithelial cells of these 3 donors.
Ten minutes after a single helical CT scan exposure, a significant increase in the number of γH2AX foci was observed for 200CLBepi, 202CLBepi, and 203CLBepi. These values represented the maximal number of γH2AX foci for these cell lines, suggesting a maximal DSB recognition rate reached early after irradiation (Fig. 7). By contrast, for the 201CLBepi cells, there was no statistically significant increase in the number of γH2AX foci after irradiation. In addition, the number of γH2AX foci at 24 h post-irradiation was lower than that of the non-irradiated controls (p < 0.001) (Fig. 7).
The 200CLBepi, 202CLBepi, and 203CLBepi cell lines showed a number of γH2AX foci at 24 h post-irradiation significantly higher than that observed in non-irradiated controls (p < 0.001) (Fig. 7).
Along our observations, some cells with more than 20 γH2AX foci appeared in certain conditions of exposure (Fig. 8). As already reported, these cells were considered highly damaged cells (HDC) [31]. A relatively small percentage of HDC were found in 200CLBepi and 201CLBepi cell lines, compared to 20% in 202CLBepi and 203CLBepi at 10 min post-irradiation (Fig. 8). The kinetic of the disappearance of HDC roughly followed that of γH2AX foci for each cell line.
The anti-pATM immunofluorescence was also performed on mammary epithelial cells, but the number of pATM foci could not be accurately assessed, mainly because of the predominant cytoplasmic localisation of this protein specific to the epithelial cells (Supplemental Fig. S3).