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Influence of cellular models and individual factor in the biological response to head CT scan exams

Influence of cellular models and individual factor in the biological response to head CT scan exams Background: While computed tomography (CT) exams are the major cause of medical exposure to ionising radiation, the radiation-induced risks must be documented. We investigated the impact of the cellular models and individual factor on the deoxyribonucleic acid double-strand breaks (DSB) recognition and repair in human skin fibroblasts and brain astrocytes exposed to current head CT scan conditions. Method: Nine human primary fibroblasts and four human astrocyte cell lines with different levels of radiosensitivity/susceptibility were exposed to a standard head CT scan exam using adapted phantoms. Cells were exposed to a single-helical (37.4 mGy) and double-helical (37.4 mGy + 5 min + 37.4 mGy) examination. DSB signalling and repair was assessed through anti-γH2AX and anti-pATM immunofluorescence. Results: Head CT scan induced a significant number of γH2AX and pATM foci. The kinetics of both biomarkers were found strongly dependent on the individual factor. Particularly, in cells from radiosensitive/susceptible patients, DSB may be significantly less recognised and/or repaired, whatever the CT scan exposure conditions. Similar conclusions were reached with astrocytes. Conclusions: Our results highlight the importance of both individual and tissue factors in the recognition and repair of DSB after current head CT scan exams. Further investigations are needed to better define the radiosensitivity/susceptibility of individual humans. Keywords: DNA breaks (double-stranded), Li-Fraumeni syndrome, Neurofibromatosis 1, Radiobiology, Tomography (x-ray computed) Key points Background To date, computed tomography (CT) scan exams, repre- Head computed tomography (CT) scan exposure sents the largest cause of medical exposure to ionising discriminates individuals with deoxyribonucleic acid radiation (IR) [1, 2]. For the last decade, the average double-strand breaks (DSB) as endpoints. annual effective dose received in a medical context has Cells from radiosensitive/susceptible patients elicit increased continuously [1, 3]. These statements raise more DSB after head CT scan exposure. questions about the justification of the medical exposure The justification of CT scans should take into to IR and the risks potentially due to CT scan exams account individual factors. [4–6]. More recently, a decree of the French National Nuclear Safety Authority encourages radiologists to * Correspondence: nicolas.foray@inserm.fr reduce or better justify the diagnosis involving IR by Institut National de la Santé et de la Recherche Médicale, U1296 Radiations taking into account individual risk factors of the patients Defense, Health and Environment Centre Léon-Bérard, 69008 Lyon, France Full list of author information is available at the end of the article to be imaged [7]. © The Author(s) under exclusive licence to European Society of Radiology. 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Devic et al. European Radiology Experimental (2022) 6:17 Page 2 of 12 Numerous radiobiological studies have been per- Methods formed to better understand and evaluate the biological Cells consequences of CT scan exposures [8–10]: Since Human untransformed fibroblasts were cultured as mono- chromosome and deoxyribonucleic acid (DNA) damage layers in the conditions detailed elsewhere [16]. The fibro- are likely involved in the response to IR, the great major- blasts were exposed at passages lower than 15. All the ity of the research groups have investigated the chromo- experiments were performed with cells in plateau phase of some and DNA damage induced by CT scan exposure in growth (95–99% in G0/G1) to overcome any cell cycle ef- in vitro or ex vivo lymphocytes [8–10]. However, the fect. Some of the fibroblast cell lines used in this study were radiation-induced (RI) cancers are not limited to leukae- provided from a collection of cells derived from radiosensi- mia and lymphocytes are not necessarily the most ap- tive and/or radiosusceptible patients, the COPERNIC collec- propriate cellular models to evaluate the RI risks linked tion [16]. This collection was approved by the regional to CT scan exposure [4, 5]. Since brain tumours are ethical committee in respect of the national regulatory pro- often cited as cancers potentially induced by CT scan cedures. Cell lines were declared under the agreement num- exams [4, 11, 12], brain astrocytes should also be used in bers DC2008-585, DC2011-1437, and DC2021-3957 to the the radiobiological characterisation of CT scan exposure. Ministry of Research. The COPERNIC database that gathers Similarly, fibroblasts, that represent the majority of cells radiobiological data of these cell lines was protected under in human body, may be also a useful cellular model. the reference IDDN.FR.001.510017.000.D.P.2014.000.10300. However, no radiobiological data has been obtained All the anonymous donors were informed and gave signed from astrocytes and fibroblasts exposed to current CT and consent according to the ethics recommendations [16]. scan exposure conditions. Among the COPERNIC cell lines, the 200CLB cell line There is increasing evidence that individual factors, derived from an apparently healthy patient and served as notably mutations of genome maintenance genes, may radioresistant control. The 01HNG, 02HNA and significantly influence the follow-up of exposed patients 13HNG cell lines derived from patients who showed se- and contribute to increase the RI risks linked to CT scan vere tissue reactions after radiotherapy [16] and served exposure. Again, few studies, if any, have raised the as representative radiosensitive examples. The RACK- question of the relative contribution of the individual HAM01, RACKHAM12, and RACKHAM39 cell lines factor in the biological response to CT scan exposures were derived from three different neurofibromatosis type +/− [13–15]. 1(NF1 mutated patients). The 85MA cell line derived +/− A unified model of the individual response to IR, rele- from a Li-Fraumeni syndrome (p53 mutated) patient vant for both high and low doses, and based on the RI was a kind gift from D. Scott (Manchester, UK). The nucleoshuttling of the ataxia-telangiectasia mutated GM03399 cell line was derived from a heterozygous +/− (ATM) protein kinase (RIANS), was proposed recently ataxia telangiectasia (ATM mutated) patient and was [16–18] (Fig. 1a). Any delay in the RIANS may lead to purchased from Coriell Institute (Camden, NJ, USA). radiosensitivity (RI adverse tissue reactions proneness), These last five cell lines served as representative radio- radiosusceptibility (RI cancer proneness), and/or radio- susceptible (high cancer risk) examples. The origin and degeneration (RI aging proneness) [15, 17]. By using im- the major clinical features of the nine fibroblast cell lines munofluorescence technique, in the frame of the RIANS have been gathered in the Table 1. model, the nuclear foci formed in irradiated cells by the Four brain cell lines, HA, HA-sp, HA-h, and HA- phosphorylated forms of the H2AX variant histone bs, providing from the same donor, were used in this (γH2AX) and ATM (pATM) proteins at the DSB sites study. These human untransformed astrocytes were may serve as useful endpoints to characterise any spe- purchased from the Sciencell Research laboratories cific exposure to IR. Particularly, the γH2AX and pATM (Carlsbad, CA,USA). HA,HA-sp,HA-h, andHA-bs foci observed early (10 min, 1 h) after irradiation provide were isolated from the cerebral cortex, spinal cord, information about the DSB recognition process while hippocampus, and brain stems, respectively. Astro- the late (24 h) ones characterise the DSB repair step cytes were cultured in the conditions recommended (Fig. 1b) [17, 22]. by the manufacturer i.e., in medium AM (#1801, This study aims to assess the DSB recognition and re- Sciencell), supplemented with 20% fetal bovine serum pair induced by current head CT scan exposure condi- (#0010; Sciencell) and penicillin/streptomycin solution tions in nine untransformed skin fibroblasts showing a (#0503; Sciencell). Astrocytes were exposed at pas- wide spectrum of radio-sensitivity/susceptibility and four sages lower than 10. All the experiments were per- untransformed brain astrocytes providing from the same formed with cells in plateau phase of growth (95–99% donor, by using γH2AX and pATM foci as endpoints. in G0/G1) to overcome any cell cycle effect. The ori- Physical dosimetry was insured by a new generation op- gin and the major clinical features of the four astro- tical scintillating fibre dosimeter [23]. cyte cell lines have been gathered in Table 1. Devic et al. European Radiology Experimental (2022) 6:17 Page 3 of 12 Fig. 1 The radiation-induced ATM nucleoshuttling: RIANS model and its current biomarkers. a In the frame of the RIANS model, IR induce the monomerisation of the ATM dimers in cytoplasm. The resulting ATM monomers diffuse in the nucleus and phosphorylate the H2AX histone variant molecular (γH2AX) at DSB sites, which triggers the formation of nuclear γH2AX foci, easily quantifiable by immunofluorescence. This is the recognition step. The recognised DSB are repaired by the non-homologous end-joining, the major DSB repair pathway in humans (see refs. [8, 16–21]). During the DSB repair process, two ATM monomers reassociate on the DSB sites and form the nuclear autophophorylated ATM (pATM) foci, also visible by immunofluorescence. b In the kinetics of γH2AX or pATM foci appearance, the early (10 min, 1 h) post-irradiation times provide information on the functionality of the DSB recognition step while the late (24 h) ones provide information on the functionality of the DSB repair step. ATM Ataxia telangiectasia mutated gene/protein, DSB Deoxyribonucleic acid double-strand breaks, RIANS Radiation-induced nucleoshuttling of the ATM protein, γH2AX Phosphorylated forms of the H2AX histone variant molecular Head CT scan exposure conditions (#353001, Falcon, Deutscher, Bernolsheim, France) (Fig. In vitro set up was characterised by a tissue-specific 1). The dose was measured with a dosimeter based on phantom. Fibroblasts were exposed on the surface of the scintillating fibre developed by the Fibermetrix company anthropomorphic head phantom and astrocytes were ex- [23–25] (Fig. 2). Spiral CT scan was performed by using posed inside the poly(methyl methacrylate) 16-cm diam- a Siemens Definition Edge apparatus (Siemens Healthi- eter phantom, both in Petri dishes 35 × 10 mm neers, Erlangen, Germany) operated at 166/230 mAs, Devic et al. European Radiology Experimental (2022) 6:17 Page 4 of 12 Table 1 Major clinical features of the cell lines used in this study Cell lines Cell type Known genes mutation Cancer proneness Radiobiological status 200CLB Fibroblast Apparently healthy ND Radioresistance +/− RACKHAM01 Fibroblast Neurofibromatosis type 1 Central and peripheral nervous system tumours Radiosensitivity and radiosusceptibility +/− RACKHAM12 Fibroblast Neurofibromatosis type 1 Central and peripheral nervous system tumours Radiosensitivity and radiosusceptibility +/− RACKHAM39 Fibroblast Neurofibromatosis type 1 Central and peripheral nervous system tumours Radiosensitivity and radiosusceptibility 01HNG Fibroblast ND (cancer patient) ND Radiosensitivity 02HNA Fibroblast ND (cancer patient) ND Radiosensitivity 13HNG Fibroblast ND (cancer patient) ND Radiosensitivity +/− GM03399 Fibroblast Ataxia-telangiectasia mutated Mainly leukaemia, lymphoma Radioresistance and radiosusceptibility +/− 85MA Fibroblast p53 Breast, brain, leukaemia, sarcoma Radioresistance and radiosusceptibility HA Astrocyte Apparently healthy ND ND HA-sp Astrocyte Apparently healthy ND ND HA-h Astrocyte Apparently healthy ND ND HA-bs Astrocyte Apparently healthy ND ND ND Not determined 120 kV, with a 1-s rotation time, a 0.6 pitch and a 3-mm mGy) to illustrate repeated helical series that can occur collimation in Centre Léon-Bérard (Lyon, France). CT during a CT exam. Additional data were also obtained at exposures were delivered with single-helical (37.4 mGy) Army Hospital Desgenettes (Lyon, France) (see “Results” or double-helical helical (37.4 mGy + 5 min + 37.4 section). Fig. 2 Representative images of the irradiation setup. a Anthropomorphic phantom used for fibroblasts irradiation. b PMMA phantom (longer width, 16 cm) used for astrocytes irradiation. In order to be clinically relevant, fibroblasts were exposed on the surface of the anthropomorphic head phantom and astrocytes were exposed inside the PMMA phantom. PMMA Poly(methyl methacrylate) Devic et al. European Radiology Experimental (2022) 6:17 Page 5 of 12 An intercomparison study with different CT scan ma- corresponding data of all fibroblasts together on the one chines was initiated. As a first step, the head CT scan ex- hand, and all astrocytes together on the other hand, for posure conditions described in Methods were applied to both single- and double-helical irradiations [31]. For a Philips Brillance iCT 256 at the Army Hospital Des- each test, the differences were considered statistically genettes (Lyon, France). By comparing data from the significant when the p value was lower than 0.050. Table Siemens Definition Edge CT scan machine at Centre S1 recapitulates all the significant differences observed. Léon-Bérard (Lyon, France), the doses assessed at the surface of the phantom were not found different (34.6 ± Results 2.4 mGy and 37.4 ± 1 mGy and, respectively (p > 0.550), Radiobiological effects of single- and double-helical head suggesting a low impact of the CT scan machines when CT scans on cutaneous fibroblasts comparing these two irradiators. The average volumetric CT dose index (CTDIvol) was 29.6 ± 1.2 mGy. The average dose-length product (DLP) Immunofluorescence analysis was 467 ± 17 mGy.cm. The average absorbed dose at The γH2AX and pATM immunofluorescence protocol for the surface of the phantom was 37.4 ± 1 mGy and 75.1 assessing DSB recognition and repair was described else- ± 2.3 mGy for the single- and double-helical conditions, where [26–28]. Briefly, cells were fixed in paraformalde- respectively. hyde for 15 min at room temperature and permeabilised Without irradiation, the radioresistant 200CLB control in detergent solution for 3 min. Primary antibody incuba- fibroblasts showed 0.31 ± 0.05 spontaneous γH2AX foci ser139 tions were performed for 1 h at 37 °C. Anti-γH2AX per cell. Among the other tested fibroblasts, 4 cell lines antibody (#05-636 Merck, Molsheim, France) was used at (GM03399, 85MA, 01HNG, 13HNG) showed signifi- ser1981 1:800, the monoclonal anti-mouse anti-pATM (#05- cantly more spontaneous γH2AX foci (p < 0.001), sug- 740 Merck) was used at 1:100. Incubations with anti- gesting a higher genomic instability, although the mouse fluorescein secondary antibodies provided by number of γH2AX foci never exceeded 1 foci per cell. Sigma-Aldrich (L’Isle d’Abeau Chesnes, France) were per- For the other cell lines, the numbers of spontaneous formed at 1:100 at 37 °C for 20 min. Slides were mounted γH2AX foci were not found different from that of con- in 4′,6′ Diamidino-2-phenyl-indole-stained Vectashield trols (Fig. 3a). (Vector Laboratories, Burlingame, CA, USA) and cells Ten minutes after a single-helical CT scan session, the were counted using an X100 objective with a fluorescence average number γH2AX foci was found to be 1.05 ± BX51 Olympus microscope (Olympus-France, Rungis, 0.15 γH2AX foci per cell in the radioresistant controls. France). For each of the three independent experiments, This value was not statistically different from the cur- 100 nuclei were analysed. The patented procedures of foci rently reported rate of DSB induced per Gy per human scoring have been detailed elsewhere [28]. diploid fibroblast (37 ± 4 γH2AX foci per Gy per cell [27]: the expected value would have been 1.38 ± 0.15 Data processing and statistics γH2AX foci per cell after 37.4 mGy) (Fig. 3a). Five cell The data and statistical analyses were processed using lines (RACKHAM01, RACKHAM12, 01HNG, 02HNA, MATLAB R2019a (MathWorks, Natick, MA, USA). and 13HNG showed a number of γH2AX foci signifi- Since each experiment is the result of 3 independent cantly lower than radioresistant controls (p < 0.001, p < replicates with 100 nuclei scored, the general mean of 0.001, p = 0.015, p = 0.005 and p = 0.020, respectively the 3 means of each replicate was given with the stand- (Fig. 3a). In the other cell lines, the early DSB recogni- ard error of the mean (SEM). By contrast, significance tion was found normal for the doses applied. tests were done by grouping the 300 nuclei data for each In the radioresistant controls, the number of γH2AX cell line and condition. To compare two conditions with foci reached its maximal value at 10 min post- each other, a non-parametric Mann-Whitney-Wilcoxon irradiation, reflecting a normal DSB recognition, de- test was used [29, 30]. For the γH2AX data, the number creased thereafter, and reached a number of residual of foci in each cell line without irradiation, 10 min and 1 γH2AX foci at 24 h post-irradiation not different from h post-irradiation was compared with the corresponding that assessed before irradiation. The γH2AX foci kinetics conditions of 200CLB for fibroblasts, and HA-h for as- of all the other cell lines differed from those of controls trocytes. The residual number of γH2AX foci was com- with a number of γH2AX foci reaching its maximal pared with the non-irradiated conditions for each cell value at 1 h post-irradiation, suggesting a delay in the line. For pATM data, the number of foci in each cell DSB recognition process and decreasing thereafter (Figs. lines without irradiation and 10 min post-irradiation was 3a and S1a). At 24 h post-irradiation, two situations compared with the corresponding conditions of 200CLB were encountered with the radio-sensitive/susceptible for fibroblasts, and HA-h for astrocytes [29, 30]. cell lines: (1) a subset of fibroblasts showed a slower rate Kruskal-Wallis test was performed to compare the of γH2AX foci disappearance and the number of γH2AX Devic et al. European Radiology Experimental (2022) 6:17 Page 6 of 12 Fig. 3 γH2AX foci in fibroblasts after single- and double-helical CT scan exposure. a Kinetics of γH2AX foci in fibroblasts after a single helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non irradiated). Each data represents the mean ± SEM of 3 independent experiments. The asterisks shown at the non-irradiated conditions and at 10 min and 1 h post-irradiation times correspond to a statistically significant difference with the radioresistant control 200CLB (p < 0.05). The asterisks shown at 24 h post-irradiation correspond to a statistically significant difference with the non-irradiated conditions for the same cell lines. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, SEM Standard error of the mean foci was found significantly higher than non-irradiated RACKHAM01, GM03399, 85MA, and 13HNG cell control, suggesting an impaired DSB repair process. This lines (Fig. 3b; p = 0.045); (2) another subset of fibro- is the case of RACKHAM01, RACKHAM39, GM03399, blasts did not show any statistically significant differ- 85MA, 01HNG, and 13HNG cell lines (Fig. 3a; p < ence when comparing residual γH2AX foci with 0.050); (2) another subset of fibroblasts did not show spontaneous ones, suggesting a normal DSB repair any statistically significant difference when comparing process. This is the case of the other cell lines (Fig. 3b). residual γH2AX foci with spontaneous ones, suggesting Table 2 recapitulates these findings. a normal DSB repair process. This is the case of RACK- In our hands, significant biological effects are observed HAM12 and 02HNA (Figs. 3a and S1a). in cells that show more than two γH2AX foci [16] (Fig. When fibroblasts were irradiated in the double-helical 4). In agreement with the data described above, all the head CT scan conditions, the number of γH2AX foci cell lines derived from radio-sensitive/susceptible pa- assessed 10 min post-irradiation increased significantly in a tients showed more than two γH2AX foci than controls, cell line-dependent manner (Figs. 3band S1b; p < 0.001). In supporting again a great variety of individual responses the radioresistant controls, there was two times more to CT scan exposure. γH2AX foci after a double-helical than after single-helical Since a normal nuclear ATM activity is required for CT scan, suggesting an additive dose-effect. By contrast, the the formation of γH2AX foci, we have also investigated γH2AX data ratio between double-helical and single-helical the number of nuclear pATM foci assessed in the same CT scan conditions was lower than 2 for RACKHAM01, experimental conditions than those described above. RACKHAM39, 02HNA and GM03399 cells, suggesting Again, accordingly with γH2AX data, the pATM data that the lack of DSB recognition is so severe that the sec- consolidated our conclusions with regard to the diversity ond helical CT scan view did not help in providing more of the responses to both single- and double-helical CT ATM monomers in the nucleus of these cells to better scan conditions (Figs. 5 and S5). The Table 2 sum- recognise DSB. Conversely, in 01HNG, 85MA and RACK- marised the results obtained in the two conditions of CT HAM12, the ratio between double-helical and single-helical scan exposure. CT scan conditions was found much higher, suggesting that the double-helical CT scan conditions may enhance Radiobiological effects of single- and double-helical head the recognition of the RI DSB or trigger a supplementary CT scans on human astrocytes induction of DSB (like the hyper-recombination The same head CT scan exposure conditions were ap- phenomenon). plied to brain astrocytes. For astrocytes, the average At 24 h after the double-helical CT scan exposure, CTDIvol was 27.3 ± 1.99 mGy. The DLP was 441 ± 36 two situations were encountered: (1) a subset of fibro- mGy.cm. The average absorbed dose in the phantom blasts showed a number of γH2AX foci significantly was 27.2 ± 2.5 mGy and 55.9 ± 5.72 mGy for the single- higher than non-irradiated control, suggesting an im- and double-helical conditions. paired DSB repair process. This is the case of Devic et al. European Radiology Experimental (2022) 6:17 Page 7 of 12 Table 2 Major clinical features of the cell lines used in this study Cell lines Status Single-helical scan Double-helical scan DSB recognition DSB repair DSB recognition DSB repair Fibroblasts 200CLB Apparently healthy + + + + +/− RACKHAM01 Neurofibromatosis type 1 −− −− +/− RACKHAM12 Neurofibromatosis type 1 − ++ + +/− RACKHAM39 Neurofibromatosis type 1 −− − + 01HNG Cancer patient −− ++ 02HNA Cancer patient − + − + 13HNG Cancer patient −− + − +/− GM03399 Ataxia-telangiectasia mutated −− −− +/− 85MA p53 −− + − Astrocytes HA Apparently healthy −− −− HA-sp Apparently healthy − + −− HA-h Apparently healthy + + − + HA-bs Apparently healthy −− − + No statistical difference was observed between the 0.10 γH2AX foci per cell after 27.2 mGy) (Fig. 6a). The numbers of spontaneous γH2AX foci assessed in the 4 three other brain astrocytes cell lines showed similar brain cell lines tested (less than 0.5 spontaneous γH2AX γH2AX foci data with less than one γH2AX foci per cell, foci was scored per cell). It is noteworthy that the num- but significantly less than the HA-h astrocytes (p < bers of spontaneous γH2AX foci were similar to that ob- 0.001) (Fig. 6a). Interestingly, while the hippocampus as- served with the radioresistant 200CLB control trocytes elicited a maximal number of γH2AX foci at 10 fibroblasts (Figs. 3a and 6a). min post-irradiation higher than the other astrocytes cell Ten minutes after a single-helical CT scan exposure, lines, the number of γH2AX foci decreased with repair the hippocampus astrocytes (HA-h) elicited 1.33 ± 0.13 time to reach the lowest number of residual γH2AX foci γH2AX foci per cell. This value was slightly higher than at 24 h post-irradiation, suggesting both complete DSB the currently reported rate of DSB induced per Gy per recognition and repair processes. All the other astrocytes human diploid fibroblast (37 ± 4 γH2AX foci per Gy per showed a maximal number of γH2AX foci at 1 h post- cell [16]: the expected value would have been 1.01 ± irradiation, suggesting a delayed in the RIANS and Fig. 4 Average number of cells with more than 2 γH2AX foci in fibroblasts after single- and double-helical CT scan exposure. Data shown in Fig. 3 but expressed as the number of cells with more than 2 γH2AX foci after single- (a) or double-helical (b) CT scan exposure. Each data represents the mean ± SEM of three independent experiments. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, SEM Standard error of the mean Devic et al. European Radiology Experimental (2022) 6:17 Page 8 of 12 Fig. 5 pATM foci in fibroblasts after single- and double-helical CT scan exposure. a Kinetics of pATM foci in fibroblasts after a single-helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non-irradiated). Each data represents the mean ± SEM of 3 independent experiments. The asterisks shown at 10 min post-irradiation times correspond to a statistically significant difference between the cell line and the radioresistant 200CLB controls. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, pATM Phosphorylated forms of the ATM protein, SEM Standard error of the mean therefore an impaired DSB recognition. At 24 h post- number of γH2AX foci at 1 h instead of 10 min post- irradiation, both cortex and brain stem astrocytes elic- irradiation, suggesting a deficient DSB recognition early ited a significant number of residual γH2AX foci (HA, p after irradiation. = 0.024; HA-bs, p = 0.044), suggesting therefore both At 24 h after a double-helical CT scan, only the HA impaired DSB recognition and DSB repair. Conversely, and HA-sp cells showed significantly higher number of spinal cord astrocytes (HA-sp) data suggested impaired residual γH2AX foci than in non-irradiated conditions, DSB recognition but normal DSB repair. suggesting an impairment in the DSB repair (Fig. 6b). After a double-helical CT scan exposure, the number Finally, the percentages of cells with more than 2 foci of γH2AX foci assessed 10 min after irradiation signifi- γH2AX (Fig. 7) and the numbers of pATM foci (Fig. 8) cantly increased for all the astrocytes cell lines (p < were assessed in all the conditions tested; the conclu- 0.001) but, like for the fibroblast cell lines tested, the sions reached with these two parameters were similar to assessed value did not correspond to the double of the those suggested by the data described above. Like for fi- number of γH2AX foci obtained after a single-helical broblasts, the Table 2 summarised the results obtained CT scan (Fig. 6b). Interestingly, in double-helical CT and revealed a large diversity of response in astrocytes scan exposure, all the astrocytes reached their maximal from the same donor. Fig. 6 γH2AX foci in astrocytes after single and double-helical CT-scan exposure. a Kinetics of γH2AX foci in astrocytes after a single helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non-irradiated). Each data represents the mean ± SEM foci per cell of 3 independent experiments. The asterisks shown at 10 min post-irradiation times correspond to a statistically significant difference between HA-h and the three other cell lines. For time 24 h, asterisks correspond to a statistically significant difference from the non-irradiated conditions for the same cell line. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant molecular, SEM Standard error of the mean Devic et al. European Radiology Experimental (2022) 6:17 Page 9 of 12 Fig. 7 Average number of cells with more than 2 γH2AX foci in astrocytes after single- and double-helical CT scan exposure. Data are shown in Fig. 6 but expressed as the number of cells with more than 2 γH2AX foci after single- (a) or double-helical (b) CT scan exposure. Each data represents the mean ± SEM of 3 independent experiments. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant molecular, SEM Standard error of the mean Discussion radiotherapy. This is particularly the case of patients +/− For the first time to our knowledge, cutaneous fibro- with Li-Fraumeni syndrome (p53 mutations), with +/− blasts from patients with different levels of radio- heterozygous ataxia telangiectasia (ATM mutations) +/− sensitivity/susceptibility, and brain astrocytes from the and neurofibromatosis type 1 (NF1 mutations) who same donor were exposed to single- and double-helical represent a non-negligible subset of patients (the cor- head CT scan sessions. Our findings suggest that indi- responding prevalence of those three syndromes is 1/ vidual factors and the nature of tissue, at least, are at the 4,000, 1/100, and 1/3,000 on average, respectively). origin of a great diversity of biological response, even at The fibroblasts derived from these syndromes show low doses and that the radio-sensitivity/susceptibility impaired DSB recognition and/or repair in CT scan may condition the functionality of DSB recognition and exposure conditions but also in radiotherapy exposure repair. Even if the number of cell lines is reduced, the conditions [32, 33]. In addition, this study also in- diversity of response should encourage us to investigate cluded three fibroblast cell lines derived from patients further the role of individual factors and tissue- showing grade 1-to-4 radiosensitivity after their anti- dependence in the final response to CT scan exposure. cancer radiotherapy (01HNG, 02HNA, 13HNG) [16]. In this study, most of the cell lines derived from Again, these patients have been submitted to CT scan patients at high risk of cancer and therefore, are sup- exposures during their anticancer treatment plan. posed to be exposed to CT scan conditions, either for Another argument for the necessity of taking into ac- routine CT diagnosis or for tumour imaging before count the individual radio-sensitivity/susceptibility status Fig. 8 pATM foci in astrocytes after single and double-helical CT-scan exposure. a Kinetics of pATM foci in astrocytes after a single helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non-irradiated). Each data represents the mean ± SEM foci per cell of three independent experiments. Asterisks for time 10 min correspond to a statistically significant difference between HA-h and the other cell lines. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, pATM Phosphorylated forms of the ATM protein, SEM Standard error of the mean Devic et al. European Radiology Experimental (2022) 6:17 Page 10 of 12 in the justification of the CT scan exams is provided by justification of the CT scan exam. However, additional the hypersensitivity to low doses phenomenon [34]. This studies are obviously needed to quantify the risk for a phenomenon shows exacerbated biological effect at a large spectrum genetic statuses and conditions and to low dose that can correspond to a 5−10 times higher better estimate the risks/benefits ratio. dose [18, 35, 36]. It was shown that this phenomenon preferentially occurs in cells with delayed RIANS. Since Abbreviations ATM: Ataxia telangiectasia mutated gene/protein; CT: Computed this is the case of all the radio-sensitive/radiosusceptible tomography; CTDIvol: Volumetric CT dose index; DLP: Dose-length fibroblast cell lines used in this study, it was important product; DNA: Deoxyribonucleic acid; DSB: DNA double-strand breaks; to recall the mechanistic model of hypersensitivity to IR: Ionizing radiation; NF1: Neurofibromatosis type 1 gene/protein/ syndrome; pATM: Phosphorylated forms of the ATM protein; low doses phenomenon: at low dose, less ATM mono- RI: Radiation-induced; RIANS: Radiation-induced nucleoshuttling of the mers are produced in cytoplasm and less DSB are in- ATM protein; SEM: Standard error of the mean; γH2AX: Phosphorylated duced in nucleus. However, if the RIANS is delayed, forms of the H2AX histone variant much less ATM monomers can diffuse to the nucleus. Consequently, few DSB, if any, are recognised and there- Supplementary Information fore repaired. The unrepaired DSB contribute therefore The online version contains supplementary material available at https://doi. to the cell lethality but also to RI gene mutations like in org/10.1186/s41747-022-00269-x. an exposure to higher doses [18, 35, 36]. Interestingly, the optimal dose range to observe such the hypersensi- Additional file 1: Figure S1. Kinetics of the γH2AX foci in excess in fibroblasts after a single helical head CT scan (a), or a double-helical head tivity to low doses phenomenon with the dose-rate ap- CT scan (b) at the indicated post-irradiation times (t0 = non-irradiated). plied in head CT scan (100 mGy/min) was found to be Data result from those shown in Fig. 2 with background subtraction in [10−50 mGy] [36], which is in very good agreement with order to show γH2AX foci in excess effectively due to CT exposure. Error bars indicate SEM. Figure S2. Kinetics of pATM foci in excess in fibro- head CT scan conditions. blasts (a) after a single helical head CT scan, (b) or a double-helical head This study involves four human astrocytes cell lines CT scan at the indicated post-irradiation times (t0 = non irradiated). Data derived from the same donor and representing differ- result from those shown in Fig. 3 with background subtraction in order to show γH2AX foci in excess effectively due to CT exposure. Error bars ent regions of the brain. Even if the number of cases indicate SEM. Figure S3. Kinetics of γH2AX foci in excess in astrocytes (a) is limited, it is noteworthy that our findings revealed, after a single helical head CT scan, or (b) a double-helical head CT scan for the first time to our knowledge, different re- at the indicated post-irradiation times (t0 = non-irradiated). Data result from those shown in Fig. 4 with background subtraction in order to show sponses to CT scan exposure according to the irradi- γH2AX foci in excess effectively due to CT exposure. Error bars indicate ated part of the brain. The data obtained at low dose SEM. Figure S4. Kinetics of the pATM foci in in excess astrocytes (a) after reflect the differences observed already at high dose a single helical head CT scan, or (b) a double-helical head CT scan at the indicated post-irradiation times (t0 = non-irradiated). Data result from (2 Gy) [37]. Notably, the astrocytes in cortex ap- those shown in Fig. 5 with background subtraction in order to show peared to be more radiosusceptible (with a high rate γH2AX foci in excess effectively due to CT exposure. Error bars indicate of misrepaired DSB) while those in hippocampus SEM. Figure S5. Distribution of the number of γH2AX foci per cell over the 300 nuclei scored for the 200CLB and 85MA cell lines at 1h after a showed more radiosensitivity (with a high rate of single-helical head CT exposure. Table S1. Statistical results and p-values unrepaired DSB) [37]. Further experiments are needed to establish an actual radiobiological cartography of the brain in order to better define the regions at risk Acknowledgements We would like to thank the radiology department of Army Hospital of RI cancers, even after low dose exposure. Desgenettes in Lyon and all the staff of the radiology department of Centre All along our investigations, absorbed doses were Léon Bérard in Lyon for their help in this work, especially Aline Riccardi- assessed by a new generation optical scintillating fibre Rousseau, Toufik Mallem, Rémi Auge, Fouzia Mesbah, and Didier Stanowski. We would like to thank Frédéric Lafay from the medical physics department dosimeter developed by the Fibermetrix company of Centre Léon Bérard in Lyon for his kind technical assistance. (Entzheim, France) and validated in the energy ranges cur- rently used in CT [23]. The dosimetry indicators generally Authors’ contributions used in the radiobiological studies involving in CT scan, Conceptualisation: CD, FP, FLM, DP, FC, MN, and NF. Data acquisition and namely CTDI and DLP, show many limitations and are methodology: CD, LS, HR, and CDC. Validation and data analysis: LB and NF. Writing—original draft preparation: CD and NF. Writing—review and editing: not representative of the dose actually delivered to cells CD, LB, LS, FP, HR, CDC, FLM, DP, FC, MN, and NF. Project administration and [10, 38]. Hence, our approach allowed us to have more ac- funding acquisition: FC, MN, and NF. All authors have read and agreed to the curate data to provide to the dose-response study. More- published version of the manuscript. over, given their tightness and their small diameter, these dosimeters permitted to measure reliably the absorbed Funding dose inside the petri dishes on the surface and inside the This work was supported by the Commissariat General à l’Investissement (CGI) (INDIRA project), the Institut National du Cancer (INCA) (PROUST poly(methyl methacrylate) phantoms (Fig. 2). project), the Centre National d’Etudes Spatiales (CNES) (BERNADOTTE Altogether, these data provide a quantitative proof that Project), EU FetOpen (SCANnTREAT project), and the Association individual factor should be taken into account in the Neurofibromatose et Recklinghausen (ANR) (RACKHAM project). Devic et al. European Radiology Experimental (2022) 6:17 Page 11 of 12 Availability of data and materials 10. Shi L, Tashiro S (2018) Estimation of the effects of medical diagnostic The data presented here are either present in a deposed database (see radiation exposure based on DNA damage. J Radiat Res 59:ii121–ii129. “Methods” section) or will be made available on reasonable request. https://doi.org/10.1093/jrr/rry006 11. Huang W-Y, Muo C-H, Lin C-Y, et al (2014) Paediatric head CT scan and subsequent risk of malignancy and benign brain tumour: a nation-wide Declarations population-based cohort study. Br J Cancer 110:2354–2360. https://doi.org/1 0.1038/bjc.2014.103 Ethics approval and consent to participate 12. de Gonzalez AB, Salotti JA, McHugh K, et al (2016) Relationship between This collection was approved by the regional ethical committee. Cell lines paediatric CT scans and subsequent risk of leukaemia and brain tumours: were declared under the numbers DC2008-585 and DC2011-1437 to the assessment of the impact of underlying conditions. Br J Cancer 114:388– Ministry of Research. The database was protected under the reference as 394. https://doi.org/10.1038/bjc.2015.415 IDDN.FR.001.510017.000.D.P.2014.000.10300. All the anonymous patients were 13. Bourguignon M, Bérard P, Bertho JM, Farah J, Mercat C, Radioprotection informed and gave signed consent according to the ethics recommenda- Editorial Board (2017) What’s next in Radioprotection? Radioprotection 52: tions. See “Methods” section 21–28. https://doi.org/10.1051/radiopro/2017006 14. Foray N, Colin C, Bourguignon M (2012) 100 Years of Individual Competing interests Radiosensitivity: How We Have Forgotten the Evidence. Radiology 264:627– Two authors (CD and MM) are employees of Fibermetrix™. NF is a member 631. https://doi.org/10.1148/radiol.12112560 of the European Radiology Experimental Editorial Board. He has not taken 15. Foray N, Bourguignon M, Hamada N (2016) Individual response to ionizing part in the review or selection process of this article. The remaining authors radiation. 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Influence of cellular models and individual factor in the biological response to head CT scan exams

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Abstract

Background: While computed tomography (CT) exams are the major cause of medical exposure to ionising radiation, the radiation-induced risks must be documented. We investigated the impact of the cellular models and individual factor on the deoxyribonucleic acid double-strand breaks (DSB) recognition and repair in human skin fibroblasts and brain astrocytes exposed to current head CT scan conditions. Method: Nine human primary fibroblasts and four human astrocyte cell lines with different levels of radiosensitivity/susceptibility were exposed to a standard head CT scan exam using adapted phantoms. Cells were exposed to a single-helical (37.4 mGy) and double-helical (37.4 mGy + 5 min + 37.4 mGy) examination. DSB signalling and repair was assessed through anti-γH2AX and anti-pATM immunofluorescence. Results: Head CT scan induced a significant number of γH2AX and pATM foci. The kinetics of both biomarkers were found strongly dependent on the individual factor. Particularly, in cells from radiosensitive/susceptible patients, DSB may be significantly less recognised and/or repaired, whatever the CT scan exposure conditions. Similar conclusions were reached with astrocytes. Conclusions: Our results highlight the importance of both individual and tissue factors in the recognition and repair of DSB after current head CT scan exams. Further investigations are needed to better define the radiosensitivity/susceptibility of individual humans. Keywords: DNA breaks (double-stranded), Li-Fraumeni syndrome, Neurofibromatosis 1, Radiobiology, Tomography (x-ray computed) Key points Background To date, computed tomography (CT) scan exams, repre- Head computed tomography (CT) scan exposure sents the largest cause of medical exposure to ionising discriminates individuals with deoxyribonucleic acid radiation (IR) [1, 2]. For the last decade, the average double-strand breaks (DSB) as endpoints. annual effective dose received in a medical context has Cells from radiosensitive/susceptible patients elicit increased continuously [1, 3]. These statements raise more DSB after head CT scan exposure. questions about the justification of the medical exposure The justification of CT scans should take into to IR and the risks potentially due to CT scan exams account individual factors. [4–6]. More recently, a decree of the French National Nuclear Safety Authority encourages radiologists to * Correspondence: nicolas.foray@inserm.fr reduce or better justify the diagnosis involving IR by Institut National de la Santé et de la Recherche Médicale, U1296 Radiations taking into account individual risk factors of the patients Defense, Health and Environment Centre Léon-Bérard, 69008 Lyon, France Full list of author information is available at the end of the article to be imaged [7]. © The Author(s) under exclusive licence to European Society of Radiology. 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Devic et al. European Radiology Experimental (2022) 6:17 Page 2 of 12 Numerous radiobiological studies have been per- Methods formed to better understand and evaluate the biological Cells consequences of CT scan exposures [8–10]: Since Human untransformed fibroblasts were cultured as mono- chromosome and deoxyribonucleic acid (DNA) damage layers in the conditions detailed elsewhere [16]. The fibro- are likely involved in the response to IR, the great major- blasts were exposed at passages lower than 15. All the ity of the research groups have investigated the chromo- experiments were performed with cells in plateau phase of some and DNA damage induced by CT scan exposure in growth (95–99% in G0/G1) to overcome any cell cycle ef- in vitro or ex vivo lymphocytes [8–10]. However, the fect. Some of the fibroblast cell lines used in this study were radiation-induced (RI) cancers are not limited to leukae- provided from a collection of cells derived from radiosensi- mia and lymphocytes are not necessarily the most ap- tive and/or radiosusceptible patients, the COPERNIC collec- propriate cellular models to evaluate the RI risks linked tion [16]. This collection was approved by the regional to CT scan exposure [4, 5]. Since brain tumours are ethical committee in respect of the national regulatory pro- often cited as cancers potentially induced by CT scan cedures. Cell lines were declared under the agreement num- exams [4, 11, 12], brain astrocytes should also be used in bers DC2008-585, DC2011-1437, and DC2021-3957 to the the radiobiological characterisation of CT scan exposure. Ministry of Research. The COPERNIC database that gathers Similarly, fibroblasts, that represent the majority of cells radiobiological data of these cell lines was protected under in human body, may be also a useful cellular model. the reference IDDN.FR.001.510017.000.D.P.2014.000.10300. However, no radiobiological data has been obtained All the anonymous donors were informed and gave signed from astrocytes and fibroblasts exposed to current CT and consent according to the ethics recommendations [16]. scan exposure conditions. Among the COPERNIC cell lines, the 200CLB cell line There is increasing evidence that individual factors, derived from an apparently healthy patient and served as notably mutations of genome maintenance genes, may radioresistant control. The 01HNG, 02HNA and significantly influence the follow-up of exposed patients 13HNG cell lines derived from patients who showed se- and contribute to increase the RI risks linked to CT scan vere tissue reactions after radiotherapy [16] and served exposure. Again, few studies, if any, have raised the as representative radiosensitive examples. The RACK- question of the relative contribution of the individual HAM01, RACKHAM12, and RACKHAM39 cell lines factor in the biological response to CT scan exposures were derived from three different neurofibromatosis type +/− [13–15]. 1(NF1 mutated patients). The 85MA cell line derived +/− A unified model of the individual response to IR, rele- from a Li-Fraumeni syndrome (p53 mutated) patient vant for both high and low doses, and based on the RI was a kind gift from D. Scott (Manchester, UK). The nucleoshuttling of the ataxia-telangiectasia mutated GM03399 cell line was derived from a heterozygous +/− (ATM) protein kinase (RIANS), was proposed recently ataxia telangiectasia (ATM mutated) patient and was [16–18] (Fig. 1a). Any delay in the RIANS may lead to purchased from Coriell Institute (Camden, NJ, USA). radiosensitivity (RI adverse tissue reactions proneness), These last five cell lines served as representative radio- radiosusceptibility (RI cancer proneness), and/or radio- susceptible (high cancer risk) examples. The origin and degeneration (RI aging proneness) [15, 17]. By using im- the major clinical features of the nine fibroblast cell lines munofluorescence technique, in the frame of the RIANS have been gathered in the Table 1. model, the nuclear foci formed in irradiated cells by the Four brain cell lines, HA, HA-sp, HA-h, and HA- phosphorylated forms of the H2AX variant histone bs, providing from the same donor, were used in this (γH2AX) and ATM (pATM) proteins at the DSB sites study. These human untransformed astrocytes were may serve as useful endpoints to characterise any spe- purchased from the Sciencell Research laboratories cific exposure to IR. Particularly, the γH2AX and pATM (Carlsbad, CA,USA). HA,HA-sp,HA-h, andHA-bs foci observed early (10 min, 1 h) after irradiation provide were isolated from the cerebral cortex, spinal cord, information about the DSB recognition process while hippocampus, and brain stems, respectively. Astro- the late (24 h) ones characterise the DSB repair step cytes were cultured in the conditions recommended (Fig. 1b) [17, 22]. by the manufacturer i.e., in medium AM (#1801, This study aims to assess the DSB recognition and re- Sciencell), supplemented with 20% fetal bovine serum pair induced by current head CT scan exposure condi- (#0010; Sciencell) and penicillin/streptomycin solution tions in nine untransformed skin fibroblasts showing a (#0503; Sciencell). Astrocytes were exposed at pas- wide spectrum of radio-sensitivity/susceptibility and four sages lower than 10. All the experiments were per- untransformed brain astrocytes providing from the same formed with cells in plateau phase of growth (95–99% donor, by using γH2AX and pATM foci as endpoints. in G0/G1) to overcome any cell cycle effect. The ori- Physical dosimetry was insured by a new generation op- gin and the major clinical features of the four astro- tical scintillating fibre dosimeter [23]. cyte cell lines have been gathered in Table 1. Devic et al. European Radiology Experimental (2022) 6:17 Page 3 of 12 Fig. 1 The radiation-induced ATM nucleoshuttling: RIANS model and its current biomarkers. a In the frame of the RIANS model, IR induce the monomerisation of the ATM dimers in cytoplasm. The resulting ATM monomers diffuse in the nucleus and phosphorylate the H2AX histone variant molecular (γH2AX) at DSB sites, which triggers the formation of nuclear γH2AX foci, easily quantifiable by immunofluorescence. This is the recognition step. The recognised DSB are repaired by the non-homologous end-joining, the major DSB repair pathway in humans (see refs. [8, 16–21]). During the DSB repair process, two ATM monomers reassociate on the DSB sites and form the nuclear autophophorylated ATM (pATM) foci, also visible by immunofluorescence. b In the kinetics of γH2AX or pATM foci appearance, the early (10 min, 1 h) post-irradiation times provide information on the functionality of the DSB recognition step while the late (24 h) ones provide information on the functionality of the DSB repair step. ATM Ataxia telangiectasia mutated gene/protein, DSB Deoxyribonucleic acid double-strand breaks, RIANS Radiation-induced nucleoshuttling of the ATM protein, γH2AX Phosphorylated forms of the H2AX histone variant molecular Head CT scan exposure conditions (#353001, Falcon, Deutscher, Bernolsheim, France) (Fig. In vitro set up was characterised by a tissue-specific 1). The dose was measured with a dosimeter based on phantom. Fibroblasts were exposed on the surface of the scintillating fibre developed by the Fibermetrix company anthropomorphic head phantom and astrocytes were ex- [23–25] (Fig. 2). Spiral CT scan was performed by using posed inside the poly(methyl methacrylate) 16-cm diam- a Siemens Definition Edge apparatus (Siemens Healthi- eter phantom, both in Petri dishes 35 × 10 mm neers, Erlangen, Germany) operated at 166/230 mAs, Devic et al. European Radiology Experimental (2022) 6:17 Page 4 of 12 Table 1 Major clinical features of the cell lines used in this study Cell lines Cell type Known genes mutation Cancer proneness Radiobiological status 200CLB Fibroblast Apparently healthy ND Radioresistance +/− RACKHAM01 Fibroblast Neurofibromatosis type 1 Central and peripheral nervous system tumours Radiosensitivity and radiosusceptibility +/− RACKHAM12 Fibroblast Neurofibromatosis type 1 Central and peripheral nervous system tumours Radiosensitivity and radiosusceptibility +/− RACKHAM39 Fibroblast Neurofibromatosis type 1 Central and peripheral nervous system tumours Radiosensitivity and radiosusceptibility 01HNG Fibroblast ND (cancer patient) ND Radiosensitivity 02HNA Fibroblast ND (cancer patient) ND Radiosensitivity 13HNG Fibroblast ND (cancer patient) ND Radiosensitivity +/− GM03399 Fibroblast Ataxia-telangiectasia mutated Mainly leukaemia, lymphoma Radioresistance and radiosusceptibility +/− 85MA Fibroblast p53 Breast, brain, leukaemia, sarcoma Radioresistance and radiosusceptibility HA Astrocyte Apparently healthy ND ND HA-sp Astrocyte Apparently healthy ND ND HA-h Astrocyte Apparently healthy ND ND HA-bs Astrocyte Apparently healthy ND ND ND Not determined 120 kV, with a 1-s rotation time, a 0.6 pitch and a 3-mm mGy) to illustrate repeated helical series that can occur collimation in Centre Léon-Bérard (Lyon, France). CT during a CT exam. Additional data were also obtained at exposures were delivered with single-helical (37.4 mGy) Army Hospital Desgenettes (Lyon, France) (see “Results” or double-helical helical (37.4 mGy + 5 min + 37.4 section). Fig. 2 Representative images of the irradiation setup. a Anthropomorphic phantom used for fibroblasts irradiation. b PMMA phantom (longer width, 16 cm) used for astrocytes irradiation. In order to be clinically relevant, fibroblasts were exposed on the surface of the anthropomorphic head phantom and astrocytes were exposed inside the PMMA phantom. PMMA Poly(methyl methacrylate) Devic et al. European Radiology Experimental (2022) 6:17 Page 5 of 12 An intercomparison study with different CT scan ma- corresponding data of all fibroblasts together on the one chines was initiated. As a first step, the head CT scan ex- hand, and all astrocytes together on the other hand, for posure conditions described in Methods were applied to both single- and double-helical irradiations [31]. For a Philips Brillance iCT 256 at the Army Hospital Des- each test, the differences were considered statistically genettes (Lyon, France). By comparing data from the significant when the p value was lower than 0.050. Table Siemens Definition Edge CT scan machine at Centre S1 recapitulates all the significant differences observed. Léon-Bérard (Lyon, France), the doses assessed at the surface of the phantom were not found different (34.6 ± Results 2.4 mGy and 37.4 ± 1 mGy and, respectively (p > 0.550), Radiobiological effects of single- and double-helical head suggesting a low impact of the CT scan machines when CT scans on cutaneous fibroblasts comparing these two irradiators. The average volumetric CT dose index (CTDIvol) was 29.6 ± 1.2 mGy. The average dose-length product (DLP) Immunofluorescence analysis was 467 ± 17 mGy.cm. The average absorbed dose at The γH2AX and pATM immunofluorescence protocol for the surface of the phantom was 37.4 ± 1 mGy and 75.1 assessing DSB recognition and repair was described else- ± 2.3 mGy for the single- and double-helical conditions, where [26–28]. Briefly, cells were fixed in paraformalde- respectively. hyde for 15 min at room temperature and permeabilised Without irradiation, the radioresistant 200CLB control in detergent solution for 3 min. Primary antibody incuba- fibroblasts showed 0.31 ± 0.05 spontaneous γH2AX foci ser139 tions were performed for 1 h at 37 °C. Anti-γH2AX per cell. Among the other tested fibroblasts, 4 cell lines antibody (#05-636 Merck, Molsheim, France) was used at (GM03399, 85MA, 01HNG, 13HNG) showed signifi- ser1981 1:800, the monoclonal anti-mouse anti-pATM (#05- cantly more spontaneous γH2AX foci (p < 0.001), sug- 740 Merck) was used at 1:100. Incubations with anti- gesting a higher genomic instability, although the mouse fluorescein secondary antibodies provided by number of γH2AX foci never exceeded 1 foci per cell. Sigma-Aldrich (L’Isle d’Abeau Chesnes, France) were per- For the other cell lines, the numbers of spontaneous formed at 1:100 at 37 °C for 20 min. Slides were mounted γH2AX foci were not found different from that of con- in 4′,6′ Diamidino-2-phenyl-indole-stained Vectashield trols (Fig. 3a). (Vector Laboratories, Burlingame, CA, USA) and cells Ten minutes after a single-helical CT scan session, the were counted using an X100 objective with a fluorescence average number γH2AX foci was found to be 1.05 ± BX51 Olympus microscope (Olympus-France, Rungis, 0.15 γH2AX foci per cell in the radioresistant controls. France). For each of the three independent experiments, This value was not statistically different from the cur- 100 nuclei were analysed. The patented procedures of foci rently reported rate of DSB induced per Gy per human scoring have been detailed elsewhere [28]. diploid fibroblast (37 ± 4 γH2AX foci per Gy per cell [27]: the expected value would have been 1.38 ± 0.15 Data processing and statistics γH2AX foci per cell after 37.4 mGy) (Fig. 3a). Five cell The data and statistical analyses were processed using lines (RACKHAM01, RACKHAM12, 01HNG, 02HNA, MATLAB R2019a (MathWorks, Natick, MA, USA). and 13HNG showed a number of γH2AX foci signifi- Since each experiment is the result of 3 independent cantly lower than radioresistant controls (p < 0.001, p < replicates with 100 nuclei scored, the general mean of 0.001, p = 0.015, p = 0.005 and p = 0.020, respectively the 3 means of each replicate was given with the stand- (Fig. 3a). In the other cell lines, the early DSB recogni- ard error of the mean (SEM). By contrast, significance tion was found normal for the doses applied. tests were done by grouping the 300 nuclei data for each In the radioresistant controls, the number of γH2AX cell line and condition. To compare two conditions with foci reached its maximal value at 10 min post- each other, a non-parametric Mann-Whitney-Wilcoxon irradiation, reflecting a normal DSB recognition, de- test was used [29, 30]. For the γH2AX data, the number creased thereafter, and reached a number of residual of foci in each cell line without irradiation, 10 min and 1 γH2AX foci at 24 h post-irradiation not different from h post-irradiation was compared with the corresponding that assessed before irradiation. The γH2AX foci kinetics conditions of 200CLB for fibroblasts, and HA-h for as- of all the other cell lines differed from those of controls trocytes. The residual number of γH2AX foci was com- with a number of γH2AX foci reaching its maximal pared with the non-irradiated conditions for each cell value at 1 h post-irradiation, suggesting a delay in the line. For pATM data, the number of foci in each cell DSB recognition process and decreasing thereafter (Figs. lines without irradiation and 10 min post-irradiation was 3a and S1a). At 24 h post-irradiation, two situations compared with the corresponding conditions of 200CLB were encountered with the radio-sensitive/susceptible for fibroblasts, and HA-h for astrocytes [29, 30]. cell lines: (1) a subset of fibroblasts showed a slower rate Kruskal-Wallis test was performed to compare the of γH2AX foci disappearance and the number of γH2AX Devic et al. European Radiology Experimental (2022) 6:17 Page 6 of 12 Fig. 3 γH2AX foci in fibroblasts after single- and double-helical CT scan exposure. a Kinetics of γH2AX foci in fibroblasts after a single helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non irradiated). Each data represents the mean ± SEM of 3 independent experiments. The asterisks shown at the non-irradiated conditions and at 10 min and 1 h post-irradiation times correspond to a statistically significant difference with the radioresistant control 200CLB (p < 0.05). The asterisks shown at 24 h post-irradiation correspond to a statistically significant difference with the non-irradiated conditions for the same cell lines. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, SEM Standard error of the mean foci was found significantly higher than non-irradiated RACKHAM01, GM03399, 85MA, and 13HNG cell control, suggesting an impaired DSB repair process. This lines (Fig. 3b; p = 0.045); (2) another subset of fibro- is the case of RACKHAM01, RACKHAM39, GM03399, blasts did not show any statistically significant differ- 85MA, 01HNG, and 13HNG cell lines (Fig. 3a; p < ence when comparing residual γH2AX foci with 0.050); (2) another subset of fibroblasts did not show spontaneous ones, suggesting a normal DSB repair any statistically significant difference when comparing process. This is the case of the other cell lines (Fig. 3b). residual γH2AX foci with spontaneous ones, suggesting Table 2 recapitulates these findings. a normal DSB repair process. This is the case of RACK- In our hands, significant biological effects are observed HAM12 and 02HNA (Figs. 3a and S1a). in cells that show more than two γH2AX foci [16] (Fig. When fibroblasts were irradiated in the double-helical 4). In agreement with the data described above, all the head CT scan conditions, the number of γH2AX foci cell lines derived from radio-sensitive/susceptible pa- assessed 10 min post-irradiation increased significantly in a tients showed more than two γH2AX foci than controls, cell line-dependent manner (Figs. 3band S1b; p < 0.001). In supporting again a great variety of individual responses the radioresistant controls, there was two times more to CT scan exposure. γH2AX foci after a double-helical than after single-helical Since a normal nuclear ATM activity is required for CT scan, suggesting an additive dose-effect. By contrast, the the formation of γH2AX foci, we have also investigated γH2AX data ratio between double-helical and single-helical the number of nuclear pATM foci assessed in the same CT scan conditions was lower than 2 for RACKHAM01, experimental conditions than those described above. RACKHAM39, 02HNA and GM03399 cells, suggesting Again, accordingly with γH2AX data, the pATM data that the lack of DSB recognition is so severe that the sec- consolidated our conclusions with regard to the diversity ond helical CT scan view did not help in providing more of the responses to both single- and double-helical CT ATM monomers in the nucleus of these cells to better scan conditions (Figs. 5 and S5). The Table 2 sum- recognise DSB. Conversely, in 01HNG, 85MA and RACK- marised the results obtained in the two conditions of CT HAM12, the ratio between double-helical and single-helical scan exposure. CT scan conditions was found much higher, suggesting that the double-helical CT scan conditions may enhance Radiobiological effects of single- and double-helical head the recognition of the RI DSB or trigger a supplementary CT scans on human astrocytes induction of DSB (like the hyper-recombination The same head CT scan exposure conditions were ap- phenomenon). plied to brain astrocytes. For astrocytes, the average At 24 h after the double-helical CT scan exposure, CTDIvol was 27.3 ± 1.99 mGy. The DLP was 441 ± 36 two situations were encountered: (1) a subset of fibro- mGy.cm. The average absorbed dose in the phantom blasts showed a number of γH2AX foci significantly was 27.2 ± 2.5 mGy and 55.9 ± 5.72 mGy for the single- higher than non-irradiated control, suggesting an im- and double-helical conditions. paired DSB repair process. This is the case of Devic et al. European Radiology Experimental (2022) 6:17 Page 7 of 12 Table 2 Major clinical features of the cell lines used in this study Cell lines Status Single-helical scan Double-helical scan DSB recognition DSB repair DSB recognition DSB repair Fibroblasts 200CLB Apparently healthy + + + + +/− RACKHAM01 Neurofibromatosis type 1 −− −− +/− RACKHAM12 Neurofibromatosis type 1 − ++ + +/− RACKHAM39 Neurofibromatosis type 1 −− − + 01HNG Cancer patient −− ++ 02HNA Cancer patient − + − + 13HNG Cancer patient −− + − +/− GM03399 Ataxia-telangiectasia mutated −− −− +/− 85MA p53 −− + − Astrocytes HA Apparently healthy −− −− HA-sp Apparently healthy − + −− HA-h Apparently healthy + + − + HA-bs Apparently healthy −− − + No statistical difference was observed between the 0.10 γH2AX foci per cell after 27.2 mGy) (Fig. 6a). The numbers of spontaneous γH2AX foci assessed in the 4 three other brain astrocytes cell lines showed similar brain cell lines tested (less than 0.5 spontaneous γH2AX γH2AX foci data with less than one γH2AX foci per cell, foci was scored per cell). It is noteworthy that the num- but significantly less than the HA-h astrocytes (p < bers of spontaneous γH2AX foci were similar to that ob- 0.001) (Fig. 6a). Interestingly, while the hippocampus as- served with the radioresistant 200CLB control trocytes elicited a maximal number of γH2AX foci at 10 fibroblasts (Figs. 3a and 6a). min post-irradiation higher than the other astrocytes cell Ten minutes after a single-helical CT scan exposure, lines, the number of γH2AX foci decreased with repair the hippocampus astrocytes (HA-h) elicited 1.33 ± 0.13 time to reach the lowest number of residual γH2AX foci γH2AX foci per cell. This value was slightly higher than at 24 h post-irradiation, suggesting both complete DSB the currently reported rate of DSB induced per Gy per recognition and repair processes. All the other astrocytes human diploid fibroblast (37 ± 4 γH2AX foci per Gy per showed a maximal number of γH2AX foci at 1 h post- cell [16]: the expected value would have been 1.01 ± irradiation, suggesting a delayed in the RIANS and Fig. 4 Average number of cells with more than 2 γH2AX foci in fibroblasts after single- and double-helical CT scan exposure. Data shown in Fig. 3 but expressed as the number of cells with more than 2 γH2AX foci after single- (a) or double-helical (b) CT scan exposure. Each data represents the mean ± SEM of three independent experiments. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, SEM Standard error of the mean Devic et al. European Radiology Experimental (2022) 6:17 Page 8 of 12 Fig. 5 pATM foci in fibroblasts after single- and double-helical CT scan exposure. a Kinetics of pATM foci in fibroblasts after a single-helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non-irradiated). Each data represents the mean ± SEM of 3 independent experiments. The asterisks shown at 10 min post-irradiation times correspond to a statistically significant difference between the cell line and the radioresistant 200CLB controls. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, pATM Phosphorylated forms of the ATM protein, SEM Standard error of the mean therefore an impaired DSB recognition. At 24 h post- number of γH2AX foci at 1 h instead of 10 min post- irradiation, both cortex and brain stem astrocytes elic- irradiation, suggesting a deficient DSB recognition early ited a significant number of residual γH2AX foci (HA, p after irradiation. = 0.024; HA-bs, p = 0.044), suggesting therefore both At 24 h after a double-helical CT scan, only the HA impaired DSB recognition and DSB repair. Conversely, and HA-sp cells showed significantly higher number of spinal cord astrocytes (HA-sp) data suggested impaired residual γH2AX foci than in non-irradiated conditions, DSB recognition but normal DSB repair. suggesting an impairment in the DSB repair (Fig. 6b). After a double-helical CT scan exposure, the number Finally, the percentages of cells with more than 2 foci of γH2AX foci assessed 10 min after irradiation signifi- γH2AX (Fig. 7) and the numbers of pATM foci (Fig. 8) cantly increased for all the astrocytes cell lines (p < were assessed in all the conditions tested; the conclu- 0.001) but, like for the fibroblast cell lines tested, the sions reached with these two parameters were similar to assessed value did not correspond to the double of the those suggested by the data described above. Like for fi- number of γH2AX foci obtained after a single-helical broblasts, the Table 2 summarised the results obtained CT scan (Fig. 6b). Interestingly, in double-helical CT and revealed a large diversity of response in astrocytes scan exposure, all the astrocytes reached their maximal from the same donor. Fig. 6 γH2AX foci in astrocytes after single and double-helical CT-scan exposure. a Kinetics of γH2AX foci in astrocytes after a single helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non-irradiated). Each data represents the mean ± SEM foci per cell of 3 independent experiments. The asterisks shown at 10 min post-irradiation times correspond to a statistically significant difference between HA-h and the three other cell lines. For time 24 h, asterisks correspond to a statistically significant difference from the non-irradiated conditions for the same cell line. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant molecular, SEM Standard error of the mean Devic et al. European Radiology Experimental (2022) 6:17 Page 9 of 12 Fig. 7 Average number of cells with more than 2 γH2AX foci in astrocytes after single- and double-helical CT scan exposure. Data are shown in Fig. 6 but expressed as the number of cells with more than 2 γH2AX foci after single- (a) or double-helical (b) CT scan exposure. Each data represents the mean ± SEM of 3 independent experiments. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant molecular, SEM Standard error of the mean Discussion radiotherapy. This is particularly the case of patients +/− For the first time to our knowledge, cutaneous fibro- with Li-Fraumeni syndrome (p53 mutations), with +/− blasts from patients with different levels of radio- heterozygous ataxia telangiectasia (ATM mutations) +/− sensitivity/susceptibility, and brain astrocytes from the and neurofibromatosis type 1 (NF1 mutations) who same donor were exposed to single- and double-helical represent a non-negligible subset of patients (the cor- head CT scan sessions. Our findings suggest that indi- responding prevalence of those three syndromes is 1/ vidual factors and the nature of tissue, at least, are at the 4,000, 1/100, and 1/3,000 on average, respectively). origin of a great diversity of biological response, even at The fibroblasts derived from these syndromes show low doses and that the radio-sensitivity/susceptibility impaired DSB recognition and/or repair in CT scan may condition the functionality of DSB recognition and exposure conditions but also in radiotherapy exposure repair. Even if the number of cell lines is reduced, the conditions [32, 33]. In addition, this study also in- diversity of response should encourage us to investigate cluded three fibroblast cell lines derived from patients further the role of individual factors and tissue- showing grade 1-to-4 radiosensitivity after their anti- dependence in the final response to CT scan exposure. cancer radiotherapy (01HNG, 02HNA, 13HNG) [16]. In this study, most of the cell lines derived from Again, these patients have been submitted to CT scan patients at high risk of cancer and therefore, are sup- exposures during their anticancer treatment plan. posed to be exposed to CT scan conditions, either for Another argument for the necessity of taking into ac- routine CT diagnosis or for tumour imaging before count the individual radio-sensitivity/susceptibility status Fig. 8 pATM foci in astrocytes after single and double-helical CT-scan exposure. a Kinetics of pATM foci in astrocytes after a single helical head CT scan, or (b) a double-helical head CT scan at the indicated times after exposure (t0 = non-irradiated). Each data represents the mean ± SEM foci per cell of three independent experiments. Asterisks for time 10 min correspond to a statistically significant difference between HA-h and the other cell lines. CT Computed tomography, γH2AX Phosphorylated forms of the H2AX histone variant, pATM Phosphorylated forms of the ATM protein, SEM Standard error of the mean Devic et al. European Radiology Experimental (2022) 6:17 Page 10 of 12 in the justification of the CT scan exams is provided by justification of the CT scan exam. However, additional the hypersensitivity to low doses phenomenon [34]. This studies are obviously needed to quantify the risk for a phenomenon shows exacerbated biological effect at a large spectrum genetic statuses and conditions and to low dose that can correspond to a 5−10 times higher better estimate the risks/benefits ratio. dose [18, 35, 36]. It was shown that this phenomenon preferentially occurs in cells with delayed RIANS. Since Abbreviations ATM: Ataxia telangiectasia mutated gene/protein; CT: Computed this is the case of all the radio-sensitive/radiosusceptible tomography; CTDIvol: Volumetric CT dose index; DLP: Dose-length fibroblast cell lines used in this study, it was important product; DNA: Deoxyribonucleic acid; DSB: DNA double-strand breaks; to recall the mechanistic model of hypersensitivity to IR: Ionizing radiation; NF1: Neurofibromatosis type 1 gene/protein/ syndrome; pATM: Phosphorylated forms of the ATM protein; low doses phenomenon: at low dose, less ATM mono- RI: Radiation-induced; RIANS: Radiation-induced nucleoshuttling of the mers are produced in cytoplasm and less DSB are in- ATM protein; SEM: Standard error of the mean; γH2AX: Phosphorylated duced in nucleus. However, if the RIANS is delayed, forms of the H2AX histone variant much less ATM monomers can diffuse to the nucleus. Consequently, few DSB, if any, are recognised and there- Supplementary Information fore repaired. The unrepaired DSB contribute therefore The online version contains supplementary material available at https://doi. to the cell lethality but also to RI gene mutations like in org/10.1186/s41747-022-00269-x. an exposure to higher doses [18, 35, 36]. Interestingly, the optimal dose range to observe such the hypersensi- Additional file 1: Figure S1. Kinetics of the γH2AX foci in excess in fibroblasts after a single helical head CT scan (a), or a double-helical head tivity to low doses phenomenon with the dose-rate ap- CT scan (b) at the indicated post-irradiation times (t0 = non-irradiated). plied in head CT scan (100 mGy/min) was found to be Data result from those shown in Fig. 2 with background subtraction in [10−50 mGy] [36], which is in very good agreement with order to show γH2AX foci in excess effectively due to CT exposure. Error bars indicate SEM. Figure S2. Kinetics of pATM foci in excess in fibro- head CT scan conditions. blasts (a) after a single helical head CT scan, (b) or a double-helical head This study involves four human astrocytes cell lines CT scan at the indicated post-irradiation times (t0 = non irradiated). Data derived from the same donor and representing differ- result from those shown in Fig. 3 with background subtraction in order to show γH2AX foci in excess effectively due to CT exposure. Error bars ent regions of the brain. Even if the number of cases indicate SEM. Figure S3. Kinetics of γH2AX foci in excess in astrocytes (a) is limited, it is noteworthy that our findings revealed, after a single helical head CT scan, or (b) a double-helical head CT scan for the first time to our knowledge, different re- at the indicated post-irradiation times (t0 = non-irradiated). Data result from those shown in Fig. 4 with background subtraction in order to show sponses to CT scan exposure according to the irradi- γH2AX foci in excess effectively due to CT exposure. Error bars indicate ated part of the brain. The data obtained at low dose SEM. Figure S4. Kinetics of the pATM foci in in excess astrocytes (a) after reflect the differences observed already at high dose a single helical head CT scan, or (b) a double-helical head CT scan at the indicated post-irradiation times (t0 = non-irradiated). Data result from (2 Gy) [37]. Notably, the astrocytes in cortex ap- those shown in Fig. 5 with background subtraction in order to show peared to be more radiosusceptible (with a high rate γH2AX foci in excess effectively due to CT exposure. Error bars indicate of misrepaired DSB) while those in hippocampus SEM. Figure S5. Distribution of the number of γH2AX foci per cell over the 300 nuclei scored for the 200CLB and 85MA cell lines at 1h after a showed more radiosensitivity (with a high rate of single-helical head CT exposure. Table S1. Statistical results and p-values unrepaired DSB) [37]. Further experiments are needed to establish an actual radiobiological cartography of the brain in order to better define the regions at risk Acknowledgements We would like to thank the radiology department of Army Hospital of RI cancers, even after low dose exposure. Desgenettes in Lyon and all the staff of the radiology department of Centre All along our investigations, absorbed doses were Léon Bérard in Lyon for their help in this work, especially Aline Riccardi- assessed by a new generation optical scintillating fibre Rousseau, Toufik Mallem, Rémi Auge, Fouzia Mesbah, and Didier Stanowski. We would like to thank Frédéric Lafay from the medical physics department dosimeter developed by the Fibermetrix company of Centre Léon Bérard in Lyon for his kind technical assistance. (Entzheim, France) and validated in the energy ranges cur- rently used in CT [23]. The dosimetry indicators generally Authors’ contributions used in the radiobiological studies involving in CT scan, Conceptualisation: CD, FP, FLM, DP, FC, MN, and NF. Data acquisition and namely CTDI and DLP, show many limitations and are methodology: CD, LS, HR, and CDC. Validation and data analysis: LB and NF. Writing—original draft preparation: CD and NF. Writing—review and editing: not representative of the dose actually delivered to cells CD, LB, LS, FP, HR, CDC, FLM, DP, FC, MN, and NF. Project administration and [10, 38]. Hence, our approach allowed us to have more ac- funding acquisition: FC, MN, and NF. All authors have read and agreed to the curate data to provide to the dose-response study. More- published version of the manuscript. over, given their tightness and their small diameter, these dosimeters permitted to measure reliably the absorbed Funding dose inside the petri dishes on the surface and inside the This work was supported by the Commissariat General à l’Investissement (CGI) (INDIRA project), the Institut National du Cancer (INCA) (PROUST poly(methyl methacrylate) phantoms (Fig. 2). project), the Centre National d’Etudes Spatiales (CNES) (BERNADOTTE Altogether, these data provide a quantitative proof that Project), EU FetOpen (SCANnTREAT project), and the Association individual factor should be taken into account in the Neurofibromatose et Recklinghausen (ANR) (RACKHAM project). Devic et al. European Radiology Experimental (2022) 6:17 Page 11 of 12 Availability of data and materials 10. Shi L, Tashiro S (2018) Estimation of the effects of medical diagnostic The data presented here are either present in a deposed database (see radiation exposure based on DNA damage. J Radiat Res 59:ii121–ii129. “Methods” section) or will be made available on reasonable request. https://doi.org/10.1093/jrr/rry006 11. Huang W-Y, Muo C-H, Lin C-Y, et al (2014) Paediatric head CT scan and subsequent risk of malignancy and benign brain tumour: a nation-wide Declarations population-based cohort study. Br J Cancer 110:2354–2360. https://doi.org/1 0.1038/bjc.2014.103 Ethics approval and consent to participate 12. de Gonzalez AB, Salotti JA, McHugh K, et al (2016) Relationship between This collection was approved by the regional ethical committee. Cell lines paediatric CT scans and subsequent risk of leukaemia and brain tumours: were declared under the numbers DC2008-585 and DC2011-1437 to the assessment of the impact of underlying conditions. Br J Cancer 114:388– Ministry of Research. The database was protected under the reference as 394. https://doi.org/10.1038/bjc.2015.415 IDDN.FR.001.510017.000.D.P.2014.000.10300. All the anonymous patients were 13. Bourguignon M, Bérard P, Bertho JM, Farah J, Mercat C, Radioprotection informed and gave signed consent according to the ethics recommenda- Editorial Board (2017) What’s next in Radioprotection? Radioprotection 52: tions. See “Methods” section 21–28. https://doi.org/10.1051/radiopro/2017006 14. Foray N, Colin C, Bourguignon M (2012) 100 Years of Individual Competing interests Radiosensitivity: How We Have Forgotten the Evidence. Radiology 264:627– Two authors (CD and MM) are employees of Fibermetrix™. NF is a member 631. https://doi.org/10.1148/radiol.12112560 of the European Radiology Experimental Editorial Board. He has not taken 15. Foray N, Bourguignon M, Hamada N (2016) Individual response to ionizing part in the review or selection process of this article. The remaining authors radiation. Mutat Res Mutat Res 770:369–386. https://doi.org/10.1016/j. declare that they have no competing interests. mrrev.2016.09.001 16. Granzotto A, Benadjaoud MA, Vogin G, et al (2016) Influence of Author details Nucleoshuttling of the ATM Protein in the Healthy Tissues Response to Institut National de la Santé et de la Recherche Médicale, U1296 Radiations Radiation Therapy: Toward a Molecular Classification of Human Defense, Health and Environment Centre Léon-Bérard, 69008 Lyon, France. Radiosensitivity. Int J Radiat Oncol 94:450–460. https://doi.org/10.1016/j. 2 3 FibermetrixTM SAS, 7 Allée de l’Europe, 67960 Entzheim, France. Radiation ijrobp.2015.11.013 Oncology Department, American University of Beirut Medical Center, Beirut 17. Berthel E, Foray N, Ferlazzo ML (2019) The nucleoshuttling of the ATM 1107 2020, Lebanon. Service de Radiologie, Centre Léon Bérard, 28 rue protein: a unified model to describe the individual response to high- and Laennec, 69008 Lyon, France. 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European Radiology ExperimentalSpringer Journals

Published: Apr 7, 2022

Keywords: DNA breaks (double-stranded); Li-Fraumeni syndrome; Neurofibromatosis 1; Radiobiology; Tomography (x-ray computed)

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