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Relation between DNA double-strand breaks and energy spectra of secondary electrons produced by different X-ray energies.

Abstract Purpose: In a radiological examination, low-energy X-radiation is used (<100 keV). For other radiological procedures, the energy used is several MeV. ICRP in publication 103 has currently considered that photons irrespective of their energy have the same radiation weighting factor. Nevertheless, there are topological differences at the nanoscale of X-ray energy deposition as a function of its energy spectrum, meaning that the different interactions with living matter could vary in biological efficacy. Materials and methods: To study these differences, we characterized our irradiation conditions in terms of initial photon energies, but especially in terms of energy spectra of secondary electrons at the cell nucleus level, using Monte Carlo simulations. We evaluated signaling of DNA damage by monitoring a large number of γH2A.X foci after exposure of G0/G1-phase synchronized human primary endothelial cells from 0.25 to 5 Gy at 40 kV, 220 kV and 4 MV X-rays. Number and spatial distribution of γH2A.X foci were explored. In parallel, we investigated cell behavior through cell death and ability of a mother cell to produce two daughter cells. We also studied the missegregation rate after cell division. Results: We report a higher number of DNA double-strand breaks signaled by γH2A.X for 40 kVp and/or 220 kVp compared to 4 MVp for the highest tested doses of 2 and 5 Gy. We observed no difference between the biological endpoint studies with 40 kVp and 220 kVp X-ray spectra. This lack of difference could be explained by the relative similarity of the calculated energy spectra of secondary electrons at the cell monolayer. Conclusion: The energy spectrum of secondary electrons seems to be more closely related to the level of DNA damage measured by γH2A.X than the initial spectrum of photon energy or voltage settings. Our results indicate that as the energy spectrum of secondary electrons increases, the DNA damage signaled by γH2A.X decreases and this effect is observable beyond 220 kVp.


Introduction
X-rays with an energy below 50 keV are frequently applied in diagnostic radiology, particularly mammography. ICRP publication 103 assigned a radiation weighting factor of 1 to this radiation quality (ICRP 2007). This has since been a topic of continuous discussion. One reason for this is a potential risk of radiation-induced cancer by mammography. An increasing amount of in vitro data obtained in the low-energy region indicates a complex dependence of biological effectiveness on photon energy (Lehnert et al. 2008). Indeed, Brenner and Amols (1989); Goodhead and Nikjoo (1990); and Kellerer (2002) have reported an increase in relative biological effectiveness of photons when their energy decreases.
In theory, this difference in biological efficacy could have its origin in the spatial distribution differences at the nanoscale of X-ray energy deposition as a function of their energy spectrum (Goodhead 1988). Indeed, as the photon energy decreases, the nature of its interaction with matter changes. In water, below an energy of 50 keV, there is a decrease in the probability of Compton interactions and an increase in photoelectric interactions. This change leads to a modification of the energy spectrum of the secondary electrons created, and thus potentially a change in the quality of the resulting radiation-induced damage, as DNA damage, misrepair of DNA double-strand breaks and, finally, cell behavior.
Most of the observations reported in the literature concern misrepair of DNA double-strand breaks, including those of Mestres et al. who showed an increase in chromosome aberrations in lymphocytes irradiated with 30 kV, 80 kV and 120 kV X-rays. Słonina et al. showed induction of micronuclei with human fibroblasts and keratinocytes irradiated with 25 kV and Lehnert et al. with human mammary epithelial cells irradiated with 25 kV and 10 kV X-rays Lehnert et al. 2006;Mestres et al. 2008;Lehnert et al. 2008). In addition, some studies have focused on clonogenic survival, including a study by Panteleeva et al. who showed a decrease in clonogenic survival with primary human epidermal keratinocytes and fibroblasts irradiated with 25 kV compared to 200 kV ). These differences in misrepair or clonogenic survival rates are likely to originate in the generation of DNA damage. DNA can be damaged by ionizing radiation in various ways, amongst others by forming double-strand breaks (DSBs). DNA DSBs are probably the most deleterious of the many different types of DNA damage that occur within the eukaryotic cell (Burma et al. 2001). An early response to DSBs is the phosphorylation of a variant form of the histone H2A designated H2A.X, located in the area of the break, at serine 139. Phosphorylated H2A.X (called cH2A.X) can be visualized as foci at break sites by immunofluorescence using phosphospecific antibodies (Paull et al. 2000); these are also known as ionizing radiation-induced foci (IRIF). cH2A.X initiates the formation of a platform to attract and retain proteins, such as Nijmegen breakage syndrome 1 (NBS1), mediator of DNA damage checkpoint protein 1 (MDC1), breast cancer susceptibility 1 (BRCA1), and p53-binding protein 1 (53BP1), and in turn these lead to the recruitment of DNA damage repair proteins (Paull et al. 2000;Kinner et al. 2008;van Attikum and Gasser 2009;Lisby and Rothstein 2009). The study of DNA damage formation seems to be a good way to evaluate possible quantitative and qualitative differences in the damage induced by low-and high-energy radiation.
Few studies have focused on characterizing the qualitative differences of DNA damage. Beyreuther et al. (2009) investigated the number of induced as well as persistent DNA damage foci to investigate late effects after exposure of medical applications such as mammography screening. They studied IRIF formation after exposure to different energy X-rays, 10 kV, 25 kV and 200 kV. They showed energy dependence with increasing DNA damage number at decreasing photon energy. Despite highlighting this difference, we currently lack a qualitative analysis of the damage and its effects on cell behavior. Moreover, these comparative studies do not usually explore the biological effects of higher energy X-rays usually used in radiotherapy. This is all the more interesting as it can be assumed that the energy spectrum of the secondary electrons of these latter radiations would be significantly different from the reference X-ray used today in most radiobiology studies (i.e. 200-220 kV).
In the present work, we deployed a multiparametric experimental strategy allowing us to study different biological endpoints in parallel, from the same pool of cells, i.e. cells from one experiment yielding simultaneously foci data, cell division measurements and quantification of chromosome missegregation events. In addition, we characterized our irradiation conditions in terms of initial photon energies, but especially in terms of energy spectra of secondary electrons at the cell nucleus level, using Monte Carlo simulations which took into account their environment at the time of irradiations. Special attention was paid to dosimetry in these experiments to ensure comparable macroscopic absorbed doses among the studied energy spectra, including the exclusive use of plastic dishes for the irradiations (Kegel et al. 2007). Photon spectra from X-ray generators of 40 kVp, 220 kVp and 4 MVp were used to determine the respective biological effects on human primary cells. We studied the quality of DNA damage through the amount and the spatial distribution in the nucleus of cH2A.X foci in primary endothelial cells with respect to post-irradiation time and X-ray energy. We explored cell behavior to compare the consequences of radiation-induced DNA damage. To explore the regulation of the cell cycle depending on the X-ray energy, the frequencies of cell division and death, and missegregation rate, such as micronuclei and/or nucleoplasmic bridges in mono-and binucleated G1 cells were analyzed.

Cell culture
Primary human umbilical vein endothelial cells (HUVECs) were from the Lonza Group (ref. C2519A, lot0000394986) and isolated by Lonza from human tissue (from 2 females and 2 males) donated after permission was obtained for its use in research applications by informed consent or legal authorization. All cells tested negative for mycoplasma, bacteria, yeast and fungi. Cell lots and donors were tested and negative for HIV-1, hepatitis B and hepatitis C. HUVECs were cultured at 37 C, with 95% humidity and 5% CO 2 in EGM-2 media optimized for the proliferation of endothelial cells and supplemented with 5% fetal bovine serum, hydrocortisone, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, gentamicin and amphotericin-B (EGM-2MV BulletKit, CC-3202, Lonza). After thawing, HUVECs were counted using the Cellometer V R K2 -Nexcelom, and seeded at 8000 cells/cm 2 . Seventy-two hours later, when they are 80% confluent, the HUVECs were trypsinized and seeded at 8000 cells/cm 2 . Ninety-six hours later, on the day of irradiation, G0/G1-phase synchronized HUVECs were obtained by contact inhibition induced in confluent culture. Synchronized cells were seeded 5 h prior to irradiation at a density of 30,000 cells/cm 2 on plastic dishes (1-well Permanox V R in Nunc V R Lab-Tek V R chamber slide systems; Thermo Fisher Scientific and Petri; BD Biosciences) and incubated at 37 C.

Irradiation platform and conditions
Cell irradiations were performed using two irradiation facilities with different energy ranges: a medical linear accelerator (Elekta Synergy V R ) and a Small Animal Radiation Research Platform (SARRP).
Elekta Synergy V R is a medical linear accelerator and was used to deliver X-rays with a maximal energy of 4 MeV (4 MVp). The dose rate measured with an ionization chamber calibrated in air kerma free was about 1 Gy min À1 at a distance of 120 cm from the source and with a 30 Â 30 cm irradiation field (Table 1).
The SARRP (XSTRAHL, Ltd., UK) is an image-guided micro irradiator, composed of a Varian X-ray tube mounted on a gantry, mainly dedicated to small animal irradiation (Wong et al. 2008). However, thanks to an adequate dosimetry protocol and irradiation configuration set-up (Dos Santos et al. 2018), it is also possible to perform cell irradiation. In this work, two configurations, using two different high voltages, were used with this platform. In both cases, inherent and additional filtrations of 0.8 and 1 mm of beryllium and aluminum, respectively, a source sample distance of 30.5 cm and a collimator leading to an 8 Â 8 cm irradiation field were used ( Table 1). The SARRP was used to deliver X-rays with a maximal energy of 40 keV (40 kVp) and 220 keV (220 kVp) at a dose rate of about 1 Gy min À1 in air kerma, in both cases. The details of the dosimetry protocol used for cell irradiation on the SARRP facility are given in Dos Santos et al. (2018). Briefly, absolute dosimetry measurements were performed inside a cell container with an ionization chamber calibrated in air kerma free-in-air and then converted into water kerma free-inair. Then, attenuation due to the cell culture medium was evaluated using measurements with EBT3 films.
For all irradiations configurations, the doses delivered to the cell monolayer were 0.25, 0.5, 1 and 2 Gy for the earlier time (30 min post-irradiation), and 5 Gy for the later time (5 h post-irradiation). Culture media were not renewed after irradiation.
The uncertainty in the dose rate measurement was about 5%, 3% and 7% for 40 kVp, 220 kVp and 4 MVp respectively at k ¼ 2.

Fluorescence image acquisition and analysis
Image acquisition and analysis were performed with the Scan Ù R platform (Olympus), as described previously (Gruel et al. 2016;Vaurijoux et al. 2017). Briefly, images were acquired on an inverted Olympus IX81 fluorescence microscope with a UPLSAPO 100XO oil immersion objective (Olympus) and an NA of 1.4; the microscope was coupled with an Orca R 2 CCD camera (Hamamatsu) and a motorized SCAN IM IX2 stage (M€ arzh€ auser). Image analysis was performed with Scan Ù R analysis software (Olympus). For each channel, images were acquired as 3 z-stack images around and including the focus plane with step size of 0.5 lm between planes. The images of the 3D stack were projected to 2D xy images using maximum intensity projection. An edge segmentation algorithm was used to detect nuclei in the DAPI channel (main object) and cH2AX foci in the Alexa Fluor V R 488 channel (sub-object 1). Cells in G0/G1 phase were selected with a 'flow cytometry-like' analysis (Gruel et al. 2016). A first selection based on the area and circularity of the nuclei was based on the definition of an adequate region on the corresponding scatter plot. This step allowed us to consider a region with only isolated nuclei, by removing from the analysis objects corresponding to nucleus clusters. A second region, based on the integrated intensity levels of DAPI fluorescence associated with the nuclei and Alexa Fluor V R 488 fluorescence associated with cH2A.X foci, was measured for each nucleus and was then used to isolate nuclei in G0/G1 phase. IRIF were analyzed only in nuclei within the gate formed by the intersection of these two regions (Vaurijoux et al. 2017).

Videomicroscopy
Following irradiation, HUVECs, seeded in Corning TM Falcon TM Easy-Grip Tissue Culture Dishes, were placed in the incubation system, allowing control of temperature, humidity and CO 2 levels, which is mounted on an inverted Olympus IX83 phase microscope with a UPLFLN 10XPH objective (Olympus). The acquisition was performed with Scan Ù R software (Olympus). Six different fields were acquired per dish, every 5 minutes, for 96 hours. Image analysis was performed with Image J, using MTrackJ and Grid Overlay plugins, allowing us to count the cells able to form two daughter cells and the dead cells. When an adherent cell produced two daughter cells, we counted this as one 'cell division' event, and when we observed the disruption of an adherent cell, it was counted as one cell death event. The frequency of division was the number of 'cell division' events observed for a given time step, divided by the initial number of cells in the live-cell imaging field (the same analysis methodology was used for 'cell death' event).

Monte Carlo simulations
Monte Carlo simulations of the secondary electron spectra were performed using the Geant4 Monte Carlo toolkit version 10.3. To this end, the modeling of the irradiation configurations was performed taking into account the initial photon spectra and the different layers of material between the source and the cells.
In particular, for the SARRP configurations, the initial photon spectra were calculated using Speckcalc software (Poludniowski et al. 2009), whereas, for the 4 MVp irradiation performed with the medical linear accelerator, the initial photon spectra were derived from Figure 10 in Sheikh- Bagheri and Rogers (2002). In both cases, the irradiated cells were modeled as a mono-volume of liquid water 5 lm in height and 125 Â 85 mm in the x-y plane in order to ensure electronic equilibrium. Between the photon source and the cell volume (or behind the cell volume), different material layers were taken into account as shown in Figure 1.
For the SAARP configurations, the photon source traverses the cell medium, approximated here by a liquid water layer 3 mm in height, then the cell volume is positioned and a polystyrene layer of 3 mm is modeled behind to represent the dish material where cells are grown.
In the medical accelerator simulation, a carbon table is located between the beam and the dish containing the cells. Therefore, in the simulation, the first layer traversed by the photon source is a 50 mm height table (2 mm of carbon fiber þ46 mm of foam þ2 mm of carbon fiber), followed by 5 mm of Plexiglas, then 3 mm of polystyrene and then the cell volume 5 lm in height. Behind the cell volume, a 3 mm liquid water volume representing the cell medium is also taken into account.
The secondary electron spectra at cell level were calculated from the phase space of the electrons entering (or produced) in the modeled cell volume. In order to obtain the phase space of the secondary electrons, photon transport in the different layers of matter was performed using the Livermore physics list (LowEnergy, option 4) in Geant4, which simulates photon interactions to a minimal energy of 250 eV. Secondary electrons produced by photons were also transported in the simulated geometry using the physical models available in this physics list. When an electron entered in the cell volume, its kinetic energy, position (x, y, z) and momentum (p x , p y , p z ) were recorded and the electrons were killed, in order to save calculation time. In rare cases, electrons were produced directly by a photon reaction in the cell volume. In this case, the same information was registered in the phase space. This information allowed us to calculate the energy spectra presented in this work and to perform a more detailed simulation of the energy deposition in the cell volume using the information described above as the irradiation source (simulations in progress).

Statistics
Student's t-test was performed to evaluate the statistical significance of any differences among measurements.

Results
Photon and electron energy spectrum Figure 2 reports the photon energy spectrum for irradiations performed on the SARRP Platform at 40 and 220 kVp obtained with the Speckcalc software (Poludniowski et al. 2009). As mentioned before, an additional filtration of 1 mm of aluminum was used for irradiation. It allows lower energy X-rays compared to the reference filtration of 0.15 mm of copper normally used for irradiation with the SARRP. The 40 kVp voltage was chosen because it is the lower voltage that can be achieved on the SARRP irradiator and is close to the photon energy spectrum used for mammography examinations. This configuration allows a large proportion of very low-energy X-rays leading to photoelectric interactions. The 220 kVp voltage was chosen because it is often considered as a reference voltage for the SARRP and also in the literature. For the 4 MVp irradiation performed with the medical linear accelerator, the initial photon spectra were derived from Figure 10 in Sheikh-Bagheri and Rogers (2002).
To overcome problems due to the modification of the energy spectrum of the emitted photons, all irradiations were performed without a lid. The mean photon energies were 25.6 keV, 70.2 keV and around 1.3 MeV, respectively, for 40 kVp, 220 kVp and 4 MVp. Figure 3 reports the secondary electron energy spectrum obtained for the three configurations. The mean energy of the produced secondary electrons increased with X-ray energy, as expected, respectively 14 keV, 20 keV and 859 keV for 40 kVp, 220 kVp and 4 MVp.

Quantification of cH2A.X foci per nucleus
We investigated the number of cH2A.X foci induced by Xrays of different energy spectra. To harmonize the DNA  content of the cells at the time of irradiation, we performed our analysis on cells in the same cell cycle state. Thus, we exposed HUVECs synchronized in the G0/G1 phase of the cell cycle to 0.25, 0.5, 1 or 2 Gy of 40 kVp, 220 kVp or 4 MVp X-rays and studied cH2A.X foci formation 30 min after exposure. All irradiations were performed exclusively on adherent cells in plastic dishes. Using automated detection of nuclei and foci, we analyzed a mean of 4200 cells for each replicate experiment so that we could screen subpopulations of cells or foci by different characteristics, such as shape, foci spatial distribution in the nuclei, and weight their representativeness within the entire population of exposed cells. All results presented here concern only cells in the G0/G1 phase of the cell cycle.
Thirty minutes post-irradiation, when analyzing dose-bydose after exposure, we observed no significant difference from 0.25 to 1 Gy in the mean number of cH2A.X foci per nucleus among the three tested X-ray energies ( In order to study the differences of resolution rate, we studied the IRIF number 5 h after exposure to 5 Gy, using the same three different qualities of radiation ( Figure 5). We analyzed a mean of 4500 cells for each condition and performed at least three replicate experiments. All results here relate only to cells in the G0/G1 phase of the cell cycle. We observed, on average, 15.2 ± 0.80, 14.3 ± 0.67 and 12.2 ± 0.54 cH2A.X foci per nucleus, respectively (mean ± SE, from at least three replicate experiments). Significant differences were seen between 40 kVp and 4 MVp (t-test, p ¼ .008) and between 220 kVp and 4 MVp voltages (t-test, p ¼ .01).

Spatial distribution of cH2A.X foci per nucleus
To investigate if this decrease in mean values could be due to differences in the spatial distribution of cH2A.X foci, we analyzed the mean distance from the nearest neighbor of cH2A.X foci as a function of their number per nuclei ( Figure 6), 30 min post-exposure at 0.25, 0.5, 1 and 2 Gy, and 5 h post-exposure at 5 Gy, among the three tested X-ray energies. As expected, the distances from the nearest neighbor were dependent on the number of cH2A.X foci per nuclei, i.e. the more cH2A.X foci, the lower the distance from the nearest neighbor will be. The mean distance was obtained by using the x, y coordinates of all cH2A.X foci, keeping the minimum distance of each cH2A.X focus from each other and calculating the average of all the minimum distances per nucleus. For all doses, 30 min and 5 h postirradiation, there was no significant difference between the three qualities of radiation (t-test, p > .05).

Cell division and mortality rate as a function of the energy spectra of irradiation
We also investigated if this difference in the mean number of foci had an impact on cell behavior like cell division and   mortality rates. We focused on the two most different energy spectra: 40 kVp and 4 MVp. To identify cells able to complete a full cell cycle after exposure, we used phase contrast livecell imaging of synchronized cells exposed to 5 Gy. All results reported here are from four replicate experiments per condition. Around 1500 and 500 events per condition (division þ death) were counted for the non-irradiated and irradiated cells, respectively. Figure 7 shows the cumulated number of cell division events per step of 6 hours, for the non-irradiated and 5 Gy irradiated cells. For the control irradiation, we observed that division events began 18 h post-plating, and seemed to be delayed by around 6 h for the exposure conditions. For the 5 Gy irradiated cells, more than a third of the initial cells achieved one cell division within 42 h following exposure. No significant difference was found between the two qualities of radiation studied, with 34.2 ± 24.2% and 40.9 ± 7.7% of division frequency 42 h post-exposure after 5 Gy of irradiation at 40 kVp and 4 MVp, respectively (mean ± SE, t-test, p > .05). Figure 8 shows the division frequency during 6-h periods over 96 hours after exposure. The post-irradiation cell cycle recovery was the same for the two ionizing radiation qualities. The cell division activity peaked between 24 and 30 h post-irradiation and stopped 60 h after exposure. No significant difference was observed between 40 kVp and 4 MVp (t-test, p > 0.05). Figure 9 shows the frequency of mortality for the 5 Gy irradiated cells at 40 kVp and 4 MVp. We observed that the mortality frequency started to increase between 24 and 30 h post-irradiation for both energies, with 0.6 ± 0.58%, and 0.6 ± 0.61% of cells that had died, at 40 kVp and 4 MVp, respectively. We observed a maximum in the relative frequency of mortality between 54 and 60 h post-irradiation, with a mortality frequency of 4.1 ± 1.92%, and 5.3 ± 0.78%, respectively, at 40 kVp and 4 MVp. There was no significant difference between the two radiation qualities (mean ± SE, t-test, p > .05). Also, we found no difference between our experimental protocol in terms of radiation-induced cell death.

Missegregation events following exposure at two radiation qualities after cell division
The synchronization of cells at the time of exposure and their ability to divide in the same proportions for both qualities of radiation allowed the comparative measurement of missegregation events observed 48 h post-irradiation. These missegregation events gave us an indication of the rate of abnormal chromosomal structures formed in each of the studied irradiation conditions. This analysis was performed on the same slides as those used for the cH2A.X foci scoring and allowed continuity to be maintained between different biological measurements. Around 4100 micronuclei and/or nucleoplasmic bridges in mono-and binucleated G1 cells were scored per condition. Among the entire population of G1 cells, the frequency of micronuclei and/or nucleoplasmic bridges in mono-and binucleated cells was not significantly Percentages of the number of nuclei were calculated in around 4400 G0/G1 cells exposed to 40 kVp (blue bar), 220 kVp (violet bar) and 4 MVp (grey bar) X-rays. Values were obtained from at least 3 experiments. Significance was tested using Student's t-test. different between 40 kVp and 4 MVp (t-test, p > .05), with 8.68 ± 3.82% and 11.78 ± 4.40% of missegregation events (mean ± SE from at least three replicate experiments), respectively (Table 3).

Discussion
The question of the biological effectiveness of X-rays as a function of their energy spectra was raised several decades ago (Brenner and Amols 1989;Kellerer 2002;Lehnert et al. 2006Lehnert et al. , 2008, but is hard to solve definitively. The main reason is that the development of irradiation configurations suitable for a biological comparison is complicated by the combination of strong dosimetric constraints related to low-energy X-rays (Kegel et al. 2007) and the need to carry out biological experiments under relevant and robust conditions. The goal is to evaluate a possible difference in effects on a biological target by ensuring that the absorbed dose at this target is equal. In other words, it is a matter of verifying whether the micro-and nanodosimetric differences between low-and high-energy X-rays are sufficient, by themselves, to lead to different biological consequences. In this complex comparison, the role of the nano-and micro-organization of the biological target of interest such as local or temporal variations in DNA density (chromatin organization and cell cycle, respectively) should not be forgotten. We sought to take into account these considerations in the experimental development of irradiation and the interpretation of our results. We also maximized the robustness of our biological quantifications by analyzing several thousand cells for each experimental condition and replicate experiments.
In the present work, we observed no difference between the biological endpoint studies with 40 kVp and 220 kVp Xray spectra. This lack of difference could be explained by the relative similarity of the calculated energy spectra of secondary electrons at the cell monolayer. Indeed, the mean energies of the two spectra of secondary electrons were 14 keV and 20 keV and the shape of these spectra is also quite similar. This emphasizes the difficulty of interpreting biological results only on the basis of X-ray irradiation voltage settings or initial photon energies. In our experimental settings, despite a significant difference in energy spectra of photons between 40 kVp and 220 kVp (mean energy of photons at 40 kVp ¼ 25.6 keV; mean energy of photons at 220 kVp ¼ 70.2 keV; Figure 2), the calculated energy spectra of secondary electrons at the cell level were close (mean energy of secondary electrons at 40 kVp ¼ 14 keV; mean energy of secondary electrons at 220 kVp ¼ 20 keV; Figure 3).    Nevertheless, we report a higher number of DNA doublestrand breaks signaled by cH2A.X for 40 kVp and/or 220 kVp compared to 4 MVp for the highest tested doses of 2 and 5 Gy. Our results seem to indicate that as the energy spectrum of secondary electrons increases, the DNA damage signaled by cH2A.X decreases and this effect is observable beyond 220 kVp. In other words, the increase in the number of cH2A.X already reported by Beyreuther et al. for lowenergy X-rays (Beyreuther et al. 2009) could still be measured in our case by taking a reference radiation of 4 MVp. In fact, Beyreuther et al. studied energy spectra below 40 kVp and reported an increase of the number of cH2A.X and 53BP1 colocated foci for 10 kVp and 25 kVp compared to 200 kVp, 2 h post-irradiation, at 0.5, 1, 2, 4 and 6 Gy (Beyreuther et al. 2009). In their experimental conditions, the reported mean energy of secondary electrons at the cell monolayer was significantly lower for the two tested low-energy X-ray conditions (3.7 keV and 7 keV for 10 kVp and 25 kVp, respectively) compared to their reference X-ray voltage of 200 kVp (21.4 keV as mean energy of secondary electrons). This confirms that the analysis of the relation between the mean number of foci and the energy of the X-rays must be made in terms of the energy of secondary electrons at the cell level and not directly on the photon spectra. This is something to keep in mind as the energy of the secondary electrons interacting with cells can be strongly influenced by the experimental set-up.
Furthermore, this effect seems also to be related to the absorbed dose. In our work, the differences between 40/ 220 kVp and 4 MVp exposures were observed only for absorbed doses higher than or equal to 2 Gy. Beyreuther et al. reported the same kind of effect. They did not observe a significant difference 24 h after exposure for doses up to 2 Gy, but they reported an elevated yield for higher doses, (i.e. 4 Gy and more) between 10/25 kVp and 200 kV. Thus, to observe a differential effect between the radiation qualities, doses above a specific threshold had to be used. This might suggest a joint role of the absorbed doses and secondary electron energy spectra in the ability to observe biological difference in terms of DNA damage signaled by cH2A.X. This could be consistent with a specific nanodosimetric explanation of these differences. This still needs to be identified, and this hypothesis is currently being explored in our laboratory.
However, there are also several methodological differences between the work of Beyreuther et al. (2009) and ours that could partly explain the differences in reported results. One of them is that we chose to work on synchronized cells in the G0/G1 phase of the cell cycle at the time of irradiation. We made this choice to generate damage in cells that are mostly in the same state of the cell cycle, with the same amount and spatial distribution of DNA. The other advantage of this choice concerns the study of late DNA damage signaling. There are different repair pathways with their own repair kinetics and risk of failure. Some depend on the state of the cell cycle in which the damage occurs. Thus the rate of persistent damage can be strongly correlated with the distribution of cells in the cell cycle at the time of irradiation. In the work by Beyreuther et al. (2009) we can assume that their cells, at the time of irradiation, were not synchronized. They do not specify their distribution among the different cell cycle phases, nor the phase of the cell cycle of the cells in which they scored DNA damage signaling. It would be particularly interesting to investigate if these experimental differences contribute to the results.
Independently of the measurements of DNA damage signaling, two groups have reported differences in the number of chromosome aberrations in lymphocytes for different Xray energy spectra. Mestres et al. showed that exposure to 30 kVp X-rays resulted in a slight increase in the acoefficient of dose-effect curves compared to 80 and 120 kVp X rays (Mestres et al. 2011). They also reported that 30 kVp X rays were more efficient in inducing complex aberrations than 120 kVp. Schmid et al. observed an increase in the frequency of dicentrics per cell after irradiation at 10 kV compared to 60 kV (Schmid et al. 2002). Once again, these increases in the rate of mostly unstable chromosome aberrations were reported for X-ray irradiation below 40 kVp. It would have been interesting to know the energy spectrum of secondary electrons corresponding to their experimental conditions. However, with our experimental set-up, no difference in the rate of missegregation was measured between 40 kVp and 4 MVp. Thus, the extra 15% of cH2A.X foci observed 30 min after 2 Gy of 40 kVp X-rays may not be sufficient to generate a detectable increase of the frequency of unstable chromosome aberrations leading to missegregation events. However, one should keep in mind that the geometry and size of lymphocytes are significantly different from those of the HUVECs used in the present study. The nuclei of HUVECs have a volume two times larger than lymphocyte nuclei, which implies a mean density of DNA twice that in the latter. So the impact on DNA damage of the spatial distribution of energy deposits for a given photon energy spectra may not be the same in lymphocytes and in endothelial cells. In addition to the difference in size, the shape of adherent cells like HUVEC compared to lymphocytes may also be involved in the probability of formation of chromosome aberrations. The involvement of these geometric parameters in differential effects of X-rays as a function of their energy is currently under investigation.
In conclusion, the results presented here indicate that the energy spectrum of secondary electrons seems to be more closely related to the level of DNA damage measured by cH2A.X than the initial spectrum of photon energy or voltage settings. They confirm the potential joint role of the absorbed doses and secondary electron energy spectra in the ability to observed biological difference in terms of DNA damage signaled by cH2A.X. This highlights the importance of identifying and characterizing specific nanodosimetric explanations of these differences. However, from a radiation protection point of view, it is important to note that differences in the level of DNA damage observed at doses of 2 Gy and over could not be associated with differential effects on downstream biological endpoints in the range of tested energies under our experimental conditions.