Nanodosimetric Calculations of Radiation-Induced DNA Damage in a New Nucleus Geometrical Model Based on the Isochore Theory.

Double-strand breaks (DSBs) in nuclear DNA represents radiation-induced damage that has been identified as particularly deleterious. Calculating this damage using Monte Carlo track structure modeling could be a suitable indicator to better assess and anticipate the side-effects of radiation therapy. However, as already demonstrated in previous work, the geometrical description of the nucleus and the DNA content used in the simulation significantly influence damage calculations. Therefore, in order to obtain accurate results, this geometry must be as realistic as possible. In this study, a new geometrical model of an endothelial cell nucleus and DNA distribution according to the isochore theory are presented and used in a Monte Carlo simulation chain based on the Geant4-DNA toolkit. In this theory, heterochromatin and euchromatin compaction are distributed along the genome according to five different families (L1, L2, H1, H2, and H3). Each of these families is associated with a different hetero/euchromatin rate related to its compaction level. In order to compare the results with those obtained using a previous nuclear geometry, simulations were performed for protons with linear energy transfers (LETs) of 4.29 keV/µm, 19.51 keV/µm, and 43.25 keV/µm. The organization of the chromatin fibers at different compaction levels linked to isochore families increased the DSB yield by 6-10%, and it allowed the most affected part of the genome to be identified. These new results indicate that the genome core is more radiosensitive than the genome desert, with a 3-8% increase in damage depending on the LET. This work highlights the importance of using realistic distributions of chromatin compaction levels to calculate radio-induced damage using Monte Carlo simulation methods.

Assessment of Radio-Induced Damage in Endothelial Cells Irradiated with 40 kVp, 220 kVp, and 4 MV X-rays by Means of Micro and Nanodosimetric Calculations.

The objective of this work was to study the differences in terms of early biological effects that might exist between different X-rays energies by using a mechanistic approach. To this end, radiobiological experiments exposing cell monolayers to three X-ray energies were performed in order to assess the yields of early DNA damage, in particular of double-strand breaks (DSBs). The simulation of these irradiations was set in order to understand the differences in the obtained experimental results. Hence, simulated results in terms of microdosimetric spectra and early DSB induction were analyzed and compared to the experimental data. Human umbilical vein endothelial cells (HUVECs) were irradiated with 40, 220 kVp, and 4 MV X-rays. The Geant4 Monte Carlo simulation toolkit and its extension Geant4-DNA were used for the simulations. Microdosimetric calculations aiming to determine possible differences in the variability of the energy absorbed by the irradiated cell population for those photon spectra were performed on 10,000 endothelial cell nuclei representing a cell monolayer. Nanodosimetric simulations were also carried out using a computation chain that allowed the simulation of physical, physico-chemical, and chemical stages on a single realistic endothelial cell nucleus model including both heterochromatin and euchromatin. DNA damage was scored in terms of yields of prompt DSBs per Gray (Gy) and per giga (109) base pair (Gbp) and DSB complexity was derived in order to be compared to experimental data expressed as numbers of histone variant H2AX (γ-H2AX) foci per cell. The calculated microdosimetric spread in the irradiated cell population was similar when comparing between 40 and 220 kVp X-rays and higher when comparing with 4 MV X-rays. Simulated yields of induced DSB/Gy/Gbp were found to be equivalent to those for 40 and 220 kVp but larger than those for 4 MV, resulting in a relative biological effectiveness (RBE) of 1.3. Additionally, DSB complexity was similar between the considered photon spectra. Simulated results were in good agreement with experimental data obtained by IRSN (Institut de radioprotection et de sûreté nucléaire) radiobiologists. Despite differences in photon energy, few differences were observed when comparing between 40 and 220 kVp X-rays in microdosimetric and nanodosimetric calculations. Nevertheless, variations were observed when comparing between 40/220 kVp and 4 MV X-rays. Thanks to the simulation results, these variations were able to be explained by the differences in the production of secondary electrons with energies below 10 keV.

Multiparametric radiobiological assays show that variation of X-ray energy strongly impacts relative biological effectiveness: comparison between 220 kV and 4 MV.

Based on classic clonogenic assay, it is accepted by the scientific community that, whatever the energy, the relative biological effectiveness of X-rays is equal to 1. However, although X-ray beams are widely used in diagnosis, interventional medicine and radiotherapy, comparisons of their energies are scarce. We therefore assessed in vitro the effects of low- and high-energy X-rays using Human umbilical vein endothelial cells (HUVECs) by performing clonogenic assay, measuring viability/mortality, counting γ-H2AX foci, studying cell proliferation and cellular senescence by flow cytometry and by performing gene analysis on custom arrays. Taken together, excepted for γ-H2AX foci counts, these experiments systematically show more adverse effects of high energy X-rays, while the relative biological effectiveness of photons is around 1, whatever the quality of the X-ray beam. These results strongly suggest that multiparametric analysis should be considered in support of clonogenic assay.

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

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.

Breast cancer stem cell-like cells generated during TGFß-induced EMT are radioresistant.

Failure of conventional antitumor therapy is commonly associated with cancer stem cells (CSCs), which are often defined as inherently resistant to radiation and chemotherapeutic agents. However, controversy about the mechanisms involved in the radiation response remains and the inherent intrinsic radioresistance of CSCs has also been questioned. These discrepancies observed in the literature are strongly associated with the cell models used. In order to clarify these contradictory observations, we studied the radiosensitivity of breast CSCs using purified CD24-/low/CD44+ CSCs and their corresponding CD24+/CD44low non-stem cells. These cells were generated after induction of the epithelial-mesenchymal transition (EMT) by transforming growth factor ß (TGFß) in immortalized human mammary epithelial cells (HMLE). Consequently, these 2 cellular subpopulations have an identical genetic background, their differences being related exclusively to TGFß-induced cell reprogramming. We showed that mesenchymal CD24-/low/CD44+ CSCs are more resistant to radiation compared with CD24+/CD44low parental cells. Cell cycle distribution and free radical scavengers, but not DNA repair efficiency, appeared to be intrinsic determinants of cellular radiosensitivity. Finally, for the first time, we showed that reduced radiation-induced activation of the death receptor pathways (FasL, TRAIL and TNF-α) at the transcriptional level was a key causal event in the radioresistance of CD24-/low/CD44+ cells acquired during EMT.

Twenty years of FISH-based translocation analysis for retrospective ionizing radiation biodosimetry.

PURPOSE: The fluorescent in situ hybridization (FISH) technique, which easily detects reciprocal translocations, is currently used to estimate doses in retrospective biological dosimetry, after suspected accidental overexposure to ionizing radiation (IR). This study of 42 cases aimed to verify the appropriateness of this assay for radiation dose reconstruction, compared to the dicentric assay, and to evaluate other limitations. MATERIAL AND METHODS: We labeled chromosomes 2, 4, and 12 by 3-color FISH painting to detect translocations on lymphocytes of patients with suspected past IR overexposure. RESULT: Translocation dose estimation showed doses significantly different from 0 Gy in 25 of the 42 cases. The lowest positive dose measured was 0.3 Gy. Several months after IR exposure, the doses measured by translocation and dicentric assays are quite similar. For a year, dose estimation by translocation assay becomes more relevant as dicentric frequency starts to decrease, coming close to 0 for more than a year after the exposure. The persistence of translocations enabled us to corroborate an overexposure 44 years earlier. Interpretation of the observed translocation yield requires the knowledge of the patient's other radiation exposures. A dose assessment by this biomarker is relevant only if the radiation exposure is confirmed. CONCLUSIONS: This technique is appropriate for corroborating a former IR exposure of individuals. When the radiation dose is greater than 1 Gy, the translocations in complex exchanges must be considered. Another relevant point is the use of an appropriate background yield of translocations. The dose assessment, however, also depends on exposure to various genotoxic agents besides IR. If no evidence about the existence of radiation exposure is available, dose assessment is not useful. For this reason, report only the translocation frequency and its comparison with the background yield by age class is preferable.

Transmission of persistent ionizing radiation-induced foci through cell division in human primary cells.

Unrepaired DNA double-strand breaks (DSBs) induced by ionizing radiation are associated with lethal effects and genomic instability. After the initial breaks and chromatin destabilization, a set of post-translational modifications of histones occurs, including phosphorylation of serine 139 of histone H2AX (γH2AX), which leads to the formation of ionizing radiation-induced foci (IRIF). DSB repair results in the disappearance of most IRIF within hours after exposure, although some remain 24h after irradiation. Their relation to unrepaired DSBs is generally accepted but still controversial. This study evaluates the frequency and kinetics of persistent IRIF and analyzes their impact on cell proliferation. We observed persistent IRIF up to 7 days postirradiation, and more than 70% of cells exposed to 5Gy had at least one of these persistent IRIF 24h after exposure. Moreover we demonstrated that persistent IRIF did not block cell proliferation definitively. The frequency of IRIF was lower in daughter cells, due to asymmetric distribution of IRIF between some of them. We report a positive association between the presence of IRIF and the likelihood of DNA missegregation. Hence, the structure formed after the passage of a persistent IRI focus across the S and G2 phases may impede the correct segregation of the affected chromosome's sister chromatids. The ensuing abnormal resolution of anaphase might therefore cause the nature of IRIF in daughter-cell nuclei to differ before and after the first cell division. The resulting atypical chromosomal assembly may be lethal or result in a gene dosage imbalance and possibly enhanced genomic instability, in particular in the daughter cells.

Characterization of gene expression profiles at low and very low doses of ionizing radiation.

The aim of the present study is to analyse by microarray techniques how gene expression is modulated after exposure to low and very low doses of ionizing radiation, to evaluate if the pattern of gene expression shows dose dependence, and to search for putative regulatory mechanisms behind the observed gene-expression modifications. For this, whole blood samples from five healthy donors were exposed in six doses between 5 and 500mGy. Total RNA extraction from CD4(+) lymphocytes was done at four different post-irradiation times. After mRNA amplification, aRNAs were hybridized on DNA microarrays. The results indicated that up-regulation was twice than down-regulation. Surprisingly, the number of modulated genes does not seem to change drastically with dose, even at the lowest dose of 5mGy. Clustering analysis revealed seven gene expression clusters with different dose dependence profiles. The functional analysis showed that the genes which increased their expression with the dose were related to p53 pathway and DNA damage response. This could be observed from 25mGy, but became very clear at doses equal or greater than 100mGy. On the other hand, genes with a constant modulation of their expression in all the tested doses were related to cellular respiration, ATP metabolic process and chromatin organization. These latter molecular mechanisms seem to be triggered at very low doses (5-25mGy). In silico promoter analysis seems to confirm the implication of transcription factors related to the pathways mentioned above.

Biological dosimetry by automated dicentric scoring in a simulated emergency.

Dicentric chromosome analysis remains the most widely used method in biodosimetry. It has a lower detection limit of about 0.1 Gy, and allows one to distinguish between whole- and partial-body exposures. A drawback of the dicentric analysis is that it is a time consuming method and maybe difficult to implement in a mass casualty event. To try to increase the analysis capacity, automatic dicentric scoring (ADS) using image analysis software is being incorporated in several laboratories. Here we present the results obtained in an emergency exercise simulating 50 victims. The ability to distinguish different radiations scenarios is evaluated. To simulate whole-body exposures peripheral blood samples were irradiated at doses between 0-4.7 Gy, and to simulate partial-body exposures irradiated and nonirradiated blood were mixed in different proportions. With the data obtained from the first slide analyzed (with about 300-400 cells), 32 of 34 simulated whole-body exposures were correctly classified according to radiation exposure levels. For simulated partial-body irradiations, it was possible to detect them as partial exposures at the end of the first slide analyzed but only at the highest doses. In all cases the classification was updated every time the analysis of one additional slide was finished. The comparison between our present results and those reported in the literature for manual scoring shows that for triage purposes the ADS based on 300-400 cells is similar in efficiency to classifying the cases based on manual scoring of 50 cells. However, if one accounts for the associated uncertainties and the time needed for ADS, we suggest that ADS triage scoring should be based on about 1,000 cells. For final dose estimations the number of cells to score will depend on the initial estimated dose, and on the information contributed from physical dose-reconstruction or clinical symptoms. At doses higher than 1 Gy, we propose analysis of 1,500 and for lower doses or suspected partial-body exposures, the number of cells to score should be 3,000.

PAI-1-dependent endothelial cell death determines severity of radiation-induced intestinal injury.

Normal tissue toxicity still remains a dose-limiting factor in clinical radiation therapy. Recently, plasminogen activator inhibitor type 1 (SERPINE1/PAI-1) was reported as an essential mediator of late radiation-induced intestinal injury. However, it is not clear whether PAI-1 plays a role in acute radiation-induced intestinal damage and we hypothesized that PAI-1 may play a role in the endothelium radiosensitivity. In vivo, in a model of radiation enteropathy in PAI-1 -/- mice, apoptosis of radiosensitive compartments, epithelial and microvascular endothelium was quantified. In vitro, the role of PAI-1 in the radiation-induced endothelial cells (ECs) death was investigated. The level of apoptotic ECs is lower in PAI-1 -/- compared with Wt mice after irradiation. This is associated with a conserved microvascular density and consequently with a better mucosal integrity in PAI-1 -/- mice. In vitro, irradiation rapidly stimulates PAI-1 expression in ECs and radiation sensitivity is increased in ECs that stably overexpress PAI-1, whereas PAI-1 knockdown increases EC survival after irradiation. Moreover, ECs prepared from PAI-1 -/- mice are more resistant to radiation-induced cell death than Wt ECs and this is associated with activation of the Akt pathway. This study demonstrates that PAI-1 plays a key role in radiation-induced EC death in the intestine and suggests that this contributes strongly to the progression of radiation-induced intestinal injury.

Detection of partial-body exposure to ionizing radiation by the automatic detection of dicentrics.

In accidental exposure to ionizing radiation, it is essential to estimate the dose received by the victims. Currently dicentric scoring is the best biological indicator of exposure. The standard biological dosimetry procedure (500 metaphases scored manually) is suitable for a few dose estimations, but the time needed for analysis can be problematic in the case of a large-scale accident. Recently, a new methodology using automatic detection of dicentrics has greatly decreased the time needed for dose estimation and preserves the accuracy of the estimation. However, the capability to detect nonhomogeneous partial-body exposures is an important advantage of dicentric scoring-based biodosimetry, and this remains to be tested with automatic scoring. Thus we analyzed the results obtained with in vitro blood dilutions and in real cases of accidental exposure (partial- or whole-body exposure) using manual scoring and automatic detection of dicentrics. We confirmed that automatic detection allows threefold quicker dicentric scoring than the manual procedure with similar dose estimations and uncertainty intervals. The results concerning partial-body exposures were particularly promising, and homogeneously exposed samples were correctly distinguished from heterogeneously exposed samples containing 5% to 75% of blood irradiated with 2 Gy. In addition, the results obtained for real accident cases were similar whatever the methodology used. This study demonstrates that automatic detection of dicentrics is a credible alternative for recent and acute cases of whole- and partial-body accidental exposures to ionizing radiation.

Cell response to ionising radiation analysed by gene expression patterns.

Following ionising radiation exposure of living cells several mechanisms are activated through gene modulations. The measurement of these modifications can be done with QT-PCR and, since about 10 years, microarray technique. The latter approach has the advantage to allow a global monitoring of the complex cellular responses to radiation-induced stress and has been proposed to be used for dose assessment. Even if some publications have identified sets of genes specific to given doses, and that some of the genes have an induction proportional to the dose, a precise estimation of the received dose seems difficult with gene expression, at least in the near future. Nevertherless, in vivo studies have shown that gene profiles of individuals chronically exposed to a cumulative dose of more than 10 mSv are significantly modified. This highlights the great potential of microarray approaches in the detection of low dose exposure.

Strategy for population triage based on dicentric analysis.

After large-scale accidental overexposure to ionizing radiation, a rapid triage of the exposed population can be performed by scoring dicentrics and ring chromosomes among 50 metaphases. This is rapid but is not accurate because the sensitivity is around 0.5 Gy. After the triage step, dose can be estimated by scoring 500 metaphases. This is lengthy but very accurate because the sensitivity is between 0.1 and 0.2 Gy. To improve the methodology, we propose the use of software for automatic dicentric scoring that was tested on victims of an accident in Dakar. Manual scoring of 50 metaphases was carried out, then manual scoring of 500 metaphases, and automatic scoring. Comparison between the dose classifications obtained with manual scoring on 50 metaphases and 500 metaphases showed 50% misclassification with the manual scoring on 50 metaphases. Comparison between the dose classifications obtained with the automatic scoring and manual scoring on 500 metaphases showed only 4.35% misclassification with the automatic scoring. The automatic scoring method is more accurate than the manual scoring on 50 metaphases and can therefore be used for triage, and in place of the manual scoring on 500 metaphases method for individual dose estimation, because it is as accurate and much faster.

Broad modulation of gene expression in CD4+ lymphocyte subpopulations in response to low doses of ionizing radiation.

To compare the responses of the different lymphocyte subtypes after an exposure of whole blood to low doses of ionizing radiation, we examined variations in gene expression in different lymphocyte subpopulations using microarray technology. Blood samples from five healthy donors were independently exposed to 0 (sham irradiation), 0.05 and 0.5 Gy of ionizing radiation. Three and 24 h after exposure, CD56+, CD4+ and CD8+ cells were negatively isolated. RNA from each set of experimental conditions was competitively hybridized on 25k oligonucleotide microarrays. Modifications of gene expression were measured after both intervals and in all cell types. Twenty-four hours after exposure to 0.5 Gy, we observed an induction of the expression of BAX, PCNA, GADD45, DDB2 and CDKN1A. However, the numbers of modulated genes greatly differed between cell types. In particular, 3 h after exposure to doses as low as 0.05 Gy, the number of down-modulated genes was 10 times greater for CD4+ cells than for all other cell types. Moreover, most of these repressed genes were taking part in the cell processes of protein biosynthesis and oxidative phosphorylation. The results suggest that several biological pathways in CD4+ cells could be sensitive to low doses of radiation. Therefore, specifically studying CD4+ cells could help to understand the mechanisms involved in low-dose response and allow their detection.

Antiapoptotic cytokines in combination with pegfilgrastim soon after irradiation mitigates myelosuppression in nonhuman primates exposed to high irradiation dose.

OBJECTIVE: Preservation of hematopoietic stem and progenitor cells from early radiation-induced apoptosis is the rationale for emergency antiapoptotic cytokine therapy (EACK) after radiation accidents. This strategy is based on the combination of stem cell factor + Flt3-ligand + thrombopoietin + interleukin 3 (SFT3). The long-term safety and efficacy of EACK in managing severe radiation exposure were evaluated. MATERIAL AND METHODS: Early administration of SFT3 + pegfilgrastim was assessed in 7-Gy gamma total body-irradiated (TBI) monkeys. Efficiency of delayed administration was also addressed after 5-Gy TBI. RESULTS: Here we showed that a single, intravenous injection of SFT3 2 hours after 7-Gy TBI reduced the period of thrombocytopenia (platelet count <20 x 10(9)/L: 0.8 +/- 1.5 day vs 23.8 +/- 15.9 days in controls; p < 0.05) and blood transfusion needs. Moreover, addition of pegfilgrastim to SFT3 treatment shortened the period of neutropenia compared with SFT3 and control groups (neutrophil count <0.5 x 10(9)/L: 7 +/- 1.4 days vs 13 +/- 3.2 days and 15.2 +/- 1.5 days; p < 0.05). In both SFT3 groups, bone marrow activity recovered earlier and, in contrast with controls, platelet count returned to baseline values from 250 days after irradiation. Furthermore, delayed (48 hours) single SFT3 administration in 5-Gy irradiated monkeys significantly reduced thrombocytopenia compared to controls. Finally, SFT3 did not increase frequency of total chromosome translocations observed in the blood lymphocytes of controls 1 year after 5 Gy TBI. CONCLUSION: These results suggest the safety and efficacy of EACK in managing severe radiation exposure.