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.

From Energy Deposition of Ionizing Radiation to Cell Damage Signaling: Benchmarking Simulations by Measured Yields of Initial DNA Damage after Ion Microbeam Irradiation.

Advances in accelerator technology, which have enabled conforming radiotherapy with charged hadronic species, have brought benefits as well as potential new risks to patients. To better understand the effects of ionizing radiation on tumor and surrounding tissue, it is important to investigate and quantify the relationship between energy deposition at the nanometric scale and the initial biological events. Monte Carlo track structure simulation codes provide a powerful tool for investigating this relationship; however, their success and reliability are dependent on their improvement and development accordingly to the dedicated biological data to which they are challenged. For this aim, a microbeam facility that allows for fluence control, down to one ion per cell nucleus, was used to evaluate relative frequencies of DNA damage after interaction between the incoming ion and DNA according to radiation quality. Primary human cells were exposed to alpha particles of three different energies with respective linear energy transfers (LETs) of approximately 36, 85 or 170 keV·µm-1 at the cells' center position, or to protons (19 keV·µm-1). Statistical evaluation of nuclear foci formation (53BP1/γ-H2AX), observed using immunofluorescence and related to a particle traversal, was undertaken in a large population of cell nuclei. The biological results were adjusted to consider the factors that drive the experimental uncertainties, then challenged with results using Geant4-DNA code modeling of the ionizing particle interactions on a virtual phantom of the cell nucleus with the same mean geometry and DNA density as the cells used in our experiments. Both results showed an increase of relative frequencies of foci (or simulated DNA damage) in cell nuclei as a function of increasing LET of the traversing particles, reaching a quasi-plateau when the LET exceeded 80-90 keV·µm-1. For the LET of an alpha particle ranging from 80-90 to 170 keV·µm-1, 10-30% of the particle hits did not lead to DNA damage inducing 53BP1 or γ-H2AX foci formation.

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.

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.

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.

Cell to Cell Variability of Radiation-Induced Foci: Relation between Observed Damage and Energy Deposition.

Most studies that aim to understand the interactions between different types of photon radiation and cellular DNA assume homogeneous cell irradiation, with all cells receiving the same amount of energy. The level of DNA damage is therefore generally determined by averaging it over the entire population of exposed cells. However, evaluating the molecular consequences of a stochastic phenomenon such as energy deposition of ionizing radiation by measuring only an average effect may not be sufficient for understanding some aspects of the cellular response to this radiation. The variance among the cells associated with this average effect may also be important for the behaviour of irradiated tissue. In this study, we accurately estimated the distribution of the number of radiation-induced γH2AX foci (RIF) per cell nucleus in a large population of endothelial cells exposed to 3 macroscopic doses of gamma rays from 60Co. The number of RIF varied significantly and reproducibly from cell to cell, with its relative standard deviation ranging from 36% to 18% depending on the macroscopic dose delivered. Interestingly, this relative cell-to-cell variability increased as the dose decreased, contrary to the mean RIF count per cell. This result shows that the dose effect, in terms of the number of DNA lesions indicated by RIF is not as simple as a purely proportional relation in which relative SD is constant with dose. To analyse the origins of this observed variability, we calculated the spread of the specific energy distribution for the different target volumes and subvolumes in which RIF can be generated. Variances, standard deviations and relative standard deviations all changed similarly from dose to dose for biological and calculated microdosimetric values. This similarity is an important argument that supports the hypothesis of the conservation of the association between the number of RIF per nucleus and the specific energy per DNA molecule. This comparison allowed us to calculate a volume of 1.6 µm3 for which the spread of the specific energy distribution could explain the entire variability of RIF counts per cell in an exposed cell population. The definition of this volume may allow to use a microdosimetric quantity to predict heterogeneity in DNA damage. Moreover, this value is consistent with the order of magnitude of the volume occupied by the hydrated sugar-phosphate backbone of the DNA molecule, which is the part of the DNA molecule responsible for strand breaks.

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.

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.

Quantification of gamma-H2AX foci in human lymphocytes: a method for biological dosimetry after ionizing radiation exposure.

Recent studies have suggested that visualization of gamma-H2AX nuclear foci can be used to estimate exposure to very low doses of ionizing radiation. Although this approach is widely used for various purposes, its suitability for individual human biodosimetry has not yet been assessed. We therefore conducted such an assessment with the help of available software for observing and automatically scoring gamma-H2AX foci. The presence of gamma-H2AX foci was evaluated in human peripheral blood lymphocytes exposed ex vivo to gamma rays in a dose range of 0.02 to 2 Gy. We analyzed the response of gamma-H2AX to ionizing radiation in relation to dose, time after exposure, and individual variability. We constructed dose-effect calibration curves at 0.5, 8 and 16 h after exposure and evaluated the threshold of detection of the technique. The results show the promise of automatic gamma-H2AX scoring for a reliable assessment of radiation doses in a dose range of 0.6 Gy to 2 Gy up to 16 h after exposure. This gamma-H2AX-based assay may be useful for biodosimetry, especially for triage to distinguish promptly among individuals the ones who have received negligible doses from those with significantly exposures who are in need of immediate medical attention. However, additional in vivo experiments are needed for validation.