RENEB Inter-Laboratory comparison 2017: limits and pitfalls of ILCs

Abstract Purpose In case of a mass-casualty radiological event, there would be a need for networking to overcome surge limitations and to quickly obtain homogeneous results (reported aberration frequencies or estimated doses) among biodosimetry laboratories. These results must be consistent within such network. Inter-laboratory comparisons (ILCs) are widely accepted to achieve this homogeneity. At the European level, a great effort has been made to harmonize biological dosimetry laboratories, notably during the MULTIBIODOSE and RENEB projects. In order to continue the harmonization efforts, the RENEB consortium launched this intercomparison which is larger than the RENEB network, as it involves 38 laboratories from 21 countries. In this ILC all steps of the process were monitored, from blood shipment to dose estimation. This exercise also aimed to evaluate the statistical tools used to compare laboratory performance. Materials and methods Blood samples were irradiated at three different doses, 1.8, 0.4 and 0 Gy (samples A, C and B) with 4-MV X-rays at 0.5 Gy min−1, and sent to the participant laboratories. Each laboratory was requested to blindly analyze 500 cells per sample and to report the observed frequency of dicentric chromosomes per metaphase and the corresponding estimated dose. Results This ILC demonstrates that blood samples can be successfully distributed among laboratories worldwide to perform biological dosimetry in case of a mass casualty event. Having achieved a substantial harmonization in multiple areas among the RENEB laboratories issues were identified with the available statistical tools, which are not capable to advantageously exploit the richness of results of a large ILCs. Even though Z- and U-tests are accepted methods for biodosimetry ILCs, setting the number of analyzed metaphases to 500 and establishing a tests’ common threshold for all studied doses is inappropriate for evaluating laboratory performance. Another problem highlighted by this ILC is the issue of the dose-effect curve diversity. It clearly appears that, despite the initial advantage of including the scoring specificities of each laboratory, the lack of defined criteria for assessing the robustness of each laboratory’s curve is a disadvantage for the ‘one curve per laboratory’ model. Conclusions Based on our study, it seems relevant to develop tools better adapted to the collection and processing of results produced by the participant laboratories. We are confident that, after an initial harmonization phase reached by the RENEB laboratories, a new step toward a better optimization of the laboratory networks in biological dosimetry and associated ILC is on the way.AFRRI’s-RBB44313 y AFR-B4-431

Effects of repetitive Iodine thyroid blocking on the foetal brain and thyroid in rats: a systems biology approach

International audienceA single administration of an iodine thyroid blocking agent is usually sufcient to protect thyroid from radioactive iodine and prevent thyroid cancer. Repeated administration of stable iodine (rKI) may be necessary during prolonged or repeated exposure to radioactive iodine. We previously showed that rKI for eight days ofers protection without toxic efects in adult rats. However, the efect of rKI administration in the developing foetus is unknown, especially on brain development, although a correlation between impaired maternal thyroid status and a decrease in intelligence quotient of the progeny has been observed. This study revealed distinct gene expression profles between the progeny of rats receiving either rKI or saline during pregnancy. To understand the implication of these diferentially expressed (DE) genes, a systems biology approach was used to construct networks for each organ using three diferent techniques: Bayesian statistics, sPLS-DA and manual construction of a Process Descriptive (PD) network. The PD network showed DE genes from both organs participating in the same cellular processes that afect mitophagy and neuronal outgrowth. This work may help to evaluate the doctrine for using rKI in case of repetitive or prolonged exposure to radioactive particles upon nuclear accidents

Development of whole brain versus targeted dentate gyrus irradiation model to explain low to moderate doses of exposure effects in mice

International audienceEvaluation of the consequences of low to moderate doses of ionizing radiation (IR) remains a societal challenge, especially for children exposed to CT scans. Appropriate experimental models are needed to improve scientific understanding of how exposure of the postnatal brain to IR affects behavioral functions and their related pathophysiological mechanisms, considering brain complex functional organization. In the brain, the dorsal and ventral hippocampal dentate gyrus can be involved in distinct major behavioral functions. To study the long term behavioral effects of brain exposure at low to moderate doses of IR (doses range 0.25–1 Gy), we developed three new experimental models in 10-day-old mice: a model of brain irradiation and two targeted irradiation models of the dorsal and ventral dentate gyrus. We used the technological properties of the SARRP coupled with MR imaging. Our irradiation strategy has been twofold endorsed. The millimetric ballistic specificity of our models was first validated by measuring gamma-H2AX increase after irradiation. We then demonstrated higher anxiety/depressive-like behavior, preferentially mediate by the ventral part of the dentate gyrus, in mice after brain and ventral dentate gyrus IR exposure. This work provides new tools to enhance scientific understanding of how to protect children exposed to IR

Effect of chronic low doses of ionizing radiations on central nervous system development of zebrafish

International audienceAfter the nuclear accidents at Chernobyl and Fukushima and due to a growing use of radionuclides, radiation protection of the environment is a major concern. Studies show an increased sensitivity to ionizing radiation (IR) during development. However, there are few data on the molecular mechanisms leading to the effects of IR on embryogenesis and moreover for chronic exposures. In this context, the objective of this thesis is to better understand the effects of exposure to IR on the development of zebrafish (Danio rerio), a model organism in ecotoxicology and in biomedicine. Fertilized eggs were exposed to gamma rays emitted by a 137Cs source at low to moderate dose rates (from 0.005 mGy/h to 50 mGy/h), then analysed from the gastrula stage (6 hours) to the larval stage (5 days) by an integrative approach from molecular effects to behavioural effects. Part of this work has focused on studying the effects of IR at an early stage of zebrafish development after exposure to IR. For this, a multi-omics approach with the study of the transcriptome and the methylome has been implemented. A transcriptomic study at the shield stage with dose rates ranging from 0.005 mGy/h to 50 mGy/h was carried out and demonstrated a moderate but significant effect on the regulation of oxidative stress and mitochondrial activity. At 5 and 50 mGy/h, the genes involved in the development of the central nervous system (CNS) and muscles are affected. The methylome study has revealed hypomethylation of the promoter sequences of genes involved in the development of the CNS and muscles, associated with a modification of gene expression. These results on early embryonic stages, highlight potential effects on the development of the CNS and muscles. The other part of this work focused on assessing the effects of IR exposure at later developmental stages using a system biology approach. For this, analyses were carried out from the molecular scale to the phenotypic scale. At the molecular level, a multi-omics analysis of the transcriptome and the proteome has highlighted the modulation of genes involved in the retinoic acid pathway, the development of the CNS and muscles in embryos and larvae exposed to 0.5; 5 and 50 mGy/h. Labelling by in-situ hybridization confirmed the misregulation of the transcription factors her4.4 in the CNS and myog in the muscles of embryos exposed to IR. At the tissue level, disruption of muscle myofibrils and alteration of neuromuscular junctions are detected in embryos exposed to 5 and 50 mGy/h. In order to assess an individual effect at phenotypic scale, the locomotor behaviour of the larvae was assessed under stress conditions. A significant decrease in larval motility was detected between 0.5 mGy/h and 50 mGy/h. This work has shown effects of IR on the development of the CNS and muscles from early development at the molecular level. These effects are confirmed at later stages of development. This study suggests that the molecular disturbances observed during early development are predictive of the effects observed at later developmental stages. In addition, this thesis work allows us to propose a model of "Adverse Outcome Pathway" where the deregulation of the retinoic acid pathway by exposure to IR will lead to effects on the development of the CNS and muscles

Importance of dosimetry protocol for cell irradiation on a low X-rays facility and consequences for the biological response

International audiencePurpose: The main objective of radiobiology is to establish links between doses and radiation-induced biological effects. In this context, well-defined dosimetry protocols are crucial to the determination of experimental protocols. This work proposes a new dosimetry protocol for cell irradiation in a SARRP and shows the importance of the modification of some parameters defined in dosimetry protocol for physical dose and biological outcomes.Materials and methods: Once all parameters of the configuration were defined, dosimetry measurements with ionization chambers and EBT3 films were performed to evaluate the dose rate and the attenuation due to the cell culture medium. To evaluate the influence of changes in cell culture volume and/or additional filtration, 6-well plates containing EBT3 films with water were used to determine the impact on the physical dose at 80 kV. Then, experiments with the same irradiation conditions were performed by replacing EBT3 films by HUVECs. The biological response was assessed using clonogenic assay.Results: Using a 0.15 mm copper filter lead to a variation of +1% using medium thickness of 0.104 cm to -8% using a medium thickness of 0.936 cm on the physical dose compare to the reference condition (0.313 cm). For the 1 mm aluminum filter, a variation of +8% to -40% for the same medium thickness conditions has been observed. Cells irradiated in the same conditions showed significant differences in survival fraction, corroborating the effects of dosimetric changes on physical dose.Conclusion: This work shows the importance of dosimetry in radiobiology studies and the need of an accurate description of the dosimetry protocol used for irradiation

Biological measure of DNA damage after single-ion microbeam irradiation and Monte Carlo simulations

International audienceBackground/Aim : The growing use of hadrons in oncology entails the need to establish more specific dosimetry concepts adapted to their biological efficacy. This requires more information on the likelihood of subcellular effects, mainly DNA damage, according to how energy is deposited within cells by ionizing particle tracks. Monte Carlo track structure simulation provides a powerful tool for investigating such relation. However, the reliability of simulation results can only be assessed by comparison with dedicated biological data.Methods : Primary human cells were exposed, with single-ion microbeam facility, to α particles and protons of different energies with LETs ranging from 19 to 170 keV·μm-1 at the cells’ center position. Statistical evaluation of nuclear foci formation (53BP1/γ-H2AX) related to a particle traversal was undertaken in a large population of cell nuclei. The biological results were adjusted for factors leading to experimental bias and compared with Geant4-DNA simulations results. Such simulations model the ionizing particle interactions on a virtual phantom of the cell nucleus with the same geometry and DNA density as the cells used in the experiments.Results : Observed relocation/modification of DNA damage signaling proteins and simulations both show an initial increase of the relative frequency of induction of DNA damage with increasing LET of the projectile. For α particles of LET ranging from 80-90 to 170 keV·μm-1, a constant DNA damage-induction frequency was found where 10-30% of the particle traversals did not lead to foci formation. Conclusions : These findings will allow for a better comprehension of the relationship between the topology of energy deposition from particles of different LET and early cell damage. They will also help obtain an accurate estimate of the probability of particle interaction to induce foci formation at DNA damage sites. Work carried out within EMRP (European Metrology Research Programme) JRP-SIB06 BioQuaRT (Biologically weighted Quantities in RadioTherapy)

Mesenchymal stem cells limit vascular and epithelial damage and restore the impermeability of the urothelium in chronic radiation cystitis

International audienceAbstract Background Cellular therapy seems to be an innovative therapeutic alternative for which mesenchymal stem cells (MSCs) have been shown to be effective for interstitial and hemorrhagic cystitis. However, the action of MSCs on chronic radiation cystitis (CRC) remains to be demonstrated. The aim of this study was to set up a rat model of CRC and to evaluate the efficacy of MSCs and their mode of action. Methods CRC was induced by single-dose localized irradiation of the whole bladder using two beams guided by tomography in female Sprague–Dawley rat. A dose range of 20–80 Gy with follow-up 3–12 months after irradiation was used to characterize the dose effect and the kinetics of radiation cystitis in rats. For the treatment, the dose of 40 Gy was retained, and in order to potentiate the effect of the MSCs, MSCs were isolated from adipose tissue. After expansion, they were injected intravenously during the pre-chronic phase. Three injections of 5 million MSCs were administered every fortnight. Follow-up was performed for 12 months after irradiation. Results We observed that the intensity and frequency of hematuria are proportional to the irradiation dose, with a threshold at 40 Gy and the appearance of bleeding from 100 days post-irradiation. The MSCs reduced vascular damage as well as damage to the bladder epithelium. Conclusions These results are in favor of MSCs acting to limit progression of the chronic phase of radiation cystitis. MSC treatment may afford real hope for all patients suffering from chronic radiation cystitis resistant to conventional treatments

Variation of 4 MV X-ray dose rate in fractionated irradiation strongly impacts biological endothelial cell response in vitro

International audienceComparisons of X-ray beam dose rates are scarce although these beams are widely used in medical diagnosis or radiotherapy. We have recently demonstrated in vitro and in vivo, that for a single dose of irradiation, the relative biological effectiveness (RBE) deviates from 1 when changing the dose rate of high energy X-ray beams. To further investigate the impact of the dose rate on RBE, in this study we performed in vitro fractionated irradiations by using the same two dose rates (0.63 and 2.5 Gy.min-1) of high-energy X-rays (both at 4 MV) on normal endothelial cells (HUVECs). We studied the viability/mortality, measured cellular senescence by flow cytometry and performed gene analysis on custom arrays. Taken together, these experiments show that the RBE of photons deviates from 1 when varying the dose rate of high-energy X-rays in fractionated irradiations. These results strengthen the interest of multiparametric analysis approaches in providing an accurate evaluation of the outcomes of irradiated cells in support of clonogenic assays, especially when such assays are not feasible

Biological measure of DNA damage after single-ion microbeam irradiation and Monte Carlo simulations

International audienceBackground/Aim : The growing use of hadrons in oncology entails the need to establish more specific dosimetry concepts adapted to their biological efficacy. This requires more information on the likelihood of subcellular effects, mainly DNA damage, according to how energy is deposited within cells by ionizing particle tracks. Monte Carlo track structure simulation provides a powerful tool for investigating such relation. However, the reliability of simulation results can only be assessed by comparison with dedicated biological data.Methods : Primary human cells were exposed, with single-ion microbeam facility, to α particles and protons of different energies with LETs ranging from 19 to 170 keV·μm-1 at the cells’ center position. Statistical evaluation of nuclear foci formation (53BP1/γ-H2AX) related to a particle traversal was undertaken in a large population of cell nuclei. The biological results were adjusted for factors leading to experimental bias and compared with Geant4-DNA simulations results. Such simulations model the ionizing particle interactions on a virtual phantom of the cell nucleus with the same geometry and DNA density as the cells used in the experiments.Results : Observed relocation/modification of DNA damage signaling proteins and simulations both show an initial increase of the relative frequency of induction of DNA damage with increasing LET of the projectile. For α particles of LET ranging from 80-90 to 170 keV·μm-1, a constant DNA damage-induction frequency was found where 10-30% of the particle traversals did not lead to foci formation. Conclusions : These findings will allow for a better comprehension of the relationship between the topology of energy deposition from particles of different LET and early cell damage. They will also help obtain an accurate estimate of the probability of particle interaction to induce foci formation at DNA damage sites. Work carried out within EMRP (European Metrology Research Programme) JRP-SIB06 BioQuaRT (Biologically weighted Quantities in RadioTherapy)

Variation of 4 MV X-ray dose rate strongly impacts biological response both in vitro and in vivo

International audienceBased on classic clonogenic assay, it is accepted by the scientific community that, whatever the energy or the dose rate, 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 dose rates are scarce. We therefore assessed in vitro the effects of high-energy X-rays at two dose rates (0.63 and 2.5 Gy/min) using normal endothelial cells (HUVECs) by using clonogenic assay, measuring viability/mortality, studying the cell cycle and cellular senescence by flow cytometry and by performing gene analysis on custom arrays. In order to consolidate these data, we performed localized irradiation of exteriorized small intestine at 0.63 and 2.5 Gy/min. Interestingly, in vivo validation has shown a significantly higher loss of weight at the higher dose when irradiating to 19 Gy a small fragment of exteriorized small intestine of C57Bl6J mice. Nevertheless, no significant differences were observed in lesioned scores between the two dose rates, while bordering epithelium staining indicated twofold greater severe damage at 2.5 Gy/min compared to 0.63 Gy/min at one week post-irradiation. Taken together, these experiments systematically show more adverse effects of high energy X-rays at 2.5 Gy/min, 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