Biological and internal dosimetry for radiation medicine: current status and future perspectives.

The International Atomic Energy Agency (IAEA) and Hiroshima International Council for Health Care of the Radiation-Exposed (HICARE) jointly organized two relevant workshops in Hiroshima, Japan, i.e. a Training Meeting 'Biodosimetry in the 21st century' (BIODOSE-21) on 10-14 June 2013 and a Workshop on 'Biological and internal dosimetry: recent advance and clinical applications' which took place between 17 and 21 February 2020. The main objective of the first meeting was to develop the ability of biodosimetry laboratories to use mature and novel techniques in biological dosimetry for the estimation of radiation doses received by individuals and populations. This meeting had a special focus on the Asia-Pacific region and was connected with the then on-going IAEA Coordinated Research Project (CRP) E35008 'Strengthening of "Biological dosimetry" in IAEA Member States: Improvement of current techniques and intensification of collaboration and networking among the different institutes' (2012-17). The meeting was attended by 25 participants, which included 11 lecturers. The 14 trainees for this meeting came from India, Indonesia, Japan, Malaysia, Philippines, Republic of Korea, Singapore, Thailand and Vietnam. During the meeting 13 lectures by HICARE and IAEA invited lecturers were delivered besides eight research reports presented by the IAEA CRP E35008 network centers from the Asia-Pacific region. Two laboratory exercises were also undertaken, one each at Hiroshima University and the Radiation Effects Research Foundation (RERF). The second training workshop aimed to discuss with the participants the use of mature and novel techniques in biological and internal dosimetry for the estimation of radiation effects by accidental, environmental and medical exposures. The workshop was attended by 19 participants from Indonesia, Jordan, Oman, Philippines, Singapore, Syrian Arab Republic, Thailand, UAE, USA and Yemen. The main outcome of both meetings was a review of the state-of-the-art of biodosimetry and internal dosimetry and their future perspectives in medical management. This report highlights the learning outcome of two meetings for the benefit of all stake-holders in the field of biological and internal dosimetry.

Clinical Applications of Biological Dosimetry in Patients Exposed to Low Dose Radiation Due to Radiological, Imaging or Nuclear Medicine Procedures.

Radiation dosimetric biomarkers have found applications beyond radiation protection area and now are actively introduced into clinical practice. Cytogenetic assays appeared to be a valuable tool for individualized quantifying radiation effects in patients, with high capability for assessing genotoxicity of various medical exposure modalities and providing meaningful radiation dose estimates for prognoses of radiation-related cancer risk. This review summarized current data on the use of biological dosimetry methods in patients undergoing various medical irradiations to low doses. The highlighted topics include basic aspects of biological dosimetry and its limitations in the range of low radiation doses, and main patterns of in vivo induction of radiation biomarkers in clinical exposure scenarios, occurring in X-ray diagnostics, computed tomography, interventional radiology, low dose radiotherapy, and nuclear medicine (internally administered 131I and other radiopharmaceuticals). Additionally, several specific issues, examined by biodosimetry techniques, are analysed, such as contrast media effect, radiation response in pediatric patients, impact of magnetic resonance imaging, evaluation of radioprotectors, detection of patients' abnormal intrinsic radiosensitivity and dose estimation in persons involved in medical radiation incidents. A prognosis of possible directions for further improvements in this area includes the automation of cytogenetic analysis, introduction of molecular biodosimeters and development of multiparametric biodosimetry platforms. A potential approach to the advanced biodosimetry of internal exposure and/or low dose external irradiation is suggested; this can be a multiparametric platform based on the combination of the γ-H2AX foci, dicentric, and translocation assays, each applied in the optimum postexposure time range, with the amalgamation of the dose estimates. The study revealed the necessity of further research, which might clarify medical radiation safety concerns for patients via using stringent biodosimetry methodology.

Prediction of the Acute or Late Radiation Toxicity Effects in Radiotherapy Patients Using Ex Vivo Induced Biodosimetric Markers: A Review.

A search for effective methods for the assessment of patients' individual response to radiation is one of the important tasks of clinical radiobiology. This review summarizes available data on the use of ex vivo cytogenetic markers, typically used for biodosimetry, for the prediction of individual clinical radiosensitivity (normal tissue toxicity, NTT) in cells of cancer patients undergoing therapeutic irradiation. In approximately 50% of the relevant reports, selected for the analysis in peer-reviewed international journals, the average ex vivo induced yield of these biodosimetric markers was higher in patients with severe reactions than in patients with a lower grade of NTT. Also, a significant correlation was sometimes found between the biodosimetric marker yield and the severity of acute or late NTT reactions at an individual level, but this observation was not unequivocally proven. A similar controversy of published results was found regarding the attempts to apply G2- and γH2AX foci assays for NTT prediction. A correlation between ex vivo cytogenetic biomarker yields and NTT occurred most frequently when chromosome aberrations (not micronuclei) were measured in lymphocytes (not fibroblasts) irradiated to relatively high doses (4-6 Gy, not 2 Gy) in patients with various grades of late (not early) radiotherapy (RT) morbidity. The limitations of existing approaches are discussed, and recommendations on the improvement of the ex vivo cytogenetic testing for NTT prediction are provided. However, the efficiency of these methods still needs to be validated in properly organized clinical trials involving large and verified patient cohorts.

Radiation Exposure Biomarkers in the Practice of Medical Radiology: Cooperative Research and the Role of the International Atomic Energy Agency (IAEA) Biodosimetry/Radiobiology Laboratory.

The strategy toward personalized medicine in radiation oncology, nuclear medicine, and diagnostic and interventional radiology demands a specific set of assays for individualized estimation of radiation load for safety concerns and prognosis of normal tissue reactions caused by ionizing radiation. Apparently, it seems reasonable to use validated radiation dosimetric biomarkers for these purposes. However, a number of gaps in knowledge and methodological limitations still have to be resolved until dosimetric biomarkers will start to play a valuable role in clinical practice beyond radiation protection and radiation medicine. An extensive international multicenter research is necessary to improve the methodology of clinical applications of biodosimetry. That became a rationale for launching the IAEA Coordinated Research Project E35010 MEDBIODOSE: "Applications of Biological Dosimetry Methods in Radiation Oncology, Nuclear Medicine, and Diagnostic and Interventional Radiology." At the 2 Coordination Meeting on MEDBIODOSE (18-22 February 2019, Recife, Brazil), participants reported progress in the usage of biological dosimetry for genotoxicity assessment and/or individualization of radiotherapy treatment plans. Another avenue of research was the prognosis of normal tissue toxicity and cancer risk prediction using biomarkers' yield measured in vivo or after ex vivo irradiation of patients' cells. Other important areas are mechanisms of cytogenetic radiation response, validation of new radiation biomarkers, development of innovative techniques, automated and high-throughput assays for biodosimetry, and the overall improvement of biodosimetry service. An important aspect of clinical application of biodosimetry is standardization of techniques and unification of approaches to data interpretation. The new IAEA Biodosimetry/Radiobiology Laboratory, which is being established, will provide support for this activity. The declared lab's mission includes, among other tasks, a harmonization of the biodosimetry applications with relevant international standards, guidelines on good laboratory practice, and the IAEA EPR-Biodosimetry manual.

Understanding High-Dose, Ultra-High Dose Rate, and Spatially Fractionated Radiation Therapy.

The National Cancer Institute's Radiation Research Program, in collaboration with the Radiosurgery Society, hosted a workshop called Understanding High-Dose, Ultra-High Dose Rate and Spatially Fractionated Radiotherapy on August 20 and 21, 2018 to bring together experts in experimental and clinical experience in these and related fields. Critically, the overall aims were to understand the biological underpinning of these emerging techniques and the technical/physical parameters that must be further defined to drive clinical practice through innovative biologically based clinical trials.

CLINICAL APPLICATIONS OF BIOMARKERS OF RADIATION EXPOSURE: LIMITATIONS AND POSSIBLE SOLUTIONS THROUGH COORDINATED RESEARCH.

Dosimetric biomarkers have been effectively and intensively used for a long time in the area of radiation protection. In contrast to that, no robust standards or widely accepted protocols for application of these end-points in radiotherapy, diagnostic and interventional radiology and nuclear medicine exist to date. The International Atomic Energy Agency (IAEA) organized the review of the available data on the possibilities of the use of dosimetric biomarkers in medical irradiation scenarios. The resultant Technical Report also contains a summary of identified problems, gaps in knowledge, limitations in methodology and recommendations for their overcoming. This work provided a conceptual background for the initiation of a new IAEA Coordinated Research Project E35010, MEDBIODOSE (2017-21), which is aimed specifically at the development and improvement of applications of biodosimetric markers in clinical practice.

The Frequency of Lymphocytes Containing Dumbbell-Shaped Nuclei Depends on Ionizing Radiation Dose and Correlates with Appearance of Chromosomal Aberrations.

Nuclear anomalies of different types appear in cells in response to the action of ionizing radiation after the passage of the first mitotic division. In this article, we present the results of the study of the frequency of occurrence of three types of nuclear anomalies ("tailed" nuclei, nucleoplasmic bridges, and dumbbell-shaped nuclei) in vitro in human lymphocytes cultured with cytochalasin B when exposed to X-rays at doses of 0.0, 0.1, 0.2, 0.4, 0.5, 0.7, 1.0, 1.5, and 2.0 Gy. To stop the cell cycle of cultured lymphocytes after the first mitotic division, a cytokinesis block was performed using cytochalasin B. Dose-dependent curves of the occurrence of lymphocytes containing "tailed" nuclei, nucleoplasmic bridges, or dumbbell-shaped nuclei after irradiation have been constructed. At the same time, frequencies of occurrence of chromosomal aberrations (dicentric and ring chromosomes) in the culture of lymphocytes exposed to the same radiation doses were studied. Comparison of the frequencies of occurrence of dicentric and ring chromosomes with frequencies of occurrence of nuclear anomalies allows us to conclude that these nuclear anomalies are formed as a result of chromosomal aberrations arising in lymphocytes under the action of ionizing radiation. More than that, most of the chromosomal aberrations are converted into dumbbell-shaped nuclei in vitro in the culture of lymphocytes in the cytochalasin block.

Biomarkers of Ionizing Radiation Exposure: A Multiparametric Approach.

Humans are exposed to ionizing radiation not only through background radiation but also through the ubiquitous presence of devices and sources that generate radiation. With the expanded use of radiation in day-to-day life, the chances of accidents or misuse only increase. Therefore, a thorough understanding of the dynamic effects of radiation exposure on biological entities is necessary. The biological effects of radiation exposure on human cells depend on much variability such as level of exposure, dose rate, and the physiological state of the cells. During potential scenarios of a large-scale radiological event which results in mass casualties, dose estimates are essential to assign medical attention according to individual needs. Many attempts have been made to identify biomarkers which can be used for high throughput biodosimetry screening. In this study, we compare the results of different biodosimetry methods on the same irradiated cells to assess the suitability of current biomarkers and push forward the idea of employing a multiparametric approach to achieve an accurate dose and risk estimation.

Non-targeted effects of ionising radiation--implications for low dose risk.

Non-DNA targeted effects of ionising radiation, which include genomic instability, and a variety of bystander effects including abscopal effects and bystander mediated adaptive response, have raised concerns about the magnitude of low-dose radiation risk. Genomic instability, bystander effects and adaptive responses are powered by fundamental, but not clearly understood systems that maintain tissue homeostasis. Despite excellent research in this field by various groups, there are still gaps in our understanding of the likely mechanisms associated with non-DNA targeted effects, particularly with respect to systemic (human health) consequences at low and intermediate doses of ionising radiation. Other outstanding questions include links between the different non-targeted responses and the variations in response observed between individuals and cell lines, possibly a function of genetic background. Furthermore, it is still not known what the initial target and early interactions in cells are that give rise to non-targeted responses in neighbouring or descendant cells. This paper provides a commentary on the current state of the field as a result of the non-targeted effects of ionising radiation (NOTE) Integrated Project funded by the European Union. Here we critically examine the evidence for non-targeted effects, discuss apparently contradictory results and consider implications for low-dose radiation health effects.

Some important questions connected with non-targeted effects.

This paper briefly reviews the highlights of experimental evidence that led to the adoption of the term "non-targeted" to describe new effects induced by ionising radiation that did not fit the classical radiobiological paradigm, principally genomic instability and bystander effect, identifying the reports that were most influential on the subsequent course of radiobiological research. The issue of appropriate terminology for the new effects is discussed. Particular emphasis is placed on the inheritance of genomic instability, where there are issues concerning which effects should be considered as transgenerational. Finally, in respect of the question as to whether these new effects are likely to have an impact on human health is addressed. It is concluded that there is a need for a clearer terminology to facilitate research progress, that real health effects cannot be ruled out and that therefore there is a need for new paradigms not only for radiobiology but also for risk assessment and radiological protection.

A (238)Pu irradiator for exposure of cultured cells with alpha-radiation: construction, calibration and dosimetry.

An alpha-particle irradiator that can facilitate investigations of alpha-radiation effects on human cells in radiation protection, carcinogenesis and radioimmunotherapy was constructed. The irradiator was based on a 1.3 GBq (238)Pu source, housed in a stainless steel tube flushed with helium. Radiation provided by (238)Pu consists mainly of alpha-particles with energy of 5.5 MeV. The alpha-particle fluence and energy spectra were measured with a silicon semiconductor detector. Monte Carlo simulations were used to estimate the mean number of alpha-particles and the mean absorbed alpha-particle dose to cells for various irradiation times and distances between cells and source. There was a linear dependence between exposure time and alpha-particle fluence for exposure times above 1s. The alpha-particle activity concentration varied with a factor 2.7 over the source area, while the variation in energy peak position was <4%. At the cell nucleus position and with a distance of 45 mm between the source and the mylar dish surface, the alpha-fluence was 4.6 x 10(4)counts/(mm(2)s), the average incident alpha-particle energy was 2.5 MeV and the average linear energy transfer was 167 keV/microm. The average dose rate to the cells, with 5 microm diameter nucleus, was 1.2 Gy/s. The (238)Pu alpha-particle irradiator is feasible for irradiation of cells and it can be used for studies of both direct effects and bystander effects of alpha-radiation.

Microbeam studies of the bystander response.

Microbeams have undergone a renaissance since their introduction and early use in the mid 60s. Recent advances in imaging, software and beam delivery have allowed rapid technological developments in microbeams for use in a range of experimental studies. The resurgence in the use of microbeams since the mid 90s has coincided with major changes in our understanding of how radiation interacts with cells. In particular, the evidence that bystander responses occur, where cells not directly irradiated can respond to irradiated neighbours, has brought about the evolution of new models of radiation response. Although these processes have been studied using a range of experimental approaches, microbeams offer a unique route by which bystander responses can be elucidated. Without exception, all of the microbeams currently active internationally have studied bystander responses in a range of cell and tissue models. Together these studies have considerably advanced our knowledge of bystander responses and the underpinning mechanisms. Much of this has come from charged particle microbeam studies, but increasingly, X-ray and electron microbeams are starting to contribute quantitative and mechanistic information on bystander effects. A recent development has been the move from studies with 2-D cell culture models to more complex 3-D systems where the possibilities of utilizing the unique characteristics of microbeams in terms of their spatial and temporal delivery will make a major impact.

Design, spectrum measurements and simulations for a 238Pu alpha-particle irradiator for bystander effect and genomic instability experiments.

Design, spectrum measurements and simulations for an alpha-particle irradiator for bystander effect and genomic instability experiments are presented. Measured alpha-particle energy spectra were used to confirm the characteristics of the source of the irradiator specified by the manufacturer of the source. The spectra were measured in vacuum with a high-resolution spectrometer and simulated with an AASI Monte Carlo code. As a next step, we simulated alpha-particle energy spectra at the target plane of the irradiator for three different source-to-target distances. In these simulations, helium was used as the medium between the source and the exit window of the irradiator; its pressure and temperature corresponded to those of the ambient air. Mean energies and full-widths at half-maximum (FWHM) were calculated for the three different helium gas tracks.

Bystander-induced differentiation: a major response to targeted irradiation of a urothelial explant model.

A ureter primary explant technique, using porcine tissue sections was developed to study bystander effects under in vivo like conditions where dividing and differentiated cells are present. Targeted irradiations of ureter tissue fragments were performed with the Gray Cancer Institute charged particle microbeam at a single location (2 microm precision) with 10 3He2+ particles (5 MeV; LET 70 keV/microm). After irradiation the ureter tissue section was incubated for 7 days allowing explant outgrowth to be formed. Differentiation was estimated using antibodies to Uroplakin III, a specific marker of terminal urothelial differentiation. Even although only a single region of the tissue section was targeted, thousands of additional cells were found to undergo bystander-induced differentiation in the explant outgrowth. This resulted in an overall increase in the fraction of differentiated cells from 63.5+/-5.4% to 76.6+/-5.6%. These changes are much greater than that observed for the induction of damage in this model. One interpretation of these results is that in the tissue environment, differentiation is a much more significant response to targeted irradiation and potentially a protective mechanism.

Classical radiation biology, the bystander effect and paradigms: a reply.

Although, in retrospect, it can be seen that the bystander effect and the related effect of genomic instability were observed well before they were recognized as such, they have not been able to be accommodated within the existing understanding of how radiation causes late effects, which provides the basis for radiological protection standards. It is argued here that before these effects can be fully researched and there can be full confidence in radiological protection, a paradigm shift that provides a framework in which these effects can be considered alongside the well established effects of radiation is needed. In particular this framework will encompass the epigenetic as well as genetic aspects of radiation biology. Examples of how this might be achieved are given.

Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away.

A central tenet in understanding the biological effects of ionizing radiation has been that the initially affected cells were directly damaged by the radiation. By contrast, evidence has emerged concerning "bystander" responses involving damage to nearby cells that were not themselves directly traversed by the radiation. These long-range effects are of interest both mechanistically and for assessing risks from low-dose exposures, where only a small proportion of cells are directly hit. Bystander effects have been observed largely by using single-cell in vitro systems that do not have realistic multicellular morphology; no studies have as yet been reported in three-dimensional, normal human tissue. Given that the bystander phenomenon must involve cell-to-cell interactions, the relevance of such single-cell in vitro studies is questionable, and thus the significance of bystander responses for human health has remained unclear. Here, we describe bystander responses in a three-dimensional, normal human-tissue system. Endpoints were induction of micronucleated and apoptotic cells. A charged-particle microbeam was used, allowing irradiation of cells in defined locations in the tissue yet guaranteeing that no cells located more than a few micrometers away receive any radiation exposure. Unirradiated cells up to 1 mm distant from irradiated cells showed a significant enhancement in effect over background, with an average increase in effect of 1.7-fold for micronuclei and 2.8-fold for apoptosis. The surprisingly long range of bystander signals in human tissue suggests that bystander responses may be important in extrapolating radiation risk estimates from epidemiologically accessible doses down to very low doses where nonhit bystander cells will predominate.