Summary of the 2021 ICRP workshop on the future of radiological protection.

The International Commission on Radiological Protection (ICRP) has embarked on a process to review and revise the current System of Radiological Protection ('the System'). To stimulate discussion, the ICRP published two open-access articles: one on aspects of the System that might require review, and another on research that might improve the scientific foundation of the System. Building on these articles, the ICRP organized a Workshop on the Future of Radiological Protection as an opportunity to engage in the review and revision of the System. This digital workshop took place from 14 October-3 November 2021 and included 20 live-streamed and 43 on-demand presentations. Approximately 1500 individuals from 100 countries participated. Based on the subjects covered by the presentations, this summary is organized into four broad areas: the scientific basis, concepts and application of the System; and the role of the ICRP. Some of the key topics that emerged included the following: classification of radiation-induced effects; adverse outcome pathway methodologies; better understanding of the dose-response relationship; holistic and reasonable approaches to optimization of protection; radiological protection of the environment; ethical basis of the System; clarity, consistency and communication of the System; application of the System in medicine and application of the principles of justification and optimization of protection.

Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise.

Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5-40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5-8 h varying exposure times; second: varying dose rates of 0.5-8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.

The 2019-2020 EURADOS WG10 and RENEB Field Test of Retrospective Dosimetry Methods in a Small-Scale Incident Involving Ionizing Radiation.

With the use of ionizing radiation comes the risk of accidents and malevolent misuse. When unplanned exposures occur, there are several methods which can be used to retrospectively reconstruct individual radiation exposures; biological methods include analysis of aberrations and damage of chromosomes and DNA, while physical methods rely on luminescence (TL/OSL) or EPR signals. To ensure the quality and dependability of these methods, they should be evaluated under realistic exposure conditions. In 2019, EURADOS Working Group 10 and RENEB organized a field test with the purpose of evaluating retrospective dosimetry methods as carried out in potential real-life exposure scenarios. A 1.36 TBq 192Ir source was used to irradiate anthropomorphic phantoms in different geometries at doses of several Gy in an outdoor open-air geometry. Materials intended for accident dosimetry (including mobile phones and blood) were placed on the phantoms together with reference dosimeters (LiF, NaCl, glass). The objective was to estimate radiation exposures received by individuals as measured using blood and fortuitous materials, and to evaluate these methods by comparing the estimated doses to reference measurements and Monte Carlo simulations. Herein we describe the overall planning, goals, execution and preliminary outcomes of the 2019 field test. Such field tests are essential for the development of new and existing methods. The outputs from this field test include useful experience in terms of planning and execution of future exercises, with respect to time management, radiation protection, and reference dosimetry to be considered to obtain relevant data for analysis.

Individual response of humans to ionising radiation: governing factors and importance for radiological protection.

Tissue reactions and stochastic effects after exposure to ionising radiation are variable between individuals but the factors and mechanisms governing individual responses are not well understood. Individual responses can be measured at different levels of biological organization and using different endpoints following varying doses of radiation, including: cancers, non-cancer diseases and mortality in the whole organism; normal tissue reactions after exposures; and, cellular endpoints such as chromosomal damage and molecular alterations. There is no doubt that many factors influence the responses of people to radiation to different degrees. In addition to the obvious general factors of radiation quality, dose, dose rate and the tissue (sub)volume irradiated, recognized and potential determining factors include age, sex, life style (e.g., smoking, diet, possibly body mass index), environmental factors, genetics and epigenetics, stochastic distribution of cellular events, and systemic comorbidities such as diabetes or viral infections. Genetic factors are commonly thought to be a substantial contributor to individual response to radiation. Apart from a small number of rare monogenic diseases such as ataxia telangiectasia, the inheritance of an abnormally responsive phenotype among a population of healthy individuals does not follow a classical Mendelian inheritance pattern. Rather it is considered to be a multi-factorial, complex trait.

BIODOSIMETRY AND BIODOSIMETRY NETWORKS FOR MANAGING RADIATION EMERGENCY.

Biological dosimetry enables individual dose reconstruction in the case of unclear or inconsistent radiation exposure situations, especially when a direct measurement of ionizing radiation is not or is no longer possible. To be prepared for large-scale radiological incidents, networking between well-trained laboratories has been identified as a useful approach for provision of the fast and trustworthy dose assessments needed in such circumstances. To this end, various biodosimetry laboratories worldwide have joined forces and set up regional and/or nationwide networks either on a formal or informal basis. Many of these laboratories are also a part of global networks such as those organized by World Health Organization, International Atomic Energy Agency or Global Health Security Initiative. In the present report, biodosimetry networks from different parts of the world are presented, and the partners, activities and cooperation actions are detailed. Moreover, guidance for situational application of tools used for individual dosimetry is given.

Human individual radiation sensitivity and prospects for prediction.

In the past few decades, it has become increasingly evident that sensitivity to ionising radiation is variable. This is true for tissue reactions (deterministic effects) after high doses of radiation, for stochastic effects following moderate and possibly low doses, and conceivably also for non-cancer effects such as cardiovascular disease, the causal pathway(s) of which are not yet fully understood. A high sensitivity to deterministic effects is not necessarily correlated with a high sensitivity to stochastic effects. The concept of individual sensitivity to high and low doses of radiation has long been supported by data from patients with certain rare hereditary conditions. However, these syndromes only affect a small proportion of the general population. More relevant to the majority of the population is the notion that some part of the genetic contribution defining radiation sensitivity may follow a polygenic model, which predicts elevated risk resulting from the inheritance of many low-penetrance risk-modulating alleles. Can the different forms of individual radiation sensitivities be inferred from the reaction of cells exposed ex vivo to ionising radiation? Can they be inferred from analyses of individual genotypes? This paper reviews current evidence from studies of late adverse tissue reactions after radiotherapy in potentially sensitive groups, including data from functional assays, candidate gene approaches, and genome-wide association studies. It focuses on studies published in 2013 or later because a comprehensive review of earlier studies was published previously in a report by the UK Advisory Group on Ionising Radiation.

Lack of Impact of Presence of Positive C4d Staining in Capillaries in Myocardial Biopsies on Long-term Survival of Heart Transplant Patients.

BACKGROUND: The long-term survival of 209 consecutive patients (mean age, 46 ± 15 years) from a single center with ≥1 diagnostic myocardial biopsy after heart transplantation was analyzed. METHODS: Patients were considered as C4d positive if a capillary staining (immunohistochemistry in paraffin samples) was observed in ≥1 myocardial biopsy. Data were analyzed according to pathologic consensus of antibody mediated rejection definition of C4d+ positivity: 2004 definition in group A and the 2013 definition in group B and compared with their respective controls, composed of patients who do not meet those criteria. Age, follow-up time, and number of biopsies were comparable between patients with C4d+ and controls in both groups. Follow-up was 100% complete with mean of observation time 2143 days. RESULTS: During the follow-up period, 62 patients died (group A: C4d+ 32% vs controls 29%; group B: C4d+ 36% vs controls 29% [P = NS]). There were no differences in survival between patients with positive staining and without C4d+ staining when Kaplan-Meier survival curves were compared. CONCLUSIONS: The presence of C4d positive staining in myocardial capillaries of heart biopsies of patients after heart transplantation, as an isolated finding, was not related to worse long-term survival.

Web-based scoring of the dicentric assay, a collaborative biodosimetric scoring strategy for population triage in large scale radiation accidents.

In the case of a large scale radiation accident high throughput methods of biological dosimetry for population triage are needed to identify individuals requiring clinical treatment. The dicentric assay performed in web-based scoring mode may be a very suitable technique. Within the MULTIBIODOSE EU FP7 project a network is being established of 8 laboratories with expertise in dose estimations based on the dicentric assay. Here, the manual dicentric assay was tested in a web-based scoring mode. More than 23,000 high resolution images of metaphase spreads (only first mitosis) were captured by four laboratories and established as image galleries on the internet (cloud). The galleries included images of a complete dose effect curve (0-5.0 Gy) and three types of irradiation scenarios simulating acute whole body, partial body and protracted exposure. The blood samples had been irradiated in vitro with gamma rays at the University of Ghent, Belgium. Two laboratories provided image galleries from Fluorescence plus Giemsa stained slides (3 h colcemid) and the image galleries from the other two laboratories contained images from Giemsa stained preparations (24 h colcemid). Each of the 8 participating laboratories analysed 3 dose points of the dose effect curve (scoring 100 cells for each point) and 3 unknown dose points (50 cells) for each of the 3 simulated irradiation scenarios. At first all analyses were performed in a QuickScan Mode without scoring individual chromosomes, followed by conventional scoring (only complete cells, 46 centromeres). The calibration curves obtained using these two scoring methods were very similar, with no significant difference in the linear-quadratic curve coefficients. Analysis of variance showed a significant effect of dose on the yield of dicentrics, but no significant effect of the laboratories, different methods of slide preparation or different incubation times used for colcemid. The results obtained to date within the MULTIBIODOSE project by a network of 8 collaborating laboratories throughout Europe are very promising. The dicentric assay in the web based scoring mode as a high throughput scoring strategy is a useful application for biodosimetry in the case of a large scale radiation accident.

Automatic scoring of dicentric chromosomes as a tool in large scale radiation accidents.

Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage in order to rapidly identify individuals who require clinical treatment. The manual dicentric assay is a highly suitable technique, but it is also very time consuming and requires well trained scorers. In the framework of the MULTIBIODOSE EU FP7 project, semi-automated dicentric scoring has been established in six European biodosimetry laboratories. Whole blood was irradiated with a Co-60 gamma source resulting in 8 different doses between 0 and 4.5Gy and then shipped to the six participating laboratories. To investigate two different scoring strategies, cell cultures were set up with short term (2-3h) or long term (24h) colcemid treatment. Three classifiers for automatic dicentric detection were applied, two of which were developed specifically for these two different culture techniques. The automation procedure included metaphase finding, capture of cells at high resolution and detection of dicentric candidates. The automatically detected dicentric candidates were then evaluated by a trained human scorer, which led to the term 'semi-automated' being applied to the analysis. The six participating laboratories established at least one semi-automated calibration curve each, using the appropriate classifier for their colcemid treatment time. There was no significant difference between the calibration curves established, regardless of the classifier used. The ratio of false positive to true positive dicentric candidates was dose dependent. The total staff effort required for analysing 150 metaphases using the semi-automated approach was 2 min as opposed to 60 min for manual scoring of 50 metaphases. Semi-automated dicentric scoring is a useful tool in a large scale radiation accident as it enables high throughput screening of samples for fast triage of potentially exposed individuals. Furthermore, the results from the participating laboratories were comparable which supports networking between laboratories for this assay.

Realising the European Network of Biodosimetry (RENEB).

In Europe, a network for biological dosimetry has been created to strengthen the emergency preparedness and response capabilities in case of a large-scale nuclear accident or radiological emergency. Through the RENEB (Realising the European Network of Biodosimetry) project, 23 experienced laboratories from 16 European countries will establish a sustainable network for rapid, comprehensive and standardised biodosimetry provision that would be urgently required in an emergency situation on European ground. The foundation of the network is formed by five main pillars: (1) the ad hoc operational basis, (2) a basis of future developments, (3) an effective quality-management system, (4) arrangements to guarantee long-term sustainability and (5) awareness of the existence of RENEB. RENEB will thus provide a mechanism for quick, efficient and reliable support within the European radiation emergency management. The scientific basis of RENEB will concurrently contribute to increased safety in the field of radiation protection.

Chromosomal aberrations in peripheral blood lymphocytes exposed to a mixed beam of low energy neutrons and gamma radiation.

Cells exposed to thermal neutrons are simultaneously damaged by radiations with high and low linear energy transfer (LET). A question relevant for the assessment of risk of exposure to a mixed beam is whether the biological effect of both radiation types is additive or synergistic. The aim of the present investigation was to calculate whether the high and low LET components of a thermal neutron field interact when damaging cells. Human peripheral blood lymphocytes were exposed to neutrons from the HB11 beam at the Institute for Energy and Transport, Petten, Netherlands, in a 37 °C water phantom at varying depths, where the mix of high and low LET beam components differs. Chromosomal aberrations were analysed and the relative biological effectiveness (RBE) values as well as the expected contributions of protons and photons to the aberration yield were calculated based on a dose response of aberrations in lymphocytes exposed to (60)Co gamma radiation. The RBE for 10 dicentrics per 100 cells was 3 for mixed beam and 7.2 for protons. For 20 dicentrics per 100 cells the respective values were 2.4 and 5.8. Within the limitations of the experimental setup the results indicate that for this endpoint there is no synergism between the high and low LET radiations.

Review of retrospective dosimetry techniques for external ionising radiation exposures.

The current focus on networking and mutual assistance in the management of radiation accidents or incidents has demonstrated the importance of a joined-up approach in physical and biological dosimetry. To this end, the European Radiation Dosimetry Working Group 10 on 'Retrospective Dosimetry' has been set up by individuals from a wide range of disciplines across Europe. Here, established and emerging dosimetry methods are reviewed, which can be used immediately and retrospectively following external ionising radiation exposure. Endpoints and assays include dicentrics, translocations, premature chromosome condensation, micronuclei, somatic mutations, gene expression, electron paramagnetic resonance, thermoluminescence, optically stimulated luminescence, neutron activation, haematology, protein biomarkers and analytical dose reconstruction. Individual characteristics of these techniques, their limitations and potential for further development are reviewed, and their usefulness in specific exposure scenarios is discussed. Whilst no single technique fulfils the criteria of an ideal dosemeter, an integrated approach using multiple techniques tailored to the exposure scenario can cover most requirements.

Inguinal hernia repair with the peduncled fascial flap: a new surgical technique.

INTRODUCTION: This paper presents a new surgical technique of inguinal hernia repair in which both crura of the aponeurosis of the external oblique abdominal muscle and transverse fascia were used for complex reconstruction of the entire musculopectineal hiatus. MATERIAL AND METHODS: Between 2nd December 2003 and 29th April 2005, 250 patients (233 male and 17 female) underwent inguinal hernia repairs using our own technique. The inguinal canal was opened together with the posterior wall, dividing the transverse fascia into two flaps. The lower flap was inserted into the femoral opening and sewn to the pectineal ligament, whereas the upper flap with both crura of the aponeurosis of the external oblique abdominal muscle were used for three-fascia reconstruction of the posterior wall of the inguinal canal. The study group was randomly chosen from patients undergoing surgery due to inguinal hernias in our hospital. Procedures were carried out under subarachnoid anaesthesia; postoperative pain was treated with methamizol or ketoprofen. Patients were discharged 48 h after surgery. RESULTS: The postoperative complications included one hernia recurrence and one testicular atrophy. The remaining complications were transient and included prolonged wound healing, transient skin hypoaesthesia around the wound or testis oedema. CONCLUSIONS: The technique used strengthens the musculopectineal hiatus, effectively preventing recurrences of inguinal as well as femoral hernias.

Micromachined "Side-Viewing" Optical Sensor Probe for Detection of Esophageal Cancers.

In this paper, we report the design, fabrication and testing of a new miniaturized optical sensor probe with "side viewing" capability for oblique incidence diffuse reflectance spectrometry. The sensor probe consists of a lithographically patterned polymer waveguides chip and two micromachined positioning substrates and source/collection fibers to achieve 45° light incidence and collection of spatially resolved diffuse reflectance. Diffuse reflectance of human esophageal surface has been successfully measured for differentiation of cancerous tissues from normal ones.

Nucleotide excision repair impairment by nodularin in CHO cell lines due to ERCC1/XPF inactivation.

The problem of toxicity of cyanobacterial toxins is of increasing concern, as the incidence of such blooms grows. Among the toxins, the most abundant in the environment are hepatotoxins known as nodularins and microcystins. These toxins are responsible for almost all known cases of fresh and brackish water intoxication and are responsible for recurrent episodes of human and animal illness and death. Moreover, they are believed to be potent tumor promoters and initiators. However, the mechanisms by which these toxins induce liver cancer are not well understood. The aim of the present study was to determine the effect of nodularin on the kinetics of nucleotide excision repair (NER) in Chinese hamster ovary (CHO) cells exposed to UV radiation. The first set of experiments was performed to define the optimal treatment conditions for nodularin to avoid the possibility of encountering false positive signals in the comet assay due to the apoptogenic activity of nodularin. Based on the analysis of apoptosis, the 6-h treatment time of cells with nodularin (1mug/ml, 10mug/ml and 20mug/ml) was chosen for the alkaline comet assay. The kinetics of NER was determined in CHO cell lines: AA8 (wild-type) and mutant cell lines: UV135 (XPG(-)), UV41 (XPF(-)) and UV20 (ERCC1(-)) exposed to 20J/m(2) UV radiation. The micronucleus assay was performed to determine a residual DNA damage in four cell lines treated with nodularin (10mug/ml) and exposed to equitoxic doses UV radiation. Radiation doses of UV producing 50% of survival for AA8, UV135, UV20 and UV41 cell lines were calculated from UV survival curves. The results show that nodularin impairs the incision/excision step of NER in CHO cells by the ERCC1/XPF inactivation and leads to an increased level of UV-induced cytogenetic DNA damage.

No induction of structural chromosomal aberrations in cylindrospermopsin-treated CHO-K1 cells without and with metabolic activation.

Cylindrospermopsin (CYN) is a cyanobacterial alkaloid that has been implicated in outbreaks of human morbidity and animal mortality. The principal mode of action for CYN is inhibition of protein and glutathione synthesis, and its toxicity seems to be mediated by cytochrome P-450-generated metabolites. It was also shown that CYN might be responsible for tumor initiation in animals; nevertheless, mechanisms leading to CYN-induced carcinogenesis are scarce and equivocal. The aim of the present study was to investigate the impact of metabolic activation on CYN-induced DNA damage. The effect of different doses of CYN (0.05-2mug/ml) on DNA damage was determined in CHO-K1 cells after 3, 16 and 21h of the treatment. The chromosome aberration assay with and without metabolic activation was applied to evaluate the clastogenic activity of CYN and its metabolite(s). In addition, the occurrence of apoptosis and necrosis was estimated by the annexin method using flow cytometry. The results revealed that CYN is not clastogenic in CHO-K1 cells irrespective of S9 fraction-induced metabolic activation. However, CYN significantly decreases the frequencies of mitotic indices and decreases proliferation irrespective of metabolic activation system. CYN increases the frequency of necrotic cells in a dose- and time-dependent manner, whereas it has a very slight impact on apoptosis. Moreover, the presence of metabolic activation influences a susceptibility to necrotic cell death but not an apoptotic one.

Inhibition of nucleotide excision repair (NER) by microcystin-LR in CHO-K1 cells.

Microcystin-LR (MC-LR), a potent inhibitor of PP1 and PP2A protein phosphatases, is related to tumor promotion and initiation. Although the genotoxic properties of this toxin have been extensively investigated with a variety of non-mammalian and mammalian test systems, the existing results are contradictory. Based on our previous results regarding the impact of MC-LR on the processes of DNA repair we decided to examine in greater detail its effect on the capacity of nucleotide excision repair (NER). CHO-K1 cells were pre-treated with increasing doses of MC-LR (1, 10 and 20 microg/ml) and then exposed to UV radiation (25 J/m(2)). Apoptosis was analyzed to exclude the possibility of false positive results in the comet assay. The results suggest that MC-LR targets the nucleotide excision repair mechanisms by interference with the incision/excision phase as well as the rejoining phase of NER and leads to an increased level of UV-induced cytogenetic DNA damage in CHO-K1 cells.

Nodularin-induced genotoxicity following oxidative DNA damage and aneuploidy in HepG2 cells.

The problem of toxicity of Nodularia spumigena to animals and people is of increasing concern, as the incidence of such blooms grows. It was shown that nodularin is a liver carcinogen possessing both initiating and tumor-promoting activities. However, the mechanisms by which this toxin damages the DNA and induces liver cancer are not well understood. The aim of the present study was to investigate the DNA damaging properties of nodularin. The effect of different doses of nodularin (1-10 microg/ml) on DNA damage was determined in HepG2 cells after 6, 12, 24 and 48 h of the treatment. The modified comet assay in conjunction with Fpg (ROS-induced DNA damage) and FISH-micronucleus assay (clastogenic and/or aneugenic activities of nodularin) were applied. In addition the occurrence of apoptosis was estimated by the morphological analysis of chromatin condensation and the annexin method using flow cytometry. We found that nodularin induces oxidative DNA damage by oxidation of purines and increases the formation of centromere positive micronuclei due to aneugenic activity. In addition to genotoxic properties, nodularin exerts a cytotoxic activity by inducing apoptosis in HepG2 cells. These results suggest a causative role for nodularin in the process leading to the accumulation of genetic alterations which may be implicated in carcinogenesis.

The repair of gamma-radiation-induced DNA damage is inhibited by microcystin-LR, the PP1 and PP2A phosphatase inhibitor.

The genotoxic activity of microcystin-LR (MC-LR) is a matter of debate. MC-LR is known to be a phosphatase inhibitor and it may be expected that it is involved in the regulation of the activity of DNA-dependent protein kinase (DNA-PK), the key enzyme involved in the repair of radiation-induced DNA damage. We studied the effect of MC-LR on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. A dose of 0.5 microg/ml of MC-LR was chosen because it induced very little early apoptosis which gives no false positive results in the comet assay. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 h and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition the frequencies of chromosomal aberrations were analysed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage and lead to enhanced frequencies of chromosomal aberrations including dicentric chromosomes. The results of a split-dose experiment, where cells were exposed to two 1.5 Gy doses of radiation separated by 3 h with or without MC-LR, confirmed that the toxin increased the frequency of dicentric chromosomes. We also determined the effect of MC-LR and ionizing radiation on the frequency of gamma-H2AX foci. The pre-treatment with MC-LR resulted in reduced numbers of gamma-H2AX foci in irradiated cells. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma MO59J and MO59K cells. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analysed by the comet assay. A strong inhibitory effect was observed in the MO59K but not in the MO59J cells. These results indicate that DNA-PK might be involved in DNA repair inhibition by MC-LR.