Search

The bottleneck in data acquisition during biological dosimetry based on a dicentric assay is the need to score dicentrics in a large number of lymphocytes. One way to increase the capacity of a given laboratory is to use the ability of skilled operators from other laboratories. This can be done using image analysis systems and distributing images all around the world. Two exercises were conducted to test the efficiency of such an approach involving 10 laboratories. During the first exercise (E1), the participant laboratories analysed the same images derived from cells exposed to 0.5 and 3 Gy; 100 images were sent to all participants for both doses. Whatever the dose, only about half of the cells were complete with well-spread metaphases suitable for analysis. A coefficient of variation (CV) on the standard deviation of ∼15 % was obtained for both doses. The trueness was better for 3 Gy (0.6 %) than for 0.5 Gy (37.8 %). The number of estimated doses classified as satisfactory according to the z-score was 3 at 0.5 Gy and 8 at 3 Gy for 10 dose estimations. In the second exercise, an emergency situation was tested, each laboratory was required to score a different set of 50 images in 2 d extracted from 500 downloaded images derived from cells exposed to 0.5 Gy. Then the remaining 450 images had to be scored within a week. Using 50 different images, the CV on the estimated doses (79.2 %) was not as good as in E1, probably associated to a lower number of cells analysed (50 vs. 100) or from the fact that laboratories analysed a different set of images. The trueness for the dose was better after scoring 500 cells (22.5 %) than after 50 cells (26.8 %). For the 10 dose estimations, the number of doses classified as satisfactory according to the z-score was 9, for both 50 and 500 cells. Overall, the results obtained support the feasibility of networking using electronically transmitted images. However, before its implementation some issues should be elucidated, such as the number and resolution of the images to be sent, and the harmonisation of the scoring criteria. Additionally, a global website able to be used for the different regional networks, like Share Points, will be desirable to facilitate worldwide communication. [ABSTRACT FROM AUTHOR]

Several studies have been carried out to evaluate the mutagenic and carcinogenic potential of atrazine, the most prevalent of triazine herbicides classified as a “possible human carcinogen”. The majority of these studies have been negative but positive responses have been also reported including mammary tumors in female Sprague-Dawley rats. Sister chromatid exchanges (SCEs) caused by the presence of DNA lesions at the moment of DNA replication have been extensively used for genotoxicity testing, but for non-cytotoxic exposures to atrazine controversial results have been reported. Even though exposures to higher concentrations of atrazine could provide clear evidence for its genotoxicity, conventional SCE analysis at metaphase cells cannot be used because affected cells are delayed in G2-phase and do not proceed to mitosis. As a result, the genotoxic potential of atrazine may have been underestimated. Since clear evidence has been recently reported relating SCEs to homologous recombinational events, we are testing here the hypothesis that high concentrations of atrazine will cause a dose-dependent increase in homologous recombinational events as quantified by the frequency of SCEs analyzed in G2-phase. Towards this goal, a new cytogenetic approach is applied for the analysis of SCEs directly in G2-phase prematurely condensed chromosomes (PCCs). The methodology enables the visualization of SCEs in G2-blocked cells and is based on drug-induced PCCs in cultured lymphocytes. The results obtained for high concentrations of atrazine do not demonstrate a dose-dependent increase in homologous recombinational events. They do not support, therefore, a genotoxic mode of action. However, they suggest that an important part in the variation of SCE frequency reported by different laboratories when conventional SCE analysis is applied after exposure to a certain concentration of atrazine, is due to differences in cell cycle kinetics of cultured lymphocytes, rather than to a true biological variation in the cytogenetic end point used. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Purpose : To investigate whether amifostine is effective at reducing the yield of chromatid breaks when present during G 2 -phase irradiation of human normal cells and cells from cancer prone patients, as well as to study the mechanisms underlying the radioprotective effect of amifostine. Materials and methods : G 2 chromosomal radiosensitivity in the presence or absence of amifostine was studied in healthy donors, cancer patients, ataxia-telangietasia (A-T) patients and five human lymphoblastoid cell lines with genes predisposing to cancer. The yield of chromatid breaks following γ -irradiation in G 2 phase was obtained at the subsequent metaphase using the G 2 assay. For scoring chromatid damage directly in G 2 or G 0 phase, premature chromosome condensation was used. Results : When amifostine was present during irradiation, the mean yield of radiation-induced chromatid breaks as visualized by the G 2 assay was significantly reduced in healthy donors ( t -test, p =0.001), in cells from cancer patients ( p =0.001) and in cell lines from patients with genes predisposing to cancer ( p =0.01) except ATM-/- (0.1 < p < 0.2). However, when chromatid breaks were scored directly in G 2 or G 0 phase using premature chromosome condensation, the presence of amifostine did not affect the yields obtained. Conclusion : Amifostine reduces the mean yield of chromatid breaks in normal cells and in cells from cancer prone patients when present during G 2 irradiation. Although the precise mechanisms of radioprotection caused by amifostine remain unclear, the results obtained using premature chromosome condensation reveal that amifostine does not act on cells only as a free radical scavenger and as a repair enhancer of DNA damage. [ABSTRACT FROM AUTHOR]

Purpose: To test the hypothesis that deficient DNA repair as measured by increased G2 chromosomal radiosensitivity results from up-regulation of cdk1/cyclinB and cell cycle control mechanisms during the G2 to M transition. Materials and methods: A total of 185 cancer patients and 25 normal individuals were tested for G2 chromosomal radiosensitivity. The chromatid breaks were analysed in metaphase using the G2 assay or directly in G0 and G2 phase using premature chromosome condensation (PCC). The activity of cdk1/cyclinB, a key regulator of the G2 to M-phase transition, was measured by histone H1 kinase activity and correlated with the development of chromatid breaks after irradiation of cell lines in vitro. Results: Based on the G2 assay, cancer patients on average showed increased chromosomal radiosensitivity above controls. When the analysis was carried out directly in G0 or G2 lymphocytes using PCC, no differences in the induction of chromosomal damage and its repair were observed between G2 assay-sensitive and G2-normal donors. Using the G2 assay to test G2 radiosensitivity in various cell lines, it was found that the higher the cdk1/cyclinB activity level of the cell line tested, the higher the yield of chromatid breaks scored. Furthermore, when mitotic cells from these cell lines were used for PCC induction in irradiated G2 lymphocytes it was observed that the higher the cdk1/cyclinB activity level of mitotic cells used, the higher was the induced yield of chromatid breaks. Conclusion: The cdk1/cyclin-B activity levels during the G2 to M transition impair DNA repair processes and play a major role in the yield of chromatid breaks induced after G2-irradiation. Regulation of cdk1/cyclinB complex activity rather than deficient repair enzymes of DNA damage may underlie the mechanisms of G2 radiosensitivity. [ABSTRACT FROM AUTHOR]

The patterning of biomolecules on semiconducting surfaces is of central importance in the fabrication of novel biodevices. In the process of patterning, it is required that the biomolecule preserves its properties and the substrate is not damaged by the chemicals, the temperatures or the patterning beams involved in the procedure. Recently, both DUV and electron beam microlithography have been used in order to deposit protein layers in predefined patterns. Various approaches have been used, some involving photoresists. Contrast between exposed and unexposed regions, resolution of adjacent features and sensitivity to dose variation, are the key issues. The approach followed in this paper consists of a direct patterning of a biotin layer, deposited on an amino-silane primed silicon nitride surface, using an electron beam. After irradiation, the surface is covered by bovine serum albumin (BSA), which acts as a blocking material to protect the exposed areas from streptavidin adsorption. Using 20 keV e-beam energy and doses, in the range 100–1000 μC/cm2, submicrometer dense lines of 1-μm pitch have been obtained. The results have been tested by fluorescence optical microscopy. [Copyright &y& Elsevier]

In the case of a radiological or nuclear event, biological dosimetry can be an important tool to support clinical decision-making. During a nuclear event, individuals might be exposed to a mixed field of neutrons and photons. The composition of the field and the neutron energy spectrum influence the degree of damage to the chromosomes. During the transatlantic BALANCE project, an exposure similar to a Hiroshima-like device at a distance of 1.5 km from the epicenter was simulated, and biological dosimetry based on dicentric chromosomes was performed to evaluate the participants ability to discover unknown doses and to test the influence of differences in neutron spectra. In a first step, calibration curves were established by irradiating blood samples with 5 doses in the range of 0-4 Gy at two different facilities in Germany (Physikalisch-Technische Bundesanstalt [PTB]) and the USA (the Columbia IND Neutron Facility [CINF]). The samples were sent to eight participating laboratories from the RENEB network and dicentric chromosomes were scored by each participant. Next, blood samples were irradiated with 4 blind doses in each of the two facilities and sent to the participants to provide dose estimates based on the established calibration curves. Manual and semiautomatic scoring of dicentric chromosomes were evaluated for their applicability to neutron exposures. Moreover, the biological effectiveness of the neutrons from the two irradiation facilities was compared. The calibration curves from samples irradiated at CINF showed a 1.4 times higher biological effectiveness compared to samples irradiated at PTB. For manual scoring of dicentric chromosomes, the doses of the test samples were mostly successfully resolved based on the calibration curves established during the project. For semiautomatic scoring, the dose estimation for the test samples was less successful. Doses &gt;2 Gy in the calibration curves revealed nonlinear associations between dose and dispersion index of the dicentric counts, especially for manual scoring. The differences in the biological effectiveness between the irradiation facilities suggested that the neutron energy spectrum can have a strong impact on the dicentric counts., (© 2023 S. Karger AG, Basel.)

In the context of improving radiation therapy, high-atomic number (Z) metallic nanoparticles and, more importantly, gold-based nanostructures are developed as radiation enhancers/radiosensitizers. Due to the diversity of cell lines, nanoparticles, as well as radiation types or doses, the resulting biological effects may differ and remain obscure. In this multiparameter study, we aim to shed light on these effects and investigate them further by employing X-irradiation and three human cancer cell lines (PC3, A549, and U2OS cells) treated by multiple techniques. TEM experiments on PC3 cells showed that citrate-capped AuNPs were found to be located mostly in membranous structures/vesicles or autophagosomes, but also, in the case of PEG-capped AuNPs, inside the nucleus as well. The colony-forming capability of cancer cells radiosensitized by AuNPs decreased significantly and the DNA damage detected by cytogenetics, γH2AX immunostaining, and by single (γH2AX) or double (γH2AX and OGG1) immunolocalization via transmission electron microscopy (TEM) was in many cases higher and/or persistent after combination with AuNPs than upon individual exposure to ionizing radiation (IR). Moreover, different cell cycle distribution was evident in PC3 but not A549 cells after treatment with AuNPs and/or irradiation. Finally, cellular senescence was investigated by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13) which showed that based on the AuNPs' concentration, an increased number of senescent cells might be observed after exposure to IR. Even though different cell lines or different types and concentrations of AuNPs may alter the levels of radiosensitization, our results imply that the complexity of damage might also be an important factor of AuNP-induced radiosensitization., Competing Interests: The authors declare no conflict of interest.

The new and increasingly studied concept of immunogenic cell death (ICD) revealed a previously unknown perspective of the various regulated cell death (RCD) modalities, elucidating their immunogenic properties and rendering obsolete the notion that immune stimulation is solely the outcome of necrosis. A distinct characteristic of ICD is the release of danger-associated molecular patterns (DAMPs) by dying and/or dead cells. Thus, several members of the DAMP family, such as the well-characterized heat shock proteins (HSPs) HSP70 and HSP90, the high-mobility group box 1 protein and calreticulin, and the thymic polypeptide prothymosin α (proTα) and its immunoreactive fragment proTα(100-109), are being studied as potential diagnostic tools and/or possible therapeutic agents. Here, we present the basic aspects and mechanisms of both ICD and other immunogenic RCD forms; denote the role of DAMPs in ICD; and further exploit the relevance of human proTα and proTα(100-109) in ICD, highlighting their possible clinical applications. Furthermore, we present the preliminary results of our in vitro studies, which show a direct correlation between the concentration of proTα/proTα(100-109) and the levels of cancer cell apoptosis, induced by anticancer agents and γ-radiation.

While rapid technological advances in radiotherapy techniques have led to a more precise delivery of radiation dose and to a decreased risk of side effects, there is still a need to evaluate the efficacy of the new techniques estimating the biological dose and to investigate the radiobiological impact of the protracted radiotherapy treatment duration. The aim of this study is to compare, at a cytogenetic level, advanced radiotherapy techniques VMAT and IMRT with the conventional 3D-CRT, using biological dosimetry. A dicentric biodosimetry assay based on the frequency of dicentrics chromosomes scored in peripheral blood lymphocytes from prostate cancer patients and PC3 human prostate cancer cell line was used. For each patient blood sample and each subpopulation of the cultured cell line, three different irradiations were performed using the 3D-CRT, IMRT, and VMAT technique. The absorbed dose was estimated with the biodosimetry method based on the induced dicentric chromosomes. The results showed a statistically significant underestimation of the biological absorbed dose of ~6% for the IMRT and VMAT compared to 3D-CRT irradiations for peripheral blood lymphocytes, whereas IMRT and VMAT results were comparable without a statistically significant difference, although slightly lower values were observed for VMAT compared to IMRT irradiation. Similar results were obtained using the PC3 cell line. The observed biological dose underestimation could be associated with the relative decreased dose rate and increase irradiation time met in modulated techniques compared to the conventional 3D-CRT irradiations.

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.

Purpose: Biological and/or physical assays for retrospective dosimetry are valuable tools to recover the exposure situation and to aid medical decision making. To further validate and improve such biological and physical assays, in 2019, EURADOS Working Group 10 and RENEB performed a field exercise in Lund, Sweden, to simulate various real-life exposure scenarios., Materials and Methods: For the dicentric chromosome assay (DCA), blood tubes were located at anthropomorphic phantoms positioned in different geometries and were irradiated with a 1.36 TBq 192 Ir-source. For each exposure condition, dose estimates were provided by at least one laboratory and for four conditions by 17 participating RENEB laboratories. Three radio-photoluminescence glass dosimeters were placed at each tube to assess reference doses., Results: The DCA results were homogeneous between participants and matched well with the reference doses (≥95% of estimates within ±0.5 Gy of the reference). For samples close to the source systematic underestimation could be corrected by accounting for exposure time. Heterogeneity within and between tubes was detected for reference doses as well as for DCA doses estimates., Conclusions: The participants were able to successfully estimate the doses and to provide important information on the exposure scenarios under conditions closely resembling a real-life situation.

The importance of nontargeted (systemic) effects of ionizing radiation is attracting increasing attention. Exploiting synchrotron radiation generated by the Imaging and Medical Beamline at the Australian Synchrotron, we studied radiation-induced nontargeted effects in C57BL/6 mice. Mice were locally irradiated with a synchrotron X-ray broad beam and a multiplanar microbeam radiotherapy beam. To assess the influence of the beam configurations and variations in peak dose and irradiated area in the response of normal tissues outside the irradiated field at 1 and 4 days after irradiation, we monitored oxidatively induced clustered DNA lesions (OCDL), DNA double-strand breaks (DSB), apoptosis, and the local and systemic immune responses. All radiation settings induced pronounced persistent systemic effects in mice, which resulted from even short exposures of a small irradiated area. OCDLs were elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, there was a trend for elevated apoptotic cell death under most irradiation conditions; however, DSBs were elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in plasma concentrations of macrophage-derived cytokine, eotaxin, IL10, TIMP1, VEGF, TGFβ1, and TGFβ2, along with changes in tissues in frequencies of macrophages, neutrophils, and T lymphocytes. Overall, our findings have implications for the planning of therapeutic and diagnostic radiation treatments to reduce the risk of radiation-related adverse systemic effects. Cancer Res; 77(22); 6389-99. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: To establish a training data set of digital images and to investigate the scoring criteria and dose assessment of the dicentric assay within the European network of biodosimetry (RENEB), a web based scoring inter-comparison was undertaken by 17 RENEB partners., Materials and Methods: Two sets of 50 high resolution images were uploaded onto the RENEB website. One set included metaphases after a moderate exposure (1.3 Gy) and the other set consisted of metaphases after a high dose exposure (3.5 Gy). The laboratories used their own calibration curves for estimating doses based on observed aberration frequencies., Results: The dose estimations and 95% confidence limits were compared to the actual doses and the corresponding z-values were satisfactory for the majority; only the dose estimations from two laboratories were too low or too high. The coefficients of variation were 17.6% for the moderate and 11.2% for the high dose. Metaphases with controversial results could be identified for training purposes., Conclusions: Overall, the web based scoring of the two galleries by the 17 laboratories produced very good results. Application of web based scoring for the dicentric assay may therefore be a relevant strategy for an operational biodosimetry assistance network.

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event., Materials and Methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners., Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes)., Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested.

Purpose: Reliable dose estimation is an important factor in appropriate dosimetric triage categorization of exposed individuals to support radiation emergency response., Materials and Methods: Following work done under the EU FP7 MULTIBIODOSE and RENEB projects, formal methods for defining uncertainties on biological dose estimates are compared using simulated and real data from recent exercises., Results: The results demonstrate that a Bayesian method of uncertainty assessment is the most appropriate, even in the absence of detailed prior information. The relative accuracy and relevance of techniques for calculating uncertainty and combining assay results to produce single dose and uncertainty estimates is further discussed., Conclusions: Finally, it is demonstrated that whatever uncertainty estimation method is employed, ignoring the uncertainty on fast dose assessments can have an important impact on rapid biodosimetric categorization.

Purpose: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios., Results: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015., Conclusions: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects.

Purpose: The European Network of Biological and Physical Retrospective Dosimetry 'RENEB' has contributed to European radiation emergency preparedness. To give homogeneous dose estimation results, RENEB partners must harmonize their processes., Materials and Methods: A first inter-comparison focused on biological and physical dosimetry was used to detect the outliers in terms of dose estimation. Subsequently, trainings were organized to improve both tools dose estimation. A second inter-comparison was performed to validate training efficiency. Simultaneously, based on ISO standards, a QA&QM manual on all dosimetry assays was produced which states a common basis and harmonized procedures for each assay. The evaluation of the agreement of RENEB partners to follow the QA&QM manual was performed through a questionnaire. The integration of new members into the network was carried out in the same way, whatever the assays., Results: The training courses on biological and physical dosimetry were judged to be successful because most of the RENEB members' dose estimates improved in the second inter-comparison. The QA&QM manual describes the consensus for the minimum requirements and the performance criteria for both dosimetry assays. The questionnaire revealed that the whole network capacity currently can manage between 15 and 3800 samples once., Conclusion: The methodology used to harmonize all dosimetry practice within the network RENEB was highly successful. The network is operational to manage a mass casualty radiation accident for immediate dose assessment.

Purpose: To identify and assess, among the participants in the RENEB (Realizing the European Network of Biodosimetry) project, the emergency preparedness, response capabilities and resources that can be deployed in the event of a radiological or nuclear accident/incident affecting a large number of individuals. These capabilities include available biodosimetry techniques, infrastructure, human resources (existing trained staff), financial and organizational resources (including the role of national contact points and their articulation with other stakeholders in emergency response) as well as robust quality control/assurance systems., Materials and Methods: A survey was prepared and sent to the RENEB partners in order to acquire information about the existing, operational techniques and infrastructure in the laboratories of the different RENEB countries and to assess the capacity of response in the event of radiological or nuclear accident involving mass casualties. The survey focused on several main areas: laboratory's general information, country and staff involved in biological and physical dosimetry; retrospective assays used, the number of assays available per laboratory and other information related to biodosimetry and emergency preparedness. Following technical intercomparisons amongst RENEB members, an update of the survey was performed one year later concerning the staff and the available assays., Conclusions: The analysis of RENEB questionnaires allowed a detailed assessment of existing capacity of the RENEB network to respond to nuclear and radiological emergencies. This highlighted the key importance of international cooperation in order to guarantee an effective and timely response in the event of radiological or nuclear accidents involving a considerable number of casualties. The deployment of the scientific and technical capabilities existing within the RENEB network members seems mandatory, to help other countries with less or no capacity for biological or physical dosimetry, or countries overwhelmed in case of a radiological or nuclear accident involving a large number of individuals.