International Comparison Exercise for Biological Dosimetry after Exposures with Neutrons Performed at Two Irradiation Facilities as Part of the BALANCE Project.

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 >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.

Resilience after a nuclear accident: readiness in using mobile phone applications to measure radiation and health indicators in various groups (SHAMISEN SINGS project).

An anonymous web-based survey was developed to check different aspects (SHAMISEN SINGS project): stakeholder awareness and perceptions of available mobile applications (apps) for measuring ionising radiation doses and health/well-being indicators; whether they would be ready to use them in the post-accidental recovery; and what are their preferred methodologies to acquire information etc. The results show that participation of the citizens would be most beneficial during post-accident recovery, providing individual measurements of external ionizing dose and health/well-being parameters, with possible follow-up. Also, participants indicated different preferences for sources to gain knowledge on ionising radiation and for the functions that an ideal app should have. The level of awareness and readiness to use apps to measure ionising radiation dose depended on two main aspects: individual differences (age & gender) and whether people were from countries affected by the previous major accidents. We concluded that stakeholders could have benefits from the data management plan: (1) it potentiates resilience at individual and community level; (2) citizens' measurements contribute to environmental monitoring and public health screening; (3) linkages between different types of data (environmental exposure, individual behavioural diaries, and measurements of health indicators) allow to perform more rigorous epidemiological studies.

Cytogenetic effects of radioiodine therapy: a 20-year follow-up study.

The purpose of this study was to compare cytogenetic data in a patient before and after treatment with radioiodine to evaluate the assays in the context of biological dosimetry. We studied a 34-year-old male patient who underwent a total thyroidectomy followed by ablation therapy with (131)I (19.28 GBq) for a papillary thyroid carcinoma. The patient provided blood samples before treatment and then serial samples at monthly intervals during the first year period and quarterly intervals for 5 years and finally 20 years after treatment. A micronucleus assay, dicentric assay, FISH method and G-banding were used to detect and measure DNA damage in circulating peripheral blood lymphocytes of the patient. The results showed that radiation-induced cytogenetic effects persisted for many years after treatment as shown by elevated micronuclei and chromosome aberrations as a result of exposure to (131)I. At 5 years after treatment, the micronucleus count was tenfold higher than the pre-exposure frequency. Shortly after the treatment, micronucleus counts produced a dose estimate of 0.47 ± 0.09 Gy. The dose to the patient evaluated retrospectively using FISH-measured translocations was 0.70 ± 0.16 Gy. Overall, our results show that the micronucleus assay is a retrospective biomarker of low-dose radiation exposure. However, this method is not able to determine local dose to the target tissue which in this case was any residual thyroid cells plus metastases of thyroidal origin.

A mouse model of cytogenetic analysis to evaluate caesium137 radiation dose exposure and contamination level in lymphocytes.

In case of external overexposure to ionizing radiation, an estimation of its genotoxic effects on exposed individuals can be made retrospectively by the measurement of radiation-induced chromosome aberrations on circulating lymphocytes. Compared with external irradiation, intakes of radionuclides may, however, lead to specific features influencing dose distribution at the scale of body, of tissue or even of cell. Therefore, in case of internal contamination by radionuclides, experimental studies, particularly using animal models, are required to better understand mechanisms of their genotoxic effects and to better estimate the absorbed dose. The present study was designed to evaluate a cytogenetic method in mouse peripheral blood lymphocytes that would allow determination of yields and complexities of chromosome aberrations after low-dose rate exposure to (137)Cs delivered in vitro either by irradiation or by contamination. By using M-FISH analysis, we compared the low-dose rate responses observed in mouse to the high-dose rate responses observed both in mouse and in human. Promising similarities between the two species in the relative biological effect evaluation show that our cytogenetic model established in mouse might be useful to evaluate various radiation exposures, particularly relevant in case of intakes of radionuclides.

Suitability of scoring PCC rings and fragments for dose assessment after high-dose exposures to ionizing radiation.

Assessment of radiation doses through measurement of dicentric chromosomes may be difficult due to the inability of damaged cells to reach mitosis. After high-dose exposures, premature chromosome condensation (PCC) has become an important method in biodosimetry. PCC can be induced upon fusion with mitotic cells, or by treatment with chemicals such as calyculin A or okadaic acid. Several different cytogenetic endpoints have been measured with chemically induced PCC, e.g., via scoring of extra chromosome pieces or ring chromosomes. The dose-effect curves published with chemically induced PCC show differences in their coefficients and in the distribution of rings among cells. Here we present a study with calyculin A to induce PCC in peripheral blood lymphocytes irradiated at nine different doses of γ-rays up to 20Gy. Colcemid was also added in order to observe metaphase cells. During microscopical analysis the chromosome aberrations observed in the different cell-cycle phases (G2/M-PCC, M/A-PCC and M cells) were recorded. The proportion of G2/M-PCC cells was predominant from 3 to 20Gy, M cells decreased above 1Gy and M/A-PCC cells remained constant at all doses and showed the highest frequencies of PCC rings. Depending on the cell-cycle phase there was a difference in the linear coefficients in the dose-effect curves of extra fragments and rings. Poisson distribution among PCC rings was observed after calyculin A+colcemid treatment, facilitating the use of this methodology also for partial body exposures to high doses. This has been tested with two simulated partial exposures to 6 and 12Gy. The estimated doses in the irradiated fraction were very close to the real dose, indicating the usefulness of this methodology.

Manual versus automated γ-H2AX foci analysis across five European laboratories: can this assay be used for rapid biodosimetry in a large scale radiation accident?

The identification of severely exposed individuals and reassurance of the 'worried well' are of prime importance for initial triage following a large scale radiation accident. We aim to develop the γ-H2AX foci assay into a rapid biomarker tool for use in accidents. Here, five laboratories established a standard operating procedure and analysed 100 ex vivo γ-irradiated, 4 or 24h incubated and overnight-shipped lymphocyte samples from four donors to generate γ-H2AX reference data, using manual and/or automated foci scoring strategies. In addition to acute, homogeneous exposures to 0, 1, 2 and 4Gy, acute simulated partial body (4Gy to 50% of cells) and protracted exposures (4Gy over 24h) were analysed. Data from all laboratories could be satisfactorily fitted with linear dose response functions. Average yields observed at 4h post exposure were 2-4 times higher than at 24h and varied considerably between laboratories. Automated scoring caused larger uncertainties than manual scoring and was unable to identify partial exposures, which were detectable in manually scored samples due to their overdispersed foci distributions. Protracted exposures were detectable but doses could not be accurately estimated with the γ-H2AX assay. We conclude that the γ-H2AX assay may be useful for rapid triage following a recent acute radiation exposure. The potentially higher speed and convenience of automated relative to manual foci scoring needs to be balanced against its compromised accuracy and inability to detect partial body exposures. Regular re-calibration or inclusion of reference samples may be necessary to ensure consistent results between laboratories or over long time periods.

Assessment methods for inter-laboratory comparisons of the dicentric assay.

PURPOSE: To test the performance of different algorithms that can be used in inter-laboratory comparisons based on dicentric chromosome analysis, and to evaluate the impact of considering a priori values different to calculate individual laboratory performance based on the ionizing radiation dose estimation. METHODS: Mean and standard deviation estimations in inter-laboratory comparisons are tested on simulated data and data from previously published inter-laboratory comparisons using three robust algorithms, Algorithm A, Algorithm B and Q/Hampel, all programmed in R-project language and implemented in a Shiny application. The simulated data were generated assuming three different probabilities to contaminate inter-laboratory comparisons samples with atypical dose values. Comparison between different algorithms was also done using published exercises where blood samples were irradiated at 0 and 0.7 Gy that represent a challenge for the assessment of an inter-laboratory comparison. RESULTS: The best performance was obtained with the Q/Hampel algorithm for the estimation of the dose mean and with the Algorithm B for the estimation of the dose standard deviation under the conditions tested in the simulations. The Q/Hampel algorithm showed the best performance when non-irradiated samples were evaluated and there was a high proportion of identical values. The presence identical values cause the Algorithm B to fail. Real examples illustrating the need to consider standard deviation priors, and the need to use algorithms resistant to a high proportion of identical values are presented. CONCLUSIONS: Q/Hampel algorithm is a serious candidate to estimate the dose mean in the inter-laboratory comparisons, and to estimate both parameters when the proportion of identical values equals or higher than the half of the results. When the proportion of identical values is less than the half of the results, the Algorithm B should be considered as a candidate to estimate the standard deviation in the inter-laboratory comparisons with small number of laboratories. We remark that special attention is needed to establish prior definitions of standard deviation in the assessment of inter-laboratory dicentric assay comparisons.

Evaluation of γ-H2AX foci distribution among different peripheral blood mononucleated cell subtypes.

INTRODUCTION: The detection of γ-H2AX foci in peripheral blood mononucleated cells (PBMCs) has been incorporated as an early assay for biological dosimetry. However, overdispersion in the γ-H2AX foci distribution is generally reported. In a previous study from our group, it was suggested that overdispersion could be caused by the fact that when evaluating PBMCs, different cell subtypes are analyzed, and that these could differ in their radiosensitivity. This would cause a mixture of different frequencies that would result in the overdispersion observed. OBJECTIVES: The objective of this study was to evaluate both the possible differences in the radiosensitivities of the different cell subtypes present in the PBMCs and to evaluate the distribution of γ-H2AX foci in each cell subtype. MATERIALS AND METHODS: Peripheral blood samples from three healthy donors were obtained and total PBMCs, and CD3+, CD4+, CD8+, CD19+, and CD56+ cells were separated. Cells were irradiated with 1 and 2 Gy and incubated at 37 °C for 1, 2, 4, and 24 h. Sham-irradiated cells were also analyzed. γ-H2AX foci were detected after immunofluorescence staining and analyzed automatically using a Metafer Scanning System. For each condition, 250 nuclei were considered. RESULTS: When the results from each donor were compared, no observable significant differences between donors were observed. When the different cell subtypes were compared, CD8+ cells showed the highest mean of γ-H2AX foci in all post-irradiation time points. The cell type that showed the lowest γ-H2AX foci frequency was CD56+. The frequencies observed in CD4+ and CD19+ cells fluctuated between CD8+ and CD56+ without any clear pattern. For all cell types evaluated, and at all post-irradiation times, overdispersion in γ-H2AX foci distribution was significant. Independent of the cell type evaluated the value of the variance was four times greater than that of the mean. CONCLUSION: Although different PBMC subsets studied showed different radiation sensitivity, these differences did not explain the overdispersion observed in the γ-H2AX foci distribution after exposure to IR.

Biodose Tools: an R shiny application for biological dosimetry.

INTRODUCTION: In the event of a radiological accident or incident, the aim of biological dosimetry is to convert the yield of a specific biomarker of exposure to ionizing radiation into an absorbed dose. Since the 1980s, various tools have been used to deal with the statistical procedures needed for biological dosimetry, and in general those who made several calculations for different biomarkers were based on closed source software. Here we present a new open source program, Biodose Tools, that has been developed under the umbrella of RENEB (Running the European Network of Biological and retrospective Physical dosimetry). MATERIALS AND METHODS: The application has been developed using the R programming language and the shiny package as a framework to create a user-friendly online solution. Since no unique method exists for the different mathematical processes, several meetings and periodic correspondence were held in order to reach a consensus on the solutions to be implemented. RESULTS: The current version 3.6.1 supports dose-effect fitting for dicentric and translocation assay. For dose estimation Biodose Tools implements those methods indicated in international guidelines and a specific method to assess heterogeneous exposures. The app can include information on the irradiation conditions to generate the calibration curve. Also, in the dose estimate, information about the accident can be included as well as the explanation of the results obtained. Because the app allows generating a report in various formats, it allows traceability of each biological dosimetry study carried out. The app has been used globally in different exercises and training, which has made it possible to find errors and improve the app itself. There are some features that still need consensus, such as curve fitting and dose estimation using micronucleus analysis. It is also planned to include a package dedicated to interlaboratory comparisons and the incorporation of Bayesian methods for dose estimation. CONCLUSION: Biodose Tools provides an open-source solution for biological dosimetry laboratories. The consensus reached helps to harmonize the way in which uncertainties are calculated. In addition, because each laboratory can download and customize the app's source code, it offers a platform to integrate new features.

Differential Radiosensitizing Effect of 50 nm Gold Nanoparticles in Two Cancer Cell Lines.

Radiation therapy is widely used as an anti-neoplastic treatment despite the adverse effects it can cause in non-tumoral tissues. Radiosensitizing agents, which can increase the effect of radiation in tumor cells, such as gold nanoparticles (GNPs), have been described. To evaluate the radiosensitizing effect of 50 nm GNPs, we carried out a series of studies in two neoplastic cell lines, Caco2 (colon adenocarcinoma) and SKBR3 (breast adenocarcinoma), qualitatively evaluating the internalization of the particles, determining with immunofluorescence the number of γ-H2AX foci after irradiation with ionizing radiation (3 Gy) and evaluating the viability rate of both cell lines after treatment by means of an MTT assay. Nanoparticle internalization varied between cell lines, though they both showed higher internalization degrees for functionalized GNPs. The γ-H2AX foci counts for the different times analyzed showed remarkable differences between cell lines, although they were always significantly higher for functionalized GNPs in both lines. Regarding cell viability, in most cases a statistically significant decreasing tendency was observed when treated with GNPs, especially those that were functionalized. Our results led us to conclude that, while 50 nm GNPs induce a clear radiosensitizing effect, it is highly difficult to describe the magnitude of this effect as universal because of the heterogeneity found between cell lines.

Lessons from past radiation accidents: Critical review of methods addressed to individual dose assessment of potentially exposed people and integration with medical assessment.

The experiences of the Chernobyl and Fukushima nuclear accidents showed that dosimetry was the essential tool in the emergency situation for decision making processes, such as evacuation and application of protective measures. However, at the consequent post-accidental phases, it was crucial also for medical health surveillance and in further adaptation to changed conditions with regards to radiation protection of the affected populations. This review provides an analysis of the experiences related to the role of dosimetry (dose measurements, assessment and reconstruction) regarding health preventive measures in the post-accidental periods on the examples of the major past nuclear accidents such as Chernobyl and Fukushima. Recommendations derived from the review are called to improve individual dose assessment in case of a radiological accident/incident and should be considered in advance as guidelines to follow for having better information. They are given as conclusions.

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

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.

RENEB/EURADOS field exercise 2019: robust dose estimation under outdoor conditions based on the dicentric chromosome assay.

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 192Ir-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.

Chromosomal aberration dynamics through the cell cycle.

DNA double-strand breaks are the crucial lesions underlying the formation of chromosomal aberrations, their formation and kinetics have been extensively studied, although dynamics of the repair process has not been fully understood. By using a combination of different cytogenetic techniques to analyze cells in G0, G2 and M phase, in the present study we perform a follow up study of the dynamics of different radiation induced chromosomal aberrations. Data here presented show that in G0 phase chromosome fragments lacking telomere signals (incomplete chromosome elements, ICE) show a slow repair, but when repair occurs tend to reconstitute the original chromosomes, and those that do not repair seem to be selected by interphase cell death and cell cycle checkpoints. In contrast, complete chromosome aberrations, as dicentrics, show a very fast formation kinetics. Similar frequencies of dicentrics were observed in G0, G2 and M cells, indicating that this chromosome-type of aberration can progress through the cell cycle without negative selection. Our study reinforce the hypothesis that ICE are strongly negatively selected from G2 to M phase. However, the G2/M checkpoint seems to be not involved in this selection. The ICE frequencies observed after G2/M abrogation by caffeine are similar to the ones without abrogation, and clearly lower to the ones observed in G2.

Redox Status, Dose and Antioxidant Intake in Healthcare Workers Occupationally Exposed to Ionizing Radiation.

The purpose of this study was to evaluate the relationship between blood redox status, dose and antioxidant dietary intake of different hospital staff groups exposed to low doses of ionizing radiation (LDIR) (Interventional Radiology and Cardiology, Radiation Oncology, and Nuclear Medicine) and non-exposed. Personal dose equivalent (from last year and cumulative), plasma antioxidant markers (total antioxidant capacity, extracellular superoxide dismutase activity, and glutathione/oxidized glutathione ratio), oxidative stress markers (nitrites and nitrates, and lipid peroxidation) and dietary intake (antioxidant capacity using ORAC values) were collected and analyzed from 28 non-exposed healthcare workers and 42 healthcare workers exposed to LDIR. Hospital staff exposed to LDIR presented a redox imbalance in blood that seems to correlate with dose. Workers from the Nuclear Medicine Unit were the most affected group with the lowest value of plasma antioxidant response and the highest value of plasma thiobarbituric acid reactive substances, TBARS (indicator of lipid peroxidation) of all four groups. Cumulative personal dose equivalent positively correlated with nitrites and negatively correlated with total antioxidant capacity in blood. The diet of healthcare workers from Nuclear Medicine Unit had higher ORAC values than the diet of non-exposed. Therefore, occupational exposure to LDIR, especially for the Nuclear Medicine Unit, seems to produce an imbalanced redox status in blood that would correlate with cumulative personal dose equivalent.

Persistence of radiation-induced chromosome aberrations in a long-term cell culture.

The aim of the present study was to evaluate the persistence of chromosome aberrations induced by X rays. FISH painting and mFISH techniques were applied to long-term cultures of irradiated cells. With painting, at 2 Gy the frequency of apparently simple translocations remained almost invariable during all the culture, whereas at 4 Gy a rapid decline was observed between the first and the second samples, followed by a slight decrease until the end of the culture. Apparently simple dicentrics and complex aberrations disappeared after the first sample at 2 and 4 Gy. By mFISH, at 2 Gy the frequency of complete plus one-way translocations remained invariable between the first and last sample, but at 4 Gy a 60% decline was observed. True incomplete simple translocations disappeared at 2 and 4 Gy, indicating that incompleteness could be a factor to consider when the persistence of translocations is analyzed. The analysis by mFISH showed that the frequency of complex aberrations and their complexity increased with dose and tended to disappear in the last sample. Our results indicate that the influence of dose on the decrease in the frequency of simple translocations with time postirradiation cannot be fully explained by the disappearance of true incomplete translocations and complex aberrations. The chromosome involvement was random for radiation-induced exchange aberrations and non-random for total aberrations. Chromosome 7 showed the highest deviations from expected, being less and more involved than expected in the first and last samples, respectively. Some preferential chromosome-chromosome associations were observed, including a coincidence with a cluster from radiogenic chromosome aberrations described in other studies.

Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint.

Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of gamma-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

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

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

Polymorphisms in MDM2 and TP53 Genes and Risk of Developing Therapy-Related Myeloid Neoplasms.

One of the most severe complications after successful cancer therapy is the development of therapy-related myeloid neoplasms (t-MN). Constitutional genetic variation is likely to impact on t-MN risk. We aimed to evaluate if polymorphisms in the p53 pathway can be useful for predicting t-MN susceptibility. First, an association study revealed that the Pro variant of the TP53 Arg72Pro polymorphism and the G allele of the MDM2 SNP309 were associated with t-MN risk. The Arg variant of TP53 is more efficient at inducing apoptosis, whereas the Pro variant is a more potent inductor of cell cycle arrest and DNA repair. As regards MDM2 SNP309, the G allele is associated with attenuation of the p53 apoptotic response. Second, to evaluate the biological effect of the TP53 polymorphism, we established Jurkat isogenic cell lines expressing p53Arg or p53Pro. Jurkat p53Arg cells presented higher DNA damage and higher apoptotic potential than p53Pro cells, after treatment with chemotherapy agents. Only p53Pro cells presented t(15;17) translocation and del(5q). We suggest that failure to repair DNA lesions in p53Arg cells would lead them to apoptosis, whereas some p53Pro cells, prone to cell cycle arrest and DNA repair, could undergo misrepair, generating chromosomal abnormalities typical of t-MN.

Cytogenetic damage induced by radiotherapy. Evaluation of protection by amifostine and analysis of chromosome aberrations persistence.

PURPOSE: To evaluate the cytogenetic damage induced by radiotherapy, the effect of concomitant amifostine and the persistence of translocations and dicentrics after the treatment. MATERIALS AND METHODS: Blood samples from 16 head and neck cancer patients were obtained at different times, just before treatment, at the 1st and 22nd sessions, at the end of radiotherapy, and one, four and 12 months later. Solid stain and fluorescent in situ hybridization (FISH) techniques were applied to analyse chromosome aberrations. RESULTS: In all the analysis the frequencies of dicentrics plus rings were slightly lower in the group of patients receiving concomitant amifostine, but in each sampling point the differences were not significant. The persistence of translocations and dicentrics one year after radiotherapy was very similar, with a decline of more than 50%. For all the chromosome aberrations considered, a negative correlation between their initial yield and the percentage of this yield remained 12 months after radiotherapy was observed (p < 0.05). CONCLUSION: No significant protection by amifostine against radiation-induced chromosome damage was observed in head and neck cancer patients treated only with radiotherapy. In these cases, the persistence of translocations and dicentrics during the first year after radiotherapy is similar and related to their initial yield.