Chromosomal damage, gene expression and alternative transcription in human lymphocytes exposed to mixed ionizing radiation as encountered in space.

Astronauts travelling in space will be exposed to mixed beams of particle radiation and photons. Exposure limits that correspond to defined cancer risk are calculated by multiplying absorbed doses by a radiation-type specific quality factor that reflects the biological effectiveness of the particle without considering possible interaction with photons. We have shown previously that alpha radiation and X-rays may interact resulting in synergistic DNA damage responses in human peripheral blood lymphocytes but the level of intra-individual variability was high. In order to assess the variability and validate the synergism, blood from two male donors was drawn at 9 time points during 3 seasons of the year and exposed to 0-2 Gy of X-rays, alpha particles or 1:1 mixture of both (half the dose each). DNA damage response was quantified by chromosomal aberrations and by mRNA levels of 3 radiation-responsive genes FDXR, CDKN1A and MDM2 measured 24 h post exposure. The quality of response in terms of differential expression of alternative transcripts was assessed by using two primer pairs per gene. A consistently higher than expected effect of mixed beams was found in both donors for chromosomal aberrations and gene expression with some seasonal variability for the latter. No synergy was detected for alternative transcription.

Minimum reporting standards about dosimetry of radiation sources used in radiation research studies.

The necessity of precise dosimetry and its documentation in research is less obvious than in medicine and in radiological protection. However, in radiation research, results can only be validated if experiments were carried out with sufficient precision and described with sufficient details, especially information regarding dosimetry. In order to ensure this, an initiative was launched to establish reproducible dosimetry reporting parameters in published studies. Minimum standards for reporting radiation dosimetry information were developed and published in parallel in the International Journal of Radiation Biology and Radiation Research. As editors of Radiation and Environmental Biophysics, we support this initiative and reproduce the agreed minimum irradiation parameters that should be reported in publications on radiation biology submitted to our journal.

Impact of fractionated cisplatin and radiation treatment on cell growth and accumulation of DNA damage in two normal cell types differing in origin.

Evidence on the impact of chemotherapy on radiotherapy-induced second malignant neoplasms is controversial. We estimated how cisplatin modulates the in vitro response of two normal cell types to fractionated radiation. AHH-1 lymphoblasts and VH10 fibroblasts were irradiated at 1 Gy/fraction 5 and 3 times per week during 12 and 19 days, respectively, and simultaneously treated with 0.1, 0.2, 0.4, 0.8, 1.7 and 3.3 µM of cisplatin twice a week. Cell growth during treatment was monitored. Cell growth/cell death and endpoints related to accumulation of DNA damage and, thus, carcinogenesis, were studied up to 21 days post treatment in cells exposed to radiation and the lowest cisplatin doses. Radiation alone significantly reduced cell growth. The impact of cisplatin alone below 3.3 µM was minimal. Except the lowest dose of cisplatin in VH10 cells, cisplatin reduced the inhibitory effect of radiation on cell growth. Delayed cell death was highest in the combination groups while the accumulation of DNA damage did not reveal a clear pattern. In conclusion, fractionated, concomitant exposure to radiation and cisplatin reduces the inhibitory effect of radiation on cell proliferation of normal cells and does not potentiate delayed effects resulting from accumulation of DNA damage.

Fluorescence in situ hybridisation for interphase chromosomal aberration-based biological dosimetry.

Metaphase spreads stained with Giemsa or painted with chromosome-specific probes by fluorescence in situ hybridisation (FISH) have been in use since long for retrospective dose assessment (biological dosimetry). However, in cases of accidental exposure to ionising radiation, the culturing of lymphocytes to obtain metaphase chromosomes and analysis of chromosomal aberrations is time-consuming and problematic after high radiation doses. Similarly, analysing chromosomal damage in G0/G1 cells or nondividing cells by premature chromosome condensation is laborious. Following large-scale radiological emergencies, the time required for analysis is more important than precision of dose estimate. Painting of whole chromosomes using chromosome-specific probes in interphase nuclei by the FISH technique will eliminate the time required for cell culture and allow a fast dose estimate, provided that a meaningful dose-response can be obtained by scoring the number of chromosomal domains visible in interphase nuclei. In order to test the applicability of interphase FISH for quick biological dosimetry, whole blood from a healthy donor was irradiated with 8 Gy of gamma radiation. Irradiated whole blood was kept for 2 h at 37°C to allow DNA repair and thereafter processed for FISH with probes specific for Chromosomes-1 and 2. Damaged chromosomal fragments, distinguished by extra color domains, were observed in interphase nuclei of lymphocytes irradiated with 8 Gy. These fragments were efficiently detected and quantified by the FISH technique utilising both confocal and single plane fluorescence microscopy. Furthermore, a clear dose-response curve for interphase fragments was achieved following exposure to 0, 1, 2, 4 and 8 Gy of gamma radiation. These results demonstrate interphase FISH as a promising test for biodosimetry and for studying cytogenetic effects of radiation in nondividing cells.

Mechanistic insights from high resolution DNA damage analysis to understand mixed radiation exposure.

Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.

The order of sequential exposure of U2OS cells to gamma and alpha radiation influences the formation and decay dynamics of NBS1 foci.

DNA double strand breaks (DSBs) are a deleterious form of DNA damage. Densely ionising alpha radiation predominantly induces complex DSBs and sparsely ionising gamma radiation-simple DSBs. We have shown that alphas and gammas, when applied simultaneously, interact in producing a higher DNA damage response (DDR) than predicted by additivity. The mechanisms of the interaction remain obscure. The present study aimed at testing whether the sequence of exposure to alphas and gammas has an impact on the DDR, visualised by live NBS1-GFP (green fluorescent protein) focus dynamics in U2OS cells. Focus formation, decay, intensity and mobility were analysed up to 5 h post exposure. Focus frequencies directly after sequential alpha → gamma and gamma → alpha exposure were similar to gamma alone, but gamma → alpha foci quickly declined below the expected values. Focus intensities and areas following alpha alone and alpha → gamma were larger than after gamma alone and gamma → alpha. Focus movement was most strongly attenuated by alpha → gamma. Overall, sequential alpha → gamma exposure induced the strongest change in characteristics and dynamics of NBS1-GFP foci. Possible explanation is that activation of the DDR is stronger when alpha-induced DNA damage precedes gamma-induced DNA damage.

The DNA damage response to radiological imaging: from ROS and γH2AX foci induction to gene expression responses in vivo.

Candidate ionising radiation exposure biomarkers must be validated in humans exposed in vivo. Blood from patients undergoing positron emission tomography-computed tomography scan (PET-CT) and skeletal scintigraphy (scintigraphy) was drawn before (0 h) and after (2 h) the procedure for correlation analyses of the response of selected biomarkers with radiation dose and other available patient information. FDXR, CDKN1A, BBC3, GADD45A, XPC, and MDM2 expression was determined by qRT-PCR, DNA damage (γH2AX) by flow cytometry, and reactive oxygen species (ROS) levels by flow cytometry using the 2', 7'-dichlorofluorescein diacetate test in peripheral blood mononuclear cells (PBMC). For ROS experiments, 0- and 2-h samples were additionally exposed to UVA to determine whether diagnostic irradiation conditioned the response to further oxidative insult. With some exceptions, radiological imaging induced weak γH2AX foci, ROS and gene expression fold changes, the latter with good coherence across genes within a patient. Diagnostic imaging did not influence oxidative stress in PBMC successively exposed to UVA. Correlation analyses with patient characteristics led to low correlation coefficient values. γH2AX fold change, which correlated positively with gene expression, presented a weak positive correlation with injected activity, indicating a radiation-induced subtle increase in DNA damage and subsequent activation of the DNA damage response pathway. The exposure discrimination potential of these biomarkers in the absence of control samples as frequently demanded in radiological emergencies, was assessed using raw data. These results suggest that the variability of the response in heterogeneous populations might complicate identifying individuals exposed to low radiation doses.

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.

Numerical Investigation of the Formation of a Failure Cone during the Pullout of an Undercutting Anchor.

Previously published articles on anchors have mainly focused on determining the pullout force of the anchor (depending on the strength parameters of the concrete), the geometric parameters of the anchor head, and the effective anchor depth. The extent (volume) of the so-called failure cone has often addressed as a secondary matter, serving only to approximate the size of the zone of potential failure of the medium in which the anchor is installed. For the authors of these presented research results, from the perspective of evaluating the proposed stripping technology, an important aspect was the determination of the extent and volume of the stripping, as well as the determination of why the defragmentation of the cone of failure favors the removal of the stripping products. Therefore, it is reasonable to conduct research on the proposed topic. Thus far, the authors have shown that the ratio of the radius of the base of the destruction cone to the anchorage depth is significantly larger than in concrete (~1.5) and ranges from 3.9-4.2. The purpose of the presented research was to determine the influence of rock strength parameters on the mechanism of failure cone formation, including, in particular, the potential for defragmentation. The analysis was conducted with the finite element method (FEM) using the ABAQUS program. The scope of the analysis included two categories of rocks, i.e., those with low compressive strength (<100 MPa) and strong rocks (>100 MPa). Due to the limitations of the proposed stripping method, the analysis was conducted for an effective anchoring depth limited to 100 mm. It was shown that for anchorage depths <100 mm, for rocks with high compressive strength (above 100 MPa), there is a tendency to spontaneously generate radial cracks, leading to the fragmentation of the failure zone. The results of the numerical analysis were verified by field tests, yielding convergent results regarding the course of the de-fragmentation mechanism. In conclusion, it was found that in the case of gray sandstones, with strengths of 50-100 MPa, the uniform type of detachment (compact cone of detachment) dominates, but with a much larger radius of the base (a greater extent of detachment on the free surface).

Modeling of dose and linear energy transfer homogeneity in cell nuclei exposed to alpha particles under various setup conditions.

PURPOSE: Different alpha exposure setups are often used to study the relation between biological responses and LET. This study aimed to estimate the dose heterogeneity and uncertainty in four exposure setups using Geant4 and PARTRAC codes. The importance of the irradiation system characteristics was shown in the context of reporting experimental results, especially in radiobiological studies at the molecular level. MATERIALS AND METHODS: Geant4 was used to estimate the dose distributions in cells grown on a disk exposed to alpha particles penetrating from above and below. The latter setup was simulated without and with a collimator. PARTRAC was used for the validation of Geant4 simulations based on distributions of the number of alpha particles penetrating a round nucleus and the deposited energy. RESULTS: The LET distributions obtained for simulated setups excluding the collimator were wide and non-Gaussian. Using a collimator resulted in a Gaussian LET distribution, but strongly reduced dose rate and dose homogeneity. Comparison between PARTRAC and Geant4 calculations for the cell nucleus exposed to alpha radiation showed an excellent agreement. CONCLUSIONS: The interpretation of results from radiobiological experiments with alpha particles should always cover the characteristics of the experimental setup, which can be done precisely with computational methods.

Short- and long-term effects of radiation exposure at low dose and low dose rate in normal human VH10 fibroblasts.

Introduction: Experimental studies complement epidemiological data on the biological effects of low doses and dose rates of ionizing radiation and help in determining the dose and dose rate effectiveness factor. Methods: Human VH10 skin fibroblasts exposed to 25, 50, and 100 mGy of 137Cs gamma radiation at 1.6, 8, 12 mGy/h, and at a high dose rate of 23.4 Gy/h, were analyzed for radiation-induced short- and long-term effects. Two sample cohorts, i.e., discovery (n = 30) and validation (n = 12), were subjected to RNA sequencing. The pool of the results from those six experiments with shared conditions (1.6 mGy/h; 24 h), together with an earlier time point (0 h), constituted a third cohort (n = 12). Results: The 100 mGy-exposed cells at all abovementioned dose rates, harvested at 0/24 h and 21 days after exposure, showed no strong gene expression changes. DMXL2, involved in the regulation of the NOTCH signaling pathway, presented a consistent upregulation among both the discovery and validation cohorts, and was validated by qPCR. Gene set enrichment analysis revealed that the NOTCH pathway was upregulated in the pooled cohort (p = 0.76, normalized enrichment score (NES) = 0.86). Apart from upregulated apical junction and downregulated DNA repair, few pathways were consistently changed across exposed cohorts. Concurringly, cell viability assays, performed 1, 3, and 6 days post irradiation, and colony forming assay, seeded just after exposure, did not reveal any statistically significant early effects on cell growth or survival patterns. Tendencies of increased viability (day 6) and reduced colony size (day 21) were observed at 12 mGy/h and 23.4 Gy/min. Furthermore, no long-term changes were observed in cell growth curves generated up to 70 days after exposure. Discussion: In conclusion, low doses of gamma radiation given at low dose rates had no strong cytotoxic effects on radioresistant VH10 cells.

Cell Type-Specific Patterns in the Accumulation of DNA Damage Following Multifractional Radiation Exposure.

Predicting the risk of second malignant neoplasms is complicated by uncertainties regarding the shape of the dose-response relationship at high doses. Limited understanding of the competitive relationship between cell killing and the accumulation of DNA lesions at high doses, as well as the effects of other modulatory factors unique to radiation exposure during radiotherapy, such as dose heterogeneity across normal tissue and dose fractionation, contribute to these uncertainties. The aim of this study was to analyze the impact of fractionated irradiations on two cell systems, focusing on the endpoints relevant for cancer induction. To simulate the heterogeneous dose distribution across normal tissue during radiotherapy, exponentially growing VH10 fibroblasts and AHH-1 lymphoblasts were irradiated with 9 and 12 fractions (VH10) and 10 fractions (AHH-1) at 0.25, 0.5, 1, or 2 Gy per fraction. The effects on cell growth, cell survival, radiosensitivity and the accumulation of residual DNA damage lesions were analyzed as functions of dose per fraction and the total absorbed dose. Residual γH2AX foci and other DNA damage markers (micronuclei, nuclear buds, and giant nuclei) were accumulated at high doses in both cell types, but in a cell type-dependent manner. The competitive relationship between cell killing and the accumulation of carcinogenic DNA damage following multifractional radiation exposure is cell type-specific.

The 2020 MELODI workshop on the effects of spatial and temporal variation in dose delivery.

A key activity of MELODI is to organise annual European meetings where scientific results and future directions and strategies of relevant research are discussed. The annual meetings, previously organised solely under the auspices of MELODI are, since 2016, jointly organised by the European platforms and referred to as European Radiation Protection Weeks (ERPW). In addition to ERPW meetings, MELODI organises and finances annual workshops dedicated to specific topics. Outputs and recommendations from the meetings are published as review articles. The 2020 workshop focussed on one of the cross cutting topics: the effects of spatial and temporal variation in dose delivery on disease risk. The current issue of REBS includes five review articles from the workshop on the effects of spatial and temporal variation in dose delivery and this editorial is a short summary of their content.

Hypothermia differentially modulates the formation and decay of NBS1, γH2AX and 53BP1 foci in U2OS cells exposed to gamma radiation.

In studies on the mechanism of DNA damage response where ionizing radiation is used as the DNA damaging agent, cells are often exposed to ionizing radiation on melting ice (corresponding to 0.8 °C). The purpose of this procedure is to inhibit cellular processes i.e. DNA repair. Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage, but its mechanisms of action are poorly understood. The aim of the study was to analyze the effect of hypothermia at the level of formation and decay of NBS1, γH2AX, and 53BP1 foci, micronuclei, survival, cell cycle progression and oxidative stress in U2OS cells. The results show that hypothermia alone induced oxidative stress and foci. When applied in combination with radiation but only during the exposure time, it potentiated the formation of γH2AX and 53BP1 but not of NBS1 foci. When applied during irradiation and subsequent repair time, 53BP1 and NBS1 foci formed and decayed, but the levels were markedly lower than when repair was carried out at 37 °C. The frequency of micronuclei was elevated in cells irradiated at 0.8 °C, but only when analysed 20 h after irradiation which is likely due to a reduced G2 cell cycle block. Hypothermia reduced cell survival, both with and without radiation exposure. The temperature effect should be considered when cooling cells on melting ice to inhibit DNA repair in the induction of DNA damage.

Determining the Effect of Rock Strength Parameters on the Breakout Area Utilizing the New Design of the Undercut/Breakout Anchor.

This paper presents the idea and provides an analysis of the rock breakout mechanism utilizing an undercut/breakout anchor. The new design is a modification of a standard undercut anchor, which is commonly found in applications involving steel-to-concrete anchorage. Of particular concern was the effect of the rock breakout strength on the anchor-pullout-induced failure of the rock mass. A numerical analysis was employed to model the effect of the changes to the shape and size of the breakout cones under varying rock strength conditions as a result of modifying the anchor design and loading pattern. The problem in question is pivotal for the potential evaluation of the effectiveness of the said anchor design under the non-standard conditions of its utilization.

Reflections on effects of low doses and risk inference based on the UNSCEAR 2021 report on 'biological mechanisms relevant for the inference of cancer risks from low-dose and low-dose-rate radiation'.

The 2021 United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) report summarises the knowledge on biological mechanisms of radiation action at low doses where, due to low statistical power of epidemiological investigations, the level of cancer risk must be inferred. It is the fourth UNSCEAR report since 1994 that looks into biological effects following low dose exposure with the aim of examining whether they support the assumption of the linear non-threshold (LNT) dose response for radiation-induced cancers. The conclusions of all four reports are affirmative. The new aspect of the 2021 report is that it focuses on the process of cancer risk inference. The aim of this article is to discuss the consequences of the conclusions regarding LNT and the possibilities of inferring risks from biological studies.

Dose-dependent expression of extracellular microRNAs in HCT116 colorectal cancer cells exposed to high-dose-rate ionising radiation.

Biomarkers of tumour response to radiotherapy may help optimise cancer treatment. The aim of the present study was to identify changes in extracellular microRNAs (miRNAs) as a biomarker of radiation-induced damage to human colorectal cancer cells. HCT116 cells were exposed to increasing doses of X-rays, and extracellular miRNAs were analysed by microarray. The results were correlated with the frequency of micronuclei. A total of 59 miRNAs with a positive correlation and 4 with a negative correlation between dose (up to 6 Gy) and extracellular miRNA expression were identified. In addition, for doses between 0 and 10 Gy, 12 miRNAs among those 59 miRNAs with a positive correlation were identified; for these extracellular miRNAs, a significantly positive correlation was observed between their expression and the frequency of micronuclei for doses up to 10 Gy. These results suggest that specific miRNAs may be considered as cell damage markers and may serve as secreted radiotherapy response biomarkers for colorectal cancer; however, the results must be further validated in serum samples collected from patients undergoing radiotherapy.