Home-made low-cost dosemeter for photon dose measurements in radiobiological experiments and for education in the field of radiation sciences.

Reliable dosimetry systems are crucial for radiobiological experiments either to quantify the biological consequences of ionizing radiation or to reproduce results by other laboratories. Also, they are essential for didactic purposes in the field of radiation research. Professional dosemeters are expensive and difficult to use in exposure facilities with closed exposure chambers. Consequently, a simple, inexpensive, battery-driven dosemeter was developed that can be easily built using readily available components. Measurements were performed to validate its readout with photons of different energy and dose rate and to demonstrate the applicability of the dosemeter. It turned out that the accuracy of the dose measurements using the developed dosemeter was better than 10%, which is satisfactory for radiobiological experiments. It is concluded that this dosemeter can be used both for determining the dose rates of an exposure facility and for educational purposes.

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.

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.

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.

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.

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.

Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy-RC) and nine cervical cancer patients (treatment with radiotherapy alone-R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

Analysis of the Applicability of microRNAs in Peripheral Blood Leukocytes as Biomarkers of Sensitivity and Exposure to Fractionated Radiotherapy towards Breast Cancer.

Biomarkers for predicting individual response to radiation and for dose verification are needed to improve radiotherapy. A biomarker should optimally show signal fidelity, meaning that its level is stable and proportional to the absorbed dose. miRNA levels in human blood serum were suggested as promising biomarkers. The aim of the present investigation was to test the miRNA biomarker in leukocytes of breast cancer patients undergoing external beam radiotherapy. Leukocytes were isolated from blood samples collected prior to exposure (control); on the day when a total dose of 2 Gy, 10 Gy, or 20 Gy was reached; and one month after therapy ended (46-50 Gy in total). RNA sequencing was performed and univariate analysis was used to analyse the effect of the radiation dose on the expression of single miRNAs. To check if combinations of miRNAs can predict absorbed dose, a multinomial logistic regression model was built using a training set from eight patients (representing 40 samples) and a validation set with samples from the remaining eight patients (15 samples). Finally, Broadside, an explorative interaction mining tool, was used to extract sets of interacting miRNAs. The most prominently increased miRNA was miR-744-5p, followed by miR-4461, miR-34a-5p, miR-6513-5p, miR-1246, and miR-454-3p. Decreased miRNAs were miR-3065-3p, miR-103a-2-5p, miR-30b-3p, and miR-5690. Generally, most miRNAs showed a relatively strong inter-individual variability and different temporal patterns over the course of radiotherapy. In conclusion, miR-744-5p shows promise as a stable miRNA marker, but most tested miRNAs displayed individual signal variability which, at least in this setting, may exclude them as sensitive biomarkers of radiation response.

Small is beautiful: low activity alpha and gamma sources for small-scale radiation protection research experiments.

PURPOSE: Uncertainties regarding the magnitude of health effects following exposure to low doses of ionizing radiation remain a matter of concern both for professionals and for the public. There is consensus within the international radiation research community that more research is required on biological effects of radiation doses below 100 mGy applied at low dose rates. Moreover, there is a demand for increasing education and training of future radiation researchers and regulators. Research, education and training is primarily carried out at universities but university-based radiation research is often hampered by limited access to radiation sources. The aim of the present report is to describe small and cost-effective low activity gamma and alpha sources that can easily be installed and used in university laboratories. METHODS AND RESULTS: A gamma radiation source was made from an euxenite-(Y) rock (Y,Ca,Ce,U,Th)(Nb,Ta,Ti)2O6) that was found in an abandoned mine in Sweden. It allows exposing cells grown in culture dishes to radiation at a dose rate of 50 µGy/h and lower. Three alpha sources were custom-made and yield a dose rate of 1 mGy/h each. The construction, dosimetry and cellular effects of the sources are described. CONCLUSIONS: We hope that the report will stimulate research and training activities in the low dose field by facilitating access to radiation sources.

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

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

HER2-positive breast cancer that resists therapeutic drugs and ionizing radiation releases sphingomyelin-based molecules to circulating blood serum.

Breast cancer is the second most common cancer in the world based on incidence, reaching more than 2 million new cases in 2018, while continuing to increase. Invasive ductal carcinoma is the most common type of this cancer, making up approximately 70-80% of all breast cancer diagnoses. In particular, the type of breast cancer overexpressing human epidermal growth factor receptor 2 (HER2) has potential of strong proliferation, migration and invasion and early treatment is necessary. The authors identified and studied a single patient displaying complete therapeutic resistance to monoclonal anti-HER2 antibody therapy, chemotherapy and radiotherapy. A patient who exhibited resistance to postoperative adjuvant therapy after mastectomy was selected from HER2-positive breast cancer, and this patient had the grade of T4bN2aM0, Stage IIIB. The patient samples, blood serum and cancer tissue, were analyzed by metabolome and immunostaining technique, respectively. The characteristics of peripheral blood serum and solid tumor were investigated, aiming to find new serum biomarker(s) using the metabolomics technique. A correlation between the appearance of HER2-positive cancer tissue and serum concentration of the sphingomyelin family was found. In addition, HER2-positive tumor tissue in both the primary and recurrent cancer express the sphingomyelinase. These results suggest that sphingomyelins from this cancer tissue leads to therapy resistance, induction of invasion and strong proliferation.

Biological effectiveness of very high gamma dose rate and its implication for radiological protection.

Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for this is the uncertainty regarding the value of the dose rate effectiveness factor (DREF) used in radiological protection. While a LDR is defined as 0.1 mGy/min or lower, anything above that is seen as HDR. In cell and animal experiments, a dose rate around 1 Gy/min is usually used as representative for HDR. However, atomic bomb survivors, the reference cohort for radiological protection, were exposed to tens of Gy/min. The important question is whether gamma radiation delivered at very high dose rate (VHDR-several Gy/min) is more effective in inducing DNA damage than that delivered at HDR. The aim of this investigation was to compare the biological effectiveness of gamma radiation delivered at VHDR (8.25 Gy/min) with that of HDR (0.38 Gy/min or 0.79 Gy/min). Experiments were carried out with human peripheral mononuclear cells (PBMC) and the human osteosarcoma cell line U2OS. Endpoints related to DNA damage response were analysed. The results show that in PBMC, VHDR is more effective than HDR in inducing gene expression and micronuclei. In U2OS cells, the repair of 53BP1 foci was delayed after VHDR indicating a higher level of damage complexity, but no VHDR effect was observed at the level of micronuclei and clonogenic cell survival. We suggest that the DREF value may be underestimated when the biological effectiveness of HDR and LDR is compared.