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The Health Effects of Cardiac Fluoroscopy and Modern Radiotherapy (photon and proton) in Pediatrics (HARMONIC) is a five-year project funded by the European Commission that aimed to improve the understanding of the long-term ionizing radiation (IR) risks for pediatric patients. In this paper, we provide a detailed overview of the rationale, design, and methods for the biological aspect of the project with objectives to provide a mechanistic understanding of the molecular pathways involved in the IR response and to identify potential predictive biomarkers of individual response involved in long-term health risks. Biological samples will be collected at three time points: before the first exposure, at the end of the exposure, and one year after the exposure. The average whole-body dose, the dose to the target organ, and the dose to some important out-of-field organs will be estimated. State-of-the-art analytical methods will be used to assess the levels of a set of known biomarkers and also explore high-resolution approaches of proteomics and miRNA transcriptomes to provide an integrated assessment. By using bioinformatics and systems biology, biological pathways and novel pathways involved in the response to IR exposure will be deciphered.

The Health Effects of Cardiac Fluoroscopy and Modern Radiotherapy (photon and proton) in Pediatrics (HARMONIC) is a five-year project funded by the European Commission that aimed to improve the understanding of the long-term ionizing radiation (IR) risks for pediatric patients. In this paper, we provide a detailed overview of the rationale, design, and methods for the biological aspect of the project with objectives to provide a mechanistic understanding of the molecular pathways involved in the IR response and to identify potential predictive biomarkers of individual response involved in long-term health risks. Biological samples will be collected at three time points: before the first exposure, at the end of the exposure, and one year after the exposure. The average whole-body dose, the dose to the target organ, and the dose to some important out-of-field organs will be estimated. State-of-the-art analytical methods will be used to assess the levels of a set of known biomarkers and also explore high-resolution approaches of proteomics and miRNA transcriptomes to provide an integrated assessment. By using bioinformatics and systems biology, biological pathways and novel pathways involved in the response to IR exposure will be deciphered.

Purpose The aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence. Materials and methods Different datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence. Results Simultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario. Conclusions This study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.

Nearly half of all cancers are treated with radiotherapy alone or in combination with other treatments, where damage to normal tissues is a limiting factor for the treatment. Radiotherapy-induced adverse health effects, mostly of importance for cancer patients with long-term survival, may appear during or long time after finishing radiotherapy and depending on the patient's radiosensitivity. Currently, there is no assay available that can reliably predict the individual's response to radiotherapy. We profiled two study sets from breast (n = 29) and head-and-neck cancer patients (n = 74) that included radiosensitive patients and matched radioresistant controls. We studied 55 single nucleotide polymorphisms (SNPs) in 33 genes by DNA genotyping and 130 circulating proteins by affinity-based plasma proteomics. In both study sets, we discovered several plasma proteins with the predictive power to find radiosensitive patients (adjusted p < 0.05) and validated the two most predictive proteins (THPO and STIM1) by sandwich immunoassays. By integrating genotypic and proteomic data into an analysis model, it was found that the proteins CHIT1, PDGFB, PNKD, RP2, SERPINC1, SLC4A, STIM1, and THPO, as well as the VEGFA gene variant rs69947, predicted radiosensitivity of our breast cancer (AUC = 0.76) and head-and-neck cancer (AUC = 0.89) patients. In conclusion, circulating proteins and a SNP variant of VEGFA suggest that processes such as vascular growth capacity, immune response, DNA repair and oxidative stress/hypoxia may be involved in an individual's risk of experiencing radiation-induced toxicity., QC 20200622

Purpose: Radiation biology is a branch of the radiation research field which focuses on studying radiation effects in cells and organisms. Radiation can be used in biological investigations for two, mutually non-exclusive reasons: (1) to study biological processes by perturbing their functioning (qualitative approach) and (2) to assess consequences of radiation-induced damage (quantitative approach). While the former approach has a basic research character, the latter has an applied character that is driven by needs of medical applications and radiological protection. Radiation protection biology is defined in the sense of the second approach. The aim of the article is to provide a historical review of how radiation protection biology developed and how it influences radiological protection.Conclusions: While radiobiological investigations started immediately after the discovery of X-rays, the qualitative approach dominated until the end of World War II. After 1945, the nuclear weapons race and nuclear energy programs initiated quantitative radiobiological research. Radiation protection biology does not provide results from which radiation risks can be directly derived. Rather, it provides data that is necessary for understanding the nature of risks. Most recent years have seen, especially in Europe, a growing interest in coordinated studies on the effects of low radiation doses.

Several studies in different species have documented that tardigrades are among the most radiation-tolerant animals on Earth, surviving doses of ionizing radiation on the order of kGy. Both low-LET and high-LET radiation have been used with no apparent differences in the tolerance of the animals. Tolerance to ionizing radiation in tardigrades also seems to be independent of whether the animal has entered a dry anhydrobiotic state or is hydrated with normal activity. However, when exposed to UV radiation, desiccated tardigrades show a higher tolerance than hydrated animals. Recent studies in several species have shown that tardigrade embryos have considerably lower tolerance to ionizing radiation compared to adults, and embryos in the early stage of development are clearly more sensitive to radiation than those in the late developmental stage. The molecular mechanisms behind radiation tolerance in tardigrades are still largely unclear, but available evidence suggests that mechanisms related to both the avoidance of DNA damage and the repair of damage are involved.

MELODI (Multidisciplinary European Low Dose Initiative) is a European radiation protection research platform with focus on research on health risks after exposure to low-dose ionising radiation. It was founded in 2010 and currently includes 44 members from 18 countries. A major activity of MELODI is the continuous development of a long-term European Strategic Research Agenda (SRA) on low-dose risk for radiation protection. The SRA is intended to identify priorities for national and European radiation protection research programs as a basis for the preparation of competitive calls at the European level. Among those key priorities is the improvement of health risk estimates for exposures close to the dose limits for workers and to reference levels for the population in emergency situations. Another activity of MELODI is to ensure the availability of European key infrastructures for research activities, and the long-term maintenance of competences in radiation research via an integrated European approach for training and education. The MELODI SRA identifies three key research topics in low dose or low dose-rate radiation risk research: (1) dose and dose rate dependence of cancer risk, (2) radiation-induced non-cancer effects and (3) individual radiation sensitivity. The research required to improve the evidence base for each of the three key topics relates to three research lines: (1) research to improve understanding of the mechanisms contributing to radiogenic diseases, (2) epidemiological research to improve health risk evaluation of radiation exposure and (3) research to address the effects and risks associated with internal exposures, differing radiation qualities and inhomogeneous exposures. The full SRA and associated documents can be downloaded from the MELODI website (http://www.melodi-online.eu/sra.html).

In September 2007 tardigrades became the first animal in history to survive the combined effect of exposure to space vacuum, cosmic radiation and ultraviolet radiation in low Earth orbit. The main results from this experiment were reported in 2008, but some of the results have remained unpublished. Here we report that descendant generations of space-exposed tardigrades of the species Milnesium tardigradum did not show reduced performance. This indicates that individual tardigrades that survived the exposure to environmental extremes in space, and were able to reproduce, did not transfer any damage to later generations. Repair of environmentally induced damage may therefore follow a make or break' rule, such that a damaged animal either fails to repair all damage and dies, or repairs damage successfully and leaves no mutations to descendants. We also report that two additional tardigrade species, Echiniscus testudo and Ramazzottius oberhaeuseri, showed high survival after exposure to space vacuum and cosmic radiation within the TARDIS experiment.

Tardigrades are highly tolerant to desiccation and ionizing radiation but the mechanisms of this tolerance are not well understood. In this paper, we report studies on dose responses of adults and eggs of the tardigrade Hypsibius dujardini exposed to gamma radiation. In adults the LD50/48h for survival was estimated at ~ 4200 Gy, and doses higher than 100 Gy reduced both fertility and hatchability of laid eggs drastically. We also evaluated the effect of radiation (doses 50 Gy, 200 Gy, 500 Gy) on eggs in the early and late embryonic stage of development, and observed a reduced hatchability in the early stage, while no effect was found in the late stage of development. Survival of juveniles from irradiated eggs was highly affected by a 500 Gy dose, both in the early and the late stage. Juveniles hatched from eggs irradiated at 50 Gy and 200 Gy developed into adults and produced offspring, but their fertility was reduced compared to the controls. Finally we measured the effect of low temperature during irradiation at 4000 Gy and 4500 Gy on survival in adult tardigrades, and observed a slight delay in the expressed mortality when tardigrades were irradiated on ice. Since H.dujardini is a freshwater tardigrade with lower tolerance to desiccation compared to limno-terrestrial tardigrades, the high radiation tolerance in adults, similar to limno-terrestrial tardigrades, is unexpected and seems to challenge the idea that desiccation and radiation tolerance rely on the same molecular mechanisms. We suggest that the higher radiation tolerance in adults and late stage embryos of H.dujardini (and in other studied tardigrades) compared to early stage embryos may partly be due to limited mitotic activity, since tardigrades have a low degree of somatic cell division (eutely), and dividing cells are known tobe more sensitive to radiation.

Radiation therapy is a cornerstone of modern cancer treatment. Understanding the mechanisms behind normal tissue sensitivity is essential in order to minimize adverse side effects and yet to prevent local cancer reoccurrence. The aim of this study was to identify biomarkers of radiation sensitivity to enable personalized cancer treatment. To investigate the mechanisms behind radiation sensitivity a pilot study was made where eight radiation-sensitive and nine normo-sensitive patients were selected from a cohort of 2914 breast cancer patients, based on acute tissue reactions after radiation therapy. Whole blood was sampled and irradiated in vitro with 0, 1, or 150 mGy followed by 3 h incubation at 37 degrees C. The leukocytes of the two groups were isolated, pooled and protein expression profiles were investigated using isotope-coded protein labeling method (ICPL). First, leukocytes from the in vitro irradiated whole blood from normo-sensitive and extremely sensitive patients were compared to the non-irradiated controls. To validate this first study a second ICPL analysis comparing only the non-irradiated samples was conducted. Both approaches showed unique proteomic signatures separating the two groups at the basal level and after doses of 1 and 150 mGy. Pathway analyses of both proteomic approaches suggest that oxidative stress response, coagulation properties and acute phase response are hallmarks of radiation sensitivity supporting our previous study on oxidative stress response. This investigation provides unique characteristics of radiation sensitivity essential for individualized radiation therapy.

A central question in radiation protection research is dose and dose-rate relationship for radiation-induced cardiovascular diseases. The response of endothelial cells to different low dose rates may contribute to help estimate risks for cardiovascular diseases by providing mechanistic understanding. In this study we investigated whether chronic low-dose-rate radiation exposure had an effect on the inflammatory response of endothelial cells and their function. Human umbilical vein endothelial cells (HUVECs) were chronically exposed to radiation at a dose of 1.4 mGy/h or 4.1 mGy/h for 1, 3, 6 or 10 weeks. We determined the pro-inflammatory profile of HUVECs before and during radiation exposure, and investigated the functional consequences of this radiation exposure by measuring their capacity to form vascular networks in matrigel. Expression levels of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1, and the release of pro-inflammatory cytokines such as MCP-1, IL-6 and TNF-alpha were analyzed. When a total dose of 2 Gy was given at a rate of 4.1 mGy/h, we observed an increase in IL-6 and MCP-1 release into the cell culture media, but this was not observed at 1.4 mGy/h. The increase in the inflammatory profile induced at the dose rate of 4.1 mGy/h was also correlated with a decrease in the capacity of the HUVECs to form a vascular network in matrigel. Our results suggest that dose rate is an important parameter in the alteration of HUVEC inflammatory profile and function., AuthorCount:7

In September 2007 tardigrades became the first animal in the history to survive the combined effect of exposure to space vacuum, cosmic radiation, and ultra-violet radiation in low Earth orbit. The main results from this experiment were reported in 2008, but some of the results have remained unpublished. Here we report that no delayed effects of the exposure to space could be detected in the descendants (up to F3 generation) of space exposed Milnesium tardigradum. This indicates that individual tardigrades that survived the damage induced by environmental agents in space, and were able to reproduce, did not transfer any delayed damage to later generations. Repair of environmentally induced damage may therefore follow a “make or break” rule, such that a damaged animal either fails to repair all damage and dies, or repairs damage successfully and leaves no mutations to descendants. We also provide previously unreported data on two tardigrade species, Echiniscus testudo and Ramazzottius oberhaeuseri, that showed high survival after exposure to space vacuum and cosmic radiation within the TARDIS experiment.

Purpose: Ionizing radiation has been recognized to increase the risk of cardiovascular diseases (CVD). However, there is no consensus concerning the dose-risk relationship for low radiation doses and a mechanistic understanding of low dose effects is needed. Material and methods: Previously, human umbilical vein endothelial cells (HUVEC) were exposed to chronic low dose rate radiation (1.4 and 4.1 mGy/h) during one, three and six weeks which resulted in premature senescence in cells exposed to 4.1 mGy/h. To gain more insight into the underlying signaling pathways, we analyzed gene expression changes in these cells using microarray technology. The obtained data were analyzed in a dual approach, combining single gene expression analysis and Gene Set Enrichment Analysis. Results: An early stress response was observed after one week of exposure to 4.1 mGy/h which was replaced by a more inflammation-related expression profile after three weeks and onwards. This early stress response may trigger the radiation-induced premature senescence previously observed in HUVEC irradiated with 4.1 mGy/h. A dedicated analysis pointed to the involvement of insulin-like growth factor binding protein 5 (IGFBP5) signaling in radiation-induced premature senescence. Conclusion: Our findings motivate further research on the shape of the dose-response and the dose rate effect for radiation-induced vascular senescence., AuthorCount:13

Purpose: Primary fibroblasts are not suitable for in vitro macrocolony assay due to their inability to form distinct colonies. Here we present a modification of agarose overlay that yielded extensive improvement in their colony formation and assessment of radiosensitivity. Materials and methods: Macrocolony formation was assessed in primary human fibroblasts VH10 and HDFn with or without overlay using 0.5% agarose in growth medium at 24 h post-seeding. Malignant human cell lines (A549, U87) and transformed nonmalignant fibroblasts (AA8 hamster, MRC5 human) were used for comparison. Results: Agarose overlay caused significant improvement marked by early appearance (one week) of distinct colonies with high cell density and multifold higher plating efficiency than conventional macrocolony assay in VH10 and HDFn human fibroblasts. Compared to conventional assay or feeder cell supplementation, agarose overlay resulted in broader cell morphology due to improved adherence, and yielded more compact colonies. Gamma-radiation dose-response survival curves could be successfully generated for both fibroblast cell lines using this method, which yielded no such effects in the transformed/malignant cell lines tested. Conclusion: This easy and inexpensive 'agarose overlay technique' significantly and selectively improves the fibroblast plating efficiency, thus considerably reducing time and effort to greatly benefit the survival studies on primary fibroblasts., AuthorCount:6

Purpose: Ionizing radiation has been recognized to increase the risk of cardiovascular diseases (CVD). However, there is no consensus concerning the dose-risk relationship for low radiation doses and a mechanistic understanding of low dose effects is needed. Material and methods: Previously, human umbilical vein endothelial cells (HUVEC) were exposed to chronic low dose rate radiation (1.4 and 4.1 mGy/h) during one, three and six weeks which resulted in premature senescence in cells exposed to 4.1 mGy/h. To gain more insight into the underlying signaling pathways, we analyzed gene expression changes in these cells using microarray technology. The obtained data were analyzed in a dual approach, combining single gene expression analysis and Gene Set Enrichment Analysis. Results: An early stress response was observed after one week of exposure to 4.1 mGy/h which was replaced by a more inflammation-related expression profile after three weeks and onwards. This early stress response may trigger the radiation-induced premature senescence previously observed in HUVEC irradiated with 4.1 mGy/h. A dedicated analysis pointed to the involvement of insulin-like growth factor binding protein 5 (IGFBP5) signaling in radiation-induced premature senescence. Conclusion: Our findings motivate further research on the shape of the dose-response and the dose rate effect for radiation- induced vascular senescence.

Tardigrades are known as one of the most radiation tolerant animals on Earth, and several studies on tolerance in adult tardigrades have been published. In contrast, very few studies on radiation tolerance of embryonic stages have been reported. Here we report a study on tolerance to gamma irradiation in eggs of the eutardigrade Richtersius coronifer. Irradiation of eggs collected directly from a natural substrate (moss) showed a clear dose-response, with a steep decline in hatchability at doses up to 0.4 kGy followed by a relatively constant hatchability around 25% up to 2 kGy, and a decline to ca. 5% at 4 kGy above which no eggs hatched. Analysis of the time required for eggs to hatch after irradiation (residual development time) showed that hatching of eggs after exposure to high doses of gamma radiation was associated with short residual development time. Since short residual development time means that the egg was irradiated at a late developmental stage, this suggests that eggs were more tolerant to radiation late in development. This was also confirmed in another experiment in which stage of development at irradiation was controlled. No eggs irradiated at the early developmental stage hatched, and only one egg at middle stage hatched, while eggs irradiated in the late stage hatched at a rate indistinguishable from controls. This suggests that the eggs are more sensitive to radiation in the early stages of development, or that tolerance to radiation is acquired only late in development, shortly before the eggs hatch, hypotheses that are not mutually exclusive. Our study emphasizes the importance of considering specific cell cycle phases and developmental stages in studies of tolerance to radiation in tardigrades, and the potential importance of embryonic studies in revealing the mechanisms behind the radiation tolerance of tardigrades and other cryptobiotic animals.