Ionizing radiation responses appear incidental to desiccation responses in the bdelloid rotifer Adineta vaga.

BACKGROUND: The remarkable resistance to ionizing radiation found in anhydrobiotic organisms, such as some bacteria, tardigrades, and bdelloid rotifers has been hypothesized to be incidental to their desiccation resistance. Both stresses produce reactive oxygen species and cause damage to DNA and other macromolecules. However, this hypothesis has only been investigated in a few species. RESULTS: In this study, we analyzed the transcriptomic response of the bdelloid rotifer Adineta vaga to desiccation and to low- (X-rays) and high- (Fe) LET radiation to highlight the molecular and genetic mechanisms triggered by both stresses. We identified numerous genes encoding antioxidants, but also chaperones, that are constitutively highly expressed, which may contribute to the protection of proteins against oxidative stress during desiccation and ionizing radiation. We also detected a transcriptomic response common to desiccation and ionizing radiation with the over-expression of genes mainly involved in DNA repair and protein modifications but also genes with unknown functions that were bdelloid-specific. A distinct transcriptomic response specific to rehydration was also found, with the over-expression of genes mainly encoding Late Embryogenesis Abundant proteins, specific heat shock proteins, and glucose repressive proteins. CONCLUSIONS: These results suggest that the extreme resistance of bdelloid rotifers to radiation might indeed be a consequence of their capacity to resist complete desiccation. This study paves the way to functional genetic experiments on A. vaga targeting promising candidate proteins playing central roles in radiation and desiccation resistance.

C. elegans: A potent model for high-throughput screening experiments investigating the FLASH effect.

This study explores the effects of UHDR irradiation on Caenorhabditis elegans embryos. UHDR proton and electron beams demonstrate a sparing effect, aligning with literature findings. This highlights C. elegans suitability as a screening model for studying the LET impact on the FLASH effect, reinforcing its potential in radiation research.

Radiation therapy-activated nanoparticle and immunotherapy: The next milestone in oncology?

Radiotherapy (RT) is a fundamental treatment at the locoregional or oligometastatic stages of cancer. In various tumors, RT effects may be optimized using synergistic combinations that enhance tumor response. Innovative strategies have been designed that explore the radiation mechanisms, at the physical, chemical and biological levels, to propose precision RT approaches. They consist in combining RT with immunotherapy to revert radiation immunosuppressive effects or to enhance radiation-induced immune defenses against the tumor to favor immunogenic cell death. Radiotherapy-activated nanoparticles are another innovation. By increasing radiation response in situ, nanoparticles improve tumor control locally, and can trigger systemic immune reactions that may be exploited to improve the systemic efficacy of RT. Strong clinical evidence of improved outcomes is now available for combinations of RT and immunotherapy on one hand and RT and nanoparticles on the other hand. The triple combination of RT, immunotherapy and nanoparticles is promising in terms of tolerance, local and systemic anti-tumor control. Yet, significant challenges remain to unravel the complexity of the multiscale mechanisms underlying response to this combination and their associated parameters. Such parameters include patient characteristics, tumor bulk and histology, radiation technique, energy, dose, fractionation, immunotherapy targets and predictive biomarkers, nanoparticle type, size, delivery (intratumoral/intravenous), distribution. The temporal combination is another critical parameter. The mechanisms of response of the combinatorial approaches are reviewed, with a focus on underlying mechanisms based on preclinical, translational and clinical studies. Opportunities for translation of current understanding into precision RT trials combined with immunotherapy and nanoparticles are also discussed.

Back to the roots, desiccation and radiation resistances are ancestral characters in bdelloid rotifers.

BACKGROUND: Bdelloid rotifers are micro-invertebrates distributed worldwide, from temperate latitudes to the most extreme areas of the planet like Antarctica or the Atacama Desert. They have colonized any habitat where liquid water is temporarily available, including terrestrial environments such as soils, mosses, and lichens, tolerating desiccation and other types of stress such as high doses of ionizing radiation (IR). It was hypothesized that bdelloid desiccation and radiation resistance may be attributed to their potential ability to repair DNA double-strand breaks (DSBs). Here, these properties are investigated and compared among nine bdelloid species collected from both mild and harsh habitats, addressing the correlation between the ability of bdelloid rotifers to survive desiccation and their capacity to repair massive DNA breakage in a phylogenetically explicit context. Our research includes both specimens isolated from habitats that experience frequent desiccation (at least 1 time per generation), and individuals sampled from habitats that rarely or never experienced desiccation. RESULTS: Our analysis reveals that DNA repair prevails in somatic cells of both desiccation-tolerant and desiccation-sensitive bdelloid species after exposure to X-ray radiation. Species belonging to both categories are able to withstand high doses of ionizing radiation, up to 1000 Gy, without experiencing any negative effects on their survival. However, the fertility of two desiccation-sensitive species, Rotaria macrura and Rotaria rotatoria, was more severely impacted by low doses of radiation than that of desiccation-resistant species. Surprisingly, the radioresistance of desiccation-resistant species is not related to features of their original habitat. Indeed, bdelloids isolated from Atacama Desert or Antarctica were not characterized by a higher radioresistance than species found in more temperate environments. CONCLUSIONS: Tolerance to desiccation and radiation are supported as ancestral features of bdelloid rotifers, with a group of species of the genus Rotaria having lost this trait after colonizing permanent water habitats. Together, our results provide a comprehensive overview of the evolution of desiccation and radiation resistance among bdelloid rotifers.

Mouse genomic associations with in vitro sensitivity to simulated space radiation.

Exposure to ionizing radiation is considered by NASA to be a major health hazard for deep space exploration missions. Ionizing radiation sensitivity is modulated by both genomic and environmental factors. Understanding their contributions is crucial for designing experiments in model organisms, evaluating the risk of deep space (i.e. high-linear energy transfer, or LET, particle) radiation exposure in astronauts, and also selecting therapeutic irradiation regimes for cancer patients. We identified single nucleotide polymorphisms in 15 strains of mice, including 10 collaborative cross model strains and 5 founder strains, associated with spontaneous and ionizing radiation-induced in vitro DNA damage quantified based on immunofluorescent tumor protein p53 binding protein (53BP1) positive nuclear foci. Statistical analysis suggested an association with pathways primarily related to cellular signaling, metabolism, tumorigenesis and nervous system damage. We observed different genomic associations in early (4 and 8 h) responses to different LET radiation, while later (24 hour) DNA damage responses showed a stronger overlap across all LETs. Furthermore, a subset of pathways was associated with spontaneous DNA damage, suggesting 53BP1 positive foci as a potential biomarker for DNA integrity in mouse models. Our results suggest several mouse strains as new models to further study the impact of ionizing radiation and validate the identified genetic loci. We also highlight the importance of future human in vitro studies to refine the association of genes and pathways with the DNA damage response to ionizing radiation and identify targets for space travel countermeasures.

Advances in the Mechanistic Understanding of Iron Oxide Nanoparticles' Radiosensitizing Properties.

Among the plethora of nanosystems used in the field of theranostics, iron oxide nanoparticles (IONPs) occupy a central place because of their biocompatibility and magnetic properties. In this study, we highlight the radiosensitizing effect of two IONPs formulations (namely 7 nm carboxylated IONPs and PEG5000-IONPs) on A549 lung carcinoma cells when exposed to 225 kV X-rays after 6 h, 24 h and 48 h incubation. The hypothesis that nanoparticles exhibit their radiosensitizing effect by weakening cells through the inhibition of detoxification enzymes was evidenced by thioredoxin reductase activity monitoring. In particular, a good correlation between the amplification effect at 2 Gy and the residual activity of thioredoxin reductase was observed, which is consistent with previous observations made for gold nanoparticles (NPs). This emphasizes that NP-induced radiosensitization does not result solely from physical phenomena but also results from biological events.

Understanding the Radiobiological Mechanisms Induced by 177Lu-DOTATATE in Comparison to External Beam Radiation Therapy.

Radionuclide Therapy (RNT) with 177Lu-DOTATATE targeting somatostatin receptors (SSTRs) in neuroendocrine tumours (NET) has been successfully used in routine clinical practice, mainly leading to stable disease. Radiobiology holds promise for RNT improvement but is often extrapolated from external beam radiation therapy (EBRT) studies despite differences in these two radiation-based treatment modalities. In a panel of six human cancer cell lines expressing SSTRs, common radiobiological endpoints (i.e., cell survival, cell cycle, cell death, oxidative stress and DNA damage) were evaluated over time in 177Lu-DOTATATE- and EBRT-treated cells, as well as the radiosensitizing potential of poly (ADP-ribose) polymerase inhibition (PARPi). Our study showed that common radiobiological mechanisms were induced by both 177Lu-DOTATATE and EBRT, but to a different extent and/or with variable kinetics, including in the DNA damage response. A higher radiosensitizing potential of PARPi was observed for EBRT compared to 177Lu-DOTATATE. Our data reinforce the need for dedicated RNT radiobiology studies, in order to derive its maximum therapeutic benefit.

Investigation of the Antibacterial Properties of Silver-Doped Amorphous Carbon Coatings Produced by Low Pressure Magnetron Assisted Acetylene Discharges.

Hospital-acquired infections are responsible for a significant part of morbidity and mortality. Among the possible modes of transmission, this study focuses on environmental surfaces by developing innovative antibacterial coatings that can be applied on interior fittings in hospitals. This work aims to optimize a coating made of an amorphous carbon matrix doped with silver (a-C:H:Ag) produced by a hybrid PVD/PECVD process and to evaluate its antibacterial activity. We present a coating characterization (chemical composition and morphology) as well as its stability in an ageing process and after multiple exposures to bacteria. The antibacterial activity of the coatings is demonstrated against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria through several bioassays. Moreover, the data suggest a crucial role of silver diffusion towards the surface and nanoparticle formation to explain the very promising anti-bacterial activities reported in this work.

Quantification of radiation-induced DNA double strand break repair foci to evaluate and predict biological responses to ionizing radiation.

Radiation-induced foci (RIF) are nuclear puncta visualized by immunostaining of proteins that regulate DNA double-strand break (DSB) repair after exposure to ionizing radiation. RIF are a standard metric for measuring DSB formation and repair in clinical, environmental and space radiobiology. The time course and dose dependence of their formation has great potential to predict in vivo responses to ionizing radiation, predisposition to cancer and probability of adverse reactions to radiotherapy. However, increasing complexity of experimentally and therapeutically setups (charged particle, FLASH …) is associated with several confounding factors that must be taken into account when interpreting RIF values. In this review, we discuss the spatiotemporal characteristics of RIF development after irradiation, addressing the common confounding factors, including cell proliferation and foci merging. We also describe the relevant endpoints and mathematical models that enable accurate biological interpretation of RIF formation and resolution. Finally, we discuss the use of RIF as a biomarker for quantification and prediction of in vivo radiation responses, including important caveats relating to the choice of the biological endpoint and the detection method. This review intends to help scientific community design radiobiology experiments using RIF as a key metric and to provide suggestions for their biological interpretation.

Considering Cell Proliferation to Optimize Detection of Radiation-Induced 53BP1 Positive Foci in 15 Mouse Strains Ex Vivo.

Due to high metabolic activity, proliferating cells continuously generate free radicals, which induce DNA double-strand breaks (DSB). Fluorescently tagged nuclear foci of DNA repair protein 53 binding protein-1 (53BP1) are used as a standard metric for measuring DSB formation at baseline and in response to environmental insults such as radiation. Here we demonstrate that the background level of spontaneous 53BP1+ foci formation can be modeled mathematically as a function of cell confluence, which is a metric of their proliferation rate. This model was validated using spontaneous 53BP1+ foci data from 72 cultures of primary skin fibroblasts derived from 15 different strains of mice, showing a ∼10-fold decrease from low to full confluence that is independent of mouse strain. On the other hand, the baseline level of spontaneous 53BP1+ foci in a fully confluent cell population was strain-dependent, suggesting genomic associations, and correlated with radiation sensitivity based on previous measurements in the same cell lines. Finally, we have developed an online open-access tool to correct for the effect of cell confluence on 53BP1+ foci-based quantification of DSB. This tool provides guidelines for the number of cells required to reach statistical significance for the detection of DSB induced by low doses of ionizing radiation as a function of confluence and time postirradiation.

53BP1 Repair Kinetics for Prediction of In Vivo Radiation Susceptibility in 15 Mouse Strains.

We present a novel mathematical formalism to predict the kinetics of DNA damage repair after exposure to both low- and high-LET radiation (X rays; 350 MeV/n 40Ar; 600 MeV/n 56Fe). Our method is based on monitoring DNA damage repair protein 53BP1 that forms radiation-induced foci (RIF) at locations of DNA double-strand breaks (DSB) in the nucleus and comparing its expression in primary skin fibroblasts isolated from 15 mice strains. We previously reported strong evidence for clustering of nearby DSB into single repair units as opposed to the classic "contact-first" model where DSB are considered immobile. Here we apply this clustering model to evaluate the number of remaining RIF over time. We also show that the newly introduced kinetic metrics can be used as surrogate biomarkers for in vivo radiation toxicity, with potential applications in radiotherapy and human space exploration. In particular, we observed an association between the characteristic time constant of RIF repair measured in vitro and survival levels of immune cells collected from irradiated mice. Moreover, the speed of DNA damage repair correlated not only with radiation-induced cellular survival in vivo, but also with spontaneous cancer incidence data collected from the Mouse Tumor Biology database, suggesting a relationship between the efficiency of DSB repair after irradiation and cancer risk.

Iron Ladies - How Desiccated Asexual Rotifer Adineta vaga Deal With X-Rays and Heavy Ions?

Space exposure experiments from the last 15 years have unexpectedly shown that several terrestrial organisms, including some multi-cellular species, are able to survive in open space without protection. The robustness of bdelloid rotifers suggests that these tiny creatures can possibly be added to the still restricted list of animals that can deal with the exposure to harsh condition of space. Bdelloids are one of the smallest animals on Earth. Living all over the world, mostly in semi-terrestrial environments, they appear to be extremely stress tolerant. Their desiccation tolerance at any stage of their life cycle is known to confer tolerance to a variety of stresses including high doses of radiation and freezing. In addition, they constitute a major scandal in evolutionary biology due to the putative absence of sexual reproduction for at least 60 million years. Adineta vaga, with its unique characteristics and a draft genome available, was selected by ESA (European Space Agency) as a model system to study extreme resistance of organisms exposed to space environment. In this manuscript, we documented the resistance of desiccated A. vaga individuals exposed to increasing doses of X-ray, protons and Fe ions. Consequences of exposure to different sources of radiation were investigated in regard to the cellular type including somatic (survival assay) and germinal cells (fertility assay). Then, the capacity of A. vaga individuals to repair DNA DSB induced by different source of radiation was investigated. Bdelloid rotifers represent a promising model in order to investigate damage induced by high or low LET radiation. The possibility of exposure both on hydrated or desiccated specimens may help to decipher contribution of direct and indirect radiation damage on biological processes. Results achieved through this study consolidate our knowledge about the radioresistance of A. vaga and improve our capacity to compare extreme resistance against radiation among living organisms including metazoan.

Gold Nanoparticles as a Potent Radiosensitizer: A Transdisciplinary Approach from Physics to Patient.

Over the last decade, a growing interest in the improvement of radiation therapies has led to the development of gold-based nanomaterials as radiosensitizer. Although the radiosensitization effect was initially attributed to a dose enhancement mechanism, an increasing number of studies challenge this mechanistic hypothesis and evidence the importance of chemical and biological contributions. Despite extensive experimental validation, the debate regarding the mechanism(s) of gold nanoparticle radiosensitization is limiting its clinical translation. This article reviews the current state of knowledge by addressing how gold nanoparticles exert their radiosensitizing effects from a transdisciplinary perspective. We also discuss the current and future challenges to go towards a successful clinical translation of this promising therapeutic approach.

Roadmap for metal nanoparticles in radiation therapy: current status, translational challenges, and future directions.

This roadmap outlines the potential roles of metallic nanoparticles (MNPs) in the field of radiation therapy. MNPs made up of a wide range of materials (from Titanium, Z = 22, to Bismuth, Z = 83) and a similarly wide spectrum of potential clinical applications, including diagnostic, therapeutic (radiation dose enhancers, hyperthermia inducers, drug delivery vehicles, vaccine adjuvants, photosensitizers, enhancers of immunotherapy) and theranostic (combining both diagnostic and therapeutic), are being fabricated and evaluated. This roadmap covers contributions from experts in these topics summarizing their view of the current status and challenges, as well as expected advancements in technology to address these challenges.

ZnO/Carbon xerogel photocatalysts by low-pressure plasma treatment, the role of the carbon substrate and its plasma functionalization.

ZnO is known to be photocatalytic, but with limited performances due to the strong electron-hole recombination after irradiation. The integration of ZnO nanomaterials on a conductive and high surface area carbon substrate is thus a potential alternative to obtain a significant improvement of the photocatalytic performance. Moreover, the carbon functionalization is expected to have a significant role in the adsorption/degradation mechanisms of dye, due to the difference in wettability or surface charge. In this view, ZnO photocatalytic nanoparticles have been deposited on high surface area carbon xerogel substrate (CXG), using a new and original plasma process, consisting in the degradation of a solid organometallic directly on the carbon substrate (no gaseous precursor). In addition to the ZnO nanoparticle formation, the plasma treatment allows the carbon functionalization. The ZnO/CXG composite has been tested for the degradation of Rhodamine B (RhB) in aqueous media and compared with and O2 or NH3 plasma-treated xerogels (without nanoparticles) to identify the significant role of the substrate and its modification in the RhB adsorption and degradation mechanism. The high photocatalytic activity of ZnO/CXG composite is attributed to (i) the formation of small (8-10 nm) and well-crystallized ZnO nanoparticles anchored to the carbon substrate and (ii) to the modification of the xerogel surface chemistry. Indeed, O2 plasma treatment of the CXG promotes the generation of hydroxyl, carbonyl and carboxyl surface functional groups, which are polar and acidic, while the NH3 plasma treatment mainly leads to the formation of polar and basic amino groups. While both plasma treatments promote the formation of polar functional groups, which enhance the CXG wettability, the formation of acidic groups is identified as beneficial for the adsorption of the RhB dye, while basic groups are detrimental.

Gold nanoparticles affect the antioxidant status in selected normal human cells.

Purpose: This study evaluates the cytotoxicity of AuNPs coated with polyallylamine (AuNPs-PAA) and conjugated or not to the epidermal growth factor receptor (EGFR)-targeting antibody Cetuximab (AuNPs-PAA-Ctxb) in normal human kidney (HK-2), liver (THLE-2) and microvascular endothelial (TIME) cells, and compares it with two cancer cell lines that are EGFR-overexpressing (A431) or EGFR-negative (MDA-MB-453). Results: Conjugation of Cetuximab to AuNPs-PAA increased the AuNPs-PAA-Ctxb interactions with cells, but reduced their cytotoxicity. TIME cells exhibited the strongest reduction in viability after exposure to AuNPs-PAA(±Ctxb), followed by THLE-2, MDA-MB-453, HK-2 and A431 cells. This cell type-dependent sensitivity was strongly correlated to the inhibition of thioredoxin reductase (TrxR) and glutathione reductase (GR), and to the depolarization of the mitochondrial membrane potential. Both are suggested to initiate apoptosis, which was indeed detected in a concentration- and time-dependent manner. The role of oxidative stress in AuNPs-PAA(±Ctxb)-induced cytotoxicity was demonstrated by co-incubation of the cells with N-acetyl L-cysteine (NAC), which significantly decreased apoptosis and mitochondrial membrane depolarization. Conclusion: This study helps to identify the cells and tissues that could be sensitive to AuNPs and deepens the understanding of the risks associated with the use of AuNPs in vivo.

Dose, LET and Strain Dependence of Radiation-Induced 53BP1 Foci in 15 Mouse Strains Ex Vivo Introducing Novel DNA Damage Metrics.

We present a comprehensive comparative analysis on the repair of radiation-induced DNA damage ex vivo in 15 strains of mice, including 5 inbred reference strains and 10 collaborative-cross strains, of both sexes, totaling 5 million skin fibroblast cells imaged by three-dimensional highthroughput conventional microscopy. Non-immortalized primary skin fibroblasts derived from 76 mice were subjected to increasing doses of both low- and high-LET radiation (X rays; 350 MeV/n 40Ar; 600 MeV/n 56Fe), which are relevant to carcinogenesis and human space exploration. Automated image quantification of 53BP1 radiation-induced foci (RIF) formation and repair during the first 4-48 h postirradiation was performed as a function of dose and LET. Since multiple DNA double-strand breaks (DSBs) are induced in a dose- and LET-dependent manner, our data suggest that when DSBs are formed within the same discrete nuclear region, referred to as the "repair domain", novel mathematical formalisms used to report RIF allowed us to conclude that multiple DSBs can be present in single RIF. Specifically, we observed that the number of RIF per Gy was lower for higher X-ray doses or higher LET particles (i.e., 600 MeV/n 56Fe), suggesting there are more DSBs per RIF when the local absorbed dose increases in the nucleus. The data also clearly show that with more DSBs per RIF, it becomes more difficult for cells to fully resolve RIF. All 15 strains showed the same dose and LET dependence, but strain differences were preserved under various experimental conditions, indicating that the number and sizes of repair domains are modulated by the genetic background of each strain.

Thioredoxin Reductase Activity Predicts Gold Nanoparticle Radiosensitization Effect.

Gold nanoparticles (GNPs) have been shown to be effective contrast agents for imaging and emerge as powerful radiosensitizers, constituting a promising theranostic agent for cancer. Although the radiosensitization effect was initially attributed to a physical mechanism, an increasing number of studies challenge this mechanistic hypothesis and evidence the importance of oxidative stress in this process. This work evidences the central role played by thioredoxin reductase (TrxR) in the GNP-induced radiosensitization. A cell type-dependent reduction in TrxR activity was measured in five different cell lines incubated with GNPs leading to differences in cell response to X-ray irradiation. Correlation analyses demonstrated that GNP uptake and TrxR activity inhibition are associated to a GNP radiosensitization effect. Finally, Kaplan-Meier analyses suggested that high TrxR expression is correlated to low patient survival in four different types of cancer. Altogether, these results enable a better understanding of the GNP radiosensitization mechanism, which remains a mandatory step towards further use in clinic. Moreover, they highlight the potential application of this new treatment in a personalized medicine context.

Antibody-functionalized gold nanoparticles as tumor-targeting radiosensitizers for proton therapy.

AIM: This study aimed at developing antibody-functionalized gold nanoparticles (AuNPs) to selectively target cancer cells and probing their potential radiosensitizing effects under proton irradiation. MATERIALS & METHODS: AuNPs were conjugated with cetuximab (Ctxb-AuNPs). Ctxb-AuNP uptake was evaluated by transmission electron microscopy and atomic absorption spectroscopy. Radioenhancing effect was assessed using conventional clonogenic assay. RESULTS & CONCLUSION: Ctxb-AuNPs specifically bound to and accumulated in EGFR-overexpressing A431 cells, compared with EGFR-negative MDA-MB-453 cells. Ctxb-AuNPs enhanced the effect of proton irradiation in A431 cells but not in MDA-MB-453 cells. These data indicate, for the first time, that combining enhanced uptake by specific targeting and radioenhancing effect, using conjugated AuNPs, is a promising strategy to increase cell killing by protontherapy.

The role of thioredoxin reductase in gold nanoparticle radiosensitization effects.

AIM: To identify new mechanisms responsible for the radiosensitization effect of gold nanoparticles (GNPs). MATERIALS & METHODS: A549 lung carcinoma cells were incubated with 10-nm GNPs during 6 or 24 h before to be exposed to 25 keV/µm protons or 225 kV x-rays. RESULTS: GNP incubation led to a time-dependent mitochondria membrane depolarization, oxidative stress and to x-ray and proton radiosensitization. Moreover, a marked inhibition of thioredoxin reductase was observed. Irradiation of cells invalidated for thioredoxin reductase evidenced a radiosensitization effect, suggesting that this enzyme is a potential GNP target. CONCLUSION: We suggest that GNPs play a radiosensitizer role by weakening detoxification systems. Altogether, these results open up promising novel strategies for the development of nanotechnologies associated to radiotherapy.