Deep underground laboratories (DULs) were originally created to host particle, astroparticle or nuclear physics experiments requiring a low-background environment with vastly reduced levels of cosmic-ray particle interference. More recently, the range of science projects requiring an underground experiment site has greatly expanded, thus leading to the recognition of DULs as truly multidisciplinary science sites that host important studies in several fields, including geology, geophysics, climate and environmental sciences, technology/instrumentation development and biology. So far, underground biology experiments are ongoing or planned in a few of the currently operating DULs. Among these DULs is the Gran Sasso National Laboratory (LNGS), where the majority of radiobiological data have been collected. Here we provide a summary of the current scenario of DULs around the world, as well as the specific features of the LNGS and a summary of the results we obtained so far, together with other findings collected in different underground laboratories. In particular, we focus on the recent results from our studies of Drosophila melanogaster, which provide the first evidence of the influence of the radiation environment on life span, fertility and response to genotoxic stress at the organism level. Given the increasing interest in this field and the establishment of new projects, it is possible that in the near future more DULs will serve as sites of radiobiology experiments, thus providing further relevant biological information at extremely low-dose-rate radiation. Underground experiments can be nicely complemented with above-ground studies at increasing dose rate. A systematic study performed in different exposure scenarios provides a potential opportunity to address important radiation protection questions, such as the dose/dose-rate relationship for cancer and non-cancer risk, the possible existence of dose/dose-rate threshold(s) for different biological systems and/or end points and the possible role of radiation quality in triggering the biological response.

In previous studies we determined a gene expression signature in baboons for predicting the severity of hematological acute radiation syndrome. We subsequently validated a set of eight of these genes in leukemia patients undergoing total-body irradiation. In the current study, we addressed the effect of intra-individual variability on the basal level of expression of those eight radiation-responsive genes identified previously, by examining baseline levels in 200 unexposed healthy human donors (122 males and 88 females with an average age of 46 years) using real-time PCR. In addition to the eight candidate genes (DAGLA, WNT3, CD177, PLA2G16, WLS, POU2AF1, STAT4 and PRF1), we examined two more genes (FDXR and DDB2) widely used in ex vivo whole blood experiments. Although significant sex- (seven genes) and age-dependent (two genes) differences in expression were found, the fold changes ranged only between 1.1–1.6. These were well within the twofold differences in gene expression generally considered to represent control values. Age and sex contributed less than 20–30% to the complete inter-individual variance, which is calculated as the fold change between the lowest (reference) and the highest Ct value minimum–maximum fold change (min–max FC). Min–max FCs ranging between 10–17 were observed for most genes; however, for three genes, min–max FCs of complete inter-individual variance were found to be 37.1 (WNT3), 51.4 (WLS) and 1,627.8 (CD177). In addition, to determine whether discrimination between healthy and diseased baboons might be altered by replacing the published gene expression data of the 18 healthy baboons with that of the 200 healthy humans, we employed logistic regression analysis and calculated the area under the receiver operating characteristic (ROC) curve. The additional inter-individual variance of the human data set had either no impact or marginal impact on the ROC area, since up to 32-fold change gene expression differences between healthy and diseased baboons were observed.

The use of targeted radionuclide therapy for cancer is on the rise. While beta-particle-emitting radionuclides have been extensively explored for targeted radionuclide therapy, alpha-particle-emitting radionuclides are emerging as effective alternatives. In this context, fundamental understanding of the interactions and dosimetry of these emitted particles with cells in the tumor microenvironment is critical to ascertaining the potential of alpha-particle-emitting radionuclides. One important parameter that can be used to assess these metrics is the S-value. In this study, we characterized several alpha-particle-emitting radionuclides (and their associated radionuclide progeny) regarding S-values in the cellular and tumor-metastasis environments. The Particle and Heavy Ion Transport code System (PHITS) was used to obtain S-values via Monte Carlo simulation for cell and tumor metastasis resulting from interactions with the alpha-particle-emitting radionuclides, lead-212 (²¹²Pb), actinium-225 (²²⁵Ac) and bismuth-213 (²¹³Bi); these values were compared to the beta-particle-emitting radionuclides yttrium-90 (⁹⁰Y) and lutetium-177 (¹⁷⁷Lu) and an Auger-electron-emitting radionuclide indium-111 (¹¹¹In). The effect of cellular internalization on S-value was explored at increasing degree of internalization for each radionuclide. This aspect of S-value determination was further explored in a cell line-specific fashion for six different cancer cell lines based on the cell dimensions obtained by confocal microscopy. S-values from PHITS were in good agreement with MIRDcell S-values (cellular S-values) and the values found by Hindié et al. (tumor S-values). In the cellular model, ²¹²Pb and ²¹³Bi decay series produced S-values that were 50- to 120-fold higher than ¹⁷⁷Lu, while ²²⁵Ac decay series analysis suggested S-values that were 240- to 520-fold higher than ¹⁷⁷Lu. S-values arising with 100% cellular internalization were two- to sixfold higher for the nucleus when compared to 0% internalization. The tumor dosimetry model defines the relative merit of radionuclides and suggests alpha particles may be effective for large tumors as well as small tumor metastases. These results from PHITS modeling substantiate emerging evidence that alpha-particle-emitting radionuclides may be an effective alternative to beta-particle-emitting radionuclides for targeted radionuclide therapy due to preferred dose-deposition profiles in the cellular and tumor metastasis context. These results further suggest that internalization of alpha-particle-emitting radionuclides via radiolabeled ligands may increase the relative biological effectiveness of radiotherapeutics.

DNA damage induced by ionizing radiation exposure is enhanced in the presence of oxygen (the “oxygen effect”). Despite its practical importance in radiotherapy, the oxygen effect has largely been excluded from models that predict DNA damage from radiation tracks. A Monte Carlo-based algorithm was developed in MATLAB software to predict DNA damage from physical and chemical tracks through a cell nucleus simulated in Geant4-DNA, taking into account the effects of cellular oxygenation (pO₂) on DNA radical chemistry processes. An initial spatial distribution of DNA base and sugar radicals was determined by spatially clustering direct events (that deposited at least 10.79 eV) and hydroxyl radical (^•OH) interactions. The oxygen effect was modeled by increasing the efficiency with which sugar radicals from direct-type effects were converted to strand breaks from 0.6 to 1, the efficiency with which sugar radicals from the indirect effect were converted to strand breaks from 0.28 to 1 and the efficiency of base-to-sugar radical transfer from ^•OH-mediated base radicals from 0 to 0.03 with increasing pO₂ from 0 to 760 mmHg. The DNA damage induction algorithm was applied to tracks from electrons, protons and alphas with LET values from 0.2 to 150 keV/μm under different pO₂ conditions. The oxygen enhancement ratio for double-strand break induction was 3.0 for low-LET radiation up to approximately 15 keV/μm, after which it gradually decreased to a value of 1.3 at 150 keV/μm. These values were consistent with a range of experimental data published in the literature. The DNA damage yields were verified using experimental data in the literature and results from other theoretical models. The spatial clustering approach developed in this work has low memory requirements and may be suitable for particle tracking simulations with a large number of cells.

The radiation-induced bystander effect is mechanistically complex, involving many different signaling components. Serotonin, present in fetal bovine serum (FBS), has been implicated in the modulation of cellular responses to radiation. However, the role of this ubiquitous signaling molecule has yet to be elucidated with regard to cell line-specific radiation responses. In this study, cell survival was measured in HCT116 p53 wild-type (HCT116^+/+) and HaCaT cell cultures treated with media containing serotonin-depleted FBS and compared to our standard FBS-supplemented media, using clonogenic assays. We utilized an enzyme-linked immunosorbent assay to quantify the difference (4.3 ± 1.3 ng/ml) in serotonin concentrations among the media. Serotonin-depleted media significantly reduced survival in both nonirradiated cell lines. Furthermore, we sought to determine the effects to cells in this media exposed to direct irradiation as well as bystander media from irradiated cells. Cell survival was significantly increased when HCT116^+/+ cells were directly irradiated in serotonin-depleted media, while HaCaT cells showed no significant difference in survival between the media. Bystander investigations demonstrated that HCT116^+/+ cells were only able to generate a bystander effect when cultured in standard media conditions containing greater serotonin levels. Conversely, HaCaT cells were unaffected by the different media in terms of producing a bystander response, generating bystander effects irrespective of the media. Previous research linking serotonin receptors to the bystander effect, together with our results, indicate that receptor heterogeneity among cell types may underlie serotonin sensitivity in direct irradiation and bystander responses through serotonin receptor-mediated cell signaling cascades.

This study analyzed the impact of galactic and solar cosmic rays on ambient dose equivalent during airline travel. A high statistic of flights are considered, which is representative of European international air traffic. Flight paths are based on the Eurocontrol Demand Data Repository and consider realistic flight plans with and without regulations or updated with radar data from the Central Flow Management Unit. Ambient dose equivalent during flights was investigated during quiet solar periods and extreme solar flare events. Thus, the statistical analyses presented here take into account route characteristics (departure, arrival, continent, etc.) and space weather conditions. The findings of this work show the important influence of flight path, particularly the latitude, which drives the cutoff rigidity variations. Moreover, dose values vary drastically during ground level enhancement events, with the route path (latitude, longitude and altitude) and the phasing of the solar event. This study highlights the importance of monitoring these solar events and developing a physical approach to obtain reliable assessment of ambient dose equivalents.

The number and energy of secondary electrons generated around the trajectories of swift protons interacting with biological materials are highly relevant in proton therapy, due to the prominent role of low-energy electrons in the production of biodamage. For a given material, electron energy distributions are determined by the proton energy; and it is imperative that the distribution of proton energy at depths around the Bragg peak region be described as accurately as possible. With this objective, we simulated the energy distributions of proton beams of clinically relevant energies (50–300 MeV) at depths around the Bragg peak in liquid water and the water-equivalent polymer poly(methyl methacrylate) (PMMA). By using a simple model, this simulation has been conveniently extended to account for nuclear fragmentation reactions, providing depth-dose curves in excellent agreement with available experimental data. Special care has been taken to describe the electronic excitation spectrum of the target, taking into account its condensed phase nature. A predictive formula has been obtained for the mean value and the width of the proton energy distribution at the Bragg peak depth, quantities which are found to grow linearly with the initial energy of the beam, in good agreement with available data. To accurately characterize (in number and energy) the electrons generated around the proton paths, the energy distributions of the latter at each depth have been convoluted with the energy-dependent ionization inverse mean free paths. This results in a number of low-energy electrons around the Bragg peak larger than when only the proton beam average energy at the given depths is considered. The convoluted ionization inverse mean free path closely resembles the Bragg curve shape. The average energy of the secondary electrons is nearly constant (∼55 eV for liquid water and ∼43 eV for PMMA) in the plateau of the Bragg curve, independent of the proton incident energy and suddenly decaying once the Bragg peak is reached. These findings highlight the importance of a precise calculation of the proton beam energy distribution as a function of the target depth to reliably characterize the secondary electrons generated around the Bragg peak region.

Cell death and tissue injury occur as a result of radiation accidents and radiotherapy. The role of endothelial cell damage in mediating radiation-induced acute tissue injury has been extensively studied. We previously demonstrated that ferulic acid (FA) mitigates radiation-induced hematopoietic injury in mice and lessens radiation-induced oxidative damage in human umbilical vein endothelial cells (HUVECs). The purpose of the current study was to determine whether FA can protect HUVECs from radiation toxicity in a cell model via the thrombomodulin (Thbd) pathway, an anti-radiation pathway with anticoagulant, anti-inflammatory and antioxidant properties. HUVEC culture media was supplemented with FA 12 h before 4 Gy ⁶⁰Co gamma irradiation. At 30 min postirradiation, the FA media was refreshed, then renewed once daily. HUVEC injury was assessed at day 5 postirradiation through cell proliferation analysis. Ferulic acid significantly ameliorated HUVEC radiation injury, as evidenced by increases in cell viability and angiogenesis and decreases in G₂/M cell cycle arrest and levels of high mobility group box 1 protein (HMGB1), interleukin (IL)-6 and IL-8. These findings can be attributed to the effect of FA on the Thbd promoter, resulting in increased expression of Thbd and activated protein C with associated radioprotection. These observations indicate that FA intervention significantly ameliorates HUVEC radiation injury via the Thbd pathway. Therefore, FA could be further developed as a potential agent to mitigate radiation-induced damage.

During the pulsed-electron beam direct grafting of neat styrene onto poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) substrate, the radiolytically-produced styryl and carbon-centered FEP radicals undergo various desired and undesired competing reactions. In this study, a high-dose rate is used to impede the undesired free radical homopolymerization of styrene and ensure uniform covalent grafting through 125-μm FEP films. This outweighs the enhancement of the undesired crosslinking reactions of carbon-centered FEP radicals and the dimerization of the styryl radicals. The degree of uniform grafting through 125-μm FEP films increases from ≈8%, immediately after pulsed electron irradiation to 33% with the subsequent thermal treatment exceeding the glass transition temperature of FEP of 39°C. On the contrary, steady-state radiolysis using ⁶⁰Co gamma radiolysis, shows that the undesired homopolymerization of the styrene has become the predominant reaction with a negligible degree of grafting. Time-resolved fast kinetic measurements on pulsed neat styrene show that the styryl radicals undergo fast decays via propagation homopolymerization and termination reactions at an observed reaction rate constant of 5 × 10⁸ l · mol^–1 · s^–1. The proton conductivity of 25-μm film at 80°C is 0.29 ± 0.01 s cm^–1 and 0.007 s cm^–1 at relative humidity of 92% and 28%, respectively. The aims of this work are: 1. electrolyte membranes are prepared via grafting initiated by a pulsed electron beam; 2. postirradiation heat-treated membranes are uniformly grafted, ideal for industry; 3. High dose rate is the primary parameter to promote the desired reactions; 4. measurement of kinetics of undesired radiation-induced styrene homopolymerization; and 5. The conductivity of prepared membranes is on par or higher than industry standards.

Until recently, patients with relapsed Hodgkin's lymphoma after brentuximab vedotin (Bv) treatments had poor treatment outcomes. Checkpoint inhibitors such as nivolumab and pembrolizumab that bind to and inhibit programmed cell death protein-1 (PD-1), have demonstrated an overall response rate of 70% in Hodgkin's lymphoma patients; however, complete response is still low at 20% with median progression-free survival of 14 months. There are ongoing clinical studies to seek out synergistic combinations, with the goal of improving the complete response rates for the cure of Hodgkin's lymphoma. Although radiotherapy has a limited survival benefit in such refractory patients, several preclinical models and anecdotal clinical evidence have suggested that combining local tumor irradiation with checkpoint inhibitors can produce systemic regression of distant tumors, an abscopal effect. Most of these reported studies on the response with local conformal radiotherapy and checkpoint inhibitors in combination with the anti-cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) antibody-ipilimumab are in melanoma. Here we report in our case series that the checkpoint inhibitors that block CTLA4 and B7-homolog 1 (B7-H1) or PD-1 in preclinical radiotherapy models have shown an increased the rate of tumor regression. Our case series demonstrates that combining local irradiation with anti-PD-1 checkpoint blockade treatment is feasible and synergistic in refractory Hodgkin's lymphoma. Correlative studies also suggest that the expression of programmed death-ligand 1 (PD-L1), DNA damage response and mutational tumor burden can be used as potential biomarkers for treatment response.