The pervasiveness of deep space radiation remains a confounding factor for the transit of humans through our solar system. Spacecraft shielding both protects astronauts but also contributes to absorbed dose through galactic cosmic ray interactions that produce secondary particles. The resultant biological effects drop to a minimum for aluminum shielding around 20 g/ cm² but increase with additional shielding. The present work evaluates for the first time, the impact of secondary pions on central nervous system functionality. The fractional pion dose emanating from thicker shielded spacecraft regions could contribute up to 10% of the total absorbed radiation dose. New results from the Paul Scherrer Institute have revealed that low dose exposures to 150 MeV positive and negative pions, akin to a Mars mission, result in significant, long-lasting cognitive impairments. These surprising findings emphasize the need to carefully evaluate shielding configurations to optimize safe exposure limits for astronauts during deep space travel.

In the present paper we numerically investigate, using Monte Carlo simulation, the theoretical results predicted by the Generalized Stochastic Microdosimetric Model (GSM²), as shown in the published companion paper. Taking advantage of the particle irradiation data ensemble (PIDE) dataset, we calculated GSM² biological parameters of human salivary gland (HSG) and V79 cell lines. Further, exploiting the TOPAS-microdosimetric extension, we simulated the microdosimetric spectra of different radiation fields of therapeutic interest generated by four different ions (protons, helium-4, carbon-12 and oxygen-16) each at three different residual ranges. We investigated the properties of the initial damage distributions as well as the cell survival curve predicted by GSM², focusing especially on the non-Poissonian effects naturally included in the model. GSM² successfully computed cell survival curves, accurately describing experimental behavior even under challenging LET and dose conditions.

The effects of long-term low-dose X-ray irradiation on the outer root sheath (ORS) cells of C3H/He mice were investigated. Mice were irradiated with a regime of 100 mGy/day, 5 days/ week, for 12 weeks (Group X) and the results obtained were compared to those in a non-irradiated control (Group C). Potential protection against ORS cells damage induced by this exposure was investigated by adding the stable nitroxide radical 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) at 1 mM to the drinking water of mice (Group X + TEMPOL). The results obtained were compared with Group C and a nonirradiated group treated with TEMPOL (Group C + TEMPOL). After fractionated X-ray irradiation, skin was removed and ORS cells were examined by hematoxylin and eosin staining and electron microscopy for an abnormal nuclear morphology and nuclear condensation changes. Fractionated X-irradiated mice had an increased number of ORS cells with an abnormal nuclear morphology as well as nuclear condensation changes. Sections were also immunohistochemically examined for the presence of TdT-mediated dUTP nick-end labeling (TUNEL), 8-hydroxy-2′-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), vascular endothelial growth factor (VEGF), nitrotyrosine, heme oxygenase 1 (HO-1), and protein gene product 9.5 (PGP 9.5). Significant increases were observed in TUNEL, 8-OHdG, and 4-HNE levels in ORS cells from mice in Group X. Electron microscopy also showed irregular shrunken ORS cells in Group X. These changes were prevented by the presence of TEMPOL in the drinking water of the irradiated mice. TEMPOL alone had no significant effects. These results suggest that fractionated doses of radiation induced oxidative damage in ORS cells; however, TEMPOL provided protection against this damage, possibly as a result of the rapid reaction of this nitroxide radical with the reactive oxidants generated by fractionated X-ray irradiation.

Low-dose radiation has been extensively employed in clinical practice, including tumor immunotherapy, chronic inflammation treatment and nidus screening. However, the damage on the spleen caused by low-dose radiation significantly increases the risk of late infection-related mortality, and there is currently no corresponding protective strategy. In the present study, a novel compound preparation named CB001 mainly constituted of Acanthopanax senticosus (AS) and Oldenlandia diffusa (OD) was developed to alleviate splenic injury caused by fractionated low-dose exposures. As our results show that, white pulp atrophy and the excessive apoptosis in spleen tissue induced by radiation exposure were significantly ameliorated by CB001. Mechanistically, BAX-caspase-3 signaling and nucleotide-binding domain and leucine-rich-repeat-containing family pyrin 3 (NLRP3) inflammasome signaling were demonstrated to be involved in the radio-protective activity of CB001 with the selective activators. Furthermore, the crosstalk between apoptosis signaling and NLRP3 inflammasome signaling in mediating the radio-protective activity of CB001 was clarified, in which the pro-apoptotic protein BAX but not the antiapoptotic protein Bcl2 was found to be downstream of NLRP3. Our study demonstrated that the use of a novel drug product CB001 can potentially facilitate the alleviation of radiation-induced splenic injury for patients receiving medical imaging diagnosis or fractionated radiation therapy.

High-linear energy transfer (LET) radiation, such as heavy ions is associated with a higher relative biological effectiveness (RBE) than low-LET radiation, such as photons. Irradiation with low- and high-LET particles differ in the interaction with the cellular matter and therefore in the spatial dose distribution. When a single high-LET particle interacts with matter, it results in doses of up to thousands of gray (Gy) locally concentrated around the ion trajectory, whereas the mean dose averaged over the target, such as a cell nucleus is only in the range of a Gy. DNA damage therefore accumulates in this small volume. In contrast, up to hundreds of low-LET particle hits are required to achieve the same mean dose, resulting in a quasi-homogeneous damage distribution throughout the cell nucleus. In this study, we investigated the dependence of RBE from different spatial dose depositions using different focused beam spot sizes of proton radiation with respect to the induction of chromosome aberrations and clonogenic cell survival. Human-hamster hybrid (A_L) as well as Chinese hamster ovary cells (CHO-K1) were irradiated with focused low LET protons of 20 MeV (LET = 2.6 keV/µm) beam energy with a mean dose of 1.7 Gy in a quadratic matrix pattern with point spacing of 5.4 × 5.4 µm² and 117 protons per matrix point at the ion microbeam SNAKE using different beam spot sizes between 0.8 µm and 2.8 µm (full width at half maximum). The dose-response curves of X-ray reference radiation were used to determine the RBE after a 1.7 Gy dose of radiation. The RBE for the induction of dicentric chromosomes and cell inactivation was increased after irradiation with the smallest beam spot diameter (0.8 µm for chromosome aberration experiments and 1.0 µm for cell survival experiments) compared to homogeneous proton radiation but was still below the RBE of a corresponding high LET single ion hit. By increasing the spot size to 1.6–1.8 µm, the RBE decreased but was still higher than for homogeneously distributed protons. By further increasing the spot size to 2.7–2.8 µm, the RBE was no longer different from the homogeneous radiation. Our experiments demonstrate that varying spot size of low-LET radiation gradually modifies the RBE. This underlines that a substantial fraction of enhanced RBE originates from inhomogeneous energy concentrations on the µm scale (mean intertrack distances of low-LET particles below 0.1 µm) and quantifies the link between such energy concentration and RBE. The missing fraction of RBE enhancement when comparing with high-LET ions is attributed to the high inner track energy deposition on the nanometer scale. The results are compared with model results of PARTRAC and LEM for chromosomal aberration and cell survival, respectively, which suggest mechanistic interpretations of the observed radiation effects.

Clustered DNA damage (cluster) or a multiply damaged site, which is a region with two or more lesions within one or two helical turns, has a high mutagenic potential and causes cell death. We quantified fluorophore-labeled lesions and estimated their proximity through fluorescence anisotropy measurements depending on Förster resonance energy transfer (FRET) among the fluorophores close to each other. pUC19 plasmid DNA (2,686 base pairs) dissolved in water or 0.2 M Tris-HCl buffer at a concentration of 10 µg/µL was irradiated by several ionizing radiations with varying linear energy transfers (LET, 0.2–1890 keV/ µm). Electrophilic carbonyls (aldehydes and ketones) at abasic sites (APs) produced in DNA were labeled with Alexa Fluor 488 fluorescent dyes with an O-amino functional group. Regardless of the presence or absence of the buffer, AP yields (the number of APs/base pair/Gy) tended to decrease with increasing LET, and the ratio of the AP yield (in 0.2 M Tris-HCl/in water) was less than 0.1 in the LET range of 0.2–200 keV/µm. However, in a higher LET range, the ratios were greater than 0.1. At a low dose, fluorescence anisotropy decreased with increasing LET in 0.2 M Tris-HCl, whereas, in water, this LET dependence was almost insignificant. These findings suggest that 1. the damage distribution on a DNA molecule formed by indirect effects (e.g., by hydroxyl radicals) does not depend on radiation quality and 2. greater LET radiation is more likely to produce a cluster and/or to produce a cluster with shorter distances between lesions by direct effects. This FRET-based proximity estimation of DNA lesions will contribute not only to the identification of clusters and their complexity in a whole genome, but also to the study of their repair mechanism by single-molecular level fluorescence microscopy.

The effect of ionizing radiation on the gastrointestinal tract is a common complication of abdominal and pelvic radiotherapy. However, the pathological features of radiation enteropathy and its effective medical intervention regimen is still a global challenge. Here, we explored the role and mechanism of enteric alpha-defensins (EαDs) in protecting against radiation enteropathy. To address this, we utilized EαDs-deficiency mice, in which the matrix metallopeptidase 7 to activate Paneth cell α-defensins was knockout (KO) mice, and the complementary wild-type (WT) control mice for this study. Remarkably, the KO mice were more susceptible to 5.0 Gy total-body irradiation, resulting in worse clinic scores and lower survival rate, compared with the wild-type mice. Histological examination indicated that the KO mice were subjected to slow recovery of intestinal villus and mucosa function, characterized by the reduced expression of TFF3, Glut1 and Muc2. In addition, compared with the wild-type controls, the KO mice experienced serious inflammation response in intestinal tissue, indicated by the remarkably increased expression level of IL-1β, IL-6 and IL-12. Using high-throughput sequencing analysis, we found that the intestinal bacterial community of the KO mice was more prone to dysbiosis than that of the WT mice, with significantly increased abundance of opportunistic pathogenic bacteria, such as Streptococcus sp. and Escherichia-Shigella sp., whereas remarkably decreased probiotics harboring Lactobacillus sp., Desulfovibrio sp. etc. Fecal metabolomics analysis indicated that the relative abundance of 31 metabolites arose significantly different between WT and KO mice on day 10 after radiation exposure. A subset of differential metabolites to regulate host metabolism and immunity, such as acetic acid, acetate, butanoic acid, was negatively correlated with the alteration of gut microbiota in the irradiated KO mice. This study provides new insight into EαDs contribution to the recovery of radiation-induced intestinal damage, and suggests a potential novel target to prevent the adverse effects of radiotherapy.

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5–2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed in vivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.