Mitochondrial reactive oxygen species impact human fibroblast responses to protracted γ-ray exposures.

Purpose: Continuous exposure to ionizing radiation at a low dose rate poses significant health risks to humans on deep space missions, prompting the need for mechanistic studies to identify countermeasures against its deleterious effects. Mitochondria are a major subcellular locus of radiogenic injury, and may trigger secondary cellular responses through the production of reactive oxygen species (mtROS) with broader biological implications. Methods and Materials: To determine the contribution of mtROS to radiation-induced cellular responses, we investigated the impacts of protracted γ-ray exposures (IR; 1.1 Gy delivered at 0.16 mGy/min continuously over 5 days) on mitochondrial function, gene expression, and the protein secretome of human HCA2-hTERT fibroblasts in the presence and absence of a mitochondria-specific antioxidant mitoTEMPO (MT; 5 µM). Results: IR increased fibroblast mitochondrial oxygen consumption (JO2) and H2O2 release rates (JH2O2) under energized conditions, which corresponded to higher protein expression of NADPH Oxidase (NOX) 1, NOX4, and nuclear DNA-encoded subunits of respiratory chain Complexes I and III, but depleted mtDNA transcripts encoding subunits of the same complexes. This was associated with activation of gene programs related to DNA repair, oxidative stress, and protein ubiquination, all of which were attenuated by MT treatment along with radiation-induced increases in JO2 and JH2O2. IR also increased secreted levels of interleukin-8 and Type I collagens, while decreasing Type VI collagens and enzymes that coordinate assembly and remodeling of the extracellular matrix. MT treatment attenuated many of these effects while augmenting others, revealing complex effects of mtROS in fibroblast responses to IR. Conclusion: These results implicate mtROS production in fibroblast responses to protracted radiation exposure, and suggest potentially protective effects of mitochondrial-targeted antioxidants against radiogenic tissue injury in vivo.

Radiation cataract in Heterogeneous Stock mice after γ-ray or HZE ion exposure.

Health effects of space radiation are a serious concern for astronauts on long-duration missions. The lens of the eye is one of the most radiosensitive tissues in the body and, therefore, ocular health risks for astronauts is a significant concern. Studies in humans and animals indicate that ionizing radiation exposure to the eye produces characteristic lens changes, termed "radiation cataract," that can affect visual function. Animal models of radiation cataractogenesis have previously utilized inbred mouse or rat strains. These studies were essential for determining morphological changes and dose-response relationships between radiation exposure and cataract. However, the relevance of these studies to human radiosensitivity is limited by the narrow phenotypic range of genetically homogeneous animal models. To model radiation cataract in genetically diverse populations, longitudinal cataract phenotyping was nested within a lifetime carcinogenesis study in male and female heterogeneous stock (HS/Npt) mice exposed to 0.4 Gy HZE ions (n = 609) or 3.0 Gy γ-rays (n = 602) and in unirradiated controls (n = 603). Cataractous change was quantified in each eye for up to 2 years using Merriam-Focht grading criteria by dilated slit lamp examination. Virtual Optomotry™ measurement of visual acuity and contrast sensitivity was utilized to assess visual function in a subgroup of mice. Prevalence and severity of posterior lens opacifications were 2.6-fold higher in HZE ion and 2.3-fold higher in γ-ray irradiated mice compared to unirradiated controls. Male mice were at greater risk for spontaneous and radiation associated cataracts. Risk for cataractogenesis was associated with family structure, demonstrating that HS/Npt mice are well-suited to evaluate genetic determinants of ocular radiosensitivity. Last, mice were extensively evaluated for cataract and tumor formation, which revealed an overlap between individual susceptibility to both cancer and cataract.

Galactic cosmic ray simulation at the NASA space radiation laboratory - Progress, challenges and recommendations on mixed-field effects.

For missions beyond low Earth orbit to the moon or Mars, space explorers will encounter a complex radiation field composed of various ion species with a broad range of energies. Such missions pose significant radiation protection challenges that need to be solved in order to minimize exposures and associated health risks. An innovative galactic cosmic ray simulator (GCRsim) was recently developed at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). The GCRsim technology is intended to represent major components of the space radiation environment in a ground analog laboratory setting where it can be used to improve understanding of biological risks and serve as a testbed for countermeasure development and validation. The current GCRsim consists of 33 energetic ion beams that collectively simulate the primary and secondary GCR field encountered by humans in space over the broad range of particle types, energies, and linear energy transfer (LET) of interest to health effects. A virtual workshop was held in December 2020 to assess the status of the NASA baseline GCRsim. Workshop attendees examined various aspects of simulator design, with a particular emphasis on beam selection strategies. Experimental results, modeling approaches, areas of consensus, and questions of concern were also discussed in detail. This report includes a summary of the GCRsim workshop and a description of the current status of the GCRsim. This information is important for future advancements and applications in space radiobiology.

Clinical Trial in a Dish for Space Radiation Countermeasure Discovery.

NASA aims to return humans to the moon within the next five years and to land humans on Mars in a few decades. Space radiation exposure represents a major challenge to astronauts' health during long-duration missions, as it is linked to increased risks of cancer, cardiovascular dysfunctions, central nervous system (CNS) impairment, and other negative outcomes. Characterization of radiation health effects and developing corresponding countermeasures are high priorities for the preparation of long duration space travel. Due to limitations of animal and cell models, the development of novel physiologically relevant radiation models is needed to better predict these individual risks and bridge gaps between preclinical testing and clinical trials in drug development. "Clinical Trial in a Dish" (CTiD) is now possible with the use of human induced pluripotent stem cells (hiPSCs), offering a powerful tool for drug safety or efficacy testing using patient-specific cell models. Here we review the development and applications of CTiD for space radiation biology and countermeasure studies, focusing on progress made in the past decade.

Reversing radiation-induced immunosuppression using a new therapeutic modality.

Radiation-induced immune suppression poses significant health challenges for millions of patients undergoing cancer chemotherapy and radiotherapy treatment, and astronauts and space tourists travelling to outer space. While a limited number of recombinant protein therapies, such a Sargramostim, are approved for accelerating hematologic recovery, the pronounced role of granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF2) as a proinflammatory cytokine poses additional challenges in creating immune dysfunction towards pathogenic autoimmune diseases. Here we present an approach to high-throughput drug-discovery, target validation, and lead molecule identification using nucleic acid-based molecules. These Nanoligomer™ molecules are rationally designed using a bioinformatics and an artificial intelligence (AI)-based ranking method and synthesized as a single-modality combining 6-different design elements to up- or downregulate gene expression of target gene, resulting in elevated or diminished protein expression of intended target. This method additionally alters related gene network targets ultimately resulting in pathway modulation. This approach was used to perturb and identify the most effective upstream regulators and canonical pathways for therapeutic intervention to reverse radiation-induced immunosuppression. The lead Nanoligomer™ identified in a screen of human donor derived peripheral blood mononuclear cells (PBMCs) upregulated Erythropoietin (EPO) and showed the greatest reversal of radiation induced cytokine changes. It was further tested in vivo in a mouse radiation-model with low-dose (3 mg/kg) intraperitoneal administration and was shown to regulate gene expression of epo in lung tissue as well as counter immune suppression. These results point to the broader applicability of our approach towards drug-discovery, and potential for further investigation of our lead molecule as reversible gene therapy to treat adverse health outcomes induced by radiation exposure.

Orthologs of human circulating miRNAs associated with hepatocellular carcinoma are elevated in mouse plasma months before tumour detection.

Research examining the potential for circulating miRNA to serve as markers for preneoplastic lesions or early-stage hepatocellular carcinoma (HCC) is hindered by the difficulties of obtaining samples from asymptomatic individuals. As a surrogate for human samples, we identified hub miRNAs in gene co-expression networks using HCC-bearing C3H mice. We confirmed 38 hub miRNAs as associated with HCC in F2 hybrid mice derived from radiogenic HCC susceptible and resistant founders. When compared to a panel of 12 circulating miRNAs associated with human HCC, two had no mouse ortholog and 7 of the remaining 10 miRNAs overlapped with the 38 mouse HCC hub miRNAs. Using small RNA sequencing data generated from serially collected plasma samples in F2 mice, we examined the temporal levels of these 7 circulating miRNAs and found that the levels of 4 human circulating markers, miR-122-5p, miR-100-5p, miR-34a-5p and miR-365-3p increased linearly as the time approaching HCC detection neared, suggesting a correlation of miRNA levels with oncogenic progression. Estimation of change points in the kinetics of the 4 circulating miRNAs suggested the changes started 17.5 to 6.8 months prior to HCC detection. These data establish these 4 circulating miRNAs as potential sentinels for preneoplastic lesions or early-stage HCC.

Transcriptomic analysis links hepatocellular carcinoma (HCC) in HZE ion irradiated mice to a human HCC subtype with favorable outcomes.

High-charge, high-energy ion particle (HZE) radiations are extraterrestrial in origin and characterized by high linear energy transfer (high-LET), which causes more severe cell damage than low-LET radiations like γ-rays or photons. High-LET radiation poses potential cancer risks for astronauts on deep space missions, but the studies of its carcinogenic effects have relied heavily on animal models. It remains uncertain whether such data are applicable to human disease. Here, we used genomics approaches to directly compare high-LET radiation-induced, low-LET radiation-induced and spontaneous hepatocellular carcinoma (HCC) in mice with a human HCC cohort from The Cancer Genome Atlas (TCGA). We identified common molecular pathways between mouse and human HCC and discovered a subset of orthologous genes (mR-HCC) that associated high-LET radiation-induced mouse HCC with a subgroup (mrHCC2) of the TCGA cohort. The mrHCC2 TCGA cohort was more enriched with tumor-suppressing immune cells and showed a better prognostic outcome than other patient subgroups.

Associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation.

The space radiation environment consists of multiple species of charged particles, including 28Si ions, that may impact brain function during and following missions. To develop biomarkers of the space radiation response, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with 28Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select pertinent brain regions were dissected for lipidomic analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. There were associations between lipids in select brain regions, plasma miRNA, and cognitive measures and behavioral following 28Si ion irradiation. Different but overlapping sets of miRNAs in plasma were found to be associated with cognitive measures and behavioral in sham and irradiated mice at the three time points. The radiation condition revealed pathways involved in neurodegenerative conditions and cancers. Levels of the dendritic marker MAP2 in the cortex were higher in irradiated than sham-irradiated mice at middle age, which might be part of a compensatory response. Relationships were also revealed with CD68 in miRNAs in an anatomical distinct fashion, suggesting that distinct miRNAs modulate neuroinflammation in different brain regions. The associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation could be used for the development of biomarker of the space radiation response.

Effects of Acute and Chronic Exposure to a Mixed Field of Neutrons and Photons and Single or Fractionated Simulated Galactic Cosmic Ray Exposure on Behavioral and Cognitive Performance in Mice.

During space missions, astronauts experience acute and chronic low-dose-rate radiation exposures. Given the clear gap of knowledge regarding such exposures, we assessed the effects acute and chronic exposure to a mixed field of neutrons and photons and single or fractionated simulated galactic cosmic ray exposure (GCRsim) on behavioral and cognitive performance in mice. In addition, we assessed the effects of an aspirin-containing diet in the presence and absence of chronic exposure to a mixed field of neutrons and photons. In C3H male mice, there were effects of acute radiation exposure on activity levels in the open field containing objects. In addition, there were radiation-aspirin interactions for effects of chronic radiation exposure on activity levels and measures of anxiety in the open field, and on activity levels in the open field containing objects. There were also detrimental effects of aspirin and chronic radiation exposure on the ability of mice to distinguish the familiar and novel object. Finally, there were effects of acute GCRsim on activity levels in the open field containing objects. Activity levels were lower in GCRsim than sham-irradiated mice. Thus, acute and chronic irradiation to a mixture of neutrons and photons and acute and fractionated GCRsim have differential effects on behavioral and cognitive performance of C3H mice. Within the limitations of our study design, aspirin does not appear to be a suitable countermeasure for effects of chronic exposure to space radiation on cognitive performance.

Tumor aggressiveness is independent of radiation quality in murine hepatocellular carcinoma and mammary tumor models.

PURPOSE: Estimating cancer risk associated with interplanetary space travel is complicated. Human exposure data to high atomic number, high-energy (HZE) radiation is lacking, so data from low linear energy transfer (low-LET) γ-ray radiation is used in risk models, with the assumption that HZE and γ-ray radiation have comparable biological effects. This assumption has been challenged by reports indicating that HZE radiation might produce more aggressive tumors. The goal of this research is to test whether high-LET HZE radiation induced tumors are more aggressive. MATERIALS AND METHODS: Murine models of mammary and liver cancer were used to compare the impact of exposure to 0.2Gy of 300MeV/n silicon ions, 3 Gy of γ-rays or no radiation. Numerous measures of tumor aggressiveness were assessed. RESULTS: For the mammary cancer models, there was no significant change in the tumor latency or metastasis in silicon-irradiated mice compared to controls. For the liver cancer models, we observed an increase in tumor incidence but not tumor aggressiveness in irradiated mice. CONCLUSION: Tumors in the HZE-irradiated mice were not more aggressive than those arising from exposure to low-LET γ-rays or spontaneously. Thus, enhanced aggressiveness does not appear to be a uniform characteristic of all tumors in HZE-irradiated animals.

Design and dosimetry of a facility to study health effects following exposures to fission neutrons at low dose rates for long durations.

PURPOSE: During extended missions into deep space, astronauts will be exposed to a complex radiation field that includes high linear energy transfer (LET) radiation from high energy, heavy ions (HZE particles) at low dose rates of about 0.5 mGy/d for long durations. About 20% of the dose is delivered by ions with LET greater than 10 keV/µm. There are sparse empirical data in any species for carcinogenic effects from whole-body exposures to external sources of mixed or high LET radiation at this level of dose rates. For the induction of solid tumors, acute exposures to HZE ions have been shown to be substantially more effective per unit dose than low LET exposures associated with photons. To determine the health effects of high LET radiation at space-relevant dose rates on experimental animals, we developed a vivarium in which rodents could be irradiated with Californium (252Cf) neutrons for protracted periods of time. MATERIALS AND METHODS: The neutron source is a panoramic irradiator containing 252Cf located in a concrete shielded vault with a footprint of 53 m2. The vault can accommodate sufficient caging to simultaneously irradiate 900 mice and 60 rats for durations up to 400 d at a dose rate of 1 mGy/d and is approved for extended animal husbandry. RESULTS: The mixed field fluence is a combination of neutrons and photons emitted directly from the source and scattered particles from the concrete walls and floor. Mixed field dosimetry was performed using a miniature GM counter and CaF2:Dy thermoluminescent dosimeters (TLD) for photons and tissue-equivalent proportional counters (TEPC) for neutrons. TEPC data provided macroscopic dose rates as well as measurements of radiation quality based on lineal energy, y, and LET. The instantaneous dose rate from the source decreases with a half-life of 2.6 years. The exposure time is adjusted weekly to yield a total dose 1 mGy/d. The photon contribution is 20% of the total dose. The uncertainty in the delivered dose is estimated to be ±20% taking into account spatial variations in the room and random position of mice in each cage. The dose averaged LET for the charged particle recoil nuclei is 68 keV/µ. CONCLUSIONS: We have developed a facility to perform high LET studies in mice and rats at space relevant dose rates and career-relevant doses using neutrons emitted from the spontaneous fission of 252Cf.

Adverse outcome pathways, key events, and radiation risk assessment.

The overall aim of this contribution to the 'Second Bill Morgan Memorial Special Issue' is to provide a high-level review of a recent report developed by a Committee for the National Council on Radiation Protection and Measurements (NCRP) titled 'Approaches for Integrating Information from Radiation Biology and Epidemiology to Enhance Low-Dose Health Risk Assessment'. It derives from previous NCRP Reports and Commentaries that provide the case for integrating data from radiation biology studies (available and proposed) with epidemiological studies (also available and proposed) to develop Biologically-Based Dose-Response (BBDR) models. In this review, it is proposed for such models to leverage the adverse outcome pathways (AOP) and key events (KE) approach for better characterizing radiation-induced cancers and circulatory disease (as the example for a noncancer outcome). The review discusses the current state of knowledge of mechanisms of carcinogenesis, with an emphasis on radiation-induced cancers, and a similar discussion for circulatory disease. The types of the various informative BBDR models are presented along with a proposed generalized BBDR model for cancer and a more speculative one for circulatory disease. The way forward is presented in a comprehensive discussion of the research needs to address the goal of enhancing health risk assessment of exposures to low doses of radiation. The use of an AOP/KE approach for developing a mechanistic framework for BBDR models of radiation-induced cancer and circulatory disease is considered to be a viable one based upon current knowledge of the mechanisms of formation of these adverse health outcomes and the available technical capabilities and computational advances. The way forward for enhancing low-dose radiation risk estimates will require there to be a tight integration of epidemiology data and radiation biology information to meet the goals of relevance and sensitivity of the adverse health outcomes required for overall health risk assessment at low doses and dose rates.

Effects of chronic exposure to a mixed field of neutrons and photons on behavioral and cognitive performance in mice.

To simulate the space radiation environment astronauts are exposed to, most studies involve acute exposures but during a space mission there will be chronic (long-lasting) exposures. To address this knowledge gap, a neutron irradiator using a 252Cf (252Californium) source was used to generate a mixed field of neutrons and photons to simulate chronic, low dose rate exposures to high LET radiation. In the present study, we assessed the effects chronic neutron exposure starting at 60 days of age on behavioral and cognitive performance of BALB/c female and C3H male mice at 600 and 700 days of age as part of an opportunistic study that took advantage of the availability of neutron and sham-irradiated mice from a radiation carcinogenesis experiment. There were profound dose- and time point-dependent effects of chronic neutron exposure. At the 600-day time point, irradiated BALB/c female mice showed improved nest building at all three doses. At the 700-day, but not 600-day, time point slightly but significantly increased body weights were seen in C3H male mice exposed to 0.118 Gy. At the 600-day time point BALB/c female mice irradiated with 0.2 Gy did, like sham-irradiated, not show preferential exploration of the novel object that was seen in mice irradiated with 0.118 or 0.4 Gy. In C3H male mice exposed to 0.4 Gy and at the 600-day time point, increased measures of anxiety were observed on days 1 and 2 in the open field. Thus, different outcome measures show distinct dose-response relationships, with some anticipated to worsen performance during space missions, like increased measures of anxiety, while other anticipated to enhance performance, such as increased nest building and object recognition.

Persistence of Gamma-H2AX Foci in Bronchial Cells Correlates with Susceptibility to Radiation Associated Lung Cancer in Mice.

The risk of developing radiation-induced lung cancer differs between different strains of mice, but the underlying cause of the strain differences is unknown. Strains of mice also differ in how quickly they repair radiation-induced DNA double-strand breaks (DSBs). We assayed mouse strains from the CcS/Dem recombinant congenic strain set for their efficacy in repairing DNA DSBs during protracted irradiation. We measured unrepaired γ-H2AX radiation-induced foci (RIF), which persisted after chronic 24-h gamma irradiation, as a surrogate marker for repair efficiency in bronchial epithelial cells for 17 of the CcS/Dem strains and the BALB/c founder strain. We observed a very strong correlation (R2 = 79.18%, P < 0.001) between the level of unrepaired RIF and radiogenic lung cancer incidence measured in the same strains. Interestingly, spontaneous levels of foci in nonirradiated mice also showed good correlation with lung cancer incidence when incidence data from male and female mice were combined. These results suggest that genetic differences in DNA repair capacity largely account for differing susceptibilities to radiation-induced lung cancer among CcS/Dem mouse strains, and that high levels of spontaneous DNA damage are also a relatively good marker of cancer predisposition. In a smaller pilot study, we found that the repair capacity measured in peripheral blood leucocytes also correlated well with radiogenic lung cancer susceptibility, raising the possibility that the assay could be used to detect radiogenic lung cancer susceptibility in humans.

Space Radiation Alters Genotype-Phenotype Correlations in Fear Learning and Memory Tests.

Behavioral and cognitive traits have a genetic component even though contributions from individual genes and genomic loci are in many cases modest. Changes in the environment can alter genotype-phenotype relationships. Space travel, which includes exposure to ionizing radiation, constitutes environmental challenges and is expected to induce not only dramatic behavioral and cognitive changes but also has the potential to induce physical DNA damage. In this study, we utilized a genetically heterogeneous mouse model, dense genotype data, and shifting environmental challenges, including ionizing radiation exposure, to explore and quantify the size and stability of the genetic component of fear learning and memory-related measures. Exposure to ionizing radiation and other external stressors altered the genotype-phenotype correlations, although different behavioral and cognitive measures were affected to different extents. Utilizing an integrative genomic approach, we identified pathways and functional ontology categories associated with these behavioral and cognitive measures.

Mimicking the effects of spaceflight on bone: Combined effects of disuse and chronic low-dose rate radiation exposure on bone mass in mice.

During spaceflight, crewmembers are subjected to biomechanical and biological challenges including microgravity and radiation. In the skeleton, spaceflight leads to bone loss, increasing the risk of fracture. Studies utilizing hindlimb suspension (HLS) as a ground-based model of spaceflight often neglect the concomitant effects of radiation exposure, and even when radiation is accounted for, it is often delivered at a high-dose rate over a very short period of time, which does not faithfully mimic spaceflight conditions. This study was designed to investigate the skeletal effects of low-dose rate gamma irradiation (8.5 cGy gamma radiation per day for 20 days, amounting to a total dose of 1.7 Gy) when administered simultaneously to disuse from HLS. The goal was to determine whether continuous, low-dose rate radiation administered during disuse would exacerbate bone loss in a murine HLS model. Four groups of 16 week old female C57BL/6 mice were studied: weight bearing + no radiation (WB+NR), HLS + NR, WB + radiation exposure (WB+RAD), and HLS+RAD. Surprisingly, although HLS led to cortical and trabecular bone loss, concurrent radiation exposure did not exacerbate these effects. Our results raise the possibility that mechanical unloading has larger effects on the bone loss that occurs during spaceflight than low-dose rate radiation.

High-energy particle beam and gamma radiation exposure, familial relatedness and cancer in mice.

BACKGROUND: Some highly penetrant familial cancer syndromes exhibit elevated leukaemia risk, and there is evidence for familial clustering of lung cancer and other common cancers. Lung cancer and leukaemia are strongly radiogenic, but there are few indications that high-energy beam irradiation is markedly more effective than lower-energy radiation. METHODS: We used a Cox model with familially structured random effects to assess 16 mortality end points in a group of 1850 mice in 47 families maintained in a circular-breeding scheme, exposed to accelerated Si or Fe ions (0.4 Gy) or 137Cs gamma rays (3 Gy). RESULTS: There is periodicity in the effect of familial relatedness, which is most pronounced for pulmonary adenoma, Harderian-gland adenoma, Harderian-gland tumour, ectodermal tumour, pulmonary adenocarcinoma and hepatocellular carcinoma (P=0.0001/0.0003/0.0017/0.0035/0.0257/0.0340, respectively) with families that are 3-4 generations apart most strongly correlated; myeloid leukaemia also exhibited a striking periodic correlation structure. The relative risks of high-energy Si or Fe ions are not significantly different and are less than for 137Cs gamma-rays for most end points at the doses used. CONCLUSIONS: There is periodicity in the effect of familial relatedness for various cancer sites. The effects per unit dose of high-energy charged particle beams are no higher than ninefold those of lower-energy gamma radiation.