Blair Stewart

Exposure to Low-Dose 56Fe-Ion Radiation Induces Long-Term Epigenetic Alterations in Mouse Bone Marrow Hematopoietic Progenitor and Stem Cells

There is an increasing need to better understand the long-term health effects of high-linear energy transfer (LET) radiation due to exposure during space missions, as well as its increasing use in clinical treatments. Previous studies have indicated that exposure to 56Fe heavy ions increases the incidence of acute myeloid leukemia (AML) in mice but the underlying molecular mechanisms remain elusive. Epigenetic alterations play a role in radiation-induced genomic instability and the initiation and progression of AML. In this study, we assessed the effects of low-dose 56Fe-ion irradiation on epigenetic alterations in bone marrow mononuclear cells (BM-MNCs) and hematopoietic progenitor and stem cells (HPSCs). Exposure to 56Fe ions (600 MeV, 0.1, 0.2 and 0.4 Gy) resulted in significant epigenetic alterations involving methylation of DNA, the DNA methylation machinery and expression of repetitive elements. Four weeks after irradiation, these changes were primarily confined to HPSCs and were exhibited as dose-dependent hypermethylation of LINE1 and SINE B1 repetitive elements [4.2-fold increase in LINE1 (P < 0.001) and 7.6-fold increase in SINE B1 (P < 0.01) after exposure to 0.4 Gy; n = 5]. Epigenetic alterations were persistent and detectable for at least 22 weeks after exposure, when significant loss of global DNA hypomethylation (1.9-fold, P < 0.05), decreased expression of Dnmt1 (1.9-fold, P < 0.01), and increased expression of LINE1 and SINE B1 repetitive elements (2.8-fold, P < 0.001 for LINE1 and 1.9-fold, P < 0.05 for SINE B1; n = 5) were observed after exposure to 0.4 Gy. In contrast, exposure to 56Fe ions did not result in accumulation of increased production of reactive oxygen species (ROS) and DNA damage, exhibited as DNA strand breaks. Furthermore, no significant alterations in cellular senescence and apoptosis were detected in HPSCs after exposure to 56Fe-ion radiation. These findings suggest that epigenetic reprogramming is possibly involved in the development of radiation-induced genomic instability and thus, may have a causative role in the development of AML.

Whole-Body Proton Irradiation Causes Long-Term Damage to Hematopoietic Stem Cells in Mice

Space flight poses certain health risks to astronauts, including exposure to space radiation, with protons accounting for more than 80% of deep-space radiation. Proton radiation is also now being used with increasing frequency in the clinical setting to treat cancer. For these reasons, there is an urgent need to better understand the biological effects of proton radiation on the body. Such improved understanding could also lead to more accurate assessment of the potential health risks of proton radiation, as well as the development of improved strategies to prevent and mitigate its adverse effects. Previous studies have shown that exposure to low doses of protons is detrimental to mature leukocyte populations in peripheral blood, however, the underlying mechanisms are not known. Some of these detriments may be attributable to damage to hematopoietic stem cells (HSCs) that have the ability to self-renew, proliferate and differentiate into different lineages of blood cells through hematopoietic progenitor cells (HPCs). The goal of this study was to investigate the long-term effects of low-dose proton irradiation on HSCs. We exposed C57BL/6J mice to 1.0 Gy whole-body proton irradiation (150 MeV) and then studied the effects of proton radiation on HSCs and HPCs in the bone marrow (BM) 22 weeks after the exposure. The results showed that mice exposed to 1.0 Gy whole-body proton irradiation had a significant and persistent reduction of BM HSCs compared to unirradiated controls. In contrast, no significant changes were observed in BM HPCs after proton irradiation. Furthermore, irradiated HSCs and their progeny exhibited a significant impairment in clonogenic function, as revealed by the cobblestone area-forming cell (CAFC) and colony-forming cell assays, respectively. These long-term effects of proton irradiation on HSCs may be attributable to the induction of chronic oxidative stress in HSCs, because HSCs from irradiated mice exhibited a significant increase in NADPH oxidase 4 (NOX4) mRNA expression and reactive oxygen species (ROS) production. In addition, the increased production of ROS in HSCs was associated with a significant reduction in HSC quiescence and an increase in DNA damage. These findings indicate that exposure to proton radiation can lead to long-term HSC injury, probably in part by radiation-induced oxidative stress.

Long-term epigenetic effects of exposure to low doses of 56Fe in the mouse lung

Despite significant progress, the long-term health effects of exposure to high charge (Z) and energy (E) nuclei (HZEs) and the underlying mechanisms remain poorly understood. Mouse studies show that space missions can result in pulmonary pathological states. The goal of this study was to evaluate the pro-fibrotic and pro-carcinogenic effects of exposure to low doses of heavy iron ions (56Fe) in the mouse lung. Exposure to 56Fe (600 MeV; 0.1, 0.2 and 0.4 Gy) resulted in minor pro-fibrotic changes, detected at the beginning of the fibrotic phase (22 weeks post exposure), which were exhibited as increased expression of chemokine Ccl3, and interleukin Il4. Epigenetic alterations were exhibited as global DNA hypermethylation, observed after exposure to 0.4 Gy. Cadm1, Cdh13, Cdkn1c, Mthfr and Sfrp1 were significantly hypermethylated after exposure to 0.1 Gy, while exposure to higher doses resulted in hypermethylation of Cdkn1c only. However, expression of these genes was not affected by any dose. Congruently with the observed patterns of global DNA methylation, DNA repetitive elements were hypermethylated after exposure to 0.4 Gy, with minor changes observed after exposure to lower doses. Importantly, hypermethylation of repetitive elements coincided with their transcriptional repression. The findings of this study will aid in understanding molecular determinants of pathological states associated with exposure to 56Fe, as well as serve as robust biomarkers for the delayed effects of irradiation. Further studies are clearly needed to investigate the persistence and outcomes of molecular alterations long term after exposure.

28Silicon Irradiation Impairs Contextual Fear Memory in B6D2F1 Mice

The space radiation environment consists of multiple species of charged particles, including 28Si, 48Ti and protons that may impact cognition, but their damaging effects have been poorly defined. In mouse studies, C57Bl6/J homozygous wild-type mice and genetic mutant mice on a C57Bl6/J background have typically been used for assessing effects of space radiation on cognition. In contrast, little is known about the radiation response of mice on a heterozygous background. Therefore, in the current study we tested the effects of 28Si, 48Ti and proton radiation on hippocampus-dependent contextual fear memory and hippocampus-independent cued fear memory in C57Bl6/J × DBA2/J F1 (B6D2F1) mice three months after irradiation. Contextual fear memory was impaired at a 1.6 Gy dose of 28Si radiation, but not cued fear memory. 48Ti or proton irradiation did not affect either type of memory. Based on earlier space radiation cognitive data in C57Bl6/J mice, these data highlight the importance of including different genetic backgrounds in studies aimed at assessing cognitive changes after exposure to space radiation.

Short- and long-term effects of 56 Fe irradiation on cognition and hippocampal DNA methylation and gene expression

BackgroundAstronauts are exposed to 56Fe ions that may pose a significant health hazard during and following prolonged missions in deep space. We showed previously that object recognition requiring the hippocampus, a structure critical for cognitive function, is affected in 2-month-old mice irradiated with 56Fe ions. Here we examined object recognition in 6-month-old mice irradiated with 56Fe ions, a biological age more relevant to the typical ages of astronauts. Moreover, because the mechanisms mediating the detrimental effects of 56Fe ions on hippocampal function are unclear, we examined changes in hippocampal networks involved in synaptic plasticity and memory, gene expression, and epigenetic changes in cytosine methylation (5mC) and hydroxymethylation (5hmC) that could accompany changes in gene expression. We assessed the effects of whole body 56Fe ion irradiation at early (2 weeks) and late (20 weeks) time points on hippocampus-dependent memory and hippocampal network stability, and whether these effects are associated with epigenetic changes in hippocampal DNA methylation (both 5mC and 5hmC) and gene expression.ResultsAt the two-week time point, object recognition and network stability were impaired following irradiation at the 0.1 and 0.4 Gy dose, but not following irradiation at the 0.2 Gy dose. No impairments in object recognition or network stability were seen at the 20-week time point at any irradiation dose used. Consistent with this pattern, the significance of pathways for gene categories for 5hmC was lower, though not eliminated, at the 20-week time point compared to the 2-week time point. Similarly, significant changes were observed for 5mC gene pathways at the 2-week time point, but no significant gene categories were observed at the 20-week time point. Only the 5hmC changes tracked with gene expression changes.ConclusionsDose- and time-dependent epigenomic remodeling in the hippocampus following 56Fe ion exposure correlates with behavioral changes.

Bi-directional and shared epigenomic signatures following proton and 56 Fe irradiation

The brain’s response to radiation exposure is an important concern for patients undergoing cancer therapy and astronauts on long missions in deep space. We assessed whether this response is specific and prolonged and is linked to epigenetic mechanisms. We focused on the response of the hippocampus at early (2-weeks) and late (20-week) time points following whole body proton irradiation. We examined two forms of DNA methylation, cytosine methylation (5mC) and hydroxymethylation (5hmC). Impairments in object recognition, spatial memory retention, and network stability following proton irradiation were observed at the two-week time point and correlated with altered gene expression and 5hmC profiles that mapped to specific gene ontology pathways. Significant overlap was observed between DNA methylation changes at the 2 and 20-week time points demonstrating specificity and retention of changes in response to radiation. Moreover, a novel class of DNA methylation change was observed following an environmental challenge (i.e. space irradiation), characterized by both increased and decreased 5hmC levels along the entire gene body. These changes were mapped to genes encoding neuronal functions including postsynaptic gene ontology categories. Thus, the brain’s response to proton irradiation is both specific and prolonged and involves novel remodeling of non-random regions of the epigenome.

Enhanced functional connectivity involving the ventromedial hypothalamus following methamphetamine exposure

Methamphetamine (MA) consumption causes disruption of many biological rhythms including the sleep-wake cycle. This circadian effect is seen shortly following MA exposure and later in life following developmental MA exposure. MA phase shifts, entrains the circadian clock and can also alter the entraining effect of light by currently unknown mechanisms. We analyzed and compared immunoreactivity of the immediate early gene c-Fos, a marker of neuronal activity, to assess neuronal activation 2 h following MA exposure in the light and dark phases. We used network analyses of correlation patterns derived from global brain immunoreactivity patterns of c-Fos, to infer functional connectivity between brain regions. There were five distinct patterns of neuronal activation. In several brain areas, neuronal activation following exposure to MA was stronger in the light than the dark phase, highlighting the importance of considering circadian periods of increased effects of MA in defining experimental conditions and understanding the mechanisms underlying detrimental effects of MA exposure to brain function. Functional connectivity between the ventromedial hypothalamus (VMH) and other brain areas, including the paraventricular nucleus of the hypothalamus and basolateral and medial amygdala, was enhanced following MA exposure, suggesting a role for the VMH in the effects of MA on the brain.

28Si total body irradiation injures bone marrow hematopoietic stem cells via induction of cellular apoptosis

Long-term space mission exposes astronauts to a radiation environment with potential health hazards. High-energy charged particles (HZE), including 28Si nuclei in space, have deleterious effects on cells due to their characteristics with high linear energy transfer and dense ionization. The influence of 28Si ions contributes more than 10% to the radiation dose equivalent in the space environment. Understanding the biological effects of 28Si irradiation is important to assess the potential health hazards of long-term space missions. The hematopoietic system is highly sensitive to radiation injury and bone marrow (BM) suppression is the primary life-threatening injuries after exposure to a moderate dose of radiation. Therefore, in the present study we investigated the acute effects of low doses of 28Si irradiation on the hematopoietic system in a mouse model. Specifically, 6-month-old C57BL/6J mice were exposed to 0.3, 0.6 and 0.9Gy 28Si (600MeV) total body irradiation (TBI). The effects of 28Si TBI on BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were examined four weeks after the exposure. The results showed that exposure to 28Si TBI dramatically reduced the frequencies and numbers of HSCs in irradiated mice, compared to non-irradiated controls, in a radiation dose-dependent manner. In contrast, no significant changes were observed in BM HPCs regardless of radiation doses. Furthermore, irradiated HSCs exhibited a significant impairment in clonogenic ability. These acute effects of 28Si irradiation on HSCs may be attributable to radiation-induced apoptosis of HSCs, because HSCs, but not HPCs, from irradiated mice exhibited a significant increase in apoptosis in a radiation dose-dependent manner. However, exposure to low doses of 28Si did not result in an increased production of reactive oxygen species and DNA damage in HSCs and HPCs. These findings indicate that exposure to 28Si irradiation leads to acute HSC damage.

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.