Histopathological studies of nonhuman primates exposed to supralethal doses of total- or partial-body radiation: influence of a medical countermeasure, gamma-tocotrienol.

Despite remarkable scientific progress over the past six decades within the medical arts and in radiobiology in general, limited radiation medical countermeasures (MCMs) have been approved by the United States Food and Drug Administration for the acute radiation syndrome (ARS). Additional effort is needed to develop large animal models for improving the prediction of clinical safety and effectiveness of MCMs for acute and delayed effects of radiation in humans. Nonhuman primates (NHPs) are considered the animal models that reproduce the most appropriate representation of human disease and are considered the gold standard for drug development and regulatory approval. The clinical and histopathological effects of supralethal, total- or partial-body irradiations (12 Gy) of NHPs were assessed, along with possible protective actions of a promising radiation MCM, gamma-tocotrienol (GT3). Results show that these supralethal radiation exposures induce severe injuries that manifest both clinically as well as pathologically, as evidenced by the noted functionally crippling lesions within various major organ systems of experimental NHPs. The MCM, GT3, has limited radioprotective efficacy against such supralethal radiation doses.

Modulation of Hematopoietic Injury by a Promising Radioprotector, Gamma-Tocotrienol, in Rhesus Macaques Exposed to Partial-Body Radiation.

Currently, no radioprotectors have been approved to mitigate hematopoietic injury after exposure to ionizing radiation. Acute ionizing radiation results in damage to both hematopoietic and immune system cells. Pre-exposure prophylactic agents are needed for first responders and military personnel. In this study, the ability of gamma-tocotrienol (GT3), a promising radioprotector and antioxidant, to ameliorate partial-body radiation-induced damage to the hematopoietic compartment was evaluated in a nonhuman primate (NHP) model. A total of 15 rhesus NHPs were divided into two groups, and were administered either GT3 or vehicle 24 h prior to 4 or 5.8 Gy partial-body irradiation (PBI), with 5% bone marrow (BM) sparing. Each group consisted of four NHPs, apart from the vehicle-treated group exposed to 5.8 Gy, which had only three NHPs. BM samples were collected 8 days prior to irradiation in addition to 2, 7, 14, and 30 days postirradiation. To assess the clonogenic ability of hematopoietic stem and progenitor cells (HSPCs), colony forming unit (CFU) assays were performed, and lymphoid cells were immunophenotyped using flow cytometry. As a result of GT3 treatment, an increase in HSPC function was evident by an increased recovery of CFU-granulocyte macrophages (CFU-GM). Additionally, GT3 treatment was shown to increase the percentage of CD34+ cells, including T and NK-cell subsets. Our data further affirm GT3's role in hematopoietic recovery and suggest the need for its further development as a prophylactic radiation medical countermeasure.

Cytogenetic and epigenetic aberrations in peripheral lymphocytes of northwest Arkansas Marshallese.

PURPOSE: Nuclear weapons testing in the northern Marshall Islands between 1946 and 1958 resulted in ionizing radiation (IR) exposure of the thousands of Marshallese. Furthermore, numerous islands were contaminated by radioactive fallout. Significant increases in cancer and metabolic syndrome incidences have been reported among Marshallese, and potential for further increases looms due to the latency of radiation-induced health effects. The purpose of this study was to investigate the genetic and epigenetic effects of exposure to IR that could be associated with radiation-induced disease among the Northwest Arkansas (NWA) Marshallese. MATERIALS AND METHODS: We performed analysis of chromosomal aberrations and DNA methylation based on residential and exposure history of NWA Marshallese. RESULTS: Analysis of chromosomal aberrations demonstrated higher incidence of genetic rearrangements in women with self-reported history of radiation exposure (95% CI: 0.10, 1.22; p=.022). Further clustering of study participants based on their residential history demonstrated that participants who spent substantial amounts of time (≥6 months) in the northern atolls (thus, in the proximity of nuclear tests) before 1980 had more chromosomal aberrations than their peers who lived only in the southern atolls (95% CI: 0.08, -0.95; p=.021), and that this difference was driven by women. A relationship between the time spent in the northern atolls and increase in chromosomal aberrations was observed: 0.31 increase in chromosomal aberrations for every 10 years spent at northern atolls (95% CI: 0.06, 0.57; p=.020). Finally, significant inverse correlations between the chromosomal aberrations and the extent of DNA methylation of four LINE-1 elements L1PA2, L1PA16, L1PREC1, and L1P4B were identified. CONCLUSIONS: The results of this study provide first evidence of the presence of stable genetic and epigenetic rearrangements in peripheral lymphocytes of NWA Marshallese and warrant further studies to analyze the role of radiation exposure in health disparities experienced by this Pacific Island nation.

Gamma-Tocotrienol Modulates Total-Body Irradiation-Induced Hematopoietic Injury in a Nonhuman Primate Model.

Radiation exposure causes acute damage to hematopoietic and immune cells. To date, there are no radioprotectors available to mitigate hematopoietic injury after radiation exposure. Gamma-tocotrienol (GT3) has demonstrated promising radioprotective efficacy in the mouse and nonhuman primate (NHP) models. We determined GT3-mediated hematopoietic recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques divided into two groups received either vehicle or GT3, 24 h prior to TBI. Four animals in each treatment group were exposed to either 4 or 5.8 Gy TBI. Flow cytometry was used to immunophenotype the bone marrow (BM) lymphoid cell populations, while clonogenic ability of hematopoietic stem cells (HSCs) was assessed by colony forming unit (CFU) assays on day 8 prior to irradiation and days 2, 7, 14, and 30 post-irradiation. Both radiation doses showed significant changes in the frequencies of B and T-cell subsets, including the self-renewable capacity of HSCs. Importantly, GT3 accelerated the recovery in CD34+ cells, increased HSC function as shown by improved recovery of CFU-granulocyte macrophages (CFU-GM) and burst-forming units erythroid (B-FUE), and aided the recovery of circulating neutrophils and platelets. These data elucidate the role of GT3 in hematopoietic recovery, which should be explored as a potential medical countermeasure to mitigate radiation-induced injury to the hematopoietic system.

Effects of Gamma-Tocotrienol on Partial-Body Irradiation-Induced Intestinal Injury in a Nonhuman Primate Model.

Exposure to high doses of radiation, accidental or therapeutic, often results in gastrointestinal (GI) injury. To date, there are no therapies available to mitigate GI injury after radiation exposure. Gamma-tocotrienol (GT3) is a promising radioprotector under investigation in nonhuman primates (NHP). We have shown that GT3 has radioprotective function in intestinal epithelial and crypt cells in NHPs exposed to 12 Gy total-body irradiation (TBI). Here, we determined GT3 potential in accelerating the GI recovery in partial-body irradiated (PBI) NHPs using X-rays, sparing 5% bone marrow. Sixteen rhesus macaques were treated with either vehicle or GT3 24 h prior to 12 Gy PBI. Structural injuries and crypt survival were examined in proximal jejunum on days 4 and 7. Plasma citrulline was assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Crypt cell proliferation and apoptotic cell death were evaluated using Ki-67 and TUNEL staining. PBI significantly decreased mucosal surface area and reduced villous height. Interestingly, GT3 increased crypt survival and enhanced stem cell proliferation at day 4; however, the effects seemed to be minimized by day 7. GT3 did not ameliorate a radiation-induced decrease in citrulline levels. These data suggest that X-rays induce severe intestinal injury post-PBI and that GT3 has minimal radioprotective effect in this novel model.

Effects of Gamma-Tocotrienol on Intestinal Injury in a GI-Specific Acute Radiation Syndrome Model in Nonhuman Primate.

The gastrointestinal (GI) system is highly susceptible to irradiation. Currently, there is no Food and Drug Administration (FDA)-approved medical countermeasures for GI radiation injury. The vitamin E analog gamma-tocotrienol (GT3) is a promising radioprotector in mice and nonhuman primates (NHP). We evaluated GT3-mediated GI recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques were divided into two groups; eight received vehicle and eight GT3 24 h prior to 12 Gy TBI. Proximal jejunum was assessed for structural injuries and crypt survival on day 4 and 7. Apoptotic cell death and crypt cell proliferation were assessed with TUNEL and Ki-67 immunostaining. Irradiation induced significant shortening of the villi and reduced mucosal surface area. GT3 induced an increase in crypt depth at day 7, suggesting that more stem cells survived and proliferated after irradiation. GT3 did not influence crypt survival after irradiation. GT3 treatment caused a significant decline in TUNEL-positive cells at both day 4 (p < 0.03) and 7 (p < 0.0003). Importantly, GT3 induced a significant increase in Ki-67-positive cells at day 7 (p < 0.05). These data suggest that GT3 has radioprotective function in intestinal epithelial and crypt cells. GT3 should be further explored as a prophylactic medical countermeasure for radiation-induced GI injury.

Methods for induction and assessment of intestinal permeability in rodent models of radiation injury.

Ionizing radiation (IR) is a significant contributor to the contemporary market of energy production and an important diagnostic and treatment modality. Besides having numerous useful applications, it is also a ubiquitous environmental stressor and a potent genotoxic and epigenotoxic agent, capable of causing substantial damage to organs and tissues of living organisms. The gastrointestinal (GI) tract is highly sensitive to IR. This problem is further compounded by the fact that there is no FDA-approved medication to mitigate acute radiation-induced GI syndrome. Therefore, establishing the animal model for studying IR-induced GI-injury is crucially important to understand the harmful consequences of intestinal radiation damage. Here, we discuss two different animal models of IR-induced acute gastrointestinal syndrome and two separate methods for measuring the magnitude of intestinal radiation damage.

Randomised single centre double-blind placebo controlled phase II trial of Tocovid SupraBio in combination with pentoxifylline in patients suffering long-term gastrointestinal adverse effects of radiotherapy for pelvic cancer: The PPALM study.

BACKGROUND: Preclinical data suggest that combined gamma-tocotrienol with pentoxifylline ameliorates radiotherapy-induced gastrointestinal damage. AIM: To test whether gastrointestinal symptoms arising after radiotherapy, and persisting after maximal medical therapy, can be improved using Tocovid SupraBio 200 mg and pentoxifylline 400 mg orally twice daily for one year. Patients stratified by severity of symptoms, and randomised to active treatment or matched placebo were assessed after 12 months. The primary end point was improvement in gastrointestinal symptoms measured using the Inflammatory Bowel Disease Questionnaire, bowel subset score. Changes in bio-markers of fibrosis were assessed. RESULTS: 62 patients, median age 66, 34(55%) treated for prostate, 21(34%) gynaecological, 6(10%) anal and one(1%) rectal cancer were recruited; 40(65%) randomised to treatment, 22(35%) to placebo, 39 months (median) after radiotherapy completion. Gamma tocotrienol was not detected in serum in 41% of treated patients, despite good compliance with study medication. Treatment was completed in 28(70%) and 17(77%) patients in the treatment and placebo groups respectively. No improvement in symptom scores nor in quality of life was identified. Thirteen serious adverse events occurred. A transient ischaemic attack, was possibly related to pentoxifylline, others were assessed as unlikely to be related to treatment. Levels of EGF, PDGF and FGF were significantly reduced and consistent trends in reduced inflammation were seen during treatment but were not sustained once treatment ended. SUMMARY: This single centre study closed prematurely and therefore data interpretation is of necessity limited. No clinical benefit was demonstrated. However, biochemical data suggest that this intervention does have anti-inflammatory and anti-fibrotic effects.

Plasma Metabolomics in a Nonhuman Primate Model of Abdominal Radiation Exposure.

The acute radiation syndrome is defined in large part by radiation injury in the hematopoietic and gastrointestinal (GI) systems. To identify new pathways involved in radiation-induced GI injury, this study assessed dose- and time-dependent changes in plasma metabolites in a nonhuman primate model of whole abdominal irradiation. Male and female adult Rhesus monkeys were exposed to 6 MV photons to the abdomen at doses ranging between 8 and 14 Gy. At time points from 1 to 60 days after irradiation, plasma samples were collected and subjected to untargeted metabolomics. With the limited sample size of females, different discovery times after irradiation between males and females were observed in metabolomics pattern. Detailed analyses are restricted to only males for the discovery power. Radiation caused an increase in fatty acid oxidation and circulating levels of corticosteroids which may be an indication of physiological stress, and amino acids, indicative of a cellular repair response. The largest changes were observed at days 9 and 10 post-irradiation, with most returning to baseline at day 30. In addition, dysregulated metabolites involved in amino acid pathways, which might indicate changes in the microbiome, were detected. In conclusion, abdominal irradiation in a nonhuman primate model caused a plasma metabolome profile indicative of GI injury. These results point to pathways that may be targeted for intervention or used as early indicators of GI radiation injury. Moreover, our results suggest that effects are sex-specific and that interventions may need to be tailored accordingly.

Differential Recovery of Small Intestinal Segments after Partial-Body Irradiation in Non-Human Primates.

In the event of a radiological attack or accident, it is more likely that the absorbed radiation dose will be heterogeneous, rather than uniformly distributed throughout the body. This type of uneven dose distribution is known as partial-body irradiation (PBI). Partial exposure of the vital organs, specifically the highly radiosensitive intestines, may cause death, if the injury is significant and the post-exposure recovery is considerably compromised. Here we investigated the recovery rate and extent of recovery from PBI-induced intestinal damage in large animals. Rhesus macaques (Macaca mulatta) were randomly divided into four groups: sham-irradiated (0 Gy), 8 Gy PBI, 11 Gy PBI and 14 Gy PBI. A single dose of ionizing radiation was delivered in the abdominal region using a uniform bilateral anteroposterior and posteroanterior technique. Irradiated animals were scheduled for euthanasia on days 10, 28 or 60 postirradiation, and sham-irradiated animals on day 60. Intestinal structural injuries were assessed via crypt depth, villus height, and mucosal surface length in the four different intestinal regions (duodenum, proximal jejunum, distal jejunum and ileum) using H&E staining. Higher radiation doses corresponded with more injury at 10 days post-PBI and a faster recovery rate. However, at 60 days post-PBI, damage was still evident in all regions of the intestine. The proximal and distal ends (duodenum and ileum, respectively) sustained less damage and recovered more fully than the jejunum.

Dietary Methionine Deficiency Enhances Genetic Instability in Murine Immune Cells.

Both cell and animal studies have shown that complete or partial deficiency of methionine inhibits tumor growth. Consequently, the potential implementation of this nutritional intervention has recently been of great interest for the treatment of cancer patients. Unfortunately, diet alteration can also affect healthy immune cells such as monocytes/macrophages and their precursor cells in bone marrow. As around half of cancer patients are treated with radiotherapy, the potential deleterious effect of dietary methionine deficiency on immune cells prior to and/or following irradiation needs to be evaluated. Therefore, we examined whether modulation of methionine content alters genetic stability in the murine RAW 264.7 monocyte/macrophage cell line in vitro by chromosomal analysis after 1-month culture in a methionine-deficient or supplemented medium. We also analyzed chromosomal aberrations in the bone marrow cells of CBA/J mice fed with methionine-deficient or supplemented diet for 2 months. While all RAW 264.7 cells revealed a complex translocation involving three chromosomes, three different clones based on the banding pattern of chromosome 9 were identified. Methionine deficiency altered the ratio of the three clones and increased chromosomal aberrations and DNA damage in RAW 264.7. Methionine deficiency also increased radiation-induced chromosomal aberration and DNA damage in RAW 264.7 cells. Furthermore, mice maintained on a methionine-deficient diet showed more chromosomal aberrations in bone marrow cells than those given methionine-adequate or supplemented diets. These findings suggest that caution is warranted for clinical implementation of methionine-deficient diet concurrent with conventional cancer therapy.

Simultaneous exposure to chronic irradiation and simulated microgravity differentially alters immune cell phenotype in mouse thymus and spleen.

Deep-space missions may alter immune cell phenotype in the primary (e.g., thymus) and secondary (e.g., spleen) lymphoid organs contributing to the progression of a variety of diseases. In deep space missions, astronauts will be exposed to chronic low doses of HZE radiation while being in microgravity. Ground-based models of long-term uninterrupted exposures to HZE radiation are not yet available. To obtain insight in the effects of concurrent exposure to microgravity and chronic irradiation (CIR), mice received a cumulative dose of chronic 0.5 Gy gamma rays over one month ± simulated microgravity (SMG). To obtain insight in a dose rate effect, additional mice were exposed to single acute irradiation (AIR) at 0.5 Gy gamma rays. We measured proportions of immune cells relative to total number of live cells in the thymus and spleen, stress level markers in plasma, and change in body weight, food consumption, and water intake. CIR affected thymic CD3+/CD335+ natural killer T (NK-T) cells, CD25+ regulatory T (Treg) cells, CD27+/CD335- natural killer (NK1) cells and CD11c+/CD11b- dendritic cells (DCs) differently in mice subjected to SMG than in mice with normal loading. No such effects of CIR on SMG as compared to normal loading were observed in cell types from the spleen. Differences between CIR and AIR groups (both under normal loading) were found in thymic Treg and DCs. Food consumption, water intake, and body weight were less after coexposure than singular or no exposure. Compared to sham, all treatment groups exhibited elevated plasma levels of the stress marker catecholamines. These data suggest that microgravity and chronic irradiation may interact with each other to alter immune cell phenotypes in an organ-specific manner and appropriate strategies are required to reduce the health risk of crewmembers.

Dietary Methionine Supplementation Exacerbates Gastrointestinal Toxicity in a Mouse Model of Abdominal Irradiation.

BACKGROUND AND PURPOSE: Identification of appropriate dietary strategies for prevention of weight and muscle loss in cancer patients is crucial for successful treatment and prolonged patient survival. High-protein oral nutritional supplements decrease mortality and improve indices of nutritional status in cancer patients; however, high-protein diets are often rich in methionine, and experimental evidence indicates that a methionine-supplemented diet (MSD) exacerbates gastrointestinal toxicity after total body irradiation. Here, we sought to investigate whether MSD can exacerbate gastrointestinal toxicity after local abdominal irradiation, an exposure regimen more relevant to clinical settings. MATERIALS AND METHODS: Male CBA/CaJ mice fed either a methionine-adequate diet or MSD (6.5 mg methionine/kg diet vs 19.5 mg/kg) received localized abdominal X-irradiation (220 kV, 13 mA) using the Small Animal Radiation Research Platform, and tissues were harvested 4, 7, and 10 days after irradiation. RESULTS: MSD exacerbated gastrointestinal toxicity after local abdominal irradiation with 12.5 Gy. This was evident as impaired nutrient absorption was paralleled by reduced body weight recovery. Mechanistically, significant shifts in the gut ecology, evident as decreased microbiome diversity, and substantially increased bacterial species that belong to the genus Bacteroides triggered proinflammatory responses. The latter were evident as increases in circulating neutrophils with corresponding decreases in lymphocytes and associated molecular alterations, exhibited as increases in mRNA levels of proinflammatory genes Icam1, Casp1, Cd14, and Myd88. Altered expression of the tight junction-related proteins Cldn2, Cldn5, and Cldn6 indicated a possible increase in intestinal permeability and bacterial translocation to the liver. CONCLUSIONS: We report that dietary supplementation with methionine exacerbates gastrointestinal syndrome in locally irradiated mice. This study demonstrates the important roles registered dieticians should play in clinical oncology and further underlines the necessity of preclinical and clinical investigations in the role of diet in the success of cancer therapy.

Methionine dietary supplementation potentiates ionizing radiation-induced gastrointestinal syndrome.

Methionine is an essential amino acid needed for a variety of processes in living organisms. Ionizing radiation depletes tissue methionine concentrations and leads to the loss of DNA methylation and decreased synthesis of glutathione. In this study, we aimed to investigate the effects of methionine dietary supplementation in CBA/CaJ mice after exposure to doses ranging from 3 to 8.5 Gy of 137Cs of total body irradiation. We report that mice fed a methionine-supplemented diet (MSD; 19.5 vs. 6.5 mg/kg in a methionine-adequate diet, MAD) developed acute radiation toxicity at doses as low as 3 Gy. Partial body irradiation performed with hindlimb shielding resulted in a 50% mortality rate in MSD-fed mice exposed to 8.5 Gy, suggesting prevalence of radiation-induced gastrointestinal syndrome in the development of acute radiation toxicity. Analysis of the intestinal microbiome demonstrated shifts in the gut ecology, observed along with the development of leaky gut syndrome and bacterial translocation into the liver. Normal gut physiology impairment was facilitated by alterations in the one-carbon metabolism pathway and was exhibited as decreases in circulating citrulline levels mirrored by decreased intestinal mucosal surface area and the number of surviving crypts. In conclusion, we demonstrate that a relevant excess of methionine dietary intake exacerbates the detrimental effects of exposure to ionizing radiation in the small intestine.NEW & NOTEWORTHY Methionine supplementation, instead of an anticipated health-promoting effect, sensitizes mice to gastrointestinal radiation syndrome. Mechanistically, excess of methionine negatively affects intestinal ecology, leading to a cascade of physiological, biochemical, and molecular alterations that impair normal gut response to a clinically relevant genotoxic stressor. These findings speak toward increasing the role of registered dietitians during cancer therapy and the necessity of a solid scientific background behind the sales of dietary supplements and claims regarding their benefits.

NZO/HlLtJ as a novel model for the studies on the role of metabolic syndrome in acute radiation toxicity.

Purpose: Growing rates of metabolic syndrome and associated obesity warrant the development of appropriate animal models for better understanding of how those conditions may affect sensitivity to IR exposure.Materials and methods: We subjected male NZO/HlLtJ mice, a strain prone to spontaneous obesity and diabetes, to 0, 5.5, 6.37, 7.4 or 8.5 Gy (137Cs) of total body irradiation (TBI). Mice were monitored for 30 days, after which proximal jejunum and colon tissues were collected for further histological and molecular analysis.Results: Obese NZO/HlLtJ male mice are characterized by their lower sensitivity to IR at doses of 6.37 Gy and under, compared to other strains. Further escalation of the dose, however, results in a steep survival curve, reaching LD100/30 values at a dose of 8.5 Gy. Alterations in the expression of various tight junction-related proteins coupled with activation of inflammatory responses and cell death were the main contributors to the gastrointestinal syndrome.Conclusions: We demonstrate that metabolic syndrome with exhibited hyperglycemia but without alterations to the microvasculature is not a pre-requisite of the increased sensitivity to TBI at high doses. Our studies indicate the potential of NZO/HlLtJ mice for the studies on the role of metabolic syndrome in acute radiation toxicity.

C/EBPδ protects from radiation-induced intestinal injury and sepsis by suppression of inflammatory and nitrosative stress.

Ionizing radiation (IR)-induced intestinal damage is characterized by a loss of intestinal crypt cells, intestinal barrier disruption and translocation of intestinal microflora resulting in sepsis-mediated lethality. We have shown that mice lacking C/EBPδ display IR-induced intestinal and hematopoietic injury and lethality. The purpose of this study was to investigate whether increased IR-induced inflammatory, oxidative and nitrosative stress promote intestinal injury and sepsis-mediated lethality in Cebpd-/- mice. We found that irradiated Cebpd-/- mice show decreased villous height, crypt depth, crypt to villi ratio and expression of the proliferation marker, proliferating cell nuclear antigen, indicative of intestinal injury. Cebpd-/- mice show increased expression of the pro-inflammatory cytokines (Il-6, Tnf-α) and chemokines (Cxcl1, Mcp-1, Mif-1α) and Nos2 in the intestinal tissues compared to Cebpd+/+ mice after exposure to TBI. Cebpd-/- mice show decreased GSH/GSSG ratio, increased S-nitrosoglutathione and 3-nitrotyrosine in the intestine indicative of basal oxidative and nitrosative stress, which was exacerbated by IR. Irradiated Cebpd-deficient mice showed upregulation of Claudin-2 that correlated with increased intestinal permeability, presence of plasma endotoxin and bacterial translocation to the liver. Overall these results uncover a novel role for C/EBPδ in protection against IR-induced intestinal injury by suppressing inflammation and nitrosative stress and underlying sepsis-induced lethality.

Changes in one-carbon metabolism and DNA methylation in the hearts of mice exposed to space environment-relevant doses of oxygen ions (16O).

Cardiovascular disease constitutes an important threat to humans after space missions beyond the Earth's magnetosphere. Epigenetic alterations have an important role in the etiology and pathogenesis of cardiovascular disease. Previous research in animal models has shown that protons and 56Fe ions cause long-term changes in DNA methylation and expression of repetitive elements in the heart. However, astronauts will be exposed to a variety of ions, including the smaller fragmented products of heavy ions after they interact with the walls of the space craft. Here, we investigated the effects of 16O on the cardiac methylome and one-carbon metabolism in male C57BL/6 J mice. Left ventricles were examined 14 and 90 days after exposure to space-relevant doses of 0.1, 0.25, or 1 Gy of 16O (600 MeV/n). At 14 days, the two higher radiation doses elicited global DNA hypomethylation in the 5'-UTR of Long Interspersed Nuclear Elements 1 (LINE-1) compared to unirradiated, sham-treated mice, whereas specific LINE-1 elements exhibited hypermethylation at day 90. The pericentromeric major satellites were affected both at the DNA methylation and expression levels at the lowest radiation dose. DNA methylation was elevated, particularly after 90 days, while expression showed first a decrease followed by an increase in transcript abundance. Metabolomics analysis revealed that metabolites involved in homocysteine remethylation, central to DNA methylation, were unaffected by radiation, but the transsulfuration pathway was impacted after 90 days, with a large increase in cystathione levels at the lowest dose. In summary, we observed dynamic changes in the cardiac epigenome and metabolome three months after exposure to a single low dose of oxygen ions.

Identification of novel breakpoints for locus- and region-specific translocations in 293 cells by molecular cytogenetics before and after irradiation.

The human kidney embryonic 293 cell line (293 cells) is extensively used in biomedical and pharmaceutical research. These cells exhibit a number of numerical and structural chromosomal anomalies. However, the breakpoints responsible for these structural chromosomal rearrangements have not been comprehensively characterized. In addition, it is not known whether chromosomes with structural rearrangement are more sensitive to external toxic agents, such as ionizing radiation. We used G-banding, spectral karyotyping (SKY), and locus- and region-specific fluorescence in situ hybridization (FISH) probes designed in our lab or obtained from commercial vendor to address this gap. Our G-banding analysis revealed that the chromosome number varies from 66 to 71, with multiple rearrangements and partial additions and deletions. SKY analysis confirmed 3 consistent rearrangements, two simple and one complex in nature. Multicolor FISH analysis identified an array of breakpoints responsible for locus- and region-specific translocations. Finally, SKY analysis revealed that radio-sensitivity of structurally rearranged chromosomes is dependent on radiation dose. These findings will advance our knowledge in 293 cell biology and will enrich the understanding of radiation biology studies.

Enhanced Survival in Mice Exposed to Ionizing Radiation by Combination of Gamma-Tocotrienol and Simvastatin.

Ionizing radiation exposure is a major concern for active military service members, as well as civilian population. Considering that the exposure is not predictable, it is imperative that strategies to counteract radiation damage must be discovered. Recent in vitro studies performed in our laboratory demonstrated that the vitamin E analog gamma-tocotrienol (GT3) in combination with cholesterol-lowering drugs (Statins), synergistically induced endothelial thrombomodulin, an anticoagulant with radio-protective efficacy. It was hypothesized that the combination of treatment with both GT3 along with Statins would provide better radiation protection in vivo than each drug individually. CD2F1 mice were injected subcutaneously with either vehicle or single dose of GT3 (200 mg/kg body weight) 24 hours before irradiation followed by oral or subcutaneous administration of various doses of simvastatin (25, 50, and 100 mg/kg body weight) before exposure to lethal doses (11.5 and 12 Gy) of Cobalt-60 (60Co) gamma-irradiation. The combined treatment group exhibited enhanced radiation lethality protection substantially, accelerated white blood cell recovery, and augmented restoration of bone marrow cellularity when compared to the animals treated with either drug exclusively. This information clearly suggests that combined treatment could be used as a safeguard for military personnel from exposure to harmful ionizing radiation.

Gamma-Tocotrienol Protects the Intestine from Radiation Potentially by Accelerating Mesenchymal Immune Cell Recovery.

Natural antioxidant gamma-tocotrienol (GT3), a vitamin E family member, provides intestinal radiation protection. We seek to understand whether this protection is mediated via mucosal epithelial stem cells or sub-mucosal mesenchymal immune cells. Vehicle- or GT3-treated male CD2F1 mice were exposed to total body irradiation (TBI). Cell death was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Villus height and crypt depth were measured with computer-assisted software in tissue sections. Functional activity was determined with an intestinal permeability assay. Immune cell recovery was measured with immunohistochemistry and Western blot, and the regeneration of intestinal crypts was assessed with ex vivo organoid culture. A single dose of GT3 (200 mg/kg body weight (bwt)) administered 24 h before TBI suppressed cell death, prevented a decrease in villus height, increased crypt depth, attenuated intestinal permeability, and upregulated occludin level in the intestine compared to the vehicle treated group. GT3 accelerated mesenchymal immune cell recovery after irradiation, but it did not promote ex vivo organoid formation and failed to enhance the expression of stem cell markers. Finally, GT3 significantly upregulated protein kinase B or AKT phosphorylation after TBI. Pretreatment with GT3 attenuates TBI-induced structural and functional damage to the intestine, potentially by facilitating intestinal immune cell recovery. Thus, GT3 could be used as an intestinal radioprotector.