One of the most likely risks astronauts on long duration space missions face is exposure to ionizing radiation associated with highly energetic and charged heavy (HZE) particles. Since access to medical expertise on such a mission is limited at best, early diagnosis and mitigation of such exposure is critical. In order to accurately determine the dosage within 1 hour post-exposure, dose-dependent biomarkers are needed. Therefore, we performed a dose-course transcriptional analysis for radiation exposure at 0, 0.3, 1.5, and 3.0 Gy with corresponding time point at 1 hour (hr) post-exposure using Affymetrix GeneChip Human Gene 1.0 ST v1 Array chips. The analysis of our data suggests a set of sensitive genetic biomarkers specific to each radiation level as well as generic radiation response biomarkers. Upregulated biomarkers can then be used within lab-on-a-chip (LOC) systems to detect exposure to ionizing radiation. A total of sixteen human samples representing radiation exposure at levels 0 Gy, 0.3 Gy, 1.5 Gy and 3.0 Gy at time point 1 hour (hr) post-exposure were constructed. Blood samples were extracted from four human volunteers, and were irradiated. Leukocytes were extracted, and gene expression was measured. Samples for all four volunteers were measured at 1 hr for all four dose levels, resulting in four replicates at each dose level. Thus, a total of 4 samples at each of the four radiation levels were sampled, yielding the total of 16 samples.

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach our previous NASA work has shown both TGF xce xb2 signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We investigated this hypothesis both in vivo and in vitro and test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach was used to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR) we used the ions energy and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To simulate high-dose radiation exposure due to solar particle events (SPE) we used an acute dose of SPE simulated beam at 1Gy which has energies ranging from 50MeV to 150MeV. C57BL/6 wild-type mice were utilized for irradiation with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions will also be irradiated. To expand on the circulating miRNA signature determined from our preliminary data we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing on serum (before IR and at time of sacrifice) liver heart and muscle tissue for all radiation groups revealed the key circulating miRNA signature (consisting of multiple miRNAs) impacting disease risk. Collectively understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation.

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach our previous NASA work has shown both TGF xce xb2 signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We investigated this hypothesis both in vivo and in vitro and test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach was used to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR) we used the ions energy and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To simulate high-dose radiation exposure due to solar particle events (SPE) we used an acute dose of SPE simulated beam at 1Gy which has energies ranging from 50MeV to 150MeV. C57BL/6 wild-type mice were utilized for irradiation with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions will also be irradiated. To expand on the circulating miRNA signature determined from our preliminary data we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing on serum (before IR and at time of sacrifice) liver heart and muscle tissue for all radiation groups revealed the key circulating miRNA signature (consisting of multiple miRNAs) impacting disease risk. Collectively understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation.

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach our previous NASA work has shown both TGF xce xb2 signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We investigated this hypothesis both in vivo and in vitro and test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach was used to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR) we used the ions energy and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To simulate high-dose radiation exposure due to solar particle events (SPE) we used an acute dose of SPE simulated beam at 1Gy which has energies ranging from 50MeV to 150MeV. C57BL/6 wild-type mice were utilized for irradiation with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions will also be irradiated. To expand on the circulating miRNA signature determined from our preliminary data we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing on serum (before IR and at time of sacrifice) liver heart and muscle tissue for all radiation groups revealed the key circulating miRNA signature (consisting of multiple miRNAs) impacting disease risk. Collectively understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation.

Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR) and in particular the high linear energy transfer (LET) heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation and primarily affected sites in open chromatin regions including enhancers promoters and edges ( shores ) of CpG islands. The 28Si ion-exposure had mixed effects inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments whereas X rays induced mostly hypomethylation primarily at sites in gene bodies and intergenic regions. Significantly the methylation status of 56Fe ion irradiation sensitive sites but not those affected by X ray or 28Si ions could discriminate tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus high LET radiation exposure leaves a lasting imprint on the epigenome and affects sites relevant to human lung cancer. The 56Fe ion signature may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space. Genome wide DNA methylation profiling of normal human bronchial epithelial cells irradiated with varying doses of 28Si-ion radiation ( 300 MeV/u at 0 0.3 1.0 Gy) 56Fe-ion radiation (600 MeV/u at 0 0.1 0.3 1.0 Gy) or X rays (320 kV at 0 1.0 Gy). Triplicate control and irradiated samples were incubated and sampled at 4 timepoints between 2 and 62 days. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across >485,000 CpGs from collected samples. Samples include: 56Fe ions 4 doses x 4 time points x 3 replicates (4 removed in QC) = 44 samples; 28Si ions = 3 doses x 4 time points x 3 replicates = 36 samples; X ray 2 doses x 4 time points x 3 replicates (2 removed in QC)= 22 samples. Overall design: Bisulphite converted DNA from the 102 samples were hybridized to the Illumina Infinium 450k Human Methylation Beadchip.

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach our previous NASA work has shown both TGF xce xb2 signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We investigated this hypothesis both in vivo and in vitro and test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach was used to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR) we used the ions energy and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To simulate high-dose radiation exposure due to solar particle events (SPE) we used an acute dose of SPE simulated beam at 1Gy which has energies ranging from 50MeV to 150MeV. C57BL/6 wild-type mice were utilized for irradiation with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions will also be irradiated. To expand on the circulating miRNA signature determined from our preliminary data we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing on serum (before IR and at time of sacrifice) liver heart and muscle tissue for all radiation groups revealed the key circulating miRNA signature (consisting of multiple miRNAs) impacting disease risk. Collectively understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation.

Densely ionizing radiation is a major component of the space radiation environment and has potentially greater carcinogenic effect compared to sparsely ionizing radiation that is prevalent in the terrestrial environment. It is unknown to what extent the irradiated microenvironment contributes to the differential carcinogenic potential of densely ionizing radiation. To address this gap, 10-week old BALB/c mice were irradiated with 100 cGy sparsely ionizing g-radiation or 10, 30, or 80 cGy of densely ionizing, 350 MeV/amu Si particles and transplanted 3 days later with syngeneic Trp53 null mammary fragments. Tumor appearance was monitored for 600 days. Tumors arising in Si-particle irradiated mice had a shorter median time to appearance, grew faster and were more likely to metastasize. Most tumors arising in sham-irradiated mice were ER-positive, pseudo-glandular and contained both basal keratin 14 and luminal keratin 8/18 cells (designated K14/18), while most tumors arising in irradiated hosts were K8/18 positive (designated K18) and ER negative. Comparison of K18 vs K14/18 tumor expression profiles showed that genes increased in K18 tumors were associated with ERBB2 and KRAS while decreased genes overlapped with those down regulated in metastasis and by loss of E-cadherin. Consistent with this, K18 tumors grew faster than K14/18 tumors and more mice with K18 tumors developed lung metastases compared to mice with K14/18 tumors. However, K18 tumors arising in Si-particle irradiated mice grew even faster and were more metastatic compared to control mice. A K18 Si-irradiated host profile was enriched in genes involved in mammary stem cells, stroma, and Notch signaling. Thus systemic responses to densely ionizing radiation enriches for a ER-negative, K18-positive tumor, whose biology is more aggressive compared to similar tumors arising in non-irradiated hosts. Key Words: ionizing radiation; breast cancer; heavy ion radiation;initiation; promotion 3 different dose of Si were used. Total RNA was extracted from mammary tumors derived from transplantations of non-irradiated p53null mammary fragments into irradiated hosts. We analyzed a total of 45 Trp53-null tumors: 18 from sham-irradiated hosts, 9 from 10 cGy Si-irradiated hosts, 10 from 30 cGy Si-irradiated hosts, and 8 from irradiated hosts.

Future space missions will include return to the Moon and long duration deep space roundtrip missions to Mars. Leaving the protection that Low Earth Orbit provides will unavoidably expose astronauts to higher cumulative doses of space radiation, in addition to other stressors, e.g. microgravity. Immune regulation is known to be impacted and it remains to be seen whether prolonged effects will be encountered in deep space that can have an adverse impact on health. In this study we investigated the effects in overall metabolism of three different low dose radiation exposures (γ-rays, 16O, and 56Fe) in spleen from male C57BL/6 mice at 1, 2, and 4 months after exposure. Forty metabolites were identified with significant enrichment in purine metabolism, tricarboxylic acid cycle, fatty acids, acylcarnitines, and amino acids. Early perturbations were more prominent in the γ irradiated samples, while longer term responses shifted towards the high energy particle effects. Regression analysis showed a positive correlation of fatty acids with time and negative association with γ-rays, while degradation of purines were positively associated with time. Taken together, there is a strong suggestion of mitochondrial implication and the possibility of long term effects in DNA repair and nucleotide pools following radiation exposure.

BACKGROUND: Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity a condition of weightlessness experienced by astronauts during space missions which could have a synergistic action on cells increasing the risk of radiation exposure. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of gamma-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover let-7i* miR-7 miR-7-1* miR-27a miR-144 miR-200a miR-598 miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles carried out on PBL of the same donors identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of Response to DNA damage is enriched when PBL are incubated in 1 g but not in MMG. Moreover some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. CONCLUSIONS/SIGNIFICANCE: On the whole by integrating the transcriptome and microRNome we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL.

BACKGROUND: Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity a condition of weightlessness experienced by astronauts during space missions which could have a synergistic action on cells increasing the risk of radiation exposure. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of gamma-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover let-7i* miR-7 miR-7-1* miR-27a miR-144 miR-200a miR-598 miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles carried out on PBL of the same donors identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of Response to DNA damage is enriched when PBL are incubated in 1 g but not in MMG. Moreover some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. CONCLUSIONS/SIGNIFICANCE: On the whole by integrating the transcriptome and microRNome we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. Overall Design: Gene expression signature was defined in PBL irradiated with gamma-rays (2.0 Gy) and incubated in modeled microgravity (mmg) and in parallel ground conditions (1g) for 24h. Five independent experiments were performed for each donor to address which mRNAs were regulated on IR stress. The level of each transcript was represented as Log2.