Data integration reveals key homeostatic mechanisms following low dose radiation exposure.

The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10 cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time - with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24-72h). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress was measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 was experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation.

Sexual dimorphism during integrative endocrine and immune responses to ionizing radiation in mice.

Exposure to cosmic ionizing radiation is an innate risk of the spaceflight environment that can cause DNA damage and altered cellular function. In astronauts, longitudinal monitoring of physiological systems and interactions between these systems are important to consider for mitigation strategies. In addition, assessments of sex-specific biological responses in the unique environment of spaceflight are vital to support future exploration missions that include both females and males. Here we assessed sex-specific, multi-system immune and endocrine responses to simulated cosmic radiation. For this, 24-week-old, male and female C57Bl/6J mice were exposed to simplified five-ion, space-relevant galactic cosmic ray (GCRsim) radiation at 15 and 50 cGy, to simulate predicted radiation exposures that would be experienced during lunar and Martian missions, respectively. Blood and adrenal tissues were collected at 3- and 14-days post-irradiation for analysis of immune and endocrine biosignatures and pathways. Sexually dimorphic adrenal gland weights and morphology, differential total RNA expression with corresponding gene ontology, and unique immune phenotypes were altered by GCRsim. In brief, this study offers new insights into sexually dimorphic immune and endocrine kinetics following simulated cosmic radiation exposure and highlights the necessity for personalized translational approaches for astronauts during exploration missions.

Quantitative phosphoproteomics identifies filaggrin and other targets of ionizing radiation in a human skin model.

Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low- and high-dose ionizing radiation-dependent signalling in a complex three-dimensional setting. Application of an isobaric labelling strategy using sham and three radiation doses (3, 10, 200 cGy) resulted in the identification of 1052 unique phosphopeptides. Statistical analyses identified 176 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1, 2 and 3) had altered phosphorylation levels following exposure to both low and high doses of radiation. Altered phosphorylation of multiple sites in profilaggrin linker domains coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to both low and high doses of ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain tissue integrity and mitigate effects of radiation exposure.

Neuro-consequences of the spaceflight environment.

As human space exploration advances to establish a permanent presence beyond the Low Earth Orbit (LEO) with NASA's Artemis mission, researchers are striving to understand and address the health challenges of living and working in the spaceflight environment. Exposure to ionizing radiation, microgravity, isolation and other spaceflight hazards pose significant risks to astronauts. Determining neurobiological and neurobehavioral responses, understanding physiological responses under Central Nervous System (CNS) control, and identifying putative mechanisms to inform countermeasure development are critically important to ensuring brain and behavioral health of crew on long duration missions. Here we provide a detailed and comprehensive review of the effects of spaceflight and of ground-based spaceflight analogs, including simulated weightlessness, social isolation, and ionizing radiation on humans and animals. Further, we discuss dietary and non-dietary countermeasures including artificial gravity and antioxidants, among others. Significant future work is needed to ensure that neural, sensorimotor, cognitive and other physiological functions are maintained during extended deep space missions to avoid potentially catastrophic health and safety outcomes.

Artificial gravity partially protects space-induced neurological deficits in Drosophila melanogaster.

Spaceflight poses risks to the central nervous system (CNS), and understanding neurological responses is important for future missions. We report CNS changes in Drosophila aboard the International Space Station in response to spaceflight microgravity (SFµg) and artificially simulated Earth gravity (SF1g) via inflight centrifugation as a countermeasure. While inflight behavioral analyses of SFµg exhibit increased activity, postflight analysis displays significant climbing defects, highlighting the sensitivity of behavior to altered gravity. Multi-omics analysis shows alterations in metabolic, oxidative stress and synaptic transmission pathways in both SFµg and SF1g; however, neurological changes immediately postflight, including neuronal loss, glial cell count alterations, oxidative damage, and apoptosis, are seen only in SFµg. Additionally, progressive neuronal loss and a glial phenotype in SF1g and SFµg brains, with pronounced phenotypes in SFµg, are seen upon acclimation to Earth conditions. Overall, our results indicate that artificial gravity partially protects the CNS from the adverse effects of spaceflight.

Confocal microscopy for modeling electron microbeam irradiation of skin.

For radiation exposures employing targeted sources such as particle microbeams, the deposition of energy and dose will depend on the spatial heterogeneity of the sample. Although cell structural variations are relatively minor for two-dimensional cell cultures, they can vary significantly for fully differentiated tissues. Employing high-resolution confocal microscopy, we have determined the spatial distribution, size, and shape of epidermal keratinocyte nuclei for the full-thickness EpiDerm™ skin model (MatTek, Ashland, VA). Application of these data to calculate the microdosimetry and microdistribution of energy deposition by an electron microbeam is discussed.

Overexpression of catalase in mitochondria mitigates changes in hippocampal cytokine expression following simulated microgravity and isolation.

Isolation on Earth can alter physiology and signaling of organs systems, including the central nervous system. Although not in complete solitude, astronauts operate in an isolated environment during spaceflight. In this study, we determined the effects of isolation and simulated microgravity solely or combined, on the inflammatory cytokine milieu of the hippocampus. Adult female wild-type mice underwent simulated microgravity by hindlimb unloading for 30 days in single or social (paired) housing. In hippocampus, simulated microgravity and isolation each regulate a discrete repertoire of cytokines associated with inflammation. Their combined effects are not additive. A model for mitochondrial reactive oxygen species (ROS) quenching via targeted overexpression of the human catalase gene to the mitochondria (MCAT mice), are protected from isolation- and/or simulated microgravity-induced changes in cytokine expression. These findings suggest a key role for mitochondrial ROS signaling in neuroinflammatory responses to spaceflight and prolonged bedrest, isolation, and confinement on Earth.

Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells.

For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. approximately 4-fold increased survival at 5Gy). The cell cycle delay induced following exposure to 2 and 5Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

High speed method for in situ multispectral image registration.

High speed data registration is required for the study of fluorescence resonance energy transfer in real time as well as fast dynamic intra- and inter-cellular signaling events. Multispectral confocal spinning disk microscopy provides a high resolution method for performing such real time live cell imaging. However, optical distortions and the physical misalignments introduced by the use of multiple acquisition cameras can obscure spatial information contained in the captured images. In this manuscript, we describe a multispectral method for real time image registration whereby the image from one camera is warped onto the image from a second camera via a polynomial correction. This method provides a real time pixel-for-pixel match between images obtained over physically distinct optical paths. Using an in situ calibration method, the polynomial is characterized by a set of coefficients, using a least squares solver. Error analysis demonstrates optimal performance results from the use of cubic polynomials. High-speed evaluation of the warp is then performed through forward differencing with fixed-point data types. Forward differencing is an iterative approach for evaluating polynomials on the condition that the function variable changes with constant steps. Image reconstruction errors are reduced through bilinear interpolation. The registration techniques described here allow for successful registration of multispectral images in real time (exceeding 15 frame/s) and have a broad applicability to imaging methods requiring pixel matching over multiple data channels.

Non-targeted bystander effects induced by ionizing radiation.

Radiation-induced bystander effects refer to those responses occurring in cells that were not subject to energy deposition events following ionizing radiation. These bystander cells may have been neighbors of irradiated cells, or physically separated but subject to soluble secreted signals from irradiated cells. Bystander effects have been observed in vitro and in vivo and for various radiation qualities. In tribute to an old friend and colleague, Anthony V. Carrano, who would have said "well what are the critical questions that should be addressed, and so what?", we review the evidence for non-targeted radiation-induced bystander effects with emphasis on prevailing questions in this rapidly developing research field, and the potential significance of bystander effects in evaluating the detrimental health effects of radiation exposure.

Ballooning for Biologists: Mission Essentials for Flying Life Science Experiments to Near Space on NASA Large Scientific Balloons.

Despite centuries of scientific balloon flights, only a handful of experiments have produced biologically-relevant results. Yet unlike orbital spaceflight, it is much faster and cheaper to conduct biology research with balloons, sending specimens to the near space environment of Earth's stratosphere. Samples can be loaded the morning of a launch and sometimes returned to the laboratory within one day after flying. The National Aeronautics and Space Administration (NASA) flies large, unmanned scientific balloons from all over the globe, with missions ranging from hours to weeks in duration. A payload in the middle portion of the stratosphere (~35 km above sea level) will be exposed to an environment similar to the surface of Mars: temperatures generally around -36 °C, atmospheric pressure at a thin 1 kPa, relative humidity levels < 1%, and a harsh illumination of ultraviolet (UV) and cosmic radiation levels (about 100 W/m2 and 0.1 mGy/d, respectively) that can be obtained nowhere else on the surface of the Earth, including environmental chambers and particle accelerator facilities attempting to simulate space radiation effects. Considering the operational advantages of ballooning and the fidelity of space-like stressors in the stratosphere, researchers in aerobiology, astrobiology, and space biology can benefit from balloon flight experiments as an intermediary step on the extraterrestrial continuum (ground, low Earth orbit, and deep space studies). Our review targets biologists with no background or experience in scientific ballooning. We will provide an overview of large balloon operations, biology topics that can be uniquely addressed in the stratosphere, and a roadmap for developing payloads to fly with NASA.

Low-LET microbeam investigation of the track-end dependence of electron-induced damage in normal human diploid fibroblasts.

Using a pulsed electron beam, we investigated the dependence of micronucleus formation on the incident electron energy in AG01522 human diploid fibroblasts after nontargeted irradiations at 25 and 80 keV. Examining the dose response, we found that 25 keV electrons are more effective than 80 keV electrons at producing biological damage for a given dose. Our results demonstrating the induction of micronuclei as a function of incident electron energy offer direct support for the hypothesis that the electron track end is responsible for the biological damage occurring in the cell.

A variable-energy electron microbeam: a unique modality for targeted low-LET radiation.

We have designed and constructed a low-cost, variable-energy low-LET electron microbeam that uses energetic electrons to mimic radiation damage produced by gamma and X rays. The microbeam can access lower regions of the LET spectrum, similar to conventional X-ray or 60Co gamma-ray sources. The device has two operating modes, as a conventional microbeam targeting single cells or subpopulations of cells or as a pseudo broad-beam source allowing for direct comparison with conventional sources. By varying the incident electron energy, the target cells can be selectively exposed to different parts of the energetic electron tracks, including the track ends.

Non-targeted effects induced by ionizing radiation: mechanisms and potential impact on radiation induced health effects.

Not-targeted effects represent a paradigm shift from the "DNA centric" view that ionizing radiation only elicits biological effects and subsequent health consequences as a result of an energy deposition event in the cell nucleus. While this is likely true at higher radiation doses (>1 Gy), at low doses (<100 mGy) non-targeted effects associated with radiation exposure might play a significant role. Here definitions of non-targeted effects are presented, the potential mechanisms for the communication of signals and signaling networks from irradiated cells/tissues are proposed, and the various effects of this intra- and intercellular signaling are described. We conclude with speculation on how these observations might lead to and impact long-term human health outcomes.

The effect of low dose ionizing radiation on homeostasis and functional integrity in an organotypic human skin model.

Outside the protection of Earth's atmosphere, astronauts are exposed to low doses of high linear energy transfer (LET) radiation. Future NASA plans for deep space missions or a permanent settlement on the moon are limited by the health risks associated with space radiation exposures. There is a paucity of direct epidemiological data for low dose exposures to space radiation-relevant high LET ions. Health risk models are used to estimate the risk for such exposures, though these models are based on high dose experiments. There is increasing evidence, however, that low and high dose exposures result in different signaling events at the molecular level, and may involve different response mechanisms. Further, despite their low abundance, high LET particles have been identified as the major contributor to health risk during manned space flight. The human skin is exposed in every external radiation scenario, making it an ideal epithelial tissue model in which to study radiation induced effects. Here, we exposed an in vitro three dimensional (3-D) human organotypic skin tissue model to low doses of high LET oxygen (O), silicon (Si) and iron (Fe) ions. We measured proliferation and differentiation profiles in the skin tissue and examined the integrity of the skin's barrier function. We discuss the role of secondary particles in changing the proportion of cells receiving a radiation dose, emphasizing the possible impact on radiation-induced health issues in astronauts.

Annexin A2 modulates radiation-sensitive transcriptional programming and cell fate.

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10-200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

Integrated experimental and computational approach to understand the effects of heavy ion radiation on skin homeostasis.

The effects of low dose high linear energy transfer (LET) radiation on human health are of concern for space, occupational, and clinical exposures. As epidemiological data for such radiation exposures are scarce for making relevant predictions, we need to understand the mechanism of response especially in normal tissues. Our objective here is to understand the effects of heavy ion radiation on tissue homeostasis in a realistic model system. Towards this end, we exposed an in vitro three dimensional skin equivalent to low fluences of neon (Ne) ions (300 MeV u(-1)), and determined the differentiation profile as a function of time following exposure using immunohistochemistry. We found that Ne ion exposures resulted in transient increases in the tissue regions expressing the differentiation markers keratin 10, and filaggrin, and more subtle time-dependent effects on the number of basal cells in the epidermis. We analyzed the data using a mathematical model of the skin equivalent, to quantify the effect of radiation on cell proliferation and differentiation. The agent-based mathematical model for the epidermal layer treats the epidermis as a collection of heterogeneous cell types with different proliferation-differentiation properties. We obtained model parameters from the literature where available, and calibrated the unknown parameters to match the observed properties in unirradiated skin. We then used the model to rigorously examine alternate hypotheses regarding the effects of high LET radiation on the tissue. Our analysis indicates that Ne ion exposures induce rapid, but transient, changes in cell division, differentiation and proliferation. We have validated the modeling results by histology and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The integrated approach presented here can be used as a general framework to understand the responses of multicellular systems, and can be adapted to other epithelial tissues.

Using imaging methods to interrogate radiation-induced cell signaling.

There is increasing emphasis on the use of systems biology approaches to define radiation-induced responses in cells and tissues. Such approaches frequently rely on global screening using various high throughput 'omics' platforms. Although these methods are ideal for obtaining an unbiased overview of cellular responses, they often cannot reflect the inherent heterogeneity of the system or provide detailed spatial information. Additionally, performing such studies with multiple sampling time points can be prohibitively expensive. Imaging provides a complementary method with high spatial and temporal resolution capable of following the dynamics of signaling processes. In this review, we utilize specific examples to illustrate how imaging approaches have furthered our understanding of radiation-induced cellular signaling. Particular emphasis is placed on protein colocalization, and oscillatory and transient signaling dynamics.

Metabolomic response of human skin tissue to low dose ionizing radiation.

Understanding how human organs respond to ionizing radiation (IR) at a systems biology level and identifying biomarkers for IR exposure at low doses can help provide a scientific basis for establishing radiation protection standards. Little is known regarding the physiological responses to low dose IR at the metabolite level, which represents the end-point of biochemical processes inside cells. Using a full thickness human skin tissue model and GC-MS-based metabolomic analysis, we examined the metabolic perturbations at three time points (3, 24 and 48 h) after exposure to 3, 10 and 200 cGy of X-rays. PLS-DA score plots revealed dose- and time-dependent clustering between sham and irradiated groups. Importantly, delayed metabolic responses were observed at low dose IR. When compared with the high dose at 200 cGy, a comparable number of significantly changed metabolites were detected 48 h after exposure to low doses (3 and 10 cGy) of irradiation. Biochemical pathway analysis showed perturbations to DNA/RNA damage and repair, lipid and energy metabolisms, even at low doses of IR.

Cell type-dependent gene transcription profile in a three-dimensional human skin tissue model exposed to low doses of ionizing radiation: implications for medical exposures.

The concern over possible health risks from exposures to low doses of ionizing radiation has been driven largely by the increase in medical exposures, the routine implementation of X-ray backscatter devices for airport security screening, and, most recently, the nuclear incident in Japan. Because of a paucity of direct epidemiological data at very low doses, cancer risk must be estimated from high dose exposure scenarios. However, there is increasing evidence that low and high dose exposures result in different signaling events and may have different response mechanisms than higher doses. We have examined the radiation-induced temporal response after exposure to 10 cGy of an in vitro three dimensional (3D) human skin tissue model using microarray-based transcriptional profiling. Cell type-specific analysis showed significant changes in gene expression with the levels of >1,400 genes altered in the dermis and >400 genes regulated in the epidermis. The two cell layers rarely exhibited overlapping responses at the mRNA level. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements validated the microarray data in both regulation direction and value. Key pathways identified relate to cell cycle regulation, immune responses, hypoxia, reactive oxygen signaling, and DNA damage repair. The proliferation status as well as the expression of PCNA was examined in histological samples. We discuss in particular the role of proliferation, emphasizing how the disregulation of cellular signaling in normal tissue may impact progression toward radiation-induced secondary diseases.