OBJECTIVES:
NASA is preparing for space exploration beyond low earth orbit (LEO). Space exploration beyond LEO poses threats to astronauts’ mental and physical health due to chronic exposure to microgravity and space radiation. Exposure to space radiation has four primary biomedical risks: degenerative tissue effects, neurological effects, carcinogenesis, and acute radiation syndrome. Exposure to microgravity compounds these risks. Modifications to cellular and molecular mechanisms because of exposure to the space environment must be understood in order to mitigate the consequences.
Endogenous Retroviruses (ERVs) are genetic elements that comprise approximately eight percent of the human genome and are considered to play a major role in human evolution. ERVs are genomic instability markers that are maintained in a dormant state by epigenetic mechanisms. However, environmental stressors can inadvertently activate expression of ERVs. Activation of ERVs have been associated with the development of cancer, autoimmune diseases, and neurodegenerative disease in humans. ERV activation may have detrimental health effects on astronauts including biological, mental, and reproductive consequences. Astronauts are exposed to exogenous stressors, microgravity and space radiation, which can cause ERV activation through genomic modifications.
The goal of this experiment was to address if microgravity and radiation could influence expression of ERVs and retroviral elements in human stem/progenitor cells in vitro and in mouse tissues in vivo. This study had the following specific aims:
- Evaluate human cells grown in a microgravity environment for evidence of ERV activation.
- Evaluate mouse tissues that have been exposed to space radiation for evidence of ERV activation.
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APPROACH:
A549 human lung fibroblasts cells used for radiation, microgravity, and combined effects (10 Gy gamma + microgravity) experiments, were cultured 3 days prior to treatment. 500 x 10(3) A549 cells were cultured per T-25 for radiation experiments using standard cell culturing techniques and media (10% FBS containing DMEM with high glucose and pyruvate). For microgravity (n=2) and combined effects (n=2) experiments, 350 x 10(3) A549 cells were cultured per Synthecon cell rotator with Cytodex microcarriers. A459 cells were exposed to gamma radiation at Duke University using the J. L. Shepherd Mark I Model 68A 137Cs Gamma irradiator (8 Gy (n=12) and 10 Gy (n=8)). Each experimental group was treated at the same time and harvested for 2-day and 5-day post-radiation assessment using flow cytometry, while A459 cells were irradiated at Brookhaven National Laboratory (BNL) and harvested at 1-day post-radiation for flow cytometry (150 cGy GCR5-ion samples (n=4)). For all experiments, untreated controls were cultured and harvested identically to their treated counterparts. Indirect flow cytometry assay was developed using the J2 primary antibody, mouse monoclonal anti-double stranded (ds) RNA antibody, and Alexa 488 conjugated anti-mouse IgG polyclonal antibody. All cells were fixed and permeabilized using eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set from Invitrogen. 10,000 events were recorded per sample and FlowJo v10.8 was used to gate singlet populations for average median fluorescence intensity (aMFI) of detected dsRNA.
RESULTS:
For individual effects-treated samples at 2-days post-radiation, only 8 Gy gamma showed a significant elevation in aMFI—a 1.30-fold increase. Combined 10 Gy gamma and microgravity effects at the 2-day post-radiation timepoint showed a 1.73-fold increase in aMFI compared to normal gravity controls and a 1.94-fold increase in aMFI compared to 10 Gy gamma only treated samples. 5-days post-radiation results, however, showed statistical significance across all experimental treatment groups; 8 Gy gamma, 10 Gy gamma, and microgravity treated samples showing a 1.33-fold increase, 2.13-fold increase, and 2.41-fold increase in aMFI, respectively. Combined effects of 10 Gy Gamma and microgravity at the 5-day post-radiation timepoint showed a 4.72-fold increase in aMFI compared to normal gravity controls and a 1.96-fold increase in aMFI compared to microgravity only treated samples. 1-day post-radiation samples irradiated with GCR5-ion displayed a 1.21-fold increase in aMFI, although it was not statistically significant
Overall some progress has been made demonstrating the increase in cells with dsRNA (and possibly endogenous retrovirus reactivation) following both gamma and GCR exposure. This project is significant in that identification of ERVs that respond to specific space conditions may function as early, surrogate markers of putative genomic change. Furthermore, expression of ERV-encoded proteins may be seen as foreign and elicit autoimmune responses.
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Hematopoietic
RNA, endogenous retroviruses (ERV)-encoded, expression (Human)
RNA, endogenous retroviruses (ERV)-encoded, expression (Mice)
RNA, retrotransposable element (Human)
RNA, retrotransposable element (Mice)
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