Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel.
Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O₂ for 8 h only (O₂), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O₂ + IR) followed by 16 h of normoxia (ambient air containing 21% O₂ and 5% CO₂) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O₂ + IR exacerbated cell death and DNA damage compared to individual exposures O₂ or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest.
Division of Pulmonary, Allergy, and Critical Care Medicine and the Department of Medicine, University of Pennsylvania Perelman School of Medicine, 3450 Hamilton Walk, Edward J. Stemmler Hall 2nd Floor, Office Suite 227, Philadelphia, PA 19104, USA. ralphp@mail.med.upenn.edu.
Ralph A
RA
Pietrofesa
Division of Pulmonary, Allergy, and Critical Care Medicine and the Department of Medicine, University of Pennsylvania Perelman School of Medicine, 3450 Hamilton Walk, Edward J. Stemmler Hall 2nd Floor, Office Suite 227, Philadelphia, PA 19104, USA. avela@mail.med.upenn.edu.
Anastasia
A
Velalopoulou
Department of Radiation Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. staceylehman87@gmail.com.
Stacey L
SL
Lehman
Division of Pulmonary, Allergy, and Critical Care Medicine and the Department of Medicine, University of Pennsylvania Perelman School of Medicine, 3450 Hamilton Walk, Edward J. Stemmler Hall 2nd Floor, Office Suite 227, Philadelphia, PA 19104, USA. evguenia@mail.med.upenn.edu.
Evguenia
E
Arguiri
Division of Pulmonary, Allergy, and Critical Care Medicine and the Department of Medicine, University of Pennsylvania Perelman School of Medicine, 3450 Hamilton Walk, Edward J. Stemmler Hall 2nd Floor, Office Suite 227, Philadelphia, PA 19104, USA. pantelis.solomides@temple.edu.
Pantelis
P
Solomides
Department of Radiation Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. kochc@mail.med.upenn.edu.
Cameron J
CJ
Koch
Division of Pulmonary, Allergy, and Critical Care Medicine and the Department of Medicine, University of Pennsylvania Perelman School of Medicine, 3450 Hamilton Walk, Edward J. Stemmler Hall 2nd Floor, Office Suite 227, Philadelphia, PA 19104, USA. mishra.o@gmail.com.
Om P
OP
Mishra
Department of Radiation Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. costas.Koumenis@uphs.upenn.edu.
Constantinos
C
Koumenis
The National Aeronautics and Space Administration (NASA) Johnson Space Center, Houston, TX 77058, USA. tgoodwin3@comcast.net.
Thomas J
TJ
Goodwin
Division of Pulmonary, Allergy, and Critical Care Medicine and the Department of Medicine, University of Pennsylvania Perelman School of Medicine, 3450 Hamilton Walk, Edward J. Stemmler Hall 2nd Floor, Office Suite 227, Philadelphia, PA 19104, USA. melpo@mail.med.upenn.edu.
Melpo
M
Christofidou-Solomidou
D056809:Alveolar Epithelial Cells / Q000378:metabolism / Q000528:radiation effects; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D002453:Cell Cycle / Q000235:genetics / Q000528:radiation effects; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D005786:Gene Expression Regulation / Q000528:radiation effects; D015971:Gene Expression Regulation, Enzymologic; D006657:Histones / Q000378:metabolism; D006801:Humans; D018496:Hyperoxia; D051379:Mice; D008954:Models, Biological; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010766:Phosphorylation; D011839:Radiation, Ionizing; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D013026:Space Flight
D000975:Antioxidants; D006657:Histones; D017382:Reactive Oxygen Species
DNA damage; and space exploration; cell cycle; extravehicular activity; hyperoxia; ionizing radiation; lung cell injury; oxidative stress; reactive oxygen species