Pietrofesa Ralph A, Velalopoulou Anastasia, Lehman Stacey L, Arguiri Evguenia, Solomides Pantelis, Koch Cameron J, Mishra Om P, Koumenis Constantinos, Goodwin Thomas J, Christofidou-Solomidou Melpo
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.
Department of Radiation Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Int J Mol Sci. 2016 Jun 16;17(6):953. doi: 10.3390/ijms17060953.
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.
太空飞行偶尔需要进行多次舱外活动（EVA），这可能会使宇航员反复暴露于环境氧气变化中，叠加在太空辐射暴露之上。因此，我们开发了一种新型体外模型系统，以测试反复暴露于辐射和高氧环境后肺细胞的损伤情况。将非致瘤性小鼠肺泡II型上皮细胞（C10）仅暴露于>95% O₂ 8小时（O₂组）、仅暴露于0.25 Gy电离γ辐射（IR组）或同时接受两种刺激的双重打击组合（O₂ + IR组），随后在常氧环境（含21% O₂和5% CO₂的空气）中培养16小时（1个周期 = 24小时，2个周期 = 48小时）。在暴露1个周期和2个周期后，评估细胞存活率、DNA损伤、细胞凋亡和氧化应激指标。我们观察到，与未暴露于高氧或辐射的细胞相比，在所有刺激条件下细胞存活率均显著降低（p < 0.05），同时通过彗星分析和H2AX磷酸化测定的DNA损伤增加，通过膜联蛋白V染色测定的细胞凋亡增加。在暴露1个周期和2个周期后，辐射和高氧暴露后DNA损伤（GADD45α和裂解的PARP）、凋亡（裂解的caspase-3和BAX）和抗氧化（HO-1和Nqo1）蛋白均增加。重要的是，与单独暴露于O₂或IR相比，暴露于O₂ + IR组合刺激会加剧细胞死亡和DNA损伤。此外，所有暴露组在两个时间点的细胞周期蛋白磷酸化p53和p21水平均显著升高，而CDK1和细胞周期蛋白B1水平降低。同样，与单独的每个应激源相比，参与细胞周期阻滞的蛋白在组合刺激下变化更为显著。这些结果与暴露于高氧条件2个周期后G2/G1期细胞比例显著增加4至6倍相关。我们已经建立了一种新型的体外双重打击模型，即低水平辐射和高氧暴露，可导致肺细胞氧化损伤、DNA损伤、细胞凋亡和细胞周期阻滞。
