Ionizing radiation-induced oxidative stress is attributed to generation of reactive oxygen species (ROS) due to radiolysis of water molecules and is short lived. Persistent oxidative stress has also been observed after radiation exposure and is implicated in the late effects of radiation. The goal of this study was to determine if long-term oxidative stress in freshly isolated mouse intestinal epithelial cells (IEC) is dependent on radiation quality at a dose relevant to fractionated radiotherapy. Mice (C57BL/6J; 6 to 8 weeks; female) were irradiated with 2 Gy of γ-rays, a low-linear energy transfer (LET) radiation, and intestinal tissues and IEC were collected 1 year after radiation exposure. Intracellular ROS, mitochondrial function, and antioxidant activity in IEC were studied by flow cytometry and biochemical assays. Oxidative DNA damage, cell death, and mitogenic activity in IEC were assessed by immunohistochemistry. Effects of γ radiation were compared to 56Fe radiation (iso-toxic dose: 1.6 Gy; energy: 1000 MeV/nucleon; LET: 148 keV/µm), we used as representative of high-LET radiation, since it's one of the important sources of high Z and high energy (HZE) radiation in cosmic rays. Radiation quality affected the level of persistent oxidative stress with higher elevation of intracellular ROS and mitochondrial superoxide in high-LET 56Fe radiation compared to unirradiated controls and γ radiation. NADPH oxidase activity, mitochondrial membrane damage, and loss of mitochondrial membrane potential were greater in 56Fe-irradiated mice. Compared to γ radiation oxidative DNA damage was higher, cell death ratio was unchanged, and mitotic activity was increased after 56Fe radiation. Taken together our results indicate that long-term functional dysregulation of mitochondria and increased NADPH oxidase activity are major contributing factors towards heavy ion radiation-induced persistent oxidative stress in IEC with potential for neoplastic transformation.

Risk of colorectal cancer (CRC) after exposure to low linear energy transfer (low-LET) radiation such as γ-ray is highlighted by the studies in atom bomb survivors. On the contrary, CRC risk prediction after exposure to high-LET cosmic heavy ion radiation exposure is hindered due to scarcity of in vivo data. Therefore, intestinal tumor frequency, size, cluster, and grade were studied in APCMin/+ mice (n = 20 per group; 6 to 8 wks old; female) 100 to 110 days after exposure to 1.6 or 4 Gy of heavy ion 56Fe radiation (energy: 1000 MeV/nucleon) and results were compared to γ radiation doses of 2 or 5 Gy, which are equitoxic to 1.6 and 4 Gy 56Fe respectively. Due to relevance of lower doses to radiotherapy treatment fractions and space exploration, we followed 2 Gy γ and equitoxic 1.6 Gy 56Fe for comparative analysis of intestinal epithelial cell (IEC) proliferation, differentiation, and β-catenin signaling pathway alterations between the two radiation types using immunoblot, and immunohistochemistry. Relative to controls and γ-ray, intestinal tumor frequency and grade was significantly higher after 56Fe radiation. Additionally, tumor incidence per unit of radiation (per cGy) was also higher after 56Fe radiation relative to γ radiation. Staining for phospho-histone H3, indicative of IEC proliferation, was more and alcian blue staining, indicative of IEC differentiation, was less in 56Fe than γ irradiated samples. Activation of β-catenin was more in 56Fe-irradiated tumor-free and tumor-bearing areas of the intestinal tissues. When considered along with higher levels of cyclin D1, we infer that relative to γ radiation exposure to 56Fe radiation induced markedly reduced differentiation, and increased proliferative index in IEC resulting in increased intestinal tumors of larger size and grade due to preferentially greater activation of β-catenin and its downstream effectors.

SignificanceCoordinated epithelial cell migration is key to maintaining functional integrity and preventing pathological processes in gastrointestinal tissue, and is essential for astronauts’ health and space mission success. Here we show that energetic heavy ions, which are more prevalent in deep space relative to low-Earth orbit, could persistently decrease intestinal epithelial cell migration, alter cytoskeletal remodeling, and increase cell proliferation with ongoing DNA damage and cell senescence, even a year after irradiation. Our study has provided the molecular underpinnings for energetic heavy-ion 56Fe radiation-induced cell migration alterations, and raises a potentially serious concern, particularly for long-term deep-space manned missions.

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Purpose There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or x-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. Methods C57BL/6J mice were irradiated with 56Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of 56Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. Results Although onset was more rapid, 56Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)50/30 (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy respectively with relative biologic effectiveness for 56Fe ions of 1.25 and 1.06 for protons. Conclusions 56Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity.

Despite recent epidemiological evidences linking radiation exposure and a number of human ailments including cancer, mechanistic understanding of how radiation inflicts long-term changes in cerebral cortex, which regulates important neuronal functions, remains obscure. The current study dissects molecular events relevant to pathology in cerebral cortex of 6 to 8 weeks old female C57BL/6J mice two and twelve months after exposure to a γ radiation dose (2 Gy) commonly employed in fractionated radiotherapy. For a comparative study, effects of 1.6 Gy heavy ion56Fe radiation on cerebral cortex were also investigated, which has implications for space exploration. Radiation exposure was associated with increased chronic oxidative stress, oxidative DNA damage, lipid peroxidation, and apoptosis. These results when considered with decreased cortical thickness, activation of cell-cycle arrest pathway, and inhibition of DNA double strand break repair factors led us to conclude to our knowledge for the first time that radiation caused aging-like pathology in cerebral cortical cells and changes after heavy ion radiation were more pronounced than γ radiation.

While acute effects of toxic radiation doses on intestine are well established, we are yet to acquire a complete spectrum of sub-lethal radiation-induced chronic intestinal perturbations at the molecular level. We investigated persistent effects of a radiation dose (2 Gy) commonly used as a daily fraction in radiotherapy on oxidants and anti-oxidants, and autophagy pathways, which are interlinked processes affecting intestinal homeostasis. Six to eight weeks old C57BL/6J mice (n=10) were exposed to 2 Gy γ-ray. Mice were euthanized two or twelve months after radiation, intestine surgically removed, and flushed using sterile PBS. Parts of the intestine from jejunal-ilial region were fixed, frozen, or used for intestinal epithelial cell (IEC) isolation. While oxidant levels and mitochondrial status were assessed in isolated IEC, autophagy and oxidative stress related signaling pathways were probed in frozen and fixed samples using PCR-based expression arrays and immunoprobing. Radiation exposure caused significant alterations in the expression level of 26 autophagy and 17 oxidative stress related genes. Immunoblot results showed decreased Beclin1 and LC3-II and increased p62, PI3K/Akt, and mTOR. Flow cytometry data showed increased oxidant production and compromised mitochondrial integrity in irradiated samples. Immunoprobing of intestinal sections showed increased 8-oxo-dG and nuclear PCNA, and decreased autophagosome marker LC3-II in IEC after irradiation. We show that sub-lethal radiation could persistently downregulate anti-oxidants and autophagy signaling, and upregulate oxidant production and proliferative signaling. Radiation-induced promotion of oxidative stress and downregulation of autophagy could work in tandem to alter intestinal functions and have implications for post-radiation chronic gastrointestinal diseases.