Significance Coordinated 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.

Abstract Proliferative gastrointestinal (GI) tissue is radiation-sensitive, and heavy-ion space radiation with its high-linear energy transfer (high-LET) and higher damaging potential than low-LET γ-rays is predicted to compromise astronauts’ GI function. However, much uncertainty remains in our understanding of how heavy ions affect coordinated epithelial cell migration and extrusion, which are essential for GI homeostasis. Here we show using mouse small intestine as a model and BrdU pulse labeling that cell migration along the crypt–villus axis is persistently decreased after a low dose of heavy-ion 56Fe radiation relative to control and γ-rays. Wnt/β-catenin and its downstream EphrinB/EphB signaling are key to intestinal epithelial cell (IEC) proliferation and positioning during migration, and both are up-regulated after 56Fe radiation. Conversely, factors involved in cell polarity and adhesion and cell–extracellular matrix interactions were persistently down-regulated after 56Fe irradiation—potentially altering cytoskeletal remodeling and cell extrusion. 56Fe radiation triggered a time-dependent increase in γH2AX foci and senescent cells but without a noticeable increase in apoptosis. Some senescent cells acquired the senescence-associated secretory phenotype, and this was accompanied by increased IEC proliferation, implying a role for progrowth inflammatory factors. Collectively, this study demonstrates a unique phenomenon of heavy-ion radiation-induced persistently delayed IEC migration involving chronic sublethal genotoxic and oncogenic stress-induced altered cytoskeletal dynamics, which were seen even a year later. When considered along with changes in barrier function and nutrient absorption factors as well as increased intestinal tumorigenesis, our in vivo data raise a serious concern for long-duration deep-space manned missions.

Ionizing radiation (IR) exposure as a risk factor for chronic gastrointestinal (GI) pathologies including colorectal cancer has been reported in atom bomb survivors and radiological workers (1⇓–3). Increasing interest in human exploration of outer space is fraught with exposure to IR, which is considerably different from radiation on Earth. Astronauts traveling on long-duration space missions, such as missions to Mars, will be exposed to energetic particle radiation, including protons and heavy ions from solar particle events and galactic cosmic radiation (4, 5). While protons are the major component of space radiation, energetic heavy ions such as 56Fe, 28Si, and 12C contribute significantly toward the dose equivalent, and ∼30% of astronauts’ cells are predicted to be hit by heavy ions during a round trip to Mars (6, 7). For NASA mission planners, heavy ions are of major concern because they are difficult to block with current shielding measures. Also, qualitatively heavy ions are high-linear energy transfer (high-LET) and densely ionizing, and deposit higher focal energy in traversed tissues relative to terrestrial low-LET sparsely ionizing γ-rays or X-rays (5). Importantly, heavy ions, such as 12C, with its higher relative biological effectiveness and better dose distribution relative to photon-based radiotherapy, are increasingly used for overcoming radioresistance and extending cancer patients’ survival (8). The GI tract with its high cell turnover is sensitive to radiation, and the effects of radiation are evident when the replacement process of cells lost during normal turnover is deregulated. In the small intestine, considered a model system to study GI epithelial cell turnover (9), differentiated epithelial cells from crypt-base stem cells migrate along the crypt–villus axis to replace cells that shed into intestinal lumen via apoptosis at the villus tip (10). While heavy ions are predicted to pose greater risk to GI tissue homeostasis, at present there is much uncertainty in understanding heavy-ion radiation-related GI risk because sufficient in vivo mechanistic data are not available. We previously demonstrated differential long-term stress responses after heavy ions compared to γ-rays in intestinal epithelial cells (IECs) (4). However, it remains to be elucidated how heavy ions modulate molecular events associated with IEC migration, which is essential for maintaining physiological integrity such as nutrient absorption and barrier function and preventing pathological processes such as inflammatory or malabsorption disorders, as well as GI cancer.

Migration of IECs is a bidirectional phenomenon, with all of the differentiated cells moving toward the lumen except Paneth cells, which move toward the crypt base. IEC migration is tightly regulated to minimize exposure time to physical and biochemical insults from luminal contents while maintaining optimal cell turnover for functional integrity (11). Wnt signaling plays key roles in maintaining intestinal cell turnover homeostasis and coordinates with other signaling pathways to regulate proliferation, differentiation, migration, and shedding of IECs (12). Furthermore, Dickkopf-1 (Dkk1), a Wnt antagonist, has been reported to attenuate directional polarization and migration of IECs (13). Importantly, the Wnt-signaling downstream effector β-catenin ensures compartmentalization of IECs in crypts and villi through transcriptional regulation of EphB/EphrinB signaling (12). Furthermore, increased transgenic expression of β-catenin has been reported to cause increased DNA damage and γH2AX foci, and activation of the DNA damage response possibly due to oncogenic stress (14). Increased DNA damage could also be induced by chronic oxidative stress, which has been reported to activate β-catenin (15). DNA damage has been reported to adversely impact the cytoskeleton (16), and efficient cytoskeletal remodeling is essential for coordinated migration of IECs along the crypt–villus axis through regulation of cell polarity, tight junction, adhesion, actomyosin, and microtubule dynamics. Movement of IECs along the crypt–villus axis also requires interaction between epithelial cells and the extracellular matrix (ECM) via integrins (17). Integrins have been reported to modulate the DNA damage response, and activation of β1-integrin decreased DNA damage-induced apoptosis (16). Migrating IECs are shed at the villus tip via apoptosis, and the actin-binding protein villin, through its differential cleavage and interaction with actin, has been shown to regulate cell turnover in the villi (18). While heavy-ion radiation is capable of inducing long-lasting cellular stress and DNA damage (4, 19), its effects on key cellular processes involved in IEC migration and pathological implications of their dysregulation on GI tissue are poorly defined.

The current study now demonstrates that IEC migration along the crypt–villus axis is decreased after a low dose of heavy-ion radiation relative to γ-rays. While heavy ions activated β-catenin and EphB/EphrinB signaling, promoted cell proliferation, and increased the number of crypts, it also induced chronic oxidative stress and DNA damage without increasing cell death up to 12 mo after 56Fe radiation. These effects correlated with higher levels of senescent cells in GI crypts and features of the senescence-associated secretory phenotype (SASP). Heavy-ion radiation induced long-term down-regulation of factors involved in cytoskeletal remodeling, cell adhesion, cellular tight junction, and microtubule dynamics without altering the direction of cell migration. Overall, our study systematically analyzed and identified differential molecular perturbations underlying heavy ion- and γ-ray–induced decreased IEC migration that could aid in developing not only risk prediction models but also risk mitigation strategies for GI tissue in astronauts and patients.

Discussion Epithelial cell turnover is important for maintaining overall GI health, and its perturbation by space radiation, as well as by heavy-ion radiotherapy, raises concerns that require an understanding of its molecular underpinnings. Using the small intestine with its crypt–villus axis as a model system, we show in WT mice that heavy ions negatively impact cell migration as well as molecular events associated with it up to 12 mo later; an overview of our results is included in SI Appendix, Table S7. While a smaller, modest effect was seen early (7 d) after the same dose of γ-rays, cell migration recovered to normal later (60 d), and effects of heavy ions on cell migration were more pronounced relative to γ-rays. Importantly, our data in APC1638N/+ mice demonstrate decreased cell migration signaling is concomitant with increased intestinal tumorigenesis. Hallmarks of increased cell senescence and the SASP were seen in crypt regions after heavy ions, including increased SA-β-gal and IL8 along with an ongoing DNA damage response (DDR), as exemplified by increased γH2AX foci; remarkably, these persisted for a year after irradiation, and actually increased with time. This was accompanied by an increased proliferative response and β-catenin activation, and also changes in intestinal nutrient absorption and barrier function factors. Overall, our results support the conclusion that even low-dose heavy-ion radiation triggers persistent stress signaling and the SASP with adverse changes in the intestine. The canonical Wnt pathway along with its central signal transducer, β-catenin, is involved in maintaining intestinal homeostasis (31). Our data demonstrate persistent up-regulation of not only β-catenin but also phospho-GSK3β, which inactivates GSK3β and scuttles β-catenin degradation, after heavy-ion radiation. Wnt signaling is fine-tuned with coordination between several agonist and antagonists, and Dkk1, a Wnt antagonist, blocks canonical Wnt signaling (13). Here we show that 56Fe radiation even after 60 d depleted Dkk1 expression in mouse intestine, which could further explain radiation-induced β-catenin up-regulation. Escape of β-catenin from degradation allows its cytoplasmic accumulation and subsequent nuclear translocation for transcriptional activation of responsive genes such as EphB3 and EphrinB1 (12). Our data show that EphrinB1 and EphrinB2 ligands and their EphB2 and EphB3 receptors are persistently up-regulated, and such events could affect IEC migration. We next queried whether heavy-ion radiation exposure is modulating β-catenin/TCF4 binding to EphrinB and EphB promoters. ChIP analysis data demonstrate enhanced recruitment of β-catenin/TCF4 to the EphB3 and EphrinB1 promoters after 56Fe radiation, and could be due to radiation-induced up-regulation of β-catenin. A spatial gradient of EphrinB/EphB along the crypt–villus axis determines the directional migration of IECs (32), and our Paneth cell staining data demonstrate that heavy-ion radiation did not alter the direction of migration, which could be due to up-regulation of both the receptors, EphB2 and EphB3, and the ligands, EphrinB1 and EphrinB2. The observed effects of increased accumulation of β-catenin and consequent up-regulation of β-catenin target genes have two general implications: first, progrowth oncogenic stress, and second, cytoskeletal dynamics perturbations; both are expected to adversely impact coordinated IEC migration. Increased expression of proproliferative oncogenes such as β-catenin leads to oncogenic stress that has been reported to induce DNA DSBs via DNA replication fork collapse-mediated replication stress (33). Oncogenic stress is interlinked with oxidative stress (33), and our current data showing increased β-catenin, DNA damage, and oxidative stress are consistent with our previous data showing persistent stress after heavy-ion radiation exposure (4). However, despite increased cellular stress, we did not observe any alteration in cell death, suggesting persistent levels of DNA damage below the apoptotic threshold, which is also consistent with our previous observations (4). Furthermore, our data support the notion that cells are proliferating in the presence of sublethal DNA damage and oxidative stress and that growing cells are accumulating in the crypt area, as evidenced by increased crypt numbers after heavy-ion radiation. It is reasonable to assume that increased β-catenin and decreased E-cadherin are altering β-catenin/E-cadherin dynamics (34), which work in tandem to affect cell–cell adhesion and thus cell migration after heavy-ion radiation. We further hypothesize that heavy-ion radiation-induced increased β-catenin and DNA damage must be altering cytoskeleton dynamics through modulation of cell–cell and cell–ECM interactions, cell polarity and positioning, and microtubule turnover and cell shedding to deregulate IEC migration (12). Cellular apicobasal polarity is established and regulated via Par3/Par6/PKC/Cdc42 complex formation (35), and our data show that the polarity-associated molecule Par3 is down-regulated after 56Fe radiation. Additionally, Cdc42 is known to regulate actin dynamics, especially actin microspike formation, and thus be influencing cell polarity and morphology (36). Therefore, heavy-ion radiation-induced decreased expression of Cdc42 along with Par3 probably contributes to altered cell polarity dynamics and thus deregulated cell migration. Cellular tight junction is regulated through Claudin1, Occludin, and the junctional adhesion molecule family of proteins. We found that 56Fe radiation down-regulated expression of Zo-1, Claudin1, and Scribe in mouse intestine, and such down-regulation could have contributed to perturbation of collective cell migration. This agrees with the recent report showing high-dose γ-radiation inhibits the expression of Zo-1, Claudin3, and E-cadherin, disrupting tight junctions, adherens junctions, and actin cytoskeleton dynamics by an oxidative stress-dependent mechanism in mouse intestine (37). Cell adhesion and microtubule dynamics are also regulated by Rock1 and Mlck through phosphorylation of myosin regulatory light chain-mediated changes in actomyosin contractility (36). In our study, increased Rock1 and decreased Mlck are expected to further destabilize cytoskeletal dynamics and thus disturb coordinated cell migration. Microtubules play a critical role in cytoskeleton remodeling during cell migration. Our data on Map1b and Tau, which mediate interaction between microtubule and other cytoskeleton elements for coordinated cell migration, suggest that heavy-ion radiation exposure compromises microtubule dynamics. Additionally, dynamic interactions between cells’ cytoskeleton and ECM through integrins are also essential for coordinated cellular movement (38). We have shown that 56Fe radiation exposure was associated with increased expression of two integrins that could contribute to decreased dynamicity between cell and ECM, ultimately contributing to persistently slower migration after heavy ions relative to γ-rays and control samples. It is also important that migrating cells upon reaching the villus tip are shed via apoptosis in a timely manner to make way for the incoming new cells (17). Our data on down-regulation of proapoptotic NH 2 -terminal villin at the villus tip are suggestive of compromised cell shedding along with decreased migration after heavy ions, which further highlights the broad impact of heavy ions on normal intestinal homeostasis. Altered migration of intestinal epithelial cells could not only compromise barrier function and nutrient absorption but could also prolong exposure of cells to luminal content and initiate stress responses with pathological consequences, including colon cancer (10). Increased DNA damage evidenced by increased 8-oxo-dG and γH2AX observed in our study could be due to decreased migration allowing increased cell luminal content contact time. DNA damage in our study progressively increased and persisted 12 mo after radiation exposure, and persistent DNA damage can be a trigger for cellular senescence (30) as well as a reflection of increased senescent cells. Indeed, the increasing number of senescent cells observed 12 mo after heavy-ion radiation correlates with persistent γH2AX foci count. Since a primary heavy-ion 56Fe radiation track hit to a cell is expected to induce irreversible damage, it is possible that initial sublethal DNA damage is induced either by secondary delta rays or by nontargeted effects, allowing intestinal stem cells to survive and propagate effects long-term (39). The initial DNA damage could also stochastically induce senescence, which also allows cells to survive without propagation but with SASP potential. Indeed, some of the senescent cells costained with IL8, indicating acquisition of the SASP, and our data align with earlier reports on radiation-induced SASP (30). Our results show altered activity of GGT and ALP involved in amino acid and fat absorption (40) as well as decreased expression of glucose, cholesterol, and fatty acid transporters and intestinal hormones, supporting the notion that heavy-ion radiation-induced decreased cell migration is affecting intestinal function. I-FABP and citrulline are produced by intestinal epithelial cells, and their serum levels have been reported as an indicator of intestinal epithelial integrity (29). Our results showing increased I-FABP and decreased citrulline suggest compromised epithelial integrity, which could be due to either a SASP-mediated chronic inflammatory stress or changes in microbiota, and is in agreement with previous observations (29). While we previously reported compromised barrier function and altered intestinal epithelial morphology, albeit at higher doses (20), the current study not only shows loss of epithelial function but also initiation of pathological processes evidenced by increased intestinal metaplasia marker at a low dose of heavy ions. Although WT mice show metaplasia, they are resistant to spontaneous intestinal tumor development, and therefore we used APC1638N/+ mice to demonstrate concurrently decreased migration signaling molecules and increased intestinal tumorigenesis. Evidence in the literature (27) along with our APC1638N/+ data led us to conclude that slow cell migration is contributing to intestinal tumor initiation and promotion after heavy-ion radiation exposure. In summary, our findings provide experimental evidence unraveling the molecular underpinnings of how high-LET heavy-ion radiation in contrast to low-LET γ-rays can deregulate IEC migration even at a low dose. Although we used the small intestine as a model to study heavy-ion radiation effects on cell migration, our observations could have wider applicability for other GI sites including the colon, which is prone to carcinogenesis. Overall, our analysis provides insight into the effects of heavy-ion radiation on molecular events involved in IEC migration including (i) cell proliferation, (ii) cell–cell interaction, (iii) cell–ECM interaction, and (iv) cell shedding. It is conceivable that chronic Wnt/β-catenin activation and oxidative stress are the two central events cooperatively working to modulate cell migration dynamics after heavy-ion radiation exposure. Given that β-catenin triggers proliferation and oxidative stress triggers the DDR, an appealing probability is that a combination of proliferative and DDR signals could be slowing down cell migration through initial generation of senescent cells by densely ionizing heavy ions, and then a feedback loop involving ongoing generation of additional senescent cells by SASP signaling and ongoing DNA damage and resultant DDR. A key question that future studies will need to address is the role of intestinal stem cells in heavy-ion radiation-induced DNA damage, senescence, SASP, deregulated cell migration, and risk of GI pathologies. We propose that persistent genotoxic and oncogenic stress is triggering cellular senescence, with some of the senescent cells acquiring the SASP with secretion of proinflammatory factors leading to intestinal pathophysiological changes (Fig. 9). We may consider whether modulation of β-catenin activity, oxidative stress, and/or SASP could be advantageous in mitigating some of the risk of heavy-ion radiation-induced GI pathology for astronauts and radiotherapy patients. Fig. 9. Proposed model for altered IEC migration and pathophysiology after heavy-ion space radiation. Unlike low-LET γ-radiation, 56Fe ions trigger long-term perturbation in IECs, including direct damage with the generation of senescent cells and long-term signaling abnormalities consistent with SASP responses. Ongoing oxidative stress can further exacerbate signaling events affecting proliferation and differentiation, cell–cell interactions, cell–ECM interaction, as well as generation of additional senescent cells. The presence of senescent cells in crypts indicates that these events impact stem cell function.

Materials and Methods Mouse Irradiation. Wild-type mice (C57BL/6J; male, 6 to 8 wk old; n = 10) were irradiated (dose: 0.5 Gy) using a simulated space radiation source at the NASA Space Radiation Laboratory (NSRL), Brookhaven National Laboratory for iron (56Fe; energy: 1,000 MeV per nucleon; LET: 148 keV/μm) irradiation, and a 137Cs source was used for γ-ray (LET: 0.8 keV/μm) whole-body irradiation of mice. Mice were euthanized either 7 d, 60 d, or 12 mo after radiation exposure. For BrdU pulse labeling, mice were intraperitoneally administered 30 mg/kg BrdU 24 h before euthanasia. Since wild-type C57BL/6J mice are resistant to tumor development, we also exposed APC1638N/+ mice to the same dose of γ- and 56Fe radiation detailed above to correlate effects on intestinal cell migration parameters with tumorigenesis after exposure to different radiation types. As described previously (5), APC1638N/+ mice were euthanized 150 d after radiation exposure. All animal procedures were performed as per protocols approved by the Institutional Animal Care and Use Committee at Brookhaven National Laboratory (Protocol#345) and at Georgetown University (Protocol#2016-1129). Our study followed the Guide for the Care and Use of Laboratory Animals (41). Additional detail about animal care is provided in SI Appendix, Section H. Sample Collection, Histology, and Immunostaining. In WT mice, intestinal tissues from the jejunal–ilial junction were surgically removed from euthanized mice and flush-cleaned using PBS. In APC1638N/+ mice, intestinal tumors were counted as described previously (5) and tumor and tumor-adjacent normal tissues were collected for further analysis. Tissues were fixed in 10% buffered formalin, paraffin-embedded, and sectioned at 5-μm thickness for hematoxylin and eosin (H&E) staining and immunostaining. Sections were deparaffinized, sequentially rehydrated, and H&E-stained using established procedures. For 8-oxo-dG and BrdU staining, rehydrated sections were treated with 2 M HCl for 20 min at 37 °C followed by neutralization with 0.1 M borate buffer for 10 min at room temperature (RT) and incubated overnight with either anti–8-oxo-dG or anti-BrdU antibodies at 4 °C. For immunostaining of other proteins, rehydrated sections were used for antigen retrieval in pH 6.0 citrate buffer (Dako) for 20 min. All sections were permeabilized in 0.3% Triton X-100 and blocked with 5% BSA at RT followed by incubation in specific primary antibody at 4 °C. Detail about the immunostaining procedure is provided in SI Appendix, Section H. Assessing IEC Migration. Sections stained with anti-BrdU antibody were scored for measuring IEC migration distance along the crypt–villus axis using a method described previously (27). Briefly, in each crypt–villus unit, we marked the highest BrdU-labeled cell and counted the number of cells between the highest-labeled cell and the lowest cell in the crypt. We counted 10 crypt–villus units in each mouse, and 10 different mice were used in each study group for 7- and 60-d time points. IEC migration measured in cell distance after irradiation is presented as percent change in migration distance relative to control (100%) per villus–crypt unit per mouse. Additional Methods, Image Analysis, and Statistical Consideration. Methods describing immunoblots, nutrient absorption and barrier function measurement, qRT-PCR for nutrient absorption, senescence, and senescence-associated secretory phenotype gene expression analysis, TUNEL assay, ChIP real-time PCR, Alcian blue staining, Cdc42 activity assay, SA-β-gal staining, tumor frequency count, and statistical analysis are provided in SI Appendix, Sections D–H. Statistical significance is set at P < 0.05 and error bars represent mean ± SEM.

Acknowledgments We are thankful to the NSRL, especially Drs. Peter Guida and Adam Rusek, for their excellent support. This study is funded through NASA Grants NNX13AD58G and NNX15AI21G. The Histopathology and Tissue Shared Resource is partially supported by NIH/National Cancer Institute (NCI) Grant P30-CA051008.

Footnotes Author contributions: S.K., S.S., and K.D. designed research; S.K. and S.S. performed research; S.K., S.S., and K.D. analyzed data; and S.K., S.S., A.J.F., and K.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807522115/-/DCSupplemental.