56Fe Radiation Research Articles

Abstract 428: Decreased RXRα dampened APC-independent proteosomal targeting of β-catenin to cause increased intestinal tumorigenesis after 56-Fe radiation.

Abstract Purpose: Radiation exposure is a risk factor for colorectal cancer (CRC) and high linear energy transfer radiation (high-LET) radiation such as 56-Fe in outer space is expected to pose greater risk of CRC to space travelers. Earlier, we have reported increased intestinal tumorigenesis in heavy ion radiation (HZE) exposed APCMin/+ mice, a well studied model of CRC, and higher tumor incidence was due to increased stabilization and activation of β-catenin signaling after 56-Fe radiation exposure. However the molecular mechanism behind β-catenin activation is not yet explored. Levels of β-catenin are tightly regulated through its targeting to proteasomal degradation mediated via adenomatous polyposis coli (APC) dependent and independent pathways. The APC independent pathway of β-catenin degradation is facilitated via retinoid-X receptor alpha (RXRα). The present study aimed to investigate alterations in RXRα mediated proteasomal pathways in intestinal tumors induced by 56Fe relative to γ radiation. Materials and Methods: Six to eight weeks old female APCMin/+ mice (n=6 mice/ group) were irradiated with 5 Gy of gamma and isotoxic doses of 56Fe (4 Gy, energy: 1 GeV/nucleon; LET: 148 keV/μ) radiation. Mice were euthanized between 100 and 110 days after radiation and intestinal tumor samples were collected. Tumor samples were analyzed to deliniate the 56-Fe-induced changes in RXRα-proteasomal pathways using activity assays, Western blot and immunohistochemistry (IHC). Results: Higher proteasomal activity was observed in 56Fe-induced tumors accompanied with increased expression of proteasomal subunit 20S-β and increased expression/activation of proteosomal activator-200 (PA200) compared to γ radiation. However, we found decreased phosphorylation and consequent reduction in ubiquitination of β-catenin, leading to its increased cellular level in 56Fe relative to gamma radiation-induced tumors. We also show both by Western blot as well as IHC that exposure to 56-Fe radiation led to decreased RXRα expression, a key element in the APC-independent β-catenin degradation pathway. Conclusions: Although 56Fe radiation led to elevated proteasomal activity via overexpression of proteasomal subunit and its activator, we observed increased rather than decreased active β-catenin. We propose that increased β-catenin activity and consequent higher frequency of intestinal tumors is due to the inability of not only the APC-dependent but also the APC-independent RXRα-mediated pathway to facilitate ubiquitination of β-catenin and its subsequent proteasomal degradation. Taken together our study shows for the first time that 56-Fe radiation exposure modulates β-catenin stability in an APC-independent manner to promote intestinal tumorigenesis. Citation Format: Shubhankar Suman, Kamal Datta, Albert J. Fornace. Decreased RXRα dampened APC-independent proteosomal targeting of β-catenin to cause increased intestinal tumorigenesis after 56-Fe radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 428. doi:10.1158/1538-7445.AM2013-428

Abstract 3600: Heavy ion radiation exposure triggered higher intestinal tumor frequency and greater β-catenin activation than γ radiation in APCMin/+ mice.

Abstract Background: Risk of colorectal cancer (CRC) after exposure to low linear energy transfer (low-LET) radiation, such as γ-rays, is highlighted by the studies in atom bomb survivors. Long duration space missions are expected to expose astronauts and future space tourists to considerable levels of cosmic radiation consisting of highly energetic charged (HZE) heavy ions such as 56-Fe, which are densely ionizing high-LET radiation. 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, and intestinal epithelial cell (IEC) proliferation and differentiation were studied in APCMin/+ mice. We also studied effects of 56-Fe radiation on the β-catenin pathway, which is frequently perturbed in CRC. Methods: Female APCMin/+ mice (n=20 mice per group; 6 to 8 wks old) were exposed to whole-body γ (2 or 5 Gy) or 56-Fe (1.6 or 4 Gy; energy-1 GeV/nucleon; LET: 148 keV/μ) radiation and dose rate was 1 Gy/min. The 56-Fe radiation doses were equitoxic to respective γ radiation doses calculated using an RBE factor of 1.25. Mice were euthanized between 100 and 110 days after radiation exposure. The small intestinal tract was surgically removed, divided into duodenum, jejunum, and ilium, flushed gently with PBS, cut open longitudinally, and tumors counted. Intestinal samples of tumor free and tumor bearing areas were either fixed in 10% buffered formalin or flash frozen in liquid nitrogen. Results: Relative to controls and γ-ray, intestinal tumor frequency was significantly higher after 56-Fe and unlike γ-ray, tumorigenesis after 56-Fe was dose dependent. Also, tumor incidence per unit of radiation (per cGy) was higher after 1.6 Gy (28 tumors/160 cGy=0.18) than after 4 Gy (52 tumors/400 cGy=0.13) suggesting that the lower dose was relatively more tumorigenic than the higher dose. We followed the 2 Gy γ and equitoxic 1.6 Gy 56-Fe samples for differential molecular pathway alterations. Staining for phospho-histone H3, indicative of proliferation, was more and alcian blue staining, indicative of differentiation, was less in 56-Fe than γ radiation. Activation of β-catenin as well as cyclin D1 was more in 56-Fe-irradiated normal-appearing and tumor tissues. Conclusions: Our study has implications for health risk estimates of not only astronauts undertaking exploratory missions into outer space and future space tourists but also for patients exposed to heavy ion radiotherapy. We have shown for the first time that 56-Fe radiation exposure led to increased incidence of intestinal tumors in APCMin/+ mice. Importantly, molecular analysis of tumor-free and tumor-bearing areas suggests that increased intestinal tumorigenesis after 56-Fe radiation was due to reduced differentiation and increased proliferation of IEC as a result of preferentially greater activation of β-catenin pathway. Citation Format: Kamal Datta, Shubhankar Suman, Albert J. Fornace. Heavy ion radiation exposure triggered higher intestinal tumor frequency and greater β-catenin activation than γ radiation in APCMin/+ mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2013-3600

Effects of 56Fe radiation on hippocampal function in mice deficient in chemokine receptor 2 (CCR2)

56Fe irradiation affects hippocampus-dependent cognition. The underlying mechanisms may involve alterations in neurogenesis, expression of the plasticity-related immediate early gene Arc, and inflammation. Chemokine receptor-2 (CCR2), which mediates the recruitment of infiltrating and resident microglia to sites of CNS inflammation, is upregulated by 56Fe irradiation. CCR2 KO and wild-type mice were used to compare effects of 56Fe radiation (600MeV, 0.25Gy) on hippocampal function using contextual fear conditioning involving tone shock pairing during training (+/+) and exposure to the same environment without tone shock pairings (−/−). In the −/− condition, irradiation enhanced habituation in WT mice, but not CCR2 KO mice, suggesting that a lack of CCR2 was associated with reduced cognitive performance. In the +/+ condition, irradiation reduced freezing but there was no genotype differences. There were no significant correlations between the number of Arc-positive cells in the dentate gyrus and freezing in either genotype. While measures of neurogenesis and gliogenesis appeared to be modulated by CCR2, there were no effects of genotype on the total numbers of newly born activated microglia before or after irradiation, indicating that other mechanisms are involved in the genotype-dependent radiation response.

Effects of whole body 56Fe radiation on contextual freezing and Arc-positive cells in the dentate gyrus

The space radiation environment contains high-energy charged particles such as 56Fe, which could pose a significant hazard to hippocampal function in astronauts during and after the mission(s). The mechanisms underlying impairments in cognition are not clear but might involve alterations in the percentage of neurons in the dentate gyrus expressing the plasticity-related immediate early gene Arc. Previously, we showed effects of cranial 56Fe irradiation on hippocampus-dependent contextual freezing and on the percentage of Arc-positive cells in the enclosed, but not free, blade. Because it is unclear whether whole body 56Fe irradiation causes similar effects on these markers of hippocampal function, in the present study we quantified the effects of whole body 56Fe irradiation (600MeV, 0.5 or 1Gy) on hippocampus-dependent and hippocampus-independent cognitive performance and determined whether these effects were associated with changes in Arc expression in the enclosed and free blades of the dentate gyrus. Whole body 56Fe irradiation impacted contextual but not cued fear freezing and the percentage of Arc-positive cells in the enclosed and free blades. In mice tested for contextual freezing, there was a correlation between Arc-positive cells in the enclosed and free blades. In addition, in mice irradiated with 0.5Gy, contextual freezing in the absence of aversive stimuli correlated with the percentage of Arc-positive cells in the enclosed blade. In mice tested for cued freezing, there was no correlation between Arc-positive cells in the enclosed and free blades. In contrast, cued freezing in the presence or absence of aversive stimuli correlated with Arc-positive cells in the free blade. In addition, in mice irradiated with 1Gy cued freezing in the absence of aversive stimuli correlated with the percentage of Arc-positive neurons in the free blade. These data indicate that while whole body 56Fe radiation affects contextual freezing and Arc-positive cells in the dentate gyrus, the enclosed blade might be more important for contextual freezing while the free blade might be more important for cued freezing.

Abstract 4422: Activation of beta-catenin/TCF-4 signaling leading to intestinal tumorigenesis in APCMin/+ mice is radiation quality dependent

Abstract Purpose: Radiation quality depends on loss of energy in tissues per unit distance travelled and could be low-linear energy transfer (low-LET) or high-linear energy transfer (high-LET). The low-LET radiation like gamma-rays and x-rays loose less and hence deposits less energy in tissues and are sparsely ionizing. However, the high-LET radiation like α-particle, neutron, and heavy ions loose more and so deposits more energy in tissues traversed. Epidemiological studies showed strong correlation between radiation exposure and colorectal cancer (CRC) and CRC is the second leading cause of cancer mortality in the USA. However, it is not known if activation of molecular pathways involved in CRC and consequent intestinal tumorigenesis is radiation quality dependent. Methods: Female APCMin/+ mice (6-8 weeks) were irradiated with 5 Gy of gamma radiation, a tumorigenic dose in these mice. After 90 days mice were euthanized, intestinal tumors counted and tumor samples were flash frozen. Tumor samples were subjected to histology, western blot and immunohistochemistry (IHC) to dissect the APC/β-catenin/TCF4 pathway, commonly altered in human CRC. Results of gamma radiation were compared to two qualitatively different types of radiation - high-energy proton (energy: 1 giga electron volt (GeV)) and iron (56-Fe) ion (energy: 1 GeV/nucleon). We used 4 Gy of 56-Fe and 4.7 Gy of proton, which are isotoxic to the gamma radiation dose of 5 Gy determined using a quality factor 1.25 and 1.06 respectively. Quantitation of IHC and western blot were performed using ImageJ v2.45 softwere. Results: Quantitative observations showed enhanced intestinal tumorigenesis after exposure to proton and 56-Fe radiation. Marked increases in β-catenin and TCF4 were associated with a decreased phospho-β-catenin and increased phospho-GSK3β in proton and 56-Fe mice. Increased β-catenin was also associated with higher levels of cyclin-D1 and c-myc in these samples. Furthermore, compared to gamma radiation, strikingly lower level of p53 along with higher MDM2 was observed in proton and 56-Fe samples. Interestingly, however, the decrease in p53 and increase in MDM2 was higher in 56-Fe. These observations were further supported by IHC staining of tumor sections. Statistical analyses to support these observations in proton and 56-Fe compared to ≤ ray or spontaneous tumors will be presented. Conclusions: Considering higher active β-catenin and inactivated GSK3β, we speculate that exposure to high-LET radiation caused stabilization of oncogenic β-catenin and upregulation of c-myc and cyclin-D1 leading to higher tumorigencity. Enhanced potential of high-LET radiation for carcinogenesis is further supported by downregulation of p53. In conclusion, this is the first evidence showing greater potential of high-LET radiation for intestinal carcinogenesis mediated via activation of β-catenin/TCF4 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4422. doi:1538-7445.AM2012-4422

Accelerated hematopoietic toxicity by high energy 56Fe radiation

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.

Abstract 1323: Sustained activation of proliferative pathways and increased mammary gland tumorigenesis with γ radiation: A mouse model study

Abstract Breast cancer is the second most commonly diagnosed cancer in women in the USA. The mammary gland is known to be particularly sensitive to radiation injury and persistent cellular changes are known to occur after exposure to ionizing radiation (IR). While measurable effects of IR have been observed in vitro and in vivo, the longer-term consequences still have considerable uncertainty. Delayed consequences of exposure to γ radiation including tumorigensis were compared to heavy ion radiation in wild-type C57BL/6J and in APC+/min mice. Studying heavy ion radiation has implications in two distinct fields - space missions and heavy ion radiotherapy. We used 137-Cs for γ radiation and 56-Fe as heavy ion at the NASA space radiation research laboratory at Brookhaven National Laboratory. Experiments were performed as per approved protocol and 6-8 weeks female C57BL/6J mice were irradiated with 2 to 7 Gy of γ and 56-Fe radiation. APC+/min mice were irradiated at 2 and 5 Gy of γ-rays and equitoxic doses of 56-Fe radiation using an RBE (relative biological effectiveness) of 1.25. Mammary glands were harvested from WT mice at 2 and 9 months postradiation for morphological, histological, transcriptomic, and proteomic analysis. Tumorigenesis was studied in APC+/min mice at 100 days post-radiation. Mammary gland morphology in whole mount revealed a more pronounced decrease in duct density and lateral branching in the 56-Fe exposed mice. H&E stained sections of 2 Gy at 9 months were near normal than 7 Gy γ radiation. The 56-Fe glands, in contrast, were markedly different even at 9 months and showed proliferative changes in the fatty component confirmed by Ki67 staining. With γ radiation proliferative activity was more evident in the ductal tree in a dose dependent manner. In contrast to 56-Fe radiation, transcriptomic analysis at 2 months showed increased estrogen signaling with γ radiation, which correlates with the urinary estradiol level. Expression profiles at 2 or 9 month post-radiation suggests that γ-rays causes greater upregulation of gene expression. In contrast to upregulation of mostly proliferative pathways by γ radiation, 56-Fe radiation showed upregulation of major metabolic pathways. Enhanced metabolic pathways with 56-Fe are consistent with the phenotypic changes in these mice. Sustained activation of proliferative pathways by γ radiation is consistent with tumorigensis in APC+/min mice. An average 40% of the γ irradiated Min mice had mammary tumors most of which were adenomas. Only 8 % of the 56-Fe mice had mammary tumor mostly desmoid cysts. In conclusion, γ radiation although less damaging than heavy ion radiation could have more carcinogenic potential and lower radiation doses are currently investigated. We speculate that reduced tumorigensis could be due to early apoptosis and removal of 56-Fe radiation-induced complex-damages-containing cells and will be further studied. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1323.

Long-term changes in rat hematopoietic and other physiological systems after high-energy iron ion irradiation

Purpose: To assess the long-term consequences of high-linear energy transfer (LET) iron ion radiation on immune and other critical body systems in the context of assessing potential effects astronauts may experience during exploratory missions.Materials and methods: Sprague-Dawley rats were nearly whole-body irradiated with 56-Fe (5 GeV/n) to total doses of 0, 1, 2, and 4 Gray (Gy) and euthanized 9 months post-exposure for analyses.Results: Irradiated groups consistently had low body mass. Numbers of circulating white blood cells (WBC), lymphocytes and monocytes were lower in the 2 Gy group compared to 0 Gy (p < 0.05); a trend for low granulocytes was also noted. Red blood cell counts, hemoglobin, and hematocrit were decreased in irradiated animals (p < 0.05), whereas platelet counts and volume were unaffected. In the spleen, WBC counts and DNA synthesis by T cells were similar among groups and there were no differences in secreted interferon-γ and interleukin-6. However, trends were noted for increased splenocyte capacity to secrete tumor necrosis factor-α and increased level of vascular endothelial cell growth factor in plasma. One or more of the irradiated groups had significant (p < 0.05) aberrations in several blood chemistry parameters associated with liver and kidney function.Conclusion: The data show that exposure to 56-Fe radiation induced pathological changes in important body systems long after exposure.

HZE radiation and dopaminergic modification of startle and prepulse inhibition in mice

C57BL/6 mice were exposed to 5 Gy 28Si or 56Fe particle radiation in order to explore the immediate or short-latency effect of exposure to high energy (HZE) particle radiation on dopaminergic modification of acoustic startle and prepulse inhibition. The radiation is representative of the type which would be encountered as galactic cosmic rays during long-duration space flight. The acoustic startle response was elicited with 120 dB white noise and prepulse inhibition of the startle response was produced with 79 dB and 86 dB stimuli presented with a 125 ms onset asynchrony. Startle reactivity was inhibited by 56Fe radiation but not by 28Si particles. Apomorphine (3 mg/kg) produced a general inhibition of startle reactivity while haloperidol (1 mg/kg) facilitated it. Apomorphine disrupted prepulse inhibition, but only in animals which were not exposed to radiation. Both 56Fe and 28Si radiation exposure attenuated the disruption of prepulse inhibition induced by apomorphine. In contrast, the facilitation of prepulse inhibition induced by haloperidol was not modified by radiation. These data are consistent with a short-latency disruption of dopaminergic systems by HZE particle radiation. We speculate that this disruption may occur as a restriction in the capacity of the dopaminergic system.

Cognitive deficits induced by 56Fe radiation exposure

Exposing rats to particles of high energy and charge (e.g., 56Fe) disrupts neuronal systems and the behaviors mediated by them; these adverse behavioral and neuronal effects are similar to those seen in aged animals. Because cognition declines with age, and our previous study showed that radiation disrupted Morris water maze spatial learning and memory performance, the present study used an 8-arm radial maze (RAM) to further test the cognitive behavioral consequences of radiation exposure. Control rats or rats exposed to whole-body irradiation with 1.0 Gy of 1 GeV/n high-energy 56Fe particles (delivered at the alternating gradient synchrotron at Brookhaven National Laboratory) were tested nine months following exposure. Radiation adversely affected RAM performance, and the changes seen parallel those of aging. Irradiated animals entered baited arms during the first 4 choices significantly less than did controls, produced their first error sooner, and also tended to make more errors as measured by re-entries into non-baited arms. These results show that irradiation with high-energy particles produces age-like decrements in cognitive behavior that may impair the ability of astronauts to perform critical tasks during long-term space travel beyond the magnetosphere.