NOTE: End date changed to 05/24/2024 per S. Mack/JSC. Original end date was 05/24/2023 (Ed., 5/22/23).

(1) Dose-response investigations were conducted to generate quantitative data on GI tumorigenesis following acute exposure to GCRsim (10-75 cGy) in both male and female Apc1638N/+ mice. The results revealed a significant increase in GI-tumorigenesis across all GCRsim doses tested compared to the control group.

(2) To assess the impact of GCRsim dose rate on GI-tumorigenesis, we conducted exposures at levels of 25, 50, and 75 cGy in both acute and chronic settings. For the chronic simulation of GCRsim exposure, mice received fractionated doses (2.08 cGy/day) over specific durations: 25 cGy (administered 6 days per week for two weeks), 50 cGy (6 days per week for four weeks), and 75 cGy (6 days per week for six weeks). Both acute and chronic exposures led to significantly elevated levels of GI tumorigenesis in male and female mice compared to the control group. These findings indicate that the occurrence of GI tumorigenesis following GCR exposure appears to be unaffected by the dose rate.

(3) GI tumor data resulting from equivalent doses of p+He (45.7 cGy), HZE (4.37 cGy), and GCRsim (50 cGy) were compared to discern the contributions of heavy ions to GI-tumorigenesis. Comparing intestinal tumor data from GCRsim with equivalent doses of heavy ions revealed a correlation between heightened GI-tumorigenesis and doses from the heavy-ion (HZE) fraction. This is evidenced by the significantly greater tumor induction per unit dose (cGy) observed for HZE compared to p+He and GCRsim exposures.

(4) Male Apc1638N/+ mice were exposed to 50 cGy of 4-ion GCRsim (60% H, 20% He, 5% O, and 5% Si), and gastric tumors were counted at 5 months post-exposure. Both 4-ion GCRsim and gamma-ray exposure caused an increase in gastric tumor (adenoma) and carcinoma development relative to the sham group. Our findings suggest that exposure to GCRsim is more likely to cause gastric cancer than low-LET radiation.

(5) Analysis of intestinal tissues and serum collected five months post-irradiation revealed a significant increase in the levels of LINE1 element-specific amplicons in serum DNA from the irradiated group compared to the control. Furthermore, there was a decrease in DNase2 and TREX1 mRNA levels in intestinal epithelial cells following irradiation, albeit with no significant change in DNase1 levels. We also observed a marked decrease in TREX1 immunostaining and protein levels in irradiated mouse intestinal tissues compared to controls. These findings underscore the impact of GCRsim exposure on the expression of key nucleases involved in DNA degradation and the maintenance of extracellular DNA levels in the mouse intestine.

(6) Exposure to acute GCRsim leads to activation of the cGAS-STING signaling pathway in the mouse intestine. Moreover, downstream signaling molecules of the cGAS-STING pathway, including phospho-TBK1 and phospho-IKKa, were also altered after radiation exposure. Notably, these alterations were more pronounced in GCRsim-exposed tissues compared to those exposed to gamma radiation.

(7) Prolonged crewed space flight poses health risks due to exposure to galactic cosmic radiation, with previous research at the NASA Space Radiation Laboratory indicating that HZE ion components of GCR lead to persistent inflammation, increased mutations, and elevated cancer rates. Utilizing the 33-beam galactic cosmic radiation simulations (GCRsim) at NSRL, experiments on a lung cancer susceptible mouse model, i.e., K-rasLA-1 mice showed increased lung adenocarcinoma incidence with higher doses and prolonged exposures, particularly when neutron exposure was added. We interpret these findings to suggest that the risks of carcinogenesis are heightened with doses anticipated during a round trip to Mars, and this risk is magnified when coupled with extra neutron exposure on the Martian surface. Overall, carcinogenesis risks are enhanced with doses expected on a round trip to Mars. We also observed that risks were reduced when the NASA official 33-beam GCR simulations are provided at high dose rates compared to low dose rates.

(8) Administration of metformin (0.5 mM) showed radioprotective effects in vitro on BJ human fibroblasts, increasing DNA damage repair, and increasing SOD1 expression in the nucleus. Importantly, metformin (200 mg/kg) pre-administration for only 3 days in wild type 129/sv mice, decreases the formation of micronuclei in bone marrow cells and DNA damage in colon and lung tissues compared to control irradiated mice at sub-lethal and lethal doses, increasing the overall survival fraction by 37% after 10 Gy total body irradiation. We next pre-treated with metformin and then exposed 129/sv mice to the 33-beam GCRsim. We found metformin pre-treatment decreases the presence of bone marrow micronuclei and DNA damage in colon and lung tissues and an increase of 8-oxoguanine DNA glycosylase-1 (OGG1) expression. We demonstrate a radioprotective effect of metformin through an indirect modulation of gene expression involved in cellular detoxification rather than its effects on mitochondria.

(9) We developed and implemented a mechanistically-motivated mathematical carcinogenesis model that includes both TE and NTE components, and applied it to the data on intestinal tumorigenesis in Apc1638N/+ tumor-prone mice. Importantly, when mixtures of several space radiation types, such as protons and different species of heavy ions, bombard the same biological target, it is not a trivial task to quantify the contribution of each radiation type and predict the overall effect of the mixture. This is the case because, when dose response shapes for the mixture components are not linear (e.g., due to NTE contributions), simply adding the predicted cancer risk contributions of each radiation type together will not correctly predict the risk from the entire mixture. In other words, the seemingly intuitive simple effect additivity (SEA) approach is known to be incorrect for curved dose responses, and more specialized alternative synergy theories such as incremental effect additivity (IEA) are required.

(10) We employed the incremental effect additivity (IEA) methodology to predict the tumorigenic effectiveness of mixtures of space radiation types, based on the fits of our model to data for individual radiation types: protons (1000 MeV/n; 50 to 120 cGy; 0.22 keV/µm, 40 mice), 4He (250 MeV/n; 5 to 50 cGy; 1.6 keV/µm, 92 mice), 12C (290 MeV/n; 10 to 200 cGy; 13 keV/µm, 60 mice), 16O (325 MeV/n; 5 to 50 cGy; 22 keV/µm, 66 mice), 28Si (300 MeV/n; 5 to 140 cGy; 69 keV/µm, 136 mice), 56Fe (1000 MeV/n; 5 to 160 cGy; 148 keV/µm, 90 mice), gamma rays (5-200 cGy, 127 mice). At the current stage of the project, a new data set on tumorigenesis after 33-ion GCRsim mixture exposures (25, 50 and 75 cGy; acute and chronic) is being assembled. This type of mixture is more detailed and more realistic of the space exposure environment. We are working on implementing our TE+NTE model formalism and the IEA synergy theory approach on this data set to provide a more accurate assessment and mechanistic explanation for the overall mixture effect, and for the contributions of its individual components.

1) Ionizing radiation dose, dose rate, and gender effects are known variables that need to be addressed for an accurate estimation of space radiation-induced tumorigenesis risk. In order to address this issue, we participated in the NSRL-22B (summer) beam campaign and conducted a comprehensive GI-tumorigenesis experiment with male and female Apc1638N/+ mice exposed to acute and chronic full-spectrum (33-ion) GCR beams. Additionally, to investigate heavy-ion (HZE) contributions in GCRsim-induced GI tumorigenesis, male Apc1638N/+ mice were exposed to p+He (45.7 cGy, i.e., HZE omitted) and HZE only (4.3 cGy, i.e., p+He omitted) beams. In addition to tumorigenesis studies, we also exposed C57BL6 and Lgr5+ mice to full-spectrum GCR beams, and samples obtained from these animals are being used for molecular studies focused on analyzing GCRsim-induced senescent cell signaling, and GI-functional changes, relative to low-LET gamma-ray exposure.

2) Dose response studies to develop quantitative GI-tumorigenesis data after acute GCRsim (10-75 cGy) exposures were completed in male and female Apc1638N/+ mice. A significant induction in GI-tumorigenesis was noted at all tested GCRsim doses, relative to the control group, which suggested a good signal-to-noise ratio for GI-tumorigenesis in both male and female Apc1638N/+ mice at 150 days post-exposure. In male Apc1638N/+ mice, a GCRsim dose-dependent increase in GI-tumorigenesis was observed up to the 50 cGy dose, while in the case of female mice, a dose-dependent increase in GI-tumorigenesis was observed up to the 25 cGy dose. Additionally, male preponderance for GCRsim-induced GI-tumorigenesis was also observed.

3) The dose rate effect was analyzed using acute and chronic GCRsim exposures. We completed 25, 50, and 75 cGy of exposures in acute and chronic exposure settings. To simulate chronic GCRsim exposure, mice were exposed to a fractionated (2.08 cGy/day) GCRsim beam, i.e., 25 cGy (6 days/week for two weeks), 50 cGy (6 days/week for four weeks), and 75 cGy (6 days/week for six weeks). Both acute and chronic exposure resulted in significantly higher GI tumorigenesis in male and female mice, relative to the control group. No significant difference in GI tumorigenesis was observed between acute and chronic GCRsim exposed male and female Apc1638N/+ mice. Our findings suggest that GI tumorigenesis after GCR exposure is independent of dose rate.

4) Current risk predictions are based on RBE values derived from in vivo studies using single-ion beams, while GCR is essentially a mixed radiation field composed of protons (p), helium (He), and heavy ions (HZE). Therefore, Apc1638N/+ mice were total-body irradiated with 33-ion GCRsim beam components, i.e., p+He (HZE omitted, i.e., LET up to 10 keV/micron) and HZE only (p+He omitted, i.e., LET >10 keV/micron) beams. Further, GI-tumor data from equivalent doses of p+He (i.e., 45.7 cGy), HZE (i.e., 4.37 cGy), and GCRsim (i.e., 50 cGy) were compared to understand the contributions of heavy ions in GI-tumorigenesis. A comparison of tumor data from GCRsim and equivalent doses of heavy ions revealed an association between higher GI-tumorigenesis and dose contributed from the HZE fraction, as the induced tumor per unit (cGy) dose was remarkably higher for HZE compared to p+He and GCRsim exposures. 5) Male C57BL6J mice (8 weeks old) were exposed to either sham, gamma-rays (50 cGy), or acute GCRsim (50 cGy), and intestinal samples were obtained 150 days post exposure. Intestinal tissue sections stained for 4-hydroxyneonenal (4-HNE), a marker of oxidative stress, indicated a significant increase in lipid peroxidation after GCRsim exposure, which was significantly higher than that in the gamma-rays and control groups. Further, DNA double-strand breaks (DSBs) were detected and quantified using gammaH2Ax. Approximately a 2-fold increase in gammaH2Ax foci was noted in the GCRsim-exposed group, compared to a 1.5-fold increase after gamma-ray exposure. In conclusion, a radiation quality-dependent increase in persistent oxidative stress and DNA damage was observed in mouse intestinal epithelial cells (IECs).

6) Male C57BL6J mice (8 weeks old) were exposed to either sham, gamma-rays (50 cGy), or acute GCRsim (50 cGy), and intestinal samples were obtained at 150 days post exposure. We analyzed SA-b-gal activity (a senescence marker) in isolated IEC using C12FDG as substrate and flow cytometry. The data show a significant increase in SA-b-gal activity in GCRsim, relative to gamma-rays and control groups. The mRNA expression analysis of cellular senescence-associated genes, i.e., p16 (upregulated during senescence) and lamin-B1 (downregulated in senescent cells), also indicated a radiation quality-dependent onset of cellular senescence in the mouse intestine. Further, decreased lamin-B1, increased p21, and IL1 protein expression was also noted in GCRsim-exposed mice, relative to gamma-rays and control groups. In conclusion, a radiation quality-dependent increase in the senescent cell population was observed in the mouse intestine.

7) Male C57BL6J mice (8 weeks old) were exposed to either sham, gamma-rays (50 cGy) or acute GCRsim (50 cGy), and intestinal samples were obtained after 150 days of radiation exposure. We investigated various senescence-associated-secretory-phenotype (SASP) molecular markers using qPCR and immunostaining. The qPCR data show at least a 3-fold increase in Cxcl10, Il6, Il1, and Icam1 mRNA levels in GCRsim-exposed mice, relative to gamma-rays and control groups. Immunostaining analysis indicated a significant increase in CXCL10 protein expression in GCRsim-exposed mice, relative to gamma-rays and control groups. In conclusion, a radiation quality-dependent increase in the SASP cell population was observed in the mouse intestine.

8) We exposed lung cancer susceptible mouse models (K-rasLA-1) at the NSRL with fast switching three ion beams: Proton (H) (120 MeV/n) 20cGy, Helium (He) (250 MeV/n) 5cGy, and Silicon (Si) (300MeV/n) 5cGy with a dose rate of 0.5 cGy/min. In these studies, we observed an increase in the incidence of lung cancer initiation and progression. Additionally, when we titrated the dose of HZE ion in the above irradiation protocol, we observed a dose-dependent effect of silicon ions delivered and observed reducing the total dose of silicon from 5cGy to 2cGy and 0.5cGy, progressively reduced cancer progression back to the background rates.

9) To more closely simulate the deep space environment, we exposed lung cancer susceptible mouse models (K-rasLA-1) to acute GCRsim lasting 1-2 hours, more prolonged exposures lasting 10-15 hours, and chronic exposure experiments up to 4-6 weeks with a total dose of 50cGy and 75cGy. We also conducted 25 and 100cGy GCRsim acute experiments. We found a non-statistically significant trend in the increase of malignant adenocarcinomas for an acute 50cGy and 75cGy total dose.

10) We developed and implemented a mechanistically-motivated mathematical carcinogenesis model that includes both TE and NTE components, and applied it to the data on intestinal tumorigenesis in Apc1638N/+ tumor-prone mice. Importantly, when mixtures of several space radiation types, such as protons and different species of heavy ions, bombard the same biological target, it is not a trivial task to quantify the contribution of each radiation type and predict the overall effect of the mixture. This is the case because, when dose response shapes for the mixture components are not linear (e.g., due to NTE contributions), simply adding the predicted cancer risk contributions of each radiation type together will not correctly predict the risk from the entire mixture. In other words, the seemingly intuitive simple effect additivity (SEA) approach is known to be incorrect for curved dose responses, and more specialized alternative synergy theories such as incremental effect additivity (IEA) are required.

11) We employed the incremental effect additivity (IEA) methodology to predict the tumorigenic effectiveness of mixtures of space radiation types, based on the fits of our model to data for individual radiation types: protons (1000 MeV/n; 50 to 120 cGy; 0.22 keV/µm, 40 mice), 4He (250 MeV/n; 5 to 50 cGy; 1.6 keV/µm, 92 mice), 12C (290 MeV/n; 10 to 200 cGy; 13 keV/µm, 60 mice), 16O (325 MeV/n; 5 to 50 cGy; 22 keV/µm, 66 mice), 28Si (300 MeV/n; 5 to 140 cGy; 69 keV/µm, 136 mice), 56Fe (1000 MeV/n; 5 to 160 cGy; 148 keV/µm, 90 mice), gamma-rays (5-200 cGy, 127 mice). At the current stage of the project, a new data set on tumorigenesis after 33-ion GCRsim mixture exposures (25, 50 and 75 cGy; acute and chronic) is being assembled. This type of mixture is more detailed and more realistic of the space exposure environment. We are working on implementing our TE+NTE model formalism and the IEA synergy theory approach on this data set to provide a more accurate assessment and mechanistic explanation for the overall mixture effect, and for the contributions of its individual components.