NOTE: End date changed to 12/31/2023 per NSSC information (Ed., 11/14/22)

NOTE: End date changed to 9/30/2022 per L. Barnes-Moten/JSC (Ed., 3/30/21)

NOTE: End date changed to 1/28/2021 per NSSC information (Ed., 2/21/2020)

The overarching hypothesis for this project is that space radiation induces molecular manifestations of a pro-inflammatory response as a function of radiation type, radiation doses, doses rates, LET value, and time. We are testing if an oral available anti-oxidant and anti-inflammatory countermeasure, already in human clinical trials with a good safety profile, CDDO, significantly reduces proton and HZE-ion exposure associated tumor initiation and progression. Based on experiments conducted at the NASA Space Radiation Laboratory (Brookhaven, NY) we demonstrate that HZE ion components of GCR (galactic cosmic radiation) result in persistent DNA damage and inflammatory signaling, increased mutations in tumor suppressor genes, and higher rates of cancer initiation and progression compared to that seen with similar doses of terrestrial radiation. While physical shielding may reduce some of the risks of space radiation, there is substantial evidence that biological countermeasures will be required to ensure that the established safety limits of increased lifetime fatal cancer risks are not exceeded. We are conducting GCR simulations consisting of fast switching between protons, helium, and silicon using a dose rate of 0.5 cGy/min and a total combined dose of between 27-30 cGy to more closely mimic the space environment on a trip to Mars and back. Finally, we are conducting experiments with the official NASA GCRsim with acute and protracted mixed fields.

References

Kim, S.B., Bozeman, R.G., Kaisani, A., Kim, W., Zhang, L., Richardson, J.A., Wright, W.E., and Shay, J.W. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. Oncogene. 2015. https://doi.org/10.1038/onc.2015.395

Norbury, J.W., Schimmerling, W., Slaba, T.C., Edouard Azzam, Francis F. Badavi, Giorgio Baiocco, Eric Benton, Veronica Bindi, Eleanor A. Blakely, Steve R. Blattnig, David A. Boothman, Thomas B. Borak, Richard A. Britten, Stan Curtis, Michael Dingfelder, Marco Durante, William Dynan, Amelia Eisch, S. Robin Elgart, Dudley T. Goodhead, Peter M. Guida, Lawrence H. Heilbronn, Christine E. Hellweg, Janice L. Huff, Amy Kronenberg, Chiara La Tessa, Derek Lowenstein, Jack Miller, Taksahi Morita, Livio Narici, Gregory A. Nelson, Ryan B. Norman, Takeo Ohnishi, Andrea Ottolenghi, Zarana S. Patel, Guenther Reitz, Adam Rusek, Ann-Sofie Schreurs, Lisa A. Scott-Carnell, Edward Semones, Jerry W. Shay, Vyacheslav A. Shurshakov, Lembit Sihver, Lisa C. Simonsen, Michael Story, Mitchell S. Turker, Yukio Uchihori, Jacqueline Williams, Cary J. Zeitlin. Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research 8:38-51, 2016. PMID: 26948012

Lutiel, K. Bozeman, R., Kaisani, A. Kim, S.B., Barron, S., Richardson, J.A., Shay, J.W. Proton radiation-induced cancer progression. Life Sciences in Space Research, 2018. https://doi.org/10.1016/j.lssr.2018.08.002

Luitel, K., Kim, S.B., Barron, S. Richardson, J.A. and Shay, J.W. Lung cancer progression using fast switching multiple ion beam irradiation and countermeasure prevention, Life Sciences in Space Research, 2019. https://doi.org/10.1016/j.lssr.2019.07.011

A single dose of metformin (0.5-1mM) on human colon epithelial cells (HCECs) and human skin fibroblasts (BJs), showed an increase in the expression of AMPK alpha subunit phosphorylation and superoxide dismutase 1 (SOD1), during the first 72 hours. Interestingly, SOD1 has a crucial role in protecting against oxidative DNA damage and its overexpression is associated with radio-resistance in human glioma cells. Furthermore, a decrease in the basal DNA damage (phosphorylation of H2AX at Ser 139 foci) and in reactive oxygen species (ROS) production was observed. Moreover, metformin treatment enhanced DNA damage responses (DDR) 24 hours after exposure to 2Gy of γ-rays. To evaluate the radioprotective effect of metformin, cells were treated one time and irradiated 72 hours later, with 2, 4, and 6 Gy doses of gamma-rays. Cells were seeded at low density (200-1000 cells) and a colony formation assay was analyzed after 21 days. Metformin showed an increase in the surviving fraction of cells compared to the irradiated controls. Next, we investigated the radioprotective effect of metformin in vivo. Wild type 129/Sv mice were injected once per day with metformin 200mg/kg, for three consecutive days prior to exposure to 7.5Gy of X-rays and sacrificed after 24 hours. Metformin pre-treatment dramatically decreased DNA damage (p53 binding protein 1 foci) in mouse lung and colon tissues as well as the number of micronuclei in bone marrow cells, compared to the irradiated controls. This suggested increased DNA repair. Notably, when mice were irradiated at the dose of 10Gy X-rays post-metformin treatment, an increase of 30% in the surviving fraction was observed. In conclusion, metformin might act as a radioprotector from GCR, potentially lowering the risk of cancer initiation or promotion in astronauts. We have now formulated metformin into lab chow and completed studies at the NASA Space Radiation Laboratory (NSRL) when mice were exposed to 75cGy full spectrum GCR (these are now published in Siteni et al. PLoS One. 2024;19(7): e0307598. https://doi.org/10.1371/journal.pone.0307598

Metformin resulted in a hyperpolarization of the mitochondrial membrane, likely induced by the inhibitory effect on the complex I. Furthermore, metformin 0.5mM was able to reduce the reactive oxygen species (ROS) production in combination with 2Gy of γ-rays. Next, the radioprotective effect of metformin in vivo was investigated. Wild type 129/Sv mice were injected once per day with metformin 200mg/kg, for three consecutive days prior to exposure of 7.5Gy of X-rays and sacrificed after 24 hours. Metformin pre-treatment dramatically decreased the DNA damage (p53 binding protein 1 foci) in mouse lung and colon tissues as well as the number of micronuclei in bone marrow cells, compared to the irradiated controls. Notably, when mice were irradiated at the dose of 10 Gy X-rays post-metformin treatment, an increase of 30% in the surviving fraction was observed. Wild type mice were fed with metformin 200mg/kg chow and then irradiated with 2Gy of X-rays and sacrificed after 100 days. Metformin pre-treatment determined the phosphorylation of the protein NF-κB p65 at serine 536, with a major increase in the nuclear fraction. The phosphorylation of the subunit p65 at serine 536 is associated with NF-κB transcriptional activity, but also increased p65 turnover, thus reducing NF-κB activity and supporting the resolution of inflammation.

We next tested the GCR spectrum at the NSRL which is composed of 33 beams and uses mixed ion species, protons, and sporadic heavy ions to better simulate the space environment. Wild type mice were fed with 200mg/kg metformin chow for 72 hours and then irradiated with 75cGy GCR. After 4 days, mice were sacrificed and cells from bone marrow were examined. Fixed bone marrow slides were then stained with Giemsa and the number of micronuclei was analyzed. We found that 75cGy GCR increases the number of micronuclei, inducing a genotoxic effect. Administration of metformin 72 hours prior to irradiation decreased the number of micronuclei significantly. Furthermore, DNA damage foci (53BP1) significantly decreased in lung and colon tissues, as well as oxidative stress (8-oxo-G), after 6 hours from irradiation. Metformin pre-administration showed a decrease in apoptosis (cleaved –PARP), and an increase in oxidative DNA damage repair enzyme OGG1 when compared to irradiation only, in colon and lung tissue. Finally, lung cancer susceptible mice K-rasLA-1 were fed with metformin 200mg/kg 4 days prior to 75cGy GCR irradiation and mice were sacrificed after 100 days. Metformin pre-treatment showed a decrease in plasma lipid peroxidation, as well as the number and area of pre-malignant lesions in lung tissue. In conclusion, our published results can be interpreted to suggest that metformin acts as an oral available and safe radioprotector, potentially lowering the risk of cancer initiation or promotion on healthy tissues.

NOTE: End date changed to 12/31/2023 per NSSC information (Ed., 11/14/22)

NOTE: End date changed to 9/30/2022 per L. Barnes-Moten/JSC (Ed., 3/30/21)

NOTE: End date changed to 1/28/2021 per NSSC information (Ed., 2/21/2020)

The overarching hypothesis for this project is that space radiation induces molecular manifestations of a pro-inflammatory response as a function of radiation type, radiation doses, doses rates, LET value, and time. We are testing if an oral available anti-oxidant and anti-inflammatory countermeasure, already in human clinical trials with a good safety profile, CDDO, significantly reduces proton and HZE-ion exposure associated tumor initiation and progression. Based on experiments conducted at the NASA Space Radiation Laboratory (Brookhaven, NY) we demonstrate that HZE ion components of GCR (galactic cosmic radiation) result in persistent DNA damage and inflammatory signaling, increased mutations in tumor suppressor genes, and higher rates of cancer initiation and progression compared to that seen with similar doses of terrestrial radiation. While physical shielding may reduce some of the risks of space radiation, there is substantial evidence that biological countermeasures will be required to ensure that the established safety limits of increased lifetime fatal cancer risks are not exceeded. We are conducting GCR simulations consisting of fast switching between protons, helium, and silicon using a dose rate of 0.5 cGy/min and a total combined dose of between 27-30 cGy to more closely mimic the space environment on a trip to Mars and back. Finally, we are conducting experiments with the official NASA GCRsim with acute and protracted mixed fields.

References

Kim, S.B., Bozeman, R.G., Kaisani, A., Kim, W., Zhang, L., Richardson, J.A., Wright, W.E., and Shay, J.W. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. Oncogene. 2015. https://doi.org/10.1038/onc.2015.395

Norbury, J.W., Schimmerling, W., Slaba, T.C., Edouard Azzam, Francis F. Badavi, Giorgio Baiocco, Eric Benton, Veronica Bindi, Eleanor A. Blakely, Steve R. Blattnig, David A. Boothman, Thomas B. Borak, Richard A. Britten, Stan Curtis, Michael Dingfelder, Marco Durante, William Dynan, Amelia Eisch, S. Robin Elgart, Dudley T. Goodhead, Peter M. Guida, Lawrence H. Heilbronn, Christine E. Hellweg, Janice L. Huff, Amy Kronenberg, Chiara La Tessa, Derek Lowenstein, Jack Miller, Taksahi Morita, Livio Narici, Gregory A. Nelson, Ryan B. Norman, Takeo Ohnishi, Andrea Ottolenghi, Zarana S. Patel, Guenther Reitz, Adam Rusek, Ann-Sofie Schreurs, Lisa A. Scott-Carnell, Edward Semones, Jerry W. Shay, Vyacheslav A. Shurshakov, Lembit Sihver, Lisa C. Simonsen, Michael Story, Mitchell S. Turker, Yukio Uchihori, Jacqueline Williams, Cary J. Zeitlin. Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research 8:38-51, 2016. PMID: 26948012

Lutiel, K. Bozeman, R., Kaisani, A. Kim, S.B., Barron, S., Richardson, J.A., Shay, J.W. Proton radiation-induced cancer progression. Life Sciences in Space Research, 2018. https://doi.org/10.1016/j.lssr.2018.08.002

Luitel, K., Kim, S.B., Barron, S. Richardson, J.A. and Shay, J.W. Lung cancer progression using fast switching multiple ion beam irradiation and countermeasure prevention, Life Sciences in Space Research, 2019. https://doi.org/10.1016/j.lssr.2019.07.011

[1] Kim SB, Bozeman RG, Kaisani A, Kim W, Zhang L, Richardson JA, Wright WE, Shay JW. Oncogene. 2016 Jun 30;35(26):3365-75. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. https://doi.org/10.1038/onc.2015.395 ; PMID: 26477319 PMCID: PMC4837107

[2] Delgado O, et al. Clin Cancer Res. 2014 Mar 15;20(6):1610-22. Radiation-enhanced lung cancer progression in a transgenic mouse model of lung cancer is predictive of outcomes in human lung and breast cancer. https://doi.org/10.1158/1078-0432.CCR-13-2589 ; PMID: 24486591; PMCID: PMC3961755

[3]. Suman S, et al. Int J Radiat Oncol Biol Phys. 2016 May 1;95(1):131-138.Relative biological effectiveness of energetic heavy ions for intestinal tumorigenesis shows male preponderance and radiation type and energy dependence in APC(1638N/+) mice. https://doi.org/10.1016/j.ijrobp.2015.10.057 ; PMID: 26725728

Kim SB, Zhan, L, Shay JW. Gravitational and Space Research. 2014 Aug 29;2(1):32-41. Oxygen and silicon ion particles induce neoplastic transformation of human colonic epithelial cells. https://doi.org/10.2478/gsr-2014-0003

[5] Luitel. K. Life Sci Space Res (Amst). 2018 Nov;19:31-42. doi: 0.1016/j.lssr.2018.08.002

NOTE: End date changed to 9/30/2022 per L. Barnes-Moten/JSC (Ed., 3/30/21)

NOTE: End date changed to 1/28/2021 per NSSC information (Ed., 2/21/2020)

The overarching hypothesis for this project is that space radiation induces molecular manifestations of a pro-inflammatory response as a function of radiation type, radiation doses, doses rates, LET value, and time. We are testing if an oral available anti-oxidant and anti-inflammatory countermeasure, already in human clinical trials with a good safety profile, CDDO, significantly reduces proton and HZE-ion exposure associated tumor initiation and progression. Based on experiments conducted at the NASA Space Radiation Laboratory (Brookhaven, NY) we demonstrate that HZE ion components of GCR (galactic cosmic radiation) result in persistent DNA damage and inflammatory signaling, increased mutations in tumor suppressor genes, and higher rates of cancer initiation and progression compared to that seen with similar doses of terrestrial radiation. While physical shielding may reduce some of the risks of space radiation, there is substantial evidence that biological countermeasures will be required to ensure that the established safety limits of increased lifetime fatal cancer risks are not exceeded. We are conducting GCR simulations consisting of fast switching between protons, helium, and silicon using a dose rate of 0.5 cGy/min and a total combined dose of between 27-30 cGy to more closely mimic the space environment on a trip to Mars and back. Finally, we are conducting experiments with the official NASA GCRsim with acute and protracted mixed fields.

References

Kim, S.B., Bozeman, R.G., Kaisani, A., Kim, W., Zhang, L., Richardson, J.A., Wright, W.E., and Shay, J.W. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. Oncogene. 2015. https://doi.org/10.1038/onc.2015.395

Norbury, J.W., Schimmerling, W., Slaba, T.C., Edouard Azzam, Francis F. Badavi, Giorgio Baiocco, Eric Benton, Veronica Bindi, Eleanor A. Blakely, Steve R. Blattnig, David A. Boothman, Thomas B. Borak, Richard A. Britten, Stan Curtis, Michael Dingfelder, Marco Durante, William Dynan, Amelia Eisch, S. Robin Elgart, Dudley T. Goodhead, Peter M. Guida, Lawrence H. Heilbronn, Christine E. Hellweg, Janice L. Huff, Amy Kronenberg, Chiara La Tessa, Derek Lowenstein, Jack Miller, Taksahi Morita, Livio Narici, Gregory A. Nelson, Ryan B. Norman, Takeo Ohnishi, Andrea Ottolenghi, Zarana S. Patel, Guenther Reitz, Adam Rusek, Ann-Sofie Schreurs, Lisa A. Scott-Carnell, Edward Semones, Jerry W. Shay, Vyacheslav A. Shurshakov, Lembit Sihver, Lisa C. Simonsen, Michael Story, Mitchell S. Turker, Yukio Uchihori, Jacqueline Williams, Cary J. Zeitlin. Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research 8:38-51, 2016. PMID: 26948012

Lutiel, K. Bozeman, R., Kaisani, A. Kim, S.B., Barron, S., Richardson, J.A., Shay, J.W. Proton radiation-induced cancer progression. Life Sciences in Space Research, 2018. https://doi.org/10.1016/j.lssr.2018.08.002

Luitel, K., Kim, S.B., Barron, S. Richardson, J.A. and Shay, J.W. Lung cancer progression using fast switching multiple ion beam irradiation and countermeasure prevention, Life Sciences in Space Research, 2019. https://doi.org/10.1016/j.lssr.2019.07.011

Using a mouse model of lung cancer, we have tested the 33 beam GCRsim in a range of doses from 25 to 100 cGy. Mice were exposed to GCRsim and one year later sacrificed to examine for advanced lung and other cancers. At doses below 75cGy we did not observe an increase in advanced cancers. However, at 75cGy provided acutely (over 2 hours) or protracted (over 6 weeks) we did observe a significance increase in the appearance of invasive cancers. We next tested CDDO and metformin as a radioprotector and experiments are ongoing.

NOTE: End date changed to 1/28/2021 per NSSC information (Ed., 2/21/2020)

The overarching hypothesis for this project is that space radiation induces molecular manifestations of a pro-inflammatory response as a function of radiation type, radiation doses, doses rates, LET value, and time. We are testing if an oral available anti-oxidant and anti-inflammatory countermeasure, already in human clinical trials with a good safety profile, CDDO, significantly reduces proton and HZE-ion exposure associated tumor initiation and progression. Based on experiments conducted at the NASA Space Radiation Laboratory (Brookhaven, NY) we demonstrate that HZE ion components of GCR (galactic cosmic radiation) result in persistent DNA damage and inflammatory signaling, increased mutations in tumor suppressor genes, and higher rates of cancer initiation and progression compared to that seen with similar doses of terrestrial radiation. While physical shielding may reduce some of the risks of space radiation, there is substantial evidence that biological countermeasures will be required to ensure that the established safety limits of increased lifetime fatal cancer risks are not exceeded. We are conducting GCR simulations consisting of fast switching between protons, helium, and silicon using a dose rate of 0.5 cGy/min and a total combined dose of between 27-30 cGy to more closely mimic the space environment on a trip to Mars and back. Finally, we are conducting experiments with the official NASA GCRsim with acute and protracted mixed fields.

References

Kim, S.B., Bozeman, R.G., Kaisani, A., Kim, W., Zhang, L., Richardson, J.A., Wright, W.E., and Shay, J.W. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. Oncogene. 2015. https://doi.org/10.1038/onc.2015.395

Norbury, J.W., Schimmerling, W., Slaba, T.C., Edouard Azzam, Francis F. Badavi, Giorgio Baiocco, Eric Benton, Veronica Bindi, Eleanor A. Blakely, Steve R. Blattnig, David A. Boothman, Thomas B. Borak, Richard A. Britten, Stan Curtis, Michael Dingfelder, Marco Durante, William Dynan, Amelia Eisch, S. Robin Elgart, Dudley T. Goodhead, Peter M. Guida, Lawrence H. Heilbronn, Christine E. Hellweg, Janice L. Huff, Amy Kronenberg, Chiara La Tessa, Derek Lowenstein, Jack Miller, Taksahi Morita, Livio Narici, Gregory A. Nelson, Ryan B. Norman, Takeo Ohnishi, Andrea Ottolenghi, Zarana S. Patel, Guenther Reitz, Adam Rusek, Ann-Sofie Schreurs, Lisa A. Scott-Carnell, Edward Semones, Jerry W. Shay, Vyacheslav A. Shurshakov, Lembit Sihver, Lisa C. Simonsen, Michael Story, Mitchell S. Turker, Yukio Uchihori, Jacqueline Williams, Cary J. Zeitlin. Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research 8:38-51, 2016. PMID: 26948012

Lutiel, K. Bozeman, R., Kaisani, A. Kim, S.B., Barron, S., Richardson, J.A., Shay, J.W. Proton radiation-induced cancer progression. Life Sciences in Space Research, 2018. https://doi.org/10.1016/j.lssr.2018.08.002

Luitel, K., Kim, S.B., Barron, S. Richardson, J.A. and Shay, J.W. Lung cancer progression using fast switching multiple ion beam irradiation and countermeasure prevention, Life Sciences in Space Research, 2019. https://doi.org/10.1016/j.lssr.2019.07.011

Metformin is a biguanide compound used in the treatment of type 2 diabetes mellitus, showing very low cytotoxic effects, that was FDA-approved in 1995. Metformin decreases oxidative stress and DNA damage in vitro and in vivo, resulting in decreased chronic inflammation. Metformin acts mainly through the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), which has pleiotropic effects on cell metabolism. Furthermore, metformin targets mitochondria, inhibiting complex I of the electron transport chain (ETC), but the mechanisms underlying this process have not been completely elucidated. Because of its antioxidant effects, we investigated the role of metformin as a radioprotective compound. One single dose of metformin (2.5 mM) on human fibroblasts (BJs), shows an increase in the expression of phosphorylated AMPK alpha subunit and of superoxide dismutase 1 (SOD1). SOD1 acts as a transcriptional factor, protecting against oxidative DNA damage and its overexpression is associated with radioresistance in human glioma cells. Metformin decreases basal DNA damage (phosphorylation of H2AX at Ser 139 foci), and reactive oxygen species (ROS) production, and mitochondrial membrane depolarization (TMRE assay). To evaluate the radioprotective effect of metformin, cells were treated one time and irradiated 72 hours later, with 2, 4, and 6 Gy doses of gamma-rays. Cells were seeded at low density (200-1000 cells) and a colony formation assay was analyzed after 21 days. Metformin showed an increase in the surviving fraction of cells compared to the irradiated controls. Next, we investigated the radioprotective effect of metformin in vivo. Wild type 129/Sv mice were injected once per day with metformin 200mg/kg, for three consecutive days prior to exposure of 7.5 Gy of X-rays and sacrificed after 24 hours. Metformin pre-treatment was able to dramatically decrease DNA damage (p53 binding protein 1 foci) in mouse lung and colon tissues as well as the number of micronuclei in bone marrow cells, compared to the irradiated controls. Notably, when mice were irradiated at the dose of 10 Gy X-rays post-metformin treatment, a 30% increase in the surviving fraction was observed. In another experiment, wild type mice were pre-treated with metformin and irradiated with 2Gy dose of X-rays and sacrificed after 100 days. We found that metformin pre-treatment induces an increase of the expression of phosphorylated nuclear factor kappa B subunit p65 (NF-KB p65) serine536. The NF-KB p65 s536 inhibits NF-KB signaling to prevent deleterious inflammation. Next, wild type mice were irradiated with 75cGy simulated-GCR, after 72 hours of metformin treatment. Metformin 72 hours pre-treatment significantly decreased the number of micronuclei in murine bone marrow cells compared to 75cGy control GCR-sim irradiated mice. In addition, persistent oxidative stress induced by 75cGy GCR-sim in lung and colon tissues was decreased. Finally, metformin pre-administration acts as a radioprotector through AMPK phosphorylation, increasing OGG1 and decreasing cleaved Poly (ADP-ribose) polymerase (PARP) expression. All together, we interpret these results to suggest that metformin is a potential GCR radioprotector, and has the potential to lower the risk of cancer initiation/promotion in astronauts.

NOTE: End date changed to 1/28/2021 per NSSC information (Ed., 2/21/2020)

The overarching hypothesis for this project is that space radiation induces molecular manifestations of a pro-inflammatory response as a function of radiation type, radiation doses, doses rates, LET value, and time. We are testing if an oral available anti-oxidant and anti-inflammatory countermeasure, already in human clinical trials with a good safety profile, CDDO, significantly reduces proton and HZE-ion exposure associated tumor initiation and progression. Based on experiments conducted at the NASA Space Radiation Laboratory (Brookhaven, NY) we demonstrate that HZE ion components of GCR (galactic cosmic radiation) result in persistent DNA damage and inflammatory signaling, increased mutations in tumor suppressor genes, and higher rates of cancer initiation and progression compared to that seen with similar doses of terrestrial radiation. While physical shielding may reduce some of the risks of space radiation, there is substantial evidence that biological countermeasures will be required to ensure that the established safety limits of increased lifetime fatal cancer risks are not exceeded. We are conducting GCR simulations consisting of fast switching between protons, helium, and silicon using a dose rate of 0.5 cGy/min and a total combined dose of between 27-30 cGy to more closely mimic the space environment on a trip to Mars and back. Finally, we are conducting experiments with the official NASA GCRsim with acute and protracted mixed fields.

References

Kim, S.B., Bozeman, R.G., Kaisani, A., Kim, W., Zhang, L., Richardson, J.A., Wright, W.E., and Shay, J.W. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. Oncogene. 2015. https://doi.org/10.1038/onc.2015.395

Norbury, J.W., Schimmerling, W., Slaba, T.C., Edouard Azzam, Francis F. Badavi, Giorgio Baiocco, Eric Benton, Veronica Bindi, Eleanor A. Blakely, Steve R. Blattnig, David A. Boothman, Thomas B. Borak, Richard A. Britten, Stan Curtis, Michael Dingfelder, Marco Durante, William Dynan, Amelia Eisch, S. Robin Elgart, Dudley T. Goodhead, Peter M. Guida, Lawrence H. Heilbronn, Christine E. Hellweg, Janice L. Huff, Amy Kronenberg, Chiara La Tessa, Derek Lowenstein, Jack Miller, Taksahi Morita, Livio Narici, Gregory A. Nelson, Ryan B. Norman, Takeo Ohnishi, Andrea Ottolenghi, Zarana S. Patel, Guenther Reitz, Adam Rusek, Ann-Sofie Schreurs, Lisa A. Scott-Carnell, Edward Semones, Jerry W. Shay, Vyacheslav A. Shurshakov, Lembit Sihver, Lisa C. Simonsen, Michael Story, Mitchell S. Turker, Yukio Uchihori, Jacqueline Williams, Cary J. Zeitlin. Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research 8:38-51, 2016. PMID: 26948012

Lutiel, K. Bozeman, R., Kaisani, A. Kim, S.B., Barron, S., Richardson, J.A., Shay, J.W. Proton radiation-induced cancer progression. Life Sciences in Space Research, 2018. https://doi.org/10.1016/j.lssr.2018.08.002

Luitel, K., Kim, S.B., Barron, S. Richardson, J.A. and Shay, J.W. Lung cancer progression using fast switching multiple ion beam irradiation and countermeasure prevention, Life Sciences in Space Research, 2019. https://doi.org/10.1016/j.lssr.2019.07.011

Previously, using the fast beam switching technology developed in NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), we used mixed ions with different energies to more closely simulate the space environment. We exposed a lung cancer susceptible mouse model (K-rasLA-1) at the NSRL with three ion beams: Protons (H) (120 MeV/n) 20 cGy, Helium (He) (250 MeV/n) 5 cGy, and Silicon (Si) (300 MeV/n) 5 cGy with a dose rate of 0.5 cGy/min and observed increases in the incidence of lung cancer initiation and progression. Additionally, when we titrated the dose of HZE ion, we observed a dose-dependent effect of silicon ions delivered. We observed reducing the total dose of silicon from 5 cGy to 2 cGy and 0.5 cGy in combination with 20 cGy protons and 5 cGy of helium, reduced cancer progression back to background rates.

With the high energy and control upgrades at the NSRL, experiments are now being conducted to better simulate the deep space environment with low fluence rates predominated by low background fluences of low-LET radiation with lower fluences of high-LET radiation. These experiments consist of chronic exposure from 2-6 weeks irradiation (6-days per week) or acute one-day exposures with continuous exposure to background protons and helium and a sporadic heavy ion exposure. The delivery dose consists of 33 ions and energy mix to even more closely approximate the deep space environment. During NSRL18B and NSRL19A an acute 50 cGy and 75 cGy total exposure to the newly developed GCR simulation was initiated. During NSRL18C a chronic/protracted (4 week exposure) 50 cGy total exposure was performed to better simulate the low dose rates expected in the deep space environment. During NSRL19C we irrradiated mice with a chronic/protracted (6 weeks exposure) 0.75 Gy total exposure. Finally, in NSRL 20B we sent mice to the NSRL and the BNL team irradiated mice with acute 25 cGy and 100 cGy full spectrum GCR simulation. Results of these experiments will be presented at the Human Research Program (HRP) 2021 meeting. The deliverables from these experiments will be increases in tumor initiation and size as well as chronic increases in plasma lipid peroxidation at 100 days post-irradiation in a subset of mice. One year post-irradiation, all remaining mice will be sacrificed for increases in invasive (more lethal cancers) and overall survival.

We continue our experiments on medical countermeasures to test safe small molecules that may reduce effects of GCR simulations associated cancers. For long-term space missions to the Moon or Mars, it might be necessary to adopt radiological countermeasures for astronauts. Thanks to the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory, it is now possible to more closely simulate galactic cosmic radiation (GCR) that occurs in space and to determine the effects on biological models. We initiated experiments to determine if and how metformin might be a promising radioprotector from cosmic radiations and compared this to our previous and still lead countermeasure, CDDO. While effects of space radiation on the development of advanced cancer may not be an acute problem during an astronauts time in space, this remains speculative and additional research is required to develop methods to reduce either short or long-term effects of space radiation going forward. Thus, we initiated experiments with metformin an approved drug that is used by millions of Americans for controlling type 2 diabetes mellitus.

Metformin is a biguanide compound and shows very low cytotoxic effects and has been FDA (Food & Drug Administration) approved over 25 years. Metformin has been reported to reduce oxidative stress and DNA damage in vitro as well as in vivo, decreasing chronic inflammation. Metformin acts mainly through the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), which has pleiotropic effects downstream on cell metabolism. Furthermore, metformin targets mitochondria, inhibiting complex I of the electron transport chain (ETC), but the mechanisms underlying this process have not been completely elucidated. Because of its antioxidant effects, we investigated the role of metformin as a radioprotective compound. One single dose of metformin (0.5-1 mM) on human colon epithelial cells (HCECs) and human skin fibroblasts (BJs), showed an increase in the expression of AMPK alpha subunit phosphorylation and superoxide dismutase 1 (SOD1), during the first 72 hours. Interestingly, SOD1 has a crucial role protecting against oxidative DNA damage. Furthermore, a decrease of basal DNA damage (phosphorylation of H2AX at Ser 139 foci) and reactive oxygen species (ROS) production was observed. Moreover, metformin treatment enhances DNA damage responses (DDR) 24 hours after exposure of 2 Gy of γ -rays. To evaluate the radioprotective effect of metformin, cells were treated one time and irradiated 72 hours later, with 2, 4, and 6 Gy doses of gamma-rays. Cells were seeded at low density (200-1000 cells) and a colony formation assay was analyzed after 21 days. Metformin showed an increase in the surviving fraction of cells compared to the irradiated controls. Next, we investigated the radioprotective effect of metformin in vivo. Wild type 129/Sv mice were injected once per day with metformin 200 mg/kg, for three consecutive days prior exposure of 7.5 Gy of X-rays and sacrificed after 24 hours. Metformin pre-treatment was able to dramatically decrease DNA damage (p53 binding protein 1 foci) in mouse lung and colon tissues as well as the number of micronuclei in bone marrow cells, compared to the irradiated controls. Notably, when mice were irradiated at the dose of 10 Gy X-rays post-metformin treatment, an increase of 30% in the surviving fraction was observed. Currently, we are investigating the long-term effects of metformin on wild type mice after exposure to 2 Gy of x-rays. In summary, metformin might act as a radioprotector from GCR, potentially lowering the risk of cancer initiation or promotion in astronauts. In the future we will directly compare both metformin and CDDO in a high throughput screen of a panel of FDA approved drugs.

The overarching hypothesis for this project is that space radiation induces molecular manifestations of a pro-inflammatory response as a function of radiation type, radiation doses, doses rates, LET value, and time. We are testing if an oral available anti-oxidant and anti-inflammatory countermeasure, already in human clinical trials with a good safety profile, CDDO, significantly reduces proton and HZE-ion exposure associated tumor initiation and progression. Based on experiments conducted at the NASA Space Radiation Laboratory (Brookhaven, NY) we demonstrate that HZE ion components of GCR (galactic cosmic radiation) result in persistent DNA damage and inflammatory signaling, increased mutations in tumor suppressor genes, and higher rates of cancer initiation and progression compared to that seen with similar doses of terrestrial radiation. While physical shielding may reduce some of the risks of space radiation, there is substantial evidence that biological countermeasures will be required to ensure that the established safety limits of increased lifetime fatal cancer risks are not exceeded. We are conducting GCR simulations consisting of fast switching between protons, helium, and silicon using a dose rate of 0.5 cGy/min and a total combined dose of between 27-30 cGy to more closely mimic the space environment on a trip to Mars and back. Finally, we are conducting experiments with the official NASA GCRsim with acute and protracted mixed fields.

References

Kim, S.B., Bozeman, R.G., Kaisani, A., Kim, W., Zhang, L., Richardson, J.A., Wright, W.E., and Shay, J.W. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. Oncogene. 2015. https://doi.org/10.1038/onc.2015.395

Norbury, J.W., Schimmerling, W., Slaba, T.C., Edouard Azzam, Francis F. Badavi, Giorgio Baiocco, Eric Benton, Veronica Bindi, Eleanor A. Blakely, Steve R. Blattnig, David A. Boothman, Thomas B. Borak, Richard A. Britten, Stan Curtis, Michael Dingfelder, Marco Durante, William Dynan, Amelia Eisch, S. Robin Elgart, Dudley T. Goodhead, Peter M. Guida, Lawrence H. Heilbronn, Christine E. Hellweg, Janice L. Huff, Amy Kronenberg, Chiara La Tessa, Derek Lowenstein, Jack Miller, Taksahi Morita, Livio Narici, Gregory A. Nelson, Ryan B. Norman, Takeo Ohnishi, Andrea Ottolenghi, Zarana S. Patel, Guenther Reitz, Adam Rusek, Ann-Sofie Schreurs, Lisa A. Scott-Carnell, Edward Semones, Jerry W. Shay, Vyacheslav A. Shurshakov, Lembit Sihver, Lisa C. Simonsen, Michael Story, Mitchell S. Turker, Yukio Uchihori, Jacqueline Williams, Cary J. Zeitlin. Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research 8:38-51, 2016. PMID: 26948012

Lutiel, K. Bozeman, R., Kaisani, A. Kim, S.B., Barron, S., Richardson, J.A., Shay, J.W. Proton radiation-induced cancer progression. Life Sciences in Space Research, 2018. https://doi.org/10.1016/j.lssr.2018.08.002

Luitel, K., Kim, S.B., Barron, S. Richardson, J.A. and Shay, J.W. Lung cancer progression using fast switching multiple ion beam irradiation and countermeasure prevention, Life Sciences in Space Research, 2019. DOI: 10.1016/j.lssr.2019.07.011

During the current reporting period, using the fast beam switching technology developed in the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), we used mixed ions with different energies to simulate the space environment. Previously, we exposed a lung cancer susceptible mouse model (K-rasLA-1) at the NSRL with three ion beams: Protons (H) (120 MeV/n) 20cGy, Helium (He) (250 MeV/n) 5 cGy, and Silicon (Si) (300MeV/n) 5cGy with a dose rate of 0.5 cGy/min. In our recent studies we observed increases in the incidence of lung cancer initiation and progression. Additionally, when we titrated the dose of HZE ion, we observed a dose-dependent effect of silicon ions delivered. Overall, we observed reducing the total dose of silicon from 5 cGy to 2 cGy and 0.5 cGy in combination with 20 cGy protons and 5 cGy of helium, reduced cancer progression back to background rates.

With the high energy and control upgrades at the NSRL, experiments are now being conducted to better simulate the deep space environment with low fluence rates predominated by low background fluences of low-LET radiation with lower fluences of high-LET radiation. These experiments consist of chronic exposure from 2-6 weeks irradiation (6-days per week) or acute one-day exposures with continuous exposure to background protons and helium and a sporadic heavy ion exposure. The delivery dose consists of 33 ions and energy mix to even more closely approximate the deep space environment. During NSRL18B and NSRL19A an acute 0.5 Gy and 0.75 Gy total exposure to the newly developed GCR simulation was initiated and first results from this acute exposure GCR simulation are currently being analyzed. During NSRL18C a chronic/protracted (4 week exposure) 0.5 Gy total exposure was performed to better simulate the low dose rates expected in the deep space environment, and results from these experiments are being finalized and results will be forthcoming.

We are also currently (NSRL19C) irradiating mice with a chronic/protracted (6 weeks exposure) 0.75 Gy total exposure. These results will be compared to the acute 0.75 Gy experimental results of NSRl19A. While we will obtain some initial results at 100 day post IR, we need to retain the mice for one year post IR to determine if there is an increase in more advanced (invasive) cancer associated with the GCR simulations.

We continue our experiments on medical countermeasures that may reduce effects of GCR simulations associated cancers. For long-term space missions to the Moon or Mars, it might be necessary to adopt radiological countermeasures for astronauts. Thanks to the NASA Space Radiation Laboratory (NSRL) at Department of Energy's Brookhaven National Laboratory, it is now possible to simulate galactic cosmic radiation (GCR) that occurs in space and to determine the effects on biological models. We have initiated new experiments to determine if and how metformin might be a promising radioprotector from cosmic radiations. While effects of space radiation on the development of advanced cancer may not be an acute problem during an astronauts time in space, this remains highly speculative and additional research is required to develop methods to reduce either short or long-term effects going forward. Thus, we initiated experiments with metformin, an approved drug that is used by millions of Americans for controlling type 2 diabetes.

By way of background, metformin is a biguanide compound used in the treatment of type 2 diabetes mellitus, showing very low cytotoxic effects and that was FDA (Food & Drug Administration) approved in the 1990s. Metformin has been reported to decrease oxidative stress and DNA damage in vitro as well as in vivo, also decreasing chronic inflammation. Metformin acts mainly through the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), which has pleiotropic effects downstream on cell metabolism. Furthermore, metformin is able to target mitochondria, inhibiting the complex I of the electron transport chain (ETC), but the mechanisms underlying this process have not been completely elucidated. Because of its antioxidant effects, we investigated the role of metformin as a radioprotective compound. One single dose of metformin (0.5-1 mM) on human colon epithelial cells (HCECs) and human skin fibroblasts (BJs), showed an increase in the expression of AMPK alpha subunit phosphorylation and superoxide dismutase 1 (SOD1), during the first 72 hours. Interestingly, SOD1 has a crucial role protecting against oxidative DNA damage and its overexpression is associated with radioresistance in human glioma cells. Furthermore, a decrease of the basal DNA damage (phosphorylation of H2AX at Ser 139 foci) and in reactive oxygen species (ROS) production was observed. Moreover, metformin treatment enhanced DNA damage responses (DDR) 24 hours after exposure of 2Gy of gamma-rays. To evaluate the radioprotective effect of metformin, cells were treated one time and irradiated 72 hours later, with 2, 4, and 6 Gy doses of gamma-rays. Cells were seeded at low density (200-1000 cells) and a colony formation assay was analyzed after 21 days. Metformin showed an increase in the surviving fraction of cells compared to the irradiated controls. Next, we investigated the radioprotective effect of metformin in vivo. Wild type 129/Sv mice were injected once per day with metformin 200 mg/kg, for three consecutive days prior exposure of 7.5 Gy of X-rays and sacrificed after 24 hours. Metformin pre-treatment was able to dramatically decrease DNA damage (p53 binding protein 1 foci) in mouse lung and colon tissues as well as the number of micronuclei in bone marrow cells, compared to the irradiated controls. Notably, when mice were irradiated at the dose of 10 Gy X-rays post-metformin treatment, an increase of 30% in the surviving fraction was observed over non protected mice. In summary, our preliminary data can be interpreted to suggest that metformin might act as a radioprotector from GCR, potentially lowering the risk of cancer initiation or promotion in astronauts.

The overarching hypothesis for this project is that space radiation induces molecular manifestations of a pro-inflammatory response as a function of radiation type, radiation doses, doses rates, LET value, and time. We are testing if an oral available anti-oxidant and anti-inflammatory countermeasure, already in human clinical trials with a good safety profile, CDDO, significantly reduces proton and HZE-ion exposure associated tumor initiation and progression. Based on experiments conducted at the NASA Space Radiation Laboratory (Brookhaven, NY) we demonstrate that HZE ion components of GCR (galactic cosmic radiation) result in persistent DNA damage and inflammatory signaling, increased mutations in tumor suppressor genes, and higher rates of cancer initiation and progression compared to that seen with similar doses of terrestrial radiation. While physical shielding may reduce some of the risks of space radiation, there is substantial evidence that biological countermeasures will be required to ensure that the established safety limits of increased lifetime fatal cancer risks are not exceeded. We are conducting GCR simulations consisting of fast switching between protons, helium, and silicon using a dose rate of 0.5 cGy/min and a total combined dose of between 27-30 cGy to more closely mimic the space environment on a trip to Mars and back. Finally, we are conducting experiments with the official NASA GCRsim with acute and protracted mixed fields.

References

Kim, S.B., Bozeman, R.G., Kaisani, A., Kim, W., Zhang, L., Richardson, J.A., Wright, W.E., and Shay, J.W. Radiation promotes colorectal cancer initiation and progression by inducing senescence-associated inflammatory responses. Oncogene DOI: 10.1038/onc2015.395, 2015.

Norbury, J.W., Schimmerling, W., Slaba, T.C., Edouard Azzam, Francis F. Badavi, Giorgio Baiocco, Eric Benton, Veronica Bindi, Eleanor A. Blakely, Steve R. Blattnig, David A. Boothman, Thomas B. Borak, Richard A. Britten, Stan Curtis, Michael Dingfelder, Marco Durante, William Dynan, Amelia Eisch, S. Robin Elgart, Dudley T. Goodhead, Peter M. Guida, Lawrence H. Heilbronn, Christine E. Hellweg, Janice L. Huff, Amy Kronenberg, Chiara La Tessa, Derek Lowenstein, Jack Miller, Taksahi Morita, Livio Narici, Gregory A. Nelson, Ryan B. Norman, Takeo Ohnishi, Andrea Ottolenghi, Zarana S. Patel, Guenther Reitz, Adam Rusek, Ann-Sofie Schreurs, Lisa A. Scott-Carnell, Edward Semones, Jerry W. Shay, Vyacheslav A. Shurshakov, Lembit Sihver, Lisa C. Simonsen, Michael Story, Mitchell S. Turker, Yukio Uchihori, Jacqueline Williams, Cary J. Zeitlin. Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research 8:38-51, 2016. PMID: 26948012

Lutiel, K. Bozeman, R., Kaisani, A. Kim, S.B., Barron, S., Richardson, J.A., Shay, J.W. Proton radiation-induced cancer progression. Life Sciences in Space Research, in press, 2018. https://doi.org/10.1016/j.lssr.2018.08.002

• Wild type (WT), n=30 and lung cancer susceptible mice (LA1 mutant) on a 129 background (n=83), mixed male and females, 8-12 weeks old

• 18 WT mice received 500 mGy GCRsim, 12 unirradiated

• 63 lung cancer susceptible mice received 500mGy GCRsim, 20 unirradiated

• Acute experiments, ~2 hrs, 0.5 cGy/min

• First end points: 100 days +/- mitigator (CDDO, initiated two weeks post-irradiation for 30 days)

• 100-day assays: weight, inflammation, oxyblot (DNP) oxidized proteins, 8-oxo-dG for number of DNA lesions, plasma lipid peroxidation (MDA assay), initial histopathology with tumor size and number

• 365-day assays (to be completed): tumor grade, median, and overall survival

In summary, the testing of acute and chronic exposures to a GCR simulation should determine if increases in more lethal cancer risks observed in acute GCR exposure are increased, decreased, or unchanged when similar overall doses are provided with low dose rates. This should determine if countermeasures will be required for astronauts to travel to Mars and back safely.

The late biological effects from mixtures of the ions present in Galactic cosmic rays (GCR) such as protons, alpha particles, and high charge (Z) and energy E (HZE) particles are poorly understood, and at the present time astronaut’s exposure to such radiation during exploration class missions represents an unacceptable level of cancer risk. Most of the work on understanding space radiation-induced cancer progression has been performed using mono-energetic single-ion beams. However, the space radiation environment consists of a wide variety of ion species with various ranges of energies. Using the fast beam switching technology recently developed in the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), we can rapidly switch ion species allowing us to use mixed ions with different energies to simulate the actual radiation environment found in space. During the past year we exposed the lung cancer susceptible mouse model (K-rasLA-1) at the NSRL with three ion beams: Proton (H) (120 MeV/n) 20 cGy, Helium (He) (250 MeV/n) 5 cGy, and Silicon (Si) (300 MeV/n) 5 cGy with a dose rate of 0.5 cGy/min. Using three ion beams we performed whole body irradiations with a total dose of 30 cGy in two different orders: 3B-1 (H›He›Si) and 3B-2 (Si›He›H) and used 30 cGy H as a reference. In this study using the K-rasLA-1 mouse model, we show that whole-body irradiation with 3B-1 increases the incidence of cancer initiation and systemic oxidative stress in mice 100 days post-irradiation compared to 3B-2 and H only irradiation. Additionally, we observed an increase in adenomas with atypia and adenocarcinomas in 3B-1 irradiated mice but not in 3B-2 and H only irradiated mice. We also found that a non-toxic anti-inflammatory, anti-oxidative radioprotector (CDDO) provided 3 days prior to irradiation and one day post-irradiation reduced 3B-1 induced oxidative stress and cancer initiation almost back to baseline 100 days post irradiation. Thus, exposure to 3B-1 elicits significant changes in colon and lung cancer initiation that can be mitigated using CDDO.

Although biological mechanisms of normal tissue radiation injury are not completely understood, the roles of specific pathways in some cell types are becoming elucidated. While cell death is generally believed to be one the main causes of tissue injury from exposure to higher doses of low and high LET radiation, the dose and dose rates likely to be encountered by an astronaut on long-term missions into deep space are unlikely to cause massive cell death. Pathological manifestations after low-dose space radiation should be strongly influenced by non-cytotoxic radiation effects, resulting in incremental small changes in cell function, immune (micro-environmental) altered responses, and changes in metabolism. To more fully understand the tissue effects of exposure to space radiation compared to background cancer on Earth, it will require a more integrated "omics” and biological end point analysis as is proposed in this focused proposal using mouse models to help form the basis of a new description of radiation quality effects and cancer risk. Our published data (Clin Cancer Research, 2014) led us to the hypothesis that protracted/fractionated high LET irradiation can have long-term effects by changing the microenvironment in tissues leading to a pro-inflammatory cancer progressing phenotype. Importantly, the microarray signatures in these published studies on the K-ras lung cancer susceptible mouse model of lung cancer were shown to be applicable to overall survival in humans with lung and breast cancer. Thus, the studies proposed are likely to be applicable to human risks. In the current proposal we will test this hypothesis rigorously with normal mice, mice susceptible to lung cancer (LA1-Kras), and a colon cancer susceptible mouse model (CPC:APC) by incorporating the countermeasure arm in already approved studies. We have already established dose responses for tumor incidence in the K-ras and CPC;APC mouse. We will conduct experiments with these mouse models of cancer susceptibility and WT mice using a GCR (galactic cosmic ray) simulation that involve fast switching of three ion Si, He, and protons using low doses (total 30cGy) and dose rates (0.5 cGy/min). We will also test if an oral deliverable countermeasure, CDDO (with a known mechanism of action) using mission relevant irradiation doses can significantly decrease tumor incidence. As another approach we will compare CDDO to aspirin during the Fall 2017 NASA Space Radiation Laboratory (NSRL) run. We will focus on acute (24-48 hrs) as well as intermediate/persistent effects (14-100 days post-IR). A subset of mice will be maintained post-IR ~150-250 days to track tumor formation. We will conduct tissue micro-dissections and “omics” analyses of normal tissues, precancerous lesions, malignant lesions, and cleared margins surrounding the precancerous lesions in mice with and without being provided the medical radioprotector, CDDO, or aspirin. We propose that using a variety of radiation qualities and biological models, we will be able to dissect the important difference between space radiation and terrestrial radiation. This will lead to improved risk quantification and development of new systems biology risk modeling approaches that can be extrapolated to human cancer risks.

Reference

Delgado O, Batten KG, Richardson JA, Xie XJ, Gazdar AF, Kaisani AA, Girard L, Behrens C, Suraokar M, Fasciani G, Wright WE, Story MD, Wistuba II, Minna JD, Shay JW. Radiation-enhanced lung cancer progression in a transgenic mouse model of lung cancer is predictive of outcomes in human lung and breast cancer. Clin Cancer Res. 2014 Mar 15;20(6):1610-22.

Contributions to Fundamental Research: This project contributes to NASA's mission by the discovery of novel tissue biomarkers resulting from space radiation effects that are different from terrestrial radiation of equivalent doses. These biomarkers should contribute to our understanding of pathways underlying radiation-associated increases in invasive (more lethal) cancers. A better understanding of these pathways should enable the development of effective countermeasures.

Contributions to Human Health: Successful completion of this project will contribute to the development of models that could predict increased risk of more lethal cancers and potentially to approaches that may mitigate the increased risk if they are higher than expected. If novel targets are discovered and shown to have biological targets that can protect tissues from radiation-induced side effects, there is likely to be interest among other academic groups and pharmaceutical companies to develop new drugs directed at those targets. Overall, this project takes an innovative approach to an important unmet medical need.

Progress:

In the LA-1 lung cancer susceptible mouse model we can make the following conclusions:

• 50cGy simulated solar particle event (sSPE) decreases the lifespan of LA-1 animals and results in a significant increase incidence of carcinoma.

• 30cGy single dose exposure to protons (0.5cGy/min) does not increase the number of initiating lesions in the LA-1 animals 100 days post IR but overall incidence of carcinoma and survival are ongoing.

• A GCR simulation of 20cGy protons, 5cGy Helium, and 5cGy Silicon (using fast switching) results in an increase in the number of initiating lesions in the LA-1 animals 100 days post IR. Incidence of carcinomas and overall survival are ongoing.

• LA-1 mice on a CDDO diet during solar particle irradiation simulations exhibit a decreased incidence in invasive carcinoma in comparison to mice on control diet.

• There are no hematological or other tissue toxicities even when mice were kept on CDDO for long periods of time (>100days)

In the colon cancer susceptible (CPC;APC) and (WT) mouse model experiments we can make the following conclusions:

• SPE simulation (sSPE) increases colon cancer initiation and progression in CPC;APC mice; • sSPE induces prolonged DNA damage and an increase in p53 mutations; • sSPE enhances senescence associated inflammation that is persistent; • CDDO provided 3 days prior to radiation exposure protects mice from sSPE induced colon cancer initiation and progression; • 5cGy (300 MeV/n) of a single dose exposure to Silicon does not increase cancer initiation or overall survival but 10cGy Silicon results in a >2-fold cancer initiation and an overall decrease in lifespan.

Based on these findings, our initial GCR simulations in WT, lung, and colon susceptible mice were conducted with 5cGy and now 2cGy of Silicon. We conducted GCR simulations consisting of protons, helium, and silicon using a dose rate of 0.5cGy/min. We used 20cGy of protons (120 MeV/n), 5cGy of helium (250 MeV/n) and 5cGy of silicon (300 MeV/n) in the initial series of experiments. Two GCR simulations were conducted to determine if the order of sequential beams influence biological outcomes. Preliminary results suggest order of ions may be important.

1) GCRsim1 = Protons, then Helium, then Silicon (total dose 30cGy); 2) GCRsim 2 = Silicon, then Protons, then Helium. (total dose 30cGy); 3) Protons alone (total dose 30cGy)

We observed GCRsim1 result in an increase in initiating lesions but not GCRsim2. Based on our early results we have a working model to test the reason why order of sequential beam may have biological consequences. In addition, we have irradiated a cohort of mice with an even lower dose of 27cGy reducing Silicon from 5cGy to 2cGy.

Overall these experiments are designed to test the hypothesis that GCR simulations even at very low doses and dose rates may increase carcinogenesis as tested in WT and cancer susceptible mice and that a biological countermeasure will reduce the incidence of development of more lethal cancers. If we observe an increase in tumor formation with GCR simulations, we will conduct RNA transcriptomics and DNA sequencing and a variety of other molecular studies to determine if cancer susceptible and wild type mice have molecular changes that may indicate an increased risk of cancer. These studies should identify potential targets for biological countermeasures.

Although biological mechanisms of normal tissue radiation injury are not completely understood, the roles of specific pathways in some cell types are becoming elucidated. While cell death is generally believed to be one the main causes of tissue injury from exposure to higher doses of low and high LET radiation, the dose and dose rates likely to be encountered by an astronaut on long-term missions into deep space are unlikely to cause massive cell death. Pathological manifestations after low-dose space radiation should be strongly influenced by non-cytotoxic radiation effects, resulting in incremental small changes in cell function, immune (micro-environmental) altered responses, and changes in metabolism. To more fully understand the tissue effects of exposure to space radiation compared to background cancer on Earth, it will require a more integrated "omics” and biological end point analysis as is proposed in this focused proposal using mouse models to help form the basis of a new description of radiation quality effects and cancer risk. Our published data (Clin Cancer Research, 2014) led us to the hypothesis that protracted/fractionated high LET irradiation can have long-term effects by changing the microenvironment in tissues leading to a pro-inflammatory cancer progressing phenotype. Importantly, the microarray signatures in these published studies on the K-ras lung cancer susceptible mouse model of lung cancer were shown to be applicable to overall survival in humans with lung and breast cancer. Thus, the studies proposed are likely to be applicable to human risks. In the current proposal we will test this hypothesis rigorously with normal mice, mice susceptible to lung cancer (LA1-Kras), and a colon cancer susceptible mouse model (CPC:APC) by incorporating the countermeasure arm in already approved studies. We have already established dose responses for tumor incidence in the K-ras and CPC;APC mouse. We will conduct experiments with these mouse models of cancer susceptibility and WT mice using a GCR (galactic cosmic ray) simulation that involve fast switching of three ion Si, He, and protons using low doses (total 30cGy) and dose rates (0.5 cGy/min). We will also test if an oral deliverable countermeasure, CDDO (with a known mechanism of action) using mission relevant irradiation doses can significantly decrease tumor incidence. We will focus on intermediate/persistent effects (14-100 days post-IR) including some long-term effects (~150-250 days). We will conduct tissue micro-dissections and “omics” analyses of normal tissues, precancerous lesions, malignant lesions, and cleared margins surrounding the precancerous lesions in mice with and without being provided the medical radioprotector, CDDO. We propose that using a variety of radiation qualities and biological models, we will be able to dissect the important difference between space radiation and terrestrial radiation. This will lead to improved risk quantification and development of new systems biology risk modeling approaches that can be extrapolated to human cancer risks.

Radioprotector Previous Studies: We have previous demonstrated that CDDO is also a potent radioprotector in vitro and in vivo with a known molecular mechanism of action. CDDO activates Nrf2, a key transcription factor that when translocated to the nucleus binds to anti-oxidant response elements increasing cytoprotective and DNA repair kinetics. Using wild type mice we observed CDDO provided in lab chow prior to a lethal dose of whole-body irradiation protected mice from acute gastrointestinal toxicity with enhanced DNA damage repair resulting in improved overall survival. Using lung (LA-1) and colon cancer (CPC;Apc) susceptible mouse models, we examined the effects of providing CDDO for up to 100 days on the spontaneous initiation and progression of tumorigenesis. While the spontaneous rate of premalignant (hyperplasias, adenomas) lesions is 100% in these mice, the LA-1 model develops 9-10% invasive cancers while the CPC;Apc mouse model develops 6-8% invasive cancers. We demonstrated CDDO dramatically prevented the development of spontaneous invasive cancers in un-irradiated cancer susceptible mice. When these mice are exposed to x-rays, protons, or GCR ions, the spontaneously rate of invasive cancer increases 2-4 fold depending on ion, doses, and dose rates used. We next tested if the LA-1 and CPC;Apc mice fed CDDO diet only 2-3 days prior to x-rays or protons provided as a single dose or as a solar particle event simulation would lead to a lower incidence of invasive carcinomas. The results were that CDDO provided as a biological countermeasure prior to irradiation reduced the percent of invasive cancers 2-3 fold. Similar results were observed when mice were irradiated with GCR ions. These results document that exposure to space radiation increases the risk of invasive cancers in cancer susceptible mouse models, and that radioprotectors such as CDDO may reduce the overall risk of fatal cancers without affecting normal cells.

Simulated Solar Particle Events (SPE) Promotes Senescence-Associated Inflammatory Responses in Colorectal Cancer Susceptible Mouse Model: While protons and high charge and energy (HZE) particles are considered to be major risk factors for humans during space missions, the mechanism underlying the biological effects of protons and HZE particles still remain to be more fully characterized. In our recent studies we simulated solar particle events (SPE) at the Brookhaven NASA Space Radiation Laboratory to characterize the biological effects of low dose rate protons in vivo. Using the colorectal cancer susceptible (CPC;Apc) mouse model, we studied colonic tumorigenesis after whole-body exposure to a simulated SPE with varying energy (50-150 MeV/n) using a total dose of 2 Gy over a 2 hour period (at an average dose rate of 1.67 cGy/min). We also exposed mice to 2 Gy of acute (50 MeV/n) proton or X-ray (250 kVp, 1mA, 1.65 mm Al filter) at a dose rate of 20 cGy/min as a reference radiation. We observed that whole-body irradiation with simulated SPE is more effective in inducing invasive adenocarcinoma incidence (4-fold increase compared to un-irradiated controls) followed by induced senescence-associated inflammatory responses (SIR), which are involved in colon cancer initiation and progression. After irradiation to SPE simulation, a subset of SIR genes (Troy, Sox17, Opg, Faim2, Lpo, Tlr2, and Ptges) and a gene known to be involved in invasiveness (Plat), along with the senescence-associated gene (P19Arf) are markedly increased. Following these changes, p53 mutations are increased compared with the same doses of acute proton or x-ray irradiation. Pretreatment with the oral available countermeasure, CDDO reduced SPE-associated SIR gene expression and tumorigenesis. Thus, exposure to SPE irradiation elicits significant changes in colorectal cancer initiation and progression that can be protected by CDDO-EA pretreatment.

Investigating Lung Cancer Risk to Solar Particle Event (SPE) Simulations: SPEs are comprised of varying energies and doses over a period of time (protracted dose of radiation), and occurrences are difficult to predict. On a mission to Mars and back it is predicted that up to 7 SPEs will occur and while shielding may partially protect astronauts, it cannot block all irradiation exposures. It is predicted that SPEs have high carcinogenic effects compared to equivalent low energy terrestrial radiation (e.g., X-rays). However, data are still required to determine more exactly the increased risk of invasive cancer with low dose rates and varying energies of proton. During our studies, we used the K-rasLA1 mouse model which mimics the human adenocarcinoma non-small cell lung cancer progression by spontaneous activation of mutant K-ras lesions. Using K-rasLA1, we studied survival and the progression of lung cancer after total body exposure to a simulated SPE with varying energies (50 – 150 MeV/n) using a total dose 0.5 Gy, 1.0 Gy, and 2.0 Gy (at an average dose rate of 1.67 cGy/min). We also exposed mice to 2 Gy of monoenergetic (50 MeV/n) proton or X-ray (250 kVp) at a dose rate of 20 cGy/min as a reference radiation exposure. The SPEs simulation, and monoenergetic proton radiation resulted in increases in invasive carcinoma as compared to the X-rays. K-rasLA1 mice exposed to 2.0 Gy of sSPE radiation and 2.0 Gy of monoenergetic acute proton (50 MeV/n or 150 MeV/n) exhibited a significant decrease in median survival compared to un-irradiated control cancer susceptible mice. We also observed there was significant increase in the average number of tumor lesions in SPEs simulated animals as compared to monoenergetic proton radiation and X-rays. To evaluate the underlying mechanistic details involved in radiation-mediated tumorigenesis in our lung cancer model, the phosphorylation status (activation) of various targets important to the process of tumorigenesis were investigated. We found K-rasLA1 mice exposed to energetic protons exhibited altered growth factor signaling compared to un-irradiated controls. We are testing if alterations in growth factors are due to the chronic oxidative stress caused by the SPEs leading to increase in invasive cancer. Further studies are in progress to understand the SPEs biological effect including DNA sequencing of candidate genes such as p53 (which we found was increased in the colon cancer susceptible mouse model). Significant biological and mechanistically data obtained from these studies may help in risk assessment of space travel and provide insights into molecular mechanisms which could be applicable in mitigating or preventing cancer initiation and progress during long-duration space travel.

Ongoing Experiments and Future Directions: Most accelerator-based space radiation experiments have been performed with single ion beams at fixed energies. However, the space radiation environment consists of a wide variety of ion species with a continuous range of energies. Due to recent developments in fast beam switching technology implemented at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), it is now possible to rapidly switch ion species and energies, allowing for the possibility to more realistically simulate the actual radiation environment found in space. We were recently approved to conduct galactic cosmic ray (GCR) simulations at NSRL, to determine if there is an increase in cancer initiation or progression following ~30cGy total exposure of three sequential beams that are based on discussions with John Norbury and the Virtual Systems Biology – Cancer Risk working group over the last year.

On a trip to Mars and back, every human cell will be traversed by a proton and most by helium and very rarely by a HZE particle such as silicon. We cannot mimic the dose protraction that occur in deep space and we recognize that there are issues of scaling from a mouse to human. However, we have already initiated our first experiments at the NSRL 16C run. We are using a simplified GCR simulation consisting of protons, helium, and silicon using a dose rate of 0.5cGy/min (to keep within the beam time approved). We will use 20cGy of protons (120 MeV/n), 5cGy of helium (250 MeV/n), and 5cGy of silicon (300 MeV/n). We have run over 200 mice with two main variables in 16C: 1) protons, then helium, then silicon (plus or minus CDDO) and 2) silicon then protons, then helium. The first group of mice were irradiated without any problems at the NSRL. These experiments are to test the hypothesis that GCR simulations even at very low doses and dose rates may increase carcinogenesis in cancer susceptible mice and that a biological countermeasure will reduce the increases in more lethal cancers. We will also conduct molecular analyses on these and wild type mice exposed to GCR simulations at various times points. If we observe an increase in tumor formation with GCR simulations, we will conduct whole genome sequencing and a variety of other molecular studies to determine and further understand the molecular mechanism involved. We will determine if cancer susceptible and wild type mice have molecular changes that may indicate an increased risk of cancer. Finally, to more closely mimic the space environments, we will reduce the dose fractions and repeat the GCR simulation using 3 sequential cycles in the Spring 17A NSRL run. The total dose will not exceed 30cGy only the dose in each cycle. We have been told we can conduct fast switching up to 10 times but that will require more beam time and are not justified at this stage. We will conduct some single and two ion experiments going forward if we observe an increase in carcinogenesis from the 16C run in order to dissect molecular mechanisms (e.g., silicon alone, or silicon plus helium, or silicon plus protons). We are also planning mitigation experiments in the future (e.g., provide CDDO after irradiation instead of prior to irradiation).

Although biological mechanisms of normal tissue radiation injury are not completely understood, the roles of specific pathways in some cell types are becoming elucidated. While cell death is generally believed to be one the main causes of tissue injury from exposure to higher doses of low and high LET radiation, the dose and dose rates likely to be encountered by an astronaut on long-term missions into deep space are unlikely to cause massive cell death. Pathological manifestations after low-dose space radiation should be strongly influenced by non-cytotoxic radiation effects, resulting in incremental small changes in cell function, immune (micro-environmental) altered responses, and changes in metabolism. To more fully understand the tissue effects of exposure to space radiation compared to background cancer on Earth, it will require a more integrated "omics” and biological end point analysis as is proposed in this focused proposal using age-appropriate mouse models to help form the basis of a new description of radiation quality effects and cancer risk. Our published data (Clin Cancer Research, 2014) led us to the hypothesis that protracted/fractionated high LET irradiation can have long-term effects by changing the microenvironment in tissues leading to a pro-inflammatory cancer progressing phenotype. Importantly, the microarray signatures in these published studies on the K-ras lung cancer susceptible mouse model of lung cancer were shown to be applicable to overall survival in humans with lung and breast cancer. Thus, the studies proposed are likely to be applicable to human risks. In the current proposal we will test this hypothesis rigorously with normal mice, inducible EGFR mutant mice susceptible to lung cancer and a colon cancer susceptible mouse model (CPC:APC) by incorporating the countermeasure arm in already approved studies. We have already established dose responses for tumor incidence in the K-ras and CPC;APC mouse models using Si, O, protons, and solar particle event (SPE) simulations and propose to demonstrate that oral deliverable CDDO (with a known mechanism of action) using mission relevant irradiation doses can significantly decrease tumor incidence (EGFR mutant mice without induction of mutant EGFR are essentially WT mice) or progression/invasiveness (doxycycline induction of mutant EGFR either before or after irradiation). We will focus on intermediate/persistent effects (14-100 days post-IR) including some long-term effects (~150-200 days). We will conduct tissue micro-dissections and “omics” analyses of normal tissues, precancerous lesions, malignant lesions, and cleared margins surrounding the precancerous lesions in mice with and without being provided the medical radioprotector, CDDO. We propose that using a variety of radiation qualities and biological models, we will be able to dissect the important difference between space radiation and terrestrial radiation. This will lead to improved risk quantification and development of new systems biology risk modeling approaches that can be extrapolated to human cancer risks.