STEM Today, November 2018, No. 38 by Abhishek Sinha

STEM TODAY November 2018, No. 38

CONTENTS Degen 7: Are there synergistic effects from other spaceflight factors (e.g. altered gravity microgravity), stress, altered immune function, altered circadian rhythms, or other) that modify space radiationinduced degenerative diseases in a clinically significant manner? Exposure to ionizing radiation is associated with an increased risk for development of heart disease, stroke, and other neurovascular and degenerative tissue diseases such as cataracts later in life or well after flight. It is currently unknown whether there are significant synergistic effects from other secondary spaceflight factors (altered gravity (μgravity), stress, immune status, bone loss, etc.) that may alter morbidity and mortality estimates for these late effects resulting from space radiation exposure. Activities todate have included retrospective data mining and flight and ground studies to identify the role of the risk factors outlined above on cardiovascular health.

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

STEM Today, November 2018, No. 38

Disclaimer ( Non-Commercial Research and Educational Use ) STEM Today is dedicated to STEM Education and Human Spaceflight. This newsletter is designed for Teachers and Students with interests in Human Spaceflight and learning about NASA’s Human Research Roadmap. The opinion expressed in this newsletter is the opinion based on fact or knowledge gathered from various research articles. The results or information included in this newsletter are from various research articles and appropriate credits are added. The citation of articles is included in Reference Section. The newsletter is not sold for a profit or included in another media or publication that is sold for a profit. Cover Page The International Space Station as of Oct. 4, 2018 iss056e201046 (Oct. 4, 2018) – The International Space Station photographed by Expedition 56 crew members from a Soyuz spacecraft after undocking. NASA astronauts Andrew Feustel and Ricky Arnold and Roscosmos cosmonaut Oleg Artemyev executed a fly around of the orbiting laboratory to take pictures of the station before returning home after spending 197 days in space. The station will celebrate the 20th anniversary of the launch of the first element Zarya in November 2018. Image Credit: NASA/Roscosmos

Back Cover The International Space Station as of Oct. 4, 2018 iss056e20132 (Oct. 4, 2018) – The International Space Station photographed by Expedition 56 crew members from a Soyuz spacecraft after undocking. NASA astronauts Andrew Feustel and Ricky Arnold and Roscosmos cosmonaut Oleg Artemyev executed a fly around of the orbiting laboratory to take pictures of the station before returning home after spending 197 days in space. The station will celebrate the 20th anniversary of the launch of the first element Zarya in November 2018. Image Credit: NASA/Roscosmos

STEM Today , November 2018

Editorial Dear Reader

STEM Today, November 2018, No. 38

All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers. According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future." STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts. Editor Mr. Abhishek Kumar Sinha

Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers. According to former President Barack Obama, " Science is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..." STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Road map. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter. Editor / Technical Advisor Mr. Martin Cabaniss

STEM Today, November 2018, No. 38

Space Radiation (SR) Degen - 7: Are there synergistic e ects from other space ight factors (e.g. altered gravity (Âľgravity), stress, altered immune function, altered circadian rhythms, or other) that modify space radiation-induced degenerative diseases in a clinically signi cant manner? Exposure to ionizing radiation is associated with an increased risk for development of heart disease, stroke, and other neurovascular and degenerative tissue diseases such as cataracts later in life or well after ight. It is currently unknown whether there are signi cant synergistic e ects from other secondary space ight factors (altered gravity (Âľ-gravity), stress, immune status, bone loss, etc.) that may alter morbidity and mortality estimates for these late e ects resulting from space radiation exposure.

Spaceflight related gene-expression changes in the whole blood of astronauts

Jennifer Barrila, C Mark Ott, Carly LeBlanc, Satish K Mehta, Aurelie Crabbe, Phillip Stafford, Duane L Pierson and Cheryl A Nickerson studied about spaceflight related gene-expression changes in the whole blood of astronauts and published their findings in the paper "Spaceflight modulates gene expression in the whole blood of astronauts". To evaluate the potential impact of the spaceflight environment on molecular pathways mediating cellular stress responses, authors performed a study to assess spaceflight-related changes in the expression of stress-response genes in the whole blood of astronauts in response to spaceflight. Transcriptional profiling of 234 well-characterized stressresponse genes was performed using total RNA isolated from whole blood obtained from six consenting astronauts 10 days before launch aboard the space shuttle and 2-3 h after return to Earth.

STEM Today, November 2018, No. 38

Four men between the ages of 38 and 47 (mean = 43 years) and two women between the ages of 38 and 44 (mean = 41 years) participated in the study. Each of these astronauts flew aboard one of four space shuttle missions ranging between 10 and 13 days duration that took place during a 2-year period. Previously published studies using blood and saliva samples from these same crew members before, during and just after spaceflight indicated that all six astronauts displayed increases in Epstein Barr virus reactivation both during and immediately post flight, which is consistent with these individuals being in a stressed state. Microarray analysis revealed some variation in the geneexpression patterns displayed across individual crew members. This is not surprising given the smaller study size due to the exceptionally rare opportunity for this type of sample collection, as well as other studies that have shown that there can be a wide range of individual and temporal variability in gene-expression patterns in human blood. High variability was also observed in gene-expression studies using astronaut hair follicles. Authors found six transcripts that displayed significant (P< 0.05) changes in expression in the crew in response to spaceflight when compared with pre-flight levels (Table 1). Two additional differentially regulated transcripts were identified following outlier removal.

No gender-specific differences or relationship to number of missions flown were observed. It is important to note that as for many human spaceflight studies, the small number of human subjects available for analysis is a limiting factor to the statistical power. False discovery rate algorithms determined no significant genes were expressed, likely due to these low study group numbers. Genes altered in expression encode proteins of known importance for DNA repair (XRCC1 and HHR23A), ox4

idative stress (GPX1), and chaperones which have key roles in protein folding and/or proteasomal degradation (HSP27 and HSP90AB1). The finding that genes encoding DNA-repair proteins are down regulated supports previous studies which showed post-flight increases in chromosomal aberrations in lymphocytes of astronauts and indications of increased DNA damage in astronauts after long-duration spaceflight. Kumari et al previously reported that exposure of human lymphocytes to simulated microgravity over the course of 7 days led to increased DNA damage.This damage was accompanied by progressive decreases in the expression of a number of representative DNA-repair genes, leading the authors to postulate that impaired DNA-repair capacity could lead to increased damage and mutations. Of particular interest was the down regulation of GPX1, which encodes for glutathione peroxidase (GPX1), an enzyme that protects cells from oxidative damage, modulates the immune response (including delayed type hypersensitivity/DTH), and has been associated with increased viral titers in herpes viral infections. Given that spaceflight depresses the DTH response, alters cellular oxidative functions, and increases herpes viral reactivation in astronauts on orbit.

STEM Today, November 2018, No. 38

The magnitude and direction of the expression/activity levels of specific redox enzymes (including GPX1)were variable across these studies (likely due to differences in experimental parameters such as the use of human versus animal subjects, mission duration, etc) the overall findings support the potential for increased oxidative stress and/or decreased antioxidant defense capacity in response to spaceflight. It is also interesting to note that GPX1 is a selenoprotein and decreased serum selenium levels have been reported in astronauts post landing as compared with pre-launch. Therefore, additional insight into GPX1 and other oxidative defense mechanisms may serve as a guiding principle for establishing nutritional requirements to ensure health safety and performance of the crew during human exploration of space. When authors compared these transcriptomic findings from blood samples to a previously published study using hair samples, they did not find similarities in the genes that were differentially regulated pre- and post flight, which could be due to a variety of experimental differences, including sample source (whole blood versus hair follicles) and mission duration (10-13 days in the present study compared with â&#x2C6;ź 6 months in the study by Terada et al). Several transcripts encoding stress response genes were suppressed in the crew after exposure to the microgravity environment, including those important for DNA repair, oxidative stress, detoxification, and protein folding/degradation. These processes are vital for maintaining human health by mediating cellular pathways that serve to protect against both environmental and physiological stressors and have been implicated in a broad spectrum of diseases. Since changes in gene expression in peripheral blood may be attributed to a number of factors, including changes in the distribution of blood cellular subsets (which has been detected post flight in the crew). This study provides an initial foundation into the molecular genetic response profiles of astronauts during spaceflight from which additional research into alterations in crew health and performance can be investigated.

NASA Twins Study Mark and Scott Kelly are identical twins; Scottâ&#x20AC;&#x2122;s DNA did not fundamentally change. What researchers did observe are changes in gene expression, which is how your body reacts to your environment. Researchers reported that 93% of Scottâ&#x20AC;&#x2122;s genes returned to normal after landing. However, the remaining 7% point to possible longer term changes in genes related to his immune system, DNA repair, bone formation networks, hypoxia, and hypercapnia. The change related to only 7 percent of the gene expression that changed during spaceflight that had not returned to pre-flight after six months on Earth. This change of gene expression is very minimal.

NASA Twins Study Confirms Preliminary Findings, NASA.

DNA damage response in space

Tao Lu, Ye Zhang, Yared Kidane, Alan Feiveson, Louis Stodieck, Fathi Karouia, Govindarajan Ramesh, Larry Rohde and Honglu Wu studied the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage and published their findings in paper "Cellular responses and gene expression profile changes due to bleomycin-induced DNA damage in human fibroblasts in space". Potential effects of microgravity on radiation-induced DNA damage repair have been investigated since the early days of human space program. Experiments aimed at addressing such effects were conducted either by exposing cells prior to the launch of samples into space, exposing samples in space, or exposing samples shortly after landing. During an early Germini-3 mission, human lymphocytes in culture were exposed to 32 P β-particles in space within one hour after reaching the microgravity condition. The cells were kept under the microgravity condition for approximately 4 hours before reentry started. Post-flight analysis of chromosome aberrations in the lymphocytes indicated an increased frequency of β-induced chromosomal deletions in the flight samples in comparison to the ground, whereas the frequencies of dicentrics and rings were similar.

STEM Today, November 2018, No. 38

However, the potential synergism between microgravity and radiation in chromosomal deletions was not confirmed in a subsequent Germini-XI flight experiment. Inflight exposure of yeast to 63 Ni β-particles was also performed onboard the Space Shuttle STS-82, with results showing no significant differences in both the induction and the repair of double strand breaks (DSB) between µg and the 1g conditions. In a number of flight experiments, DNA damages were induced by high doses of radiation pre-flight. The cells were then kept at low temperature after irradiation so that repair of the damages did not occur until they reached orbit. Investigating survival in S. cerevisiae following this scenario, Pross and colleagues reported possible reduced repair under microgravity in an experiment conducted onboard STS-42, but a repeated experiment conducted onboard STS-76 showed that repair of pre-induced DSB in yeast was not impaired by microgravity. A similar pre-flight exposure experiment using E. coli and human fibroblasts also found no differences between the flown and ground samples in the kinetics of DNA strand break rejoining. As part of a pre-post flight study, authors exposed blood samples collected from an astronaut to γ rays of a set of doses before flight and also within 24 hours after the 9-day STS-103 mission. Comparison of the dose response for total chromosomal exchanges between pre- and post-flight samples did not show an effect of spaceflight. Interestingly, however, Greco and colleagues reported an enhancement of approximately 1.2 - 2.8 fold in the chromosome aberration frequency in a post-flight astronaut blood sample compared to parallel pre-flight data after exposure to ground based X-rays. Early studies on the combined effects of microgravity and radiation have been reviewed by Horneck, and by Pross and Keifer, with conclusions that spaceflight does affect the development of organisms, but the majority of spaceflight experiments showed little effects of microgravity on the repair of radiation-induced DNA damage. The experiments have also been conducted using microgravity simulators on the ground, particularly the rotating wall vessels (RWV) for cultured cells in suspension, to explore synergisms between microgravity and radiation. In contrast to the flight studies, the majority of ground based experiments demonstrated compromised repair of radiation-induced DNA damages under the simulated microgravity condition. These synergistic effects have been observed in human lymphocytes and human fibroblasts as measured by their survival, and the induction of micronuclei and HPRT mutation, as well as repair kinetics of γ-H2AX signals. However, no effects of simulated microgravity on proton-induced chromosome aberrations in human lymphocytes were observed. Combined effects of radiation and simulated microgravity have also been investigated in rodents using the hindlimb suspension model. To find out whether the true microgravity condition affects DNA repair with high confidence requires a radiation source in space to induce high levels of DNA damages. Alternatively, high levels of DNA damages can be induced in space by radiomimetic chemicals. In this study, authors flew human fibroblasts (AG1522) to the International Space Station (ISS) and treated the cells with bleomycin in orbit for 3 hrs. The cells were then fixed for DNA damage analysis and for gene expressions. The cells were then fixed for DNA damage analysis and for quantifying expressions of genes. These 6

cells have been widely used in investigations of radiation damages, and were able to be synchronized in mostly the G1 phase upon confluence. In this study, authors intentionally induced damages in cells in G1 to minimize the effects of cell growth condition differences between ground and space. Bleomycin is a known chemotherapy drug that induces DNA damages including DSB, and has been used previously in spaceflight experiments. The present study is the first to investigate gene expressions in response to DNA damages induced intentionally in space.

STEM Today, November 2018, No. 38

On April 18, 2014, our experimental payload was launched from NASA Kennedy Space Center (KSC) in Florida. The cells were kept at 20◦ C until they reached the ISS. On April 22, the two habitats containing the cells were then transferred to another CGBA preset at 37◦ C . On April 25, the cells were removed from the CGBA incubator, and the BioCell cell culture chamber was injected with bleomycin at a final concentration of 1 µg/ml or placebo using the same amount of PBS. This concentration is equivalent to exposure of about 0.4 Gy γ-radiation, and resulted in a balanced distribution of three types of DNA damages in our ground experiment (Fig 2).

The cells were then transferred back to CGBA. After 3 hr incubation with bleomycin, a total of 8 BioCells containing bleomycin or placebo treated cells were removed from the CGBA, washed with PBS, and fixed with RNALater II at a final concentration of approximately 90%. In addition, one BioCell from each treatment group was washed with PBS and fixed with a final concentration of 2% paraformaldehyde (PFA) for 30 min at room temperature before being washed with PBS. Immediately after fixation, the samples were transferred to a 4◦ C refrigerator onboard the ISS. On May 18, 2014, the samples were returned to Earth. The samples were kept at 4◦ C until they arrived at NASA Johnson Space Center (JSC) in Houston, Texas. Total RNA was then isolated using miRNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The residual genomic DNA was removed using Qiagen RNase free DNase kit. Processing of the samples on the ISS was performed by an astronaut using a glove bag. The ground control experiment was performed in the same manner, but with a 6-hour offset from the flight schedule at KSC. Cells fixed with paraformaldehyde from spaceflight and ground experiments were rinsed with 1xPBS. Each cell culture plate was separated into 12 pieces and stored in methanol at -20◦ C. Results DNA damage quantification Fig 2B shows the percentages of different staining types for γ-H2AX in AG1522 cells after bleomycin treatment of 0.1, 1.0 and 10.0 µg/ml concentration on the ground. As the bleomycin concentration increased, the per7

STEM Today, November 2018, No. 38

centage of Type I and Type II cells also increased, indicating a correlation between these types of staining and the levels of DNA damage. The relative contribution of different types of Îł-H2AX stained cells after treatment of bleomycin of 1.0 Âľg/ml for 3 hr are depicted in Fig 3A for both the flight and ground samples. Actual counts and percentages are given in Table 2.

Note that all untreated samples exhibited only Type III staining. For bleomycin-treated samples, it can be seen

in Fig 3A that the distributions of damage types were very similar for ground and space. Even with the large number of cells in both of these groups (total of 2456, Table 2), there was no evidence of a spaceflight effect on the type of damage (p = 0.58, chi-squared test). As expected, treatment with bleomycin substantially increased the number of Type III foci; from a median count of 3 per cell (untreated, ground) to 28 (treated, ground), and from a median of 3 (untreated, space) to 31 (treated, space). Fig 3B shows the distribution of the number of foci in cell nuclei after bleomycin treatment for both ground and flight. There was evidence that overall, treated cells in space had more damage than their counterparts on the ground (p = 0.020; Mann-Whitney test), with the difference being mainly in the lower quantiles of the two count distributions (Table 3, Fig 3B). For untreated cells, there also appeared to be more damage in space than on the ground, but the difference was not significant (p = 0.125).

STEM Today, November 2018, No. 38

AG1522 cells in exponentially growing conditions and in confluence were also treated on the ground with 1 Âľg/ml bleomycin for 3 hrs, and the percentages of different Îł-H2AX staining types are shown in Fig 4A. With the same concentration of bleomycin, exponentially growing cells exhibited higher level of Type I staining (11.5%), in comparison to 4.3% Type I staining in confluent cells. However, the Type II stained cells were similar between the two cell growth conditions (14.2% and 14.4%). In Type III cells, the distribution of foci number was shifted in the exponentially growing cells, similar to the flight samples in the present study (Fig 4B). Microarray analysis For the FT_vs_FC contrast, 48 genes with known functions had expressions significantly altered as a consequence of bleomycin treatment in space. On the other hand, in ground cells treated with bleomycin (GT_vs_GC), 40 genes showed significant expression changes. Among these genes, 24 were common between both contrasts as shown in the Venn diagram (Fig 5A).

STEM Today, November 2018, No. 38

The heatmap of genes having significant expression changes in either the ground or the flight samples is shown in Fig 5B. Hierarchical clustering of the total of 64 significant genes based on expression values revealed that bleomycin treated samples (irrespective of flight or ground) are grouped together and they are clearly segregated from the control samples. All 64 genes and their fold changes from bleomycin treatment, are listed in Table 4. Despite this clear evidence that genes were responding to the damage caused by bleomycin, there was 10

STEM Today, November 2018, No. 38

no indication of any genes responding differently to bleomycin in flight as compared to ground samples (GTGC) vs. (FT-FC).

Pathway analyzes using IPA demonstrated that p53 was the primary pathway responding to the bleomycin treatment in both the flown and ground cells, as shown in Fig 6. In addition, TP73 and RELA were involved in both the flown and ground cells in response to bleomycin treatment. These pathways were among the top 5 significant upstream regulators (Table 5). E2F1 and CDKN2A were also potentially activated as upstream transcription factors. These two upstream regulators have been shown to involve in early responses to ultraviolet radiation-induced DNA damage by directly promoting DNA repair and activating p53 tumor suppressor. The IPA analysis also revealed that bleomycin produced similar pathway cascade responses as other genotoxins that induce DNA damage, such as hydrogen peroxide, cisplatin and mitomycin C (Data not shown). Canonical pathway analysis indicated that other DNA damage signaling pathways, such as ATM and the cell cycle checkpoint, also play a role under both gravity conditions (Fig 7). Although several pathways, such as CREB1, CTNNB1, and HES1, were recognized when the unique genes identified in either flight or ground (Table 4) were input into IPA, these pathways failed to reach any statistical significance. PCR array analysis of DNA damage signaling genes To validate the gene microarray data, PCR arrays were performed for a set of genes specifically involved in DNA damage signaling pathways. Of the 84 genes, BBC3, CDKN1A, PCNA and PPM1D were the only four genes having significant expression changes in both flight and ground samples after bleomycin treatment. The fold changes of expression values and p values of these DNA damage signaling genes are listed in Table 1. Comparison of the genes having significant expression changes in the microarray analysis is shown in Fig 8, indicating an agreement between the microarray and PCR array data. In this flight study, authors induced DNA damage intentionally on the ISS with bleomycin and investigated early responses by measuring the phosphorylation of histone protein H2AX for quantification of DNA damages, and by analyzing gene expressions using both the microarray and the PCR array methods. By classifying γ-H2AX staining patterns in different types, we found a clear increase in the percentage of cell nuclei with pan-nucleus staining (Type I) as the concentration of bleomycin increased (Fig 2). Comparison of different types of γ-H2AX staining patterns in cells after bleomycin treatment between flight and ground revealed a similar percentages of Type I (∼6%) and Type II (∼15%) cells, and the percentages were not significantly different (Tables 2). However, detailed counting of the number of foci in the countable γ-H2AX stained cells (Type III)indicated differences in the low quantiles of the distribution in cells after bleomycin treatment (Table 3, Fig 2B). In this study, the cells were fixed at one time point of 3 hours after bleomycin treatment, due primarily to the limitation of samples allowed in a spaceflight experiment. This is typically the time point optimal for investigations of gene expressions. With acute damage to cells by ionizing radiation, it is known that the γ-H2AX intensity would peak within one hour post irradiation, and decrease as the DNA damage is repaired. In this study, the cells were treated continuously with bleomycin, and the γ-H2AX signals reflected a mix of accumulated damage over the 3 hr period and the repair of some of the damages. Unlike ionizing radiation, γ-H2AX foci induced by toxic chemicals or UV may not necessarily represent DNA double strand breaks. These

STEM Today, November 2018, No. 38

cells in G1 phase of the cell cycle do not go through apoptosis.

In a previous publication, authors reported that AG1522 cells in space proliferated slightly faster. To determine whether cell growth condition was responsible for the Îł-H2AX foci count in Type I nuclei between flight and ground, we treated exponentially growing and confluent cells, on the ground, with bleomycin. Authors showed that levels of bleomycin-induced DNA damages varied with the cell growth condition in that the percentage of Type I Îł-H2AX stained cells were significantly higher in exponentially growing cells (Fig 4A). Differences in the foci number distribution was also observed between the cells in these two different growth

STEM Today, November 2018, No. 38

conditions. Although cells in space in our flight experiment were far from exponential growing conditions, cell growth may be partly responsible for the different foci distributions between flight and ground as shown in Fig 3B. It has been reported that human colon carcinoma cells treated with 0.5 Âľg/ml bleomycin for 2 days in space on board Space Shuttle STS-95 showed a 4-fold increased mutation rate in comparison to the ground controls, but it was not clear whether the different response in space was due to the cell growth condition. It should also be noted that in the colon carcinoma study, the cells were incubated with bleomycin even during the reentry before the samples were harvested on the ground. In this study, faster proliferation in space may be the result of microgravity, or hypergravity and vibration during launch, or a combination of different factors.

In this study, authors also investigated gene expression profiles using the microarray technique in cells after bleomycin treatment with the intention of identifying distinct responses at the molecular level to DNA damage in space. Hierarchical clustering of genes with significant expression changes (Table 4, Fig 5) revealed that bleomycin treated samples for both the flight and ground are grouped together and they are clearly segregated from the control samples. In both the ground and flown cells, activation of the p53 pathway was the top response (Fig 6, Table 5). p53, as well as its downstream regulators such as CDKN1A, is known to be induced in cultured cells or in animals after DNA damage. Cancer cells deficient in the p53 function are also known to have altered cell killing effects of

STEM Today, November 2018, No. 38

bleomycin. In addition to p53 signaling, other signaling pathways that are known to respond to DNA damages, including ATM, G1/S and G2/M cell cycle checkpoint signaling, were also found to be common in the flown and ground cells in response to the bleomycin treatment (Fig 7). The similar effects of bleomycin for both flight and ground samples were confirmed quantitatively in that there were no genes with significant differences with respect to the interaction contrast (GT-GC) vs (FT-FC).

Authors also analyze the expressions of genes involved in DNA damage response using PCR arrays, and found that only BBC3, CDKN1A, PCNA and PPM1D were upregulated after bleomycin treatment in either the ground or flown samples treatment; a finding which agreed well with the microarray data analysis results (Fig 7). The expressions of BCL2 Binding Component 3 (BBC3), Cyclin Dependent Kinase Inhibitor 1A (CDKN1A, p21), Proliferating Cell Nuclear Antigen (PCNA), and Protein Phosphatase, Mg2+/Mn2+ Dependent 1D (PPM1D) are all induced in a p53-dependent manner in response to various environmental stresses. BBC3 encoded protein is a member of the BCL-2 family, and is an essential mediator for p53-dependent and p53-independent apoptosis.

PCNA is expressed in the nucleus, and is involved in a number of DNA repair pathways including the RAD6dependent DNA repair pathway and non-homologous end-joining repair pathway. Cells treated with bleomycin have been reported to display PCNA foci. PPM1D is a member of the PP2C family of serine/ threonine pro14

STEM Today, November 2018, No. 38

tein phosphatases. This phosphatase reduces the phosphorylation of p53, negatively regulates the activity of p38 MAP kinase, and suppresses p53-mediated transcription and apoptosis. It is activated in human cells in response to damage from ionizing radiation and UV. CDKN1A is a potent cyclin-dependent kinase inhibitor and cell cycle regulator. Its encoded protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of DNA damages. This protein can interact with PCNA to play a regulatory role in S phase DNA replication and DNA damage repair.

Although H2AX was phosphorylated in both the flight and ground samples after bleomycin treatment, no significant increase of the RNA level of the H2A histone family X gene (H2AFX) was observed. Together with the microarray data, our results suggest no differential responses at the molecular level to bleomycin-induced DNA damages in confluent human fibroblasts between flight and ground. Previously, authors reported that in AG1522 cells flown to the ISS without treatment of toxic chemicals, cells in space showed activation of NF-ÎşB which triggered activation of a number of growth factors such as HGF and VEGF. Here, authors also compared the gene expressions in the flight samples in respect to the ground controls, either treated or untreated with bleomycin. Using the same fold change and FDR thresholds (Fold > 1.3 and FDR <0.1), a total number of 697 and 432 genes had significant expression changes in space in the untreated (FC-GC) and treated samples (FT-GT), respectively. Analysis of these genes using IPA confirms that NF-ÎşB was the top canonical pathways in both the treated and untreated groups (Table 6). It should be noted that in Zhang et al., cells were removed from the incubator and fixed immediately, whereas in the present study, the cells were removed from incubator, treated with placebo or bleomycin, transferred back to incubator for 3 hrs, and fixed afterwards. The RNA levels in the PCR array analysis were also compared between the flight and ground samples for determination of changes of gene expressions due to the spaceflight condition alone. Of the 84 genes, none had significant expression changes in space in comparison to the ground, either in bleomycin or placebo treated samples, indicating that spaceflight condition did not alter the baseline level of these RNAs involved in DNA damage signaling. In human fibroblasts, spaceflight alone had affected the pathways mostly involved in cell proliferation.

DNA damage induction

SIMULATED MICROGRAVITY STUDIES DNA damage induced by simulated microgravity As opportunities for true spaceflight experiments are rare, various studies have been conducted using ground-based devices that simulate certain aspects of microgravity. Common analogs for microgravity include rotating wall vessels (RWV), and 2D and 3D clinostats for cultured cells, and the hind limb suspension model for rodents. Other approaches, such as airplanes flying in a parabolic pattern or free drop towers also offer true microgravity for a short duration. For humans, the bed rest model is used to simulate the effects of microgravity on various physiological systems, especially for studies of bone, muscle and the cardiovascular system. Using these microgravity analogs, researchers have reported a range of data concerning the induction of DNA damage by simulated microgravity. For instance, simulated microgravity has been reported to induce DNA single 15

strand breaks in human retinal pigment epithelial cells (hRPE) cultured in RWV. At 48 h after returning to the 1 g gravity condition, this DNA damage persisted and the production of the inflammatory marker prostaglandin E2 (PGE2) increased. However, hRPE cells previously treated with the anti-inflammatory agent cysteine showed less DNA damage and no PGE2 release. The bed rest model for studying the effect of microgravity on human physiology has also revealed an increase of 8-oxo-7,8 dihydro-2â&#x20AC;&#x2122; deoxyguanosine (8-oxo-HdG), which is considered a marker of oxidative DNA damage. Taken together, these results from cell models and human subjects suggest that various forms of ground-based microgravity analogs may indeed induce oxidative DNA damage. Many studies that reported measurements of simulated microgravity-induced reactive oxygen species (ROS), but not specific DNA damage markers, are not included here. Simulated microgravity alone does not appear to induce double strand breaks (DSB) in cells with normal genetic background. However, DSBs were found in mouse embryonic stem cells that were deficient in DDR and cultured under RWV,suggesting that perhaps some DSB type damage is induced by simulated microgravity, but is typically repaired in DDR competent organisms.

STEM Today, November 2018, No. 38

Effects of simulated microgravity on DDR In addition to the aforementioned DNA damage, there is evidence indicating that key elements of the DDR machinery are also affected under simulated microgravity alone. For instance, DNA damage, as well as decreased expression of DNA repair genes involved in mismatch repair, base excision repair, nucleotide excision repair, and the down regulation of p53, was observed in proliferating lymphocytes grown in simulated microgravity. Furthermore, p21 up regulation occurred rapidly in lymphocytes exposed to real microgravity from parabolic flights and under simulated microgravity in 2D clinostats, suggesting a p53-independent mechanism. Additionally, the activity of the DNA strand break sensor poly (ADP-ribose) polymerase 1 significantly increased under simulated microgravity. Further studies on mouse embryonic stem cells cultured in a 3D clinostat showed that simulated microgravity alone did not induce DNA damage, but it did affect radiation-induced DNA repair. Most of the studies on the effects of simulated microgravity on the DDR have used low-linear energy transfer (LET) X-rays or Îł rays to generate high levels of DNA damage. These damaged cells were then allowed to repair under different gravity conditions. However, our review of the studies performed under this experimental design has revealed conflicting results. For example, human lymphocytes exposed to 1.5 Gy of X-rays and cultured in a clinostat showed a higher number of X-ray induced chromosome aberrations in comparison to control cells cultured under the static 1 g condition.

But, researchers using the NASA-designed RWV bioreactor found no significant difference in high energy proton radiation-induced (60 MeV protons or 250kVp X-rays in the dose ranges of 0-6 Gy) chromosome aberrations between human lymphocytes cultured in normal vs. simulated microgravity conditions, indicating that DNA

repair was not affected. Even so, more studies do suggest that simulated microgravity has an effect on DDR. Specifically, in lymphoblastoid TK6 cells irradiated with γ rays and incubated for 24 h in a simulated microgravity environment, a significant reduction in apoptotic cells, increased number of cells in G1-phase, and higher frequencies of micronucleated cells and mutations were reported in comparison to cells that were exposed to the same doses of radiation while maintained in 1 g. Greater mutant frequency was also found in human lymphocytes after ionizing radiation exposure in simulated microgravity.

STEM Today, November 2018, No. 38

In both TK6 cells and peripheral blood mononuclear cells (PBMCs), the increase in mutation rate was associated with a reduced rate of radiation-induced apoptosis, again indicating a DDR change. Meanwhile, Omics studies such as transcriptome and microRNome analysis have provided evidence that simulated microgravity affects the DDR to ionizing radiation in human PBMCs by causing downregulation of DDR pathways associated miRNAs like miR-7 and miR-27a. Ultimately, simulated microgravity delayed DNA repair of radiation-induced DSBs in human lymphocytes, and as a consequence, the genotoxic effects of ionizing radiation increased. It should be noted, however, that few studies have employed high-LET charged particles to induce DNA damage under simulated microgravity conditions. In one such study, simulated microgravity was shown to decrease carbon ion radiation-induced cell survival and increase apoptosis in human B lymphoblasts. Such effects of simulated microgravity were associated with increased heavy ion radiation-induced intracellular ROS generation. In another study, increased apoptosis and DNA damage were found in the sperm of mice that was exposed simultaneously to carbon ions and simulated microgravity (Hindlimb suspension). Together, these results suggest that, in most reported studies, simulated microgravity alters the expression of genes involved in DDR, and indeed affect the cellular response to radiationinduced damage (Table 1). More work is needed, however, to better understand the effects of microgravity on cellular repair processes in response to high LET radiation-induced DNA damage. SPACEFLIGHT STUDIES DNA damage from spaceflight In contrast to the large number of studies that have been conducted on the ground using particles generated in accelerators, results describing DNA damage from direct exposure to natural space radiation are few. Detection of direct biological damage by space radiation is challenging not only due to the low dose and the low dose rate nature of the space environment, but also due to the possible synergistic effects of microgravity. In one such attempt, fixed human cervical carcinoma (HeLa) cells were flown in the Russian MIR space station for 40 days or on the Space Shuttle for 9 days. The resulting DNA damage levels, as measured by enzymatic incorporation of [3 H]-dATP from terminal deoxyribo-nucleotidyl transferase, correlated with space flight duration, suggesting that the measured DNA damage was caused by space radiation and was dependent on the length of the space flight. In another experiment, human lymphoblastoid TK6 cells were sent to the International Space Station (ISS) and kept frozen in space for 134 days so that the damage could accumulate (while the impact of microgravity was simultaneously minimized). The cells flown to space showed an increase in thymidine kinase deficient (TK(-)) mutations over the ground controls. In a similar study, frozen TK6 cells were also analyzed for DSBs by measuring the phosphorylation of the histone H2AX (γ-H2AX). The induced DSBs appeared as a dense track of ionizations and excitations along the particle path. Similarly, during the Foton-M3 Mission (total 12 days), normal human dermal fibroblasts fixed after 4 days in orbit were found to have increased DSBs. The γ-H2AX assay for detecting DSBs has also been performed on human fibroblasts cultured at 37 ◦ C on the ISS for 14 days. Although the average number of γ-H2AX foci was similar between the flight samples and ground controls, the flown cells exhibited several track shaped foci that were similar to those induced by high-LET space radiation analogs on the ground. Together, these results suggest that DNA damage can be directly attributed to the space radiation environment. Another effect attributed to space radiation is the induction of chromosome aberrations in white blood cells of astronauts after returning from space. Such damage can be observed only after a 3-6 month duration mission, but not a 2-week shuttle mission. Similarly, analysis of chromosomal aberrations in blood cells from one Italian and eight Russian cosmonauts were analyzed following missions of different duration on the MIR space station and the ISS. Although an increase in chromosome damage was observed in some cases, the authors did not detect a correlation between flight history and chromosome damage. DNA damage induced by spaceflight has also been detected using other biomarkers. At times, however, it has been difficult to properly isolate the effects of space radiation from other space environmental factors. For instance, urine samples collected from astronauts have been analyzed for oxidative DNA damage by measuring 8-hydroxy-2’-deoxyguanosine (8-OHdG) before, during, and after spaceflight missions. 8-OHdG excretion was 17

unchanged during spaceflight but increased after flight. The same study found no changes in 8-OHdG excretion either during or after a short-duration spaceflight, suggesting that elevated 8-OHdG excretion may depend on the length of the mission. Even though astronauts were exposed to space radiation during the mission, the radiation level was so low that the oxidative damage was perhaps caused by microgravity in LEO or hypergravity experienced during re-entry. Due to the limited in vivo space-based studies, and other factors that may contribute to 8-OHdG changes, it is evident that further work is needed to understand the increased 8-OHdG measured in astronauts. Effects of spaceflight on DNA damage response (DDR) Potential effects of spaceflight on the DDR have been investigated since the early days of the human space program. Experiments aimed at addressing such effects have been conducted according to several experimental scenarios: (i) pre-flight induction of high levels of DNA damage with radiation prior to launch, (ii) exposing samples to short-ranged particles in space, or (iii) exposing samples to radiation shortly after landing.

STEM Today, November 2018, No. 38

In these studies, the combined spaceflight factors include not only microgravity and the background space radiation, but also factors, such as hypergravity experienced during launch or re-entry. The experiments carried out in the Gemini III and the Gemini XI manned spaceflight missions were designed to test synergistic effects between ionizing radiation and other stress factors associated with spaceflight. During these missions, cultured human lymphocytes were exposed to 32 Pb particles in space after reaching the microgravity condition. Chromosomes analysis after Gemini III mission showed an increased frequency of Î˛-induced deletions compared to the ground controls, whereas the frequencies of dicentrics and rings were similar. However, the synergistic effect observed after Gemini III could not be confirmed after Gemini XI. Early studies on the combined effects of microgravity and radiation have been reviewed previously by Horneck, and by Keifer and Pross, who concluded that spaceflight does affect the development of organisms, but argued that the majority of spaceflight experiments showed little effects of microgravity on the repair of radiation-induced DNA damage. More recently, attempts at understanding the combined effects of space radiation and microgravity were implemented during the STS-91 Shuttle mission. In the Shuttle experiment, researchers showed no effect of microgravity on either the biochemical reactions involved in DNA damage repair by T4 ligase or on the repair and replication carried out by Taq polymerase and Polymerase III in response to chemically induced DNA damage. Beyond these in vitro enzymatic studies, experiments involving bacteria and yeast have provided additional results. During the Spacelab mission IML-2, frozen Escherichia coli and Bacillus subtilis bacteria were exposed to X-ray and ultraviolet (UV) irradiation before launch. Once in Spacelab, the bacteria were thawed for up to 4.5 h and frozen again until landing, and then assessed for DNA repair. Ultimately, no significant differences were found either in the rejoining of DNA strand breaks or in the survival curve between microgravity conditions and 1 g controls, suggesting that cells were able to repair radiationinduced DNA damage under real, albeit brief, microgravity. In another study (STS-84), the temperature dependent repair mutant rad54-3 of Saccharomyces cerevisiae yeast was irradiated with Ni Î˛ particles and allowed time to repair under different temperatures. By measuring the amount of remaining, unrepaired breaks, researchers demonstrated that there was no difference in double-strand break repair between the flight and ground control samples, suggesting no significant impact of the real microgravity condition on this process. While these studies on bacteria and yeast did not show impaired DNA repair in microgravity, studies on more complex organisms and human cells have yielded very different results. For instance, a study on Caenorhabditis elegans showed that several DDR genes were differentially expressed during the 16.5-day Shenzhou-8 space mission, suggesting possible enhanced DDR under microgravity. Overall, most of these studies suggest no effect of space flight on the DDR (Table 2). Further studies in space have also been conducted using bleomycin, a chemotherapy drug that is known to induce DNA damage, including DSBs. It has been reported that human colon carcinoma cells (HCT-116) treated with bleomycin for 2 days in space (Space Shuttle STS-95) showed no difference in frequencies of microsatellite mutations when compared to the ground controls. In a more recent experiment, confluent human fibroblasts, which were arrested in the G1 phase of the cell cycle, were also treated with bleomycin on the ISS and fixed after 3 h. In this study, analysis of bleomycin-induced DSBs and gene expression changes showed no significant difference between the flight and ground-treated samples, indicating a lack of microgravity effect.

STEM Today, November 2018, No. 38

Another experimental design compares how human blood cells respond to ground-based artificially induced DNA damage before and after a space mission. In one study, blood was collected from an astronaut before flight and within 24 h after the 9-day STS-103 mission. After collection, both samples were exposed to Îł rays of doses between 0 and 3 Gy. Comparison of the dose response for total chromosomal exchanges showed no differences between pre-flight and post-flight samples suggesting that microgravity had no lasting effect on DNA repair. Alternatively, Greco and colleagues reported an enhancement of approximately 1.2-2.8 -fold in the chromosome aberration frequency in a post-flight cosmonaut blood sample compared to parallel pre-flight data after exposure to ground based X-rays. Interestingly however, for cosmonauts involved in more than one space flight, the amount of chromosomal aberrations was in the range of the background before the mission started and did not depend on the total duration of flights. Whether exposure to space radiation during prior space missions caused this effect by impacting the response to damages incurred in subsequent missions has been investigated in astronauts as well. Analysis of chromosome damage in blood lymphocytes collected from five astronauts before and after their first and second long duration space flights detected an increase in chromosome aberrations after both flights, with no significant impact of prior space travel. Although these results are far from conclusive, taken together, studies from a variety of model systems and humans subjects suggest that spaceflight may indeed play a role in altering the DDR to radiation-induced damage. When faced with DNA damage, programmed cell death is an important component of the DDR. Therefore, in addition to the studies aimed at detecting DNA damage levels, investigators have also analyzed endpoints, such as apoptosis. Spaceflight studies conducted on human lymphocytes have shown an increase in apoptosisrelated markers, such as DNA fragmentation, cleavedpoly (ADP-ribose) polymerase, and elevated mRNA levels of p53 and calpain after 48 h on board the ISS. Furthermore, cell cyclerelated genes, such as p21, were inhibited in the muscle of rats from space flights compared to rats on the ground. Such studies indicate that spaceflight factors may indeed influence the rate of apoptosis and/or cell cycle regulation.

STEM Today, November 2018, No. 38

References R

Pierson, D. L., Stowe, R. P., Phillips, T. M., Lugg, D. J. and Mehta, S. K. Epstein-Barr virus shedding by astronauts during space flight. Brain Behav Immun 19, 235-242 (2005).

Pierson, D. L., Mehta, S. K. and Stowe, R. P. In: Psychoneuroimmunology Vol. II (ed Ader R.) 851-868 (Academic Press, 2007).

Terada, M. et al. Effects of a closed space environment on gene expression in hair follicles of astronauts in the International Space Station. PloS One 11, e0150801 (2016).

Whitney, A. R. et al. Individuality and variation in gene expression patterns in human blood. Proc Natl Acad Sci USA 100, 1896-1901 (2003).

Obe, G. et al. Chromosomal aberrations in blood lymphocytes of astronauts after long-term space flights. Int J Rad Biol 72, 727-734 (1997).

Smith, S. M., Zwart, S. R., Block, G., Rice, B. L. and Davis-Street, J. E. The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station. J Nutr 135, 437-443 (2005).

Kumari, R., Singh, K. P. and Dumond, J. W. Jr. Simulated microgravity decreases DNA repair capacity and induces DNA damage in human lymphocytes. J Cell Biochem 107, 723-731 (2009).

Barrila, J., Ott, C.M., LeBlanc, C., Mehta, S.K., Crabbe, A., Stafford, P., Pierson, D.L., and Nickerson, C.A. (2016). Spaceflight modulates gene expression in the whole blood of astronauts. npj Microgravity.

Montague TG, Almansoori A, Gleason EJ, Copeland DS, Foley K, Kraves S, et al. (2018) Gene expression studies using a miniaturized thermal cycler system on board the International Space Station. PLoS ONE 13(10): e0205852.

Lu T, Zhang Y, Kidane Y, Feiveson A, Stodieck L, Karouia F, et al. (2017) Cellular responses and gene expression profile changes due to bleomycin-induced DNA damage in human fibroblasts in space. PLoS ONE 12(3): e0170358.

Backup P, Westerlind K, Harris S, Spelsberg T, Kline B, Turner R. Spaceflight results in reduced mRNA levels for tissue-specific proteins in the musculoskeletal system. Am J Physiol. 1994; 266: E567-73.

Barrila J, Ott CM, LeBlanc C, Mehta SK, Crabbe A, Stafford P, et al. Spaceflight modulates gene expression in the whole blood of astronauts. NPJ Microgravity. 2016; 2: 16039.

Moreno-Villanueva, Maria; Wong, Michael; Lu, Tao; Zhang, Ye; and Wu, Honglu, ,"Interplay of space radiation and microgravity in DNA damage and DNA damage response." npj Microgravityvolume 3, Article number: 14 (2017).