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Hypoxia occurs in 80% of non-small cell lung carcinoma (NSCLC) cases, leading to treatment resistance. Hypoxia’s effects on NSCLC energetics are not well-characterized. We evaluated changes in glucose uptake and lactate production in two NSCLC cell lines under hypoxia in conjunction with growth rate and cell cycle phase distribution. The cell lines A549 (p53 wt) and H358 (p53 null) were incubated under hypoxia (0.1% and 1% O2) or normoxia (20% O2). Glucose and lactate concentrations in supernatants were measured using luminescence assays. Growth kinetics were followed over seven days. Cell nuclei were stained with DAPI and nuclear DNA content was determined by flow cytometry to determine cell cycle phase. Gene expression under hypoxia was determined by RNA sequencing. Glucose uptake and lactate production under hypoxia were greater than under normoxia. They were also significantly greater in A549 compared to H358 cells. Faster energy metabolism in A549 cells was associated with a higher growth rate in comparison to H358 cells under both normoxia and hypoxia. In both cell lines, hypoxia significantly slowed down the growth rate compared to proliferation under normoxic conditions. Hypoxia led to redistribution of cells in the different cycle phases: cells in G1 increased and the G2 population decreased. Glucose uptake and lactate production increase under hypoxia in NSCLC cells indicated greater shunting of glucose into glycolysis rather than into oxidative phosphorylation compared to normoxia, making adenosine triphosphate (ATP) production less efficient. This may explain the redistribution of hypoxic cells in the G1 cell cycle phase and the time increase for cell doubling. Energy metabolism changes were more prominent in faster-growing A549 cells compared to slower-growing H358 cells, indicating possible roles for the p53 status and inherent growth rate of different cancer cells. In both cell lines, genes associated with cell motility, locomotion and migration were upregulated under chronic hypoxia, indicating a strong stimulus to escape hypoxic conditions.

The use of high linear energy transfer (LET) ionizing radiation (IR) is progressively being incorporated in radiation therapy due to its precise dose localization and high relative biological effectiveness. At the same time, these benefits of particle radiation become a high risk for astronauts in the case of inevitable cosmic radiation exposure. Nonetheless, DNA Damage Response (DDR) activated via complex DNA damage in healthy tissue, occurring from such types of radiation, may be instrumental in the induction of various chronic and late effects. An approach to elucidating the possible underlying mechanisms is studying alterations in gene expression. To this end, we identified differentially expressed genes (DEGs) in high Z and high energy (HZE) particle-, γ-ray- and X-ray-exposed healthy human tissues, utilizing microarray data available in public repositories. Differential gene expression analysis (DGEA) was conducted using the R programming language. Consequently, four separate meta-analyses were conducted, after DEG lists were grouped depending on radiation type, radiation dose and time of collection post-irradiation. To highlight the biological background of each meta-analysis group, functional enrichment analysis and biological network construction were conducted. For HZE particle exposure at 8–24 h post-irradiation, the most interesting finding is the variety of DNA repair mechanisms that were downregulated, a fact that is probably correlated with complex DNA damage formation. Simultaneously, after X-ray exposure during the same hours after irradiation, DNA repair mechanisms continue to take place. Finally, in a further comparison of low- and high-LET radiation effects, the most prominent result is that autophagy mechanisms seem to persist and that adaptive immune induction seems to be present. Such bioinformatics approaches may aid in obtaining an overview of the cellular response to high-LET particles. Understanding these response mechanisms can consequently aid in the development of countermeasures for future space missions and ameliorate heavy ion treatments.

Human exploration of the Moon is associated with substantial risks to astronauts from space radiation. On the surface of the Moon, this consists of the chronic exposure to galactic cosmic rays and sporadic solar particle events. The interaction of this radiation field with the lunar soil leads to a third component that consists of neutral particles, i.e., neutrons and gamma radiation. The Lunar Lander Neutrons and Dosimetry experiment aboard China's Chang'E 4 lander has made the first ever measurements of the radiation exposure to both charged and neutral particles on the lunar surface. We measured an average total absorbed dose rate in silicon of 13.2 ± 1 μGy/hour and a neutral particle dose rate of 3.1 ± 0.5 μGy/hour.

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by &gamma, H2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced &gamma, H2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.

ZusammenfassungDer alte Menschheitstraum, nach den Sternen zu greifen und den Fuß auf unbewohnte Planeten zu setzen, rückt für den heutigen Menschen in unmittelbare Nähe. Die technisch mögliche Reise zu Mond oder Mars kann für den „Homo spatialis“ mit Sonnenstürmen und Partikelbeschuss gepflastert sein und birgt medizinische Risiken: Vor allem die erhöhte Strahlendosis der solaren und galaktischen Komponenten der Weltraumstrahlung (Elektronen, Protonen und hochenergetische schwere Teilchen) kann zu einem bedrohlichen Gesundheitsrisiko werden. Je nach Missionsdesign besteht die Möglichkeit, durch unvorhersehbare Sonnenstürme akute Strahlenschäden zu erleiden und, bedingt durch die erhöhte biologische Wirksamkeit der galaktischen Strahlung, Langzeitschäden zu entwickeln. Abschätzungen zeigen, dass Astronauten während einer Marsreise mit einer Strahlenbelastung zu rechnen haben, die weit über dem amtlichen Grenzwert von jährlich 20 mSv für beruflich strahlenexponierte Personen in Europa liegt. Die Karrieredosis von Astronauten kann möglicherweise zu einem 3–4 % erhöhten zusätzlichen Krebsrisiko führen. Des Weiteren ist mit einem früheren und häufigeren Auftreten von Katarakten zu rechnen. Das Risiko für neurodegenerative Effekte ist zurzeit schwer einschätzbar. Gegenmaßnahmen umfassen Abschirmung, Warnsysteme für Sonnenstürme und unter Umständen auch diätetische und pharmakologische Mittel.

Chang'E 4 is the first mission to the far side of the Moon and consists of a lander, a rover, and a relay spacecraft. Lander and rover were launched at 18:23 UTC on December 7, 2018 and landed in the von K��rm��n crater at 02:26 UTC on January 3, 2019. Here we describe the Lunar Lander Neutron \& Dosimetry experiment (LND) which is part of the Chang'E 4 Lander scientific payload. Its chief scientific goal is to obtain first active dosimetric measurements on the surface of the Moon. LND also provides observations of fast neutrons which are a result of the interaction of high-energy particle radiation with the lunar regolith and of their thermalized counterpart, thermal neutrons, which are a sensitive indicator of subsurface water content., 38 pages, submitted to Space Science Reviews

Passive and active dosimeters for space missions are available and constantly improved. The radiation environment of the International Space Station (ISS) is well characterized and radiation detectors are measuring on the surface of Mars and Moon. Concepts of storm shelters are implemented in the upcoming Artemis missions to the Moon. The first human phantom experiment beyond LEO will bring about the depth dose distribution in the female body. Space radiobiological research currently shifts from single ion experiments to combinations of several ions and from classical radiobiological endpoints such as cellular survival and chromosomal aberrations to tissue- and organ-specific responses that are relevant for degenerative diseases. Furthermore, in addition to monolayer cell cultures, more complex model systems such as organoids are used for accelerator-based research. Other spaceflight environmental factors than microgravity—stress, isolation, sleep deprivation—are coming into the research focus for combined effects. For countermeasure development, tests with well-known drugs are suggested.

The galactic cosmic radiation (GCR) results constant exposure of astronauts to charged particles of various energies at a low-dose rate. A traversal of a charged particle through a cell nucleus can result in (complex) DNA damage which initiates the DNA damage response (DDR). During this response, the cell might arrest in the cell cycle in order to gain time for DNA repair. Depending on damage severity, cell type, and other factors, different outcomes such as cell death, premature differentiation, senescence, or chromosomal aberrations are possible. In addition to effects on the main target molecule, the DNA, non-targeted effects contribute to overall outcome for a GCR exposed organism. Compared to X- or γ-rays, heavy charged particles can have a high relative biological effectiveness (RBE) for inducing different biological outcomes, including cancer-relevant outcomes and cancer in rodents. Primarily, late tissue sequels like genetic alterations, cancer and non-cancer effects, i.e., cataracts and degenerative diseases, e.g., of the central nervous system, are potential risks for space travelers. Cataracts were observed to occur with earlier onset and more frequently in astronauts exposed to even low doses of GCR. In addition, the continuously existing risk of acute exposure to high proton fluxes during a solar particle events (SPE) implies to threaten immediate survival of the astronauts in case of insufficient shielding by eliciting the acute radiation syndrome (ARS).

Different countermeasures against deleterious effects of space radiation on human health are required for galactic cosmic rays (GCR) and solar particle events (SPE). In case of SPE, efficient shielding is possible in a radiation shelter. The crew has to enter the shelter in case of increasing proton fluxes during an SPE to avoid high-dose exposures within hours or days which might lead to the acute radiation syndrome (ARS) in worst case scenarios. ARS treatment strategies are available but difficult to implement on a space mission. The chronic exposure to GCR is associated with various health risks (cancer, cataract, CNS decrements, cardiovascular disease) that can reduce the healthy life span after return to Earth. Shielding of GCR in free space is very limited and therefore, the time in free space should be minimized by fast propulsion. On planetary and moon surfaces, the habitats should provide optimal shielding in order to reduce GCR dose accumulated during the mission. Much effort is placed on the development of nutritional and pharmaceutical countermeasures for chronic and late radiation effects. Free radical scavengers such as amifostine are ruled out because of severe side effects. Encouraging results were reported in rodent studies with berries or dried plums. A concerted action of ground-based studies and space experiments is required to expand radiobiological knowledge about space radiation to improve countermeasure development. Crew selection is a sensitive topic, but from a radiation protection point of view, the lower radiosensitivity of older crew members might be considered.

The assessment of risks associated with space radiation exposure requires determination of the expected radiation exposure during the space mission, knowledge of the risks associated with such exposures and of modifying factors which are all to be incorporated in a dedicated radiation risk model. To determine the radiation exposure, the radiation field and its variation during the solar cycle, its interaction with matter and the dose distribution in the human body are considered. Disease risks are derived from large epidemiological studies on radiation-exposed populations, predominantly the Japanese atomic bomb survivors. Extrapolation from high dose rate to low-dose rate exposure, from low linear energy transfer (LET) to high-LET radiation and from the Japanese to other populations requires the use of extrapolation factors. Accordingly, predictions of cancer risk and acceptable radiation exposure in space are subject to many uncertainties including the relative biological effectiveness (RBE) of space radiation (especially heavy ions), dose rate effects and possible interaction with microgravity and other spaceflight environmental factors.

The radiation field in space is highly variable in time and space. Different sources contribute to the total exposure. In interplanetary space, the field is dominated by the omni-present galactic cosmic radiation (GCR) and sporadic solar particle events (SPE) can contribute. On the International Space Station (ISS) in low Earth orbit (LEO), on the other hand, the contribution of SPE can be neglected and GCR are modulated along the station’s trajectory due to the shielding effect of the geomagnetic field against charged particles. On planetary surfaces, for instance, on Mars, albedo particles from underground and secondary particles from interactions with the atmosphere, if present, are added to the radiation field. Secondary particles, especially neutrons, can contribute significantly to the exposure. In all cases, the field can be further modified by the potential shielding environment and the resulting particle fluxes lead to the exposure of humans under the given conditions. The exposure is calculated as the energy deposition in tissue weighted with corresponding quality factors or relative biological effectiveness and organ weighting factors. In most cases, if measured, the dose rate is determined from the energy deposition in silicon detectors and corresponding corrections have to be applied to estimate the dose in tissue. Additionally, self-shielding of the body has to be taken into account if organ doses are determined.

Epidemiological studies show that there is a link between urban water pollution and increase in human morbidity and mortality. With the increase in number of new substances arising from the chemical, pharmaceutical, and agricultural industries, there is an urgent need to develop biological test systems for fast evaluation of potential risks to humans and the environmental ecosystems. Here, a combined cellular reporter assay based on the cellular survival and the stress-induced activation of the survival-promoting factor nuclear factor κB (NF-κB) and its use for the detection of cytotoxicity and cancer-related stress responses is presented. A total of 14 chemicals that may be found in trace-amounts in ground water levels are applied and tested with the presented assay. The project is embedded within the joint research project TOX-BOX which aims to develop a harmonized testing strategy for risk management of anthropogenic trace substances in potable water. The assay identified carbendazim as a NF-κB-activating agent in mammalian cells.

Background/Aims: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs) under simulated microgravity within a fast-rotating clinostat (clinorotation) and capture of microarray-based gene signatures. Methods: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. Results: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g) after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs). Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. Conclusion: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The most significant simulated microgravity-affected genes, signal transduction pathways, and biological processes which are relevant for mESCs differentiation have been identified and discussed below.

The overall response to tumor radiation therapy results from direct radiation damage and indirect bystander effects (RIBE) mediated by secreted molecules and paracrine transfer of short-lived mediators. RIBE can have both detrimental and protective actions on cancerous as well as healthy tissues. Nuclear Factor κB (NF-κB), which governs immune responses, is a prime candidate for mediating RIBE. NF-κB also modulates DNA repair while promoting cellular survival as part of the DNA damage response. After exposure to ionizing radiation NF-κB is activated in directly targeted and non-targeted bystander cells. We tested the hypothesis that the NF-κB status is relevant for induction of RIBE, using a mouse embryonal fibroblasts (MEF) knock-out variant that is unable to activate NF-κB (NF-κB essential modulator knock-out (NEMO ko)).

Training young researchers in the field of space life sciences is essential to vitalize the future of spaceflight. In 2009, the DLR Institute of Aerospace Medicine established the Helmholtz Space Life Sciences Research School (SpaceLife) in cooperation with several universities, starting with 22 doctoral candidates. SpaceLife offered an intensive three-year training program for early-stage researchers from different fields (biology, biomedicine, biomedical engineering, physics, sports, nutrition, plant and space sciences). The candidates passed a multistep selection procedure with a written application, a self-presentation to a selection committee, and an interview with the prospective supervisors. The selected candidates from Germany as well as from abroad attended a curriculum taught in English. An overview of space life sciences was given in a workshop with introductory lectures on space radiation biology and dosimetry, space physiology, gravitational biology and astrobiology. The yearly Doctoral Students’ Workshops were also interdisciplinary. During the first Doctoral Students’ Workshop, every candidate presented his/her research topic including hypothesis and methods to be applied. The progress report was due after ∼1.5 years and a final report after ∼3 years. The candidates specialized in their subfield in advanced lectures, Journal Clubs, practical trainings, lab exchanges and elective courses. The students attended at least one transferable skills course per year, starting with a Research Skills Development course in the first year, a presentation and writing skills course in the second year, and a career and leadership course in the third year. The whole program encompassed 303 h and was complemented by active conference participation. In this paper, the six years’ experience with this program is summarized in order to guide other institutions in establishment of structured Ph.D. programs in this field. The curriculum including elective courses is documented. The applicants’ statistics revealed that personal contacts and the DLR website were most important the recruitment of doctoral candidates. The evaluation of the application and selection procedure revealed that prediction of thesis success based on master thesis mark or evaluation by the selection committee is difficult. SpaceLife Doctoral Students greatly contributed to the scientific output in terms of peer-reviewed publications of the Institute of Aerospace Medicine with a peak in the fourth year after start of the thesis and they continuously received awards for their scientific work.

Nuclear factor kappaB (NF-κB) is a central transcription factor in the immune system and modulates cell survival in response to radiotherapy. Activation of NF-κB was shown to be an early step in the cellular response to ultraviolet A (UVA) and ionizing radiation exposure in human cells. NF-κB activation by the genotoxic stress-dependent sub-pathway after exposure to different radiation qualities had been evaluated to a very limited extent. In addition, the resulting gene expression profile, which shapes the cellular and tissue response, is unknown. Therefore, in this study the activation of NF-κB after exposure to low- and high-linear energy transfer (LET) radiation and the expression of its target genes were analyzed in human embryonic kidney (HEK) cells. The activation of NF-κB via canonical and genotoxic stress-induced pathways was visualized by the cell line HEK-pNF-κB-d2EGFP/Neo L2 carrying the destabilized enhanced green fluorescent protein (d2EGFP) as reporter. The NF-κB-dependent d2EGFP expression after irradiation with X rays and heavy ions was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after irradiation with X rays (significant NF-κB activation for doses4 Gy) and heavy ions (significant NF-κB activation at doses as low as 1 Gy), it was expected that radiation quality (LET) played an important role in the cellular radiation response. In addition, the relative biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival were compared for heavy ions having a broad LET range (∼0.3-9,674 keV/μm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real-time reverse transcriptase quantitative PCR (RT-qPCR). The maximal RBE for NF-κB activation and cell killing occurred at an LET value of 80 and 175 keV/μm, respectively. There was a dose-dependent increase in expression of NF-κB target genes NF-κB1A and CXCL8. A qPCR array of 84 NF-κB target genes revealed that TNF and a set of CXCL genes (CXCL1, CXCL2, CXCL8, CXCL10), CCL2, VCAM1, CD83, NF-κB1, NF-κB2 and NF-κBIA were strongly upregulated after exposure to X rays and neon ions (LET 92 keV/μm). After heavy-ion irradiations, it was noted that the expression of NF-κB target genes such as chemokines and CD83 was highest at an LET value that coincided with the LET resulting in maximal NF-κB activation, whereas expression of the NF-κB inhibitory gene NFKBIA was induced transiently by all radiation qualities investigated. Taken together, these findings clearly demonstrate that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ∼50-200 keV/μm. The upregulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, CXCL8/IL-8 and TNF) could be important for cell-cell communication among hit as well as nonhit cells (bystander effect).

Energetic, charged particles elicit an orchestrated DNA damage response (DDR) during their traversal through healthy tissues and tumors. Complex DNA damage formation, after exposure to high linear energy transfer (LET) charged particles, results in DNA repair foci formation, which begins within seconds. More protein modifications occur after high-LET, compared with low-LET, irradiation. Charged-particle exposure activates several transcription factors that are cytoprotective or cytodestructive, or that upregulate cytokine and chemokine expression, and are involved in bystander signaling. Molecular signaling for a survival or death decision in different tumor types and healthy tissues should be studied as prerequisite for shaping sensitizing and protective strategies. Long-term signaling and gene expression changes were found in various tissues of animals exposed to charged particles, and elucidation of their role in chronic and late effects of charged-particle therapy will help to develop effective preventive measures.

Several Escherichia coli deletion mutants of the Keio collection were selected for analysis to better understand which genes may play a key role in copper or silver homeostasis. Each of the selected E. coli mutants had a deletion of a single gene predicted to encode proteins for homologous recombination or contained functions directly linked to copper or silver transport or transformation. The survival of these strains on pure copper surfaces, stainless steel, and alloys of aluminum, copper and/or silver was investigated. When exposed to pure copper surfaces, E. coli ΔcueO was the most sensitive, whereas E. coli ΔcopA was the most resistant amongst the different strains tested. However, we observed a different trend in sensitivities in E. coli strains upon exposure to alloys of the system Al–Ag–Cu. While minor antimicrobial effects were detected after exposure of E. coli ΔcopA and E. coli ΔrecA to Al–Ag alloys, no effect was detected after exposure to Al–Cu alloys. The release of copper ions and cell-associated copper ion concentrations were determined for E. coli ΔcopA and the wild-type E. coli after exposure to pure copper surfaces. Altogether, compared to binary alloys, ternary eutectic alloys (Al–Ag–Cu) had the highest antimicrobial effect and thus, warrant further investigation.

Microbial biofilms can lead to persistent infections and degrade a variety of materials, and they are notorious for their persistence and resistance to eradication. During long-duration space missions, microbial biofilms present a danger to crew health and spacecraft integrity. The use of antimicrobial surfaces provides an alternative strategy for inhibiting microbial growth and biofilm formation to conventional cleaning procedures and the use of disinfectants. Antimicrobial surfaces contain organic or inorganic compounds, such as antimicrobial peptides or copper and silver, that inhibit microbial growth. The efficacy of wetted oxidized copper layers and pure copper surfaces as antimicrobial agents was tested by applying cultures of Escherichia coli and Staphylococcus cohnii to these metallic surfaces. Stainless steel surfaces were used as non-inhibitory control surfaces. The production of reactive oxygen species and membrane damage increased rapidly within 1 h of exposure on pure copper surfaces, but the effect on cell survival was negligible even after 2 h of exposure. However, longer exposure times of up to 4 h led to a rapid decrease in cell survival, whereby the survival of cells was additionally dependent on the exposed cell density. Finally, the release of metal ions was determined to identify a possible correlation between copper ions in suspension and cell survival. These measurements indicated a steady increase of free copper ions, which were released indirectly by cells presumably through excreted complexing agents. These data indicate that the application of antimicrobial surfaces in spaceflight facilities could improve crew health and mitigate material damage caused by microbial contamination and biofilm formation. Furthermore, the results of this study indicate that cuprous oxide layers were superior to pure copper surfaces related to the antimicrobial effect and that cell density is a significant factor that influences the time dependence of antimicrobial activity. Key Words: Contact killing-E. coli-S. cohnii-Antimicrobial copper surfaces-Copper oxide layers-Human health-Planetary protection. Astrobiology 17, 1183-1191.

Charged particles, such as carbon ions, bear the promise of a more effective cancer therapy. In human spaceflight, exposure to charged particles represents an important risk factor for chronic and late effects such as cancer. Biological effects elicited by charged particle exposure depend on their characteristics, e.g., on linear energy transfer (LET). For diverse outcomes (cell death, mutation, transformation, and cell-cycle arrest), an LET dependency of the effect size was observed. These outcomes result from activation of a complex network of signaling pathways in the DNA damage response, which result in cell-protective (DNA repair and cell-cycle arrest) or cell-destructive (cell death) reactions. Triggering of these pathways converges among others in the activation of transcription factors, such as p53, nuclear factor κB (NF-κB), activated protein 1 (AP-1), nuclear erythroid-derived 2-related factor 2 (Nrf2), and cAMP responsive element binding protein (CREB). Depending on dose, radiation quality, and tissue, p53 induces apoptosis or cell-cycle arrest. In low LET radiation therapy, p53 mutations are often associated with therapy resistance, while the outcome of carbon ion therapy seems to be independent of the tumor’s p53 status. NF-κB is a central transcription factor in the immune system and exhibits pro-survival effects. Both p53 and NF-κB are activated after ionizing radiation exposure in an ataxia telangiectasia mutated (ATM)-dependent manner. The NF-κB activation was shown to strongly depend on charged particles’ LET, with a maximal activation in the LET range of 90–300 keV/μm. AP-1 controls proliferation, senescence, differentiation, and apoptosis. Nrf2 can induce cellular antioxidant defense systems, CREB might also be involved in survival responses. The extent of activation of these transcription factors by charged particles and their interaction in the cellular radiation response greatly influences the destiny of the irradiated and also neighboring cells in the bystander effect.

International audience; Purpose: Risk assessment of radiation exposure during long-term space missions requires the knowledge of the relative biological effectiveness (RBE) of space radiation components. Few data on gene transcription activation by different heavy ions are available, suggesting a dependence on linear energy transfer. The transcription factor Nuclear Factor kB (NF-kB) can be involved in cancerogenesis. Therefore, NF-kB activation by accelerated heavy ions of different linear energy transfer (LET) was correlated to survival.Materials and methods: NF-kB-dependent gene induction after exposure to heavy ions was detected in stably transfected human embryonic kidney 293 cells (HEK-pNF-kB-d2EGFP/Neo cells carrying a neomycin resistance), using the destabilized Enhanced Green Fluorescent Protein (d2EGFP) as reporter.Results: Argon (LET 272 keV/mm) and neon ions (LET 91 keV/mm) had the highest potential to activate NF-kB, resulting in a RBE of 8.9 in comparison to 150 kV X-rays. The RBE for survival also reached its maximum in this LET range, with a maximal value of 2.Conclusions: NF-kB might be involved in modulating survival responses of cells hit by heavy ions in the LET range of 91–272 keV/mm and could therefore become a factor to be considered for risk assessment of radiation exposure during space travel.

Carbon-ion cancer therapy offers several physical and radiobiological advantages over conventional photon cancer therapy. The molecular mechanisms that determine cellular outcome, including the activation of transcription factors and the alteration of gene expression profiles, after carbon-ion exposure are still under investigation. We have previously shown that argon ions (LET 272 keV/µm) had a much higher potential to activate the transcription factor nuclear factor κB (NF-κB) than X rays. NF-κB is involved in the regulation of cellular survival, mostly by antiapoptosis and cell cycle-regulating target genes, which are important in the resistance of cancer cells to radiotherapy. Therefore, activation of the NF-κB pathway by accelerated carbon ions (LET 33 and 73 keV/µm) was examined. For comparison, cells were exposed to 150 kV X rays and to accelerated carbon ions. NF-κB-dependent gene induction after exposure was detected in stably transfected human 293 reporter cells. Carbon ions and X rays had a comparable potential to activate NF-κB in human cells, indicating a comparable usefulness of pharmacological NF-κB inhibition during photon and carbon-ion radiotherapy.

Radiation is a potentially limiting factor for manned long-term space missions. Prolonged exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. Since apoptosis may be a mechanism the body uses to eliminate damaged cells, the induction by cosmic radiation of the nuclear anti-apoptotic transcription factor nuclear factor κB (NF-κB) could influence the cancer risk of astronauts exposed to cosmic radiation by improving the survival of radiation-damaged cells. In previous studies using a screening assay for the detection of NF-κB-dependent gene induction (HEK-pNF-κB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown [Baumstark-Khan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527–530, 2005]. Studies with NF-κB inhibitors can map functional details of the NF-κB pathway and the influence of radiation-induced NF-κB activation on various cellular outcomes such as survival or cell cycle arrest. In this work, the efficacy and cytotoxicity of four different NF-κB inhibitors, caffeic acid phenethyl ester (CAPE), capsaicin, the proteasome inhibitor MG-132, and the cell permeable peptide NF-κB SN50 were analyzed using HEK-pNF-κB-d2EGFP/Neo cells. In the recommended concentration range, only CAPE displayed considerable cytotoxicity. CAPE and capsaicin partially inhibited NF-κB activation by the cytokine tumor necrosis factor α. MG-132 completely abolished the activation and was therefore used for experiments with X-rays. NF-κB SN-50 could not reduce NF-κB dependent expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP). MG-132 entirely suppressed the X-ray induced NF-κB activation in HEK-pNF-κB-d2EGFP/Neo cells. In conclusion, the degradation of the inhibitor of NF-κB (IκB) in the proteasome is essential for X-ray induced NF-κB activation, and MG-132 will be useful in studies of the NF-κB pathway involvement in the cellular response to heavy ion exposure and other space-relevant radiation qualities.

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1 keV/μm, α-particles 116 keV/μm and 36Ar ions 270 keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.

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 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. The present paper discusses a variety of issues related to implementation of galactic cosmic ray (GCR) simulation at NSRL, especially for experiments in radiobiology. Advantages and disadvantages of different approaches to developing a GCR simulator are presented. In addition, issues common to both GCR simulation and single beam experiments are compared to issues unique to GCR simulation studies. A set of conclusions is presented as well as a discussion of the technical implementation of GCR simulation.

Cellular bioassays for detection of cyto- and genotoxicity are useful in the risk assessment of space environmental factors. Such bioassay systems have the potential complement the physical detector systems used in space, insofar as they yield intrinsically biologically weighted measures of cellular responses. The experiment Cellular Responses to Radiation in Space (CERASP) has been selected by NASA/ESA to be performed on the International Space Station. It will supply basic information on the cellular response to radiation applied in microgravity. One of the biological endpoints under investigation will be survival reflected by radiation-dependent reduction of constitutive expression of the enhanced variant of green fluorescent protein (EGFP), originally isolated from the bioluminescent jellyfish Aequorea victoria. In this ground based study, the usefulness of this approach in comparison to standard techniques (colony forming ability test, MTT test) is shown.

Space programmes are shifting towards planetary exploration and, in particular, towards missions by human beings to the Moon and to Mars. Radiation is considered to be one of the major hazards for personnel in space and has emerged as the most critical issue to be resolved for long-term missions both orbital and interplanetary. The two cosmic sources of radiation that could impact a mission outside the Earth's magnetic field are solar particle events (SPE) and galactic cosmic rays (GCR). Exposure to the types of ionizing radiation encountered during space travel may cause a number of health-related problems, but the primary concern is related to the increased risk of cancer induction in astronauts. Predictions of cancer risk and acceptable radiation exposure in space are extrapolated from minimal data and are subject to many uncertainties. The paper describes present-day estimates of equivalent doses from GCR and solar cosmic radiation behind various shields and radiation risks for astronauts on a mission to Mars.

Within the next decades, an increasing number of human beings will be brought into space to carry out technical and scientific tasks. There, they will be exposed simultaneously to combined stimuli, especially microgravity and radiation. In the endeavour to assess the risks for humans during long-duration space missions, it is necessary to understand already at the cellular level the complex interplay of these parameters. Cellular stress protection responses lead to an increased transcription of several genes via the modulation of transcription factors. The activation of the nuclear factor κB (NF-κB) pathway as a possible anti-apoptotic route represents such an important cellular stress response. It is hypothesized that the activation of NF-κB and the subsequent expression of NF-κB-dependent genes is involved in the cellular response to components of the cosmic radiation. Irradiation of human embryonic kidney cells (HEK/293) with α-particles (2.1 MeV, LET ∼160 keV/μm) was performed at the PTB, Braunschweig, Germany. Using the microbeam facility, cells were exposed to nuclear hits or, for the purpose of comparison, to a diffuse irradiation of the whole cell. After irradiation the following biological endpoints were determined: (i) cell survival (by means of the colony forming ability test), and (ii) quantitative RT-PCR analysis of selected NF-κB target genes (IκBα GADD45β, bcl-2, and bcl-XL). One nuclear α-particle traversal reduces the probability to survive to ∼75%. Exposure to two α-particles per nucleus resulted in an upregulation of the expression of the GADD45β gene. After exposure of HEK cells to five nuclear hits, about 43% of the irradiated cells survived, and the transcriptional response was not significant. Ten nuclear hits activated the IκBα expression, this increased IκBα production might be involved in the termination of the radiation-induced NF-κB activation. Diffuse irradiation increased the transcription of IκBα and GADD45β. Expression of the antiapoptotic genes bcl-2 and bcl-XL remained almost unchanged after α-particle irradiation. These findings suggest a role for GADD45β in the cellular radiation response to low fluences, while IκBα is enrolled at higher fluences.

Research on the effects of ionizing radiation exposure involves the use of real-time reverse transcription polymerase chain reaction (qRT-PCR) for measuring changes in gene expression. Several variables need to be controlled for gene expression analysis, such as different amounts of starting material between the samples, variations in enzymatic efficiencies of the reverse transcription step, and differences in RNA integrity. Normalization of the obtained data to an invariant endogenous control gene (reference gene) is the elementary step in relative quantification strategy. There is a strong correlation between the quality of the normalized data and the stability of the reference gene itself. This is especially relevant when the samples have been obtained after exposure to radiation qualities inducing different amounts and kinds of damage, leading to effects on cell cycle delays or even on cell cycle blocks. In order to determine suitable reference genes as internal controls in qRT-PCR assays after exposure to ionizing radiation, we studied the gene expression levels of nine commonly used reference genes which are constitutively expressed in A549 lung cancer cells. Expression levels obtained for ACTB, B2M, GAPDH, PBGD, 18S rRNA, G6PDH, HPRT, UBC, TFRC and SDHA were determined after exposure to 2 and 6 Gy X-radiation. Gene expression data for Growth arrest and damage-inducible gene 45 (GADD45α) and Cyclin-dependent kinase inhibitor 1A (CDKN1A/p21CIP1) were selected to elucidate the influence of normalization by using appropriate and inappropriate internal control genes. According to these results, we strongly recommend the use of a panel of reference genes instead of only one.

The transcription factor nuclear factor kappaB (NF-kappaB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF-kappaB in epithelial cells in response to different stresses, a cell-based screening assay for activation of NF-kappaB-dependent gene transcription in human embryonic kidney cells (HEK/293) was developed. This assay allows detection of NF-kappaB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell-based assay, activation of the pathway by several agents, for example, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF-kappaB activation by TNF-alpha were examined. NF-kappaB was activated by TNF-alpha, IL-1beta, PMA, and camptothecin in a dose-dependent manner, but not by LPS. TNF-alpha results in the strongest induction of NF-kappaB-dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20-35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF-kappaB activation or direct communication via gap junctions in the cell layer diminishing the TNF-alpha response.

Kunftige bemannte Weltraummissionen zum Mond und zu den Planeten unseres Sonnensystems erfordern die Entwicklung von Strategien zum Schutz vor moglichen gesundheitsschadigenden Effekten der kosmischen Strahlung. Abhangig von der Dosis kann Strahlenexposition das akute oder das chronische Strahlensyndrom oder Langzeitfolgen wie die Entstehung von Tumoren oder Katarakten auslosen. Eine vollstandige Abschirmung der kosmischen Strahlung bei Weltraummissionen ist technisch nicht machbar. Der beste Strahlenschutz im Weltraum basiert somit auf einer umsichtigen Planung der Mission bezuglich ihrer Dauer und der Zeit ihrer Durchfuhrung bezuglich des solaren Aktivitatszyklus. Fur eine Risikoabschatzung ist neben der Kenntnis der Korperdosis und deren Verteilungsmuster im Korper die Kenntnis der relativen biologischen Wirksamkeit (RBW) der Weltraumstrahlung notwendig. Fur diese dicht ionisierende Strahlung ist bisher wenig uber die zellularen Reaktionsketten bekannt.

Baumstark-Khan, C., Hellweg, C. E., Arenz, A. and Meier, M. M. Cellular Monitoring of the Nuclear Factor κB Pathway for Assessment of Space Environmental Radiation. Radiat. Res. 164, 527–530 (2005). A screening assay for the detection of NF-κB-dependent gene induction using the destabilized variant of the reporter protein enhanced green fluorescent protein (d2EGFP) is used for assessing the biological effects of accelerated heavy ions as a model of space environmental radiation conditions. The time course of d2EGFP expression and therefore of activation of NF-κB-dependent gene expression was measured after treatment with TNFA or after heavy-ion exposure using flow cytometry. The reported experiments clearly show that accelerated argon ions (95 MeV/nucleon, LET 230 keV/μm) induce the NF-κB pathway at low particle densities (1–2 particle hits per nucleus), which result in as few as 5–50 induced DSBs per cell.

High LET radiations, such as heavy ions or neutrons, have an increased biological effectiveness compared to X-rays for gene mutation, genomic instability and carcinogenesis. Estimating the biological risks from space radiation encountered by cosmonauts will continue to influence long term duration in space, such as the planned mission to Mars. The human radiation responsive genes CDKN1A (p21/WAF), GADD45α (GADD45), GADD45β (MyD118), RRM2b (p53R2) and BRCA2 (FancD1), involved in cell cycle control or damage repair, were screened for gene expression changes in MCF-7 cells by quantitative real-time reverse transcription PCR (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after irradiation with accelerated doses of 36-argon ions and X-rays. Examination of the expression profiles 2 and 12 h after exposure reveals a pattern consistent with a population of cells in the early response to DNA damage and invoking cell stress responses. Interesting new data showing different expression patterns according to the gene and the type of ionizing radiation used could be obtained. Results show, that the signaling and repair activities induced after heavy ion or X-ray exposure are not the same and gene expression patterns may become useful indicators for distinguishing different types of radiation in relation to their biological effects.