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The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ−1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV μ−1 no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification. [Copyright &y& Elsevier]

In the 21st century, an increasing number of astronauts will visit the International Space Station (ISS) for prolonged times. Therefore it is of utmost importance to provide necessary basic knowledge concerning risks to their health and their ability to work on the station and during extravehicular activities (EVA) in free space. It is the aim of one experiment of the German project TRIPLE-LUX (to be flown on the ISS) to provide an estimation of health risk resulting from exposure of the astronauts to the radiation in space inside the station as well as during extravehicular activities on one hand, and of exposure of astronauts to unavoidable or as yet unknown ISS-environmental genotoxic substances on the other. The project will (i) provide increased knowledge of the biological action of space radiation and enzymatic repair of DNA damage, (ii) uncover cellular mechanisms of synergistic interaction of microgravity and space radiation and (iii) examine the space craft milieu with highly specific biosensors. For these investigations, the bacterial biosensor SOS-LUX-LAC-FLUORO-Toxicity-test will be used, combining the SOS-LUX-Test invented at DLR Germany (Patent) with the commercially available LAC-FLUORO-Test. The SOS-LUX-Test comprises genetically modified bacteria transformed with the pBR322-derived plasmid pPLS-1. This plasmid carries the promoterless lux operon of Photobacterium leiognathi as a reporter element under control of the DNA-damage dependent SOS promoter of ColD as sensor element. This system reacts to radiation and other agents that induce DNA damages with a dose dependent measurable emission of bioluminescence of the transformed bacteria. The analogous LAC-FLUORO-Test has been developed for the detection of cellular responses to cytotoxins. It is based on the constitutive expression of green fluorescent protein (GFP) mediated by the bacterial protein expression vector pGFPuv (Clontech, Palo Alto, USA). In response to cytotoxic agents, this system reacts with a dose-dependent reduction of GFP-fluorescence. Currently, a fully automated miniaturized hardware system for the bacterial set up, which includes measurements of luminescence and fluorescence or absorption and the image analysis based evaluation is under development. During the first mission of the SOS-LUX-LAC-FLUORO-Toxicity-Test on the ISS, a standardized, DNA-damaging radiation source still to be determined will be used as a genotoxic inducer. A panel of recombinant Salmonella typhimurium strains carrying either the SOS-LUX plasmid or the fluorescence-mediating lac-GFPuv plasmid will be used to determine in parallel on one microplate the genotoxic and the cytotoxic action of the applied radiation in combination with microgravity. Either in addition to or in place of the fluorometric measurements of the cytotoxic agents, photometric measurements will simultaneously monitor cell growth, giving additional data on survival of the cells. The obtained data will be available on line during the TRIPLE-LUX mission time. Though it is the main goal during the TRIPLE-LUX mission to measure the radiation effect in microgravity, the SOS-LUX-LAC-FLUORO-Toxicity-test in principle is also applicable as a biomonitor for the detection and measurement of genotoxic substances in air or in the (recycled) water system on the ISS or on earth in general. [Copyright &y& Elsevier]

Abstract: 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 (2.1MeV, LET ∼160keV/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. [Copyright &y& Elsevier]

Abstract: 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 6Gy 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. [Copyright &y& Elsevier]

Abstract: The combined action of ionizing radiation and microgravity will continue to influence future manned space missions, with special risks for astronauts on the Moon surface or for long duration missions to Mars. There is increasing evidence that basic cellular functions are sensitive not only to radiation but also to microgravity. Previous space flight experiments gave contradictory results: from inhibition of DNA repair by microgravity to enhancement, whereas others did not detect any influence of microgravity on repair. At the Radiation Biology Department of the German Aerospace Center (DLR), recombinant bacterial and mammalian cell systems were developed as reporters for cellular signal transduction modulation by genotoxic environmental conditions. The space experiment “Cellular Responses to Radiation in Space” (CERASP) to be performed at the International Space Station (ISS) will make use of such reporter cell lines thereby supplying basic information on the cellular response to radiation applied in microgravity. One of the biological endpoints will be survival reflected by radiation-dependent reduction of constitutive expression of the enhanced variant of green fluorescent protein (EGFP). A second end-point will be gene activation by space flight conditions in mammalian cells, based on fluorescent promoter reporter systems using the destabilized d2EGFP variant. The promoter element to be investigated reflects the activity of the nuclear factor kappa B (NF-κB) pathway. The NF-κB family of proteins plays a major role in the inflammatory and immune response, cell proliferation and differentiation, apoptosis and tumor genesis. Results obtained with X-rays and accelerated heavy ions produced at the French heavy ion accelerator GANIL imply that densely ionizing radiation has a stronger potential to activate NF-κB dependent gene expression than sparsely ionizing radiation. The correlation of NF-κB activation to negative regulation of apoptosis could favor survival of cells with damaged DNA. A third endpoint to be examined will be DNA damage induced by combined exposure to radiation and microgravity and its repair. In the current work, preparatory experiments for the space experiment CERASP were performed. For radiation exposure on the ISS, an artificial radiation source is necessary since long-term exposure to cosmic radiation of frozen cells for damage accumulation will not be feasible. The biological activity of the designated space radiation source, the β-emitter promethium-147, was evaluated. Different shielding scenarios according to the experiment and safety requirements were evaluated. As growth surface for the human embryonic kidney cells, polytetrafluoroethylene and polyolefin foils were tested. For protection issues, the shielding effect of titanium foils was evaluated. With the prototype Pm-147 radiation source, the requirements of CERASP can be fulfilled with cells growing on the polytetrafluoroethylene foil and titanium foils for safety issues. In this setting, β-rays activated NF-κB-dependent reporter gene expression in human embryonic kidney cells. Regarding cell survival and NF-κB activation, the Pm-147 radiation source meets the requirements of the space experiment CERASP. [Copyright &y& Elsevier]

Abstract: Space exploration programmes, currently under discussion in the US and in Europe, foresee human missions to Mars to happen within the first half of this century. In this context, the European Space Agency (ESA) has conducted a study on the human responses, limits and needs for such exploratory missions, the so-called HUMEX study (ESA SP-1264). Based on a critical assessment of the limiting factors for human health and performance and the definition of the life science and life support requirements performed in the frame of the HUMEX study, the following major critical items have been identified: (i) radiation health risks, mainly occurring during the interplanetary transfer phases and severely augmented in case of an eruption of a solar particle event; (ii) health risks caused by extended periods in microgravity with an unacceptable risk of bone fracture as a consequence of bone demineralisation; (iii) psychological risks as a consequence of long-term isolation and confinement in an environment so far not experienced by humans; (iv) the requirement of bioregenerative life support systems complementary to physico-chemical systems, and of in situ resource utilisation to reach a closure of the life support system to the highest degree possible. Considering these constraints, it has been concluded that substantial research and development activities are required in order to provide the basic information for appropriate integrated risk managements, including efficient countermeasures and tailored life support. Methodological approaches should include research on the ISS, on robotic precursors missions to Mars, in ground-based simulation facilities as well as in analogue natural environments on Earth. [Copyright &y& Elsevier]

The European Space Agency has recently initiated a study of the human responses, limits and needs with regard to the stress environments of interplanetary and planetary missions. Emphasis has been laid on human health and performance care as well as advanced life support developments including bioregenerative life support systems and environmental monitoring. The overall study goals were as follows: (i) to define reference scenarios for a European participation in human exploration and to estimate their influence on the life sciences and life support requirements; (ii) for selected mission scenarios, to critically assess the limiting factors for human health, wellbeing, and performance and to recommend relevant countermeasures; (iii) for selected mission scenarios, to critically assess the potential of advanced life support developments and to propose a European strategy including terrestrial applications; (iv) to critically assess the feasibility of existing facilities and technologies on ground and in space as testbeds in preparation for human exploratory missions and to develop a test plan for ground and space campaigns; (v) to develop a roadmap for a future European strategy towards human exploratory missions, including preparatory activities and terrestrial applications and benefits. This paper covers the part of the HUMEX study dealing with lunar missions. A lunar base at the south pole where long-time sunlight and potential water ice deposits could be assumed was selected as the Moon reference scenario. The impact on human health, performance and well being has been investigated from the view point of the effects of microgravity (during space travel), reduced gravity (on the Moon) and abrupt gravity changes (during launch and landing), of the effects of cosmic radiation including solar particle events, of psychological issues as well as general health care. Countermeasures as well as necessary research using ground-based test beds and/or the International Space Station have been defined. Likewise advanced life support systems with a high degree of autonomy and regenerative capacity and synergy effects were considered where bioregenerative life support systems and biodiagnostic systems become essential. Finally, a European strategy leading to a potential European participation in future human exploratory missions has been recommended. [Copyright &y& Elsevier]

Human missions to Mars are planned to happen within this century. Activities associated therewith will interact with the environment of Mars in two reciprocal ways: (i) the mission needs to be protected from the natural environmental elements that can be harmful to human health, the equipment or to their operations; (ii) the specific natural environment of Mars should be protected so that it retains its value for scientific and other purposes. The following environmental elements need to be considered in order to protect humans and the equipment on the planetary surface: (i) cosmic ionizing radiation, (ii) solar particle events; (iii) solar ultraviolet radiation; (iv) reduced gravity; (v) thin atmosphere; (vi) extremes in temperatures and their fluctuations; and (vii) surface dust. In order to protect the planetary environment, the requirements for planetary protection as adopted by COSPAR for lander missions need to be revised in view of human presence on the planet. Landers carrying equipment for exobiological investigations require special consideration to reduce contamination by terrestrial microorganisms and organic matter to the greatest feasible extent. Records of human activities on the planet''s surface should be maintained in sufficient detail that future scientific experimenters can determine whether environmental modifications have resulted from explorations. [Copyright &y& Elsevier]