Christa Baumstark-Khan

Getting ready for the manned mission to Mars: the astronauts’ risk from space radiation

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.

The Influence of Microgravity on Repair of Radiation Induced DNA Damage in Bacteria and Human Fibroblasts

The influence of the space flight environment, above all microgravity, on the repair of radiation-induced DNA damage was examined during the Spacelab mission IML-2 as (1) rejoining of DNA strand breaks induced by X irradiation in cells of Escherichia coli B/r (120 Gy) and (2) in human fibroblasts (5 and 10 Gy); (3) induction of the SOS response after gamma irradiation (300 Gy) of cells of Escherichia coli PQ37; and (4) survival of spores of Bacillus subtilis HA 101 after UV irradiation (up to 340 J m(-2)). Cells were irradiated prior to the space mission and were kept frozen (E. coli and fibroblasts) until incubation for defined periods (up to 4.5 h) in orbit; thereafter they were frozen again for laboratory analysis. Germination and growth of spores of B. subtilis on membrane filters was initiated by humidification in orbit. Controls were performed in-flight (1g reference centrifuge) and on the ground (1g and 1.4g). We found no significant differences between the microgravity samples and the corresponding controls in the kinetics of DNA strand break rejoining and of the induction of the SOS response as well as in the survival curves (as proven by Students t test, P < or = 0.1). These observations provide evidence that in the microgravity environment cells are able to repair radiation-induced DNA damage almost normally. The results suggest that a disturbance of cellular repair processes in the microgravity environment might not be the explanation for the reported synergism of radiation and microgravity.

Generation of Stably Transfected Mammalian Cell Lines as Fluorescent Screening Assay for NF- B Activation-Dependent Gene Expression

Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor κB (NF-κB) pathway as a possible antiapoptotic route represents one important cellular stress response. To identify conditions that are capable of modifying this pathway, a screening assay for detection of NF-κB-dependent gene activation using the reporter protein Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) was developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing 4 copies of the NF-κB response element. Treatment with tumor necrosis factor α (TNF-α) gave rise to substantial EGFP/d2EGFP expression in up to 90% of the cells and was therefore used to screen different stably transfected clones for induction of NF-κB-dependent gene expression. The time course of NF-κB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-κB-ELISA. NF-κB binding in-creased after 15-min incubation with TNF-α. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-κB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-α to be caused by the activation of NF-κB. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening. (Journal of Biomolecular Screening 2003:511-521)

Life under Conditions of Ionizing Radiation

Life on Earth, throughout its almost 4 billion years history, has been shaped by interactions of the organisms with their environment. It has developed with an ever present radiation background. As a powerful mutagen it has contributed to biological evolution, however, it is potentially destructive for individual cells and organisms [1]. Radiation response differs extremely for different organisms and there is not necessarily a correlation between species radiation resistance and levels of exposure in the natural environment [2].

Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair‐induced DNA Strand Breaks in UV‐irradiated Mammalian Cells¶

Abstract Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray–induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20°C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).

HUMEX, a study on the survivability and adaptation of humans to long-duration exploratory missions, part I: lunar missions.

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.

Microscale application of the SOS-LUX-TEST as biosensor for genotoxic agents

The bacterial biodetection system SOS-LUX-TEST has been developed for rapid detection of environmental genotoxins. This cellular bioassay is based on the receptor reporter principle with the SOS system as receptor sensitive to DNA damage and the bioluminescence system giving the optical signal which is registered by an appropriate detector. The lux-operon of a marine photobacteria (Photobacterium leiognathi) containing the whole information for the synthesis of the bacterial luciferase and its substrate was cloned downstream of a SOS-controlled promoter and introduced on a plasmid into E. coli. This system reacts in a dose-dependent manner with bioluminescence to any agent like radiation or chemicals which produce damages to the DNA molecules inside these cells. For the measurements of the genotoxic potential of chemical compounds a microplate luminometer was employed which allows continuous incubation and shaking during the registration of the signal. The kinetics of bioluminescence was always monitored over a period of at least 5 h. Typical model compounds with various DNA damaging potencies were tested.

Application of the Lux-Fluoro test as bioassay for combined genotoxicity and cytotoxicity measurements by means of recombinant Salmonella typhimurium TA1535 cells ☆

Abstract The Lux-Fluoro test is a bioassay, which coincidentally measures the cytotoxic and genotoxic potency of a given substance based on the receptor reporter principle. For genotoxicity testing, bioluminescence occurs as a response to the presence of DNA-damaging agents. It is brought about by the induction of the promoterless luxCDABFE genes of Photobacterium leiognathi as reporter component under the control of a strong SOS promoter as receptor component. At concentrations of DNA-damaging agents which only scarcely affect cell survival, a high level of light production is induced. For testing cytotoxic agents viability of the bacteria is included as a test parameter describing the cytotoxicity of the agents. For that reason, Salmonella typhimurium TA1535 cells transformed with the bacterial protein expression vector pGFPuv were employed. This plasmid controls green fluorescent protein (GFP) expression by the lac promoter in TA1535 cells constitutively. A panel of recombinant S. typhimurium strains carrying either the SOS-Lux-plasmid (TA1535-pPLS-1) or the fluorescence mediating lac-GFPuv plasmid (TA1535-pGFPuv) was used to record in parallel agents (mitomycin C, chloramphenicol, doxorubicin hydrochloride, bleomycin sulphate and hydrogen peroxide) that effect the genetic material conveying either genotoxic, cytotoxic or geno- and cytotoxic effects. The light and fluorescence transmissions of untreated and chemical-treated cells were measured in a microtiter plate reader and luminescence induction factors (Fi) as well as fluorescence deduction factors (Fd) were calculated for the genotoxic and cytotoxic potential of the applied agents.

Cellular Monitoring of the Nuclear Factor κB Pathway for Assessment of Space Environmental Radiation

Abstract 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.

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

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 micrometers-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 micrometers-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.