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For the safety of astronauts and to ensure the stability and integrity of the genome of microorganisms and plants used in bioregenerative life support systems, it is important to improve our knowledge of the combined action of (space) radiation and microgravity. The SOS-LUX-TOXICITY test, as part of the TRIPLE-LUX project (accepted for flight at Biolab in Columbus on the International Space Station, (ISS)), will provide an estimation of the health risk resulting from exposure of astronauts to the radiation environment of space in microgravity. The project will: (i) increase our knowledge of biological/health threatening action of space radiation and enzymatic DNA repair; (ii) uncover cellular mechanisms of synergistic interaction of microgravity and space radiation; (iii) provide specified biosensors for spacecraft milieu examination; and (iv) provide experimental data on stability and integrity of bacterial DNA in spacecrafts. In the bacterial biosensor "SOS-LUX-Test" developed at DLR (patent), bacteria are transformed with the pBR322-derived plasmid pPLS-1 or the similar, advanced plasmid SWITCH, both carrying the promoterless lux operon of Photobacterium leiognathi as the reporter element controlled by a DNA damage-dependent SOS promoter as sensor element. A short description of the space experiment is given, and the current status of adaptation of the SOS-LUX-Test to the ISS, i.e. first results of sterilization, biocompatibility and functional tests performed with the already available hardware and bread board model of the automated space hardware under development, is described here.

A screening assay for the detection of NF-kappaB-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-kappaB-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/microm) induce the NF-kappaB pathway at low particle densities (1-2 particle hits per nucleus), which result in as few as 5-50 induced DSBs per cell.

For applications in field experiments, the recombinant strain Salmonella typhimurium TA1535 was immobilized to permit its immediate utilization after long storage periods. Salmonella typhimurium TA1535 cells contain the plasmid that has an inducible SOS promoter fused to a promoterless luxCDABFE operon from Photobacterium leiognathi. The induction of bioluminescence occurs in the presence of the DNA-damaging agent mitomycin C which stimulates the bacterial SOS response. Early stationary phase cells were immobilized at a cell concentration of 10(10) CFU/ml in microtiter plates and stored up to 6 weeks at 4 degrees C in a sealed container. Even after 4 weeks of storage, the bioluminescence kinetics and yield in response to different concentrations of mitomycin C were not significantly different from those of freshly prepared samples.

The recombinant Salmonella typhimurium TA1535 strain carrying the SWITCH plasmid (combined construct of the SOS-Lux plasmid pPLS-1 and the LAC-Fluoro plasmid pGFPuv: pSWITCH) was treated with control substances for genotoxic (2-aminoanthracene) and cytotoxic (aureomycin) potency as well as with 18 environmental samples (groundwater, river water, sediments) provided at the SENSPOL Technical Meeting on Problems Related to Diffuse Pollution Sources (Characterization of Sediment, Dredged Material, and Groundwater) organized by the Federal Institute of Hydrology in Koblenz, Germany, during late October 2003. For metabolic activation the samples were treated with S9 mix (5% S9 fraction in cofactor mix, Aroclor 1254-induced rat liver microsomes, Moltox Inc., Mol, Belgium). Simultaneously determined cytotoxicity and genotoxicity data were derived through the Multilabel Counter 1420 Victor (PerkinElmer, Boston, MA, USA), by sequential measurement of luminescence, absorbance, and fluorescence. The newly designed SWITCH test, as it was applied at the Koblenz meeting, displays a comparable sensitivity for test samples with known cyto- and genotoxic potential. Groundwater samples from the wells at the former gas plant site Kiel Canal and the agricultural area at Niederwerth expressed neither genotoxic nor cytotoxic responses of the bacteria for both metabolic conditions (+/-S9). Spiked groundwater samples from the Niederwerth well BW1 and the Urmitz well U12 located on the river Rhine were identified to be positive in terms of genotoxicity for the direct and the metabolic approach. Samples from the lake Tiefer See in the city of Potsdam showed a reduction of GFPuv expression as an indication for cytotoxicity, while luminescence output of incubated bacteria remained unaffected. This reflects the well-known presence of contaminations (especially cyanides) in the lake sediment as well as in the acetonic extract. The results obtained at the SENSPOL Technical Meeting show the SWITCH test to be of major relevance not only for the analysis of chemicals under laboratory conditions but also for environmental samples polluted by diffuse industrial sources.

Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor kappaB (NF-kappaB) 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-kappaB-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-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) 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-kappaB-dependent gene expression. The time course of NF-kappaB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-kappaB-ELISA. NF-kappaB binding in-creased after 15-min incubation with TNF-alpha. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-kappaB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-alpha to be caused by the activation of NF-kappaB. 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.

Spores of Bacillus subtilis were exposed to space in the BIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite. The spores were exposed either in dry layers without any protecting agent, or mixed with clay, red sandstone, Martian analogue soil or meteorite powder, in dry layers as well as in so-called 'artificial meteorites', i.e. cubes filled with clay and spores in naturally occurring concentrations. After about 2 weeks in space, their survival was tested from the number of colony formers. Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases < or = 10(-6)). The same low survival was obtained behind a thin layer of clay acting as an optical filter. The survival rate was increased by 5 orders of magnitude and more, if the spores in the dry layer were directly mixed with powder of clay, rock or meteorites, and up to 100% survival was reached in soil mixtures with spores comparable to the natural soil to spore ratio. These data confirm the deleterious effects of extraterrestrial solar UV radiation. Thin layers of clay, rock or meteorite are only successful in UV-shielding, if they are in direct contact with the spores. The data suggest that in a scenario of interplanetary transfer of life, small rock ejecta of a few cm in diameter could be sufficiently large to protect bacterial spores against the intense insolation; however, micron-sized grains, as originally requested by Panspermia, may not provide sufficient protection for spores to survive. The data are also pertinent to search for life on Mars and planetary protection considerations for future missions to Mars.

In the endeavour to assess radiation risks for humans in space the concerted action of all stimuli (e.g. radiation and microgravity) has to be known already at a cellular level. The introduction of reporter genes into mammalian cells which allows the visualisation of modified gene expression levels, signal transduction rates and cell metabolism activities will supply basic information on the cellular response to space radiation. The cloning of the gene for green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its subsequent expression in heterologous systems has established GFP as a unique genetic reporter system for use in a variety of organisms. Unlike other reporters, GFP fluorescence emerges in the absence of substrates or cofactors and allows for non-invasive monitoring in living and in paraformaldehyde-fixed cells. Enhancement of wild-type GFP by human codon optimisation and fluorophore mutation (EGFP) resulted in higher expression levels in mammalian cells and brighter fluorescence. The suitability of EGFP for gene expression studies to be performed on the ISS is shown for recombinant mammalian cells in response to UVC exposure.

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 degrees 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).

DNA damage in X-irradiated CHO cells was measured by alkaline filter elution and compared to fluorometric analysis of DNA unwinding (FADU). The FADU method proved to be as sensitive as the alkaline filter elution technique in detecting X-ray induced DNA breaks. Strand break induction was also measured after treatment with four radical generating chemicals (hydrogen peroxide, bleomycin, mitomycin C and methyl viologen) using the FADU technique.

The effect of aphidicolin, a specific inhibitor of DNA polymerases alpha and delta, on recovery and repair of X-irradiated human fibroblasts was investigated. Aphidicolin concentrations from 0.5 to 5 micrograms/ml reduce DNA synthesis in exponentially growing cells after short incubation periods (1 h), longer incubation times (24 h) enhance this effect. Colony forming ability of unirradiated fibroblasts, treated in confluent growth state with aphidicolin, was not reduced. After incubation with aphidicolin contact-inhibited fibroblasts show reduced PLD recovery. For the short preincubation time (1 microgram/ml) the effect depends on aphidicolin concentration, for the long preincubation period the effect is independent of concentration. DNA double-strand break induction and rejoining was investigated after combined action of aphidicolin and X-rays using the neutral filter elution technique. The yield of initial DNA double-strand breaks was not affected by aphidicolin. After a 90-min recovery time, however, DNA double-strand break rejoining was markedly reduced. This effect was dependent on concentration for the short incubation time (1 h) but not for the long preincubation (24 h).

A staining procedure that detects sister chromatid exchanges has been used to examine the response of chromosomes in cultured human fibroblasts to a series of chemical and physical mutagens. The test represents a sensitive and reproducible method for detecting mutagenicity on the chromosome level. It provides a powerful method for the detection of environmental mutagens and can be used in ocular toxicity studies.

Formation of the complete spindles during the budding process of Saccharomyces uvarum was investigated by fluorescence microscopy of protoplasted cells. Protoplasts were treated with anti-tubulin antibodies and DAPI, a fluorescent dye staining DNA. Thus, both chromatin and spindles could be visualized. Duplication as well as formation of separated spindle pole bodies during the different stages of budding are documented, demonstrating the occurrence and behaviour of microtubules during yeast cell cycle.

X-irradiated yeast cells (Saccharomyces uvarum) grown in liquid media stop mitosis and form giant cells. Chitin ring formation, being a prerequisite for cell separation, was studied by fluorescence microscopy using calcofluor white, a chitin specific dye. Experiments with inhibitors of DNA synthesis (hydroxyurea) and chitin synthesis (polyoxin D) demonstrate chitin ring formation to be dependent on DNA synthesis, whereas bud formation is independent of DNA synthesis and chitin ring formation respectively. Basing on these results the formation of X-ray induced giant cells implies one DNA replication which in turn induces the formation of only one chitin ring between mother cell and giant bud. Obviously no septum can be formed. Thus cell separation does not occur, but the bud already formed, produces another bud demonstrating that bud formation itself is independent of DNA synthesis.

Thin sections of budding yeast cells and giant gells grown after X-irradiation have been examined by electron microscopy. The different steps of cross-wall formation during budding were documented with unirradiated cells. With X-ray induced giant cells cytokinesis was shown to be absent. Neither primary nor secondary septae appeared thus cell separation did not occur. Despite this fact both macromolecular synthesis and bud growth continued, giving rise to the formation of giant cells.

Spindle formation and nuclear division of budding and irradiated yeast cells (Saccharomyces uvarum) was investigated by fluorescence microscopy of protoplasted cells. Protoplasts were treated with antitubulin antibodies and DAPI, a fluorescent dye staining DNA. In budding yeast cells, duplication of spindle pole bodies as well as formation of complete 1-micron spindles and elongated 8-micron spindles were documented. In X-irradiated cells, spindle pole bodies were duplicated as well, forming the complete 1-micron spindle. Nuclei of giant cells have lost the elongation ability and remain in a "normal" G2-phase state, thus preventing nuclear as well as cellular division.