Overview of the Liulin type instruments for space radiation measurement and their scientific results.

Ionizing radiation is recognized to be one of the main health concerns for humans in the space radiation environment. Estimation of space radiation effects on health requires the accurate knowledge of the accumulated absorbed dose, which depends on the global space radiation distribution, solar cycle and local shielding generated by the 3D mass distribution of the space vehicle. This paper presents an overview of the spectrometer-dosimeters of the Liulin type, which were developed in the late 1980s and have been in use since then. Two major measurement systems have been developed by our team. The first one is based on one silicon detector and is known as a Liulin-type deposited energy spectrometer (DES) (Dachev et al., 2002, 2003), while the second one is a dosimetric telescope (DT) with two or three silicon detectors. The Liulin-type instruments were calibrated using a number of radioactive sources and particle accelerators. The main results of the calibrations are presented in the paper. In the last section of the paper some of the most significant scientific results obtained in space and on aircraft, balloon and rocket flights since 1989 are presented.

Sensitivity to polychromatic UV-radiation of strains of Deinococcus radiodurans differing in their DNA repair capacity.

PURPOSE: To characterize the ultraviolet (UV) sensitivity and establish the UV-induced DNA damage profile of cells of four Deinococcus radiodurans strains. The investigated strains differ in their radiation susceptibility, leading to a classification into a UV-sensitive (UVS78 and 1R1A) and a UV-resistant class (wild type strain R1 and 262). MATERIALS AND METHODS: Deinococcus radiodurans cells were exposed in suspension to monochromatic 254 nm (UV-C) and polychromatic UV radiations; the surviving fraction was determined by assessing the ability of the bacteria to form colonies. The UV-induced DNA lesions were measured quantitatively using an accurate and highly specific assay that involves the combination of high performance liquid chromatography (HPLC) with tandem mass spectrometry detection. RESULTS: Analysis of the DNA photoproducts showed that the TC (6-4) photoproduct and the TT and TC cyclobutane dimers were the major lesions induced by UV-C and UV-(>200 nm)-radiation. The UV-sensitive class was approx. 10 times more susceptible to UV-C and UV-(>200 nm)-radiations than the resistant class. Interestingly, the survival curves of all investigated strains become similar with longer UV wavelengths in the UV-(>315 nm)-radiation range. This observation suggests that the repair mechanisms of the UV-resistant class are not specifically effective for damage produced by UV of the >315 nm range. However, the initial amount of DNA photoproducts produced upon irradiation was found to be the same in resistant and sensitive strains for each wavelength range. CONCLUSION: Compared to mammalian cells, the DNA of Deinococcus radiodurans cells is less susceptible to the photo-induced formation of thymine cyclobutane dimers as inferred from comparative analysis. The ongoing investigations may contribute to a better understanding of the mechanism of DNA photoprotection against the direct effects of UV radiation. This may be of interest in the present context of a possible continuous decrease in the ozone layer thickness.

Biological space experiments for the simulation of Martian conditions: UV radiation and Martian soil analogues.

The survivability of resistant terrestrial microbes, bacterial spores of Bacillus subtilis, was investigated in the BIOPAN facility of the European Space Agency onboard of Russian Earth-orbiting FOTON satellites (BIOPAN I -III missions). The spores were exposed to different subsets of the extreme environmental parameters in space (vacuum, extraterrestrial solar UV, shielding by protecting materials like artificial meteorites). The results of the three space experiments confirmed the deleterious effects of extraterrestrial solar UV radiation which, in contrast to the UV radiation reaching the surface of the Earth, also contains the very energy-rich, short wavelength UVB and UVC radiation. Thin layers of clay, rock or meteorite material were shown to be only successful in UV-shielding, if they are in direct contact with the spores. On Mars the UV radiation climate is similar to that of the early Earth before the development of a protective ozone layer in the atmosphere by the appearance of the first aerobic photosynthetic bacteria. The interference of Martian soil components and the intense and nearly unfiltered Martian solar UV radiation with spores of B. subtilis will be tested with a new BIOPAN experiment, MARSTOX. Different types of Mars soil analogues will be used to determine on one hand their potential toxicity alone or in combination with solar UV (phototoxicity) and on the other hand their UV protection capability. Two sets of samples will be placed under different cut-off filters used to simulate the UV radiation climate of Mars and Earth. After exposure in space the survival of and mutation induction in the spores will be analyzed at the DLR, together with parallel samples from the corresponding ground control experiment performed in the laboratory. This experiment will provide new insights into the principal limits of life and its adaptation to environmental extremes on Earth or other planets which and will also have implications for the potential for the evolution and distribution of life.

Critical issues in connection with human missions to Mars: protection of and from the Martian environment.

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.

Exposure of arctic field scientists to ultraviolet radiation evaluated using personal dosimeters.

During July 2000 we used an electronic personal dosimeter (X-2000) and a biological dosimeter (Deutsches Zentrum für Luft- und Raumfahrt: Biofilm) to characterize the UV radiation exposure of arctic field scientists involved in biological and geological fieldwork. These personnel were working at the Haughton impact structure on Devon Island (75 degrees N) in the Canadian High Arctic under a 24 h photoperiod. During a typical day of field activities under a clear sky, the total daily erythemally weighted exposure, as measured by electronic dosimetry, was up to 5.8 standard erythemal dose (SED). Overcast skies (typically 7-8 okta of stratus) reduced exposures by a mean of 54%. We estimate that during a month of field activity in July a typical field scientist at this latitude could potentially receive approximately 80 SED to the face. Because of body movements the upper body was exposed to a UV regimen that often changed on second-to-second time-scales as assessed by electronic dosimetry. Over a typical 10 min period on vehicle traverse, we found that erythemal exposure could vary to up to 87% of the mean exposure. Time-integrated exposures showed that the type of outdoor field activities in the treeless expanse of the polar desert had little effect on the exposure received. Although absolute exposure changed in accordance with the time of day, the exposure ratio (dose received over horizontal dose) did not vary much over the day. Under clear skies the mean exposure ratio was 0.35 +/- 0.12 for individual activities at different times of the day assessed using electronic dosimetry. Biological dosimetry showed that the occupation was important in determining daily exposures. In our study, scientists in the field received an approximately two-fold higher dose than individuals, such as medics and computer scientists, who spent the majority of their time in tents.

Critical issues in connection with human planetary missions: protection of and from the environment.

Activities associated with human missions to the Moon or to Mars will interact with the environment 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 the Moon or 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; (vii) surface dust; (viii) impacts by meteorites and micrometeorites. 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. Grant numbers: 14056/99/NL/PA.

The history of the UV radiation climate of the earth--theoretical and space-based observations.

In the Archean era (3.8-2.5 Ga ago) the Earth probably lacked a protective ozone column. Using data obtained in the Earth's orbit on the inactivation of Bacillus subtilis spores we quantitatively estimate the potential biological effects of such an environment. We combine this practical data with theoretical calculations to propose a history of the potential UV stress on the surface of the Earth over time. The data suggest that an effective ozone column was established at a pO2 of approximately 5 x 10(-3) present atmospheric level. The improvement in the UV environment on the early Proterozoic Earth might have been a much more rapid event than has previously been supposed, with DNA damage rates dropping by two orders of magnitude in the space of just a few tens of millions of years. We postulate that a coupling between reduced UV stress and increased pO2 production could have contributed toward a positive feedback in the production of ozone in the early Proterozoic atmosphere. This would contribute to the apparent rapidity of the oxidation event. The data provide an evolutionary perspective on present-day Antarctic ozone depletion.

Enhanced green fluorescent protein (EGFP) for space radiation research using mammalian cells in the International Space Station.

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.

Microgravity inhibits intestinal calcium absorption as shown by a stable strontium test.

BACKGROUND: Little is known about the onset and degree of biochemical and functional alterations in calcium metabolism during microgravity. OBJECTIVE: To evaluate the effect of microgravity on intestinal calcium absorption and calcium-regulating hormones under metabolic ward conditions. MATERIALS AND METHODS: Fractional calcium absorption (Fc240 in percentage of dose administered) was determined pre-flight, in-flight and post-flight, by use of a stable strontium test in one cosmonaut who spent 20 days in space. Moreover, a sequence of blood samples was collected for the determination of serum parathyroid hormone (PTH), 25-hydroxyvitamin D, calcitriol and serum C-telopeptide (CTx, biomarker of bone resorption) levels. During all periods of data collection, calcium intake was held constant at a minimum level of 1.000 mg day(-1) and a daily supplement of 16.6 microg vitamin D2 was given. Personal ultraviolet (UV) light exposure was measured during the whole mission using a biologically weighting UV dosimeter. RESULTS: Fc240 was markedly reduced on flight day 19 (4.4%) as compared to pre-flight and post-flight data (13.4% and 17.2%, respectively). Serum calcitriol levels fell from 40.6 pg mL(-1) (mean pre-flight level) to 1.3 pg mL(-1) on flight day 18 and returned into the normal range after recovery. Serum CTx increased during the flight, while serum PTH and 25-hydroxyvitamin D levels did not change significantly. CONCLUSIONS: Intestinal calcium absorption can be diminished after only three weeks of microgravity. Changes are associated with a severe suppression of circulating calcitriol levels, but are independent of exogenous vitamin D supply and serum PTH levels.

Comparisons of spore dosimetry and spectral photometry of solar-UV radiation at four sites in Japan and Europe.

In order to develop monitoring and assessment systems of biologically effective doses of solar-UV radiation, concurrent measurements of spectral photometry and spore dosimetry were conducted in summer months at four sites in Japan and Europe. Effectiveness spectra were derived by multiplying spectral irradiance in 0.5 nm steps between 290 and 400 nm with the inactivation efficiency of the spores determined using monochromatic radiation of fine wavelength resolution. Shapes of the effectiveness spectra were very similar at the four sites exhibiting major peaks at 303.5, 305.0, 307.5 and 311.0 nm. The dose rates for spore inactivation from direct survival measurements and from calculations by the integration of the effectiveness spectra were compared for 174 data points. The ratios (observed/calculated) of the two values were concordant with a mean of 1.26 (+/- 0.24 standard deviation [SD]). The possible causes for the variations and slightly larger observed values are discussed.

Quantification of biologically effective environmental UV irradiance.

To determine the impact of environmental UV radiation on human health and ecosystems demands monitoring systems that weight the spectral irradiance according to the biological responses under consideration. In general, there are three different approaches to quantify a biologically effective solar irradiance. (i) weighted spectroradiometry where the biologically weighted radiometric quantities are derived from spectral data by multiplication with an action spectrum of a relevant photobiological reaction, e.g. erythema, DNA damage, skin cancer, reduced productivity of terrestrial plants and aquatic foodweb, (ii) wavelength integrating chemical-based or physical dosimetric systems with spectral sensitivities similar to a biological response curve, and (iii) biological dosimeters that directly weight the incident UV components of sunlight in relation to the effectiveness of the different wavelengths and to interactions between them. Most biological dosimeters, such as bacteria, bacteriophages, or biomolecules, are based on the UV sensitivity of DNA. If precisely characterized, biological dosimeters are applicable as field and personal dosimeters.

Spore dosimetry of solar UV radiation: applications to monitoring of daily irradiance and personal exposure.

Environmental UV radiation can be quantified using spore dosimetry, which measures the inactivation of repair-deficient Bacillus subtilis spores dried on a membrane filter. The system exhibits highly selective sensitivity to UV radiation, not being affected by various environmental adversities, such as high and low temperature and humidity. Biologically-effective dose rate and cumulative dose of ambient radiation are measurable under various conditions at various places on the earth, including tropical, temperate, and polar sites. Applications to monitor the exposure at the surface of organisms including humans and plants have also been advanced.

Biologically weighted measurement of UV radiation in space and on Earth with the biofilm technique.

Biological dosimetry has provided experimental proof of the high sensitivity of the biologically effective UVB doses to changes in atmospheric ozone and has thereby confirmed the predictions from model calculations. The biological UV dosimeter 'biofilm' whose sensitivity is based on dried spores of B. subtilis as UV target weights the incident UV radiation according to its DNA damaging potential. Biofilm dosimetry was applicated in space experiments as well as in use in remote areas on Earth. Examples are long-term UV measurements in Antarctica, measurements of diurnal UV profiles parallel in time at different locations in Europe, continuous UV measurements in the frame of the German UV measurement network and personal UV dosimetry. In space biofilms were used to determine the biological efficiency of the extraterrestrial solar UV, to simulate the effects of decreasing ozone concentrations and to determine the interaction of UVB and vitamin D production of cosmonauts in the MIR station.

Biological UV dosimeters in the assessment of the biological hazard from environmental radiation.

To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage.

Intrinsic and extrinsic biomarkers for the assessment of risks from environmental UV radiation.

In the last decades the knowledge of the effects of UV radiation on human health, especially in skin cancerogenesis, but also in immunsuppression, photoaging, eye damages, has enlarged strongly. The increasing solar UV radiation and changes in life style strengthen the necessity to identify and quantitate intrinsic biomarkers which are indicative for the individual UV susceptibility and the accumulated individual UV burden. For the risk assessment of potentially deleterious UV effects extrinsic biomarkers have to be developed and tested as personal biological UV dosimeters. One example for such a well characterized biological UV dosimeter is the DLR-biofilm which consists of spores of the bacterium Bacillus subtilis as UV sensitive target.

Biological monitoring of radiation exposure.

Complementary to physical dosimetry, biological dosimetry systems have been developed and applied which weight the different components of environmental radiation according to their biological efficacy. They generally give a record of the accumulated exposure of individuals with high sensitivity and specificity for the toxic agent under consideration. Basically three different types of biological detecting/ monitoring systems are available: (i) intrinsic biological dosimeters that record the individual radiation exposure (humans, plants, animals) in measurable units. For monitoring ionizing radiation exposure, in situ biomarkers for genetic (e.g. chromosomal aberrations in human lymphocytes, germ line minisatellite mutation rates) or metabolic changes in serum, plasma and blood (e.g. serum lipids, lipoproteins, lipid peroxides, melatonin, antibody titer) have been used. (ii) Extrinsic biological dosimeters/indicators that record the accumulated dose in biological model systems. Their application includes long-term monitoring of changes in environmental UV radiation and its biological implications as well as dosimetry of personal UV exposure. (iii) Biological detectors/biosensors for genotoxic substances and agents such as bacterial assays (e.g. Ames test, SOS-type test) that are highly sensitive to genotoxins with high specificity. They may be applicable for different aspects in environmental monitoring including the International Space Station.

Biological dosimetry to determine the UV radiation climate inside the MIR station and its role in vitamin D biosynthesis.

The vitamin D synthesis in the human skin, is absolutely dependent on UVB radiation. Natural UVB from sunlight is normally absent in the closed environment of a space station like MIR. Therefore it was necessary to investigate the UV radiation climate inside the station resulting from different lamps as well as from occasional solar irradiation behind a UV-transparent quartz window. Biofilms, biologically weighting and integrating UV dosimeters successfully applied on Earth (e.g. in Antarctica) and in space (D-2, Biopan I) were used to determine the biological effectiveness of the UV radiation climate at different locations in the space station. Biofilms were also used to determine the personal UV dose of an individual cosmonaut. These UV data were correlated with the concentration of vitamin D in the cosmonaut's blood and the dietary vitamin D intake. The results showed that the UV radiation climate inside the Mir station is not sufficient for an adequate supply of vitamin D, which should therefore be secured either by vitamin D supplemental and/or by the regular exposure to special UV lamps like those in sun-beds. The use of natural solar UV radiation through the quartz window for 'sunbathing' is dangerous and should be avoided even for short exposure periods.

Localisation and distance between ABL and BCR genes in interphase nuclei of bone marrow cells of control donors and patients with chronic myeloid leukaemia.

Quantitative measurements of the nuclear localisation of the ABL and BCR genes and the distance between them were performed in randomly oriented bone marrow cells of control donors and patients with chronic myeloid leukaemia (CML). Most ABL and BCR genes (75%) are located at a distance of 20-65% of the local radius from the nuclear centre to the nuclear membrane. A chimeric BCR-ABL gene located on a derivative chromosome 22 resulting from t(9;22)(q34;q11) [the so-called Philadelphia (Ph) chromosome] as well as the intact ABL and BCR genes of patients suffering from chronic myeloid leukaemia are also located mostly in this region, which has a mean thickness of 2 microns in bone marrow cells. We have not found any significant differences in the location of the two genes in the G1 and G2 phases of the cell cycle, nor between bone marrow cells and stimulated lymphocytes. Irradiation of lymphocytes with a dose of 5 Gy of gamma-rays results in a shift of both genes to the central region of the nucleus (0-20% of the radius distant from the nuclear centre) in about 15% of the cells. The minimum distance between one ABL and one BCR gene is less than 1 micron in 47.5% of bone marrow cells of control donors. Such a small distance is found between homologous ABL and between homologous BCR genes in only 8.1% and 8.4% of cells, respectively. It is possible that the relative closeness of nonhomologous ABL and BCR genes in interphase nuclei of bone marrow cells could facilitate translocation between these genes. In 16.4% of bone marrow cells one ABL and one BCR gene are juxtaposed (the distance between them varies from 0-0.5 micron) and simulate the Ph chromosome. This juxtaposition is the result of the projection of two genes located one above another into a plane, as follows from the probability calculation.

Distribution of ABL and BCR genes in cell nuclei of normal and irradiated lymphocytes.

Using dual-color fluorescence in situ hybridization (FISH) combined with two-dimensional (2D) image analysis, the locations of ABL and BCR genes in cell nuclei were studied. The center of nucleus-to-gene and mutual distances of ABL and BCR genes in interphase nuclei of nonstimulated and stimulated lymphocytes as well as in lymphocytes stimulated after irradiation were determined. We found that, after stimulation, the ABL and BCR genes move towards the membrane, their mutual distances increase, and the shortest distance between heterologous ABL and BCR genes increases. The distribution of the shortest distances between ABL and BCR genes in the G0 phase of lymphocytes corresponds to the theoretical distribution calculated by the Monte-Carlo simulation. Interestingly, the shortest ABL-BCR distances in G1 and S(G2) nuclei are greater in experiment as compared with theory. This result suggests the existence of a certain regularity in the gene arrangement in the G1 and S(G2) nuclei that keeps ABL and BCR genes at longer than random distances. On the other hand, in about 2% to 8% of lymphocytes, the ABL and BCR genes are very close to each other (the distance is less than approximately 0.2 to 0.3 microm). For comparison, we studied another pair of genes, c-MYC and IgH, that are critical for the induction of t(8;14) translocation that occurs in the Burkitt's lymphoma. We found that in about 8% of lymphocytes, c-MYC and IgH are very close to each other. Similar results were obtained for human fibroblasts. gamma-Radiation leads to substantial changes in the chromatin structure of stimulated lymphocytes: ABL and BCR genes are shifted to the nuclear center, and mutual ABL-BCR distances become much shorter in the G1 and S(G2) nuclei. Therefore, we hypothesize that the changes of chromatin structure in the irradiated lymphocytes might increase the probability of a translocation during G1 and S(G2) stages of the cell cycle. The fact that the genes involved in the t(8;14) translocation are also located close together in a certain fraction of cells substantiates the hypothesis that physical distance plays an important role in the processes leading to the translocations that are responsible for oncogenic transformation of cells.

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 Student's 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.