UV photobiochemistry under space conditions

The response of spores of Bacillus subtilis, cells of Deinococcus radiodurans and conidia of Aspergillus ochraceus to actual and simulated space conditions (UV in combination with long-term exposure to extremely dry conditions, including vacuum) has been studied: The following effects have been analyzed: decrease of viability, occurrence of DNA double strand breaks, formation of DNA-protein cross-links and DNA-DNA cross-links. All organisms show an increased sensitivity to UV light in extreme dryness (dry argon or vacuum) compared to an irradiation in aqueous suspension. The UV irradiation leads in all cases to a variety of DNA lesions. Very conspicuous is the occurrence of double strand breaks. Most of these double strand breaks are produced by incomplete repair of other lesions, especially base damages. The increase in DNA lesions can be correlated to the loss in viability. The specific response of the chromosomal DNA to UV irradiation in extreme dryness, however, varies from species to species and depends on the state of dehydration. The formation of DNA double strand breaks and DNA-protein cross-links prevails in the case of B. subtilis spores. In cells of Deinococcus radiodurans DNA-DNA cross-links often predominate, in conidia of Aspergillus ochraceus double strand breaks. The results obtained by direct exposure to space conditions (EURECA mission and D2 mission) largely agree with the laboratory data.     

Biological responses to space: results of the experiment "Exobiological Unit" of ERA on EURECA I.

Spores of different strains of Bacillus subtilis and the Escherichia coli plasmid pUC19 were exposed to selected conditions of space (space vacuum and/or defined wavebands and intensities of solar ultraviolet radiation) in the experiment ER 161 "Exobiological Unit" of the Exobiology Radiation Assembly (ERA) on board of the European Retrievable Carrier (EURECA). After the approximately 11 months lasting mission, their responses were studied in terms of survival, mutagenesis in the his (B. subtilis) or lac locus (pUC19), induction of DNA strand breaks, efficiency of DNA repair systems, and the role of external protective agents. The data were compared with those of a simultaneously running ground control experiment. The survival of spores treated with the vacuum of space, however shielded against solar radiation, is substantially increased, if they are exposed in multilayers and/or in the presence of glucose as protective, whereas all spores in "artificial meteorites", i.e. embedded in clays or simulated Martian soil, are killed. Vacuum treatment leads to an increase of mutation frequency in spores, but not in plasmid DNA. Extraterrestrial solar ultraviolet radiation is mutagenic, induces strand breaks in the DNA and reduces survival substantially; however, even at the highest fluences, i.e. 3 x 10(8) J m-2, a small but significant fraction of spores survives the insolation. Action spectroscopy confirms results of previous space experiments of a synergistic action of space vacuum and solar UV radiation with DNA being the critical target.     

Neoplastic cell transformation by high-LET radiation: molecular mechanisms.

Experimental data on molecular mechanisms are essential for understanding the bioeffects of radiation and for developing biophysical models, which can help in determining the shape of dose-response curves at very low doses, e.g., doses less than 1 cGy. Although it has been shown that ionizing radiation can cause neoplastic cell transformation directly, that high-LET heavy ions in general can be more effective than photons in transforming cells, and that the radiogenic cell transformation is a multi-step process [correction of processes], we know very little about the molecular nature of lesions important for cell transformation, the relationship between lethal and transformational damages, and the evolution of initial damages into final chromosomal aberrations which alter the growth control of cells. Using cultured mouse embryo cells (C3H10T1/2) as a model system, we have collected quantitative data on dose-response curves for heavy ions with various charges and energies. An analysis of these quantitative data suggested that two DNA breaks formed within 80 angstroms may cause cell transformation and that two DNA breaks formed within 20 angstroms may be lethal. Through studies with restriction enzymes which produce DNA damages at specific sites, we have found that DNA double strand breaks, including both blunt- and cohesive-ended breaks, can cause cell transformation in vitro. These results indicate that DNA double strand breaks can be important primary lesions for radiogenic cell transformation and that blunt-ended double strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship is similar for HGPRT gene mutation, chromosomal deletion, and cell transformation, suggesting common lesions may be involved in these radiation effects. The high RBE of high-LET radiation for cell killing and neoplastic cell transformation is most likely related to its effectiveness in producing DNA double strand breaks in mammalian cells. At present the role of oncogenes in radiation cell transformation is unclear.     

UV photobiochemistry of anhydrobiotic organisms at extremely low temperatures

Spores of Bacillus subtilis (TKJ 3412), cells of Deinococcus radiodurans R₁ (wild type) and conidia of Aspergillus ochraceus (strain 3174) have been UV irradiated (254 nm) in the dry state (3% relative humidity, argon) or in aqueous suspension at room temperature, at −55°C to −70°C and at −165°C to −170°C. The following effects have been analyzed: decrease in viability, occurrence of DNA strand breaks (pulsed-field gel electrophoresis) and production of DNA-protein cross-links (membrane filter method). The loss in viability is usually more pronounced at around −70°C than at room temperature, but it is lowest around −170°C. The kind of prevailing DNA damage varies from organism to organism. The amount of UV induced DNA-protein cross-link products steadily decreases with the temperature and is lowest at −170°C. The decrease in highly polymeric DNA by double strand breaks follows no universal pattern. The observed hypersensitivity of the three very different species at −70°C can therefore not be simply explained on the basis of the number of DNA lesions analyzed in the course of this work. We suggest that also the changing state of cellular water below and above about −130°C significantly contributes to the change in photosensitivity.     

The influence of radiation quality on the formation of DNA breaks.

We have aimed to present a comprehensive review of our understanding to date of the formation of DNA strand breaks induced by high LET radiation. We have discussed data obtained from DNA in solution as well as from the formation and "repair" of strand breaks in cell DNA. There is good agreement, qualitatively, between these two systems. Results were evaluated for two parameters: (1) effectivity per particle, the cross section (sigma) in micrometers 2/particle; and (2) the strand break induction frequency as number of breaks per Gy per unit DNA (bp or dalton). A series of biological effects curves (one for each Z-number) is obtained in effectivity versus LET plots. The relationships between induction frequencies of single-strand breaks, or double-strand breaks, or the residual "irrepairable" breaks and LET-values have been evaluated and discussed for a wide spectrum of heavy ions, both for DNA in solution and for DNA in the cell. For radiation induced total breaks in cell DNA, the RBE is less than one, while the RBE for the induction of DSBs can be greater than one in the 100-200 keV/micrometers range. The level of irrepairable strand breaks is highest in this same LET range and may reach 25 percent of the initial break yield. The data presented cover results obtained for helium to uranium particles, covering a particle incident energy range of about 2 to 900 MeV/u with a corresponding LET range of near 16 to 16000 keV/micrometers.     

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.     

Experimental models for cellular radiation targets: LET, RBE and radioprotectors.

The topics of this presentation are: a brief review of the early research, the ideas it stimulated and the ways they are used in current efforts to explain cellular radiosensitivity; an analysis of the strengths and weaknesses of two experimental models used in vitro for simulating the induction of double strand breaks (DSB) and single strand breaks (SSB) in situ. Note that when alkali is used to denature cellular DNA for the determination of strand breaks, both overt SSB and the SSB that result from DSB in the denaturation process are recorded as total strand breaks (TSB). All information is taken from published literature.     

Heavy ion induced double strand breaks in bacteria and bacteriophages.

DNA damage induced by heavy ions in bacterial cells and bacteriophages such as Bacillus subtilis, E. coli and Bacteriophage T1 were investigated by analyzing the double strand breaks in the chromosomal DNA. This kind of lesion is considered as one of the main reasons for lethal events. To analyze double strand breaks in long molecules of DNA--up to some Mbp in length--the technique of pulse field agarose gel electrophoresis has been used. This allows the detection of one double strand break per genome. Cell lysis and DNA isolation were performed in small agarose blocks directly. This procedure secured minimum DNA destruction by shearing forces. After running a gel, the DNA was stained with ethidium bromide. The light intensity of ethidium bromide fluorescence for both the outcoming (running) DNA and the remaining intact DNA were measured by scanning. The mean number of double strand breaks was calculated by determining the quotient of these intensities. Strand break induction after heavy ion and X-ray irradiation was compared.     

DNA-lesion and cell death by alpha-particles and nitrogen ions.

When the natural logarithm of the surviving fraction is plotted against the dose of radiation, curves with shoulders at relatively high survival levels are obtained after gamma-rays. The curves were practically linear in case of HMV-I and HA-1 cells irradiated by charged particle beams. These cells were derived from human malignant melanoma and Chinese hamster cells, respectively. The amount of DNA single strand breaks (ssb) by gamma-rays or nitrogen-ions (LET=530KeV/micrometers) in HMV-I cells increases linearly with increment in dose, when the ssb is detected using the alkaline elution technique. There is no close relationship between the dose-response curve of the ssb and the dose-survival curves after gamma-rays or N-ions. The amount of DNA double strand breaks (dsb) by gamma-rays increases quadratically with increment of dose, in both HMV-I cells and HA-1 cells, when the dsb is detected using the neutral elution technique. The survival fraction for HA-1 cells is slightly higher than that for HMV-I cells, at the same dose, and the amount of dsb for HA-1 cells is considerably greater than that for HMV-I cells. These results suggest that the radiosensitivities to gamma-rays in different cell lines do not correspond to the number of DNA strand breaks. The amount of both non-repairable ssb and dsb also increases quadratically with increment of dose for gamma-rays and almost linearly with increment of dose for N-ions and alpha-particles (LET=36keV/micrometers for HA-1 cells and LET=77keV/micrometers for HMV-I cells). The dose-response curves for non-repairable dsb in case of these radiations seemed to mirror image the dose-survival curves for these radiations, in both cell lines. The number of non-repairable DNA strand breaks in the two cell lines, at the same level of survival was much the same. These results show the close relationship between the induction of non-repairable DNA strand breaks and cell killing.     

Induction of DNA breaks in SV40 by heavy ions.

Simian virus (SV40) DNA was used to study the induction of DNA strandbreaks by heavy ions varying in LET. DNA was exposed to X-rays and to accelerated particles either in dilute solution or in the presence of different radical scavengers. Relative proportions of the intact supercoiled DNA, nicked form arising from single strand breaks (SSB) and linear molecules produced by double strandbreaks (DSB) were quantified on the base of their electrophoretic mobility in agarose gels. Cross sections for the induction of SSBs and DSBs were calculated from the slope of dose effect curves. Mercaptoethanol was found to protect more efficiently against DNA strand breakage than Tris. When the biological efficiency, i.e. the number of strand breaks per unit dose and molecule weight was evaluated as a function of LET, curves for SSB induction always showed a continuous decrease. For DSB induction, an increase in the yield of DSBs with a maximum around 500 keV/micrometer was observed in the presence of radical scavenger. This peak of biological efficiency gradually disappeared when the radiosensitivity of the system was increased, and was no longer apparent in the dilute buffer system, where DNA showed a high susceptibility to strand breakage. When the relative biological efficiency was plotted versus LET, the curve for DSB induction observed in a low radical scavenging environment paralleled the curve obtained for SSB induction.     

Survival in extreme dryness and DNA-single-strand breaks.

A wide variety of organisms (the so-called "anhydrobiotes') is able to survive long periods of time in a state of utmost dehydration and can thus survive in extremely dry environments including artificially imposed or space vacuum. Known strategies of survival include the accumulation of certain polyols, especially disaccharides, which help prevent damage to membranes and proteins. Here we report that DNA in vacuum-dried spores is damaged to a very substantial degree by processes leading to DNA strand breaks. Most of these lesions are obviously repaired during germination, but extensive damage to DNA and enzymes after long exposure times (months to years) finally diminish the chances of survival.     

Survival rates of some terrestrial microorganisms under simulated space conditions.

In connection with planetary quarantine, we have been studying the survival rates of nine species of terrestrial microorganisms (viruses, bacteria, yeasts, fungi, etc.) under simulated interstellar conditions. If common terrestrial microorganisms cannot survive in space even for short periods, we can greatly reduce expenditure for sterilizing space probes. The interstellar environment in the solar system has been simulated by low temperature, high vacuum (77 k, 4 x 10(-6) torr), and protons irradiation from a Van de Graaff generator. After exposure to a barrage of protons corresponding to about 250 years of irradiation in solar space, Tobacco mosaic virus, Bacillus subtilis spores, Aspergillus niger spores and Clostridiun mangenoti spores showed survival rates of 82%, 45%, 28%, and 25%, respectively. Furthermore. pathogenic Candida albicans showed 7% survival after irradiation corresponding to about 60 years in space.     

Bacterial survival in response to desiccation and high humidity at above zero and subzero temperatures

Earthly microorganisms might have contaminated Mars for millions of years by intellectual activities or natural transfer. Knowledge on the preservation of microorganisms may help our searching for life on outer planets, particularly Mars-contaminated earthly microorganisms at ancient time. Extreme dryness is one of the current Mars characteristics. However, a humid or watery Mars at earlier time was suggested by evidence accumulated in recent decades. It raises the question that whether water helps preservation of the microorganisms or not, particularly those with high possibility of interplanetary transfer like spores and Deinococci. In this study, we examined the effects of desiccation and high humidity on survival and DNA double strand breaks (DSB) of Escherichia coli, Deinococcus radiodurans and spores of Bacillus pumilus at 25, 4 and −70 °C. They exhibited different survival rates and DSB patterns under desiccation and high humidity. Higher survival and less DSB occurred at lower temperature. We suggest that some Mars-contaminated bacteria might have been viably preserved on cold Mars regions for long periods, regardless of water availability. It is more likely to find ancient spores than ancient Deinococci on Mars. In our search for preserved extraterrestrial life, priority should be given to the Mars Polar Regions.     

Microorganisms and biomolecules in space environment experiment ES 029 on Spacelab-1.

Bacterial spores are proper test organisms for studying problems of space biology and exobiology. During the Spacelab 1 mission, studies on the limiting factors for survival of Bacillus subtilis spores in free space have been performed. An exposure tray on the pallet of Spacelab 1 accomodated 316 samples of dry spores for treatment with space vacuum and/or the following selected wavelengths of solar UV: > 170 nm, 220 nm, 240nm, 260nm and 280 nm. After recovery, inactivation, mutation induction, reparability, and photochemical damages in DNA and protein have been studied. The results contribute to the understanding of the mechanisms of increased UV sensitivity of bacterial spores in vacuo and to a better assessment of the chance of survival of resistant forms in space and of interplanetary transfer of life.     

Survival of microorganisms in space protected by meteorite material: results of the experiment 'EXOBIOLOGIE' of the PERSEUS mission.

During the early evolution of life on Earth, before the formation of a protective ozone layer in the atmosphere, high intensities of solar UV radiation of short wavelengths could reach the surface of the Earth. Today the full spectrum of solar UV radiation is only experienced in space, where other important space parameters influence survival and genetic stability additionally, like vacuum, cosmic radiation, temperature extremes, microgravity. To reach a better understanding of the processes leading to the origin, evolution and distribution of life we have performed space experiments with microorganisms. The ability of resistant life forms like bacterial spores to survive high doses of extraterrestrial solar UV alone or in combination with other space parameters, e.g. vacuum, was investigated. Extraterrestrial solar UV was found to have a thousand times higher biological effectiveness than UV radiation filtered by stratospheric ozone concentrations found today on Earth. The protective effects of anorganic substances like artificial or real meteorites were determined on the MIR station. In the experiment EXOBIOLOGIE of the French PERSEUS mission (1999) it was found that very thin layers of anorganic material did not protect spores against the deleterious effects of energy-rich UV radiation in space to the expected amount, but that layers of UV radiation inactivated spores serve as a UV-shield by themselves, so that a hypothetical interplanetary transfer of life by the transport of microorganisms inside rocks through the solar system cannot be excluded, but requires the shielding of a substantial mass of anorganic substances.     

Biochemical constraints for survival under Martian conditions.

A wide variety of terrestrial organisms, the so-called "anhydrobiotes," has learned to survive in a state of extreme dehydration in dry environments. Strategies for survival include the accumulation of certain polyols and nonreducing saccharides, which help to prevent damage to membranes and proteins, but at low water partial pressure DNA is also progressively damaged by various lesions, including strand breaks and cross-linking to proteins. These lesions, if they are not too numerous, can be repaired before the first replication step after rehydration, but long-term exposure to dry conditions finally diminishes the chances of survival as these lesions accumulate. If an organism has no chance to repair the accumulated DNA damage during intermittent periods of active life, survival will not exceed a few decades. The restriction of survival by dryness-induced DNA lesions is corroborated by new data on conidia of Aspergillus and the free plasmid pBR 322. Our results will be discussed with respect to the chance of finding dormant life or biochemical fossils on the surface of Mars.     

Detection of martian amino acids by chemical derivatization coupled to gas chromatography: in situ and laboratory analysis.

If there is, or ever was, life in our solar system beyond the Earth, Mars is the most likely place to search for. Future space missions will have then to take into account the detection of prebiotic molecules or molecules of biological significance such as amino acids. Techniques of analysis used for returned samples have to be very sensitive and avoid any chemical or biological contamination whereas in situ techniques have to be automated, fast and low energy consuming. Several possible methods could be used for in situ amino acid analyses on Mars, but gas chromatography would likely be the most suitable. Returned samples could be analyzed by any method in routine laboratory use such as gas chromatography, already successfully performed for analyses of organic matter including amino acids from martian meteorites. The derivatization step, which volatilizes amino acids to perform both in situ and laboratory analysis by gas chromatography, is discussed here.     

Heavy-ion effects on cellular and subcellular systems: inactivation, chromosome aberrations and strand breaks induced by iron and nickel ions.

A broad spectrum of particles and energies has been used in the last years to study the influence of the radiation quality i.e. of the physical parameters of the particle beam on the biological effectiveness ?2?12?. In these measurements a common structure of the functional dependence of the induction probability per particle (cross section) from the linear energy transfer is observed for different biological endpoints. Because of the relevance for space research, we present in this report our data from experiments with iron and nickel particles, in particular. Our experiments were designed to investigate the relationship between the inactivation and chromosome aberration in mammalian cells and the induction of single and double strand breaks in SV40 DNA in respect to the parameters of the track formation like LET and particle energy.     

Heavy ion induced DNA double strand breaks in cells of E. coli.

Vegetative cells of E. coli differing in their radiosensitivity have been used in heavy ion irradiation experiment. Besides inactivation measurements also the induction of DNA double strand breaks (DSB) have been measured using the method of pulse-field gel electrophoresis. This method allows to separate linear DNA with length up to 8 Mio base pairs. After irradiation with heavy ions we find a higher amount of low molecular weight fragments when compared to sparsely ionizing radiation. This agrees with the idea that heavy ions as a structured radiation have a high probability to induce more than one strand break in a DNA molecule if the particle hits the DNA. The amount of intact DNA remaining in the agarose plugs decreases exponentially for increasing radiation doses or particle fluences. From these curves cross sections for the induction of DSB after heavy ion irradiation have been determined. These results will be discussed in comparison to the results for cell survival.     

Radiobiology and photobiology on Earth and in space: points of encounter and protection considerations.

All radiations originate in space, and the spectrum of radiations reaching the troposphere is limited only because of their range and absorption by the ozone layer above the atmosphere. Ultraviolet-C and the very heavy ions are therefore produced on earth only artificially, by special lamps and in accelerators. The range of biological effects of the different UV radiations and low and high LET radiations have been studied extensively, yet only recently new facts such as the production of DNA strand breaks by long wave UV light were established, adding to the various points of encounter existing between ionizing and nonionizing radiations. There are some similarities in radiation products, and the resulting effects of insult by radiation on biological systems very often are similar, if not the same. A common phenomenon that exists in all healthy biological cells is the ability to repair damage to DNA and thus either survive or mutate, and although the specific mechanisms of repair are somewhat different, the end result is the same. Recently a mechanism of improved radioprotection was found to involve an effect of certain radioprotective compounds on DNA repair. It is suggested that improved, and nontoxic, modes of protection may be offered by employing such compounds as biological response modifiers and natural substances. Further research is needed and is under way.