Inactivation of individual Bacillus subtilis spores in dependence on their distance to single cosmic heavy ions.

For radiobiological experiments in space, designed to investigate biological effects of the heavy ions of the cosmic radiation field, a mandatory requirement is the possibility to spatially correlate the observed biological response of individual test organisms to the passage of single heavy ions. Among several undertakings towards this goal, the BIOSTACK experiments in the Apollo missions achieved the highest precision and therefore the most detailed information on this question. Spores of Bacillus subtilis as a highly radiation resistant and microscopically small test organism yielded these quantitative results. This paper will focus on experimental and procedural details, which must be included for an interpretation and a discussion of these findings in comparison to control experiments with accelerated heavy ions.     

Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions.

Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology. Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g. Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects. For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve. The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast. Lethal damage produced at LET values < 2000 MeV cm2/g is reparable. Recombination repair is more effective than excision repair. At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied. In addition, at these high LET values, the frequency of induced mutations was drastically decreased. The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.     

Inactivation probability of heavy ion-irradiated Bacillus subtilis spores as a function of the radial distance to the particle's [correction of paricle's] trajectory.

The understanding of the radiobiological action of heavy ions requires the knowledge of the dependence of the inactivation probability on the distance between the particle's trajectory and the biological test organism (the impact parameter). Spores of Bacillus subtilis with a cytoplasmic core of about 0.22 micrometer cross section are suitable test objects for the study of this radial inactivation probability in its microscopic details. The spores are irradiated at low fluences of some 10(6) ions/cm2 with very heavy ions at different specific energies up to 10 MeV per atomic mass unit u while in fixed contact with visual nuclear track detectors. The methods are described by which the biological response of individual cells can be evaluated and the impact parameter be determined with an accuracy typically better than 0.2 micrometer. The results demonstrate that the common characteristics of inactivation, e.g., an effective range of inactivation extending to at least 3 micrometers, a nonmonotonic dependence of the inactivation probabilities on the radial distance, and the fact that the inactivation probability even for direct central hits on the cytoplasmic core is substantially below one, are nearly independent of the particle energy and type. The results are incompatible with the assumption that the radiobiological effectiveness can be attributed to the dose of secondary electrons as currently understood. They also demonstrate that the widely held notion of an "overkill" at low impact parameters does not apply for the spores even with the most densely ionizing ions.     

Double strand breaks in the DNA of Bacillus subtilis cells irradiated by heavy ions.

Cells of Bacillus subtilis strain TKJ 8431 in stationary phase were irradiated with X-rays (150 kV at DLR) or heavy ions (Ne, Ar, Pb with residual energies between 3 and 15 MeV/u at GSI). The action cross section for the formation of double strand breaks in the DNA of the irradiated cells follows a similar dependence on mass and energy of the ions as has been found for various biological endpoints, e.g. inactivation, mutagenesis and repair efficacy.     

Physical events in the track structure of heavy ions and their relation to alterations of biomolecules.

Heavy charged particles interacting with biological cells can produce a wide variety of different physical, chemical and biological consequences. A rigorous identification of relevant chemical and biological alterations of biomolecules in cells, however, is still lacking and, thus, it is difficult to identify the potential biological importance of different early physical events. In addition, due to experimental and theoretical problems also little is known about the details of energy transfer, -absorption and -decay from projectiles to atoms/molecules in condensed targets; this is particularly true for not completely stripped heavy ions. Nevertheless, one might conclude from available data that higher densities of physical energy absorption events have a significantly higher probability to lead to qualitatively more severe biochemical alterations as regards the induction of DNA double strand breaks and of chromatin damage. It is not very likely that energy migration along the DNA molecule in biological cells over long distances plays a significant role as contributor to these biological radiation effects.     

Effect of heavy ions on neuro-endocrine regulations.

During American and Russian short and long-term space flights neuroimmune dysregulations have been observed in man and rats for up to three months after the return. During Extra-Vehicular Activity, radiation exposure risk is greater to elicit short and/or long-term deleterious effects on the functional capacity of the neuroimmune system. In order to assess the effects of high LET events on neuroimmune networks, our preliminary ground-based study was to investigate brain inflammatory responses in mouse after low dose radiation exposure with high LET particles (12C, 95MeV/u, 42 mGy). Plasma corticosterone levels were rapidly (6 hours) increased by two-fold, then decreased 24 hours post-irradiation. At 3 days plasma corticosterone and ACTH concentrations were also two- to three-fold increased. Plasma ACTH levels were still elevated up to seven days to two months. Furthermore immune functions are under current assessment. The results of this study should allow a greater understanding of the effects of high LET particles on neuroimmune system.     

LET dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions.

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.     

Mutagenic effects of heavy ions in bacteria.

Various mutagenic effects by heavy ions were studied in bacteria, irradiated at accelerators in Dubna, Prague, Berkeley or Darmstadt. Endpoints investigated are histidine reversion (B. subtilis, S. typhimurium), azide resistance (B. subtilis), mutation in the lactose operon (E. coli), SOS chromotest (E. coli) and lambda-prophage induction (E. coli). It was found that the cross sections of the different endpoints show a similar dependence on energy. For light ions (Z < or = 4) the cross section decreases with increasing energy. For ions of Z = 10, it is nearly independent of energy. For heavier ions (Z > or = 26) it increases with energy up to a maximum or saturation. The increment becomes steeper with increasing Z. This dependence on energy suggests a "mutagenic belt" inside the track that is restricted to an area where the density of departed energy is low enough not to kill the cell, but high enough to induce mutations.     

Mutagenic effects of heavy ions in bacteria.

The peculiarities and mechanisms of the mutagenic action of gamma-rays and heavy ions on bacterial cells have been investigated. Direct mutations in the lac-operon of E. coli in wild type cells and repair deficient strains have been detected. Furthermore, the induction of revertants in Salmonella tester strains was measured. It was found that the mutation rate was a linear-quadratic function of dose in the case of both gamma-rays and heavy ions with LET up to 200 keV/micrometer. The relative biological effectiveness (RBE) increased with LET up to 20 keV/micrometer. Low mutation rates were observed in repair deficient mutants with a block of SOS-induction. The induction of SOS-repair by ionizing radiation has been investigated by means of the "SOS-chromotest" and lambda-prophage induction. It was shown that the intensity of the SOS-induction in E. coli increased with increasing LET up to 40-60 keV/micrometer.     

Effects of heavy ions on cycling stem cells.

Murine marrow stem cells assayed with the spleen colony assay have been shown to be largely in a noncycling state, Go. In the unirradiated animal where these spleen-colony forming units (CFUs) transit normally between a non-proliferative state and active proliferation, exposure to a sufficient dose of ionizing radiation increases the frequency (probability) of this transition. For low-LET irradiation, marrow stem cells are not induced into cycle until a threshold dose is achieved. This dose appears to be in the range 50 to 100 cGy, inducing proliferation in an all-or-nothing manner. For irradiation with heavy charged-particles, however, the threshold dose is dependent on mass and energy. Irradiation with particles of sufficient mass and energy stimulates active proliferation even at the smallest doses tested, 5 cGy. Further, this response does not appear to result from an all-or-nothing effect. Rather, individual animals with intermediate levels of stem cell cycling have been observed. These data support the notion that locally controlled hemopoiesis can be affected by local deposition of radiation damage.     

Simultaneous exposure of mammalian cells to heavy ions and X-rays.

Crews of space missions are exposed to a mixed radiation field, including sparsely and densely ionizing radiation. To determine the biological effectiveness of mixed high-/low-LET radiation fields, mammalian cells were exposed in vitro simultaneously to X-rays and heavy ions, accelerated at the HIMAC accelerator. X-ray doses ranged from 1 to 11 Gy. At the same time, cells were exposed to either 40Ar (550 MeV/n, 86 keV/micrometers), 28Si (100 MeV/n, 150 keV/micrometers), or 56Fe (115 MeV/n, 442 keV/micrometers) ions. Survival was measured in hamster V79 fibroblasts. Structural aberrations in chromosome 2 were measured by chemical-induced premature chromosome condensation combined with fluorescence in situ hybridization in isolated human lymphocytes. For argon and silicon experiments, measured damage in the mixed radiation field was consistent with the value expected using an additive function for low- and high-LET separated data. A small deviation from a simple additive function is observed with very high-LET iron ions combined to X-rays.     

Low energy ions in the heavy ions in space (HIIS) experiment on LDEF.

We present data from the Lexan top stacks in the Heavy Ions In Space (HIIS) experiment which was flown for six years (April 1984-Jan 1990) onboard the LDEF spacecraft in 28.5 degrees orbit at about 476 km altitude. HIIS was built of passive (i.e. no timing resolution) plastic track detectors which collected particles continuously over the entire mission. In this paper we present data on low energy heavy ions (10 < or = Z, 20MeV/nuc < E < 200 MeV/nuc). These ions are far below the geomagnetic cutoff for fully ionized ions in the LDEF orbit even after taking into account the severe cutoff suppression caused by occasional large geomagnetic storms during the LDEF mission. Our preliminary results indicate an unusual elemental composition of trapped particles in the inner magnetosphere during the LDEF mission, including both trapped anomalous cosmic ray species (Ne, Ar) and other elements (such as Mg and Fe) which are not found in the anomalous component of cosmic rays. The origin of the non-anomalous species is not understood, but they may be associated with the solar energetic particle events and geomagnetic disturbances of 1989.     

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.     

Microdosimetric considerations of effects of heavy ions on E. coli K-12 mutants.

The inactivation cross sections of E. coli K-12 recombination-deficient mutants, JC1553 (recA) and AB2470 (recB), for several MeV/u alpha-particles and N ions have been successfully analyzed by Katz's target theory in which radiosensitivity parameter E0 is assumed to be LET independent and equal to D37 for gamma-rays. For E. coli K-12 wild type, AB1157 (rec+, uvr+), however, it is impossible to interpret the inactivation cross section data by an LET-independent E0-value. In the latter case, as in the case of B. subtilis spore, it is necessary to assume that the radiosensitivity of the target for the core of a heavy ion is higher than that for delta-electrons. As well as Waligorski, Hamm and Katz's dose, the dose around the trajectory of an ion based on Tabata and Ito's energy deposition algorithm for electrons has been used in the course of analysis.     

Rapid development of corneal lesions in rats produced by heavy ions.

Scanning electron micrographs of heavy ion irradiated corneas demonstrate a significant correlation with the heavy ion beam: The average number of plasma membrane lesions per unit area of corneal surface is correlated with the particle fluence of the beam. This observation corroborates what has already been suggested theoretically about heavy ion tracks and what has been shown experimentally through etched plastics, developed emulsions, and bubble chambers. But the new data indicate that particle tracks occur in biological tissues as well, and that a single heavy ion is responsible for each membrane lesion.     

Direct radiation action of heavy ions on DNA as studied by ESR-spectroscopy.

The effects of heavy ions on the mechanisms of free radical formation in DNA-constituents as compared to low-LET irradiation are investigated by means of Electron Spin Resonance (ESR) spectroscopy. Dose-yield curves were measured at low (T < 100 K) and ambient temperatures in order to obtain the G-value, that is number of radicals formed per 100 eV absorbed energy. These G-values show a characteristic LET-dependence and are one to two orders of magnitude lower than for low-LET irradiation. Measurements on 2'Deoxycytidine at 300 K using combined heavy ion and X-ray irradiation methods suggested that this effect can be partially explained by a destruction of radicals during the irradiation.     

Thymine photoproduct formation and inactivation of intact spores of Bacillus subtilis irradiated with short wavelength UV (200-300nm) at atmospheric pressure and in vacuo.

Vacuum exposure renders the survival of spores of Bacillus subtilis approximately five times more sensitive to ultraviolet light irradiation than exposure under atmospheric conditions. The photoproduct formation in spores irradiated under ultrahigh vacuum (UHV) conditions is compared to the photoproduct formation in spores irradiated at atmospheric pressure. Compared to irradiation at atmospheric pressure, where only the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine (TDHT) can be detected, two additional photoproducts, known as the c,s and t,s isomers of thymine dimer (T<>T) are produced in vacuo. The spectral efficiencies for photoproduct formation in spores under atmospheric and vacuum conditions are compared. Since there is no increased formation of TDHT after irradiation in vacuum, TDHT cannot be made responsible for the observed vacuum effect. "Vacuum specific" photoproducts may cause a synergistic response of spores to the simultaneous action of ultraviolet light (UV) and UHV. Three different mechanisms are discussed for the enhanced sensitivity of B. subtilis spores to UV radiation in vacuum. The experiments described contribute valuable research information on the chance for survival of microorganisms in outer space.     

Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1.

Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1. There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar). The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.     

Effects of heavy ions on visual function and electrophysiology of rodents: the ALTEA-MICE project.

ALTEA-MICE will supplement the ALTEA project on astronauts and provide information on the functional visual impairment possibly induced by heavy ions during prolonged operations in microgravity. Goals of ALTEA-MICE are: (1) to investigate the effects of heavy ions on the visual system of normal and mutant mice with retinal defects; (2) to define reliable experimental conditions for space research; and (3) to develop animal models to study the physiological consequences of space travels on humans. Remotely controlled mouse setup, applied electrophysiological recording methods, remote particle monitoring, and experimental procedures were developed and tested. The project has proved feasible under laboratory-controlled conditions comparable in important aspects to those of astronauts' exposure to particle in space. Experiments are performed at the Brookhaven National Laboratories [BNL] (Upton, NY, USA) and the Gesellschaft für Schwerionenforschung mbH [GSI]/Biophysik (Darmstadt, FRG) to identify possible electrophysiological changes and/or activation of protective mechanisms in response to pulsed radiation. Offline data analyses are in progress and observations are still anecdotal. Electrophysiological changes after pulsed radiation are within the limits of spontaneous variability under anesthesia, with only indirect evidence of possible retinal/cortical responses. Immunostaining showed changes (e.g. increased expression of FGF2 protein in the outer nuclear layer) suggesting a retinal stress reaction to high-energy particles of potential relevance in space.     

Biophysical effect of cosmic heavy ions of distinct LET-classes in a plant model system.

Results presented from recent space flight BION 9 show biological effects of different LET-classes of HZE-particles in different target regions of the seed (meristem and the whole embryo) of Arabidopsis thaliana (L.) Heyhn. HZE-one hit events and non-hit events, i.e. only hit by the low-LET background radiation, and their combined effects on the biological damage endpoint lethality are distinguished. This procedure is opening the opportunity of an approach to comparative studies of the biological effects induced by cosmic HZE-particles of different LET-ranges interacting in the complex cosmic radiation spectrum and with other space flight conditions.