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

In order to understand radiation mechanisms of heavy ions in detail, it is necessary to study effects of single ions on individual biological test objects. Spores of Bacillus subtilis have been used as a suitable small biological test system to measure the inactivation in dependence on the radial distance to the tracks of charged particles. Accelerator experiments have been performed using a modified Biostack technique--biological objects sandwiched between nuclear track detectors. Results of these experiments using ions differing in their energy and atomic number will be discussed under following aspects: (i) methodological differences between the experiments and their possible influences on the results, (ii) common features which are independent on the particle type and energy, (iii) theoretical expectations and problems to find solid theoretical concepts which explain the results.     

Unique radiobiological aspects of high-LET radiation.

Since the beg inning of manned space flight the potentially unique radiobiological properties of the heavy ions of the cosmic radiation had been, apart from possible interactions of radiation effects with biological effects of weightlessness, of major concern with respect to the assessment of radiation hazards in manned space flight. Radiobiological findings obtained from space flight experiments and ground based experiments with densely ionizing radiation are discussed, which suggest qualitative differences between the radiobiological mechanisms of sparsely ionizing and densely ionizing radiation. These findings comprise the observation of a long lateral range of radiobiological effectiveness around tracks of single heavy ions, the observation of micro lesions induced in biological targets by the penetration of heavy ions, the nonadditivity of radiobiological effects from sparsely and densely ionizing radiation, the different kinetics for the expression of late effects induced by sparsely or densely ionizing radiation, and the observation of a reversed dose rate effect for early and late effects induced by densely ionizing radiation. These findings bear on the radiation protection standards to be installed for a general public in manned space flight and on the design of experiments, which intend to contribute to their specification.     

Ground-based research with heavy ions for space radiation protection.

Human exposure to ionizing radiation is one of the acknowledged potential showstoppers for long duration manned interplanetary missions. Human exploratory missions cannot be safely performed without a substantial reduction of the uncertainties associated with different space radiation health risks, and the development of effective countermeasures. Most of our knowledge of the biological effects of heavy charged particles comes from accelerator-based experiments. During the 35th COSPAR meeting, recent ground-based experiments with high-energy iron ions were discussed, and these results are briefly summarised in this paper. High quality accelerator-based research with heavy ions will continue to be the main source of knowledge of space radiation health effects and will lead to reductions of the uncertainties in predictions of human health risks. Efforts in materials science, nutrition and pharmaceutical sciences and their rigorous evaluation with biological model systems in ground-based accelerator experiments will lead to the development of safe and effective countermeasures to permit human exploration of the Solar System.     

Particle trajectories in seeds of Lactuca sativa and chromosome aberrations after exposure to cosmic heavy ions on Cosmos Biosatellites 8 and 9.

The potentially specific importance of the heavy ions of the galactic cosmic radiation for radiation protection in manned spaceflight continues to stimulate in situ, i.e., spaceflight experiments to investigate their radiobiological properties. Chromosome aberrations as an expression of a direct assault on the genome are of particular interest in view of cancerogenesis being the primary radiation risk for man in space. In such investigations the establishment of the geometrical correlation between heavy ions' trajectories and the location of radiation sensitive biological substructures is an essential task. The overall qualitative and quantitative precision achieved for the identification of particle trajectories in the order of approximately 10 micrometers as well as the contributing sources of uncertainties are discussed. We describe how this was achieved for seeds of Lactuca sativa as biological test organisms, whose location and orientation had to be derived from contact photographies displaying their outlines and those of the holder plates only. The incidence of chromosome aberrations in cells exposed during the COSMOS 1887 (Biosatellite 8) and the COSMOS 2044 (Biosatellite 9) mission was determined for seeds hit by cosmic heavy ions. In those seeds the incidence of both single and multiple chromosome aberrations was enhanced. The results of the Biosatellite 9 experiment, however, are confounded by spaceflight effects unrelated to the passage of heavy ions.     

Recent results of comparative radiobiological experiments with short and long term expositions of arabidopsis seed embryos

Comparison of experimental data obtained from short (SDEF) and long duration exposure flights (LDEF) recently led to results, which will contribute for the estimation of genetic risk for long and/or repeated stay of man in space. Under orbital conditions biological stress and damage are induced in test subjects by cosmic radiation, especially the high energetic, densely ionizing component of heavy ions. Plant seeds were successful model systems for a biotest in studying the physiological damages and mutagenic effects caused by ionizing radiation in particular stem cells. In this article we present an overview of our space experiments with Arabidopis thaliana seeds. We present first results of investigations on certain damage endpoints (seed germination, plant survival, mutation frequencies), caused by cosmic ionizing radiation in dry dormant plant seeds of Arabidopsis thaliana after different short term (e.g. IML-1 and D-2) and long term (e.g. EURECA and LDEF-1) space exposures. Total dose effects of heavy ions and the other components of the mixed radiation field on damage endpoints and survival after space exposure and gamma-ray preirradiation were obtained. A new method of total dose spectrometry by neutron activation has been applied.     

Radiation biology in space: a critical review.

A short summary of the results of radiobiological studies in space or on respective particles on ground will be given. Among the various types of radiation in space, the effect of heavy ions with high energy (HZE-particles) are most essential. Thus, radiobiology in space concerns mostly to the effect of these particles, in cells and in whole organism. Cell death, mutation and malignant transformation are the relevant endpoints, with can be studied on ground with heavy ions of different energy with suitable accelerators or in space, especially by the BIOSTACK concept. In space, however, the effect of microgravity has to be considered as well and there are hints, that under weightlessness the biological effect of radiation may be enhanced. There are still open questions to be answered concerning radioprotection of man in space. Further experiments are necessary.     

Advanced biostack: experiment 1 ES 027 on Spacelab-1.

The radiobiological properties of the heavy ions of cosmic radiation were investigated on Spacelab 1 by use of biostacks, monolayers of biological test organisms sandwiched between thin foils of different types of nuclear track detectors. Biostacks were exposed to cosmic radiation at several locations with different shielding environments in the module and on the pallet. Evaluations of the physical and biological components of the experiment to date indicate that in general they survived the spaceflight in good condition. Dosimetric data are presented for the different shielding environments.     

Radiation quality and risk estimation in relation to space missions.

While Q is specified as a function of linear energy transfer (LET) in practice the Q for neutrons has been selected by a judgment decision based on the relative biological effectiveness (RBE) to induce stochastic effects. There are no RBE values for tumor induction by heavy ions or protons in humans. Thus, selection of Q values has been based either on LET (or lineal energy) or RBEs from animal experiments. Estimates of Q for heavy ions in low earth orbit (LEO) range from about 5 to 14. The average Q value of all radiation in LEO has been estimated to be about 1.3. There is a lack of experimental data for RBEs for heavy ions but RBE increases as a function of LET. In the case of the Harderian gland the RBE reaches a maximum of 25-30 between about 100-200 keV/micrometer but does not appear to decrease at higher LETs. The International Commission of Radiological Protection have proposed the use of radiation weighting factors in lieu of quality factors. The weighting factors will range from 1 to 20.     

Cell inactivation, repair and mutation induction in bacteria after heavy ion exposure: results from experiments at accelerators and in space.

To understand the mechanisms of accelerated heavy ions on biological matter, the responses of spores of B. subtilis to this structured high LET radiation was investigated applying two different approaches. 1) By the use of the Biostack concept, the inactivation probability as a function of radial distance to single particles' trajectory (i.e. impact parameter) was determined in space experiments as well as at accelerators using low fluences of heavy ions. It was found that spores can survive even a central hit and that the effective range of inactivation extends far beyond impact parameters where inactivation by delta-ray dose would be effective. Concerning the space experiment, the inactivation cross section exceeds those from comparable accelerator experiments by roughly a factor of 20. 2) From fluence effect curves, cross sections for inactivation and mutation induction, and the efficiency of repair processes were determined. They are influenced by the ions characteristics in a complex manner. According to dependence on LET, at least 3 LET ranges can be differentiated: A low LET range (app. < 200 keV/micrometers), where cross sections for inactivation and mutation induction follow a common curve for different ions and where repair processes are effective; an intermediate LET range of the so-called saturation cross section with negligible mutagenic and repair efficiency; and a high LET range (>1000 keV/micrometers) where the biological endpoints are majorly dependent on atomic mass and energy of the ion under consideration.     

Cosmic ionizing radiation effects in plant seeds after short and long duration exposure flights.

Recently, comparison of biophysical data obtained from orbital flights of short and long duration led to results which will be significant for long and/or repeated stay of man in space. Under orbital conditions biological stress is induced in dry seeds of Arabidopsis thaliana by cosmic radiation especially its high energetic, densely ionizing component, the heavy ions (HZE). For comparison of radiation impact during different space flights a biological attempt at estimating the impact of single particles with high mass and energy (HZE-particles) on seeds was developed. Subdivision into LET-groups showed a remarkable contribution of an intermediate group (LET = 35 to 100 keV/micrometer) due to medium heavy ions (Z = 6 to 10). Efficiency factors for radiation damage experimentally determined and assigned to different LET-classes were compared to radiation quality factors discussed in literature.     

Mortality and morphological anomalies related to the passage of cosmic heavy ions through the smallest flowering aquatic plant Wolffia arrhiza

Radiobiological effects of single cosmic heavy ions on individual, actively metabolizing test organisms, plants of Wolffia arrhiza, have been explored in an experiment flown aboard the Russian Biosatellite 10. Mortality induced during space flight, population dynamics during subsequent cultivation, and morphological anomalies occurring in the plants of these cultures were investigated. Correlation of these effects with the passage of a heavy ion was achieved by inserting monolayers of plants in a stack of surrounding plastic nuclear track detectors (BIO-STACK). Enhanced initial mortality and delayed decline of induced anomalies have been significantly associated with the passage of single heavy ions, in particular if ions penetrated the budding region of the plants. The prolonged persistence of anomalies in filial generations as an indication of delayed genetic damage has been detected for the first time as the consequence of the hit by a single heavy ion. Regarding radiation protection of space crew during prolonged missions, especially outside the magnetosphere, this appears to be a significant finding.     

Preliminary total dose measurements on LDEF.

After spending nearly six years in Earth orbit twenty stacks consisting of radiation detectors and biological objects are now back on Earth. These stacks (Experiment A0015 Free Flyer Biostack) are part of the fifty seven science and technology experiments of the Long Duration Exposure Facility (LDEF) of NASA. The major objectives of the Free Flyer Biostack experiments are to investigate the biological effectiveness of single heavy ions of the cosmic radiation in various biological systems and to provide information about the spectral composition of the radiation field and the total dose received in the LDEF orbit. The Biostacks are mounted in two different locations of the LDEF. Up to three layers of Lithium fluoride thermoluminescence dosimeters (TLD) of different isotopic composition were located at different depths of some Biostacks. The preliminary analysis of the TLD yields maximum absorbed dose rates of 2.24 mGy day-1 behind 0.7 g cm-2 shielding and 1.17 mGy day-1 behind 12 g cm-2 shielding. A thermal neutron fluence of 1.7 n cm-2 s-1 is determined from the differences in absorbed dose for different isotopic mixtures of Lithium. The results of this experiment on LDEF are especially valuable and of high importance since LDEF stayed for about six years in the prospected orbit of the Space Station Freedom. There is no knowledge about the effectiveness of the space radiation in long-term spaceflights and the dosimetric data in this orbit are scarce.     

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.     

Results on Artemia cysts, lettuce and tobacco seeds in the Biobloc 4 experiment flown aboard the Soviet Biosatellite Cosmos 1129.

Artemia cysts, lettuce and tobacco seeds were flown aboard the Cosmos 1129 for 19 days. A correlative method was used in order to determine the passage of cosmic heavy ions (HZE particles) through the biological test objects. This space flight resulted in a decrease on hatchability, nucleic acid and protein synthesis in hydrated Artemia cysts. HZE particle effects on plant cellular chromosomes are confirmed. In tobacco seeds, a stimulating effect on germination rate and a higher frequency of abnormalities were observed. Dormant biological objects are a very suitable material to study cosmic ray effects: these objects can be arranged in monolayers and sandwiched between visual track detectors in order to determine the passage of the cosmic heavy ions (HZE particles). On the other hand this method allows us to study effects of microgravity and those of the protonic component of cosmic rays in the objects not hit by the HZE articles.     

The influence of dose, dose-rate and particle fragmentation on cataract induction by energetic iron ions.

Because activities in space necessarily involve chronic exposure to a heterogeneous charged particle radiation field it is important to assess the influence of dose-rate and the possible modulating role of heavy particle fragmentation on biological systems. Using the well-studied cataract model, mice were exposed to plateau 600 MeV/amu 56Fe ions either as acute or fractionated exposures at total doses of 5 - 504 cGy. Additional groups of mice received 20, 360 and 504 cGy behind 50 mm of polyethylene, which simulates body shielding. The reference radiation consisted of 60Co gamma radiation. The animals were examined by slit lamp biomicroscopy over their three year life spans. In accordance with our previous observations with heavy particles, the cataractogenic potential of the 600 MeV/amu 56Fe ions was greater than for low-LET radiation and increased with decreasing dose relative to gamma-rays. Fractionation of a given dose of 56Fe ions did not reduce the cataractogenicity of the radiation compared to the acute regimen. Fragmentation of the beam in the polyethylene did not alter the cataractotoxicity of the ions, either when administered singly or in fractions.     

Chromosomal aberrations induced by high-energy iron ions with shielding.

Biophysical models are commonly used to evaluate the effectiveness of shielding in reducing the biological damage caused by cosmic radiation in space flights. To improve and validate these codes biophysical experiments are needed. We have measured the induction of chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to 500 MeV/n iron ion beams (dose range 0.1-1 Gy) after traversing shields of different material (lucite, aluminium, or lead) and thickness (0-11.3 g/cm2). For comparison, cells were exposed to 200 MeV/n iron ions and to X-rays. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid cell-cycle selection produced by the exposure to high-LET heavy-ion beams. Aberrations were scored in chromosomes 1, 2, and 4 following fluorescence in situ hybridization. The fraction of aberrant lymphocytes has been evaluated as a function of the dose at the sample position, and of the fluence of primary 56Fe ions hitting the shield. The influence of shield thickness on the action cross-section for the induction of exchange-type aberrations has been analyzed, and the dose average-LET measured as a function of the shield thickness. These preliminary results prove that the effectiveness of heavy ions is modified by shielding, and the biological damage is dependent upon shield thickness and material.     

Biological effects of heavy ions from the standpoint of target theory.

The biological effect of heavy ions is best described through the action cross section, as a function of the end-point of interest and the charge and speed of the ion. In track theory this is called the "ion-kill" cross section, for it is the effect produced by a single heavy ion and its delta rays. As with nuclear emulsions the biological track structure passes from the grain count regime to the track width regime to the thindown region with an increase in LET. With biological cells, as with any detector capable of storing sublethal damage, with low LET irradiation the action cross section (in the ion-kill mode) is increasingly obscured by the effect of "gamma-kill", by the influence of overlapping delta rays from neighboring heavy ions. Thus at low LET response is dominated by the gamma-kill mode, so that the RBE approaches 1. The theory requires 4 radiosensitivity parameters for biological detectors, extracted from survival curves at several high LET bombardments passing through the grain count regime, and at high doses. Once these are known the systematic response of biological detectors to all high LET bombardments can be unfolded separating ion kill from gamma kill, predicting the response to a mixed radiation environment, and predicting low dose response even at the level of a single heavy ion. Cell killing parameters are now available for a variety of cell lines. Newly added is a set of parameters for cell transformation.     

Analysis of secondary electron emission spectra of equal-LET protons and alpha particles for purposes of radiation quality and spaceflight hazard assessment.

Amongst the great variety of heavy particles present in the galactic and solar cosmic ray spectra, hydrogen and helium nuclei are significantly more abundant than all other heavier ions and, as such, represent a major radiation hazard to humans in space. Experimental data have suggested that differences in relative biological effectiveness (RBE) exist between the two species at the same value of linear energy transfer (LET). This has consequences for heavily ionising radiation protection procedures, which currently still assume a simple dependence of radiation quality on LET. By analysing the secondary electron (delta-ray) emission spectra of protons and alpha particles, in terms of the spatial characteristics of energy deposition in cellular targets and the likelihood of complex lesion formation, a numerical quantity representing biological effectiveness is generated. When expressed relative to a reference radiation, this quantity is found to differ for protons and a particles of the same LET, demonstrating not only the ion-specific nature of RBE but also the inadequacy of specifying radiation quality as a function of LET only. Such a method for numerically assessing radiation quality may have implications for procedures for heavy ion protection in space at low doses and for understanding the initial mechanisms of radiation action.     

Microlesions: theory and reality.

Efforts to assess radiation risk in space have been complicated by the considerable unknowns regarding the biological effects of the heavy ion component (HZE particles) of the cosmic rays. The attention has focused primarily on the assignation of a quality factor (Q) which would take into account the greater effectiveness of heavy ions vis-a-vis other forms of ionizing radiation. If however, as the so-called "Microlesion Theory" allows, the passage of HZE particles through living tissue produces unique biological damage, the traditional use of Q becomes meaningless. Therefore, it is critical to determine if microlesions, in fact, do exist. While the concept does not necessarily require detectable morphological damage, "tunnel-lesions" or holes in ocular tissues have been cited as evidence of microlesions. These data, however, are open to reinterpretation. On-going light, scanning and transmission electron microscopic studies of the corneas, lenses and retinas of rat eyes exposed to 450 MeV/amu 56Fe ions thus far have not revealed tunnel-lesion damage. The morphological effects of the heavy ions have been found to be qualitatively similar to the changes following other kinds of ionizing radiation.