Track structure in biological models.

High-energy heavy ions in the galactic cosmic radiation (HZE particles) may pose a special risk during long term manned space flights outside the sheltering confines of the earth's geomagnetic field. These particles are highly ionizing, and they and their nuclear secondaries can penetrate many centimeters of body tissue. The three dimensional patterns of ionizations they create as they lose energy are referred to as their track structure. Several models of biological action on mammalian cells attempt to treat track structure or related quantities in their formulation. The methods by which they do this are reviewed. The proximity function is introduced in connection with the theory of Dual Radiation Action (DRA). The ion-gamma kill (IGK) model introduces the radial energy-density distribution, which is a smooth function characterizing both the magnitude and extension of a charged particle track. The lethal, potentially lethal (LPL) model introduces lambda, the mean distance between relevant ion clusters or biochemical species along the track. Since very localized energy depositions (within approximately 10 nm) are emphasized, the proximity function as defined in the DRA model is not of utility in characterizing track structure in the LPL formulation.     

Development of human epithelial cell systems for radiation risk assessment.

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.     

Cellular parameters for track structure modeling of radiation hazard in space.

Based on irradiation with 45 MeV/u N and B ions and with Co-60 gamma rays, cellular parameters of Katz's track structure model have been fitted for the survival of V79-379A Chinese hamster lung fibroblasts. Cellular parameters representing neoplastic transformations in C3H10T/1/2 cells after their irradiation with heavy ion beams, taken from earlier work, were also used to model the radiation hazard in deep space, following the system for evaluating, summing and reporting occupational exposures proposed in 1967 by a subcommittee of NCRP. We have performed model calculations of the number of transformations in surviving cells, after a given fluence of heavy charged particles of initial energy 500 MeV/u, penetrating thick layers of cells. We take the product of cell transformation and survival probabilities, calculated along the path lengths of charged particles using cellular survival and transformation parameters, to represent a quantity proportional to the "radiation risk factor" discussed in the NCRP document. The "synergistic" effect of simultaneous charged particle transfers is accounted for by the "track overlap" mode inherent in the model of Katz.     

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.     

Early and late damages induced by heavy charged particle irradiation in embryonic tissue of Arabidopsis seeds.

Early and late effects of accelerated heavy ions (HZE) on the embryonic tissue of Arabidopsis thaliana seeds were investigated seeing that initial cells of the plant eumeristems resemble the original cells of animal and human tissues with continuous cell proliferation. The endpoints measured were lethality and tumorization in the M1-generation for early effects and embryonic lethality in the M2-generation for late effects. The biological endpoints are plotted as functions of the physical parameters of the irradiation i.e. ion fluence (p/cm2), dose (Gray), charge Z and linear energy transfer (LET). The results presented contribute to the estimation of the principles of biological HZE effects and thus may help to develop a unified theory which could explain the whole sequence from physical and chemical reactions to biological responses connected with heavy ion radiation. Additionally, the data of this paper may be used for the discussion of the quality factor for heavy ion irradiation needed for space missions and for HZE-application in radio-therapy by use of accelerators (UNILAC, (SIS/ESR), BEVALAC).     

On the quantitative interpretation of cellular heavy ion action.

Current analyses of heavy ion action assume that the survival probability of a cell hit by a heavy ion depends only on the energy absorbed in its critical site. It is known, however, that the efficiency to produce a biological effect depends also on the spatial pattern of energy deposition. This has to be included in the quantitative evaluation of heavy ion action. Based on recent models of lesion formation by ionizing radiation (Goodhead and Brenner, Phys. Med. Biol. 28, 485, 1983) data with lighter ions (LET < 500 keV/micrometer) were re-analysed. It is shown that the behaviour of various cell systems can be described by a common curve which can be used to estimate the contribution of "non-linear" components (i.e. where the distribution of energy deposition plays a role) with heavy ions. It is concluded that even with Uranium ions the regions of non-linear effects does not extend beyond 50 nm from the trade core. These data will be used to assess quantitatively survival curves obtained with very heavy ion exposure.     

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.     

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.     

Microscopic track structure of equal-LET heavy ions.

The spatial distributions of ionization and energy deposition produced by high-velocity heavy ions are crucial to an understanding of their radiation quality as exhibited eg., in track segment experiments of cell survival and chromosome aberrations of mammalian cells. The stopping power (or LET) of a high velocity ion is proportional to the ratio z2/v2, apart from a slowly varying logarithmic factor. The maximum delta-ray energy that an ion can produce is proportional to v2 (non-relativistically). Therefore, two HZE ions having the same LET, but in general differing z and v will have different maximum delta-ray energies and consequently will produce different spatial patterns of energy deposition along their paths. To begin to explore the implications of this fact for the microscopic dosimetry of heavy ions, we have calculated radial distributions in energy imparted and ionization for iron and neon ions of approximately equal LET in order to make a direct comparison of their delta-ray track structure. Monte Carlo techniques are used for the charged particle radiation transport simulation.     

Induction of chromosome aberrations in mammalian cells after heavy ion exposure.

The induction of chromosome aberrations by heavy charged particles was studied in V79 Chinese hamster cells over a wide range of energies (3-100 MeV/u) and LET (20-16000 keV/micrometer). For comparison, X-ray experiments were performed. Our data indicate quantitative and qualitative differences in the response of cells to particle and x-ray irradiation. For the same level of cell survival the amount of damaged cells which can be observed is smaller in heavy ion (11.4 MeV/u Ar) irradiated samples. The highest yield of damaged cells is found 8 to 12 hours after particle irradiation and 4 hours after x-irradiation. Differences in the amount of damaged cells are attributed to cell cycle perturbations which interfere with the expression of damage. After heavy ion exposure the amount of cells reaching mitosis (mitotic index) decreases drastically and not all damaged cells reach mitosis within 48 hours after exposure. A portion of cells die in interphase. Cell cycle delays induced by x-ray irradiation are less pronounced and all cells reach the first post-irradiation mitosis within 24 hours after irradiation. Additionally, the damage produced by charged particles seems to be more severe. The disintegration of chromosomes was only observed after high LET radiation: an indication of the high and local energy deposition in the particle track. Only cross sections for the induction of chromosome aberrations in mitotic cells were reported in this paper because of the problems arising from the drastic cell cycle perturbations. In this case, cells were irradiated in mitosis and assayed immediately.     

Heavy ion measurement on LDEF.

A stack of CR-39 track detectors was exposed on the NASA satellite LDEF and recovered after almost 6 years in space. The quick look analysis yielded heavy ion tracks on a background of low energy secondaries from proton interactions. The detected heavy ions show a steep energy spectrum which indicates a radiation belt origin.     

Do heavy ions cause microlesions in cell membranes?

Heavy ions are a hazard in manned deep space missions. It has been theoretically postulated that when they interact with cells, localized damage in the forms of "microlesions" may occur. Purported morphological evidence of these lesions, however, has not been confirmed in the most extensively studied tissue so far, the cornea. Recent morphological evidence from rat corneas demonstrated that holes in membranes do not form as consequence of heavy ion irradiation. This does not mean, however, that some other form of damage is excluded. For example such damage may be physiological in nature, impairing the ability of cells or tissues to function properly. In order to uncover any physiological effects, we investigated the microlesion question by monitoring the electrical potential difference across the endothelium of rat corneas in vitro before, during, and after irradiation. When the corneas were exposed to 1 Gy of 56Fe ions (450 and 600 MeV/a.m.u.), we detected no effect on this parameter. These results suggest that direct physical damage to cell membranes, as predicted by the microlesion theory, does not take place.     

Microdosimetric measurements of heavy ion tracks.

The biological effectiveness of radiations depends on the spatial pattern of ionizations and excitations produced by the charged particle tracks involved. Ionizations produced by both the primary ion and by energetic delta rays may contribute to the production of biologically relevant damage and to the concentration of damage which may effect the probability of repair. Although average energy concentration (dose) can be calculated using homogeneous track models, the energy is actually concentrated in small volumes containing segments of the ion and delta ray tracks. These local concentrations are studied experimentally using low pressure proportional counters, and theoretically, using Monte Carlo methods. Small volumes near an ion track may be traversed by a delta ray. If they are, the energy deposited will be similar to that produced by a single electron track in a low-energy x-ray irradiation. The probability of a delta ray interaction occurring decreases with the square of the radial distance from the track. The average energy deposited is the product of this probability and the energy deposited in an interaction. Average energy deposited calculated from measured interaction probability is in good agreement with the results of homogeneous track models.     

Multiple cell hits by particle tracks in solid tissues.

Relative Biological Effectiveness (RBE) and Quality Factor (Q) at extreme values of Linear Energy Transfer (LET) have been determined on the basis of experiments with single-cell systems and specific tissue responses. In typical single cell systems, each heavy particle (Ar or Fe) passes through a single cell or no cell. In tissue end-point experiments each heavy particle passes through several cells, and the LET can exceed 200 keV/micrometer in every cell. In most laboratory animal tissue systems, however, only a small portion of the hit cells are capable of expressing the end-point of interest to the investigator, such as cell killing, mutation or carcinogenesis. The following question must therefore be addressed: Do RBE's and Q factors derived from single-cell experiments properly account for the increased probability of multiple-cell damage by HZE tracks? A model is offered in which measured radiation effects and known tissue properties are combined to estimate the value of a multiplier of damage effectiveness on the basis of number of cells at risk, p3n, per track containing a hit cell, where n is the number of cells per track, based on tissue and organ geometry, and P3 is the probability that a cell in the track is capable of expressing the experimental end-point.     

On the quality of mutations in mammalian cells induced by high LET radiations

The deleterious effects of accelerated heavy ions as component of the space radiation environment on living cells are of increasing importance for long duration human space flight activities. The most important aspect of such densely ionizing particle radiation is attributed to the type and quality of biological damage induced by them. This issue is addressed by investigating cell inactivation and mutation induction at the Hprt locus (coding for hypoxanthine-guanine-phosphoribosyl-transferase) of cultured V79 Chinese hamster cells exposed to densely ionizing radiation (accelerated heavy ions with different LETs from oxygen to gold, specific energies ranging from 1.9 to 69.7 MeV/u, corresponding LET values range from 62 to 13,223 keV/μm) and to sparsely ionizing radiation (200 kV X-rays). 30 spontaneous, 40 X-ray induced and 196 heavy ion induced 6-thioguanine resistant Hprt mutant colonies were characterized by Southern technique using the restriction enzymes EcoRI, PstI and BglII and a full length Hprt cDNA probe isolated from the plasmid pHPT12. Restriction patterns of the spontaneous Hprt mutants were indistinguishable from the wild type pattern, as these mutants probably contain only small deletions or even point mutations in the Hprt locus. In contrast, the overall spectrum of heavy ion induced mutations revealed a majority of partial or total deletions of the Hprt gene. With constant particle fluence (3 × 10⁶ particles/cm²) the quality of heavy ion induced mutations in the Hprt locus depends on physical parameters of the beam (atomic number, specific energy, LET). This finding suggests a relationship between the type of DNA damage and track structure. The fraction of mutants with severe deletions in the Hprt locus after exposure to oxygen ions increases from 65% at 60 keV/μm up to a maximum (100%) at 300 keV/μm and declines with higher LET values to 75% at 750 keV/μm. With heavier ions (Ca- and Au-ions) and even higher LET-values this mutant fraction decreases to 58% at 13,200 keV/μm. Heavy ion induced DNA break points in the Hprt locus are not randomly distributed.     

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.     

Ground-based measurements of galactic cosmic ray fragmentation in shielding.

The mean free path for nuclear interactions of galactic cosmic-rays is comparable to shielding and tissue thicknesses present in human interplanetary exploration, resulting in a significant fraction of nuclear reaction products at depth. In order to characterize the radiation field, the energy spectrum, the angular distribution, and the multiplicity of each type of secondary particles must also be known as a function of depth. Reactions can take place anywhere in a thick absorber; therefore, it is necessary to know these quantities as a function of particle energy for all particles produced. HZE transport methods are used to predict the radiation field; they are dependent on models of the interaction of man-made systems with the space environment to an even greater extent than methods used for other types of radiation. Hence, there is a major need to validate these transport codes by comparison with experimental data. The most cost-effective method of validation is a comparison with ground-based experimental measurements. A research program to provide such validation measurements using neon, iron and other accelerated heavy ion beams will be discussed and illustrated using results from ongoing experiments and their comparison with current transport codes. The extent to which physical measurements yield radiobiological predictions will be discussed.     

On the radiosensitivity of man in space.

Astronauts' radiation exposure limits are based on experimental and epidemiological data obtained on Earth. It is assumed that radiation sensitivity remains the same in the extraterrestrial space. However, human radiosensitivity is dependent upon the response of the hematopoietic tissue to the radiation insult. It is well known that the immune system is affected by microgravity. We have developed a mathematical model of radiation-induced myelopoiesis which includes the effect of microgravity on bone marrow kinetics. It is assumed that cellular radiosensitivity is not modified by the space environment, but repopulation rates of stem and stromal cells are reduced as a function of time in weightlessness. A realistic model of the space radiation environment, including the HZE component, is used to simulate the radiation damage. A dedicated computer code was written and applied to solar particle events and to the mission to Mars. The results suggest that altered myelopoiesis and lymphopoiesis in microgravity might increase human radiosensitivity in space.     

Mechanisms underlying cellular radiosensitivity and R.B.E.: introductory remarks.

A general outline of the symposium titled "Mechanisms underlying cellular radiosensitivity and R.B.E." will be given in the introduction. The essential topics of molecular radiation biology are described with respect to the damage, repair and mutagenesis caused by high-LET irradiation to cellular DNA. The importance of clustered DNA lesions (locally multiply damaged sites) formed in vivo is discussed. This symposium is devoted to the mechanisms of the biological effects of radiation with high LET, especially with regard to the effects of heavy ions and neutrons which may cause possible risks in space flight, (e.g. carcinogenesis and mutagenesis). Detailed understanding of these risks, however, demands knowledge of the molecular mechanisms involved in the biological effects of high-LET radiations. Thus, it was the organizers' idea to hold a symposium dealing with primary physical and chemical events caused in cellular deoxyribonucleoproteins by densely-ionizing radiations and to relate them to track structures and energy transfer processes. The mechanisms of DNA damage were regarded from different points of view including those considering DNA repair and mutagenesis. Problems associated with cell survival and radiation protection were discussed as well. Our knowledge of the molecular mechanisms of high-LET radiation actions, however, is limited compared to what we know about low-LET radiation effects (e.g. from gamma-rays or X-rays). To emphasize this statement, I would like to summarize briefly the open questions in molecular radiation biology, what we know already about low-LET effects and what is lacking describing the effect of high-LET radiation.     

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.