Neoplastic cell transformation by energetic heavy ions and its modification with chemical agents.

For many years we have been interested in understanding the potential carcinogenic effects of cosmic rays. We have studied the oncogenic effects of cosmic rays with accelerator-produced heavy particle radiation and with a cultured mammalian cell system--C3H10T1/2 cells. Our quantitative data obtained with carbon, neon, silicon, and iron particles showed that RBE is both dose and LET dependent for neoplastic cell transformation. RBE is higher at lower dose, and RBE increases with LET up to about 200 keV/micrometer. In nonproliferation confluent cells, heavy-ion induced transformation damage may not be repairable, although a dose modifying factor of about 1.7 was observed for X-ray radiation. Our recent studies with super-heavy high-energy particles, e.g., 960 MeV/U U235 ions (LET = 1900 keV/micrometer), indicate that these ions with a high inactivation cross-section can cause neoplastic cell transformation. The induction of cell transformation by radiation can be modified with various chemicals. We have found that the presence of DMSO (either during or many days after irradiation) decreased the transformation frequency significantly. It is, therefore, potentially possible to reduce the oncogenic effect of cosmic rays in space through some chemical protection.     

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.     

Radiation effects in space.

The radiation protection guidelines of the National Aeronautics and Space Administration (NASA) are under review by Scientific Committee 75 of the National Council Protection and Measurements. The re-evaluation of the current guidelines is necessary, first, because of the increase in information about radiation risks since 1970 when the original recommendations were made and second, the population at risk has changed. For example, women have joined the ranks of the astronauts. Two types of radiation, protons and heavy ions, are of particular concern in space. Unfortunately, there is less information about the effects on tissues and cancer by these radiations than by other radiations. The choice of Quality Factors (Q) for obtaining dose equivalents for these radiations, is an important aspect of the risk estimate for space travel. There are not sufficient data for the induction of late effects by either protons or by heavy ions. The current information suggests a RBE for the relative protons of about 1, whereas, a RBE of 20 for tumor induction by heavy ions, such as iron-56, appears appropriate. The recommendations for the dose equivalent career limits for skin and the lens of the eye have been reduced but the 30-day and annual limits have been raised.     

Neoplastic transformation of hamster embryo cells by heavy ions

We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/μm. Exponentially growing cells were irradiated with ¹²C or ²⁸Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/μm, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.     

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.     

In vivo and in vitro measurements of complex-type chromosomal exchanges induced by heavy ions.

Heavy ions are more efficient in producing complex-type chromosome exchanges than sparsely ionizing radiation, and this can potentially be used as a biomarker of radiation quality. We measured the induction of complex-type chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to accelerated H-, He-, C-, Ar-, Fe- and Au-ions in the LET range of approximately 0.4-1400 keV/micrometers. Chromosomes were analyzed either at the first post-irradiation mitosis, or in interphase, following premature condensation by phosphatase inhibitors. Selected chromosomes were then visualized after FISH-painting. The dose-response curve for the induction of complex-type exchanges by heavy ions was linear in the dose-range 0.2-1.5 Gy, while gamma-rays did not produce a significant increase in the yield of complex rearrangements in this dose range. The yield of complex aberrations after 1 Gy of heavy ions increased up to an LET around 100 keV/micrometers, and then declined at higher LET values. When mitotic cells were analyzed, the frequency of complex rearrangements after 1 Gy was about 10 times higher for Ar- or Fe- ions (the most effective ions, with LET around 100 keV/micrometers) than for 250 MeV protons, and values were about 35 times higher in prematurely condensed chromosomes. These results suggest that complex rearrangements may be detected in astronauts' blood lymphocytes after long-term space flight, because crews are exposed to HZE particles from galactic cosmic radiation. However, in a cytogenetic study of ten astronauts after long-term missions on the Mir or International Space Station, we found a very low frequency of complex rearrangements, and a significant post-flight increase was detected in only one out of the ten crewmembers. It appears that the use of complex-type exchanges as biomarker of radiation quality in vivo after low-dose chronic exposure in mixed radiation fields is hampered by statistical uncertainties.     

Mutation induction by heavy ions.

Mutation induction by heavy ions is compared in yeast and mammalian cells. Since mutants can only be recovered in survivors the influence of inactivation cross sections has to be taken into account. It is shown that both the size of the sensitive cellular site as well as track structure play an important role. Another parameter which influences the probability of mutation induction is repair: Contrary to naive assumptions primary radiation damage does not directly lead to mutations but requires modification to reconstitute the genetic machinery so that mutants can survive. The molecular structure of mutations was analyzed after exposure to deuterons by amplification with the aid of polymerase chain reaction. The results--although preliminary--demonstrate that even with densely ionizing particles a large fraction does not carry big deletions which suggests that point mutations may also be induced by heavy ions.     

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.     

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.     

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.     

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.     

Early and late mammalian responses to heavy charged particles.

This overview summarizes murine results on acute lethality responses, inactivation of marrow CFU-S and intestinal microcolonies, testes weight loss, life span shortening, and posterior lens opacification in mice irradiated with heavy charged particles. RBE-LET relationships for these mammalian responses are compared with results from in vitro studies. The trend is that the maximum RBE for in vivo responses tends to be lower and occurs at a lower LET than for inactivation of V79 and T-1 cells in culture. Based on inactivation cross sections, the response of CFU-S in vivo conforms to expectations from earlier studies with prokaryotic systems and mammalian cells in culture. Effects of heavy ions are compared with fission spectrum neutrons, and the results are consistent with the interpretation that RBEs are lower than for fission neutrons at about the same LET, probably due to differences in track structure. Issues discussed focus on challenges associated with assessments of early and late effects of charged particles based on dose, RBE and LET, and with the concordance or discordance of results obtained with in vivo and in vitro model systems. Models for radiation damage/repair and misrepair should consider effects observed with in vivo as well as in vitro model systems.     

Mutation induction in mammalian cells by very heavy ions.

V79 Chinese hamster cells were exposed to heavy ions (O to U) and assayed for mutants at the HGPRT-locus by incubation in selective medium containing 6-thioguanine. The LET ranged from 300 to 18000 keV/micrometer. Mutants could be recovered from all particle radiation but the effectivity per deposited energy decreased with atomic numbers greater than 8. The results are discussed with regard to fundamental processes of cell reactions to very heavy ions and with respect to possible implications for hazard estimations.     

Mutation induction in yeast by very heavy ions.

Resistance to canavanine was studied in haploid yeast after exposure to heavy ions (argon to uranium) of energies between 1 and 10 MeV/u covering a LET-range up to about 10000 keV/micrometer. Mutations were found in all instances but the induction cross sections increased with ion energy. This is taken to mean that the contribution of penumbra electrons plays an important role. The probability to recover surviving mutants is highest if the cell is not directly hit by the particle. The experiments demonstrate that the geometrical dimensions of the target cell nucleus as well as its sensitivity in terms of survival have a critical influence on mutation induction with very heavy ions.     

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.     

A model of the effects of heavy ion radiation on human tissue

In heavy ion radiotherapy and space travel humans are exposed to energetic heavy ions (C, Si, Fe and others). This type of irradiation often produces more severe biological effects per unit dose than more common X-rays. A new Monte Carlo model generates a physical space with the complex geometry of human tissue or a cell culture based model of tissue, which is affected by the passage of ionizing radiation. For irradiation, the model relies on a physical code for the ion track structure; for tissues, cellular maps are derived from two- or three-dimensional confocal microscopy images using image segmentation algorithm, which defines cells as pixilated volumes. The model is used to study tissue-specific statistics of direct ion hits and the remote ion action on cells. As an application of the technique, we considered the spatial pattern of apoptotic cells after heavy ion irradiation. The pattern of apoptosis is modeled as a stochastic process, which is defined by the action cross section taken from available experimental data. To characterize the degree of apoptosis, an autocorrelation function that describes the spatial correlation of apoptotic cells is introduced. The values of the autocorrelation function demonstrate the effect of the directionality of the radiation track on the spatial arrangements of inactivated cells in tissue. This effect is intrinsic only to high linear-energy-transfer radiation.     

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.     

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.     

Biological features of an early-maturity mutant of sweet sorghum induced by carbon ions irradiation and its genetic polymorphism

It is well known that heavy ions irradiation is characterized by a high linear energy transfer (LET) and relative biological effectiveness (RBE). These characters are believed to increase mutation frequency and mutation spectrum of plants or mammalian cells irradiated by heavy ions. Here we describe an early-maturity mutant of sweet sorghum induced by carbon ion irradiation. The growth period of this mutant was shortened by about 20 days compared to the wild type. The proline content of the mutant was increased by 11.05% while the malondialdehyde content was significantly lower than that of wild type. In addition, the RAPD analysis indicated that the percentage of polymorphism between the mutant KFJT-1 and the control KFJT-CK reached 5.26%. The gain of early-maturity might solve the problem in the northwest region of China where seeds of sweet sorghum cannot be mature because of early frost. The early-maturity mutant may be important for future space cultivation.     

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.