Mutational effects of space flight on Zea mays seeds.

The growth and development of more than 500 Zea mays seeds flown on LDEF were studied. Somatic mutations, including white-yellow stripes on leaves, dwarfing, change of leaf sheath color or seedling color were observed in plants developed from these seeds. When the frequency of white-yellow formation was used as the endpoint and compared with data from ground based studies, the dose to which maize seeds might be exposed during the flight was estimated to be equivalent to 635 cGy of gamma rays. Seeds from one particular holder gave a high mutation frequency and a wide mutation spectrum. White-yellow stripes on leaves were also found in some of the inbred progenies from plants displayed somatic mutation. Electron microscopy studies showed that the damage of chloroplast development in the white-yellow stripe on leaves was similar between seeds flown on LDEF and that irradiated by accelerated heavy ions on ground.     

G2-chromosome aberrations induced by high-LET radiations.

We report measurement of initial G2-chromatid breaks in normal human fibroblasts exposed to various types of high-LET particles. Exponentially growing AG 1522 cells were exposed to gamma rays or heavy ions. Chromosomes were prematurely condensed by calyculin A. Chromatid-type breaks and isochromatid-type breaks were scored separately. The dose response curves for the induction of total chromatid breaks (chromatid-type + isochromatid-type) and chromatid-type breaks were linear for each type of radiation. However, dose response curves for the induction of isochromatid-type breaks were linear for high-LET radiations and linear-quadratic for gamma rays. Relative biological effectiveness (RBE), calculated from total breaks, showed a LET dependent tendency with a peak at 55 keV/micrometer silicon (2.7) or 80 keV/micrometer carbon (2.7) and then decreased with LET (1.5 at 440 keV/micrometer). RBE for chromatid-type break peaked at 55 keV/micrometer (2.4) then decreased rapidly with LET. The RBE of 440 keV/micrometer iron particles was 0.7. The RBE calculated from induction of isochromatid-type breaks was much higher for high-LET radiations. It is concluded that the increased production of isochromatid-type breaks, induced by the densely ionizing track structure, is a signature of high-LET radiation exposure.     

Dose protraction studies with low- and high-LET radiations on neoplastic cell transformation in vitro.

A major objective of our heavy-ion research is to understand the potential carcinogenic effects of cosmic rays and the mechanisms of radiation-induced cell transformation. During the past several years, we have studied the relative biological effectiveness of heavy ions with various atomic numbers and linear energy transfer on neoplastic cell transformation and the repair of transformation lesions induced by heavy ions in mammalian cells. All of these studies, however, were done with a high dose rate. For risk assessment, it is extremely important to have data on the low-dose-rate effect of heavy ions. Recently, with confluent cultures of the C3H10T1/2 cell line, we have initiated some studies on the low-dose-rate effect of low- and high-LET radiation on cell transformation. For low-LET photons, there was a decrease in cell killing and cell transformation frequency when cells were irradiated with fractionated doses and at low dose rate. Cultured mammalian cells can repair both subtransformation and potential transformation lesions induced by X rays. The kinetics of potential transformation damage repair is a slow one. No sparing effect, however, was found for high-LET radiation. There was an enhancement of cell transformation for low-dose-rate argon (400 MeV/u; 120 keV/micrometer) and iron particles (600 MeV/u; 200 keV/micrometer). The molecular mechanisms for the enhancement effect is unknown at present.     

DNA damage and repair in oncogenic transformation by heavy ion radiation.

Energetic heavy ions are present in galactic cosmic rays and solar particle events. One of the most important late effects in risk assessment is carcinogenesis. We have studied the carcinogenic effects of heavy ions at the cellular and molecular levels and have obtained quantitative data on dose-response curves and on the repair of oncogenic lesions for heavy particles with various charges and energies. Studies with repair inhibitors and restriction endonucleases indicated that for oncogenic transformation DNA is the primary target. Results from heavy ion experiments showed that the cross section increased with LET and reached a maximum value of about 0.02 micrometer2 at about 500 keV/micrometer. This limited size of cross section suggests that only a fraction of cellular genomic DNA is important in radiogenic transformation. Free radical scavengers, such as DMSO, do not give any effect on induction of oncogenic transformation by 600 MeV/u iron particles, suggesting most oncogenic damage induced by high-LET heavy ions is through direct action. Repair studies with stationary phase cells showed that the amount of reparable oncogenic lesions decreased with an increase of LET and that heavy ions with LET greater than 200 keV/micrometer produced only irreparable oncogenic damage. An enhancement effect for oncogenic transformation was observed in cells irradiated by low-dose-rate argon ions (400 MeV/u; 120 keV/micrometer). Chromosomal aberrations, such as translocation and deletion, but not sister chromatid exchange, are essential for heavy-ion-induced oncogenic transformation. The basic mechanism(s) of misrepair of DNA damage, which form oncogenic lesions, is unknown.     

Heavy-ion induced genetic changes and evolution processes.

On Moon and Mars, there will be more galactic cosmic rays and higher radiation doses than on earth. Our experimental studies showed that heavy ion radiation can effectively cause mutation and chromosome aberrations and that high-LET heavy-ion induced mutants can be irreversible. Chromosome translocations and deletions are common in cells irradiated by heavy particles, and ionizing radiations are effective in causing hyperploidy. The importance of the genetic changes in the evolution of life is an interesting question. Through evolution, there is an increase of DNA content in cells from lower forms of life to higher organisms. The DNA content, however, reached a plateau in vertebrates. By increasing DNA content, there can be an increase of information in the cell. For a given DNA content, the quality of information can be changed by rearranging the DNA. Because radiation can cause hyperploidy, an increase of DNA content in cells, and can induce DNA rearrangement, it is likely that the evolution of life on Mars will be effected by its radiation environment. A simple analysis shows that the radiation level on Mars may cause a mutation frequency comparable to that of the spontaneous mutation rate on Earth. To the extent that mutation plays a role in adaptation, radiation alone on Mars may thus provide sufficient mutation for the evolution of life.     

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.     

DNA fragmentation in mammalian cells exposed to various light ions.

Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/micrometer protons, 123 keV/micrometer helium-4 ions and gamma rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respect to that induced by comparable doses of gamma rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for gamma rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage repairability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by gamma rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.     

On the origin of trapped heavy ions at L = 1.4-1.6.

Aboard the NASA satellite Long Duration Exposure Facility (LDEF) heavy ions of nuclear charge Z = 8-26 were detected with energies between 15 and 50 MeV/nuc which are far below the cutoff energy required of fully stripped ions to reach the LDEF orbit. The arrival directions and the falling energy spectra of these particles are consistent with a trapped component incident in the South Atlantic Anomaly at L = 1.4-1.6. The trapped oxygen, neon and argon ions probably originate from the anomalous cosmic rays, whereas the origin of the other particles like magnesium, silicon and iron is not yet solved but may be associated with the October 89 solar energetic particle events.     

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.     

Morphological and molecular changes of maize plants after seeds been flown on recoverablf satellite

Dry seeds of Zea mays, heterozygous for Lw₁/lw₁ alleles, sandwiched between nuclear track detectors aboard Chinese satellite for 15 days, were recovered and mutations in morphological characters on plants developed from these seeds, as well as their selected progenies, were investigated. The dosimetric results indicated that 85% of the seeds received at least 1 hit with Z≥20. About 10% of plants developed from flown seeds and 40% of observed selfed lines from the first generation plants showed some morphological changes, such as yellow stripes displayed on leaves, dwarf, anomogensis of floral organs and yellow-green seedlings, when compared with those from ground control. Using yellow stripes on leaves as the main endpoint for evaluating mutation induced in space environment, the frequency of stripe occurrence was 4.6% in the first generation plants, comparable with the results obtained from Long Duration Exposure Facility (LDEF) mission (Mei et al., 1994), but much lower than those from ground based ⁶⁰ Co-gamma treatment at a dose of 100 Gy, which reached 35.5% in the selfed lines of the second generation. One hundred and ten random primers were screened in RAPD analysis to detect the variation on genomic DNA of plants with stripes on leaves. Of these primers, 10.9% were able to generate polymorphic bands between mutated plants and control, also, common band patterns in several progenies with the same mutation phenotype were observed. These results demonstrated that space radiation environment could induce inheritable mutagenic effects on plant seeds, and verified the change in genetic material in the mutants. Further study will be needed for a better understand of the nature and mechanism of this induction of mutation.     

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 exposure to different types of radiation on behaviors mediated by peripheral or central systems.

The effects of exposure to ionizing radiation on behavior may result from effects on peripheral or on central systems. For behavioral endpoints that are mediated by peripheral systems (e.g., radiation-induced conditioned taste aversion or vomiting), the behavioral effects of exposure to heavy particles (56Fe, 600 MeV/n) are qualitatively similar to the effects of exposure to gamma radiation (60Co) and to fission spectrum neutrons. For these endpoints, the only differences between the different types of radiation are in terms of relative behavioral effectiveness. For behavioral endpoints that are mediated by central systems (e.g., amphetamine-induced taste aversion learning), the effects of exposure to 56Fe particles are not seen following exposure to lower LET gamma rays or fission spectrum neutrons. These results indicate that the effects of exposure to heavy particles on behavioral endpoints cannot necessarily be extrapolated from studies using gamma rays, but require the use of heavy particles.     

DNA fragmentation induced by Fe ions in human cells: shielding influence on spatially correlated damage.

Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used gamma rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by gamma rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions.     

Early and delayed reproductive death in human cells exposed to high energy iron ion beams.

The aim of this research was to determine the biological effectiveness for early and delayed effects of high energy, high linear energy transfer (LET) charged particles. Survival and delayed reproductive death were measured in AG1522 human fibroblast cells exposed to Fe-ion beams of energies between 0.2 and 1 GeV/n, 0.97 GeV/n Ti-ion and 0.49 GeV/n Si-ion beams. The cells were irradiated at the HIMAC accelerator in Chiba, Japan (0.2 and 0.5 GeV/n Fe and 0.49 GeV/n Si) and at the NASA Space Radiation Laboratory in Brookhaven, USA (1 GeV/n Fe and 0.97 GeV/n Ti ions). The dose-effect curves were measured in the dose range between 0.25 and 2 Gy. For comparison cells were exposed to 60Co gamma rays. Analysis of the dose-effect curves show that all the heavy ion beams induce inactivation and delayed reproductive death more effectively than 60Co gamma rays. The only exception is the 0.2 GeV/n Fe-ion beam at low doses. The progeny of the irradiated cells show delayed damage in the form of reproductive death with all the heavy ion beams with the 1 GeV/n Fe-ion beam being the most effective. The relative biological effectiveness at low doses of the iron beams is highest for LET values between 140 and 200 keV/micrometers with values of 1.6 and 3 for early and delayed reproductive death, respectively. Analysis of the fluence-effect curves shows that the cross-sections for early and delayed inactivation increase with increasing LET up to 442 keV/micrometers.     

Mutation induction in human lymphoid cells by energetic heavy ions.

One of the concerns for extended space flight outside the magnetosphere is exposure to galactic cosmic radiation. In the series of studies presented herein, the mutagenic effectiveness of high energy heavy ions is examined using human B-lymphoblastoid cells across an LET range from 32keV/micrometer to 190 keV/micrometer. Mutations were scored for an autosomal locus, thymidine kinase (tk), and for an X-linked locus, hypoxanthine phosphoribosyltransferase (hprt). For each of the radiations studied, the autosomal locus is more sensitive to mutation induction than is the X-linked locus. When mutational yields are expressed in terms of particle fluence, the two loci respond quite differently across the range of LET. The action cross section for mutation induction peaks at 61 keV/micrometer for the tk locus and then declines for particles of higher LET, including Fe ions. For the hprt locus, the action cross section for mutation is maximal at 95 keV/micrometer but is relatively constant across the range from 61 keV/micrometer to 190 keV/micrometer. The yields of hprt-deficient mutants obtained after HZE exposure to TK6 lymphoblasts may be compared directly with published data on the induction of hprt-deficient mutants in human neonatal fibroblasts exposed to similar ions. The action cross section for induction of hprt-deficient mutants by energetic Fe ions is more than 10-fold lower for lymphoblastoid cells than for fibroblasts.     

The influence of space flight factors on viability and mutability of plants.

The experiments with air-dried Crepis capillaris seeds aboard the Soyuz 16 spaceship and the orbital stations Salyut 5, 6, 7 have revealed an increase in the frequency of aberrant cells in seedlings grown from flight-exposed seeds during the flight (experiment) and after the flight on Earth (flight control) as compared to the ground-based control. The increase in seedlings grown during the flight is more significant than in the flight control. During the flight Arabidopsis thaliana developed from cotyledons to the flowering stage. Analysis of seeds setting on these plants after the flight has shown a reduction in the fertility of these plants and an increase in the frequency of recessive mutants ("Light block-1"). An increased frequency of mutants was also retained in the progeny of plants which had passed through a complete cycle of development during the flight ("Fiton-3"). Suppression of embryo viability was observed in all experiments and expressed itself in reduced germinating ability of seeds from the exposed plants and in the early death of seedlings. Damages resulting from chromosome aberrations are eliminated in the first postflight generation and damages resulting from gene mutations and micro-aberrations are preserved for a longer time.     

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.     

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.     

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.     

Oncogenic transformation of mammalian cells by ultrasoft X-rays and alpha particles.

For a better understanding of oncogenic cell transformation by ionizing radiation, we conducted experiments with ultrasoft X rays and low energy alpha particles. Confluent C3H10T1/2 cells were irradiated by Al-K (1.5 keV) X rays or alpha particles from plutonium through a thin mylar sheet, on which the cells attached and grew. Our results indicated that Al-K X rays were more effective in causing cell inactivation and oncogenic transformation than 60Co gamma rays but less effective than 1.0 and 3.7 MeV alpha particles. There was no significant difference between 1.0 and 3.7 MeV alpha particles in transforming cells although the latter were slightly more effective than the former in producing lethal effect. These results indicated that track structure is important in causing biological effects by ionizing radiation.