M-FISH analysis of chromosome aberrations in human fibroblasts exposed to energetic iron ions in vitro.

Confluent human fibroblast cells were exposed to 6 Gy gamma-rays or 200 MeV/nucleon Fe ions at 0.7 or 3 Gy. The cells were allowed to repair for 24 hours after exposure and chromosomes were collected using a premature chromosome condensation technique with calyculin-A. Chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique that allows identification of both complex and truly incomplete exchanges. Results showed that both doses of the Fe ions produced higher ratios of complex to simple exchanges and lower ratio of complete to incomplete exchanges than the 6 Gy gamma-exposure. The ratios of aberration yields were similar for the two doses of Fe ions. After 0.7 Gy of Fe ions, most complex aberrations were found to involve three or four chromosomes, indicating this is the maximum number of chromosome domains traversed by a single Fe ion track.     

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.     

Effect of heavy ions on bacterial spores.

Inactivation of B. subtilis spores has been studied using accelerated He, C, N, O and Ne ions. The energy dependence of the inactivation cross sections for heavy ions was very weak and the mean cross sections for carbon ions (0.6-4.7 MeV/amu), nitrogen ions (0.6-4.1 MeV/amu), oxygen ions (0.8-1.1 MeV/amu), and neon ions (2.2-3.7 MeV/amu) were found to be about 0.22, 0.23, 0.26, and 0.33 micrometer2 , respectively. Analysis was carried out along lines similar to Katz's target theory but the parameters were chosen so that they have an experimental basis.     

Radiation effects on late cytopathological parameters in the murine lens relative to particle fluence.

Lenses of mice irradiated with 250 MeV protons, 670 MeV/amu 20Ne, 600 MeV/amu 56Fe, 600 MeV/amu 93Nb and 593 MeV/amu 139La ions were evaluated by analyzing cytopathological indicators which have been implicated in the cataractogenic process. The LETs ranged from 0.40 keV/micrometer to 953 keV/micrometer and fluences from 1.31 10(3)/mm2 to 4.99 x 10(7)/mm2. 60Co gamma-rays were used as the reference radiation. The doses ranged from 10 to 40 cGy. The lenses were assessed 64 weeks post irradiation in order to observe the late effects of LET and dose on the target cell population of the lens epithelium. Our study shows that growth dependent pathological changes occur at the cellular level as a function of dose and LET. The shapes of the RBE-LET and RBE-dose curves are consistent with previous work on eye and other biological systems done in both our laboratory and others. The RBEmax's were estimated, for the most radiation cataract related cytological changes, MN frequency and MR disorganization, by calculating the ratio of the initial slopes of dose effect curve for various heavy ions to that of 60Co gamma-ray. For each ion studied, the RBEmax derived from micronucleus (MN) frequency is similar to that derived from meridional row (MR) disorganization, suggesting that heavy ions are equally efficient at producing each type of damage. Furthermore, on a per particle basis (particle/cell nucleus), both MN frequency and MR disorganization are LET dependent indicating that these classic precataractogenic indicators are multi-gene effects. Poisson probability analysis of the particle number traversing cell nuclei (average area = 24 micrometers2) suggested that single nuclear traversals determine these changes. By virtue of their precataractogenic nature the data on these endpoints intimate that radiation cataract may also be the consequence of single hits. In any case, these observations are consistent with the current theory of the mechanism of radiation cataractogenesis, which proposes that genomic damage to the epithelial cells surviving the exposure is responsible for opacification.     

Late skin damage in rabbits and monkeys after exposure to particulate radiations.

Skin biopsies were taken from the central regions of the ears of New Zealand white rabbits following localized exposure of one ear of each rabbit to 530 MeV/amu Ar or 365 MeV/amu Ne ions. The unirradiated ears served as controls. Biopsies were taken also from the chests and inner thighs of rhesus monkeys after whole-body exposure to 32 MeV protons and from unirradiated control animals. The linear energy transfers (LET infinity's) for the radiations were 90 +/- 5, 35 +/- 3, and approximately 1.2 keV/micrometer, respectively. In the rabbit studies, explants were removed with a 2 mm diameter dermal punch at post-irradiation times up to five years after exposure. Similar volumes of monkey tissue were taken from skin samples excised surgically 16-18 years following proton irradiation. Fibroblast cultures were initiated from the explants and were propagated in vitro until terminal senescence (cessation of cell division) occurred. Cultures from irradiated tissue exhibited decreases in doubling potential that were dependent on radiation dose and LET infinity and seemed to reflect damage to stem cell populations. The implications of these results for astronauts exposed to heavy ions and/or protons in space include possible manifestations of residual effects in the skin many years after exposure (e.g. unsatisfactory responses to trauma or surgery).     

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.     

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.     

Chromosomal intrachanges induced by swift iron ions.

We measured the induction of structural aberrations in human chromosome 5 induced by iron ions using the novel technique of multicolor banding in situ hybridization (mBAND). Human lymphocytes isolated from whole blood were exposed in vitro to 500 MeV/n (LET=200 keV/micrometers, doses 1 or 4 Gy) Fe nuclei at the HIMAC accelerator in Chiba (Japan). Chromosomes were prematurely condensed by calyculin A after 48 h in culture and slides were painted by mBAND. We found a frequency of 0.11 and 0.57 residual breakpoints per chromosome 5 after 1 and 4 Gy Fe-ions, respectively. Inter-chromosomal exchanges were the prevalent aberration type measured at both doses, followed by terminal deletions, and by intra-chromosomal exchanges. Among intra-chromosomal exchanges, intra-arm events were more frequent than inter-arm, but a significant number of intra-changes was associated to inter-changes involving the same chromosome after 4 Gy of iron ions. These events show that the complexity of chromosomal exchanges induced by heavy ions can be higher than expected by previous FISH studies.     

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.     

Effects of temperature, CO2/O2 concentrations and light intensity on cellular multiplication of microalgae, Euglena gracilis.

Microalgae culture is likely to play an important role in aquatic food production modules in bioregenerative systems for producing feeds for fish, converting CO2 to O2 and remedying water quality as well as aquatic higher plants. In the present study, the effects of culture conditions on the cellular multiplication of microalgae, Euglena gracilis, was investigated as a fundamental study to determine the optimum culture conditions for microalgae production in aquatic food production modules including both microalgae culture and fish culture systems. E. gracilis was cultured under conditions with five levels of temperatures (25-33 degrees C), three levels of CO2 concentrations (2-6%), five levels of O2 concentrations (10-30%), and six levels of photosynthetic photon flux (20-200 micromoles m-2 s-1). The number of Euglena cells in a certain volume of solution was monitored with a microscope under each environmental condition. The multiplication rate of the cells was highest at temperatures of 27-31 degrees C, CO2 concentration of 4%, O2 concentration of 20% and photosynthetic photon flux of about 100 micromoles m-2 s-1. The results demonstrate that E. gracilis could efficiently produce biomass and convert CO2 to O2 under relatively low light intensities in aquatic food production modules.     

Late effects from particulate radiations in primate and rabbit tissues.

Optic tissues in groups of New Zealand white rabbits were irradiated locally at different stages throughout the median life span of the species with a single dose (9 Gy) of 425 MeV/amu Ne ions (LET infinity approximately 30 keV/micrometer) and then inspected routinely for the progression of radiation cataracts. The level of early cataracts was found to be highest in the youngest group of animals irradiated (8 weeks old), but both the onset of late cataracts and loss of vision occurred earlier when animals were irradiated during the second half of the median life span. This age response can have serious implications in terms of space radiation hazards to man. Rhesus monkeys that had been subjected to whole-body skin irradiation (2.8 and 5.6 Gy) by 32 MeV protons (range in tissue approximately 1 cm) some twenty years previously were analysed for radiation damage by the propagation of skin fibroblasts in primary cultures. Such propagation from skin biopsies in MEM-alpha medium (serial cultivation) or in supplemented Ham's F-10 medium (cultivation without dilution) revealed late damage in the stem (precursor) cells of the skins of the animals. The proton fluxes employed in this experiment are representative of those occurring in major solar flares.     

Induction of asymmetrical type of chromosomal aberrations in cultured human lymphocytes by ion beams of different energies at varying let from HIMAC and RRC

Frequencies of asymmetrical type of chromosome aberration were scored in cultured human blood lymphocytes irradiated with carbon and neon beams. Blood cells were irradiated with various doses to establish dose response curves for chromosome aberration frequency vs. dose, and chromosome preparation was made by conventional method. Dose response curves for the per cell frequencies of the dicentrics and centric rings as well as the excess amount of acentric fragments were described for 7 different qualities (LET= 22.4, 40.0, 41.5, 69.9, 70.0, 100.0 and 150 KeV/μm) of carbon and neon beams with three different energies, 135, 290 and 400 MeV/u. From the analysis of those dose response curves, the maximum effect was found in the region of LET value at near 70 KeV/μm together with linear expression in the response from all endpoints examined. The 135 MeV/u of carbons (69.9 KeV/μm) and neons (70.0 KeV/μm) showed linear response. The 290 MeV/u of carbons (100 KeV/m) and neons (150 KeV/μm) showed medium effects with different shape of response, linear with a plateau and upward concavity. The 2 carbon beams (41.5 and 40 KeV/μm) from 2 different accelerators showed much discrepancy in the response. RBE-LET relationship was also described by comparing the coefficient of the 7 different dose responses. The peak (near 70 KeV/m) was localized colse to that (80 KeV/m) for the survivals of dsb repair deficient cells (Eguchi-Kasai et al. 1998), but in different position from that previously reported in many other studies (100–200 KeV/mm). Identification of the RBEmax in the present study has yet to be definitive.     

W-reactivation and W-mutagenesis in phage phi X 174.

W-reactivation (WR) and W-mutagenesis (WM) were observed after irradiation of phage chi X 174 with He and N ions (4.6 MeV/amu). The relative amount of phage RFI DNA (RARFI) extracted from the infected cells was determined by agarose gel electrophoresis. It was found that RARFI was increased by prior UV-irradiation of host cells and this increase corresponded to the efficiency of WR.     

Gravitaxis in unicellular microorganisms.

Orientation of organisms with respect to the gravitational field of the Earth has been studied for more than 100 years in a number of unicellular microorganisms including flagellates and ciliates. Several hypotheses have been developed how the weak stimulus is perceived. Intracellular statoliths have been found to be involved in gravitaxis of Loxodes, while no specialized organelles have been detected in other ciliates, e.g. Paramecium. Also in the slime mold Physarum no specialized gravireceptors have been identified yet. In the flagellate Euglena gracilis the whole cell body, which is denser than the surrounding medium, seems to act as a statolith pressing onto the lower membrane where it activates mechanosensitive ion channels. Similar results were obtained for the ciliate Paramecium. In contrast to the flagellate Euglena, several ciliates have been found to show gravikinesis, which is defined as a dependence of the swimming velocity on the direction of movement in the gravity field.     

Mutagenic effects of heavy ion radiation in plants.

Genetic and developmental effects of heavy ions in maize and rice were investigated. Heavy particles with various charges and energies were accelerated at the BEVALAC. The frequency of occurrence of white-yellow stripes on leaves of plants developed from irradiated maize seeds increased linearly with dose, and high-LET heavy charged particles, e.g., neon, argon, and iron, were 2-12 times as effective as gamma rays in inducing this type of mutation. The effectiveness of high-LET heavy ion in (1) inhibiting rice seedling growth, (2) reducing plant fertility, (3) inducing chromosome aberration and micronuclei in root tip cells and pollen mother cells of the first generation plants developed from exposed seeds, and (4) inducing mutation in the second generation, were greater than that of low-LET gamma rays. All effects observed were dose-dependent; however, there appeared to be an optimal range of doses for inducing certain types of mutation, for example, for argon ions (400 MeV/u) at 90-100 Gy, several valuable mutant lines with favorable characters, such as semidwarf, early maturity and high yield ability, were obtained. Experimental results suggest that the potential application of heavy ions in crop improvement is promising. RFLP analysis of two semidwarf mutants induced by argon particles revealed that large DNA alterations might be involved in these mutants.     

Mutagenic effects of heavy ions in bacteria.

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

Effects of 40Ar and 56Fe ions on retinal photoreceptor cells of the rabbit: implications for manned missions to Mars.

Losses of photoreceptor cells (rods) from the retinas of New Zealand white (NZW) rabbits were detectable within 2 years after localized acute irradiation of optic and proximal tissues with > or = 7 Gy of 530 MeV u-1 40Ar ions or > or = 2 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition. Those limits were determined only from an analysis of variance of dose groups because the shapes of the dose response curves at early post-irradiation times are not known, a concern being addressed by experiments in progress. Losses of photoreceptor cells for the period 0.5-2.5 years post-irradiation, determined by provisional linear regression analysis, were approximately 1.7% Gy-1 and 2.5% Gy-1 for 40Ar and 56Fe ions, respectively.     

Induction of genomic instability after an acute whole-body exposure of mice to Fe ions

The purpose of this study was to evaluate dose–response relationships for the in vivo induction of micronuclei (MN) as a measure of both initial radiation damage and the induction of genomic instability. These measurements were made in mouse blood erythrocytes as a function of radiation dose, radiation quality, time after irradiation, and the genetic background of exposed individuals. Blood samples were collected from two strains of mouse (CBA/CaJ and C57BL/6J) at different times up to 3 months following a whole-body exposure to various doses of 1 GeV/amu ⁵⁶Fe ions (0, 0.1, 0.5 and 1.0 Gy, at the dose rate of a 1 Gy/min) or ¹³⁷Cs gamma rays (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min). Blood-smear slides were stained with acridine orange (AO). The frequencies of MN were measured in mature normochromatic-erythrocytes (MN-NCEs) and in immature polychromatic-erythrocytes (MN-PCEs). Effects of both types of radiation on erythropoiesis were also evaluated. As a measure of cell progression delay, a dose-dependent decrease in numbers of PCEs was observed at day 2 post-exposure in both strains, regardless of radiation quality. Subsequently, the levels of PCEs increased in all exposed mice, reaching control levels (or higher) by day 7 post-exposure. Further, at day 2 after the exposure, there was no increase in the frequency of MN-PCEs in CBA/CaJ mice exposed to ⁵⁶Fe ions while the frequency of MN-PCEs elevated as a function of dose in the C57BL/6J mice. At day 4, there was no dose related increase in MN-NCEs in either strain of mouse exposed to ¹³⁷Cs gamma rays. Additionally, at the early sacrifice times (days 2 and 4), ⁵⁶Fe ions were slightly more effective (per unit dose) in inducing MN-NCEs than ¹³⁷Cs gamma rays in CBA/CaJ mice. However, there was no increase in the frequency of MN-NCEs at late times after an acute exposure to either type of radiation. In contrast, both types of radiation induced increased MN-PCEs frequencies in irradiated CBA/CaJ mice, but not C57BL/6J mice, at late times post-exposure. This finding indicates the potential induction of genomic instability in hematopoietic cells of CBA/CaJ mice by both types of radiation. The finding also demonstrates the influence of genetic background on radiation-induced genomic instability in vivo.     

Damage to the photoreceptor cells of the rabbit retina from 56Fe ions: effect of age at exposure, 1.

Optic and proximate tissues of New Zealand white (NZW) rabbits at ages (approximately 3.5 years) near the middle of their median lifespan (5-7 years) were given 0.5-3.5 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition at a linear energy transfer (LET infinity) of 220 +/- 31 keV micrometer-1. Dose-dependent losses of retinal photoreceptor cells (rods) occurred until 1-2 years after irradiation, the period of this interim report. Similar cumulative losses of photoreceptor cells were seen during the period 1-2 years post-irradiation for rabbits given comparable exposures when young (6-9 weeks old). Since losses of photoreceptor cells at early times had not been determined previously, the current experiment, which was designed to simulate the responses of mature astronauts, redressed that deficiency.