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.     

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.     

Chromosome aberrations in human lymphocytes induced by 250 MeV protons: effects of dose, dose rate and shielding.

Although the space radiation environment consists predominantly of energetic protons, astronauts inside a spacecraft are chronically exposed to both primary particles as well as secondary particles that are generated when the primary particles penetrate the spacecraft shielding. Secondary neutrons and secondary charged particles can have an LET value that is greater than the primary protons and, therefore, produce a higher relative biological effectiveness (RBE). Using the accelerator facility at Loma Linda University, we exposed human lymphocytes in vitro to 250 MeV protons with doses ranging from 0 to 60 cGy at three different dose rates: a low dose rate of 7.5 cGy/h, an intermediate dose rate of 30 cGy/h and a high dose rate of 70 cGy/min. The effect of 15 g/cm2 aluminum shielding on the induction of chromosome aberrations was investigated for each dose rate. After exposure, lymphocytes were incubated in growth medium containing phytohemagglutinin (PHA) and chromosome spreads were collected using a chemical-induced premature chromosome condensation (PCC) technique. Aberrations were analyzed using the fluorescence in situ hybridization (FISH) technique with three different colored chromosome-painting probes. The frequency of reciprocal and complex-type chromosome exchanges were compared in shielded and unshielded samples.     

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.     

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

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

On the parameterization of the biological effect in a mixed radiation field.

The exposure of astronauts and electronics to the cosmic radiation especially to the particle component pose a major risk to all space flights. Up to now it is not possible to quantify this risk within acceptable limits of accuracy. This uncertainty is not only caused by difficulties in the more or less exact prediction of the incidence of the cosmic radiation but depends also on the problem of the quantification of the radiation field and the correlation of the biological effect. Usually the biological action of a mixed radiation field is estimated as product of the measured dose with an average quality factor, the relative biological efficiency. Because of the large variation in energy and atomic number of the cosmic particles, average values of the quality factor are not precise for risk estimation. A more appropriate way to treat the biological effects of mixed radiation is the concept of particle fluence and action cross section.     

Chromosome aberrations in astronauts

A review of currently available data on in vivo induced chromosome damage in the blood lymphocytes of astronauts proves that cytogenetic biodosimetry analyses on blood collected within a week or two of return from space provides a reliable estimate of equivalent radiation dose and risk after protracted exposure to space radiation of a few months or more. Recent studies indicate that biodosimetry estimates from single spaceflights lie within the range expected from physical dosimetry and biophysical models, but very large uncertainties are associated with single individual measurements and the total sample population remains low. Retrospective doses may be more difficult to estimate because of the fairly rapid time-dependent loss of “stable” aberrations in blood lymphocytes. Also, biodosimetry estimates from individuals who participate in repeated missions, or very long (interplanetary) missions, may be complicated by an adaptive response to space radiation and/or changes in lymphocyte survival and repopulation. A discussion of published data is presented and specific issues related to space radiation biodosimetry protocols are discussed.     

Radiosensibility of higher plant seeds after space flight.

The influence of long-term storage of higher plant seeds under space flight conditions (49 to 827 days) on their radiosensibility was studied in the experiments on the orbital stations Salyut 6 and 7. Short-term storage has been proved to have no effect on radiosensitivity of Crepis capillaris seeds. Only in the case of maximal exposure duration the frequency of chromosome aberrations in post-flight irradiated seeds significantly exceeded the chromosome aberration frequency in the ground-based irradiated control. A statistically significant increase in the number of cells with multiple chromosome aberrations was also observed in this experiment. After gamma-irradiation of Arabidopsis thaliana seeds the germinating ability and survival rate of plants decreased depending on the duration of seed storage. Flight-exposed seeds were more sensitive to irradiations with respect to these parameters. A statistically significant increase in the frequency of recessive lethal mutations was observed only in two experiments of long exposure duration.     

Cytogenetic examination of cosmonauts for space radiation exposure estimation

Purpose

To evaluate radiation induced chromosome aberration frequency in peripheral blood lymphocytes of cosmonauts who participated in flights on Mir Orbital Station and ISS (International Space Station).

Materials and methods

Cytogenetic examination which has been performed in the period 1992–2008 included the analysis of chromosome aberrations using conventional Giemsa staining method in 202 blood samples from 48 cosmonauts who participated in flights on Mir Orbital Station and ISS.

Results

Space flights led to an increase of chromosome aberration frequency. Frequency of dicentrics plus centric rings (Dic+Rc) depend on the space flight duration and accumulated dose value. After the change of space stations (from Mir Orbital Station to ISS) the radiation load of cosmonauts based on data of cytogenetic examination decreased. Extravehicular activity also adds to chromosome aberration frequency in cosmonauts’ blood lymphocytes. Average doses after the first flight, estimated by the frequency of Dic+Rc, were 227 and 113 mGy Eq for long-term flights (LTF) and 107 and 53 mGy Eq for short-term flights (STF).

Conclusion

Cytogenetic examination of cosmonauts can be applied to assess equivalent doses.     

Possible effects of protracted exposure on the additivity of risks from space radiations.

Conventional radiation risk assessments are presently based on the additivity assumption. This assumption states that risks from individual components of a complex radiation field involving many different types of radiation can be added to yield the total risk of the complex radiation field. If the assumption is not correct, the summations and integrations performed to obtain the presently quoted risk estimates are not appropriate. This problem is particularly important in the area of space radiation risk evaluation because of the many different types of high- and low-LET radiation present in the galactic cosmic ray environment. For both low- and high-LET radiations at low enough dose rates, the present convention is that the addivity assumption holds. Mathematically, the total risk, Rtot is assumed to be Rtot = summation (i) Ri where the summation runs over the different types of radiation present. If the total dose (or fluence) from each component is such that the interaction between biological lesions caused by separate single track traversals is negligible within a given cell, it is presently considered to be reasonable to accept the additivity assumption. However, when the exposure is protracted over many cell doubling times (as will be the case for extended missions to the moon or Mars), the possibility exists that radiation effects that depend on multiple cellular events over a long time period, such as is probably the case in radiation-induced carcinogenesis, may not be additive in the above sense and the exposure interval may have to be included in the evaluation procedure. It is shown, however, that "inverse" dose-rate effects are not expected from intermediate LET radiations arising from the galactic cosmic ray environment due to the "sensitive-window-in-the-cell-cycle" hypothesis.     

Cytogenetic studies of blood lymphocytes from cosmonauts after long-term space flights on Mir station.

Long-term space missions may increase risks of unfavorable consequences for cosmonauts as a result of radiation effects. This paper presents results of a study of cytogenetic damage in cosmonauts' peripheral blood lymphocytes induced by space radiation. Cultivation of lymphocytes and analysis of chromosomal aberrations were made according to generally accepted methods. It is shown that the yields of dicentrics and centric rings scored after long-term space flights are considerably higher than those scored prior to the flights. An attempt was made to assess individual doses received by cosmonauts. Individual biodosimetry doses received by cosmonauts who showed a reliable increase in the yields of chromosomal-type aberrations after their first flights were estimated to be from 0.02 to 0.28 Gy.     

Biological monitoring of radiation exposure.

Complementary to physical dosimetry, biological dosimetry systems have been developed and applied which weight the different components of environmental radiation according to their biological efficacy. They generally give a record of the accumulated exposure of individuals with high sensitivity and specificity for the toxic agent under consideration. Basically three different types of biological detecting/ monitoring systems are available: (i) intrinsic biological dosimeters that record the individual radiation exposure (humans, plants, animals) in measurable units. For monitoring ionizing radiation exposure, in situ biomarkers for genetic (e.g. chromosomal aberrations in human lymphocytes, germ line minisatellite mutation rates) or metabolic changes in serum, plasma and blood (e.g. serum lipids, lipoproteins, lipid peroxides, melatonin, antibody titer) have been used. (ii) Extrinsic biological dosimeters/indicators that record the accumulated dose in biological model systems. Their application includes long-term monitoring of changes in environmental UV radiation and its biological implications as well as dosimetry of personal UV exposure. (iii) Biological detectors/biosensors for genotoxic substances and agents such as bacterial assays (e.g. Ames test, SOS-type test) that are highly sensitive to genotoxins with high specificity. They may be applicable for different aspects in environmental monitoring including the International Space Station.     

Effects of prolonged exposure to space flight factors for 175 days on lettuce seeds

We have studied the effects of prolonged (up to 175 days) exposure of $\text{Lactuca sativa}$ seeds to space flight factors, including primary cosmic radiation heavy ions. The data obtained evidence a significant fourfold increase ofs pontaneous mutagenesis in seeds both with regard to the total number of aberrant cells as well as the formation of single cells with multiple aberrations. Comparison of the present experiment with earlier works shows that the frequency of such aberrations increases with the duration of the flight.     

Simultaneous measurement of multiple radiation-induced protein expression profiles using the Luminex(TM) system.

Space flight results in the exposure of astronauts to a mixed field of radiation composed of energetic particles of varying energies, and biological indicators of space radiation exposure provides a better understanding of the associated long-term health risks. Current methods of biodosimetry have employed the use of cytogenetic analysis for biodosimetry, and more recently the advent of technological progression has led to advanced research in the use of genomic and proteomic expression profiling to simultaneously assess biomarkers of radiation exposure. We describe here the technical advantages of the Luminex(TM) 100 system relative to traditional methods and its potential as a tool to simultaneously profile multiple proteins induced by ionizing radiation. The development of such a bioassay would provide more relevant post-translational dynamics of stress response and will impart important implications in the advancement of space and other radiation contact monitoring.     

Role of chromosome instability in long term effect of manned-space missions.

Astronauts are exposed to heavy ions during space missions and heavy ion induced-chromosome damages have been observed in their lymphocytes. This raises the problem of the consequence of longer space flights. Recent studies show that some alterations can appear many cell generations after the initial radiation exposure as a delayed genomic instability. This delayed instability is characterized by the accumulation of cell alterations leading to cell transformation, delayed cell death and mutations. Chromosome instability was shown in vitro in different model systems (Sabatier et al., 1992; Marder and Morgan, 1993, Kadhim et al., 1994 and Holmberg et al., 1993, 1995). All types of radiation used induce a chromosome instability, however, heavy ions cause the most damage. The period of chromosome instability followed by the formation of clones with unbalanced karyotypes seems to be shared by cancer cells. The shortening of telomere sequences leading to the formation of telomere fusions is an important factor in the appearance of this chromosome instability.     

Analytical-HZETRN model for rapid assessment of active magnetic radiation shielding

The use of active radiation shielding designs has the potential to reduce the radiation exposure received by astronauts on deep-space missions at a significantly lower mass penalty than designs utilizing only passive shielding. Unfortunately, the determination of the radiation exposure inside these shielded environments often involves lengthy and computationally intensive Monte Carlo analysis. In order to evaluate the large trade space of design parameters associated with a magnetic radiation shield design, an analytical model was developed for the determination of flux inside a solenoid magnetic field due to the Galactic Cosmic Radiation (GCR) radiation environment. This analytical model was then coupled with NASA’s radiation transport code, HZETRN, to account for the effects of passive/structural shielding mass. The resulting model can rapidly obtain results for a given configuration and can therefore be used to analyze an entire trade space of potential variables in less time than is required for even a single Monte Carlo run. Analyzing this trade space for a solenoid magnetic shield design indicates that active shield bending powers greater than ∼15 Tm and passive/structural shielding thicknesses greater than 40 g/cm² have a limited impact on reducing dose equivalent values. Also, it is shown that higher magnetic field strengths are more effective than thicker magnetic fields at reducing dose equivalent.     

Chromosomal data relevant for Q values.

It has been known for many years that relationships between absorbed dose and biological effect vary with the type of radiation. In particular, neutrons and alpha particles are more damaging than x or gamma radiations. This applies to a range of biological effects such as cell killing, chromosome aberrations, cell mutation, cell transformation as well as life shortening and cancer induction in animals. The application of this knowledge to devise a scheme for specifying the quality factor (Q) in radiological protection has been the subject of much debate. There are no tumour data in humans from which the quality factor may be derived. The problems of using animal and cell transformation data which are probably the next best choice are discussed. The extensive data base on chromosomal aberrations in human lymphocytes is described and discussed in terms of relevance to deducing quality factors. Particular emphasis is placed on data obtained at low doses and low dose rates.     

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.