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

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

Heavy-ion effects on cellular and subcellular systems: inactivation, chromosome aberrations and strand breaks induced by iron and nickel ions.

A broad spectrum of particles and energies has been used in the last years to study the influence of the radiation quality i.e. of the physical parameters of the particle beam on the biological effectiveness ?2?12?. In these measurements a common structure of the functional dependence of the induction probability per particle (cross section) from the linear energy transfer is observed for different biological endpoints. Because of the relevance for space research, we present in this report our data from experiments with iron and nickel particles, in particular. Our experiments were designed to investigate the relationship between the inactivation and chromosome aberration in mammalian cells and the induction of single and double strand breaks in SV40 DNA in respect to the parameters of the track formation like LET and particle energy.     

Multiple cell hits by particle tracks in solid tissues.

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

Microdosimetric measurements of heavy ion tracks.

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

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.     

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.     

Biological effects of galactic radiation HZE particles in experiments on the orbital station Salyut 7.

Lettuce (Lactuca sativa) seeds were flown on-board the orbital station Salyut 7 for 66-457 days. It was found that a single heavy charged particle (HZE) hitting a seed only slightly affects the subsequent plant growth. However, morphological anomalies of varying type in primordial leaves and roots were observed that show good correlation with the location of the particle track. The most severe damage detected by light and an electron microscopy were "channels" in dry and soaked seeds. The appearance of "channels" seems to be related to the LET of the incident particle. This finding is of considerable importance for assessment of space flight radiation hazard.     

Single ion actions: the induction of micronuclei in V79 cells exposed to individual protons.

Understanding the effects of single-particles from conventional radiation biology experiments is problematic due to the stochastics of particle tracks. This complicates the determinations of risk associated with low doses. We have developed a charged particle microbeam, which allows individually counted particles to be delivered to precise cellular locations. The system is capable of delivering a single charged particle with > 99% efficiency. Of these particles 90% are delivered with a resolution of +/- 2 micrometers and 96% with a resolution of +/- 5 micrometers. We have carried out preliminary studies in Chinese hamster V79 cells to monitor the effectiveness of low energy protons at inducing cytological damage. We have used the micronucleus assay as a measure of predominantly lethal chromosome damage. The effects of a single 3.2 MeV proton delivered individually to cells could be measured, with less than 2% of the exposed cells producing micronuclei 24 hours later. The yield of micronuclei formation was essentially linear up to the highest dose (30 particles per cell nucleus) delivered. Ultimately, the ability to target particles to different parts of the cell nucleus may start to impact on models available for chromosome aberration formation and chromosomal Organisation and mechanisms underlying genomic instability.     

Stochastics of HZE-induced microlesions.

Fundamental biological experiments with bacteria, yeast, and mammalian cells irradiated with ions heavier than helium indicate that maximal probability of single-hit inactivation does not occur when the ion has LET below about 100-200 keV/micrometer. Theoretical treatments of cell inactivation data and the radiation chemistry in particle tracks are consistent with this finding. If a "microlesion" is defined as a linear array, within a tissue, of cells inactivated with maximum probability, surrounded by non-lethally damaged cells, then, by this definition, there must be an LET below which "microlesion" damage cannot be expected. In a retrospective survey of experimental literature in which single-particle effects in tissues were sought, it was found that little or no evidence has been reported supporting single-particle effects in tissues when LET was below 200 keV/micrometer, while some experimenters who irradiated tissues with particles having LET greater than 200 keV/micrometer reported effects that could be attributed to single-particle tracks.     

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.     

HZE effects on mammalian cells.

In track segment experiments cell survival and chromosome aberrations of mammalian cells have been measured for various heavy ion beams between helium and uranium in the energy range between 0.5 and 960 MeV/u, corresponding to a velocity range of 0.03 to 0.87 C, and an LET spectrum from 10 to 15 000 keV/micrometers. At low LET, the cross section (sigma) for cell killing increases with increasing LET and shows a common curve for all ions regardless of the atomic number. This indicates that in this region the track structure of the different ions is of only a minor influence, and it is rather the total energy transfer, which is important for cell killing. At higher LET values, deviations from a common sigma-LET curve can be observed which indicate a saturation effect. The saturation of the lighter ions occurs at lower LET values than for the heavier ions. These findings are also confirmed by the chromosome data, where the efficiency for the induction of chromosomal aberrations for high LET particles depends on the track structure and is nearly independent of LET. In the heavier beams (Z > or = 10) individual particles cause multiple chromosome breaks in mitotic cells.     

Inactivation probability of heavy ion-irradiated Bacillus subtilis spores as a function of the radial distance to the particle's [correction of paricle's] trajectory.

The understanding of the radiobiological action of heavy ions requires the knowledge of the dependence of the inactivation probability on the distance between the particle's trajectory and the biological test organism (the impact parameter). Spores of Bacillus subtilis with a cytoplasmic core of about 0.22 micrometer cross section are suitable test objects for the study of this radial inactivation probability in its microscopic details. The spores are irradiated at low fluences of some 10(6) ions/cm2 with very heavy ions at different specific energies up to 10 MeV per atomic mass unit u while in fixed contact with visual nuclear track detectors. The methods are described by which the biological response of individual cells can be evaluated and the impact parameter be determined with an accuracy typically better than 0.2 micrometer. The results demonstrate that the common characteristics of inactivation, e.g., an effective range of inactivation extending to at least 3 micrometers, a nonmonotonic dependence of the inactivation probabilities on the radial distance, and the fact that the inactivation probability even for direct central hits on the cytoplasmic core is substantially below one, are nearly independent of the particle energy and type. The results are incompatible with the assumption that the radiobiological effectiveness can be attributed to the dose of secondary electrons as currently understood. They also demonstrate that the widely held notion of an "overkill" at low impact parameters does not apply for the spores even with the most densely ionizing ions.     

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.     

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.     

Cellular and subcellular effect of heavy ions: a comparison of the induction of strand breaks and chromosomal aberration with the incidence of inactivation and mutation.

Radiobiological effects of heavy charged particles are compared for a large variety of ions from Helium to Uranium and energies between 1 and 1000 MeV/u which correspond to LET values between 10 and 16000 keV/micrometers. The different cross section for the induction of strand breaks and chromosomal aberrations as well as for inactivation and mutation induction exhibit striking similarities when compared as function of the linear energy transfer (LET). At LET values below 100 keV/micrometers all data points of one specific effect form one single curve as a function of LET, independent of the atomic number of the ion. In this LET range, the biological effects are independ from the particle energy or track structure and depend only on the energy transfer. Therefore, LET is a good parameter in this regime. For LET values greater than 100 keV/micrometers, the curves for the different ions separate from the common curve in order of increasing atomic numbers. In this regime LET is no longer a good parameter and the physical parameters of the formation of particle tracks are important. The similarity of the sigma-LET curves for different endpoints indicates that the 'hook-structure' is produced by physical and chemical effects which occur before the biologically relevant lesions are formed. However, from the existing data of biological effects, it can be concluded that the efficiencies for cell killing are always smaller than those extrapolated from X-ray data on the basis of the energy deposition only. Therefore, cells which are directly hit by an HZE particle are not killed and undergo a finite risk of mutation and transformation.     

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.     

Repair of DNA double-strand breaks and its effect on RBE.

DNA double-strand breaks (DSB) are induced linearly with absorbed dose both for sparsely and densely ionizing radiations. By enzymatic repair the linear relationship between the number of DSB and absorbed dose is converted into a non linear one. Furthermore, the RBE-values of high LET radiations for residual DSB increase with increasing amount of DSB repair especially in the low dose range. Unrepaired and/or misrepaired DSB are supposed to be responsible for chromosomal aberrations, cell killing, oncogenic cell transformation and gene mutation. At low doses, for these endpoints much higher RBE-values than those for initial DSB are observed. However, with increasing doses the RBE-values for these endpoints approach those for initial DSB. These observations are likely to be interpreted using the following two parameters of the energy deposition structure: 1. The distribution of clusters with respect to their size at the nm-scale and to the number of ionizations per cluster (cluster distribution). 2. The distribution of distances between clusters of definite size and with definite number of ionizations (distance distribution of clusters). For the induction of DSB solely the ionization density in clusters of nm-dimensions (i.e. the cluster distribution) is important. For unrepaired or misrepaired DSB (responsible for chromosome aberrations, cell killing, oncogenic cell transformation and gene mutation) both the cluster distribution and the distance distribution of clusters are relevant. At low doses the distance distribution of clusters along a single particle track determines the RBE-value. However, with increasing dose the distribution of clusters produced by all particles traversing the cell nucleus becomes increasingly determinant. Here, solely the cluster distribution is important as it is the case for the induction of DSB.     

Biological dosimetry in Russian and Italian astronauts.

Large uncertainties are associated with estimates of equivalent dose and cancer risk for crews of long-term space missions. Biological dosimetry in astronauts is emerging as a useful technique to compare predictions based on quality factors and risk coefficients with actual measurements of biological damage in-flight. In the present study, chromosomal aberrations were analyzed in one Italian and eight Russian cosmonauts following missions of different duration on the MIR and the international space station (ISS). We used the technique of fluorescence in situ hybridization (FISH) to visualize translocations in chromosomes 1 and 2. In some cases, an increase in chromosome damage was observed after flight, but no correlation could be found between chromosome damage and flight history, in terms of number of flights at the time of sampling, duration in space and extra-vehicular activity. Blood samples from one of the cosmonauts were exposed in vitro to 6 MeV X-rays both before and after the flight. An enhancement in radiosensitivity induced by the spaceflight was observed.     

The microlesion concept in HZE particle dosimetry.

High energy, high-Z (HZE) particles are present in high-altitude and high-inclination satellite orbits. Most of the HZE dose above LET = 200 keV/micrometer is due to Fe nuclei. Individual HZE particles can damage several cells adjacent to one another along the particle track in tissue. The outcome has been described as a "microlesion" by D. Grahn. The present study attempts to define conditions for microlesions in specific tissues, to seek biological evidence that microlesions are produced, and to evaluate the microlesion as a potentially useful unit of dose in assessing hazards to spaceworkers. Microlesions in individuals cells and hair follicles have been described. Microbial studies have provided some evidence for independent secondary electron action. Whether or not a few hundred microlesions would be damaging to the whole organism depends upon the nature of damage to critical tissues. For example, cancer may occur if microlesions kill several cells in a straight line and mutate other cells alongside the particle track. Fe particle irradiation of the mouse Harderian gland (Fry et al., this issue) produces tumors efficiently. Microlesions in the lens, cornea, and retina need to be considered. Further dialogue is required before a final decision can be made concerning the most appropriate way to assess the HZE hazard.