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.     

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.     

No significant level of inheritable interchromosomal aberrations in the progeny of bystander primary human fibroblasts after alpha particle irradiation

A major concern for bystander effects is the probability that normal healthy cells adjacent to the irradiated cells become genomically unstable and undergo further carcinogenesis after therapeutic irradiation or space mission where astronauts are exposed to low dose of heavy ions. Genomic instability is a hallmark of cancer cells. In the present study, two irradiation protocols were performed in order to ensure pure populations of bystander cells and the genomic instability in their progeny were investigated. After irradiation, chromosomal aberrations of cells were analyzed at designated time points using G₂ phase premature chromosome condensation (G₂-PCC) coupled with Giemsa staining and with multiplex fluorescent in situ hybridization (mFISH). Our Giemsa staining assay demonstrated that elevated yields of chromatid breaks were induced in the progeny of pure bystander primary fibroblasts up to 20 days after irradiation. mFISH assay showed no significant level of inheritable interchromosomal aberrations were induced in the progeny of the bystander cell groups, while the fractions of gross aberrations (chromatid breaks or chromosomal breaks) significantly increased in some bystander cell groups. These results suggest that genomic instability occurred in the progeny of the irradiation associated bystander normal fibroblasts exclude the inheritable interchromosomal aberration.     

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.     

Repair and misrepair of heavy-ion-induced chromosomal damage.

The premature chromosome condensation (PCC) technique was used to investigate chromosomal damage, repair, and misrepair in the G phase of a human/hamster hybrid cell line that contains a single human chromosome. Plateau-phase cell cultures were exposed to either x-rays or a 425 MeV/u beam of neon ions near the Bragg peak where the LET is 183 kev/micrometers. An in situ hybridization technique coupled to fluorescent staining of PCC spreads confirmed the linearity of the dose response for initial chromatin breakage in the human chromosome to high doses (1600 cGy x-ray or 1062 cGy Ne). On Giemsa-stained slides, initial chromatin breakage in the total genome and the rejoining kinetics of these breaks were determined. As a measure of chromosomal misrepair, ring PCC aberrations were also scored. Ne ions were about 1.5 x more effective per unit dose compared to x-rays at producing the initially measured chromatin breakage. 90% of the x-ray-induced breaks rejoined in cells incubated at 37 degrees C after exposure. In contrast, only 50% of Ne-ion-induced breaks rejoined. In the irradiated G1 cells, ring PCC aberrations increased with time apparently by first order kinetics after either x-ray or Ne exposures. However, far fewer rings formed in Ne-irradiated cells after a dose giving a comparable initial number of chromatin breaks. Following x-ray exposures, the yield of rings formed after long repair times (6 to 9 hrs) fit a quadratic dose-response curve. These results indicate quantitative and qualitative differences in the chromosomal lesions induced by low- and high-LET radiations.     

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.     

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.     

Particle trajectories in seeds of Lactuca sativa and chromosome aberrations after exposure to cosmic heavy ions on Cosmos Biosatellites 8 and 9.

The potentially specific importance of the heavy ions of the galactic cosmic radiation for radiation protection in manned spaceflight continues to stimulate in situ, i.e., spaceflight experiments to investigate their radiobiological properties. Chromosome aberrations as an expression of a direct assault on the genome are of particular interest in view of cancerogenesis being the primary radiation risk for man in space. In such investigations the establishment of the geometrical correlation between heavy ions' trajectories and the location of radiation sensitive biological substructures is an essential task. The overall qualitative and quantitative precision achieved for the identification of particle trajectories in the order of approximately 10 micrometers as well as the contributing sources of uncertainties are discussed. We describe how this was achieved for seeds of Lactuca sativa as biological test organisms, whose location and orientation had to be derived from contact photographies displaying their outlines and those of the holder plates only. The incidence of chromosome aberrations in cells exposed during the COSMOS 1887 (Biosatellite 8) and the COSMOS 2044 (Biosatellite 9) mission was determined for seeds hit by cosmic heavy ions. In those seeds the incidence of both single and multiple chromosome aberrations was enhanced. The results of the Biosatellite 9 experiment, however, are confounded by spaceflight effects unrelated to the passage of heavy ions.     

空间重粒子击中卤虫卵的定位技术及其生物效应

为了能够估价空间重粒子对单个生物细胞的放射生物学效应,我们设计、制成了卤虫卵-核乳胶夹层式生物包.经搭载我国“8885”卫星飞行数天后回收.用图象分析仪研究了空间重离子在核乳胶上留下的径迹及其分布;并实验观察了卤虫卵的前期发育情况.看到:随星飞行过的卤虫卵与地面对照组相比,其发育能力明显降低,表现为孵化率低且发育时间推迟.实验表明:生物样品板-核乳胶影象定位技术能清楚而比较准确地给出卤虫卵(或其他生物细胞)受重离子作用的信息,是重离子生物效应及其作用机理研究的可行实验手段及分析方法之一.     

Physical events in the track structure of heavy ions and their relation to alterations of biomolecules.

Heavy charged particles interacting with biological cells can produce a wide variety of different physical, chemical and biological consequences. A rigorous identification of relevant chemical and biological alterations of biomolecules in cells, however, is still lacking and, thus, it is difficult to identify the potential biological importance of different early physical events. In addition, due to experimental and theoretical problems also little is known about the details of energy transfer, -absorption and -decay from projectiles to atoms/molecules in condensed targets; this is particularly true for not completely stripped heavy ions. Nevertheless, one might conclude from available data that higher densities of physical energy absorption events have a significantly higher probability to lead to qualitatively more severe biochemical alterations as regards the induction of DNA double strand breaks and of chromatin damage. It is not very likely that energy migration along the DNA molecule in biological cells over long distances plays a significant role as contributor to these biological radiation effects.     

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.     

Induction of chromosomal damage in CHO-K1 cells and their repair-deficient mutant XRS5 by X-ray and particle irradiation.

The cytogenetic effects of X-rays and Au ions were investigated in repair-proficient CHO-K1 cells and their radiosensitive mutant strain xrs5, which shows a defect in the rejoining of DNA double-strand breaks. Both cell lines were synchronized by mitotic shake off, irradiated in G1-phase with either 250 kV X-rays or 780 MeV/u Au ions (LET: 1150 keV/micrometer) and chromosome aberrations were analyzed in first post-irradiation metaphases. Isoeffective doses of X-rays for the induction of aberrant cells and aberrations per cell were about 14 times lower for xrs5 than for CHO-K1 cells. After high LET radiation the difference in the cytogenetic response of both cell lines was drastically diminished. Furthermore, the analysis of the aberration types induced by sparsely and densely ionizing radiation showed for both cell lines specific changes in the spectrum of aberration types as LET increases. The experimental results are discussed with respect to the different types of lesions induced by sparsely and densely ionizing radiation.     

Neoplastic cell transformation by high-LET radiation: molecular mechanisms.

Experimental data on molecular mechanisms are essential for understanding the bioeffects of radiation and for developing biophysical models, which can help in determining the shape of dose-response curves at very low doses, e.g., doses less than 1 cGy. Although it has been shown that ionizing radiation can cause neoplastic cell transformation directly, that high-LET heavy ions in general can be more effective than photons in transforming cells, and that the radiogenic cell transformation is a multi-step process [correction of processes], we know very little about the molecular nature of lesions important for cell transformation, the relationship between lethal and transformational damages, and the evolution of initial damages into final chromosomal aberrations which alter the growth control of cells. Using cultured mouse embryo cells (C3H10T1/2) as a model system, we have collected quantitative data on dose-response curves for heavy ions with various charges and energies. An analysis of these quantitative data suggested that two DNA breaks formed within 80 angstroms may cause cell transformation and that two DNA breaks formed within 20 angstroms may be lethal. Through studies with restriction enzymes which produce DNA damages at specific sites, we have found that DNA double strand breaks, including both blunt- and cohesive-ended breaks, can cause cell transformation in vitro. These results indicate that DNA double strand breaks can be important primary lesions for radiogenic cell transformation and that blunt-ended double strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship is similar for HGPRT gene mutation, chromosomal deletion, and cell transformation, suggesting common lesions may be involved in these radiation effects. The high RBE of high-LET radiation for cell killing and neoplastic cell transformation is most likely related to its effectiveness in producing DNA double strand breaks in mammalian cells. At present the role of oncogenes in radiation cell transformation is unclear.     

Heavy ion induced double strand breaks in bacteria and bacteriophages.

DNA damage induced by heavy ions in bacterial cells and bacteriophages such as Bacillus subtilis, E. coli and Bacteriophage T1 were investigated by analyzing the double strand breaks in the chromosomal DNA. This kind of lesion is considered as one of the main reasons for lethal events. To analyze double strand breaks in long molecules of DNA--up to some Mbp in length--the technique of pulse field agarose gel electrophoresis has been used. This allows the detection of one double strand break per genome. Cell lysis and DNA isolation were performed in small agarose blocks directly. This procedure secured minimum DNA destruction by shearing forces. After running a gel, the DNA was stained with ethidium bromide. The light intensity of ethidium bromide fluorescence for both the outcoming (running) DNA and the remaining intact DNA were measured by scanning. The mean number of double strand breaks was calculated by determining the quotient of these intensities. Strand break induction after heavy ion and X-ray irradiation was compared.     

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.     

Double strand breaks in the DNA of Bacillus subtilis cells irradiated by heavy ions.

Cells of Bacillus subtilis strain TKJ 8431 in stationary phase were irradiated with X-rays (150 kV at DLR) or heavy ions (Ne, Ar, Pb with residual energies between 3 and 15 MeV/u at GSI). The action cross section for the formation of double strand breaks in the DNA of the irradiated cells follows a similar dependence on mass and energy of the ions as has been found for various biological endpoints, e.g. inactivation, mutagenesis and repair efficacy.     

The effect of track structure on cell inactivation and chromosome damage at a constant LET of 120 keV/micrometer.

The influence of track structure on chromosome damage and cell inactivation are being investigated. Plateau-phase normal human fibroblast cultures were irradiated with gamma rays, and He, Ne and Ar ions. Particle velocities were chosen so that all beams had an LET of 120 keV/micrometer. In this constant-LET experimental design, the radial distribution of excitations and ionizations about the particle track is the most significant variable. Using premature chromosome condensation, chromatin breaks were measured at two time points, promptly after irradiation and after a prolonged incubation to allow for repair. These measurements give an indication of both initial chromosomal damage and also residual damage that is either not repaired or is misrepaired. Survival was measured under the same conditions. Results indicate that the RBEs for both cell inactivation and, to a lesser extent, chromosome damage decrease as particle energy increases.     

DNA fragmentation by charged particle tracks.

High-LET (linear energy transfer) charged particles induce DNA double-strand breaks (DSB) in a non-random fashion in mammalian cells. The clustering of DSB, probably determined by track structure as well as chromatin conformation, results in an excess of small- and intermediate-sized DNA fragments. DNA fragmentation in normal human fibroblasts (GM5758) was analyzed by pulsed-field gel electrophoresis after irradiation with photons (60Co) or 125 keV/micrometers nitrogen ions. Compared to conventional DSB analysis, i.e. assays only measuring the fraction of DNA smaller than a single threshold, the relative biological effectiveness (RBE) for DSB induction increased with 100%. Further, the size distribution of DNA fragments showed a significant dependence on radiation quality, with an excess of fragments up to 1 Mbp. Irradiation of naked genomic DNA without histone proteins increased the DSB yields 25 and 13 times for photons and nitrogen ions, respectively. The results suggest possible roles of both track structure and chromatin organization in the distribution of DNA double-strand breaks along the chromosome.     

Heavy ion induced DNA double strand breaks in cells of E. coli.

Vegetative cells of E. coli differing in their radiosensitivity have been used in heavy ion irradiation experiment. Besides inactivation measurements also the induction of DNA double strand breaks (DSB) have been measured using the method of pulse-field gel electrophoresis. This method allows to separate linear DNA with length up to 8 Mio base pairs. After irradiation with heavy ions we find a higher amount of low molecular weight fragments when compared to sparsely ionizing radiation. This agrees with the idea that heavy ions as a structured radiation have a high probability to induce more than one strand break in a DNA molecule if the particle hits the DNA. The amount of intact DNA remaining in the agarose plugs decreases exponentially for increasing radiation doses or particle fluences. From these curves cross sections for the induction of DSB after heavy ion irradiation have been determined. These results will be discussed in comparison to the results for cell survival.     

Mutation induction in mammalian cells by very heavy ions.

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