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.     

Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1.

Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1. There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar). The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.     

Cell cycle delays induced by heavy ion irradiation of synchronous mammalian cells.

Cell cycle effects of very high LET particles on synchronous V79 Chinese Hamster cells have been studied in a track segment experiment by means of flow cytometric methods. Cells were irradiated with 10 MeV/u Pb-ions (LET = 13500 keV/micrometers) at an average fluence of 2 particles per cell nucleus, corresponding to a survival level of about 25%. Instantaneous drastic reductions of cell proliferation in all cycle phases have been observed, which affect the cell cycle for at least 50 hours after exposure to heavy ions. These findings are in clear contrast to the results from low LET radiation experiments, where significant delays can only be observed in S-phase and G2M-phase and for comparatively short time intervals of a few hours. Additionally, high LET radiation gives rise to prolonged DNA synthesis bypassing cell division, which leads to cells with DNA content greater than that of G2M-cells.     

Effects of heavy ion radiation on the brain vascular system and embryonic development.

Using neonatal rats as a model system, we investigated the response of the brain vascular system to ionizing radiation and found that distinct petechial hemorrhages developed in the cerebral cortex within a few hours after irradiation, reached a maximum about 13 to 24 hours, and decreased exponentially with time. No brain hemorrhage was found in neonatal rats 12 days after irradiation. Our experimental results indicate that a dose of a few hundred rad of X rays can induce a significant number of hemorrhages in the brain, and the number of lesions increases exponentially with dose. Heavy ions induce more hemorrhages than X rays for a given dose, and the RBE for 670 MeV/u neon particles ranges from about 2.0 for low doses to about 1.4 for high doses. A histological study of the hemorrhages indicates that a large number of red blood cells leak from the blood vessels. The radiation-induced hemorrhages may be a result of some capillary membrane damages or reproductive death of some blood vessel epithelial cells. The fast onset of hemorrhage after irradiation suggest that some membrane damage may be involved. The effect of heavy-ion radiation on the embryonic development was studied with energetic iron particles. Pregnant mice were whole-body irradiated with 600 MeV/u iron particles on day 6 of gestation and were sacrificed 12 days after irradiation. Various physical abnormalities were observed, and embryos irradiated with 1 rad iron particles showed retardation of body development.     

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

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

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

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

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.     

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

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

A model of the effects of heavy ion radiation on human tissue

In heavy ion radiotherapy and space travel humans are exposed to energetic heavy ions (C, Si, Fe and others). This type of irradiation often produces more severe biological effects per unit dose than more common X-rays. A new Monte Carlo model generates a physical space with the complex geometry of human tissue or a cell culture based model of tissue, which is affected by the passage of ionizing radiation. For irradiation, the model relies on a physical code for the ion track structure; for tissues, cellular maps are derived from two- or three-dimensional confocal microscopy images using image segmentation algorithm, which defines cells as pixilated volumes. The model is used to study tissue-specific statistics of direct ion hits and the remote ion action on cells. As an application of the technique, we considered the spatial pattern of apoptotic cells after heavy ion irradiation. The pattern of apoptosis is modeled as a stochastic process, which is defined by the action cross section taken from available experimental data. To characterize the degree of apoptosis, an autocorrelation function that describes the spatial correlation of apoptotic cells is introduced. The values of the autocorrelation function demonstrate the effect of the directionality of the radiation track on the spatial arrangements of inactivated cells in tissue. This effect is intrinsic only to high linear-energy-transfer radiation.     

Biological effects of heavy ions from the standpoint of target theory.

The biological effect of heavy ions is best described through the action cross section, as a function of the end-point of interest and the charge and speed of the ion. In track theory this is called the "ion-kill" cross section, for it is the effect produced by a single heavy ion and its delta rays. As with nuclear emulsions the biological track structure passes from the grain count regime to the track width regime to the thindown region with an increase in LET. With biological cells, as with any detector capable of storing sublethal damage, with low LET irradiation the action cross section (in the ion-kill mode) is increasingly obscured by the effect of "gamma-kill", by the influence of overlapping delta rays from neighboring heavy ions. Thus at low LET response is dominated by the gamma-kill mode, so that the RBE approaches 1. The theory requires 4 radiosensitivity parameters for biological detectors, extracted from survival curves at several high LET bombardments passing through the grain count regime, and at high doses. Once these are known the systematic response of biological detectors to all high LET bombardments can be unfolded separating ion kill from gamma kill, predicting the response to a mixed radiation environment, and predicting low dose response even at the level of a single heavy ion. Cell killing parameters are now available for a variety of cell lines. Newly added is a set of parameters for cell transformation.     

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts.

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.     

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.     

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.     

Theoretical consideration of the chemical pathways for radiation-induced strand breaks.

A theoretical approach to the understanding of the biochemical mechanisms of indirect action of ionizing radiation on SV40 DNA in aqueous solution is presented. The extent of OH attack on the sugar moiety and bases has been calculated. A realistic model for the DNA (in B form) based on available X-ray diffraction data is used and specific reaction sites for the OH radicals are obtained. A Monte Carlo scheme is used to follow the diffusion and reaction of the OH radicals. Effects of track structure have been considered and the single strand break D37 values for 14 MeV electrons (low-LET) and 670 MeV/u and 40 MeV/u neon particles are presented. Calculated results are in agreement with available experimental data. It has been found that regardless of the qualities of radiation, 80% of the OH attack on DNA is on the bases and 20% is on the deoxyribose. From probability considerations only, it appears that the number of double strand breaks varies linearly with dose.     

The role of DNA repair on cell killing by charged particles.

It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderated RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.     

Neoplastic transformation of mouse C3H 10T1/2 and Syrian hamster embryo cells by heavy ions.

C3H 10T1/2 mouse-embryo fibroblasts were used for transformation experiments to study the effectiveness of various heavy ions with energies up to 20 MeV/u and LET values from 170 to 16,000 keV/micrometers. The transformation frequency per unit absorbed dose decreased with increasing ionization density; at the highest values of LET we found a decrease even of the transformation efficiency per unit fluence. Uranium ions at energies of 5, 9, and 16.3 MeV/u did not induce any transformation. In additional studies primary Syrian hamster embryo cells (SHE) were exposed to heavy ions in order to characterize cytological and molecular changes which may be correlated with neoplastic transformation. Growth behaviour, chromosomal status, tumorigenicity in nude mice, and expression of oncogenes of transformed cell lines were examined     

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.     

The effect of space radiation of the nervous system.

The long-term effects of irradiation by accelerated heavy ions on the structure and function of the nervous system have not been studied extensively. Although the adult brain is relatively resistant to low LET radiation, cellular studies indicate that individual heavy ions can produce serious membrane lesions and multiple chromatin breaks. Capillary hemorrhages may follow high LET particle irradiation of the developing brain as high RBE effects. Evidence has been accumulating that the glial system and blood-brain barrier (BBB) are relatively sensitive to injury by ionizing radiation. While DNA repair is active in neural systems, it may be assumed that a significant portion of this molecular process is misrepair. Since the expression of cell lethality usually requires cell division, and nerve cells have an extremely low rate of division, it is possible that some of the characteristic changes of premature aging may represent a delayed effect of chromatin misrepair in brain. Altered microcirculation, decreased local metabolism, entanglement and reduction in synaptic density, premature loss of neurons, myelin degeneration, and glial proliferation are late signs of such injuries. HZE particles are very efficient in producing carcinogenic cell transformation, reaching a peak for iron particles. The promotion of viral transformation is also efficient up to an energy transfer of approximately 300 keV/micron. The RBE for carcinogenesis in nerve tissues remains unknown. On the basis of available information concerning HZE particle flux in interplanetary space, only general estimates of the magnitude of the effects of long-term spaceflight on some nervous system parameters may be constructed.     

Experiment "Seeds" on Biokosmos 9. Dosimetric part.

The aim of the experiment "Seeds" on the Sowjetic satellite Biokosmos 9 was the observation of mutagenic effects caused at special loci of seeds of Arabidopsis thaliana and assigned to particles of the Cosmic radiation. Two types of exposure units were flown: A low-shielding unit Type I, mounted at the surface of the satellite (1.4 g/cm2 shielding) and, for comparison, an identical item inside (16 g/cm2 shielding), using nuclear emulsion as track detectors. A Type II unit, flown inside (18g/cm2 shielding) was mounted with AgCl track detectors. The layout will be briefly described. A first set of dosimetric data from the physical evaluation of the experiment will be presented. The subdivision into charge- and LET-groups shows a rather high contribution of the intermediate LET-group (350-1000 MeV/cm) due to medium heavy particles (Z = 6-10) and to enders of light (p, alpha) particles.     

The effect of alpha particles on bacteriophage T4Br+.

It is generally accepted that heavy charged particles play an important part in generating the secondary flux of nuclear particles formed by the interaction of space hadrons with nuclei. It is assumed that these particles are responsible for the high biological efficiency of space hadrons in causing cellular damage by their strong interactions. To examine this assumption we investigated the effects of 5.3 MeV alpha particles on bacteriophage T4. This energy provides a LET value of 88.6 KeV/micrometer lying in the range of the highest biological efficiency.