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.     

DNA double strand break production and rejoining in V79 cells irradiated with light ions.

Low energy protons and other densely ionizing light ions are known to have RBE>1 for cellular end points relevant for stochastic and deterministic effects. The occurrence of a close relationship between them and induction of DNA dsb is still a matter of debate. We studied the production of DNA dsb in V79 cells irradiated with low energy protons having LET values ranging from 11 to 31 keV/micrometer, i.e. in the energy range characteristic of the Bragg peak, using the sedimentation technique. We found that the initial yield of dsb is quite insensitive to proton LET and not significantly higher than that observed with X-rays, in agreement with recent data on V79 cells irradiated with alpha particles of various LET up to 120 keV/micrometer. By contrast, RBE for cell inactivation and for mutation induction rises with the proton LET. In experiments aimed at evaluating the rejoining of dsb after proton irradiation we found that the amount of dsb left unrepaired after 120 min incubation is higher for protons than for sparsely ionizing radiation. These results indicate that dsb are not homogeneous with respect to repair and give support to the hypothesis that increasing LET leads to an increase in the complexity of DNA lesions with a consequent decrease in their repairability.     

Quantitation of heavy ion damage to the mammalian brain: some preliminary findings.

Histological preparations of brains from rabbits and mice exposed to different doses of various HZE particles or to low-LET photons have been subjected to preliminary quantitation of radiation-induced morphometric changes. Computer assisted measurements of several brain structures and cell types have been made using the KONTRON Automated Interactive Measurement System (IBAS, Carl Zeiss, Inc., Thornwood, N.Y. 10594 U.S.A.). New Zealand white rabbits irradiated at approximately 6 weeks of age were euthanatized 6.5-25 months after exposure to 60Co gamma photons (LET infinity = approximately 0.3 keV/micrometer, 20Ne particles (LET infinity = 35 +/- 3 keV/micrometer), or 40Ar particles (LET infinity = 90 +/- 5 keV/micrometer). Measurements of stained sections of the olfactory bulbs of those animals indicate that the mean size (volume) of olfactory glomeruli is reduced in a dose-dependent (and perhaps an LET-dependent) manner as soon as 6.5 months after irradiation. Differences between mean volumes of additional structures have been noted when histological preparations of control mouse brains were compared with irradiated specimens. Quantitation of intermediate and late changes in nervous (and other) tissues exposed to low- and high-LET radiations will improve our ability to predict late effects in tissues of astronauts and others exposed to the radiation hazards of the space environment.     

Mutagenic effects of heavy ions in bacteria.

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

LET dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions.

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.     

Correlation between cell death and induction of non-rejoining PCC breaks by carbon-ion beams.

We have shown a correlation between cell death and induction of non-rejoining chromatin breaks in two normal human cells and three human tumor cell lines irradiated by carbon-ion beams and X rays. Non-rejoining chromatin breaks were measured by counting the number of remaining chromatin fragments detected by the premature chromosome condensation (PCC) technique. Carbon-ion beams were accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC). The cells were irradiated by two different mono-LET beams (LET = 13 keV/micrometer and 77 keV/micrometer ) and 200 kV X rays. The RBE values of cell death for carbon-ion beams relative to X rays were 1.1 to 1.4 for 13 keV/micrometer beams and 2.5 to 2.9 for 77 keV/micrometer beams. The induction rate of non-rejoining PCC breaks per cell per Gy was found to be highest for the 77 keV/micrometer beams for all of the cell lines.The results found in this study show that there is a good correlation between cell death and induction of non-rejoining PCC breaks for these human cell lines.     

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.     

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.     

Induction and rejoining of DNA double-strand breaks in CHO cells after heavy ion irradiation.

DNA double-strand breaks (DSBs) are the crucial events ultimately leading to cell inactivation. Aimed at understanding the biological action of the charged particle component of cosmic radiation, the induction of DSBs and their repairability was evaluated in Chinese hamster ovary (CHO-K1) cells after exposure to accelerated particles. Irradiations were performed with various ion species including O, Ni and Ca, covering a LET range from 20 to 2000 keV/micrometer. DSBs were determined for plateau-phase cells using the electrophoretic elution of radiation-induced DNA fragments in a static electric field combined with fluorescence scanning of ethidium bromide stained gels. Assuming a DSB yield of 22 DSB per Gy per cell, as derived from X-irradiation, cross-sections for DSB production were calculated from the corresponding fluence-effect curves at a fraction of 0.7 of DNA retained. The same ordinate was used as a reference for the calculation of relative biological efficiency (RBE) for DSB induction. At low LETs (< or = 20 keV/micrometer) RBE values slightly above unity were obtained, but a decrease of RBE was observed with increasing LET. In the region of 100-200 keV/micrometer the RBE for initial DSB induction was clearly below unity. Rejoining of DSBs was assessed by measuring the fraction of DNA retained following post-irradiation incubation of cells under culture conditions. After exposure to Ca ions, DSB rejoining was considerably impaired compared to X-rays.     

Repair of DNA double-strand breaks and cell killing by charged particles.

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing (Kelland et al., 1988; Radford, 1986). We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/micrometer and were even smaller than unity for the LET region greater than 300 keV/micrometer. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/micrometer, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main muse of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.     

RBE: mechanisms inferred from cytogenetics.

Cyclotron-accelerated heavy ion beams provide a fine degree of control over the physical parameters of radiation. Cytogenetics affords a view into the irradiated cell at the resolution of chromosomes. Combined they form a powerful means to probe the mechanisms of RBE. Cytogenetic studies with high energy heavy ion beams reveal three LET-dependent trends for 1) level of initial damage, 2) distribution of damage among cells, and 3) lesion severity. The number of initial breaks per unit dose increases from a low-LET plateau to a peak at approximately 180 keV/micrometer and declines thereafter. Overdispersion of breaks is significant above approximately 100 keV/micrometer. Lesion severity, indicated by the level of chromosomal fragments that have not restituted even after long repair times, increases with LET. Similar studies with very low energy 238Pu alpha particles (120 keV/micrometer) reveal higher levels of initial breakage per unit dose, fewer residual fragments and a higher level of misrepair when compared to high energy heavy ions at the same LET. These observations would suggest that track structure is an important factor in genetic damage in addition to LET.     

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.     

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.     

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.     

Early and delayed reproductive death in human cells exposed to high energy iron ion beams.

The aim of this research was to determine the biological effectiveness for early and delayed effects of high energy, high linear energy transfer (LET) charged particles. Survival and delayed reproductive death were measured in AG1522 human fibroblast cells exposed to Fe-ion beams of energies between 0.2 and 1 GeV/n, 0.97 GeV/n Ti-ion and 0.49 GeV/n Si-ion beams. The cells were irradiated at the HIMAC accelerator in Chiba, Japan (0.2 and 0.5 GeV/n Fe and 0.49 GeV/n Si) and at the NASA Space Radiation Laboratory in Brookhaven, USA (1 GeV/n Fe and 0.97 GeV/n Ti ions). The dose-effect curves were measured in the dose range between 0.25 and 2 Gy. For comparison cells were exposed to 60Co gamma rays. Analysis of the dose-effect curves show that all the heavy ion beams induce inactivation and delayed reproductive death more effectively than 60Co gamma rays. The only exception is the 0.2 GeV/n Fe-ion beam at low doses. The progeny of the irradiated cells show delayed damage in the form of reproductive death with all the heavy ion beams with the 1 GeV/n Fe-ion beam being the most effective. The relative biological effectiveness at low doses of the iron beams is highest for LET values between 140 and 200 keV/micrometers with values of 1.6 and 3 for early and delayed reproductive death, respectively. Analysis of the fluence-effect curves shows that the cross-sections for early and delayed inactivation increase with increasing LET up to 442 keV/micrometers.     

Unique biological aspects of radiation hazards--an overview.

Low orbit, geostationary, and deep-space flights differ from one another with respect to particle radiation environment, participating population size, mission duration, and biological risks other than radiation. It is proposed that all of these factors be considered in the setting of safety standards and, in particular, that the rem-dose concept is applicable only to radiations having low and intermediate linear energy transfer (electrons, protons, and helium ions), whereas the incidence of microlesions is a more meaningful indicator of the hazard due to higher-Z, high energy (HZE) particles. A microlesion is the biological injury inflicted in a specific tissue by a single HZE particle, and it is still in need of quantitative biological definition for specific mammalian tissues. If for example, a microlesion is taken as due to a HZE particle track 10 cell diameters long with LET > 200 KeV/micrometer in its core and > 25 rad dose in its penumbra at a distance of 10 micrometers, then the microlesion dose rate in geostationary orbit, for example, is about 9,000 microlesions per cm3 of tissue per month.     

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.     

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.     

Cataractogenesis from high-LET radiation and the Casarett model.

Space radiations, especially heavy ions, constitute significant hazards to astronauts. These hazards will increase as space missions lengthen. Moreover, the dangers to astronauts will be enhanced by the persistence, or even the progression, of biological damage throughout their subsequent life spans. To assist in the assessment of risks to astronauts, we are investigating the long-term effects of heavy ions on specific animal tissues. In one study, the eyes of rabbits of various ages were exposed to a single dose of Bragg plateau 20Ne ions (LET infinity approximately equals 30 keV/micrometer). The development of cataracts has shown a pronounced age-related response during the first year after irradiation, and will be followed for two more years. In other studies, mice were exposed to single or fractionated doses of 12C ions (4-cm spread-out Bragg peak; dose-averaged LET infinity = 70-80 keV/micrometer) or 60Co gamma-photons (LET infinity = 0.3 keV/micrometer). Measurements of the frequency of posterior lens opacification have shown that the tissue sparing observed with dose fractionation of gamma-photons was absent when 12C-ion doses were fractionated. Development of posterior lens cataracts was also followed for long periods (up to 21 months) in mice exposed to single doses of Bragg plateau HZE particles (40Ar, 20Ne and 12C ions: LET infinity approximately equals 100, 30 and 10 keV/micrometer, respectively) or 225 kVp X-rays. Based on average cataract levels at the different observation times, the RBE's (RBE = relative biological effectiveness) for the ions were circa 5, 3 and 1-2, respectively, over the range of doses used (0.05-0.9 Gy). Investigations of cataractogenesis are useful for exploring the model of radiation damage proposed by Casarett and by Rubin and Casarett with a tissue not connected directly to the vasculature.