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.     

The influence of radiation quality on the formation of DNA breaks.

We have aimed to present a comprehensive review of our understanding to date of the formation of DNA strand breaks induced by high LET radiation. We have discussed data obtained from DNA in solution as well as from the formation and "repair" of strand breaks in cell DNA. There is good agreement, qualitatively, between these two systems. Results were evaluated for two parameters: (1) effectivity per particle, the cross section (sigma) in micrometers 2/particle; and (2) the strand break induction frequency as number of breaks per Gy per unit DNA (bp or dalton). A series of biological effects curves (one for each Z-number) is obtained in effectivity versus LET plots. The relationships between induction frequencies of single-strand breaks, or double-strand breaks, or the residual "irrepairable" breaks and LET-values have been evaluated and discussed for a wide spectrum of heavy ions, both for DNA in solution and for DNA in the cell. For radiation induced total breaks in cell DNA, the RBE is less than one, while the RBE for the induction of DSBs can be greater than one in the 100-200 keV/micrometers range. The level of irrepairable strand breaks is highest in this same LET range and may reach 25 percent of the initial break yield. The data presented cover results obtained for helium to uranium particles, covering a particle incident energy range of about 2 to 900 MeV/u with a corresponding LET range of near 16 to 16000 keV/micrometers.     

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.     

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.     

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.     

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.     

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.     

Radiation effects on late cytopathological parameters in the murine lens relative to particle fluence.

Lenses of mice irradiated with 250 MeV protons, 670 MeV/amu 20Ne, 600 MeV/amu 56Fe, 600 MeV/amu 93Nb and 593 MeV/amu 139La ions were evaluated by analyzing cytopathological indicators which have been implicated in the cataractogenic process. The LETs ranged from 0.40 keV/micrometer to 953 keV/micrometer and fluences from 1.31 10(3)/mm2 to 4.99 x 10(7)/mm2. 60Co gamma-rays were used as the reference radiation. The doses ranged from 10 to 40 cGy. The lenses were assessed 64 weeks post irradiation in order to observe the late effects of LET and dose on the target cell population of the lens epithelium. Our study shows that growth dependent pathological changes occur at the cellular level as a function of dose and LET. The shapes of the RBE-LET and RBE-dose curves are consistent with previous work on eye and other biological systems done in both our laboratory and others. The RBEmax's were estimated, for the most radiation cataract related cytological changes, MN frequency and MR disorganization, by calculating the ratio of the initial slopes of dose effect curve for various heavy ions to that of 60Co gamma-ray. For each ion studied, the RBEmax derived from micronucleus (MN) frequency is similar to that derived from meridional row (MR) disorganization, suggesting that heavy ions are equally efficient at producing each type of damage. Furthermore, on a per particle basis (particle/cell nucleus), both MN frequency and MR disorganization are LET dependent indicating that these classic precataractogenic indicators are multi-gene effects. Poisson probability analysis of the particle number traversing cell nuclei (average area = 24 micrometers2) suggested that single nuclear traversals determine these changes. By virtue of their precataractogenic nature the data on these endpoints intimate that radiation cataract may also be the consequence of single hits. In any case, these observations are consistent with the current theory of the mechanism of radiation cataractogenesis, which proposes that genomic damage to the epithelial cells surviving the exposure is responsible for opacification.     

Induction of DNA breaks in SV40 by heavy ions.

Simian virus (SV40) DNA was used to study the induction of DNA strandbreaks by heavy ions varying in LET. DNA was exposed to X-rays and to accelerated particles either in dilute solution or in the presence of different radical scavengers. Relative proportions of the intact supercoiled DNA, nicked form arising from single strand breaks (SSB) and linear molecules produced by double strandbreaks (DSB) were quantified on the base of their electrophoretic mobility in agarose gels. Cross sections for the induction of SSBs and DSBs were calculated from the slope of dose effect curves. Mercaptoethanol was found to protect more efficiently against DNA strand breakage than Tris. When the biological efficiency, i.e. the number of strand breaks per unit dose and molecule weight was evaluated as a function of LET, curves for SSB induction always showed a continuous decrease. For DSB induction, an increase in the yield of DSBs with a maximum around 500 keV/micrometer was observed in the presence of radical scavenger. This peak of biological efficiency gradually disappeared when the radiosensitivity of the system was increased, and was no longer apparent in the dilute buffer system, where DNA showed a high susceptibility to strand breakage. When the relative biological efficiency was plotted versus LET, the curve for DSB induction observed in a low radical scavenging environment paralleled the curve obtained for SSB induction.     

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.     

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.     

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 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.     

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.     

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.     

Late effects from particulate radiations in primate and rabbit tissues.

Optic tissues in groups of New Zealand white rabbits were irradiated locally at different stages throughout the median life span of the species with a single dose (9 Gy) of 425 MeV/amu Ne ions (LET infinity approximately 30 keV/micrometer) and then inspected routinely for the progression of radiation cataracts. The level of early cataracts was found to be highest in the youngest group of animals irradiated (8 weeks old), but both the onset of late cataracts and loss of vision occurred earlier when animals were irradiated during the second half of the median life span. This age response can have serious implications in terms of space radiation hazards to man. Rhesus monkeys that had been subjected to whole-body skin irradiation (2.8 and 5.6 Gy) by 32 MeV protons (range in tissue approximately 1 cm) some twenty years previously were analysed for radiation damage by the propagation of skin fibroblasts in primary cultures. Such propagation from skin biopsies in MEM-alpha medium (serial cultivation) or in supplemented Ham's F-10 medium (cultivation without dilution) revealed late damage in the stem (precursor) cells of the skins of the animals. The proton fluxes employed in this experiment are representative of those occurring in major solar flares.     

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.     

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.     

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.     

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.