Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. III. Human diploid fibroblasts.

The induction of inactivation and mutation to thioguanine-resistance in cultured human diploid fibroblasts was studied after exposure to ionising radiations with LET's in the range 20--470 keV micrometer-1. Unique r.b.e. values were obtained for inactivation and mutation induction with nine different qualities of radiation. The plot of r.b.e. verus LET gave humped curves for both endpoints; r.b.e. maxima were in the LET range 90--200 keV micrometer-1 but the maximum r.b.e. value for mutation induction was almost twice that for inactivation. The accuracy of estimates of mutation induction are discussed with regard to possible selective effects against mutants during post-irradiation growth.     

Effectiveness of 0.3 keV carbon ultrasoft X-rays for the inactivation and mutation of cultured mammalian cells.

Carbon K characteristic ultrasoft X-rays of energy 0.278 keV were found to be effective in inducing inactivation and mutation to thioguanine resistance in cultured V79 Chinese hamster cells and human diploid fibroblasts. These X-rays act as a probe of the sensitive sites within the cells since they produce low-energy photoelectron tracks of range about 7 nm; this is an order of magnitude smaller than those produced by the 1.5 keV aluminium X-rays used in previous studies. A detailed interpretation of the results requires assumptions to be made about the positions of the sensitive sites within the cells but, for any reasonable set of assumptions, the carbon X-rays are found to be more effective than gamma-rays and are probably at least as effective as long tracks of helium ions of similar LET. These observations extend the conclusions previously drawn from the observed effectiveness of aluminium X-rays regarding the sizes of the subcellular sites involved in inactivation and mutation. They imply that the sensitive sites smaller than about 7 nm, and that highly localized energy depositions consisting of less than or approximately 14 ionizations are sufficient to produce biological effects. These results are also in contradiction to models of radiation action which require relatively large sites, such as the usual form of the 'theory of dual radiation action'.     

Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays. II. Dose-responses of Chinese hamster and human diploid cells to aluminium X-rays and radiations of different LET.

The induction of inactivation and mutation to thioguanine-resistance of two types of cultured mammalian cells, V79 Chinese hamster and HF19 human diploid, was studied after irradiation with aluminium K characteristic ultrasoft X-rays, helium ion track intersections of different LET, 42 MeV d-Be neutrons, and hard X- or gamma-rays. The form of the dose-response curves was different for the two cell-types, and there was an overall difference in radiosensitivity, the human cells being the more sensitive to all radiations. However, for both inactivation and mutation-induction, the relative responses of both cell-types to these radiations was similar. Aluminium X-rays were considerably more effective than hard X- or gamma-rays and were at least as effective as helium ions of 20-28 keV micron-1, although aluminium X-rays produce tracks of very limited range (less than about 0.07 micron). Single track effects by aluminium X-rays cannot, therefore, extend beyond about 0.07 micron, and the subcellular sites involved in inactivation and mutation cannot be greater than this dimension or else the effectiveness of aluminium X-rays would be similar to that of low-LET radiations. This observation is in contradiction to models of radiation action which require relatively large sensitive sites; for example the 'theory of dual radiation action' requires a site diameter of about 0.4 micron to explain the shape of the dose-response curves for V79 hamster cells.     

His+ reversions caused in Salmonella typhimurium by different types of ionizing radiation

The yield of his+ reversions in the Ames Salmonella tester strain TA2638 has been determined for 60Co gamma rays, 140 kV X rays, 5.4 keV characteristic X rays, 2.2 MeV protons, 3.1 MeV alpha particles, and 18 MeV/U Fe ions. Inactivation studies were performed with the same radiations. For both mutation and inactivation, the maximum effectiveness per unit absorbed dose was obtained for the characteristic X rays, which have a dose averaged linear energy transfer (LET) of roughly 10 keV/micron. The ratio of the effectiveness of this radiation to gamma rays was 2 for inactivation and about 1.4 for the his+ reversion. For both end points the effectiveness decreases substantially at high LET, i.e., for the alpha particles and the Fe ions. The composition of the bottom and the top agar was the one recommended by Maron and Ames [Mutat. Res. 113, 173-215 (1983)] for application in chemical mutagenicity tests. The experiments with the less penetrating radiations differed from the usual protocol by utilization of a technique of plating the bacteria on the surface of the top agar. As in an earlier study [Roos et al., Radiat. Res. 104, 102-108 (1985)] greatly enhanced yields of mutations, relative to the spontaneous reversion rate, were obtained in these experiments by performing the irradiations 6 h after plating, which differs from the conventional procedure to irradiate the bacteria shortly after plating.     

Heavy ion effects on yeast: inhibition of ribosomal RNA synthesis

Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated.     

Transient thermal and mechanical response of water subject to ionizing radiation.

The ultrafast transient (10(-14) to 10(-12)S) thermal and mechanical response of water subject to ionizing radiations of different linear energy transfers has been investigated in order to understand the initial events which lead to cell mutation and lethality. Based on computational fluid dynamics, the production of a "thermal spike" around the trajectory of a charged particle and subsequent diffusion of deposited heart are calculated for particles with linear energy transfer (LET) of 4, 40, and 400 keV/microns. A radiation damage region (that is, the so-called "thermal core") is identified, and the transient behavior of the thermal core is studied. The local and transient environment has a dimension of nanometers, a scale which is of critical interest in understanding mechanisms of radiation damage in cells. The radius of the thermal core, Dd, at temperatures (or internal energy density) of up to 1,000 K, is observed to increase with LET, L, as Dd (in nanometers) = C4.L (in keV/microns)0.6, where, for example, C4 = 0.50 for T = 800 degrees C.     

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/microm to 440 keV/microm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/microm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.     

Induction of gene and deletion mutations by accelerated heavy charged particles in Escherichia coli cells

The results of the induction of the point and the deletion mutations by the radiation with broad region of linear energy transfer (LET) ox Escherichia coli cells. The linear-quadratic function for point mutation induction was shown in comparison with linear dependence for deletion mutations. The relative biological effectiveness (RBE) is described as a function of LET by dependence with a local maximum. The greatest RBE coefficients for the lethal effects, gene and deletion mutation induction realize under different LET of heavy charged particles.     

Inactivation action spectra of Bacillus subtilis spores with monochromatic soft X rays (0.1-0.6 nm) of synchrotron radiation.

Five types of Bacillus subtilis spores differing in DNA repair and recombinational capacities were exposed in vacuum to monochromatic soft X rays from synchrotron radiation. The inactivation rate constants were obtained from exposure-survival curves upon irradiations at 12 wavelengths in the range of 0.1000 nm (12.40 keV) to 0.6000 nm (2.066 keV). Spores of two repair-deficient strains, UVS (uvrA ssp) and UVP (uvrA ssp polA), exhibited almost equal sensitivities to those of wild-type UVR+, while those of two recombination-deficient strains, RCE (recE) and RCF (recF), exhibited higher sensitivities in the whole wavelength range. This suggested that the repair of DNA damage produced by soft X rays was dependent on the recombinational capabilities. Inactivation action spectra based on photon fluence showed that the effectiveness of the radiation increased as the wavelengths became longer. Abrupt changes in the effectiveness occurred around the wavelengths corresponding to the absorption edges of K-shell electrons of phosphorus and calcium. In both cases, the sensitivity was the highest at the wavelengths of the resonance absorption peak, the next highest at those of the higher energy, and the lowest at the lower energy. Mass energy absorption coefficients of spores were obtained from the transmission of a flake made of spores. They were used to derive inactivation action spectra based on absorbed doses. In these spectra, basal levels of the sensitivity seemed constant, and enhancements of the sensitivity were observed consistent with the absorption by calcium and phosphorus. Thus calcium and phosphorus atoms were the predominant targets for the absorption events leading to the inactivation of spores in the wavelength range examined.     

Long-term consequences of radiation-induced bystander effects depend on radiation quality and dose and correlate with oxidative stress

Widespread evidence indicates that exposure of cell populations to ionizing radiation results in significant biological changes in both the irradiated and nonirradiated bystander cells in the population. We investigated the role of radiation quality, or linear energy transfer (LET), and radiation dose in the propagation of stressful effects in the progeny of bystander cells. Confluent normal human cell cultures were exposed to low or high doses of 1GeV/u iron ions (LET ～ 151 keV/μm), 600 MeV/u silicon ions (LET ～ 51 keV/μm), or 1 GeV protons (LET ～ 0.2 keV/μm). Within minutes after irradiation, the cells were trypsinized and co-cultured with nonirradiated cells for 5 h. During this time, irradiated and nonirradiated cells were grown on either side of an insert with 3-μm pores. Nonirradiated cells were then harvested and allowed to grow for 20 generations. Relative to controls, the progeny of bystander cells that were co-cultured with cells irradiated with iron or silicon ions, but not protons, exhibited reduced cloning efficiency and harbored higher levels of chromosomal damage, protein oxidation and lipid peroxidation. This correlated with decreased activity of antioxidant enzymes, inactivation of the redox-sensitive metabolic enzyme aconitase, and altered translation of proteins encoded by mitochondrial DNA. Together, the results demonstrate that the long-term consequences of the induced nontargeted effects greatly depend on the quality and dose of the radiation and involve persistent oxidative stress due to induced perturbations in oxidative metabolism. They are relevant to estimates of health risks from exposures to space radiation and the emergence of second malignancies after radiotherapy.     

Charged-particle mutagenesis. 1. Cytotoxic and mutagenic effects of high-LET charged iron particles on human skin fibroblasts.

Cytotoxic and mutagenic effects of high-LET charged iron (56Fe) particles were measured quantitatively using primary cultures of human skin fibroblasts. Argon and lanthanum particles and gamma rays were used in comparative studies. The span of LETs selected was from 150 keV/microns (330 MeV/u) to 920 keV/microns (600 MeV/u). Mutations were scored at the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus using 6-thio-guanine (6-TG) for selection. Exposure to these high-LET charged particles resulted in exponential survival curves. Mutation induction, however, was fitted by the linear model. The relative biological effectiveness (RBE) for cell killing ranged from 3.7 to 1.3, while that for mutation induction ranged from 5.7 to 0.5. Both the RBE for cell killing and the RBE for mutagenesis decreased with increasing LET over the range of 1.50 to 920 keV/microns. The inactivation cross section (sigma i) and the action cross section for mutation induction (sigma m) ranged from 32.9 to 92.0 microns2 and 1.45 to 5.56 X 10(-3) microns2; the maximum values were obtained by 56Fe with an LET of 200 keV/microns. The mutagenicity (sigma m/sigma i) ranged from 2.05 to 7.99 X 10(-5) with an inverse relationship to LET.     

A microdosimetric-kinetic model for the effect of non-Poisson distribution of lethal lesions on the variation of RBE with LET

The microdosimetric-kinetic (MK) model for cell killing by ionizing radiation is summarized. An equation based on the MK model is presented which gives the dependence of the relative biological effectiveness in the limit of zero dose (RBE1) on the linear energy transfer (LET). The relationship coincides with the linear relationship of RBE1 and LET observed for low LET, which is characteristic of a Poisson distribution of lethal lesions among the irradiated cells. It incorporates the effect of deviation from the Poisson distribution at higher LET. This causes RBE1 to be less than indicated by extrapolation of the linear relationship to higher LET, and to pass through a maximum in the range of LET of 50 to 200 keV per micrometer. The relationship is compared with several experimental studies from the literature. It is shown to approximately fit their results with a reasonable choice for the value of a cross-sectional area related to the morphology and ultrastructure of the cell nucleus. The model and the experiments examined indicate that the more sensitive cells are to radiation at low LET, the lower will be the maximum in RBE they attain as LET increases. An equation that portrays the ratio of the sensitivity of a pair of cell types as a function of LET is presented. Implications for radiotherapy with high-LET radiation are discussed.     

Responses of synchronous L5178Y S/S cells to heavy ions and their significance for radiobiological theory

Synchronous suspensions of the radiosensitive S/S variant of the L5178Y murine leukaemic lymphoblast at different positions in the cell cycle were exposed aerobically to segments of heavy-ion beams (20Ne, 28Si, 40Ar, 56Fe and 93Nb) in the Bragg plateau regions of energy deposition. The incident energies of the ion beams were in the range of 460 +/- 95 MeV u-1, and the calculated values of linear energy transfer (LET infinity) for the primary nuclei in the irradiated samples were 33 +/- 3, 60 +/- 3, 95 +/- 5, 213 +/- 21 and 478 +/- 36 keV microns-1, respectively; 280 kVp X-rays were used as the baseline radiation. Generally, the maxima or inflections in relations between relative biological effectiveness (RBE) and LET infinity were dependent upon the cycle position at which the cells were irradiated. Certain of those relations were influenced by post-irradiation hypothermia. Irradiation in the cell cycle at mid-G1 to mid-G1 + 3 h, henceforth called G1 to G1 + 3 h, resulted in survival curves that were close approximations to simple exponential functions. As the LET infinity was increased, the RBE did not exceed 1.0, and by 478 keV microns-1 it had fallen to 0.39. Although similar behaviour has been reported for inactivation of proteins and certain viruses by ionizing radiations, so far the response of the S/S variant is unique for mammalian cells. The slope of the survival curve for X-photons (D0: 0.27 Gy) is reduced in G1 to G1 + 3 h by post-irradiation incubation at hypothermic temperatures and reaches a minimum (Do: 0.51 Gy) at 25 degrees C. As the LET infinity was increased, however, the extent of hypothermic recovery was reduced progressively and essentially was eliminated at 478 keV microns-1. At the cycle position where the peak of radioresistance to X-photons occurs for S/S cells, G1 + 8 h, increases in LET infinity elicited only small increases in RBE (at 10% survival), until a maximum was reached around 200 keV microns-1. At 478 keV microns-1, what little remained of the variation in response through the cell cycle could be attributed to secondary radiations (delta rays) and smaller nuclei produced by fragmentation of the primary ions.     

Induction of minisatellite mutations in the mouse germline by low-dose chronic exposure to gamma-radiation and fission neutrons

Germline mutation induction at mouse minisatellite loci by paternal low-dose (0.125-1 Gy) exposure to chronic (1.66 x 10(-4) Gy min(-1)) low-linear energy transfer (low-LET) gamma-irradiation and high-LET fission neutrons (0.003 Gy min(-1)) was studied at pre-meiotic stages of spermatogenesis. Both types of radiation produced linear dose-response curves for mutation of the paternal allele. In contrast to previous results using higher doses, the pattern of induction of minisatellite mutation after chronic gamma-irradiation was similar to acute (0.5 Gy min(-1)) exposure to X-rays, indicating that the elevated mutation rate was independent of the ability of the cell to repair damage induced immediately or over a period of up to 100 h. Chronic exposure to fission neutrons was more effective than acute or chronic low-LET exposure (relative biological effectiveness, RBE=3.36). The data also provide strong support for the previous conclusion that increases in minisatellite mutation rate are not caused by radiation-induced DNA damage at minisatellite loci themselves, but rather from damage induced by ionising radiation elsewhere in the genome/cell.     

Influence of radiation quality on the effectiveness of small doses for induction of reproductive death and chromosome aberrations in mammalian cells.

An analysis and interpretation is presented of published data concerning the dependence of radiobiological effectiveness on the radiation quality of photons, neutrons and heavy ions for the induction of these two effects in different types of mammalian cell. The results of this analysis suggest that chromosome aberrations observable at mitosis show a stronger dependence on YF or LET infinity than cell inactivation. At high YF, observable abberrations provide a major contribution to cell reproductive death induced by small doses. At low YF the effectiveness of small doses for cell death depends mainly on another type of damage, possibly also induced in the chromosomes, but not observable at mitosis. This type of damage depends less of YF or LET infinity than observable aberrations. The implications of these differences in damage in relation to radiation quality for the extrapolation of data on other types of damage to small doses of interest in radiation protection are discussed in relation to maximum r.b.e values observed.     

The effect of accelerated argon ions on the retina

It has been postulated that high energy heavy ions cause a unique form of damage in living tissue, which results from the high linear energy transfer of accelerated single particles. We have searched for these single-particle effects, so-called "microlesions," in composite electron micrographs of retinas of rats which had been irradiated with a dose of 1 Gy of 570 MeV/amu argon ions. The calculated rate of energy deposition of the radiation in the retina was about 100 keV/micron and the influence was four particles per 100 micron 2. Different areas of the irradiated retinas which combined would have been expected to be traversed by approximately 2400 particles were examined. We were unable to detect ultrastructural changes in the irradiated retinas distinct from those of controls. The spatial cellular densities of pigment epithelial and photoreceptor cells remained within the normal range when examined at 24 h and at 6 months after irradiation. These findings suggest that the retina is relatively resistant to heavy-ion irradiation and that under the experimental conditions the passage of high energy argon ions does not cause retinal microlesions that can be detected by ultrastructural analysis.     

Heavy ion-induced DNA double-strand breaks in yeast

Induction of DSBs in the diploid yeast, Saccharomyces cerevisiae, was measured by pulsed-field gel electrophoresis (PFGE) after the cells had been exposed on membrane filters to a variety of energetic heavy ions with values of linear energy transfer (LET) ranging from about 2 to 11,500 keV/microm, (241)Am alpha particles, and 80 keV X rays. After irradiation, the cells were lysed, and the chromosomes were separated by PFGE. The gels were stained with ethidium bromide, placed on a UV transilluminator, and analyzed using a computer-coupled camera. The fluorescence intensities of the larger bands were found to decrease exponentially with dose or particle fluence. The slope of this line corresponds to the cross section for at least one double-strand break (DSB), but closely spaced multiple breaks cannot be discriminated. Based on the known size of the native DNA molecules, breakage cross sections per base pair were calculated. They increased with LET until they reached a transient plateau value of about 6 x 10(-7) microm(2) at about 300-2000 keV/microm; they then rose for the higher LETs, probably reflecting the influence of delta electrons. The relative biological effectiveness for DNA breakage displays a maximum of about 2.5 around 100-200 keV/microm and falls below unity for LET values above 10(3) keV/microm. For these yeast cells, comparison of the derived breakage cross sections with the corresponding cross section for inactivation derived from the terminal slope of the survival curves shows a strong linear relationship between these cross sections, extending over several orders of magnitude.     

DNA complex lesions induced by protons and ⋅-particles: track structure characteristics determining linear energy transfer and particle type dependence

The yield of DNA double-strand breaks (dsb) and DNA complex lesions induced by protons and α-particles of various energies was simulated using a Monte Carlo track structure code (MOCA15) and a simple model of the DNA molecule. DNA breaks of different complexity were analysed. The linear energy transfer (LET) and particle-type dependence of lesions of higher complexity seems to confirm the importance of clustered damage in DNA as a relevant step leading to biological endpoints such as cell inactivation. The detailed structure of proton and α-particle tracks was analysed to identify the main characteristics possibly responsible for such a dependence. The role of the primary ion and of its secondary electrons in inducing dsb and complex lesions is described, showing that the relative contribution of secondary electron tracks alone in inducing clustered lesions is almost negligible at high LET, but tends to dominate below ≈10 keV/μm. This is consistent with the observed similar effectiveness of low-LET fast particle radiation and sparsely ionizing radiation such as x-rays. The dependence on LET and particle type is mainly due to energy deposition events of the primary ion together with short range electrons surrounding the ion track; the yield of complex lesions due to secondary electron tracks alone is substantially LET independent. The radial distributions of the energy contributing to the induction of complex lesions were analyzed and compared with the radial distributions of energy deposition of the full tracks. The results suggest that the stochastic behaviour (i.e. cluster properties) of the energy deposition pattern within a radius of a few nanometers around the ion track plays a relevant role in determining the biological radiation effectiveness. Received: 20 December 1996 / Accepted in revised form: 5 March 1997     

DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation

Carbon beams (5.16MeV/u, LET=290keV/μm) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ-rays and carbon ion-irradiation. A549 cells were irradiated with 1Gy carbon or γ-rays. Carbon beam was found to be three times more cytotoxic than γ-rays despite the fact that the numbers of γ-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with γ-rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike γ-rays irradiated cells and prosurvival ERK pathway was activated after γ-rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored.     

LET dependence of lethality in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> irradiated by heavy ions

To clarify the effect of heavy ions in plants, dry seeds of Arabidopsis were irradiated with carbon, neon, and argon ions with various linear energy transfer (LET) values. The relative biological effectiveness (RBE) for lethality peaked at LET values over 350 keV/microns for neon and argon ions. This LET giving the peak RBE was higher than the LET of 100-200 keV/microns which was reported to have a maximum RBE for other types of cells, such as mammalian cells. Furthermore, sterility showed a higher RBE at an LET of 354 keV/microns with neon ions than that at an LET of 113 keV/microns with carbon ions. Lethality and sterility are both considered to be caused by damage to DNA. The results indicate that the LET having a maximum of RBE for lethality is higher in Arabidopsis seeds than in other unicellular systems. The most likely explanation for this shift of LET is that the DNA in dry seeds has a different chemical environment and/or hydration state than the DNA in cells in culture.