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.     

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.     

Inactivation of aerobic and hypoxic cells from three different cell lines by accelerated (3)He-, (12)C- and (20)Ne-ion beams

The LET-RBE spectra for cell killing for cultured mammalian cells exposed to accelerated heavy ions were investigated to design a spread-out Bragg peak beam for cancer therapy at HIMAC, National Institute of Radiological Sciences, Chiba, prior to clinical trials. Cells that originated from a human salivary gland tumor (HSG cells) as well as V79 and T1 cells were exposed to (3)He-, (12)C- and (20)Ne-ion beams with an LET ranging from approximately 20-600 keV/micrometer under both aerobic and hypoxic conditions. Cell survival curves were fitted by equations from the linear-quadratic model and the target model to obtain survival parameters. RBE, OER, alpha and D(0) were analyzed as a function of LET. The RBE increased with LET, reaching a maximum at around 200 keV/micrometer, then decreased with a further increase in LET. Clear splits of the LET-RBE or -OER spectra were found among ion species and/or cell lines. At a given LET, the RBE value for (3)He ions was higher than that for the other ions. The position of the maximum RBE shifts to higher LET values for heavier ions. The OER value was 3 for X rays but started to decrease at an LET of around 50 keV/micrometer, passed below 2 at around 100 keV/micrometer, and then reached a minimum above 300 keV/micrometer, but the values remained greater than 1. The OER was significantly lower for (3)He ions than the others.     

Protein synthesis modulates the biological effectiveness of the combined action of hyperthermia and high-LET radiation.

The combined effects of heavy-ion radiation and hyperthermia on the survival of CHO-SC1 cells and its temperature-sensitive (ts) mutant tsH1 cells were studied using accelerated neon ions followed by mild heating at 41.5 degrees C. The sequence of application of heat and high-LET radiation is significant to cell-killing effects. Heat applied to cells prior to irradiation with neon plateau ions (LET = 32 keV/microns) was less effective than heat applied immediately after irradiation. The ability of cells to synthesize new proteins plays a key role in this sequence-dependent thermal sensitization. When protein synthesis was shut down in tsH1 cells, the thermal enhancement of cell killing by high-LET radiation was the same regardless of the sequence. The thermal enhancement of radiation-induced cell killing was LET-dependent for the SC1 cells, but this was not clearly demonstrated in the tsH1 cells. Furthermore, the RBE of heated SC1 cells varied with LET and reached a maximum of greater than 3 at 80 keV/microns. In the absence of protein synthesis, the maximum RBE value was reduced to 2.6. These results suggest that the accumulation of cellular damage caused by exposure to densely ionizing particles with increasing LETs can be potentiated with active protein synthesis during postirradiation heat treatment.     

不同LET的重离子辐射对DSB诱导的影响

利用γ射线和不同LET的碳离子辐照小鼠B16黑色素瘤细胞的脱蛋白DNA,采用脉冲场凝胶电泳结合荧光扫描技术研究了DNA双链断裂（DSB）与LET之间的关系。结果表明：不同LET重离子诱导的PR都随剂量的增加而增加,并在超过一定的剂量之后逐渐趋于一个准阈值;而不同LET的重离子诱导的L值都与剂量呈线性关系;对于诱导DSB的RBE值则随着LET的增加先呈上升趋势,在LET超过100ke/μm后下降。     

Relative biological effectiveness of high-energy iron ions for micronucleus formation at low doses

Dose-response curves for micronucleus (MN) formation were measured in Chinese hamster V79 and xrs6 (Ku80(-)) cells and in human mammary epithelial MCF10A cells in the dose range of 0.05-1 Gy. The Chinese hamster cells were exposed to 1 GeV/nucleon iron ions, 600 MeV/nucleon iron ions, and 300 MeV/nucleon iron ions (LETs of 151, 176 and 235 keV/microm, respectively) as well as with 320 kVp X rays as reference. Second-order polynomials were fitted to the induction curves, and the initial slopes (the alpha values) were used to calculate RBE. For the repair-proficient V79 cells, the RBE at these low doses increased with LET. The values obtained were 3.1 +/- 0.8 (LET = 151 keV/microm), 4.3 +/- 0.5 (LET = 176 keV/microm), and 5.7 +/- 0.6 (LET = 235 keV/microm), while the RBE was close to 1 for the repair-deficient xrs6 cells regardless of LET. For the MCF10A cells, the RBE was determined for 1 GeV/nucleon iron ions and was found to be 5.5 +/- 0.9, slightly higher than for V79 cells. To test the effect of shielding, the 1 GeV/nucleon iron-ion beam was intercepted by various thicknesses of high-density polyethylene plastic absorbers, which resulted in energy loss and fragmentation. It was found that the MN yield for V79 cells placed behind the absorbers decreased in proportion to the decrease in dose both before and after the iron-ion Bragg peak, indicating that RBE did not change significantly due to shielding except in the Bragg peak region. At the Bragg peak itself with an entrance dose of 0.5 Gy, where the LET is very high from stopping low-energy iron ions, the effectiveness for MN formation per unit dose was decreased compared to non-Bragg peak areas.     

Microdosimetric measurements and estimation of human cell survival for heavy-ion beams

The microdosimetric spectra for high-energy beams of photons and proton, helium, carbon, neon, silicon and iron ions (LET = 0.5-880 keV/microm) were measured with a spherical-walled tissue-equivalent proportional counter at various depths in a plastic phantom. Survival curves for human tumor cells were also obtained under the same conditions. Then the survival curves were compared with those estimated by a microdosimetric model based on the spectra and the biological parameters for each cell line. The estimated alpha terms of the liner-quadratic model with a fixed beta value reproduced the experimental results for cell irradiation for ion beams with LETs of less than 450 keV/microm, except in the region near the distal peak.     

Induction of DNA double-strand breaks by 1H and 4He lons in primary human skin fibroblasts in the LET range of 8 to 124 keV/microm.

Yields of DNA double-strand breaks were determined in primary human skin fibroblasts exposed to 1H and 4He ions at various linear energy transfers (LETs) and to 15 MeV electrons as the reference radiation. The values obtained for the relative biological effectiveness (RBE) were 2.03, 1.45 and 1.36 for 1H ions at LETs of 35, 23 and 7.9 keV/microm, respectively, and 1.2, 1.18, 1.38 and 1.31 for 4He ions at LETs of 124, 76, 35 and 27 keV/microm, respectively. The data were obtained using pulsed-field gel electrophoresis of DNA released from cells using the chromosomes of the yeast Saccharomyces cerevisiae as length markers and fitting the experimental mass distributions of fragmented DNA to those obtained by computer simulation of the random breakage of human chromosomes. The RBE values for induction of DSBs in mammalian cells cannot be fitted to a common RBE-LET relationship for electrons and 1H, 4He and light ions. Comparison of the RBEs for mammalian cells with the corresponding RBEs obtained for yeast cells shows similar RBEs of electrons for yeast and mammalian cells; however, for 4He and light ions in the LET range of 100 to 1000 keV/microm, the RBEs for yeast are significantly higher compared with mammalian cells. These characteristics of the RBE-LET relationships for yeast and mammalian cells are attributed to the fraction of small DNA fragments induced by particles when traversing the higher-order chromatin structures which are different to some extent in these two cell types.     

The effect of 238Pu alpha-particles on the mouse fibroblast cell line C3H 10T1/2: characterization of source and RBE for cell survival

Considerable interest has been aroused in recent years by reports that the transforming and carcinogenic effectiveness of low doses of high LET radiations can be increased by reducing the dose rate, especially for transformation of 10T1/2 cells in vitro by fission-spectrum neutrons. We report on conditions which have been established for irradiation of 10T1/2 cells with high LET monoenergetic alpha-particles (energy of 3.2 MeV, LET of 124 keV microns-1) from 238Pu. The alpha-particle irradiator allows convenient irradiation of multiple dishes of cells at selectable high or low dose rates and temperatures. The survival curves of irradiated cells showed that the mean lethal dose of alpha-particles was 0.6 Gy and corresponded to an RBE, at high dose rates, of 7.9 at 80 per cent survival and 4.6 at 5 per cent survival, relative to 60Co gamma-rays. The mean areas of the 10T1/2 nuclei, perpendicular to the incident alpha-particles, was measured as 201 microns2, from which it follows that, on average, only one in six of the alpha-particle traversals through a cell nucleus is lethal. Under the well-characterized conditions of these experiments the event frequency of alpha-particle traversals through cell nuclei is 9.8 Gy-1.     

The relative biological effectiveness of 670 MeV/A neon as a function of depth in water for a tissue model

Linear energy transfer (LET infinity) spectra of identified charge fragments and primaries, produced by nuclear interactions of 670 MeV/A neon in water, were measured along the unmodulated Bragg curve of the neon beam. The relative biological effectiveness (RBE) values for spermatogonial cell killing, as reported on the basis of weight loss assay of mouse testes irradiated with beams of approximately constant single LET infinity, were summed over the particle LET infinity spectra to obtain an effective RBE for each charged-particle species, as a function of water absorber thickness. The resultant values of effective RBE were combined to obtain an effective RBE for the mixed radiation field. The RBE calculated in this way was compared with experimental RBEs obtained for spermatogonial cell killing in the mixed radiation field produced by neon ions traversing a thick water absorber. Discrepancies of 10-40% were observed between the calculated RBE and the RBE measured in the mixed radiation field. Part of this discrepancy can be attributed to undetected low-Z fragments, whose contribution is not included in the calculation, leading to an overestimated value for the calculated RBE. On the other hand, calculated values 10% greater than the measured RBE are explained as track structure effects due to the higher radial ionization density near neon tracks relative to the ionization density near the silicon tracks used to fit the RBE vs LET infinity data.     

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.     

LET and ion species dependence for cell killing in normal human skin fibroblasts

We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/microm. The RBE of carbon ions at 98 keV/microm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/microm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/beta2 (where Z* is the effective charge and beta is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/beta2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/beta2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.     

Heavy ion effects on cellular DNA: strand break induction and repair in cultured diploid lens epithelial cells

The relative biological effectiveness (RBE) for the induction of DNA strand breaks and the efficiency of repair of these breaks in cultured diploid bovine lens epithelial cells was measured, using accelerated heavy ions in the linear energy transfer (LET)-range up to 16,200 keV/micron. At LET values above 800 keV/micron, the number of DNA strand breaks induced per particle increases both with the atomic number of the projectile and with its kinetic energy. About 90 per cent or more of the strand breaks induced by ions with an LET of less than 10,000 keV/micron are repaired within 24 h. Repair kinetics show a dependence on the particle fluence (irradiation dose). At higher particle fluences a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At any LET value, repair is much slower after heavy-ion exposure than after X-irradiation. This is especially true for low energetic particles with a very high local density of energy deposition within the particle track. At the highest LET value (16,200 keV/micron), no significant repair is observed.     

Radioprotection by DMSO of mammalian cells exposed to X-rays and to heavy charged-particle beams

Populations of G1-phase Chinese hamster cells in stirred suspensions containing various concentrations of DMSO were irradiated with 250 kV X-rays or various heavy charged-particle beams. Chemical radioprotection of cell inactivation was observed for all LET values studied. When cell survival data were resolved into linear and quadratic components, the extent and concentration dependence of DMSO protection were found to be different for the two mechanisms. The chemical kinetics of radioprotection for single-events were similar for LET values up to those which gave the maximum RBE. DMSO protected to a lesser extent against energetic argon ions at an median LET of approximately 220 keV/micron. These data could indicate the contribution of indirect action by hydroxyl radicals and hydrogen atoms to cell inactivation by single-hit and double-hit mechanisms for various radiation qualities. The decrease in RBE observed at very high LET may result, in part, from reduced yields of water radicals at 10(-9)-10(-8) s resulting from radical recombination mechanisms within the charged particle tracks.     

Influence of radiation quality on the yield of DNA strand breaks in SV40 DNA irradiated in solution.

Using highly energetic particles to irradiate plasmid DNA in aerobic aqueous solution, we have compiled an extensive database on how yields of DNA single- and double-strand breaks (SSBs and DSBs) vary with radiation quality. This study was performed in a low-scavenging buffer system and covers a wide range of ion species (helium to uranium) and LETs (5 to 16,000 keV/microm). For LETs up to around 40 keV/microm for SSBs and 400 keV/microm for DSBs, the total energy deposition determines cross section. At higher LET, cross sections level off and individual plateaus for particles of different atomic numbers are observed. For each ion species this is more pronounced and occurs at lower LET for SSBs than for DSBs, leading to an increase in the DSB:SSB ratio from 1:70 for X rays to 1:6 at 500 keV/microm. At this LET, the influence of track structure becomes evident, with high local concentrations of ionization events favoring the formation of DSBs and also intratrack recombination reactions. For lower-energy ions, a saturation in production of measurable DSBs is apparent, due to correlated lesion induction within densely ionizing particle tracks. For very heavy low-energy ions, both SSB and DSB cross sections decrease with particle velocity at nearly constant LET, forming individual hooked curves when plotted as a function of LET.     

Neoplastic cell transformation by heavy ions

We have studied the induction of morphological transformation by heavy ions. Golden hamster embryo cells were irradiated with 95 MeV 14N ions (530 keV/microns), 22 MeV 4He ions (36 keV/microns), and 22 MeV 4He ions with a 100-microns Al absorber (77 keV/microns) which were generated by a cyclotron at the Institute of Physical and Chemical Research in Japan. Colonies were considered to contain neoplastically transformed cells when the cells were densely stacked and made a crisscross pattern. It was shown that the induction of transformation was much more effective with 14N and 4He ions than with gamma or X rays. The relative biological effectiveness (RBE) relative to 60Co gamma rays was 3.3 for 14N ions, 2.4 for 4He ions, and 3.3 for 4He ions with a 100-microns Al absorber. The relationship between RBE and linear energy transfer was qualitatively similar for both cell death and transformation.     

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to (137)Cs gamma-rays. The mutation frequency increased up to 105 keV/microm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/mum showed all or partial deletions of exons, while among gamma-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.     

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.     

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.