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.     

Heavy ion induced membrane damage: Hemolysis of erythrocytes and changes in erythrocyte membrane fluidity

Human erythrocytes were irradiated with heavy ions of energies between 4 and 18 MeV/u having linear energy transfer (LET) values between 92 and 14000 keV/µm. Hemolysis has been studied as a macroscopic parameter for membrane damage and changes of the fluidity as a more microscopic parameter. The membrane fluidity changed in a characteristic dose-dependent manner as detected by electron spin resonance employing 12-doxylstearic acid methyl ester spin label (SL 12). Lysis cross sections and RBE values were determined from dose effect curves. The results demonstrate a high hemolytic efficiency of heavy ions compared to X rays. With increasing LET values the measured relative biological efficiency (RBE) values increase continuously. In the complete LET range the cross sections formed one common curve as function of LET and no saturation effects are observed. This is in direct contrast to other biological endpoints such as cell inactivation or DNA damage.     

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.     

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.     

The RBE-LET relationship for rodent intestinal crypt cell survival, testes weight loss, and multicellular spheroid cell survival after heavy-ion irradiation.

This report presents data for survival of mouse intestinal crypt cells, mouse testes weight loss as an indicator of survival of spermatogonial stem cells, and survival of rat 9L spheroid cells after irradiation in the plateau region of unmodified particle beams ranging in mass from 4He to 139La. The LET values range from 1.6 to 953 keV/microns. These studies examine the RBE-LET relationship for two normal tissues and for an in vitro tissue model, multicellular spheroids. When the RBE values are plotted as a function of LET, the resulting curve is characterized by a region in which RBE increases with LET, a peak RBE at an LET value of 100 keV/microns, and a region of decreasing RBE at LETs greater than 100 keV/microns. Inactivation cross sections (sigma) for these three biological systems have been calculated from the exponential terminal slope of the dose-response relationship for each ion. For this determination the dose is expressed as particle fluence and the parameter sigma indicates effect per particle. A plot of sigma versus LET shows that the curve for testes weight loss is shifted to the left, indicating greater radiosensitivity at lower LETs than for crypt cell and spheroid cell survival. The curves for cross section versus LET for all three model systems show similar characteristics with a relatively linear portion below 100 keV/microns and a region of lessened slope in the LET range above 100 keV/microns for testes and spheroids. The data indicate that the effectiveness per particle increases as a function of LET and, to a limited extent, Z, at LET values greater than 100 keV/microns. Previously published results for spread Bragg peaks are also summarized, and they suggest that RBE is dependent on both the LET and the Z of the particle.     

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.     

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.     

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.     

The difference in LET and ion species dependence for induction of initially measured and non-rejoined chromatin breaks in normal human fibroblasts

We studied the LET and ion species dependence of the induction of chromatin breaks measured immediately after irradiation as initially measured breaks and after 24 h postirradiation incubation (37 degrees C) as non-rejoined breaks in normal human fibroblasts with different heavy ions, such as carbon, neon, silicon and iron, generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS). Chromatin breaks were measured as an excess number of fragments of prematurely condensed chromosomes using premature chromosome condensation (PCC). The results showed that the number of excess fragments per cell per Gy for initially measured chromatin breaks was dependent on LET in the range from 13.3 to 113.1 keV/mum but was not dependent on ion species. On the other hand, the number of non-rejoined chromatin breaks detected after 24 h postirradiation incubation was clearly dependent on both LET and ion species. No significant difference was observed in the cross section for initially measured breaks, but a statistically significant difference was observed in the cross section for non-rejoined breaks among carbon, neon, silicon and iron ions. This suggests that the LET-dependent structure in the biological effects is reflected in biological consequences of repair processes.     

Radiation effects in Caenorhabditis elegans, mutagenesis by high and low LET ionizing radiation

The nematode C. elegans was used to measure the effectiveness of high-energy ionized particles in the induction of 3 types of genetic lesions. Recessive lethal mutations in a 40-map unit autosomal region, sterility, and X-chromosome nondisjunction or damage were investigated. Induction rates were measured as a function of linear energy transfer, LET infinity, for 9 ions of atomic number 1-57 accelerated at the BEVALAC accelerator. Linear kinetics were observed for all 3 types of lesions within the dose/fluence ranges tested and varied strongly as a function of particle LET infinity. Relative Biological Effectiveness (RBE) values of up to 4.2 were measured and action cross sections were calculated and compared to mutagenic responses in other systems.     

Effects of heavy ion to the primary [correction of rimary] culture of mouse brain cells.

To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.     

A Monte Carlo code for the simulation of heavy-ion tracks in water

TILDA, a new Monte Carlo track structure code for ions in gaseous water that is valid for both high-LET (approximately 10(4) keV/microm) and low-LET ions, is presented. It is specially designed for a comparison of the patterns of energy deposited by a large range of ions. Low-LET ions are described in a perturbative frame, whereas heavy ions with a very high stopping power are treated using the Lindhard local density approximation and the Russek and Meli statistical method. Ionization cross sections singly differential with energy compare well with the experiment. As an illustration of the non-perturbative interaction of high-LET ions, a comparison between the ion tracks of light and heavy ions with the same specific energy is presented (1.4 MeV/nucleon helium and uranium ions). The mean energy for ejected electrons was found to be approximately four times larger for uranium than for helium, leading to a much larger track radius in the first case. For electrons, except for the excitation cross sections that are deduced from experimental fits, cross sections are derived analytically. For any orientation of the target molecule, the code calculates multiple differential cross sections as a function of the ejection and scattering angles and of the energy transfer. The corresponding singly differential and total ionization cross sections are in good agreement with experimental data. The angular distribution of secondary electrons is shown to depend strongly on the orientation of the water molecule.     

重离子射线照射对家蚕的生物影响

为解明重离子射线的生物影响,调查了氖、碳及氦(20Ne8+,LET=300keV/μm;12C5+,LET=116keV/μm和4He2+,LET=16.2keV/μm)等重离子射线照射家蚕(Bombyxmori)后的存活率及形态变化。重离子射线照射不同发育时期的幼虫后所引起的生物影响不同,幼虫的发育时期越早,照射后引起的生物影响越大;对同一时期的幼虫,随着剂量的增加,照射的生物影响加大;以化蛹率和羽化率为指标的放射线感受性在供试的3种射线间具有相似的变化倾向,只是射线的射程越长,照射的生物影响越大;对熟蚕卵巢存在部位的局部照射也显示相似的结果。同一射线的不同LET轨迹位置对家蚕的卵巢及真皮细胞的生物影响不同,用Mylar薄膜覆盖调节碳离子射线的射程,卵巢及真皮细胞越是接近射线高LET的Bragg峰,照射个体的鳞毛及卵的形成被强烈抑制。因此,重离子射线对家蚕的生物影响与细胞及植物种子等小个体不同,对于全体照射,重离子射线的射程长短所造成的生物影响比射线的LET大小所引起的生物影响要大;而对于局部照射,目的器官越是接近射线的高LET轨迹,照射的生物影响越大。     

Heavy ion effects on yeast cells: induction of canavanine-resistant mutants

The induction of forward mutations (resistance to canavanine) by heavy ion bombardment was investigated in wild type haploid yeast Saccharomyces cerevisiae. Accelerated ions of argon, titanium, nickel, krypton, xenon, lead and uranium with specific energies between 1.7 and 9.25 MeV/u were obtained from the UNILAC machine at the Gesellschaft für Schwerionenforschung, Darmstadt/Germany. LET-values ranged from 1200 to about 15 000 keV/microns. There was no unequivocal dependence of mutation induction cross section on either LET or Z*2/beta 2, but also a prominent influence of ion specific energy. This is explained by the action of long-ranging delta-electrons.     

Response of colony-forming units-spleen to heavy charged particles

Survival of colony-forming units-spleen (CFU-S) was measured after single doses of photons or heavy charged particles from the BEVALAC. The purposes were to define the radiosensitivity to heavy ions used medically and to evaluate relationships between relative biological effectiveness (RBE) and dose-averaged linear energy transfer (LET infinity). In in vitro irradiation experiments. CFU-S suspensions were exposed to 220 kVp X rays or to 20Ne (372 MeV/micron) or 40Ar (447 MeV/micron) particles in the plateau portion of the Bragg curve. In in vivo irradiation experiments, donor mice from which CFU-S were harvested were exposed to 12C (400 MeV/micron). 20Ne (400 or 670 MeV/micron), or 40Ar (570 MeV/micron) particles in Bragg peaks spread to 4 or 10 cm by spiral ridge filters. Based on RBE at 10 survival, the maximum RBE of 2.1 was observed for 40Ar particles characterized by an LET infinity of approximately 100 keV/micron. Lower RBEs were determined at lower or higher estimated values of LET infinity and ranged from 1.1 for low energy 40Ar particles to 1.5-1.6 for low energy 12C and 20Ne. The responses of CFU-S are compared with responses of other model systems to heavy charged particles and with the reported sensitivity of CFU-S to neutrons of various energies. The maximum RBE reported here, 2.1 for high energy 40Ar particles, is somewhat lower than values reported for fission-spectrum neutrons, and is appreciably lower than values for monoenergetic 0.43-1.8 MeV neutrons. Low energy 12C and 20Ne particles have RBEs in the range of values reported for 14.7 MeV neutrons.     

Effects of low dose particle radiation to mouse neonatal neurons in culture.

To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions generated by HIMAC at NIRS and BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis focusing on the dependencies of (1) the animal strains with different radiation sensitivities, and (2) LET with different nuclei. Of the three mouse strains, SCID, B6 and C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to both X-ray and carbon ion ( 290 MeV/n) as evaluated by 10% apoptotic criterion. However, the sensitivity differences among the strains were much smaller in case of carbon ion comparing to that of X-ray. Regarding the LET dependency, the sensitivity was compared with using C3H and B6 cells between the carbon (13 keV/micrometers) and neon (70 keV/micrometers) ions. Carbon (290 MeV/n) did not give a detectable LET dependency from the criterion whereas the neon (400 MeV/n) showed 1.4 fold difference for both C3H and B6 cells. Although a LET dependency was examined by using the most sensitive SCID cells, no significant difference was detected.     

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.     

Cell killing, nuclear damage and apoptosis in Chinese hamster V79 cells after irradiation with heavy-ion beams of (16)O, (12)C and (7)Li

Chinese hamster V79 cells were exposed to high LET (linear energy transfer) (16)O-beam (625keV/mum) radiation in the dose range of 0-9.83Gy. Cell survival, micronuclei (MN), chromosomal aberrations (CA) and induction of apoptosis were studied as a follow up of our earlier study on high LET radiations ((7)Li-beam of 60keV/mum and (12)C-beam of 295keV/mum) as well as (60)Co gamma-rays. Dose dependent decline in surviving fraction was noticed along with the increase of MN frequency, CA frequency as well as percentage of apoptosis as detected by nuclear fragmentation assay. The relative intensity of DNA ladder, which is a useful marker for the determination of the extent of apoptosis induction, was also increased in a dose dependent manner. Additionally, expression of tyrosine kinase lck-1 gene, which plays an important role in response to ionizing radiation induced apoptosis, was increased with the increase of radiation doses and also with incubation time. The present study showed that all the high LET radiations were generally more effective in cell killing and inflicting other cytogenetic damages than that of low LET gamma-rays. The dose response curves revealed that (7)Li-beam was most effective in cell killing as well as inducing other nuclear damages followed by (12)C, (16)O and (60)Co gamma-rays, in that order. The result of this study may have some application in biological dosimetry for assessment of genotoxicity in heavy ion exposed subjects and in determining suitable doses for radiotherapy in cancer patients where various species of heavy ions are now being generally used.     

Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.     

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.