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.     

Production of superoxide radicals with pulse radiolysis of water with high linear energy transfer

The radiolysis of water with heavy ions of high linear energy transfer (LET) (-dE/dx) is characterized, in deaerated medium, by the production of superoxide anions, the radiolytic yields of which increase with the LET. Radiobiological interest in such radical species comes from the oxidative stress which may be generated by their dismutation in O2 and H2O2 in anoxic medium (radiotherapy with heavy ions). A brief review of the measurements of superoxide free radicals in aqueous solution by indirect or direct methods is presented. Moreover, some experimental results obtained by pulse radiolysis with Ar18+ ions (TEL = 290 keV x microm(-1)), are described. The interpretation of the kinetics takes into account the superoxide absorbance and that of hydrogen peroxide, which is present at the millisecond time scale.     

Different contributions of the indirect effects of γ-rays on the cytotoxicity in M10 and XRCC4 transfected M10 cells

The protective effects of dimethyl sulfoxide (DMSO) against cell killing by ¹³⁷Cs γ-rays were investigated in XRCC4-deficient cell line M10, XRCC4-complemented M10 and the parental mouse leukemia cell line L5178Y. Cell survival was determined by the colony-forming ability. M10 cells were more sensitive to γ-ray-induced cell death than L5178Y and complemented M10 cells. Cell survival was increased in both M10 and L5178Y in the presence of DMSO. However, estimation of the DMSO-protectable fraction revealed a smaller protectable fraction for M10 cells than for L5178Y cells, indicating that indirect effects contributed in a smaller extent to the cytotoxicity in M10 than that in L5178Y. This effect is due to XRCC4 deficiency, since transfection of XRCC4 cDNA into M10 cells restored the radioprotective effects of DMSO to the level seen in L5178Y. In M10 cells, the killing effects of high LET radiation (Auger electrons from ¹²⁵I-antipyrine, carbon ions with an LET of 166 keV μm⁻¹) were similar to those of low LET radiation (¹³⁷Cs γ-rays, characteristic X-rays from ¹²⁵I-bovine serum albumin). We discuss that lethal lesions produced by indirect actions in L5178Y and XRCC4-complemented M10 cells may differ, at least in part, from DNA double-strand breaks repairable by non-homologous end joining.     

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.     

ESR spin trapping of hydroxyl radicals in aqueous solution irradiated with high-LET carbon-ion beams

The aim of this study was to quantify the hydroxyl radicals (*OH) produced when aqueous solutions are decomposed by high-linear energy transfer (LET) 290 MeV/nucleon carbon-ion beams using an electron spin resonance (ESR) spectrometer. Aerated cell culture medium containing 200 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was irradiated with doses of 0 to 20 Gy with an LET of 20 to 90 keV/ micro m. We were able to obtain ESR spectra 10 min after irradiation, and the formation of *OH and hydrogen atoms was confirmed by radiolysis of deuterium oxide and ethanol containing DMPO. Our results showed that the yield of *OH by carbon-ion radiolysis increased in proportion to the absorbed dose over the range of 0 to 20 Gy. Furthermore, we discovered that the yield of *OH decreased linearity as LET increased logarithmically from 20 to 90 keV/ micro m. The generation of *OH by carbon-ion radiolysis at LETs of 20, 40, 60, 80 and 90 keV/ micro m was 64, 58, 52, 49 and 50%, respectively, of that for low-LET X radiolysis. These unique findings provide a further understanding of the indirect effect of high-LET radiation.     

Track structure in DNA irradiated with heavy ions

The spatial properties of trapped radicals produced in heavy-ion-irradiated solid DNA at 77 K have been probed using pulsed electron paramagnetic double resonance (PELDOR or DEER) techniques. Salmon testes DNA hydrated to 12 water molecules per nucleotide was irradiated with 40Ar ions of energy 100 MeV/nucleon and LET ranging from 300 to 400 keV/microm. Irradiated samples were maintained at cryogenic temperature at all times. PELDOR measurements were made using a refocused echo detection sequence that allows dipolar interaction between trapped radicals to be observed. The EPR spectrum is attributed to electron loss/gain DNA base radicals and neutral carbon-centered radicals that likely arise from sugar damage. We find a radical concentration of 13.5 x 10(18) cm(-3) in the tracks and a track radius of 6.79 nm. The cross section of these tracks is 144 nm2, yielding a lineal radical density of 2.6 radicals/nm. Based on the yields determined previously for particles having calculated LET values of 300-400 keV/microm and our measured lineal density, we obtain an LET of 270 keV/microm, which is in good agreement with the calculated range of values. These measurements of radical density and spatial extent provide the first direct experimental determination of track characteristics in irradiated DNA.     

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.     

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.     

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.     

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.     

Characterization of hydroxyl radical-induced damage after sparsely and densely ionizing irradiation

The extent of hydroxyl radical mediated cell inactivation was measured for a variety of particle beams ranging from 8.5 Me V/u neon ions to 570 Me V/u argon ions. In general, the fraction of the total radiosensitivity caused by OH decreases from close to 60 per cent at low ionization density or low linear energy transfer (low LET) to close to 25 per cent at high LET for aerobically irradiated mammalian cells. The extent of OH induced cell lethality can be explained in terms of LET infinity only for low energy or low atomic number particles where fragmentations and complicated track structures do not contaminate the characteristic particle LET. For example, at a calculated LET infinity of 100 ke V/micron, the OH mediated fraction of the total radiation damage is about 25 per cent for low energy carbon but close to 40 per cent for high energy carbon ions. For low energy charged nuclei of approximately the same energy, as the 5.4-13.4 MeV/u He, Li, C and Ne ions in this report, there is a predictable diminution of the OH mediated effect with increasing LET infinity; however, the biological effect cannot be predicted accurately from calculated LET infinity values for high energy particle irradiation, nor indeed from a variety of low energy charged particles of quite different energies (incident velocities). This illustrates the unsuitability of using LET as a unifying parameter, except under specific circumstances. As more is learned about the energy deposition for energized charged particles in terms of track structure (core and penumbra), it may be possible to characterize the radiobiological data with a better physical parameter than LET infinity.     

High LET heavy ion radiation induces lower numbers of initial chromosome breaks with minimal repair than low LET radiation in normal human cells

We investigated the earliest possible chromosome break and repair process in normal human fibroblasts irradiated with low and high LET (linear energy transfer) heavy ion radiation using the modified premature chromosome condensation (PCC) technique utilizing wortmannin (WM) during the fusion incubation period [M. Okada, S. Saito, R. Okayasu, Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation, Mutat. Res., 562 (2004) 11-17]. The initial numbers of breaks were approximately 10/cell/Gy in X-irradiated samples, followed by carbon (LET: 70 keV/microm), neon, and the number was around 5/cell/Gy in silicon (LET: 70 and 200 keV/microm) and iron (LET: 200 keV/microm) samples. If WM was not used, the initial numbers of breaks with silicon and iron were higher than those of X-rays. To quantify these data, we used initial repair ratio (IRR) defined as the number of G1 PCC breaks with WM divided by the number of breaks without WM. X-irradiation gave the maximum IRR ( approximately 2.0), while iron as well as silicon irradiation showed the minimum IRR ( approximately 1.0), suggesting almost no rejoining at the initial stage. Although there is a comparatively good correlation between the IRR value and the cell survival, the survival fraction with the repair data at 2 or 6h correlates better statistically. Our data indicate that high LET heavy ion irradiation induces a lower number of initial chromosome breaks with minimal repair when compared with low LET irradiation. These results at the chromosome level substantiate and extend the notion that high LET radiation produces complex-type DNA double strand breaks (DSBs).     

Yield of OH radicals in water under high-density energy deposition by heavy-ion irradiation

The purpose of the present study was to evaluate the dependence of the OH radical yield on the atomic number and the energy of the heavy ions to understand chemical reactions of aqueous solutions. The total yields of oxidized products from phenol in water increased superlinearly as the incident energy increased from 5 MeV/nucleon to 18 MeV/nucleon for carbon and neon ions. The radiolytic yields of OH radicals produced by the ions were determined by analyzing the relationships of the oxidation yields of phenol to the incident energies up to 18 MeV/nucleon for ions in the range of LET from 110 eV/nm to 550 eV/nm and from 320 eV/nm to 1100 eV/nm for carbon and neon ions, respectively. The yields of the OH radicals increased with the specific energy for the same kind of ion and decreased with the atomic number for different ions used at the same specific energy.     

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.     

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.     

Accumulation of the cell cycle regulators TP53 and CDKN1A (p21) in human fibroblasts after exposure to low- and high-LET radiation

The accumulation of the cell cycle regulators TP53 and CDKN1A (p21/CIP1/WAF1) was investigated after exposure to X rays and carbon ions (170 keV microm(-1)) and xenon, bismuth and uranium ions (8900-15,000 keV microm(-1)) in normal human fibroblasts. The influence of the overall dose and the LET of these radiation types was studied systematically and the kinetics of the cell response was followed up to 24 h after exposure. The accumulation of TP53 protein was dependent on the dose and the LET, and TP53 levels declined to lower levels for all radiation types within 24 h after exposure. CDKN1A levels increased and peaked at 3 to 6 h after exposure. The persisting level of this protein at 24 h was strongly dependent on the dose and the LET for X rays and carbon ions. The exposure to very high-LET ions (8900-15,000 keV microm(-1)) did not lead to a further increase in CDKN1A, suggesting a saturation effect for the induction of this protein. The cellular effects of elevated CDKN1A after particle irradiation are discussed.     

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.     

Simulation of DNA damage after proton irradiation

The biophysical radiation track simulation model PARTRAC was improved by implementing new interaction cross sections for protons in water. Computer-simulated tracks of energy deposition events from protons and their secondary electrons were superimposed on a higher-order DNA target model describing the spatial coordinates of the whole genome inside a human cell. Induction of DNA double-strand breaks was simulated for proton irradiation with LET values between 1.6 and 70 keV/microm and various reference radiation qualities. The yield of DSBs after proton irradiation was found to rise continuously with increasing LET up to about 20 DSBs per Gbp and Gy, corresponding to an RBE up to 2.2. About half of this increase resulted from a higher yield of DSB clusters associated with small fragments below 10 kbp. Exclusion of experimentally unresolved multiple DSBs reduced the maximum DSB yield by 30% and shifted it to an LET of about 40 keV/microm. Simulated fragment size distributions deviated significantly from random breakage distributions over the whole size range after irradiation with protons with an LET above 10 keV/microm. Determination of DSB yields using equations derived for random breakage resulted in an underestimation by up to 20%. The inclusion of background fragments had only a minor influence on the distribution of the DNA fragments induced by radiation. Despite limited numerical agreement, the simulations reproduced the trends in proton-induced DNA DSBs and fragment induction found in recent experiments.     

High-LET ion radiolysis of water: visualization of the formation and evolution of ion tracks and relevance to the radiation-induced bystander effect

Ionizing radiation-induced bystander effects, commonly observed in cell populations exposed to high-linear energy transfer (LET) radiations, are initiated by damage to a cellular molecule which then gives rise to a toxic signal exported to neighboring cells not directly hit by radiation. A major goal in studies of this phenomenon is the identification of this initial radiation-induced lesion. Liquid water being the main constituent of biological matter, reactive species produced by water radiolysis in the cellular environment are likely to be major contributors to the induction of this lesion. In this context, the radiation track structure is of crucial importance in specifying the precise location and identity of all the radiolytic species and their subsequent signaling or damaging effects. We report here Monte Carlo track structure simulations of the radiolysis of liquid water by four different impacting ions 1H+, 4He2+, 12C6+ and 20Ne10+, with the same LET ( approximately 70 keV/ microm). The initial radial distribution profiles of the various water decomposition products (eaq(-), *OH, H*, H2 and H2O2) for the different ions considered are presented and discussed briefly in the context of track structure theory. As an example, the formation and temporal evolution of simulated 24 MeV 4He2+ ion tracks (LET approximately 26 keV/microm) are reported for each radiolytic species from 1 ps to 10 micros. The calculations reveal that the ion track structure is completely lost by approximately 1 micros.     

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.