Inactivation Cross Sections Research Articles

Mutation induction in mammalian cells by accelerated heavy ions

The deleterious effects of accelerated heavy ions on living cells are of increasing importance for long duration human space flight activities. An important aspect of this field is attributed to the type and quality of biological damage induced by these densely ionizing particles. To address this aspect, cell inactivation and mutation induction at the HPRT locus (coding for hypoxanthine guanine phosphoribosyltransferase) was investigated in cultured V79 Chinese Hamster Cells irradiated with accelerated heavy ions (16-O, 40-Ca, 197-Au, and 238-U) and X-rays. Specific energies of the ions ranged from 1.9 to 69.7 MeV/u and corresponding LET values were between 62 and 15,580 keV μm −1. While inactivation cross-sections ( σ i) rise over the whole LET range, mutation induction cross-sections ( σ m) increase up to approximately 300 keV μm −1 (O-ions) but decline with heavier ions and more extreme LET values. A similar behavior is seen with mutation frequency dependent on particle fluence. After irradiation with accelerated uranium ions (8.8 MeV/u, 15,580 keV μm −1) a significant decrease of mutation frequency was found with higher particle fluences (3 × 10 6 particles cm −2). Nearly no mutants were recovered with 8 × 10 6 particles cm −2.

Number of Fe Ion Traversals Through a Cell Nucleus for Mammalian Cell Inactivation Near the Bragg Peak

HeLa and CHO-K1 cells were irradiated with Fe ions (1.14 MeV/nucleon) near the Bragg peak to determine how many ion traversals through a cell nucleus are necessary to induce cell inactivation. The ion traversals through a cell nucleus were visualized by immunostaining the phosphorylated histone H2AX (gamma-H2AX), as an indicator of DNA double strand breaks (DSBs), to confirm that DSBs are actually induced along every Fe ion traversal through the nucleus. The survival curves after irradiation with Fe ions decreased exponentially with the ion fluence without a shoulder. The inactivation cross sections calculated from the slope of the survival curves and the standard errors were 96.9 +/- 1.8 and 57.9 +/- 5.4 microm2 for HeLa and CHO-K1 cells, respectively, corresponding to 0.442 and 0.456 of the mean value of each cell nucleus area. Taking the distribution of the cell nucleus area into consideration with an equation proposed by Goodhead et al. (1980), which calculates the average number of lesions per single ion track through the average area of a sensitive organelle (mainly nucleus), these two ratios were converted to 0.705 and 0.659 for HeLa and CHO-K1 cells, respectively. These ratios were less than one, suggesting that the average numbers of lethal hits per cell produced by a single ion traversal were less than one. We thus considered two possible explanations for ion traversals of more than one, necessary for cell inactivation.

Exploration of `Over Kill Effect' of High-LET Ar- and Fe-ions by Evaluating the Fraction of Non-hit Cell and Interphase Death

The reason why RBE for cell killing fell to less than unity (1.0) with very high-LET heavy-ions ((40)Ar: 1,640 keV/microm; (56)Fe: 780, 1,200, 2,000 keV/microm) was explored by evaluating the fraction of non-hit cell (time-lapse observation) and cells undergoing interphase death (calculation based on our previous data). CHO cells were exposed to 4 Gy (30% survival dose) of Ar (1,640 keV/microm) or Fe-ions (2,000 keV/microm). About 20% of all cells were judged to be non-hit, and about 10% cells survived radiation damage. About 70% cells died after dividing at least once (reproductive death) or without dividing (interphase death). RBE for reproductive (RBE[R]) and interphase (RBE[I]) death showed a similar LET dependence with maximum around 200 keV/microm. In this LET region, at 30% survival level, about 10% non-survivors underwent interphase death. The corresponding value for very high-LET Fe-ions (2,000 keV/microm) was not particularly high (approximately 15%), whereas that for X-rays was less than 3%. However, reproductive death (67%) predominated over interphase death (33%) even in regard to rather severely damaged cells (1% survival level) after exposure to Fe-ions (2,000 keV/microm). These indicate that interphase death is a type of cell death characteristic for the cells exposed to high-LET radiation and is not caused by "cellular over kill effect". Both NHF37 (non-hit fraction at 37% survival) and inactivation cross-section for reproductive death (sigma[R]) began to increase when LET exceeded 100 keV/microm. The exclusion of non-hit fraction in the calculation of surviving fraction partially prevented the fall of RBE[R] when LET exceeded 200 keV/microm. On the other hand, the mean number of lethal damage per unit dose (NLD/Gy) showed the same LET-dependent pattern as RBE[R]. These suggest that the increase in non-hit fraction and sigma[R] with an increasing LET is caused by enhanced clustering of ionization and DNA damage which lowers the energy efficiency for producing damage and RBE.

Inactivation cross sections for mammalian cells exposed to charged particles: a phenomenological approach.

Published data on inactivation of V79 cells irradiated with monoenergetic proton and ion beams (He, C, O, Ne) have been analysed. Values for RBE alpha, RBE10% and the inactivation cross section sigma have been evaluated in the LET range between 5 and 400 keV.micron-1. RBE against LET curves and inactivation cross sections against LET and against Z*2/beta 2 curves have been studied in a comparative approach with respect to the different ion types. RBE-LET curves depend strongly on the type of ion for LET > 30 keV.micron-1. At LET < 30 keV.micron-1 and low doses protons show the greatest effectiveness; at LET > 30 keV.micron-1 and high doses He ions provide the most effective radiation. Apart from protons, separation among the various ion curves is less marked in the sigma against Z*2/beta 2 plot than in the sigma against LET plot. sigma against Z*2/beta 2 curves for ions with 2 < or = Z < or = 10 and 200 < Z*2/beta 2 < 1500 show a common trend independent of Z and are well represented by a linear relationship.

Generalized concept of the LET-RBE relationship of radiation-induced chromosome aberration and cell death.

The frequency of chromosome aberrations per traversal of a nucleus by a charged particle at the low dose limit increases proportionally to the square of the linear energy transfer (LET), peaks at about 100 keV/micron and then decreases with further increase of LET. This has long been interpreted as an excessive energy deposition over the necessary energy required to produce a biologically effective event. Here, we present an alternative interpretation. Cell traversed by a charged particle has certain probability to receive lethal damage leading to direct death. Such events may increase with an increase of LET and the number of charged particles traversing the cell. Assuming that the lethal damage is distributed according to a Poisson distribution, the probability that a cell has no such damage is expressed by e-cLx, where c is a constant, L is LET, and x is the number of charged particles traversing the cell. From these assumptions, the frequency of chromosome aberration in surviving cells can be described by Y = alpha SD + beta S2D2 with the empirical relation Y = alpha D + beta D2 in the low LET region, where S = e-cL, alpha is a value proportional to LET, beta is a constant, and D is the absorbed dose. This model readily explains the empirically established relationship between LET and relative biological effectiveness (RBE). The model can also be applied to clonogenic survival. If cells can survive and they have neither unstable chromosome aberrations nor other lethal damage, the LET-RBE relationship for clonogenic survival forms a humped curve. The relationship between LET and inactivation cross-section becomes proportional to the square of LET in the low LET region when the frequency of a directly lethal events is sufficiently smaller than unity, and the inactivation cross-section saturates to the cell nucleus cross-sectional area with an increase in LET in the high LET region.

Monte-Carlo simulation of ion track structure in water: ionization clusters and biological effectiveness

The biological action of swift ions decreases for increasing stopping power (LET), and the loss of efficiency is particularly high for heavy ions when the LET reaches its maximum value. In one of the models developed in the literature to account for this behavior, the biological action of light ions (and X rays) is attributed to clusters of energy deposition by low energy electrons. We have performed Monte-Carlo simulations to test this model for heavy and light ions (here uranium and helium ions). For helium ions, the experimental inactivation cross sections variations with LET are well reproduced when clusters with E⩾340 eV in spheres of radius R≈ 4.54 nm are considered, with an efficiency ≈4%, in very good agreement with the literature. However, these parameters cannot be applied to uranium ions (here 1.4 MeV/u U32+): the calculated number of clusters is ≈5 times larger than expected, a result which sets a limit to the cluster model.

Repair of DNA double-strand breaks and cell killing by charged particles

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing (Kelland et al., 1988; Radford, 1986). We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/μm and were even smaller than unity for the LET region greater than 300 keV/μm. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/μm, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main cause of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.

Dependence of Induction of Interphase Death of Chinese Hamster Ovary Cells Exposed to Accelerated Heavy Ions on Linear Energy Transfer

Induction of interphase death was examined in Chinese hamster ovary cells exposed to accelerated heavy ions (carbon, neon, argon and iron) of various linear energy transfers (LETs) (10-2000 keV/microm). The fraction of cells that underwent interphase death was determined by observing individual cells with time-lapse photography (direct method) as well as by counting cells undergoing interphase death made visible by the addition of caffeine (indirect method). After exposure to X rays, interphase death increased linearly with dose above a threshold of about 10 Gy, whereas it increased at a higher rate without a threshold after exposure to high-LET heavy ions. The relative biological effectiveness (RBE) compared to X rays, as determined at the 50% level of induction, increased with LET, reached a maximum at an LET of approximately 230 keV/microm and then decreased with further increase in LET. The range of LET values corresponding to the maximum RBE appears to be narrower for interphase death than for reproductive death (120-230 keV/microm), as assayed using loss of colony-forming ability as a criterion. The inactivation cross section for interphase cell death reached a plateau of 5-10 microm2. This means that the probability for the induction of interphase death by traversal of a single heavy-ion track through the nucleus (size: about 130 microm2) is about 0.04-0.08.

Risk Scaling Factors from Inactivation to Chromosome Aberrations, Mutations and Oncogenic Transformations in Mammalian Cells

Analyses of bio-effect mechanisms of damage to mammalian cells in terms of the quality parameter 'mean free path for primary ionisation', for heavy charged particles, strongly suggests that there is a common mechanism for the biological endpoints of chromosome aberrations, mutations and oncogenic transformation. The lethal lesions are identified as unrepaired double-strand breaks in the intracellular DNA. As data for the various endpoints studied can be represented in a unified scheme, for any radiation type, it follows that radiation risk factors can be determined on the basis of simple ratios to the inactivation cross sections. There are intrinsic physical reasons why neutrons can never reach the saturation level of heavier particles for equal fluences. The probabilities of risk with respect to inactivation, for chromosome dicentrics, mutation of the HPRT gene and of oncogenic transformation are respectively 0.24, 5.8 x 10 -5 , and 4.1 x 10 -3 .

Inactivation of individual mammalian cells by single alpha-particles

Purpose : To measure clonogenic death of Chinese hamster V79 cells following exposure to a defined number of 4.3 MeV alpha particles (track-averaged LET 105 keV/ mu m). Materials and methods : Cells were irradiated at the radiobiological facility installed at the TTT-3 Tandem accelerator in Naples by using a 'Biostack' approach, which allows the positions of incident tracks relative to cells to be carefully determined. Subcellular structure was identified by fluorescence microscopy, while tracks were visualized by LR-115 solid state nuclear track detectors. Results : Particle hits in the cytoplasm did not significantly affect cell survival, yet survival probability decreased exponentially as a function of the number of nuclear traversals. Measured probability of surviving to exactly one 4.3 MeV alpha -particle traversal in the cell nucleus was 0.67 0.10. Inactivation cross-section was substantially higher than expected from conventional survival curves. However, folding of the data with Poisson statistics showed that survival level expected if a mean of one alpha -particle goes through a nucleus is higher than the measured value after exactly one particle traversal. Conclusions : V79 cells have about 67% probability to survive a single alpha -particle traversal in the cell nucleus. Single-particle survival curves are consistent with conventional dose-survival relationships, once Poisson distribution of traversals is taken into account.

Effects of heavy ions on the germination and survival of Arabidopsis thaliana

Inhibition of germination and reduction in survival of Arabidopsis thaliana were investigated to study the effects of heavy ions on a multicellular system. Dry seeds of Col and Ler ecotypes were exposed to He, C, Ar and Ne ions with linear energy transfer (LET) in the range of 17-549 keV/ mu m and to electrons (LET 0.2 keV/ mu m). The relative biological effectiveness (RBE) for the survival of both ecotypes showed the same pattern of variation with a maximum RBE of 11-12 at 252 keV/ mu m. For germination, RBE increased with increasing LET in Ler but not in Col, showing different sensitivities between the plant ecotypes. Inactivation cross sections of survival increased linearly in the range of 0.2-17 keV/ mu m and proceeded more steeply in the range of 113-252 keV/ mu m. At higher LET, cross sections appeared to reach a plateau at a little less than the size of the cell nucleus. When the value for survival was plotted against LET, it decreased steeply in the range about 113-252 keV/ mu m, indicating that heavy ions may have similar effects on both the shoulder and slope of the survival curve.

Effects of track structure and cell inactivation on the calculation of heavy ion mutation rates in mammalian cells.

It has long been suggested that inactivation severely effects the probability of mutation by heavy ions in mammalian cells. Heavy ions have observed cross sections of inactivation that approach and sometimes exceed the geometric size of the cell nucleus in mammalian cells. In the track structure model of Katz the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated using the dose-response of the system to gamma-rays and the radial dose of the ions and may be equal to unity at small impact parameters for some ions. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections from heavy ions in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT mutations in Chinese hamster cells and good agreement is found. The resulting calculations qualitatively show that mutation cross sections for heavy ions display minima at velocities where inactivation cross sections display maxima. Also, calculations show the high probability of mutation by relativistic heavy ions due to the radial extension of ions track from delta-rays in agreement with the microlesion concept. The effects of inactivation on mutations rates make it very unlikely that a single parameter such as LET or Z*2/beta(2) can be used to specify radiation quality for heavy ion bombardment.

Heavy ion inactivation in Escherichia coli cells: experiment and theory.

Cross sections for inactivation of stationary cells of E. coli strains (B(s-1) and B/r) by heavy ions (Z = 8-92) with energies <15 MeV/u were determined from survival curves and compared with track structure calculations. Whereas for low energies, inactivation cross sections for both strains are similar, and thus do not reflect their difference in X-ray sensitivity, the ratios of cross sections of the two strains approach the ratio of X-rays sensitivities for heavy ions at high energies. The essential features of the experimental data are reproduced well by the track structure calculations, which do not use fitted values for the model parameters but independently determined values for the X-ray sensitivities and the size of the target structure. Quantitatively, the calculations underestimate the cross sections for heavier ions as well as their ratios at higher energies. Some possible reasons for these discrepancies are discussed.

Track structure and the calculation of biological effects of heavy charged particles

A new approach for the calculation of biological effects of heavy charged particles is discussed. In contrast to other models, the biological effect is determined locally as a function of the local dose deposited by the charged particle tracks. Based on measurable quantities like the X-ray survival curve, the radial dose profile within a track and a geometrical description of the cell nucleus, the model is able to predict several high LET specific properties of charge particle beams, like • • the relationship of inactivation cross sections vs. LET and vs. specific energy; • • the RBE as a function of energy and atomic number of the particles, including the Z-dependent shift of RBE-maxima from 30 keV/μ m for protons to 300 keV/μ m for carbon ions; • • the transition from exponential to shouldered survival curves, depending on the size of the biological object and the corresponding particle fluences.

K-ionization and biological effect

Experimental inactivation cross sections are compared to vacancy production in inner shell (K-shell) of so-called strategic C, N, O atoms of the DNA: in a trial calculation, atoms on the DNA backbone or just contiguous to it are considered as strategic. Various processes are considered: ionization and electron capture by the primary particle, ionization by secondary electrons. The last phenomenon is shown to be important for highly charged and medium velocity ions. A strong similarity is observed between variations versus LET of inactivation and K-vacancy cross sections, especially with regards to LET values for which they maximize: for K-ionization these maxima occur when the ion impact velocity roughly matches the K electron velocity. It is shown that the “K-hypothesis” allows a quantitative fit of experimental inactivation cross sections if a 4% mean lethal efficiency is attributed to ion-induced K-vacancies in strategic C, N, O atoms.

Track structure and radiation transport model for space radiobiology studies

Radiobiology experiments performed in space are deemed necessary for validation of risk-assessment methods. The understanding of space radiobiology experiments must combine knowledge of the space radiation environment, radiation transport, and models of biological response. The heavy ion transport code HZETRN has recently been combined with improved models of the galactic cosmic rays (GCR) and extensive comparisons made to measurements on the space shuttle with a tissue equivalent, proportional counter. HZETRN was also coupled with track-structure models of biological damage from heavy ions. Track-structure calculations using improved models of the radial dose distribution around the path of heavy ions provide a good description of ground-based experiments for inactivation cross sections. Therefore, we use these models to predict inactivation of Bacillus Subtilis spores in space. Calculations consider single-particle effects, as well as the background from low linear energy transfer ions of the GCR and trapped radiations on the radial distributions of effects measured in plastic detectors.

Induction of HPRT- mutants in Chinese hamster V79 cells after heavy ion exposure.

The induction of resistance to 6-thioguanine by heavy ion exposure was investigated with various accelerated ions (oxygen-uranium) up to linear energy transfer (LET) values of about 15,000 keV/microns. Survival curves are exponential with fluence; mutation induction shows a linear dependence. Cross-sections (sigma i: inactivation, sigma m: mutation) were derived from the respective slopes. Generally, sigma i rises over the whole LET range, but separates into different declining curves for single ions with LET values above 200 keV/microns. Similar behaviour is seen for sigma m. The new SIS facility at GSI, Darmstadt, makes it possible to study the effects of ions with the same LET but very different energies and track structures. Experiments using nickel and oxygen ions (up to 400 MeV/u) showed that inactivation cross-sections do not depend very much on track structure, i.e. similar values are found with different ions at the same LET. This is not the case for mutation induction, where very energetic ions display considerably smaller induction cross-sections, compared with low-energy ions of identical LET. Preliminary analyses using the polymerase chain reaction (PCR) demonstrate that even heavy ions cause "small alterations" (small deletions or base changes). The proportion of the total deletions seems to increase with LET.

Micronucleus induction and reproductive death in a human cell line exposed to low-energy argon beam

Little is known about the action of charged particles of very high linear energy transfer (LET) on human cells and, in particular, the relationship between DNA damage and reproductive death. The aim of this study was to measure the biological efficiency of a low-energy argon beam (E = 7.1 MeV/nucleon, LET = 1590 keV/micron) produced at GSI, Darmstadt, on a human melanoma cell line (CAL4) established in our Institute. Two different methods were used: the micronucleus (MN) test and the colony-forming assay. The MN test, using the cytochalasin-block method, is a measure of genotoxic damage. MN are scored in binucleate cells (BNC) and are formed from acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis. The colony-forming assay quantifies reproductive death. Parallel experiments were run with cobalt gamma-rays for comparison. After Co irradiation, the MN-free BNC dose-response curve coincided with that of the loss of colony-forming ability, suggesting the potential of the former as a predictive test of cell killing. After Ar irradiation, there was a dissociation between the two effects, especially at high doses: cell death was greater than the frequency of BNC with MN. The inactivation cross-section was 74 microns2; it was 39 microns2 for MN yield. Therefore, the relative biological effectiveness (RBE) was higher for cell killing than for MN yield (0.8 and 0.5, respectively at a Co dose of 3 Gy). The total MN count in BNC followed the same pattern of response as the fraction of BNC with MN.(ABSTRACT TRUNCATED AT 250 WORDS)

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/μm to 975 KeV/gmm with particle energy (on the cells) between 94 – 603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase ( hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/μm. The inactivation cross-section (αi) and the action-section for mutant induction (αm) ranged from 2.2 to 92.0 μm 2 and 0.09 to 5.56 × 10 −3 μm 2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/μm. The mutagenicity (αm/αi) ranged from 2.05 to 7.99 × 10 −5 with the maximum value at 150 keV/μm. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

Mutation induction by heavy ions

Mutation induction by heavy ions is compared in yeast and mammalian cells. Since mutants can only be recovered in survivors the influence of inactivation cross sections has to be taken into account. It is shown that both the size of the sensitive cellular site as well as track structure play an important role. Another parameter which influences the probability of mutation induction is repair: Contrary to naive assumptions primary radiation damage does not directly lead to mutations but requires modification to reconstitute the genetic machinery so that mutants can survive. The molecular structure of mutations was analyzed after exposure to deuterons by amplification with the aid of polymerase chain reaction. The results-although preliminary-demonstrate that even with densely ionizing particles a large fraction does not carry big deletions which suggests that point mutations may also be induced by heavy ions.