Sylvia Ritter

RBE for carbon track-segment irradiation in cell lines of differing repair capacity

PURPOSE The LET position of the RBE maximum and its dependence on the cellular repair capacity was determined for carbon ions. Hamster cell lines of differing repair capacity were irradiated with monoenergetic carbon ions. RBE values for cell inactivation at different survival levels were determined and the differences in the RBE-LET patterns were compared with the individual sensitivity to photon irradiation of the different cell lines. MATERIAL AND METHODS Three hamster cell lines, the wild-type cell lines V79 and CHO-K1 and the radiosensitive CHO mutant xrs5, were irradiated with carbon ions of different energies (2.4-266.4 MeV/u) and LET values (13.7-482.7 keV/microm) and inactivation data were measured in comparison to 250 kV x-rays. RESULTS For the repair-proficient cell lines a RBE maximum was found at LET values between 150 and 200 keV/microm. For the repair-deficient cell line the RBE failed to show a maximum and decreased continuously for LET values above 100 keV/microm. CONCLUSIONS The carbon RBE LET relationship for inactivation is shifted to higher LET values compared with protons and alpha-particles. RBE correlated with the repair capacity of the cells.

Comparison of chromosomal damage induced by X-rays and Ar ions with an LET of 1840 keV/ mum in G1 V79 cells

Synchronous V79 Chinese hamster cells were exposed in G1 to either X-rays or 4. 6 MeV/u Ar-ions (LET = 1840 keV/mum) and the induction of chromosomal damage was measured at five sampling times ranging from 14 to 30 h after treatment. To distinguish between cells in the first and second post-irradiation cycle the fluorescence-plusGiemsa technique was applied. The experiment showed that the time-course of the appearance of damaged cells was markedly influenced by radiation-induced cell cycle delays and depended on both radiation quality and dose. The yield of aberrant metaphases and the number of aberrations per metaphase was found to increase with sampling time, but this increase was more pronounced for Ar ions. These differences in the yield-time profiles of X-ray and Ar ion induced chromosomal damage are particularly important for an accurate determination of the RBE for particles. Our data clearly indicate that meaningful RBEs can only be obtained if chromosomal damage is analysed at several post-irradi...

Heavy-ion induced chromosomal aberrations: a review.

Heavy-ion radiobiology is attracting increasing interest for its implications in radiation oncology and space radiation protection. The analysis of chromosome aberrations induced by heavy-ions started already in the 1960s, but the new FISH-painting methodologies are revealing unique features of the action of the heavy charged particles. Heavy-ions induce a high fraction of complex-type exchanges, and possibly unique chromosome rearrangements. The relative biological effectiveness for the induction of cytogenetic damage is strongly dependent on the time between irradiation and chromosome harvest, due to cell-cycle delays and loss of heavily damaged cells. In this review we will concentrate on recent data obtained with multicolor FISH methods in mammalian chromosomes exposed to heavy-ions, and the open questions that remain to be addressed.

Analysis of chromosome damage based on the time course of aberrations.

PURPOSE To describe a method for the analysis of radiation induced chromosome aberrations which will in particular be suitable for the comparison of different radiation qualities. The method is applied to previously published data. METHODS The basic idea of the approach is to use the time-course of aberrations up to the time when all cells have passed through first mitosis. Instead of comparing data obtained at a single sampling time, the number of aberrant cells or aberrations is integrated over several sampling times. The integral represents a measure of the total fraction of cells showing aberrations and the total amount of aberrations induced in the irradiated cell population. In addition, a correction factor is applied which takes into account the dilution of heavily damaged cells at late sampling times by cell division of undamaged or less severely damaged cells. RESULTS AND CONCLUSIONS Previously published data on the induction of chromosome aberrations by 4.6 MeV/u Ar ions and X-rays are re-analysed using the new approach. It is demonstrated that this method allows a better comparison of experiments using extremely different radiation qualities and consequently different cell cycle perturbations.

Cell cycle arrest and aberration yield in normal human fibroblasts. I. Effects of X‐rays and 195 MeV u−1 C ions

Purpose: To examine the relationship between cell proliferation and the expression of chromosomal damage in normal human skin fibroblasts after X‐ray and particle irradiation. Materials and methods: Confluent G0/G1 AG1522B cells were exposed to X‐rays or 195 MeV u−1 C ions with a linear energy transfer of 16.6 keV µm−1 in the dose range 1–4 Gy. Directly after irradiation, cells were reseeded at a low density in medium containing 5‐bromo‐2′‐deoxyuridine. At multiple time points post‐irradiation, the cumulative BrdU‐labelling index, mitotic index and aberration frequency were measured. Based on these data, the total amount of damage induced within the entire cell population was estimated by means of mathematical analysis. Results: Both types of radiation exposure exert a pronounced effect on the cell cycle progression of fibroblasts. They result in delayed entry of cells into S‐phase and into the first mitosis, and cause a dramatic reduction in mitotic activity. Measurement of chromosomal damage in first‐cycle cells at multiple time points post‐irradiation shows that the frequencies of aberrant cells and aberrations increase with time up to twofold for the lower doses. However, for the higher doses, this effect is less pronounced or even disappears. When the data for the whole cell population are analysed, it becomes evident that only a few damaged fibroblasts can progress to the first mitosis, a response attributable at least in part to a long‐term arrest of injured cells in the initial G0/G1‐phase. As observed in other investigations, the effectiveness of 195 MeV u−1 C ions was similar or slightly higher than X‐rays for all endpoints studied leading to a relative biological effectiveness in the range 1.0–1.4. Conclusions: Cell cycle arrests affect the aberration yield observable in normal human fibroblasts at mitosis. The data obtained for the cell population as a whole reveal that injured cells are rapidly removed from the mitotically active population through a chronic cell cycle arrest, which is consistent with other studies that indicate that this response is a specific strategy of fibroblasts to minimize the fixation and propagation of genetic alterations.

High-LET-induced chromosome aberrations in V79 cells analysed in first and second post-irradiation metaphases

Purpose : As an extension of previous studies, the time-course of high-LET-induced chromosomal damage was investigated in first- and second-cycle V79 Chinese hamster cells. Materials and methods : Cells were exposed in G 1 to 10.4MeV/u Ar ions (LET=1226keV/mum) and chromosomal damage was measured at 2h sampling intervals between 10h and 34h after irradiation. To distinguish between cells in different post-irradiation cycles, the fluorescence-plus-Giemsa technique was applied. Results : For first- and second-generation cells, the number of aberrant metaphases and aberrations per metaphase were found to increase markedly with sampling time, demonstrating that cell cycle progression was delayed according to the number of lesions carried by the cell. To account for the time-dependent expression of chromosomal damage a mathematical approach was used based on the integrated flux of aberrant cells entering mitosis. Moreover, the analysis of Ar ion-induced chromosome lesions confirmed that high-LET radiation results in specific changes in the spectrum of aberration types. In particular, an increased rate of chromatid-type aberrations as well as a high frequency of chromosomal breaks was found, although the cells were exposed in G 1. Conclusions : Due to the fact that cells collected at one sampling time are not representative of the entire population, the complete time-course of chromosomal damage has to be taken into account for the determination of a meaningful RBE value. Otherwise, the analysis of chromosomal damage can result in a pronounced over- or underestimation of the RBE depending on the subpopulation of cells entering mitosis at that particular sampling time.PURPOSE As an extension of previous studies, the time-course of high-LET-induced chromosomal damage was investigated in first- and second-cycle V79 Chinese hamster cells. MATERIALS AND METHODS Cells were exposed in G1 to 10.4 MeV/u Ar ions (LET = 1226 keV/microm) and chromosomal damage was measured at 2h sampling intervals between 10 h and 34 h after irradiation. To distinguish between cells in different post-irradiation cycles, the fluorescence-plus-Giemsa technique was applied. RESULTS For first- and second-generation cells, the number of aberrant metaphases and aberrations per metaphase were found to increase markedly with sampling time, demonstrating that cell cycle progression was delayed according to the number of lesions carried by the cell. To account for the time-dependent expression of chromosomal damage a mathematical approach was used based on the integrated flux of aberrant cells entering mitosis. Moreover, the analysis of Ar ion-induced chromosome lesions confirmed that high-LET radiation results in specific changes in the spectrum of aberration types. In particular, an increased rate of chromatid-type aberrations as well as a high frequency of chromosomal breaks was found, although the cells were exposed in G1. CONCLUSIONS Due to the fact that cells collected at one sampling time are not representative of the entire population, the complete time-course of chromosomal damage has to be taken into account for the determination of a meaningful RBE value. Otherwise, the analysis of chromosomal damage can result in a pronounced over- or underestimation of the RBE depending on the subpopulation of cells entering mitosis at that particular sampling time.

Analysis of Ar-ion and X-ray-induced chromatin breakage and repair in V79 plateau-phase cells by the premature chromosome condensation technique

Purpose : The premature chromosome condensation technique has been used to compare chromatin breakage and repair in noncycling V79 cells following high and low LET radiation. Materials and methods : Plateau-phase V79 cells were exposed to graded doses of low energy Ar ions (LET 1233 keV / μ m) and X-rays. Cells were fused to mitotic V79 cells immediately after exposure to examine initial chromatin breakage or after various time intervals of post-irradiation incubation to investigate the kinetics of chromatin break rejoining as well as the fraction of unrejoined fragments. Results and conclusions : For both radiation qualities an average initial number of about 2.4 excess PCC fragments per cell per Gy was found increasing linearly with dose. The distributions of PCC chromosomes plus excess fragments among cells followed Poisson statistics after X-ray irradiation, while an overdispersion of the frequencies was observed after Ar-irradiation indicating that a single particle traversal through a cell nucleus can produce multiple chromatin lesions. Moreover, for both radiation types the rejoining of excess fragments has been examined. Both data sets could be fitted well to first-order kinetics with a single component. Despite similar rates of rejoining cellular repair was noticeably less effective for Ar ions than for X-rays. While after 10h of post-irradiation incubation 60% of Ar ion induced excess fragments remained unrejoined, only 14% of X-ray-induced lesions were not rejoined. Furthermore, comparison of the residual number of excess PCC fragments with recently published data on the yield of chromosome aberrations in first post-irradiation metaphases shows that for both radiation types more aberrations are detected in interphase than in metaphase cells. Yet, for comparable doses this difference is more pronounced for Ar ions indicating that scoring of high LET induced aberrations in metaphase cells might result in a significant underestimation of the produced damage.PURPOSE The premature chromosome condensation technique has been used to compare chromatin breakage and repair in noncycling V79 cells following high and low LET radiation. MATERIALS AND METHODS Plateau-phase V79 cells were exposed to graded doses of low energy Ar ions (LET 1233 keV/microm) and X-rays. Cells were fused to mitotic V79 cells immediately after exposure to examine initial chromatin breakage or after various time intervals of post-irradiation incubation to investigate the kinetics of chromatin break rejoining as well as the fraction of unrejoined fragments. RESULTS AND CONCLUSIONS For both radiation qualities an average initial number of about 2.4 excess PCC fragments per cell per Gy was found increasing linearly with dose. The distributions of PCC chromosomes plus excess fragments among cells followed Poisson statistics after X-ray irradiation, while an overdispersion of the frequencies was observed after Ar-irradiation indicating that a single particle traversal through a cell nucleus can produce multiple chromatin lesions. Moreover, for both radiation types the rejoining of excess fragments has been examined. Both data sets could be fitted well to first-order kinetics with a single component. Despite similar rates of rejoining cellular repair was noticeably less effective for Ar ions than for X-rays. While after 10 h of post-irradiation incubation 60% of Ar ion induced excess fragments remained unrejoined, only 14% of X-ray-induced lesions were not rejoined. Furthermore, comparison of the residual number of excess PCC fragments with recently published data on the yield of chromosome aberrations in first post-irradiation metaphases shows that for both radiation types more aberrations are detected in interphase than in metaphase cells. Yet, for comparable doses this difference is more pronounced for Ar ions indicating that scoring of high LET induced aberrations in metaphase cells might result in a significant underestimation of the produced damage.

Response of human hematopoietic stem and progenitor cells to energetic carbon ions

Purpose: To characterise the radiation response of human hematopoietic stem and progenitor cells (HSPC) with respect to X and carbon ion irradiation. Materials and methods: HSPC from peripheral blood of healthy donors treated with granulocyte-colony stimulating factor (G-CSF) were enriched for the transmembrane glycoprotein CD34 (cluster of differentiation) and irradiated with X rays or carbon ions (29 keV/μm monoenergetic beam and 60-85 keV/μm spread-out Bragg peak), mimicking radiotherapy conditions. Apoptotic cell death, cell cycle progression and the frequency of chromosomal aberrations were determined. Results: After radiation exposure no inhibition in the progression of the cell cycle was detected. However, an enhanced frequency of apoptotic cells and an increase in aberrant cells were observed, both effects being more pronounced for carbon ions than X rays, resulting in a relative biological effectiveness (RBE) of 1.4–1.7. The fraction of complex-type aberrations was higher following carbon ion exposure. Conclusions: RBE values of carbon ions are low, as expected for radiosensitive cells. The observed frequencies of apoptotic cells and chromosome aberrations in HSPC are similar to those reported for human peripheral blood lymphocytes suggesting that at least with respect to apoptosis and chromosomal aberrations mature lymphocytes reflect the respective radiation responses of their proliferating progenitors.

Induction of chromosomal damage in CHO-K1 cells and their repair-deficient mutant xrs5 by X-ray and particle irradiation

The cytogenetic effects of X-rays and Au ions were investigated in repair-proficient CHO-K1 cells and their radiosensitive mutant strain xrs5, which shows a defect in the rejoining of DNA double-strand breaks. Both cell lines were synchronized by mitotic shake off, irradiated in G1-phase with either 250 kV X-rays or 780 MeV/u Au ions (LET: 1150 keV/micrometer) and chromosome aberrations were analyzed in first post-irradiation metaphases. Isoeffective doses of X-rays for the induction of aberrant cells and aberrations per cell were about 14 times lower for xrs5 than for CHO-K1 cells. After high LET radiation the difference in the cytogenetic response of both cell lines was drastically diminished. Furthermore, the analysis of the aberration types induced by sparsely and densely ionizing radiation showed for both cell lines specific changes in the spectrum of aberration types as LET increases. The experimental results are discussed with respect to the different types of lesions induced by sparsely and densely ionizing radiation.

Complex exchanges are responsible for the increased effectiveness of C-ions compared to X-rays at the first post-irradiation mitosis

The purpose of the present study was to investigate as to what extent differences in the linear energy transfer (LET) are reflected at the chromosomal level. For this study human lymphocytes were exposed to 9.5 MeV/u C-ions (1 or 2 Gy, LET=175 keV/microm) or X-rays (1-6 Gy), harvested at 48, 72 or 96 h post-irradiation and aberrations were scored in first cycle metaphases using 24 color fluorescence in situ hybridization (mFISH). Additionally, in selected samples aberrations were measured in prematurely condensed G2-phase cells. Analysis of the time-course of aberrations in first cycle metaphases showed a stable yield of simple and complex exchanges after X-ray irradiation. In contrast, after C-ion exposure the yields profoundly increased with harvesting time complicating the estimation of the frequency of aberrations produced by high LET particles within the entire cell population. This is especially true for the yield of complex exchanges. Complex aberrations dominate the aberration spectrum produced by C-ions. Their fraction was about 50% for the two measured doses. In contrast, isodoses of X-rays induced smaller proportions of complex aberrations (i.e. 5% and 15%, respectively). For both radiation qualities the fraction of complexes did not change with harvesting time. As expected from the different dose deposition of high and low LET radiation, complex exchanges produced by high LET C-ions involved more breaks and more chromosomes than those induced by isodoses of X-rays. Noteworthy, C-ions but not X-rays induced a small number of complex chromatid-isochromatid exchanges that are not expected for cells exposed in the G0-phase. The results obtained so far for cells arrested in G2-phase confirm these patterns. Altogether our data show that the increased effectiveness of C-ions for the induction of aberrations in first cycle cells is determined by complex exchanges, whereas for simple exchanges the relative biological effectiveness is about one.