DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study

Purpose : To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). Materials and methods : V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). Results : The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. Conclusions : The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.     

DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study

PURPOSE: To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). MATERIALS AND METHODS: V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). RESULTS: The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2 h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. CONCLUSIONS: The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.     

Correlation between cell killing and residual chromatin breaks measured by PCC in six human cell lines irradiated with different radiation types

PURPOSE: To examine the relationship between cell killing and residual chromatin breaks after irradiation with qualitatively different types of radiation in six human cell lines. MATERIALS AND METHODS: Six human tumour cell lines and normal human cells were irradiated with 200 kV X-rays and two carbon-ion beams accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) differing in LET. At the sample position the carbon-ion beams had LET infinity values of 13.1 and 77.5 +/- 0.4 keV/microm. Cell inactivation was documented by a colony assay. Residual chromatin breaks were measured by counting the number of residual chromatin fragments at 24h, detected by the premature chromosome condensation (PCC) technique. RESULTS: The cell lines covered a broad range of radiosensitivity. D10 values ranged from 3.53 to 8.12 Gy for X-rays, 2.56 to 7.41 Gy for the lower LET carbon ions and 1.17 to 3.85 Gy for the higher LET carbon ions. The results for residual chromatin breaks indicate that the more radiosensitive cell lines showed a greater induction of residual chromatin breaks either by X-rays or carbon-ions, and that an X-ray resistant cell line also showed resistance to carbon-ions. Cellular radiosensitivity correlated with the frequency of residual chromatin breaks. CONCLUSION: The detection of residual chromatin breaks by the PCC technique could be used to predict cellular radiosensitivity among qualitatively different types of radiation.     

Correlation between cell killing and residual chromatin breaks measured by PCC in six human cell lines irradiated with different radiation types

Purpose : To examine the relationship between cell killing and residual chromatin breaks after irradiation with qualitatively different types of radiation in six human cell lines. Materials and methods : Six human tumour cell lines and normal human cells were irradiated with 200kV X-rays and two carbonion beams accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) differing in LET. At the sample position the carbon-ion beams had LET2 values of 13.1 and 77.5 ±0.4keV/ μ m. Cell inactivation was documented by a colony assay. Residual chromatin breaks were measured by counting the number of residual chromatin fragments at 24 h, detected by the premature chromosome condensation (PCC) technique. Results : The cell lines covered a broad range of radiosensitivity. D 10 values ranged from 3.53 to 8.12 Gy for X-rays, 2.56 to 7.41 Gy for the lower LET carbon ions and 1.17 to 3.85Gy for the higher LET carbon ions. The results for residual chromatin breaks indicate that the more radiosensitive cell lines showed a greater induction of residual chromatin breaks either by X-rays or carbon-ions, and that an X-ray resistant cell line also showed resistance to carbon-ions. Cellular radiosensitivity correlated with the frequency of residual chromatin breaks. Conclusion : The detection of residual chromatin breaks by the PCC technique could be used to predict cellular radiosensitivity among qualitatively different types of radiation.     

Heavy ion-induced chromosome breakage studied by premature chromosome condensation (PCC) in Syrian hamster embryo cells.

We have studied induction and repair of chromosome damage induced by high linear energy transfer (LET) heavy ions in G1/G0 interphase Syrian golden hamster embryo (SHE) cells as revealed by the premature chromosome condensation (PCC) technique. The number of chromosome breaks in condensed chromosomes induced by high LET heavy ions was higher than those induced by 137Cs gamma-rays. Compared with 137Cs gamma rays, the relative biological effectiveness (RBE) for PCC breaks was 1.5 for 35 keV/microns 4He ions, 1.9 for 77keV/microns 4He ions, and 2.5 for 530keV/microns 14N ions. Although 95% of the PCC breaks induced by gamma-rays rejoined during 8 h post-irradiation incubation, only 35-45% of fragments induced by high LET radiations rejoined in the same time. These results suggest that there is a difference, spatial or qualitative, in the initial chromosome damage produced by high LET radiations and low LET radiations.     

Impact of radiation quality on the spectrum of induced chromosome exchange aberrations

PURPOSE: To study the impact of radiation quality on the spectrum of chromosome exchange aberrations in human lymphocytes using chromosome arm-specific and telomeric probes. The analysis is focused on: (1) incomplete exchanges, (2) interstitial fragments, (3) interarm intrachanges and (4) the complexity of the aberration patterns. The present data after neutron exposure are compared with previously obtained data after X-irradiation. MATERIALS AND METHODS: Isolated human lymphocytes from three donors were irradiated with 1 MeV fast neutrons (0.25, 0.5, 1.0, 1.5, 2.0 Gy). Analysis was performed on first post-irradiation metaphases with arm-specific probes for chromosome 1 in combination with a pan-centromeric probe, or with telomeric and centromeric PNA probes. RESULTS: In comparison with X-rays, exposure to neutrons leads to: (1) similar frequencies of incomplete exchanges or terminal deletions, (2) a significantly higher induction of both inter- and intraarm intrachanges, (3) a higher proportion of complex aberrations, and (4) aberrations with a higher degree of complexity, i.e. derived from more chromosome breaks which interact more frequently in a non-reciprocal fashion. Essentially no dose dependence was found for the yield ratios between the various types of chromosomal aberrations. CONCLUSIONS: Despite the reduced rejoining deficiency of DNA double-strand breaks induced by high-LET radiation, exposure to neutrons does not lead to enhanced levels of unrejoined chromosome breaks that can be observed as incomplete exchanges in cells that have reached mitosis. Proximity effects are more pronounced after densely ionizing radiation than after sparsely ionizing radiation. Clustered damage produced by neutron tracks results in a high proportion of complex aberrations and in non-reciprocal interactions of chromosome breaks. Most of the exchanges occur within one neutron track and little interaction seems to take place between the breaks formed in different tracks.     

Impact of radiation quality on the spectrum of induced chromosome exchange aberrations

Purpose: To study the impact of radiation quality on the spectrum of chromosome exchange aberrations in human lymphocytes using chromosome arm-specific and telomeric probes. The analysis is focused on: (1) incomplete exchanges, (2) interstitial fragments, (3) interarm intrachanges and (4) the complexity of the aberration patterns. The present data after neutron exposure are compared with previously obtained data after X-irradiation. Materials and methods: Isolated human lymphocytes from three donors were irradiated with 1 MeV fast neutrons (0.25, 0.5, 1.0, 1.5, 2.0Gy). Analysis was performed on first post-irradiation metaphases with arm-specific probes for chromosome 1 in combination with a pan-centromeric probe, or with telomeric and centromeric PNA probes. Results: In comparison with X-rays, exposure to neutrons leads to: (1) similar frequencies of incomplete exchanges or terminal deletions, (2) a significantly higher induction of both inter- and intraarm intrachanges, (3) a higher proportion of complex aberrations, and (4) aberrations with a higher degree of complexity, i.e. derived from more chromosome breaks which interact more frequently in a non-reciprocal fashion. Essentially no dose dependence was found for the yield ratios between the various types of chromosomal aberrations. Conclusions: Despite the reduced rejoining eyciency of DNA double-strand breaks induced by high-LET radiation, exposure to neutrons does not lead to enhanced levels of unrejoined chromosome breaks that can be observed as incomplete exchanges in cells that have reached mitosis. Proximity effects are more pronounced after densely ionizing radiation than after sparsely ionizing radiation. Clustered damage produced by neutron tracks results in a high proportion of complex aberrations and in non-reciprocal interactions of chromosome breaks. Most of the exchanges occur within one neutron track and little interaction seems to take place between the breaks formed in different tracks.     

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 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.     

Rejoining of double-stranded DNA-fragments studied in different size-intervals.

PURPOSE: To measure rejoining of radiation-induced doublestranded DNA-fragments of different sizes and to evaluate the effects of size-resolution in the analysis of rejoining. MATERIAL AND METHODS: Normal human fibroblasts (GM5758) were irradiated with photons or accelerated nitrogen ions (linear energy transfer, LET = 125 keV microm(-1)) and incubated for repair for 0-22 h. Double-stranded DNA-fragments from the irradiated cells were separated by pulsed-field gel electrophoresis in the range approximately 5 kbp to 5.7 Mbp. RESULTS: For cells irradiated with high LET nitrogen ions, there was an increase in the fast half-time from approximately 5 min for fragments < 400 kbp to 10 min when all fragments < 5.7 Mbp were measured. Further, the fraction of fragments rejoined by the slow-rejoining phase increased significantly for increased threshold sizes. The fraction of unrejoined fragments after 22 h and the half-time for the slow-rejoining phase remained constant for all threshold sizes. For cells irradiated with lower doses of low LET radiation the rejoining was shifted towards a slower kinetics when fragments up to 10 Mbp were excluded in the analysis. CONCLUSION: DNA exclusion-size and resolution may affect the estimates of DNA double-strand break rejoining. Using a low-resolution technique that does not detect small fragments will result in an underestimation, or even disappearance of the fast-rejoining phase. This is due to substantial rejoining of fragments taking place before the fragments are of sufficient size to be monitored.     

Effect of LET on the yield and quality of chromosomal damage in metaphase cells: a time-course study

PURPOSE: To investigate further the effect of linear energy transfer (LET) on the yield and quality of aberrations at different post-irradiation sampling times. MATERIALS AND METHODS: V79 G(1)-cells were exposed to either 10.6 MeV u-1 Ne ions (360 keV microm-1) or 11.1 MeV u-1 Kr ions (3980 keV microm-1) and chromosomal damage was measured in metaphase cells at several 2-h sampling intervals up to 30 h post-irradiation. To differentiate between cells in the first and second post-irradiation cycle, the fluorescence-plus-Giemsa technique was applied. RESULTS: In both experiments, an increase in the yield of aberrant cells as well as the number of aberrations per cell was observed in first- and second-cycle metaphases. Yet, the increase in the number of aberrations per cells was more pronounced for Kr ions and at comparable fluences Kr ions produced more aberrations than Ne ions. Because no sampling time was representative for the whole cell population, the total amount of Ne and Kr ion-induced chromosomal damage was determined by means of a mathematical approach and used for the comparison of data. Furthermore, in accordance with previous studies, LET-dependent changes in the spectrum of aberration types were detected, i.e. with increasing LET a higher fraction of chromatid-type aberrations was observed, although cells had been exposed in G1. In addition, more chromosomal breaks and less exchange-type aberrations were found. CONCLUSIONS: The observation that cell-cycle progression is related to the amount of aberrations harboured by a cell demonstrates that the routinely applied method to measure aberration frequencies in metaphase cells at only one post-irradiation sampling time will unavoidably result in an under- or overestimation of the cytogenetic effects of particles. Consequently, for a meaningful quantification of chromosomal damage, multiple fixation regimes should be used so that the complete time-course of aberrations can be taken into account. Moreover, to avoid bias, all aberration types should be recorded and included in the analysis since the aberration spectrum changes with LET.     

Correlation between mitotic delay and aberration burden, and their role for the analysis of chromosomal damage

The aim was to investigate further the relationship between radiation-induced mitotic delay and the expression of chromosome damage in V79 cells. Recently published data on the time-course of chromosome aberrations in V79 first-cycle metaphases after exposure to 10.4 MeV u(-1) Ar ions (LET = 1226 keV microm(-1)) were supplemented and reanalysed. A statistical analysis of the distribution of aberrations among cells was performed. Furthermore, cells were grouped into subpopulations carrying 0, 1 -2, 3-4, 5- 6 and 7 or more aberrations. Then, based on the mitotic index, the flux of each subgroup through the first mitosis was determined and the average entrance time to mitosis was estimated. For comparison, the flux of aberrant V79 cells generated by X-irradiation was analysed. Analysis of the Ar ion data revealed that the flux of each subpopulation through the first mitosis is strongly affected by its aberration burden, i.e. a positive correlation between the mitotic delay and the number of aberrations carried by a cell was observed. The distribution of aberrations among cells could be well described by Neyman-type A statistics; the corresponding fit parameters also reflect the damage-dependent mitotic delay. Interestingly, comparison of the flux of Ar ion and X-ray-irradiated V79 cells through mitosis revealed (1) that a direct correlation exists between the number of aberrations carried by a cell and its average entrance time to mitosis, and (2) that this effect is independent of the linear energy transfer. The role of these observations for radiation cytogenetics is discussed.     

Induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells by carbon ions

PURPOSE: To study the induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells exposed to carbon ions in vitro. MATERIALS AND METHODS: X-ray-resistant colon carcinoma cells (WiDr) were used. Confluent G0/G1 cells were irradiated in vitro with graded doses of 100/200/400 MeV u(-1) carbon ions and carbon ions from the middle of a 1 cm extended Bragg peak, and 200 kV X-rays for comparison. Cells were harvested in their first post-irradiation division and aberrations were analysed either by the Giemsa/Hoechst 33258-staining technique or by the fluorescent in-situ hybridization technique involving whole chromosome hybridization and 4',6-diaminido-2-phenylidole (DAPI)-staining. Whole chromosome probes were used for chromosomes 2, 4 and 5, and the chromosome painting patterns were classified according published protocols. Reproductive cell survival was determined by a standard clonogenic assay. RESULTS: With respect to the induction of reproductive cell death and chromosome aberrations, carbon ions of different energies were more effective than 200 kV X-rays. As expected, irradiation in the extended Bragg peak was the most efficient mode. For cell killing, relative biological effectiveness increased with linear energy transfer up to 2.9. The frequencies of total dicentrics and excess acentric fragments as determined in Giemsa-stained cells were higher in cells irradiated with carbon ions than in cells with X-rays. For 100 MeV u(-1) ions, the dose dependence of apparently simple dicentrics as determined for chromosomes 2, 4 and 5 by single-colour fluorescent in-situ hybridization was linear up to 4 Gy, and linear-quadratic for excess acentric fragments and apparently simple translocations. After irradiation with D=4 Gy carbon ions with energy of 100 MeV u(-1) and from the extended Bragg peak, 12 and 54% of cells displayed complex exchanges, respectively. In contrast, after irradiation with D=4 Gy X-rays, only 1% of cells displayed complex aberrations. Hence, the number of cells with complex exchange aberrations increased strongly after irradiation with carbon ions. CONCLUSION: An increased biological efficiency of carbon ions could be confirmed in radioresistant tumour cells with respect to the induction of reproductive cell death and of unstable as well as stable chromosome aberrations. Relative biological effectiveness reached 2.9 for cell killing by carbon ions from the extended Bragg peak. The yields of apparently simple dicentrics as well as of total dicentrics, i.e. simple dicentrics plus dicentrics belonging to complex exchanges, evaluated in Giemsa-stained metaphases as observed in first post-irradiation mitoses were rather low. In contrast, apparently simple translocations displayed yields systematically higher than simple dicentrics in WiDr cells irradiated with either X-rays or 100 MeV u(-1) or Bragg peak carbon ions. Frequencies o f cells containing complex aberrations increased dramatically after carbon ion irradiation, reaching a maximum for ions from the extended Bragg peak.     

High-LET radiation-induced aberrations in prematurely condensed G2 chromosomes of human fibroblasts

PURPOSE: To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIAL AND METHODS: Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290MeV/u), silicon (490MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. RESULTS: The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. CONCLUSIONS: Compared with gamma-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.     

High-LET radiation-induced aberrations in prematurely condensed G2 chromosomes of human fibroblasts

Purpose : To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. Material and methods : Exponentially growing human fibroblast cells AG1522 were irradiated with γ-rays, energetic carbon (290 MeV/u), silicon (490 MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. Results : The kinetics of chromatid break rejoining following low-or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10 min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. Conclusions : Compared with γ-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.     

Rejoining of DNA fragments produced by radiations of different linear energy transfer

Purpose : To analyse the rejoining of DNA double-strand breaks (dsb) produced by high-linear energy transfer (LET) ions, with the specific focus on the influence, on the rejoining estimates, of the way dsb are distributed along chromosomes. Material and methods : Low passages of normal human fibroblasts (GM5758) were irradiated with 60 Co photons, 40 keV/mum helium ions or nitrogen ions with LETs of 80, 125, 175 and 225 keV/mum. The double-stranded DNA fragment distributions, ranging from 5 kbp to 5.7 Mbp, were assayed by pulsed-field gel electrophoresis after repair incubation for 0–22 h. Results : The rejoining was biphasic and the half-times of the two phases were 15 min and 2–3 h, respectively, and were independent of LET. Although the majority of breaks were rejoined by the fast phase, both the fraction of dsb rejoined by the slow phase and the fraction of unrejoined dsb at 20–22 h increased with increasing LET. Conclusions : DNA fragment analysis detected LET-dependent differences in the amount of rejoining while the half-times were independent of LET. The majority of dsb were rapidly rejoined even after high-LET irradiation. If fragment-size distribution is not taken into account, both the fraction of breaks rejoined by slow kinetics, and the fraction of unrejoined breaks, can be overestimated when the LET is increased.     

Rejoining of DNA fragments produced by radiations of different linear energy transfer

PURPOSE: To analyse the rejoining of DNA double-strand breaks (dsb) produced by high-linear energy transfer (LET) ions, with the specific focus on the influence, on the rejoining estimates, of the way dsb are distributed along chromosomes. MATERIAL AND METHODS: Low passages of normal human fibroblasts (GM5758) were irradiated with 60Co photons, 40 keV/microm helium ions or nitrogen ions with LETs of 80, 125, 175 and 225 keV/microm. The double-stranded DNA fragment distributions, ranging from 5 kbp to 5.7 Mbp, were assayed by pulsed-field gel electrophoresis after repair incubation for 0-22 h. RESULTS: The rejoining was biphasic and the half-times of the two phases were 15 min and 2-3h, respectively, and were independent of LET. Although the majority of breaks were rejoined by the fast phase, both the fraction of dsb rejoined by the slow phase and the fraction of unrejoined dsb at 20-22h increased with increasing LET. CONCLUSIONS: DNA fragment analysis detected LET-dependent differences in the amount of rejoining while the half-times were independent of LET. The majority of dsb were rapidly rejoined even after high-LET irradiation. If fragment-size distribution is not taken into account, both the fraction of breaks rejoined by slow kinetics, and the fraction of unrejoined breaks, can be overestimated when the LET is increased.     

Reduction of survival and induction of chromosome aberrations in tobacco irradiated by carbon ions with different linear energy transfers

Purpose : To determine the relationship between linear energy transfer (LET) and the relative biological effectiveness (RBE) for survival reduction and chromosome aberration induction in plants. Materials and methods : Tobacco seeds were exposed to carbon ions having LET ranging from 92 to 260 keV μ m -1. Survival rate was determined at 7 weeks after sowing. Chromosome aberrations were observed when the root length reached about 0.5mm (immediately after radicle emergence), 3 and 10 mm. Results : The RBE for both endpoints increased with increasing LET and showed the highest value at 230 keV μ m -1. The highest RBE was 65.0 for survival reduction and 52.5 for chromosome aberration induction. The types and yield ratio of chromosome aberrations such as fragments and bridges were not affected by radiation type at 0.5mm root length. As the roots elongated from 0.5 to 10 mm, the frequency of aberrant cells gradually decreased. The number of cells with fragments decreased faster than the number of cells with bridges. The decrement of chromosome aberrations appeared to be slower in roots irradiated by carbon ions than in roots irradiated by γ -rays. Conclusions : The results show a close relationship between survival reduction and chromosome aberration induction in plants. The types and yield ratio of initial chromosome aberrations did not differ among γ -rays and carbon ions with different LET.     

Reduction of survival and induction of chromosome aberrations in tobacco irradiated by carbon ions with different linear energy transfers

PURPOSE: To determine the relationship between linear energy transfer (LET) and the relative biological effectiveness (RBE) for survival reduction and chromosome aberration induction in plants. MATERIALS AND METHODS: Tobacco seeds were exposed to carbon ions having LET ranging from 92 to 260 keV microm(-1). Survival ratc was determined at 7 weeks after sowing. Chromosome aberrations were observed when the root length reached about 0.5 mm (immediately after radicle emergence), 3 and 10 mm. RESULTS: The RBE for both endpoints increased with increasing LET and showed the highest value at 230 keV um(-1). The highest RBE was 65.0 for survival reduction and 52.5 for chromosome aberration induction. The types and yield ratio of chromosome aberrations such as fragments and bridges were not affected by radiation type at 0.5mm root length. As the roots elongated from 0.5 to 10 mm, the frequency of aberrant cells gradually decreased. The number of cells with fragments decreased faster than the number of cells with bridges. The decrement of chromosome aberrations appeared to be slower in roots irradiated by carbon ions than in roots irradiated by gamma-rays. CONCLUSIONS: The results show a close relationship between survival reduction and chromosome aberration induction in plants. The types and yield ratio of initial chromosome aberrations did not differ among gamma-rays and carbon ions with different LET.     

Transient DNA Double-Strand Breakage in 4-Hydroperoxyifosfamide-Treated Mammalian Cells in Vitro Does Not Interact with the Rejoining of Radiation-Induced Double-Strand Breaks

BACKGROUND: 4-Hydroxyifosfamide is the primary metabolite in vivo of the bifunctional alkylating cytostatic ifosfamide. DNA interstrand cross-linking induced by bifunctional alkylators may be repaired through an intermediate with unligated repair patches on both strands which should uncover analytically as DNA double-strand breaks and allow to measure the rejoining kinetic of this repair intermediate. Additionally, the combined effects of drug and radiation treatment on rejoining of double-strand breaks was investigated with two different mammalian cell lines. MATERIAL AND METHODS: V79 (rodent fibroblasts) and Widr (human colon carcinoma) cells were treated for 2 hours with 4-hydroperoxyifosfamide which rapidly decays to 4-hydoxyifosfamide in aqueous solution or were exposed in combination with ionizing radiation followed by incubation for repair with or without the drug. DNA double-strand breakage was measured by pulsed-field electrophoresis. RESULTS: The 2 hours 4-hydroperoxyifosfamide treatment (30 micrograms/ml) resulted in a pronounced DNA fragmentation that, 2-4 hours after drug removal, declined with an estimated half-live of about 4 hours for both cell lines. When the cells were additionally irradiated with 10 Gy given in the middle of drug exposure, the residual fragmentation after 12 or 24 hours incubation for repair was only marginally increased, roughly corresponding to the respective value after radiation, alone. A continuous drug exposure of 6 hours (at 10 micrograms/ml) resulted in a fragmentation that was independent of a preirradiation with a high dose of 30 Gy, immediately before drug addition. CONCLUSIONS: The present data support the idea that unligated/unrejoined double-stranded DNA ends are generated during the repair of lesions from bifunctional alkylators. The rate of subsequent rejoining is in the order of magnitude of the slow rejoining of radiation-induced double-strand breaks. Processing of double-stranded DNA damage from either 4-hydroperoxyifosfamid or radiation exposure is apparently unaffected in combined treatments.     

Role of DNA-PKcs in the bystander effect after low- or high-LET irradiation

PURPOSE: To investigate the role of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the medium-mediated bystander effect for chromosomal aberrations induced by low-linear energy transfer (LET) X-rays and high-LET heavy ions in normal human fibroblast cells. MATERIALS AND METHODS: The recipient cells were treated for 12 h with conditioned medium, which was harvested from donor cells at 24 h after exposure to 10 Gy of soft X-rays (5 keV/microm) and 20Ne ions (437 keV/microm), followed by analyses of chromosome aberrations in recipient cells with premature chromosome condensation methods. To examine the role of DNA-PKcs and nitric oxide (NO), cells were treated with its inhibitor LY294002 (LY) and its scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO), respectively. RESULTS: Increased frequency of chromosome aberrations in recipient cells treated with conditioned medium from irradiated but not from un-irradiated donor cells was observed which was independent of radiation type. Bystander induction of chromosome aberrations in recipient cells was mitigated when donor cells were treated with LY before irradiation and with c-PTIO after irradiation, and was enhanced when recipient cells were treated with LY before treatment of recipient cells with conditioned medium from irradiated donor cells. CONCLUSION: Irradiated normal human cells secrete NO and other molecules which in turn transmit radiation signals to unirradiated bystander cells, leading to the induction of bystander chromosome aberrations partially repairable by DNA-PKcs-mediated DNA damage repair machinery, such as non-homologous end-joining repair pathways.