22 research outputs found
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Lack of Bystander Effects From High LET Radiation For Early Cytogenetic Endpoints.
The aim of this work was to study radiation-induced bystander effects for early cytogenetic end points in various cell lines using the medium transfer technique after exposure to high- and low-LET radiation. Cells were exposed to 20 MeV/ nucleon nitrogen ions, 968 MeV/nucleon iron ions, or 575 MeV/nucleon iron ions followed by transfer of the conditioned medium from the irradiated cells to unirradiated test cells. The effects studied included DNA double-strand break induction, {gamma}-H2AX focus formation, induction of chromatid breaks in prematurely condensed chromosomes, and micronucleus formation using DNA repair-proficient and -deficient hamster and human cell lines (xrs6, V79, SW48, MO59K and MO59J). Cell survival was also measured in SW48 bystander cells using X rays. Although it was occasionally possible to detect an increase in chromatid break levels using nitrogen ions and to see a higher number of {gamma}-H2AX foci using nitrogen and iron ions in xrs6 bystander cells in single experiments, the results were not reproducible. After we pooled all the data, we could not verify a significant bystander effect for any of these end points. Also, we did not detect a significant bystander effect for DSB induction or micronucleus formation in these cell lines or for clonogenic survival in SW48 cells. The data suggest that DNA damage and cytogenetic changes are not induced in bystander cells. In contrast, data in the literature show pronounced bystander effects in a variety of cell lines, including clonogenic survival in SW48 cells and induction of chromatid breaks and micronuclei in hamster cells. To reconcile these conflicting data, it is possible that the epigenetic status of the specific cell line or the precise culture conditions and medium supplements, such as serum, may be critical for inducing bystander effects
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Relative Biological Effectiveness of HZE Fe Ions for Induction of Micro-Nuclei at Low Doses
Dose-response curves for induction of micro-nuclei (MN) was 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/u Fe ions, 600 MeV/u Fe ions, and 300 MeV/u Fe ions (LETs of 151, 176 and 235 keV/{micro}m 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 (LET=151 keV/{micro}m), 4.3 (LET = 176 keV/{micro}m) and 5.7 (LET = 235 keV/{micro}m), 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/u Fe ions and found to be 5.4, slightly higher than for V79 cells. To test the effect of shielding, the 1 GeV/u Fe ion beam was intercepted by various thickness 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 Fe ion Bragg peak (excluding the area around the Fe-ion Bragg peak itself), indicating that RBE did not change significantly due to shielding. At the Bragg peak the effectiveness for MN formation per unit dose was decreased, indicating an 'overkill' effect by low-energy very high-LET Fe ions
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Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP
Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP
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Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts
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Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts
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Relative Biological Effectiveness of HZE Fe Ions for Induction of Micro-Nuclei at Low Doses
Relative Biological Effectiveness of High-Energy Iron Ions for Micronucleus Formation at Low Doses
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Measurement of prompt DNA double-strand breaks in mammalian cells without including heat-labile sites. Results for cells deficient in non-homologous end joining
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Dose-dependent misrejoining of radiation-induced DNA double-strand breaks in human fibroblasts: Experimental and theoretical study for high and low LET radiation
Misrejoining of DNA double-strand breaks (DSBs) was measured in human primary fibroblasts after exposure to X-rays and high LET particles (He, N and Fe) in the dose range 10-80 Gy. To measure joining of wrong DNA ends, the integrity of a 3.2 Mbp restriction fragment was analyzed directly after exposure and after 16 hr of repair incubation. It was found that the misrejoining frequency for X-rays was non-linearly related to dose, with less probability of misrejoining at low doses than at high doses. The dose dependence for the high LET particles, on the other hand, was closer to being linear, with misrejoining frequencies higher than for X-rays particularly at the lower doses. These experimental results were simulated with a Monte-Carlo approach that includes a cell nucleus model with all 46 chromosomes present, combined with realistic track structure simulations to calculate the geometrical positions of all DSBs induced for each dose. The model assumes that the main determinant for misrejoining probability is the distance between two simultaneously present DSBs. With a Gaussian interaction probability function with distance, it was found that both the low and high LET data could be fitted with an interaction distance (sigma of the Gaussian curve) of 0.25 {micro}m. This is half the distance previously found to best fit chromosomal aberration data in human lymphocytes using the same methods (Holley et al. Radiat. Res . 158, 568-580 (2002)). The discrepancy may indicate inadequacies in the chromosome model, for example insufficient chromosomal overlap, but may also partly be due to differences between fibroblasts and lymphocytes. Although the experimental data was obtained at high doses, the Monte Carlo calculations could be extended to lower doses. It was found that a linear component of misrejoining versus dose dominated for doses below 1 Gy for all radiations, including X-rays. The calculated relative biological efficiency (RBE) for misrejoining at this low dose region was 31 for the He-ions, 28 for the N-ions and 19 for Fe-ions