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Title: Low-LET Microbeam Investigation of the Track-End Dependence of Electron-Induced Damage in Normal Human Diploid Fibroblasts

Abstract

Using the electron microbeam located at Pacific Northwest National Laboratory, we have investigated the incident electron energy dependence on micronuclei formation in human diploid fibroblasts, AG01522, following non-targeted irradiations at 25 and 80 keV. Examining the dose response, we find that 25 keV electrons are more effective than 80 keV electrons at producing biological damage for a given dose. Our results demonstrating the induction of micronuclei as a function of incident electron energy offer direct support for the hypothesis that the track end is responsible for the biological damage occurring in the cell.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
876871
Report Number(s):
PNNL-SA-42273
Journal ID: ISSN 0033-7587; RAREAE; 6697; KP1303000; TRN: US0601526
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Radiation Research; Journal Volume: 164; Journal Issue: 5
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRONMENTAL POLLUTANT EFFECTS ON LIVING ORGANISMS AND BIOLOGICAL MATERIALS; ELECTRON BEAMS; ENERGY DEPENDENCE; FIBROBLASTS; BIOLOGICAL RADIATION EFFECTS; HUMAN POPULATIONS; DOSE-RESPONSE RELATIONSHIPS; Environmental Molecular Sciences Laboratory

Citation Formats

Sowa, Marianne B., Kathmann, Loel E., Holben, Brooke A., Thrall, Brian D., and Kimmel, Greg A. Low-LET Microbeam Investigation of the Track-End Dependence of Electron-Induced Damage in Normal Human Diploid Fibroblasts. United States: N. p., 2005. Web. doi:10.1667/RR3464.1.
Sowa, Marianne B., Kathmann, Loel E., Holben, Brooke A., Thrall, Brian D., & Kimmel, Greg A. Low-LET Microbeam Investigation of the Track-End Dependence of Electron-Induced Damage in Normal Human Diploid Fibroblasts. United States. doi:10.1667/RR3464.1.
Sowa, Marianne B., Kathmann, Loel E., Holben, Brooke A., Thrall, Brian D., and Kimmel, Greg A. Tue . "Low-LET Microbeam Investigation of the Track-End Dependence of Electron-Induced Damage in Normal Human Diploid Fibroblasts". United States. doi:10.1667/RR3464.1.
@article{osti_876871,
title = {Low-LET Microbeam Investigation of the Track-End Dependence of Electron-Induced Damage in Normal Human Diploid Fibroblasts},
author = {Sowa, Marianne B. and Kathmann, Loel E. and Holben, Brooke A. and Thrall, Brian D. and Kimmel, Greg A.},
abstractNote = {Using the electron microbeam located at Pacific Northwest National Laboratory, we have investigated the incident electron energy dependence on micronuclei formation in human diploid fibroblasts, AG01522, following non-targeted irradiations at 25 and 80 keV. Examining the dose response, we find that 25 keV electrons are more effective than 80 keV electrons at producing biological damage for a given dose. Our results demonstrating the induction of micronuclei as a function of incident electron energy offer direct support for the hypothesis that the track end is responsible for the biological damage occurring in the cell.},
doi = {10.1667/RR3464.1},
journal = {Radiation Research},
number = 5,
volume = 164,
place = {United States},
year = {Tue Nov 01 00:00:00 EST 2005},
month = {Tue Nov 01 00:00:00 EST 2005}
}
  • The conventional paradigm in radiation biology has been that DNA is the primary target for energy deposition following exposure to ionizing radiation. However, studies focusing on the non-target effects of radiation, i.e. effects occurring in cells not directly exposed to radiation, imply that the target of exposure is larger than what has traditionally been assumed and could have significant implications for radiation health risks. We have conducted an extensive study of the low-LET bystander effect including multiple cell lines and endpoints and various radiation sources and exposure scenarios. In no instance do we see evidence of a low-LET induced bystandermore » effect. However, direct comparison for alpha particle exposure showed a statistically significant media transfer bystander effect for high-LET but not for low-LET radiation. From our results it is evident that there are many confounding factors mitigating bystander responses as reported in the literature and for the cell lines we studied that there is a LET dependence for the observed responses. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.« less
  • The occurrence of DNA repair in uv- (254 nm) and x-irradiated normal human diploid fibroblasts maintained in a quiescent, nondividing state using low serum (0.5%) medium was ascertained. Techniques that detect different steps of the excision repair process were used so that the extent of completion of repair at single sites could be determined. These included measuring the disappearance of pyrimidine dimers by chromatography, detecting repair synthesis by density-gradient and autoradiographic methods and detecting the rejoining of repaired regions and repair of x-ray-induced single-strand DNA breaks using alkaline sucrose gradients. Results show that dimer excision occurs and the subsequent stepsmore » of repair synthesis and ligation are completed. About 50% of the dimers formed by exposure to 20 J/m/sup 2/ is excised in the initial 24-h post-uv period. DNA repair (unscheduled DNA synthesis) can be detected through a 5-d post-uv period. The fraction of damaged sites eventually repaired is not known. X-ray-induced single-strand DNA breaks are repaired rapidly.« less
  • In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of thesemore » inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process.« less
  • To determine whether chromosome breaks produced by {alpha} particles are processed differently from those produced by X rays, the premature chromosome condensation technique was used to follow chromosome rejoining after irradiation. Doses of 90 and 200 keV/{mu}m {alpha} particles (2.7 Gy) and 250 kVp X rays (6 Gy) were chosen to produce approximately the same number of initial chromosome breaks (about 30 excess fragments per cell). Frequencies of excess fragments were assessed at eight times to 24 h after irradiation with the final yields being about 2, 4 and 8 excess fragments per cell for 250 kVp X rays andmore » 200 and 90 keV/{mu}m {alpha} particles, respectively. For each radiation the time for the initial measured fragment frequency per cell to be halved (i.e. to about 15) was the same (about 100 min). The results were fitted to three models of kinetics of the rejoining, and the initial and residual number of excess chromosome fragments as well as the rate of rejoining were determined. Even with eight times, discrimination between the models of the kinetics was not possible, such that a single-component first-order reaction could not be rejected for either X-ray- or {alpha}-particle-induced breaks. Although rejoining proceeds at similar rates, the probability of {open_quotes}correct{close_quotes} rejoining is apparently reduced for {alpha}-particle-irradiated cells. 25 refs., 6 figs., 7 tabs.« less
  • 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,more » 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.« less