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DOE Lowdose Radiation Program Workshop IV

Abstract

Title: Induction of Genomic Instability in vivo by Low Doses of 137Cs y rays,

Authors: K. Rithidech1, E.B. Whorton2, M. Tungjai1, E. Ar-Bab1, S.R. Simon1, M. Tawde3 and C.W. Anderson3.

Institutions: 1Pathology Department, Stony Brook University, NY 11794-8691, USA, 2University of Texas Medical Branch at Galveston, TX 77550-1047,3Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000.

Information on potential health hazards of radiation at doses below or equal to the level traditionally requiring human radiation protection (less than or equal to 10 cGy) is currently lacking. It is therefore important to characterize early and subsequent in vivo biological response induced by low doses of ionizing radiation because such data should provide information that can help determine whether radiation at this dose level causes substantial biological damage. In this study several endpoints are used to examine the effects of low doses of 137Cs y rays on bone marrow cells collected at different times post irradiation of mice with different genetic backgrounds (BALB/cJ, C57BL/6J and Scid mice).

The specific aims of this study are to:

  1. Use an in vivo approach to determine if low doses (equal or <10 cGy) of low LET radiation can induce genomic instability in the target bone marrow cells of exposed mice.
  2. Analyze the type and frequency of chromosome aberrations involved in radiation induced genomic instability and the clonal expansion.
  3. Evaluate the impact of genetic background on the induction of genomic instability in vivo.
  4. Compare changes in gene expression related to activation of nuclear factor-kappa B (NFkappa B) and activity of DNA-PKcs following exposure to low doses of low LET radiation.

In this workshop, we are presenting data obtained from bone marrow cells collected from BALB/cJ mice (a radiosensitive strain). Four groups of 20 male BALB/cJ mice (10-12 weeks old) were each given a whole body with a single dose of 0, 5, 10 or 100 cGy 137Cs y rays. Mice exposed to 0 cGy serve as sham-controls. There were four harvest times. At each harvest time (1 and 4 hours, 1 and 6 months), bone marrow cells were collected from 5 mice/dose for chromosome aberration assay. Levels of NF-kappa B activation reflected in expression levels of genes known to be under NF-kappa B regulation, and DNA-PKcs activity were measured at 1 and 4 hours post-irradiation. Genomic instability is determined by the delayed appearance of chromosome aberrations in bone marrow cells collected at different times up to six months post-irradiation, using three-color FISH to score stable and unstable chromosome aberrations involving mouse chromosomes 1, 2, and 3. All other visible aberrations involving non-painted chromosomes also were analyzed in the same cells scored for aberrations involving painted chromosomes.

NF-kappa B activation in bone marrow cells isolated 1 hour post-exposure of mice to 10 or 100 cGy increased significantly above that in sham-controls. In contrast, no difference in the levels of DNA-PKcs activity was observed at this time at these doses. In samples isolated 4 hours postirradiation, however, no significant level of activated NF-kappa B signal was detected while a marked increase in DNA-PKcs activity was observed in bone marrow cells of mice, regardless of the radiation dose to which the mice had been exposed.

We initiated pilot studies examining differential expression of NF-kappa B regulated genes in spleen cells isolated from these exposed mice. The spleen was selected for the study of gene expression because it contains hematopoietic cells. Our preliminary results indicated a significant level of in vivo expression of genes coding cytokines and cytokine moderators (as compared to the level of expression of beta-actin gene) regardless of the radiation dose to which the mice have been exposed. A significant level of expression of several genes was detected as early as 1 hour post-irradiation in samples exposed to a high dose level (100 cGy). In contrast, a high level of gene expression in samples collected from mice exposed to 5 or 10 cGy was observed at 4 hours post-irradiation indicating a delay in expression of genes in samples from mice exposed to low doses of 137Cs y rays. The results also indicated different patterns of gene expression in response to low and high doses of radiation, suggesting that low-dose extrapolation of health risk from high-dose response might be inappropriate. Although our pilot studies provide important information on potential differences in molecular response to high and low doses of radiation, these pilot studies have limitations because only a small number of genes involving a specific transduction pathway were included in the analysis. In order to characterize molecular markers of dose exposure accurately, we hope to incorporate technologies that enable the study of large number of genes simultaneously such as the microarray technology.

With respect to genomic instability, we have completed the analysis of chromosome instability in bone marrow cells collected from BABL/cJ mice at 1 hr, 4 hr, 1 month, and 6 months following exposure. Statistical analyses using two-factor ANOVA were performed to assess the significances of the dose and harvest time on four different aberration types: (1) abnormal cells, (2) chromatid breaks, (3) chromosome breaks, (4) chromosomal exchanges. The analysis, per aberration frequency type, was based on the number of specific aberration frequencies observed per mouse. The average square root transformation was applied to each animal’s measured aberration frequency to achieve reasonable normality and reasonably homogeneous inter-animal variability within exposed groups. Due to a low mitotic index, we pooled data obtained from 1 and 4 hours post irradiation, and have designated these data as “early time point”. We did the analysis to examine whether there is a dose related increase in the frequency of each type of aberration (mentioned above) with or without the inclusion of the data obtained from mice exposed to 100 cGy 137Cs gamma rays. This dual analysis was necessary because there were very few metaphase cells available for analysis in samples collected from mice in the 100- cGy exposed mice at both 1 and 4 hours post-irradiation, perhaps due to cell cycle arrest. The results from both analyses demonstrated a statistically significant dose-dependent increase in the frequencies of abnormal cells and all types of aberrations. The dose-effect was more pronounced when the data from mice exposed to 100 cGy were included in the analysis (p< 0.01 and =0.02 with and without the 100-cGy data, respectively).

Mitotic indexes were high in all treatment groups in samples collected at 1 and 6 months, resulting in sufficient metaphase cells for the analysis (at least 1,500 cells per treatment group). Chromatid breaks were detected in cells collected at 1 or 6 months post-irradiation, regardless of radiation dose to which the mice had been exposed, suggesting the induction of genomic instability. Similar to the early harvest time data, we examined whether a dose-effect relationship exists. With the inclusion of the 100-cGy data, there were statistically significant increases in the frequencies of abnormal cells, chromatid breaks, chromosome breaks, and chromosomal exchanges (0.05>p<0.01). A trend of an increase in abnormal cells and different types of chromosome aberration was suggested when the analysis was performed without the inclusion of the 100-cGy data but this increase was not statistically significant. To detect statistically very small increases associated with very low dose exposure levels would require exceptionally large sample sizes (number of cells) in every exposure group. A statistically significant reduction in number of chromatid breaks in bone marrow cells collected at six month-post irradiation from mice exposed to 5 cGy compared to all radiation doses including control was observed. Our data clearly showed that a high dose of 137Cs y rays (100 cGy) induced genomic instability in bone marrow cells of acutely exposed mice (p<0.05).

Research supported by DOE Low Dose Grant #DE-FG02-02ER63311.

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