Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop V

2005 Abstract

Title: Induction of Genomic Instability in vivo by Low Doses of 137Cs γ rays.

Authors: K. Noy Rithidech, C, Leotchutinat, L. Honikel, S.R. Simon and E.B. Whorton

Institutions: Pathology Department, Stony Brook University, NY, USA, University of Texas Medical Branch at Galveston, TX

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) remain uncertain. It is therefore important to characterize early and subsequent in vivo biological response induced by low doses of radiation because such data should provide information that can help determine whether radiation at this dose level causes substantial long term health effects. In this project, we have established several endpoints to examine the effects of ¹³⁷Cs γ rays (0, 5, 10, and 100 cGy) in bone marrow (BM) cells of exposed male mice with different genetic backgrounds (BALB/cJ and C57BL/6J mice) collected at different times post-irradiation (i.e. 1 hr, 4 hrs, 1 month and 6 months). A total of five mice per dose per strain were sacrificed at each time point for sample collection. The magnitude of genomic instability manifested in vivo as chromosome aberrations was measured by using three-color FISH to score stable and unstable chromosome aberrations involving mouse chromosomes 1, 2, and 3. All other chromosomal aberrations involving non-painted chromosomes also have been analyzed in the same cells being scored for aberrations in the painted chromosomes. Mitotic indexes (MI) in BM cells collected at all harvest times also were examined as an expeditious measure of response to radiation. Levels of nuclear factor-kappa B (NF-κB) activation and expression of genes known to be regulated through NF-κB activation have been determined in cells at 1 and 4 hrs following irradiation (indicative of early responses).

We detected a significant increase in levels of activated NF-κB dimers in lysates of BM cells isolated from both mouse strains at 1 hour post-exposure to 10 and 100 cGy. These elevated levels in the BM then precipitously declined by 4 hrs post-exposure of radiosensitive BALB/cJ mice but persisted in the BM cells of exposed radioresistant C57BL/6J mice. Although our study is the first to provide evidence for in vivo activation of NF-κB induced by radiation of mice with low does of ¹³⁷Cs γ rays, followed by subsequent strain-specific post-exposure diminution, it is limited to the analysis of cells collected at only two early time points following irradiation. As a result, the extent to which this differential response contributes to the difference in sensitivity to radiation induced genomic instability between these two mouse strains remains unclear.

We further examined the expression pattern of mRNAs which have previously been identified as being under transcriptional control by NF-κB in the spleen isolated from these mice as a function of radiation dose and time post-irradiation. In BALB/cJ mice, significant increased levels of expression of several genes were detected as early as 1 hour post-irradiation in samples from mice exposed to a high dose level (100 cGy). In contrast, high levels of gene expression in samples collected from mice exposed to 5 or 10 cGy were not observed until 4 hrs post-irradiation. The results indicate a delay in expression of genes in samples from mice exposed to low doses of ¹³⁷Cs γ rays. Expression of the anti-apoptotic genes Bcl2a1a and Bcl2l was not increased in samples collected from mice exposed to the high dose level (100 cGy), but was increased in samples collected from mice exposed to low doses of ¹³⁷Cs γ rays (5 and 10 cGy). The results suggest a different molecular mechanism in response to high or low doses of low LET radiation of bone marrow cells exposed in vivo. In addition, significant levels of expression of three genes coding for cytokines (Il10, Il11, and Pdgfb) were also found in sham-control samples at both 1 and 4 hours following irradiation, possibly reflecting an innate immune response of mice to stress triggered by the brief restraint during sham exposure. In contrast to our findings in the BALB/cJ mice, however, when we examined the splenic tissues of irradiated C57BL/6J mice, we observed no significant increase in expression of anti-apoptotic genes, regardless of radiation dose and the subsequent post-exposure interval (1 and 4 hrs after irradiation). Rather, there was a trend of increased expression of genes involved in apoptosis (such as Fas-L, p53) in spleen cells collected at 1 hr from mice exposed to 100 cGy. Although only a single transduction pathway was examined, the data suggest that, at a similar dose of radiation, up-regulation of genes involved in apoptotic pathways is more likely to occur in radiation-resistant C57BL/6J mice as compared to radiation-sensitive BALB/cJ mice.

We found a statistically significant difference in levels of mitotic indexes (MI) in response to a high dose (100 cGy) of ¹³⁷Cs γ rays between BALB/cJ and C57BL/6J mice at 1 hr post-irradiation (p<0.01, Student’s test). Our data clearly demonstrate that the MI assay is useful for detecting differences in radiosensitivity of mice with different genetic backgrounds at early times post-exposure. A significant increase in MI was detected in all samples collected at late times (1 & 6 months) from both strains, regardless of the radiation dose to which mice were exposed. However, the MI assay is unable to distinguish between surviving cells with or without damage.

With respect to the three-color FISH of mouse chromosomes, we applied the average square root transformation (ASQT) to the aberration frequency measured in each mouse prior to statistical analysis to achieve reasonable normality and reasonably homogeneous inter-animal variability within treatment combination groups. Analysis of variance methods appropriate for two factor studies (post-exposure time and radiation dose in this case) was used to evaluate the resulting data for each strain, along with a regression test for a positive linear dose related trend. A dose-dependent increase (p<0.001) in number of cells with chromosomal damage (breaks and exchanges) was detected as a function of dose at early time (1 & 4 hrs) post- irradiation in both strains of mouse. At late times (1 & 6 months) post-irradiation, there were significant decreases in the levels of chromosomal damage in both mouse strains as compared to those observed at early times. However, the residual levels of persistent chromosomal damage appeared to remain elevated for up to 6 months in BM cells collected from BALB/cJ mice only but not in the C57BL/6J mouse. It also should be noted that the C57BL/cJ mouse has a higher spontaneous rate of chromosomal damage compared to that of the BALB/cJ mouse. A statistically significant reduction (p<0.05) in the frequency of chromosome breaks was found in BM cells collected at 6 months post-irradiation from both strains of mouse exposed to 5 cGy. Mechanisms responsible for these selective decreases are unknown.

Our data indicated a trend of persistent elevation in all types of chromosomal damage in cells collected at 1 & 6 months post-irradiation from BALB/cJ mice exposed to low doses (5 and 10 cGy) although this increase was not statistically significant. It is clear, however, that there is a statistically significant increase (p<0.05) in the frequency of chromosome aberrations in cells collected at these late times from BALB/cJ mice exposed to 100 cGy. In contrast, no increase in any type of chromosomal damage (including the number of abnormal cells) was detected in BM cells collected from C57BL/6J mice at these late times post-irradiation regardless of radiation dose, suggesting no in vivo induction of sustained genomic instability in the C57BL/6J mouse by the radiation doses used in our study. The differences in response to radiation between the BALB/cJ and C57BL/6J mice may reflect differences in repair systems, removal of damaged cells by apoptosis or the cell turnover that removes damaged cells from the populations.

In summary, our findings indicate that a strain-specific difference in radiosensitivity should be considered when selecting strains of mice for evaluating in vivo biological effects of radiation.

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

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