Acute and late effects of combined internal and external radiation exposures on the hematopoietic system

Abstract

Purpose: Incidents, such as nuclear facility accidents and the release of a 'dirty bomb', might result in not only external irradiation of personnel, but additional internal exposures through concomitant inhalation and/or ingestion of radioactive particulates. The purpose of this study was to define the impact of such a combination of radiation injuries on the hematopoietic niche.Material and methods: To assess changes in the murine hematopoietic system, we used a combined exposure of total body irradiation (TBI, 6 Gy) followed immediately by an internal (intraperitoneal) administration of 100 µCi of soluble ¹³⁷Cs. We then evaluated acute survival in combined versus single modality exposure groups, as well as assessing hematopoietic function at 12 and 26 week time points.Results: Acutely, the combination of external and internal exposures led to an unexpected delay in excretion of ¹³⁷Cs, increasing the absorbed dose in the combined exposure group and leading to mortality from an acute hematopoietic syndrome. At 12 weeks, all exposure paradigms resulted in decreased numbers of phenotypic hematopoietic stem cells (HSCs), particularly the short-term HSCs (ST-HSC); long-term HSCs (LT-HSC) were depleted only in the internal and combined exposure groups. At 26 weeks, there was significant anemia in both the TBI alone and combined exposure groups. There were decreased numbers in both the LT- and ST-HSCs and decreased functionality, as measured by competitive repopulation, was seen in all radiation groups, with the greatest effects seen in the internal and combined exposure groups.Conclusions: Our data indicate that a combined injury of sublethal external irradiation with internal contamination induces significant and persistent changes in the hematopoietic system, as may have been predicted from the literature and our own group's findings. However, a novel observation was that the combined exposure led to an alteration in the excretion kinetics of the internal contamination, increasing the acute effects beyond those anticipated. As a result, we believe that a combined exposure poses a unique challenge to the medical community during both the acute and, possibly, delayed recovery stages.

Keywords: Hematopoiesis; bone marrow late effects; combined radiation injury; internal contamination.

Figure 1:

Measurements of radioactivity counts over the first 30 days following 0 Gy + IP cesium, 6 Gy external TBI ± internal (IP) 100μCi ¹³⁷Cs.

Figure 2:

Kaplan-Meier survival curve of C57BL/6J mice exposed to sham irradiation + IP cesium (controls: n=23), sham irradiation followed by 100μCi ¹³⁷Cs (hot-Cs: n=25), 6 Gy TBI followed by IP cesium (TBI; n=25) or 6 Gy TBI followed by IP 100μCi ¹³⁷Cs (TBI+hot-Cs; n=21).

Figure 3:

Peripheral blood analysis of 3 individual moribund mice (from TBI+hot-Cs group) compared to 3 controls (pooled) at day 28 post-exposure. WBC: white blood cells; RBC: red blood cells.

Figure 4:

Flow cytometry strategy for phenotypic analysis of hematopoietic stem cells: A) representative flow cytometry plots for phenotypic analysis of hematopoietic stem cells from all four experimental groups at 12 weeks post treatment; B) flow cytometric analysis of hematopoietic stem cells (HSCs) at 12 weeks post-exposure for: C) bone marrow (BM) LSKs; D) long-term (LT)-HSCs; E) short-term (ST)-HSCs; F) multipotent progenitor (MPP)-2; G) MPP3; and H) MPP4. Each dot indicates an individual animal; * denotes a significance of p<0.05; ** denotes a significance of p<0.01; *** denotes a significance of p<0.005.

Figure 5:

A) Schematic representation of transplantation experiment. B) Representative flow cytometry plots for phenotypic analysis of total cell engraftment from control and TBI+hot-Cs experimental groups at 12 weeks post treatment. Using the competitive repopulation assay, % engraftment rates from bone marrow taken from experimental groups at 12 weeks post-exposure for: C) Total cells; D) B cells; E) myeloid; and F) T cells. n=12–15 donors/group; 19–25 recipients/group. Results are represented as means ± SEM of 3 separate analyses. Double arrows indicate group comparisons by Bonferroni post-test: ** denotes a significance of p<0.01; *** denotes a significance of p<0.005; **** denotes a significance of p<0.0001. Statistics on individual time points is provided in the text.

Figure 6:

Late (26 weeks) effects of radiation exposure on: A) bone marrow cellularity and B) hemoglobin. *** denotes a significance of p<0.005.

Figure 7:

Late (26 weeks) effects of radiation exposure on erythro-myeloid progenitors: A) colony forming unit (CFU)-myeloid; B) burst forming unit (BFU)-erythroid; C) and CFU-erythroid. * denotes a significance of p<0.05; ** denotes a significance of p<0.01; *** denotes a significance of p<0.005.

Figure 8:

A) Representative flow cytometry plots for phenotypic analysis (using the strategy presented in Figure 4A) of hematopoietic stem cells from all four experimental groups at 26 weeks post treatment. Flow cytometric analysis of hematopoietic stem cells (HSCs) at 26 weeks post-exposure for: B) bone marrow (BM) LSKs; C) short-term (ST)-HSCs; and D) long-term (LT)-HSCs. Each dot indicates an individual animal; * denotes a significance of p<0.05; ** denotes a significance of p<0.01; *** denotes a significance of p<0.005.

Figure 9:

Using the competitive repopulation assay, % engraftment rates from bone marrow taken from experimental groups at 26 weeks post-exposure for: A) total cells, B) B cells; C) myeloid; and D) T cells. n=12–15 donors/group; 19–25 recipients/group. Results are represented as means ± SEM of 3 separate analyses. Double arrows indicate group comparisons by Bonferroni post-test: **** denotes a significance of p<0.0001. Statistics on individual time points is provided in the text.

Figure 10:

Quantification of adipocytes per region in the diaphyseal (A) and metaphyseal (B) regions at 12 and 26 weeks post-exposure. (C) Representative H&E stains from histologic sections of the metaphyseal region of femurs from irradiated mice at 26 weeks post-exposure.