Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: dose-response and time-course profiles measured by flow cytometry

Abstract

A flow cytometric, anti-CD71-based method was used to measure peripheral blood reticulocyte and micronucleated reticulocyte frequencies in response to (137)Cs total body irradiation (TBI). In three independent experiments, groups of five female C57BL/6N mice were irradiated at graded doses up to 3 Gy, and peripheral blood specimens were collected at 43 h post-irradiation. Whereas the frequency of reticulocytes declined over the range of doses studied, micronucleated reticulocyte incidence was observed to increase in a dose-dependent manner up to 1 Gy. At doses greater than approximately 1 Gy, micronucleated reticulocyte frequencies declined with increasing exposure. These responses were highly reproducible, with significant effects on reticulocyte and micronucleated reticulocyte frequencies observed for the lowest dose studied (0.125 Gy). A time-course experiment was performed to test whether radiation-induced cell cycle delay may explain saturation of the micronucleated reticulocyte endpoint at doses >1 Gy. For this experiment, groups of four female C57BL/6N mice were exposed to 1, 1.5, or 2 Gy TBI, and blood collection occurred at 12h intervals from 43 to 115 h post-exposure. Reduced reticulocyte frequencies were observed for each dose studied, and the recovery of reticulocytes was increasingly delayed with higher radiation doses. Maximal micronucleated reticulocyte frequencies were observed at 43 or 55 h, with progressively lower values at later time points. At no time did micronucleated reticulocyte frequencies induced by 1.5 or 2 Gy significantly exceed that observed for 1 Gy at 43 h. These time-course data suggest that radiation-induced cell cycle delay cannot account for the micronucleated reticulocyte downturn phenomenon observed at doses greater than 1 Gy. An alternate hypothesis is discussed whereby apoptotic elimination of severely damaged bone marrow erythroid precursors plays a dominant role in saturating the radiation-induced micronucleated reticulocyte response observed for C57BL/6N mice.

Figure 1

Representative 0, 0.5 and 1.0 Gy specimens (panels A, B, and C, respectively) illustrate fluorescent staining characteristics of four erythrocyte subpopulations: lower left quadrant = mature erythrocytes; lower right = micronucleated mature erythrocytes; upper left = CD71-positive reticulocytes (RETs); and upper right = micronucleated CD71-positive reticulocytes (MN-RETs). Note that only gated events are shown (i.e., platelets and nucleated cells have been excluded based on their high anti-CD61-PE and propidium iodide-associated fluorescence signals, respectively).

Figure 2

Mean percentage peripheral blood reticulocyte frequencies (%RET) are graphed as a function of gamma radiation dose. All blood samples were collected 43 hrs post-total body irradiation.

Figure 3

Mean percentage peripheral blood micronucleated reticulocyte frequencies (%MN-RET) are graphed as a function of gamma radiation dose. All blood samples were collected 43 hrs post-total body irradiation.

Figure 4

Mean percentage peripheral blood reticulocyte frequencies (%RET) are graphed as a function of time. Blood samples were collected between 43 and 115 hrs post gamma irradiation at 12 hrs intervals.

Figure 5

Mean percentage peripheral blood micronucleated reticulocyte frequencies (%MN-RET) are graphed as a function of time. Blood samples were collected between 43 and 115 hrs post gamma irradiation at 12 hrs intervals.

Figure 6

The kinetics by which radiation-induced MN-RET enter the blood stream does not appear to explain the downturn that is evident at doses > 1 Gy. The alternate model depicted here invokes selection against highly damaged precursors and progenitors. As illustrated above, relatively less damaged cells are seen to proceed through the process of division and differentiation. Conversely, those that have been more severely damaged and therefore more likely to express micronuclei are disproportionately eliminated via apoptosis. Preliminary experiments with bone marrow specimens collected 24 hrs post-irradiation support this model, as erythroid precursor and progenitor numbers are both severely reduced at doses > 1 Gy (data not shown).