Preferential Elimination of Older Erythrocytes in Circulation and Depressed Bone Marrow Erythropoietic Activity Contribute to Cadmium Induced Anemia in Mice

Abstract

Feeding cadmium chloride (50 or 1000 ppm CdCl2 in drinking water, ad libitum) to C57BL/6 mice resulted in a significant and sustained fall in blood erythrocyte count and hemoglobin levels that started 4 and 3 weeks after the start of 50 and 1000 ppm cadmium doses respectively. A transient yet significant reticulocytosis occurred during the first 4 weeks of cadmium treatment. Using the recently developed double in vivo biotinylation (DIB) technique, turnover of erythrocyte cohorts of different age groups was simultaneously monitored in control and cadmium treated mice. A significant accumulation of younger erythrocytes and a concomitant decline in the relative proportions of older erythrocytes in circulation was observed in both 50 and 1000 ppm cadmium groups indicating that older erythrocytes were preferentially eliminated in cadmium induced anemia. A significant increase in the erythropoietin levels in plasma was seen in mice exposed to 1000 ppm cadmium. Levels of inflammatory cytokines (IL1A, IL6, TNFα, IFNγ) were however not significantly altered in cadmium treated mice. A significant increase in cellular levels of reactive oxygen species (ROS) was observed in older erythrocytes in circulation but not in younger erythrocytes. Erythropoietic activity in the bone marrows and spleens of cadmium treated mice was examined by monitoring the relative proportion of cells belonging to the erythroid line of differentiation in these organs. Erythroid cells in bone marrow declined markedly (about 30%) in mice in the 1000 ppm cadmium group but the decline was not significant in the 50 ppm cadmium group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Decline of erythroid cells was essentially confined to pro-erythroblast and erythroblast-A, along with a concurrent increase in the splenic erythroid population indicating a stress response. In short cadmium exposure causes preferential clearance of older erythrocytes from circulation along with a depressed erythropoietic activity at higher doses.

Identification of Stages of Erythroid Differentiation in Bone Marrow and Erythrocyte Subpopulations in Blood Circulation that Are Preferentially Lost in Autoimmune Hemolytic Anemia in Mouse

“…Double in vivo biotinylation (DIB) of erythrocytes was done as described previously [13–19]. The DIB technique involves two steps of biotinylation of circulating erythrocytes by intravenous ( i .…”

“…Splenic and BM cells were strained through a fine sieve, pelleted by centrifugation, and resuspended at desired concentration in RPMI with 10% FBS. For delineating erythroid precursors at different stages of differentiation, freshly prepared single cell suspensions from BM or spleen were first incubated with anti-CD16/32 monoclonal antibody (Fc block, 1 μg/10 6 cells in 50 μl of PBS + 2% FBS) for 10 mins followed by staining with anti-mouse CD71-PE/FITC and anti-mouse Ter-119-APC for 20 mins at 4°C [19,26–27]. …”

“…Intercellular reactive oxygen species (ROS) level was assessed as described before [19, 28–30]. Briefly, erythrocytes from peripheral blood and primary cells from BM and spleen were washed and resuspended in pre-warmed PBS supplemented with 2% FBS and incubated with CM-H 2 DCFDA (5(and 6-)-chloromethyl-2,7-dichloro-dihydrofluorescein diacetate) stain (5 μM) in the dark for 30 minutes at 37°C in an atmosphere of 5% CO 2 in air.…”

“…Briefly, erythrocytes from peripheral blood and primary cells from BM and spleen were washed and resuspended in pre-warmed PBS supplemented with 2% FBS and incubated with CM-H 2 DCFDA (5(and 6-)-chloromethyl-2,7-dichloro-dihydrofluorescein diacetate) stain (5 μM) in the dark for 30 minutes at 37°C in an atmosphere of 5% CO 2 in air. The oxidative conversion of CM-H 2 DCFDA to its fluorescent product by ROS was measured immediately by flow cytometry [19,28,30]. Intracellular ROS generation in different subpopulations of erythrocytes and erythroid precursor cells were determined by gating the cells on the basis of biotin label and CD71 stain, or Ter119 and CD71 stain, respectively.…”

“…It has been difficult to enumerate and study at any given time point the proportions of erythrocytes of various age groups in blood circulation. A new technique of double in vivo biotinylation (DIB technique) of erythrocytes developed recently in our laboratory has however made it possible to simultaneously enumerate and study erythrocytes of different age groups in blood circulation [13–19]. Aim of the present study was to look at the complete life cycle of erythroid cells including different stages of differentiation in bone marrow and spleen as well as erythrocytes of different age groups in blood circulation in order to identify stages that are preferentially destroyed in autoimmune hemolytic anemia.…”