Elimination of Young Erythrocytes from Blood Circulation and Altered Erythropoietic Patterns during Paraquat Induced Anemic Phase in Mice

Abstract

Paraquat a widely used herbicide causes a variety of toxic effects on humans and animals. The present study is focused on the interaction of paraquat with the mouse erythroid system. Administration of paraquat (10 mg/kg body weight i.p. on alternate days in C57Bl/6 mice) induced a significant fall in blood erythrocyte count on 7, 14, and 21 day time points but the erythrocyte count reverted back to normal by 28th day indicating the emergence of refractoriness to paraquat. A marked surge in the blood reticulocyte count was observed in paraquat treated mice that also subsided by 28th day. Young erythrocytes in circulation were randomly eliminated from blood circulation in paraquat treated mice and a significant elevation in the level of reactive oxygen species (ROS) was also observed maximally the erythrocytes of this age group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were identified and enumerated flow cytometrically based on their expression of Ter119 and transferrin (CD71) receptor. Proliferative activity of erythroid cells, their relative proportion as well as their absolute numbers fell significantly in bone marrow of paraquat treated mice but all these parameters were significantly elevated in spleens of paraquat treated mice. These changes were essentially restricted to the cells belonging to the two earliest stages of erythroid differentiation. Taken together our results indicate that paraquat treatment causes a transient anemia in mice resulting from random elimination of young circulating erythrocytes as well as depressed erythropoietic activity in bone marrow. Spleen erythropoietic activity however was elevated in paraquat treated mice.

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

“…We therefore examined ROS generation in the erythrocytes in mice with experimentally induced AIHA. ROS levels in circulating erythrocytes in control and AIHA-induced mice were estimated by staining with CM-H 2 DCFDA [19,28,30]. Results indicate a significant increase in the MFI of ROS fluorescence in the whole blood erythrocyte population in AIHA-induced mice (Fig 5 panels A and B).…”

“…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.…”

A Double In Vivo Biotinylation Technique to Assess Erythrocyte Turnover in Blood Circulation

“…In our experiment mice were administered repeated doses of Paraquat (Paraquat dichloride hydrate freshly dissolved in phosphate buffered saline (PBS), 10 mg/kg of body weight) intraperitoneally (i.p.) on alternate days [18,43]. Control mice received vehicle alone.…”

“…However in the case of anemia, where the homeostasis between erythrocyte destruction and erythropoiesis is disturbed, the erythrocyte turnover pattern may completely alter. To understand the fate of erythrocyte turnover in conditions of anemia, we studied four mouse models of experimental anemia, namely anemia induced by (a) toxic heavy metal cadmium (Cd) ions [17], (b) herbicide Paraquat (PQ) [18], (c) carbon nanotubes (CNTs) [19], and (d) autoantibody in autoimmune hemolytic anemia (AIHA) [20].…”