Lead uptake and toxicity in human erythroid precursor cells

Abstract

The mechanism of lead uptaket and toxicity in human erythroid precursor cells (EPCs) are the main aims of this research. Abnormal development of EPCs, the immature red blood cells, may involve with lead induced anemia. Human EPCs, the model in this study, were prepared by two-phase liquid culture (TPLC) technique. Highly purified EPCs (>90%) and substantial numbers of the cells (30.46+-19.48x10x10x10x10x10x10 cells/blood unit) were obtained from this technique. The late stage of EPCs obtained in day 12 of secondary phase were used for lead uptake study. The study was compared between human EPCs and erythrocytes in 1%FBS/alpha-MEM (pH 7.4) at 37 ํC, and showed that lead was rapidly incorporated into EPCs and reached the maximum value at 30 minutes. The lead content in EPCs was about 3-4 folds higher than that in erythrocytes. The rate of lead uptake into EPCs increased with extracellular lead concentration and time. Transferrin (Tf), plasma iron transport protein, could enhance lead uptake in EPCs but not erythrocytes. These findings indicate that besides iron, Tf may bind and deliver lead to EPCs through the receptor-mediated endocytosis. However, the increase of lead content by Tf in EPCs was only 26% (from 3.23 to 4.08 ng/10x10x10x10x10 cells). Thus, the mechanism involved with Tf may not be the major route of lead transport into these cells. The study of inhibitory effect of microtubule inhibitors which prevent if endocytosis (colchicine and vinblastine) and anion transport inhibitor (4,4'-Diisothiocyanostilbene-2,2'-disulfonate) showed that these inhibitors could inhibit lead uptake in EPCs. This suggests that at least two mechanisms have been involved in lead uptake in these cells. The first may be through Tf receptor-mediated endocytosis. The second route may be the anion exchanger system. However, the anion transport inhibitor provided a slight inhibition of lead uptake, suggesting that this may not be the major route of lead uptake in EPCs. By using TPLC system, the early stage of EPCs obtained on day 7 of secondary phase were cultured in the presence of lead acetate. Morphological study showed that lead could inhibit EPC survival by inducing the cell cytolysis and apoptosis. The inhibition was time and dose-dependent manner. Marked effect of lead on EPC survival was at lead acetate concentration >1 ppm. Flow cytometric analysis was used to detect apoptotic cells by monitoring the binding of fluorescence labeled annexin V to phosphatidylserine on the outer membrane of apoptotic cells. The study showed that lead could induce apoptosis in EPCs in time and dose-dependent manner at lead concentration >1 ppm. The findings in this study suggest new aspect of lead induced anemia besides the impairment of hemoglobin synthesis and shortened life span of erythrocytes, lead induced apoptosis in human EPCs resulting in the inhibition of EPC survival may be another mechanism of lead induced anemia.