Gladys Pérez

MORPHOLOGIC AND FUNCTIONAL ALTERATIONS OF ERYTHROID CELLS INDUCED BY LONG-TERM INGESTION OF ALUMINIUM

Anaemia has been associated with aluminium (Al) accumulation in plasma and/or bone tissue in patients with chronic renal insufficiency. Nevertheless, in previous works, we have found shortened red-cell life span, increased osmotic resistance and inhibition of colony-forming units-erythroid (CFU-E) development in Al-overloaded rats with normal renal function. To elucidate further the action of Al on in vivo erythropoiesis, aluminium citrate was provided to Sprague Dawley rats (n = 18) in the drinking water for 8 months. Significant decreases in haematocrit (38.8 +/- 4.29 versus 43.1 +/- 3.58%, p < 0.05) and blood haemoglobin concentration (137 +/- 10.1 versus 148 +/- 8.5 g/l, p < 0.05), reticulocytosis (1.8/1.3-4.2 versus 1.2/0.4-3.7%, p < 0.05), and severe inhibition of CFU-E growth (670/120-950 versus 1530/810-2440 CFU-E/2 x 10(5) cells, p < 0.005) were found. Anysocytosis, poikilocytosis and schistocytosis were detected in peripheral blood stained films. Scanning electron microscopy revealed the presence of erythrocytes with abnormal shape, including crenated and target cells. Aluminium was localised specially inside the schistocytes by EDAX analysis. Decreased haptoglobin concentration (107/83-127 versus 139/89-169 mg/l, p < 0.05) supports the assumption of haemolytic nature of the anaemia. Rats were not iron depleted, as plasma iron concentration and total iron binding capacity were found in the range of control values, and sideroblasts and haemosiderin deposits were observed in bone marrow smears. Total 59Fe uptake and 59Fe incorporated to haem by the bone marrow cells were found decreased. In conclusion, the erythropoiesis impairment induced by Al may be a combined effect of direct action on circulating erythrocytes and interference with the cellular iron metabolism in erythroid progenitors.

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein.

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.

Interference of aluminium on iron metabolism in erythroleukaemia K562 cells.

It has been suggested that aluminium (Al) has a deleterious effect on erythropoiesis. However, there is still uncertainty as to its action mechanism. The present work was designed to determine how Al could affect the iron (Fe) metabolism in the human erythroleukaemia cell line K562. These cells, that express surface transferrin receptors (TfRs), were induced to erythroid differentiation by either haemin or hydroxyurea in 72 h cultures in media containing apotransferrin (apoTf). In the presence of aluminium citrate, the number of benzidine-positive cells decreased 18% when the cultures were induced by haemin, and 30% when hydroxyurea was the inducer. Cell viability was always unaffected. From competition assays, surface binding of 125I-Tf-Fe2 was found to be inversely related (p < 0.05) to Tf-Al2 concentration (from 2.5 to 10 nM). The dissociation constants (Kd) of the binding reaction between TfRs and the ligands Tf-Fe2 and Tf-Al2 were calculated. Kd values of the same order of magnitude demonstrated that TfR has a similar affinity for Tf-Fe2 (Kd = 1.75 x 10(-9) M) and Tf-Al2 (Kd = 1.37 x 10(-9) M). The number of surface TfRs, measured by kinetic 125I-Tf-Fe2 binding assays, was higher in induced cells cultured in the presence of Al. Nevertheless, in spite of the inhibition of cell haemoglobinization observed, 59Fe incorporation values were not different from those measured in control cultures for 72 h. As a consequence, it can be suggested that cellular Fe utilisation, and not Fe uptake, might be the main metabolic pathway impaired by Al.

Disturbance of cellular iron uptake and utilisation by aluminium.

Aluminium (Al) affects erythropoiesis but the real mechanism of action is still unknown. Transferrin receptors (TfR) in K562 cells are able to bind Tf, when carrying either iron (Fe) or Al, with similar affinity. Then, the aim of this work was to determine whether Al could interfere with the cellular Fe uptake and utilisation. K562 cells were induced to erythroid differentiation by either haemin (H) or sodium butyrate (B) and cultured with and without Al. The effect of Al on cellular Fe uptake, Fe incorporation to haem and cell differentiation was studied. H- and B-stimulated cells grown in the presence of 10 microM Al showed a reduction in the number of haemoglobinised cells (by 18% and 56%, respectively) and high amounts of Al content. Al(2)Tf inhibited both the (59)Fe cellular uptake and its utilisation for haem synthesis. The removal of Al during the (59)Fe pulse, after a previous incubation with the metal, allowed the cells to acquire Fe quantities in the normal range or even exceeding the amounts incorporated by the respective control cells. However, the Fe incorporated to haem could not reach control values in B-stimulated cells despite enough Fe acquisition was observed after removing Al. Present results suggest that Al might exert either reversible or irreversible effects on the haemoglobin synthesis depending on cellular conditions.

Oral aluminum administration to rats with normal renal function. 2. Body distribution

Aluminum (Al) has no known physiological function and is not considered an essential dietary compound. Nowadays, it is recognized as an element that can produce adverse effects on biological systems. The present study determined Al partitioning in the body compartments of rats that have been orally exposed for 15 weeks. Three experimental groups were studied: Controls (C, n=19), TAl-1 (n=10) rats receiving daily doses of Al citrate (1.0 mmol/g body weight) by gavage and TA1-2 (n=13), receiving Al citrate with the drinking water (100 mmol/ l). At the end of the experimental period, the Al contents of organs and sera were determined. Results are expressed as median and range values. Comparing the TAl-2 rats with the control ones, remarkable Al accumulation could be observed in serum (4.8/2.7 - 16.3 vs 0.4/0.2 - 1.2 mmol/l, P50.001), bone (3.33/1.78 - 4.85 vs 1.00/0.48 - 1.59 mmol/ g, P50.001), kidney (2.33/0.96 - 3.15 vs 0.52/0.22 - 2.07 mmol/g, P50.001), spleen (2.22/0.70 - 4.19 vs 0.27/ 0.11 - 0.36 mmol/g, P50.001) and liver (0.60/0.42 - 0.91 vs 0.24/0.14 - 0.78 mmol/g, P50.01) while brain Al content was not significantly increased. Aluminum levels were raised in the TAl-1 group only in serum (2.8/1.3 - 10.4 mmol/ g, P50.001), bone (1.85/1.00 - 3.41 mmol/g, P50.001) and kidney (1.74/0.96 - 2.07 mmol/g, P50.01). Bone Al concentration increased in a dose-dependent manner (TAl-2 vs TAl-1, P50.001). The results demonstrate different tissue Al accumulation in rats chronically exposed to Al citrate, irrespective of their intact renal function.

Modulation of protein tyrosine phosphatase 1B by erythropoietin in UT-7 cell line.

Background/ Aims: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. Methods: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. Results: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). Conclusion: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo.