Graciela Garbossa

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.

Oral aluminum administration to rats with normal renal function. 1. Impairment of erythropoiesis

Aluminum (Al) toxicity has been mainly investigated in uremic patients although healthy subjects and patients without renal insufficiency are not exempt from its potential deleterious effects. This experimental study aims to elucidate the action of different doses of Al citrate on in vivo erythropoiesis and find out whether the metal exerts a local toxic effect upon the bone marrow late erythroid progenitor cells. The groups in the first experimental series were: C1 (n=5) controls and TAl-1 (n=5) rats receiving 1 mmol Al citrate/g body weight/day by gavage. Colony-forming units-erythroid (CFU-E) development was inhibited in the TAl-1 group, but the median osmotic fragility (MOF) and hematocrit (Ht) values were similar to those of the C1 group. The groups in the second series were C2 (n=5) controls and TAl-2 (n=5) rats receiving Al citrate in drinking water (100 mmol/l). The TAl-2 group showed decreased Ht, hemoglobin concentration, MOF and red blood-cell life-span values (P50.05), and a marked inhibition of the CFU-E development (P50.01). Serum and bone Al concentrations were increased in both Al-treated groups (P50.01). There was a dose-dependent increase in bone Al levels (P50.01) and a dose-dependent decrease of CFU-E development (P50.05). The CFU-E development was inversely correlated with the bone Al content (r=70.79; P50.05). The results demonstrate that even very low doses of Al citrate impair erythropoiesis in vivo and higher doses exert a deleterious action on both CFU-E and mature erythrocytes. This might show a local effect of Al on CFU-E caused by the bone sensitivity to the metal accumulation.

Glycosaminoglycans in Urolithiasis

To determine if there are differences in urinary glycosaminoglycan (GAG) concentrations, 43 stone-forming patients and 37 healthy control subjects of both sexes were studied. Urinary concentrations of calcium, magnesium, creatinine, uric acid and GAGs were determined. GAGs were measured by the Di Ferrante precipitation procedure followed by the Bitter and Muir reaction. Urinary GAG concentration and daily output were significantly lower in stone-forming patients. The present study clearly demonstrates the decreased urinary GAG concentration and excretion in stone-forming patients and suggests an interaction between GAGs and urate that could modify the inhibitory potency of GAGs.

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.

Aluminium accumulation in chronic renal failure affects erythropoiesis

Summary: Aluminium (Al) toxicity has been associated with anaemia in exposed patients with chronic renal failure (CRF). the present study was undertaken to determine whether the ingestion of Al citrate was able to affect erythropoiesis in rats with normal or impaired renal function. the renal insufficiency was induced by surgical procedures and control rats were sham operated. Twenty‐four rats were allocated to four groups of six rats each: (A) Sham; (B) Sham+Al; (C) CRF; and (D) CRF+Al. the groups B and D received daily doses of Al citrate (0.5 μmol/g bodyweight) and the groups A and C, deionized water, via the intragastric route. At the end of the experimental period (15 weeks) cultures of late erythroid progenitor cells (CFU‐E) stimulated with erythropoietin were performed and haematological parameters determined. the liver, kidney, brain, bone and serum Al amounts were quantified. the results are expressed as median and interquartile range. the CFU‐E growth was found inhibited in B and D groups (A: 100; B: 74/54‐83; C: 86/54‐98; D: 46/39‐53 %). the haematocrit values were significantly diminished in rats with renal insufficiency when compared to controls (A: 42/40‐43; B: 45/42‐46; C: 37/32‐40 and D: 37/24‐39 %). Serum Al accumulation was observed in B and D groups receiving Al (A: 8/5‐12; B: 36/36‐44; C: 5/5‐6; D: 45/26‐132 μg Al/l). No differences among groups were found in the liver and kidney Al contents, but uraemic state favoured Al accumulation in brain (A: 6/5.0‐9.0; B: 4/3.8‐4.3; C: 2/1.0‐3.0; D: 15/12.0‐21.0 μg Al/g tissue) and bone (A: 29/27‐31; B: 30/29‐39; C: 42/33‐48; D: 68/56‐79 μg Al/g tissue). We suggest that the heavy accumulation of Al in the bone compartment may result in a protracted endogenous exposure of bone marrow cells, affecting the erythropoiesis in vivo.

Social and environmental health determinants and their relationship with parasitic diseases in asymptomatic children from a shantytown in Buenos Aires, Argentina

Abstract Health inequities are a common problem for all countries and are the result of not only adverse social conditions but also poor public policies. Today chronic diseases represent the most relevant threats and are a current challenge. Parasitic infections, a leading cause of child morbidity affecting low-income populations, can be transmitted because of an unhealthy environment. Notwithstanding, scarce data have been published on the epidemiological profile of intestinal parasitoses in asymptomatic children living in shantytowns. Vulnerable populations settled in slums are growing in Argentina, particularly in Buenos Aires city. Consequently, this work intended to screen healthy carriers of enteric parasites and determine the epidemiologic profile in asymptomatic children residing in one of those communities, to explore risk factors associated with the transmission of parasites, and to initiate a basic health education campaign to promote healthy behavior in the community. Fecal samples (n = 138) were analyzed by conventional parasitological methods and a survey gathered data on symptoms, family composition, and environmental and hygiene-related variables. High prevalence of feco-orally-transmitted parasitoses (83·3%) and polyparasitism were remarkable findings. The main environmental health determinants were those related to excreta disposal and water provision. Health promotion actions were performed through the diffusion of a set of posters with iconic images and brief messages for health education. Results suggest the need for an environmental sanitation policy to complement health promotion actions. It is essential to spread the results of investigations that address inequities and social determinants of health in order to integrate data with local political processes and alert on acceptable actions for developing appropriate interventions.

Dissimilar behavior of lymph cells in response to the action of aluminum. In vitro and in vivo studies

In order to detect possible immunological effects of aluminum (Al) on lymph cells, mice were orally overloaded with pharmacological doses for 22 weeks. The in vitro response of lymph cells to mitogens (phytohemmaglutinin, PHA and concanavaline A, Con A) was examined at the end of the treatment. The chronic ingestion of Al affected the lymphatic nodes that were found to be 2- to 10-fold larger than those of the control mice. Concurrently, the in vitro proliferation of lymphatic node cells was found enhanced, while spleen cell cultures were unaffected. An acute direct action of Al on lymph cells from different sources was also examined. The blastogenic response to PHA of human peripheral lymphocytes was not disturbed by the presence of Al concentrations ranging from 0.09 to 900 microM. However, the response of mouse lymph cells was quite different, given that an Al dose-dependent inhibition was observed for lymphatic node cells, whereas for spleen cells the inhibition was only detected at Al concentrations higher than 90 microM. This work shows that Al might induce alterations in cell immune responses. The opposite results observed in mouse lymphatic node cells after in vitro and in vivo Al treatment, let us suggest that either the stimulating or suppressing effects of Al on the immune system might depend on the dose, route of administration and length of exposure, as well as on the cell population assayed.