How arsenic exposure seriously affects the lifespan of erythrocytes
Arsenic is a ubiquitous metalloid, which can be found in the atmosphere, soils, rocks and natural waters. Arsenic contamination in groundwater used for irrigation as well as human consumption or industrial activities has outgrown into an epidemiological problem, especially in some countries such as India, Bangladesh and South-East Asia.
A complete perception of arsenic contamination is complicated by the fact that arsenic exists in multiple oxidation states, each having a particular toxicology: there are mainly two toxic forms of inorganic arsenic, pentavalent arsenate (AsV) and trivalent arsenite (AsIII), which – after entering the human body by ingestion, inhalation or skin absorption – go through a biotransformation by reduction, methylation (resulting in the production of monomethylarsenate, MMA, and dimethylarsenate, DMA) and excretion of the metabolites into urine and bile since the main methylation products, MMA and DMA, are rapidly excreted (Relationship of urinary arsenic to intake estimates and a biomarker of effect, bladder cell micronuclei, 1997).
Chronic consumption of arsenic-contaminated drinking water is linked to many toxic effects, among which peripheral vascular disease and anaemia should be remembered: most of the ingested and inhaled arsenic is well absorbed through gastrointestinal tract and lungs into the bloodstream (A review of arsenic poisoning and its effects on human health, 1999), first of all targeting and attacking erythrocytes. In fact, clinical experience is consistent with the phenomenon of decreased haematocrit and intravascular haemolysis.
Since the Indian populations exposed to arsenic are mainly composed by farmers, businessmen, housewives, daily wage earners and students, drinking water is known to be the principal source of arsenic exposure, and thus it has been possible to study (Mechanism of erythrocyte death in human population exposed to arsenic through drinking water, 2008) the effects of arsenic exposure on people consuming well water having far more arsenic content than the current maximum contamination level laid down by WHO, i.e. 10 µg/l. As a parameter of evaluation for these effects, the urine arsenic level was chosen, and the results showed that it was significantly (p < 0.001) elevated in the exposed group in comparison to the unexposed group. As expected, haemoglobin (Hb) concentration was significantly lower (p < 0.001) in arsenic exposed individuals as compared with the control level.
Toxicity to erythrocytes is associated with alteration of cell shape, which leads to loss of cellular deformability (Increased resistance to membrane deformation of shape-transformed human red blood cells, 1987). Arsenic toxicity results in profound changes in the ultrastructure of erythrocytes: in the analysed exposed population, in fact, the normal discoid shape of the red cells is often lost since erythrocytes under stress condition adapt an anti-haemolytic tactic by changing their shape into the evaginated form with protruded spicules, known as echinocytes. This adaptive compensatory strategy of erythrocytes strives to increase the ratio between their membrane surface area and their cell volume, which helps the cells to accumulate large volume of water (Occurrence of echinocytes in circulating RBC of black bullhead, Ictalurus melas (Rafinesque), following exposure to an anionic detergent at sublethal concentrations, 2002). However, this strategy fails in the later stages of anaemia, when the spicules bud off irreversibly forming extracellular vesicles, leaving behind a more or less spherical body (spheroechinocyte) with decreased surface area to volume ratio.
Taking into account Bessis’s morphological classification (Red cell shapes. An illustrated classification and its rationale, 1973), we can see the recurring shape transformation of discocytic cells (normal, d) to echinocytes (e) and irreversibly altered spheroechinocytes (s) in the blood samples collected from the exposed population belonging to St 3 group. Distribution of transformed cells is shown in Fig. 1B: only discocytes are found in the unexposed control samples, whereas – along with the increase in the exposure – the number of echinocytes raises while the discocytes are decreasing. Being exposed to higher arsenic levels results in an aggravation of anaemia, with a substantial decrease in discocytes and a progressive appearance of spheroechinocytes. The appearance of spheroechinocytes (along with membrane vesiculation) in St 3 indicates the passage of the red cells of this group into their pre-lytic state. Moreover, positive correlation (Fig. 1C) between urine arsenic level and morphologic alteration of the affected cells (quantified by the percentage of echinocytes among the erythrocytes) clearly shows the arsenic-induced progressive structural alteration in erythrocytes, which is likely to increase their propensity towards haemolysis during chronic arsenic exposure.
Under physiological condition, red cells undergo marked reversible deformations as a visco-elastic material. Erythrocyte deformability is a basic rheological property of the cells and refers to the ability of the red cells to change their shape during their flow in the microcirculation (Changes in erythrocyte microrheology in patients with psoriasis, 2004). Arsenic exposure triggers changes in the cellular deformability, either as a result of increased membrane rigidity or decreased surface area to volume ratio. For this reason, one of the most important parameters is membrane microviscosity (η), which depends to a great extent on the orderly distribution of membrane lipids. A significant increase of membrane microviscosity due to arsenic exposure indicates loss of membrane fluidity (Fig. 2A), and this makes the membrane less deformable in the exposed group in comparison to that of the unexposed control. Under pathological conditions, such as arsenic exposure, selective decrease in membrane fluidity of the outer leaflet (owing to cholesterol enrichment) leads to the expansion of this leaflet in relation to the inner leaflet and therefore changes the curvature of cell membrane (Influence of cholesterol content on red cell membrane viscoelasticity and fluidity, 1983). The proportion between cholesterol and PLP (phospholipid) contents of cell membrane is responsible for regulating its flexibility: arsenic exposure leads to a significant (Fig. 2B, p < 0.05) elevation of cholesterol/PLP ratio as compared to the control value, with consequently enhanced membrane rigidity and, therefore, higher microviscosity and susceptibility to haemolysis.
Like in all cell membranes, the PLP of normal erythrocyte membrane maintain an asymmetric distribution in the lipid bilayer. By using a fluorescent dye (MC540) which binds to the PLP domain of the outer leaflet of the lipid bilayer, it has been possible to monitor the molecular packing of PLP in the outer leaflet of the red cell membrane during exposure to arsenic: the dye binds preferentially to the membrane with loosely packed lipids but not to more ordered lipid bilayers of normal erythrocytes (Merocyanine 540, a fluorescent probe sensitive to lipid packing, 1983). Significant increase (p < 0.001) in the binding of the dye with the cell membrane of the exposed population (Fig. 3B) when compared to those from the controls shows how arsenic induces loss of lipid asymmetry in the red cells, thus making the outer monolayer loosely packed in favour of MC540 binding. The substantial intercalation of the dye into the disordered outer layer of the exposed red cell membrane, as shown in Fig. 3A, confirms the disruption of lipid packing in these cells: these results suggest a link between the shape transformations in erythrocytes and the altered transbilayer asymmetry of membrane PLP in the exposed population. In these people’s erythrocytes, in fact, (phosphatidylserine (PS) is exposed on the outer face of the cell membrane, which is a typical marker resulting from perturbation of membrane aminophospholipid asymmetry.
The exposure of PS at the cell surface provides a signal for their recognition by macrophages, which stimulates eryptosis, an apoptotic-like process present in erythrocytes (Mechanisms of suicidal erythrocyte death, 2005). Macrophages are equipped with PS receptor, which mediate the engulfment of the eryptotic cells and trigger their removal from circulation (for further detail see Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages, 1997, and Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia, 1998). Perturbed erythrocyte membrane structure following arsenic exposure increases surface accessibility of PS and provides a signal for eryptosis. This can be explained according to bilayer couple concept of erythrocyte shape and enhanced transbilayer mobility of PLP (Erythrocytes: death of a mummy, 2001). Arsenic-mediated transformation of discocytic cells into echinocytes and spheroechinocytes might also be related to the reorientation of endofacial aminophospholipids to the outer leaflet of the bilayer. Cell shrinkage and PS exposure mimic some traits of apoptosis in nucleated cells (Physiology of apoptosis, 2000) and contribute to the process of erythrocyte cell death during arsenic exposure.
Significant decrease in ATP content (Fig. 4A) in the erythrocytes of the exposed participants as compared to their control level (p < 0.005) depicts the adverse effect of arsenic on energy status of red cells which is likely to induce cell membrane scrambling in the exposed population. Also a decline of red cell GSH after arsenic exposure (p < 0.005) can be recognised (Fig. 4B), thus pointing to an impaired antioxidant defense system as a predictable outcome of energy depletion.
Considering the contribution of energy depletion in the stimulation of eryptosis (Enhanced erythrocyte apoptosis in sickle cell anemia, thalassemia and glucose-6-phosphate dehydrogenase deficiency, 2002), the mechanism underlying arsenic-induced «eryptotic» removal of red cells has been thought to be the decrease of cytosolic ATP. Metabolic starvation due to loss of ATP has reflection on the impaired shape recovery of defective red cells, which is dependent on a continuous re-internalization of phosphatidilserine by means of an ATP-dependent phospholipid translocase (Electric field pulses induce reversible shape transformation of human erythrocytes, 1997). Development of altered morphology and decreased deformability of red cells in consequence to arsenic-induced ATP depletion might be associated with decreased blood flow, loss of oxygen and tissue damage caused by microvascular occlusion, which may eventually lead to arsenic-mediated circulatory disorders (for further detail see Metabolic dependence of red cell deformability, 1969, and Alteration of membrane deformability in haemolytic anemias, 1970). The alteration of cellular ATP metabolism due to toxic effects of arsenic is followed by significant reduction in intracellular GSH level, implicating a diminution in the effectiveness of antioxidant defense systems. Exhaustion of GSH levels plays a leading role in allowing oxidative stress to induce functional disintegrity in erythrocytes (Peroxidative reactions in red cell biology, 1982), making cell death inevitable during arsenic exposure.
As said above, a gradual fall in ATP and GSH levels is strictly related to arsenic exposure (for a graphical analysis see Fig. 4C). Significant enhancement (p < 0.01) of protein carbonylation (Fig. 5A) and lipid peroxidation (Fig. 5B) in the erythrocyte membranes of exposed individuals reflects the extent of oxidative damages as compared to the control levels. Fig. 5C validates the association between arsenic toxicity and oxidation of red cell membrane components during chronic exposure.
Therefore, energy depletion, cell shrinkage along with loss of lipid asymmetry and PS exposure appear to be the necessary preconditions, which are likely to activate eryptosis during arsenic exposure. However, mechanisms of arsenic-induced cell death also includes oxidative stress, morphologic alterations and loss of cell deformability which may interfere with the integrity of the cell membrane leading to membrane destabilization, cell swelling and Hb release: this triggers some events which eventually lead to a kind of necrotic (or necrotic-like) cell death.
In order to explore the contribution of membrane destabilization and red cell apoptosis in arsenic-induced abbreviation of red cell life span, it is possible to measure – as a suitable marker – Hb release and PS exposure: Fig. 6A and 6B reveal concurrent enhancement of both PS exposure (p < 0.001) and Hb release (p < 0.002) in comparison to their respective controls. Fig. 6C strengthens these observations and focus on the role of arsenic in the disruption of cell membrane integrity, which promotes a haemolytic response leading to the development of anaemia.
In order to conclude this item, we could summarise the concepts discussed above with this table, which schematically represents how altered cellular metabolism and membrane perturbation in erythrocytes lead – both through an apoptotic pattern and a necrotic one – to the development of anaemia during arsenic exposure.
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