Morphological Changes In Erythrocytes Research Articles

Hemolytic anemia, thrombosis, and infarction in male and female F344 rats following gavage exposure to 2-butoxyethanol

2-butoxyethanol (BE; ethylene glycol monobutyl ether) is used extensively in the manufacture of a wide range of domestic and industrial products which may result in human exposure and toxicity. BE causes severe hemolytic anemia in male and female rats and mice. In a recent report, female F344 rats exposed to 500 ppm BE by inhalation and sacrificed moribund on day 4 of treatment exhibited disseminated thrombosis associated with infarction in several organs. In contrast, no such lesions were observed in male rats similarly exposed to BE. Additional studies were therefore undertaken to compare the effects of BE in rats of both sexes. Rats received 250 mg BE/kg/day by gavage for 1, 2 or 3 days and were sacrificed 24 or 48 hr after the last dose. Control rats received 5 ml/kg water. Progressive time-dependent hemolytic anemia--macrocytic, hypochromic, and regenerative--was observed in both sexes of rats exposed to BE. Additionally, BE caused significant morphological changes in erythrocytes, first observed 24 hr after a single dose, including stomatocytosis, macrocytosis with moderate rouleaux formation, and spherocytosis. These morphological changes became progressively more severe as BE dosing continued and included the occasional occurrence of schistocytes and ghost cells, rouleaux formation in rats of both sexes, and an increased number of red blood cells with micronuclei in female rats. Overall, the progression of hemolytic anemia and morphological changes as a function of the number of days of exposure varied with gender and suggested a faster onset of hemolysis in female rats. The range of BE-related histopathological changes noted in both sexes was comparable; however, while these lesions were observed in female rats following a single dose, similar effects were first observed in males after 3 consecutive days of exposure to BE. Pathological changes involved disseminated thrombosis in the lungs, nasal submucosa, eyes, liver, heart, bones and teeth, with evidence of infarction in the heart, eyes, teeth and bones. Hemoglobinuric nephrosis and splenic extramedullary hematopoiesis were also noted. An apparent correlation between the severity of hemolytic anemia and subsequent disseminated thrombosis in BE-treated rats is proposed. Thrombosis may be related to intravascular hemolysis, which could be triggered by procoagulant release and/or alterations in erythrocyte morphology, as well as increased rigidity.

Rat erythrocyte morphological changes after gavage dosing with 2-butoxyethanol: a comparison with the in vitro effects of butoxyacetic acid on rat and human erythrocytes.

Rats exposed to 2-butoxyethanol (2-BE) develop hemolysis preceded by red blood cell swelling and shape changes. In this study effects on red blood cell morphology of dosing rats with 2-BE by gavage were compared with the effects of incubation of rat erythrocytes in vitro with the principal metabolite of 2-BE, butoxyacetic acid (BAA). Morphology was assessed by bright-field and phase microscopy of Wright's stained blood smears and glutaraldehyde-fixed cells suspended in plasma or buffer. In vivo exposure to 2-BE resulted in stomatocytosis and spherocytosis in blood smears and cup-shaped cells and spherocytosis in the fixed samples. In vitro incubation with BAA produced erythrocytes with cup shapes, spherocytosis and red blood cell ghosts in fixed samples. The stomatocytes observed in the blood smears appear to be the morphological equivalents of the cup-shaped cells observed in fixed samples. A variable degree of echinocytosis was observed in blood smears from animals exposed to 2-BE and in the in vitro experiments with BAA. Stomatocytes, cup-shaped cells, and spherocytes are the principal morphological features of erythrocytes from rats exposed to 2-BE or in vitro exposure to BAA. In comparison, human red blood cells incubated with up to 2.0 mM BAA exhibited none of the morphological changes observed in rat erythrocytes. 2-Butoxyethanol in vivo and BAA in vitro cause similar changes in rat red blood cell morphology, adding further evidence to support the primary role of BAA in the hemolytic effect of 2-BE exposure in the rat.

Toxicity of 4-chloroaniline in early life stages of zebrafish (Danio rerio): II. Cytopathology and regeneration of liver and gills after prolonged exposure to waterborne 4-chloroaniline.

Ultrastructural alterations in liver and gills of embryonic and larval zebrafish (Danio rerio) following prolonged exposure to waterborne 0.05, 0.5, and 5 mg/L 4-chloroaniline for up to 31 days as well as after a 14-day regeneration period were investigated by means of light and electron microscopy. Acute toxicity was also tested at 25 and 50 mg/L. Survival of zebrafish embryos and larvae was only impaired from 25 mg/L 4-chloroaniline, but-after a transient stimulation following exposure to 0.5 mg/L-4-chloroaniline hatching was retarded after exposure to >/=5 mg/L, and fish displayed increasing rates of abnormal development and pigmentation. In contrast, hepatocytes displayed a time- and dose-dependent response from 0.05 mg/L 4-chloroaniline, including changes in nuclei, mitochondria, peroxisomes, endoplasmic reticulum, Golgi fields, lysosomes, and hepatic glycogen and lipid stores, as well as invasion of macrophages. In gills, dose-dependent effects were evident from 0.5 mg/L 4-chloroaniline and included deformation of secondary lamellae due to vacuolization and desquamation of respiratory epithelial cells in conjunction with dilation of intercellular spaces. Respiratory epithelial cells displayed progressive mitochondrial changes, induction of cytoplasmic myelinated structures, augmentation of lysosomes, and modifications of Golgi fields. Erythrocytes were severely deformed. A 14-day regeneration period was sufficient for almost complete recovery of pathological symptoms in both liver and gills. Only minor volumetric changes in hepatocellular organelles and a limited number of myelinated bodies, lysosomes, and cytoplasmic vacuoles were reminiscent of prior 4-chloroaniline exposure. In both qualitative and quantitative terms, most effects in hepatocytes after exposure of embryonic and larval zebrafish to waterborne 4-chloroaniline are comparable to the reaction of hepatocytes in adult zebrafish liver after prolonged sublethal exposure as well as in larval zebrafish after microinjection. Morphological changes in erythrocytes indicate disturbance of respiration as an additional mode of action of 4-chloroaniline.

Induction of Acetylcholinesterase Release from Erythrocytes in the Presence of Liposomes

When human erythrocytes are incubated with liposomes, the release of acetylcholinesterase (AChE) occurs following an induction period [Cook et al. (1980) Biochemistry 19, 4601-4607]. However, the mechanism of the induction has not been elucidated. We examined the relationships among the lipid transfer from liposomes to erythrocytes, the morphological change of erythrocytes, the fluidity of the erythrocyte membrane and the start of AChE release. The AChE release into the liposomes and into shed-vesicle fractions started simultaneously after an induction period. The morphological index (MI) of erythrocytes was approximately 2.8 at the beginning of the release, regardless of the induction period. AChE was not released from the erythrocytes of index 2.8 even in the presence of liposomes if the MI remained at 2.8. Therefore, for the release, erythrocytes needed a further increase of the MI from 2.8. As the rate of lipid transfer increased, the induction period became shorter. No significant lipid release from erythrocytes was detected during the induction period. The initiation of the AChE release was not simply affected by the change in the membrane fluidity of erythrocytes upon interaction with liposomes. These results first demonstrate that AChE release into the shed-vesicle and liposome fractions is triggered by a further increase of the MI from 2.8, which is induced by lipid transfer from liposomes to erythrocytes.

Subchronic Toxicity of Sodium Chlorite in the Rat

Chlorine dioxide (ClO2) is currently used in many countries as a drinking water disinfectant. The chlorite ion (ClO2-) is the primary degradation product of ClO2 when it is used to treat drinking water. In this study, sodium chlorite was administered daily by gavage to rats for 13 weeks at dose levels of 0, 10, 25, or 80 mg/kg/day. At the conclusion of the study, all surviving animals were killed and subjected to a gross necropsy. Hematology, blood chemistry evaluations and urinalysis were performed. At 80 mg/kg/day, there were several treatment-related deaths and morphological changes in erythrocytes were observed. Mean erythrocyte count was decreased in both sexes. In males, hematocrit and hemoglobin levels were decreased and methemoglobin levels were increased. Splenic extramedullary hemopoiesis was observed in some animals at 80 mg/kg/day. At 25 mg/kg/day, methemoglobin was increased in males. No adverse toxicological effects were noted at 10 mg/kg/day.

Effect of chronic halothane exposure on lipid peroxidation, osmotic fragility and morphology of rat erythrocytes

Effect of chronic halothane exposure on hepatic and erythrocyte lipid peroxidation and erythrocyte osmotic fragility and morphology were determined in rats exposed to 0.4% halothane, 8 h per day for 40 days. Hepatic lipid peroxidation was increased in the halothane-treated group compared to controls. Lipid peroxidation was not increased by halothane exposure in erythrocytes without hydrogen peroxide, but after peroxide supplementation lipid peroxidation increased more in the erythrocytes of halothane-exposed rats than in control rats. We have observed significant morphological changes in erythrocytes from halothane-treated rats. In addition, erythrocytes of halothane-treated rats were more fragile in saline solutions compared to those of controls. Our results suggest that chronic halothane exposure is not only hepatotoxic but also affects erythrocyte membrane structure and stability.

Anaemia and changes in erythrocyte morphology associated with copper and selenium deficiencies and dietary restriction in rats

Both selenium and copper deficiencies in rats caused morphological changes in erythrocytes but only copper deficiency resulted in anaemia. Similar but less severe morphological changes occurred when rats consuming selenium- and copper-adequate diets had their food intake restricted to that of the copper-deficient animals. Since selenium deficiency did not affect the severity of changes due to copper deficiency this suggests that alterations of erythrocyte morphology may not be entirely due to impaired functions of selenium and copper in cell antioxidant systems. Thus, erythrocyte structure was sensitive to several dietary changes and such alterations are probably independent of the anaemia induced by copper deficiency. This possibility should be considered when changes in erythrocyte morphology are attributed to copper or selenium deficiency in animals which also have a restricted food intake.

Changes in ovine erythrocyte morphology due to sphingomyelin degradation by Corynebacterium pseudotuberculosis phospholipase D

Corynebacterium pseudotuberculosis produces a sphingomyelin-specific phospholipase D exotoxin that is a major determinant in the pathogenesis of caseous lymphadenitis. We assessed the changes induced in the morphology and sphingomyelin concentration of ovine erythrocytes after incubation with broth culture filtrates or purified exotoxin. Sphingomyelin was hydrolysed nearly in parallel with shape change. Morphologically, spherostomatocytes were seen initially, and later these contained numerous vacuoles at the periphery. Vacuoles seen in thin sections with transmission electron microscopy appeared as pits with scanning electron microscopy. Pitting became progressively worse with time, leading to extensive scalloping of the membrane surface. Chemically, significant decreases ( P ⩽ 0.05) in erythrocyte membrane sphingomyelin content and significant increases ( P ⩽ 0.05) in the content of glycerophospholipids (i.e. phosphatidylglycerol) were observed in erythrocytes incubated with broth culture filtrates or purified exotoxin. These alterations may contribute to the pathophysiology of acute infections induced by C. pseudotuberculosis.

Effects of l-carnitine and palmitoylcarnitine on membrane fluidity of human erythrocytes

Amphiphilic compounds such as long-chain acyl carnitine accumulate in ischemic myocardium and potentially contribute to the myocardial damage, and the role of carnitine in protecting the heart against ischemic damage is interesting. It has been reported that palmitoylcarnitine causes alterations in the membrane molecular dynamics, so this study was designed to investigate whether l-carnitine had a stabilizing effect of membrane fluidity using the spin-label technique. Human erythrocytes were spin-labeled with 5-doxylstearic acids, and membrane fluidity was quantified by measuring the change in the order parameter S. The administration of palmitoylcarnitine (100 μM) altered the membrane fluidity of erythrocytes and caused significant morphological changes. l-carnitine (2 mM) decreased the alteration of the fluidity of erythrocytes incubated with palmitoylcarnitine (100 μM), and improved the morphological changes in erythrocytes. These results show that l-carnitine has a stabilizing effect of membrane fluidity as a result of interaction with the palmitoylcarnitine which has a detergent effect.

Effect of pH on the velocity of erythrocyte aggregation.

The effect of pH on the velocity of aggregation of human erythrocytes was quantitatively examined with a rheoscope combined with a video-camera, an image analyzer and a computer, in relation to the morphological changes of erythrocytes and their aggregates. (i) With increasing pH of the medium, the velocity of erythrocyte aggregation increased. (ii) The rouleaux formed at high pH were longer in shape and more stable against the increase of shear rate than those formed at low pH. (iii) With increasing pH, the diameter of erythrocyte increased, the (maximum) thickness decreased, and the cell volume decreased. The pH dependency of erythrocyte aggregation may be mainly due to the morphological change of erythrocytes, and partly due to the changes of erythrocyte deformability and of interaction with macromolecules.

Morphological changes in erythrocytes induced by malarial parasites.

Host cell alterations induced by Plasmodium falciparum, P. brasilianum, P. vivax and P. malariae were described by electron microscopy and post-embedding immunoelectron microscopy. P. falciparum infection induces knobs, electron-dense material and clefts in the erythrocyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythrocyte membrane. P. falciparum antigen is present in knobs which adhere to endothelial cells causing the blockage of cerebral capillaries and ensuing pathological changes in cerebral tissues. P. brasilianum infection induces knobs, short and long clefts and electron-dense material. These structures appear to contain different P. brasilianum antigens. This indicates that each structure functions independently in trafficking P. brasilianum protein to the erythrocyte surface. P. vivax infection induces caveola-vesicle complexes and clefts in the erythrocyte. These structures are also involved in trafficking P. vivax protein from the parasite to the erythrocyte membrane. P. malariae induces caveolae, electron-dense material, vesicles, clefts and knobs in the erythrocyte. Although vesicles and caveolae are seen in the erythrocyte cytoplasm, they do not form caveola-vesicle complexes as seen in P. vivax-infected erythrocytes. They also appear to be involved in trafficking of malaria antigens. These studies, therefore, indicate that host cell changes occur in order to facilitate the transport of malarial antigens to the host cell membrane. The significance of these phenomena is still not clear.

The role of the cytoskeletal protein ankyrin in the discocyte—echinocyte morphological transition of human erythrocytes

Adriamycin is an aminoglycoside antibiotic that has potent antitumour activity in man. Although traditionally classified as a DNA intercalator, it can also interact with the plasma membrane of tumour cells (Tritton & Hickman, 1985) and there is evidence to suggest that these membrane interactions may contribute to its mechanism of action (Tritton & Yee, 1982). It is not clear how an antiproliferative agent may exert its effect via the plasma membrane. As a model of the interaction between Adriamycin and the plasma membrane, we have studied the effect of the drug on human erythrocytes. Currently, there is considerable interest in the mechanism whereby changes in erythrocyte morphology are controlled. The erythrocyte is converted from the discocyte to echinocyte form whether either ATP is depleted or calcium is introduced into the cell. We have found Adriamycin to be a potent inhibitor of this morphological transition under either condition (Chahwala & Hickman, 1985). Recent find-

Inhibition of inositol lipid metabolism by adriamycin

The aminoglycoside antibiotic adriamycin has potent antitumour activity in man. Although traditionally classified as a DNA intercalator, it is also able to interact with membranes, including the plasma membrane of tumour cells (Tritton & Hickman, 1985). Evidence to support the hypothesis that these membrane interactions may contribute to its mechanism of action comes from experiments using polymerimmobilized drug which was unable to interact with DNA, yet displayed a cytotoxicity equal to or greater than that of the free drug (Tritton & Yee, 1982). It is not clear how an antiproliferative agent may exert its effects via the plasma membrane. As a model of the interaction between adriamycin and the plasma membrane, we have studied the effect of the drug on erythrocytes. We have shown that adriamycin inhibits the morphological transition from the discocyte to echinocyte form undergone by erythrocytes under conditions of either ATP depletion or Ca2+ loading, in a concentrationand cell density-dependent manner (Chahwala & Hickman, 1985). There is considerable interest in the mechanism whereby changes in erythrocyte morphology are controlled (Gratzer, 1984). Recent findings suggest that the maintenance of phosphatidylinositol 4,s-bisphosphate [PtdIns(4,5)PZ] levels in the inner leaflet of the membrane may be necessary for the retention of discocyte morphology (Feuell& Heustis, 1984; Anderson & Marchesi, 1985). Adriamycin has a particular avidity for negatively charged phospholipids (Goormaghtigh & Ruysschaert, 1984) and since another aminoglycoside antibiotic, neomycin, is known to bind to and prevent the metabolism of PtdIns(4,5)PZ including that of the erythrocyte (Downes & Michell, 1981), we have investigated whether the maintenance of discocyte morphology by adriamycin was due to an inhibition of inositol lipid metabolism. Initially, we examined whether adriamycin was able to prevent phosphodiesteratic cleavage of the phosphoinositides in red blood cell vesicles (Downes & Michell, 1981). Separation of the inositol phosphates after stimulation of the phosphodiesterase for IOmin showed that in this assay, (0.6 mg of membrane protein/sample) adriamycin prevented inositol bisand tris-phosphate release (IC, .v 5 0 0 ~ ~ ) . Thus, having initially demonstrated that adriamycin was capable of preventing phosphoinositide breakdown in red cell vesicles, we wished to relate changes in morphology of intact cells to changes in phosphoinositides in response to adriamycin under conditions of ATP depletion. Phosphoinositides were labelled to isotopic equilibrium with 32P and the erythrocytes were then transferred to a substratefree medium and incubated for 12 or 48 h. Phosphoinositides were then extracted and analysed. After 12h of ATP depletion, PtdIns(4,5)P2 levels fell to 58 f 6% (n = 4) of the control level and after 48 h they fell to 7 6% (n = 2). Phosphatidylinositol4-phosphate levels did not change significantly after a 12 h period of ATP depletion but decreased after 48 h to 39 f 6% (n = 2) of their original level. The 12 and 48 h periods of ATP depletion both produced a change

Clinical and haematological features of haemangiosarcoma in dogs

Canine haemangiosarcoma was studied retrospectively in 31 cases recorded among 2,871 dogs presented for necropsy (1.08%). The German Shepherd breed was more frequently represented than other breeds. Affected dogs were older than 5 years (mean 9.1 years). Nineteen were males. Presenting signs often included episodic lethargy and weakness, with depression, anorexia and mucosal pallor. Spleen and lungs were the most frequently affected sites. Haematological findings in 9 dogs with splenic or hepatic haemangiosarcoma included a mild to moderate normochromic anaemia, neutrophilia, thrombocytopaenia, poikilocytosis and increased target cells. Acanthocytes occurred in 90% of cases, schizocytes in 80% and keratocytes and metarubyricytes in 70%. Fibrin split products were increased in 2 of the 3 cases in which they were measured. The changes in erythrocyte morphology are considered to be useful diagnostic features of canine haemangiosarcoma.

Ca<sup>++</sup> and Morphological Changes in Erythrocytes of stroke-prone SHR

Ca<sup>++</sup> and Morphological Changes in Erythrocytes of stroke-prone SHR

Is there any connection between heat inactivation of spectrin‐dependent ATPase and loss of smooth biconcave shape of red cells?

Spectrin-dependent ATPase activity was measured in membranes from native human erythrocytes and erythrocytes heated for 20 min at different temperatures. This activity was found to decline when the erythrocytes were heated at 48 degrees C and higher. The break in ATPase activity corresponds to morphological changes in erythrocytes found by Crome and Mollison [Brit. J. Haematol. (1964) 10, 137]. The role of spectrin-dependent ATPase in erythrocyte shape maintenance is discussed.

Morphological changes of erythrocytes in patients and carriers of Duchenne disease.

The morphology of erythrocytes was investigated in 7 patients with Duchenne dystrophy and in 6 carriers of the same disease. Upon immediate fixation, the erythrocytes showed significant stomatocytosis which was observed in a scanning electron microscope. The mean value of the % of stomatocytes was significantly different in carriers compared to healthy people (P less than 0.01).

Release of vesicles containing acetylcholinesterase from erythrocyte membranes by treatment with dilauroylglycerophosphocholine.

The shedding of acetylcholinesterase-enriched vesicles from erythrocytes of various species of animals occurred when cells were treated with C12:0PC. The response was observed shortly after a morphological change of erythrocytes without any accompanying detectable K+ leakage or hemolysis. The vesiculation was inhibited by the presence of serum albumin or by the incorporation of cholesterol into C12:0PC liposomes, indicating that the insertion of C12:0PC into the erythrocyte membrane causes the vesiculation. The ratio of C12:0PC to total phospholipid determined in vesicle fractions was almost the same as that observed in non-hemolyzed cell fractions. This finding suggests that the vesicles were not shed from portions of membranes rich in C12:0PC. The vesicles showed similar characteristics to those generated by ATP depletion; their diameter is 150-200 nm and they are enriched with acetylcholinesterase activity. Erythrocytes became denser when they lost acetylcholinesterase activity on treatment with C12:0PC.

Ageing of blood in hereditary spherocytosis.

Blood from 5 patients suffering from hereditary spherocytosis (HS) was stored in acid-citrate-dextrose anticoagulant at 4 degrees C for up to 6 weeks. The erythrocyte morphology and susceptibility to lysis by phospholipase C (Bacillus cereus) were examined at 2-weekly intervals and compared with erythrocytes from 6 different donations of stored normal blood. the hereditary spherocytes went through essentially the same series of morphological changes as did normal erythrocytes. Fresh hereditary spherocytes were very resistant to lysis by phospholipase C, but, like normal erythrocytes, became progressively more susceptible to lysis upon storage. In terms of erythrocyte morphological changes and development of susceptibility to lysis by the enzyme, blood from HS patients aged less rapidly than did normal blood. Splenectomy appeared to have no marked effect on the storage properties of blood from these patients as indicated by erythrocyte morphology and susceptibility to lysis by phospholipase C.

Lysis of erythrocytes from stored human blood by phospholipase C (Bacillus cereus).

The ability of phospholipase C (Bacillus cereus) to lyse erythrocytes from human blood that had been stored under Transfusion Service conditions for up to 16 weeks has been examined. When incubated at 20 degrees C with enzyme (0.03 mg/ml, 55 units/ml) for up to 1 h fresh erythrocytes were not lysed. After about 4 weeks of storage a population of very readily lysed erythrocytes appeared. The morphological changes in erythrocytes from blood stored up to 16 weeks were examined by scanning electron microscopy. The proportion of very readily lysed erythrocytes correlated well with the proportion of spheroechinocytes I. This morphological form was shown to be preferentially removed by phospholipase C and before lysis a transient appearance of smooth spheres occurred. The decrease in blood ATP concentrations on storage was measured and found to correlate with the disappearance of discoid erythrocyte forms, but not directly with the increased susceptibility of the erythrocytes to lysis by the enzyme. However, erythrocytes of up to at least 15 weeks of age could be made less susceptible to lysis by pre-incubation in a medium designed to cause intracellular regeneration of ATP. During the lysis of spheroechinocytes I by electrophoretically pure recrystallized phospholipase C a rapid degradation of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphatidylinositol) occurred together with a slower degradation of sphingomyelin.