Influence of green tea on surface charge density and phospholipids composition of erythrocytes membrane in ethanol intoxicated rats

Ethanol is oxidized to acetaldehyde and then to acetic acid and these processes are acompanied by free radical generation. This paper reports the effect of green tea on electric charge and phospholipids composition of erythrocytes membrane from rats intoxicated with ethanol. Electrophoresis technique and HPLC have been applied to above-menthioned studies. Ethanol administration caused increase in erythrocyte membrane surface charge density and phospholipid composition. Ingestion of green tea with ethanol partially prevented changes in structure and function of membrane caused by chronic ethanol intoxication.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Mechanical lysis of human erythrocytes. Membrane stabilization by plasma proteins]

Mechanical properties of erythrocyte membranes play an important role in red cell functions. Stability of human erythrocytes under deforming mechanical tensions which occur in the rapidly moving fluid is studied. The activation energy of the mechanical hemolysis determined by the temperature dependence of the hemolysis rate is 55 + 7 kJ/mol. The fragility of erythrocytes rises sharply as the salt concentrations increase. Glutaric dialdehyde forms a certain number of interprotein bonds which increase the fragility of erythrocytes. The mechanical stability of the erythrocyte membrane falls at high (0.5 M) ethanol concentrations. Blood plasma proteins, particularly human serum albumin, have a pronounced stabilizing effect. The hemolysis occurring during the rapid mixing is not probably associated with an osmotic mechanism since high sucrose concentrations do not prevent this process. The mechanical hemolysis depends both on the deforming tension arising in the membrane and on the state of the erythrocyte membrane.     

Effect of hypervitaminosis A on hemolysis and lipid peroxidation in the rat

Erythrocytes from rats fed large doses of Vitamin A alone, or large doses of vitamin A and vitamin E or diphenyl-p-phenylene diamine (DPPD) were studied for H2O2-induced hemolysis. The vitamin A-dosed rats were more susceptible than normal rats to H2O2-induced hemolysis. Hemolysis was not accompanied by lipid peroxidation. Nevertheless, the antioxidants vitamin E and DPPD inhibited hemolysis in erythrocytes from vitamin A-dosed rats. These antioxidants had the same inhibitory effect when they were included in the diet or added to erythrocyte suspensions in vitro. Erythrocytes from vitamin A-dosed rats with or without added vitamin E or DPPD were less susceptible than the erythrocytes from normal rats to osmotic challenge, showing that vitamin A was present in levels sufficient to alter the structure of the erythrocyte membrane. These studies show that oxidative hemolysis occurs when the erythrocyte membrane is modified. Furthermore, this oxidative hemolysis is unrelated to lipid peroxidation.     

The protective effects of lidocaine on human erythrocytes stored for seven days at 04 degrees C

Erythrocyte storage may result in cell damage due to an alteration of membrane integrity, which results in potassium efflux and hemolysis. Lidocaine has been shown to protect erythrocytes from oxidative stress by a possible membrane effect. We conducted this study to examine the effects of lidocaine on human erythrocyte storage. Erythrocytes were kept for seven days at 04 degrees C in the absence or in presence of plasma, and of lidocaine at 36.9 and 221.6 microM. Cell damage was assessed by measuring potassium efflux in the supernatant after seven days, and studying potassium efflux and hemolysis induced by oxidative stress. As expected, erythrocyte storage in the presence of plasma was less deleterious. Lidocaine decreased potassium efflux after 7 days' storage. Resistance toward oxidative stress was greater when the erythrocytes had been kept in the presence of plasma. Considering that lidocaine is widely used in various clinical situations, this data may be of clinical relevance.     

Changes in the blood of Wistar rats in acute poisoning with ethanol and acetaldehyde

1. The purpose of the study was to investigate the effect of ethanol and acetaldehyde on the erythrocyte and leucocyte system of Wistar rats. 2. Administration of the ethanol or acetaldehyde caused a considerable drop in the number of erythrocytes, haemoglobin level and haematocrit value in rats. 3. The mean erythrocyte volume was smaller after only 0.5 hr of exposure to ethyl alcohol. 4. The solutions used caused changes in the leucocyte system expressed in distinct neutrophilic leucocytosis. 5. Changes in the leucogram were reflected in the increase in the leucocyte index. 6. The degree of intensity of changes in both the erythrocyte and leucocyte system point to the greater toxicity of ethyl alcohol intoxication than is the case of acetaldehyde in a toxically corresponding dose (i.e. 0.5 and 5 g/kg body wt respectively).     

Ethanol metabolism in suspension cultured carrot cells

Ethanol production in plant tissues deprived of oxygen is a well known process. Nevertheless, little information is available on the toxic effects of ethanol on plant cells and tissues, or on the possible role of acetaldehyde, the first oxidative product of ethanol, in inducing toxic effects in plants. Data on the metabolism of ethanol in suspension cultured cells of carrot ( Daucus carola L. cv. S. Valery, cell line T22), a system highly sensitive to the presence of ethanol in the culture medium, indicate that carrot cells oxidize only small amounts of ethanol to CO₂. Instead, they convert ethanol mainly to acetaldehyde, which accumulates in the culture medium. This suggests a possible role of acetaldehyde in causing ethanol-induced injury to carrot cells.     

Erythrocyte and plasma protein modification in alcoholism: a possible role of acetaldehyde

Analysis of the oxidative modification of plasma and erythrocyte ghost proteins of chronic alcoholic subjects and healthy non-alcoholics has been performed. It was found that increased levels of protein carbonyls in both plasma and erythrocyte ghosts from alcoholic subjects occurred in comparison to the levels found in preparations from non-alcoholics. Plasma proteins from alcoholic subjects did not show evidence of cross-linking, although plasma protein concentration and composition were changed. In alcoholic subjects who displayed no evidence of abnormal erythrocyte morphology no cross-linking of erythrocyte ghost proteins was detectable, whereas the ghosts obtained from alcoholic subjects who displayed morphologically abnormal erythrocytes contained cross-linked proteins. The in vitro treatment with acetaldehyde of erythrocytes from non-alcoholics caused increased levels of protein carbonyls and cross-linking products in erythrocyte ghost preparations which were similar to those found in severe alcoholics. It is concluded that chronic alcohol consumption can cause abnormal erythrocyte morphology and increased erythrocyte fragility as a result of oxidation and cross-linking of erythrocyte ghost proteins. These effects can be ascribed, in part, to exposure of erythrocytes to circulatory acetaldehyde which is a product of ethanol metabolism.     

Hypoosmotic hemolysis of erythrocytes by active carbonyl forms

Low-molecular-weight dicarbonyls formed during free radical peroxidation of polyene lipids (malondialdehyde) and autooxidation (glyoxal) or other oxidative transformations of glucose (methylglyoxal) are able to modify the structure of lipid-protein supramolecular complexes of cells. We investigated changes in the erythrocyte membrane structure after an 18-h exposure of human red blood cells in the presence of various natural dicarbonyls. The changes in the mechanical properties of the membrane after membrane modification by carbonyls were evaluated by the susceptibility of erythrocytes to hypoosmotic hemolysis. It has been shown that treatment of red blood cells with malondialdehyde increases the resistance of these cells to hypoosmotic hemolysis, whereas the malondialdehyde isomer, methylglyoxal, in contrast, makes red blood cells more sensitive to the action of hypoosmotic solutions. Paradoxically, a homologue of malondialdehyde, glyoxal, has no effect on hemolysis of red blood cells in hypoosmotic solutions. The findings point to the possibility of the multidirectional effect of low-molecular-weight dicarbonyls with similar structures on the structure and function of biological membranes.     

Effect of chronic ethanol or acetaldehyde on hepatic alcohol and aldehyde dehydrogenases, aminotransferases and glutamate dehydrogenase

Ethanol or acetaldehyde orally administered (15% and 2% respectively in drinking water) to male Wistar rats for three months induced alterations in the main liver enzymes responsible for ethanol metabolism, aspartate and alanine aminotransferases and NAD glutamate dehydrogenase. Ethanol produced a significant decrease in the activity of soluble alcohol dehydrogenase, while acetaldehyde induced alterations both in soluble and mitochondrial aldehyde dehydrogenases: soluble activity was significantly higher than in the control and ethanol-treated groups, and mitochondrial activity was significantly diminished. Both soluble aspartate and alanine aminotransferases showed pronounced increases by the chronic effect of acetaldehyde, while mitochondrial activities were practically unchanged by the effect of ethanol or acetaldehyde. Mitochondrial NAD glutamate dehydrogenase showed a rise in its activity both by the effect of chronic ethanol and acetaldehyde consumption. The level of metabolites assayed in liver extracts showed marked differences between ethanol and acetaldehyde treatment which indicates that ethanol produced a remarkable increase in glutamate, aspartate and free ammonia together with marked decrease in pyruvate and 2-oxoglutarate concentrations. Acetaldehyde consumption induced a significant decrease in 2-oxoglutarate and pyruvate concentrations. These observations suggest that ethanol has an important effect on the urea cycle enzymes, while the effect of acetaldehyde contributes to the impairment of the citric acid cycle.     

Toxicity of ethanol and acetaldehyde in hepatocytes treated with ursodeoxycholic or tauroursodeoxycholic acid

In hepatocytes ethanol (EtOH) is metabolized to acetaldehyde and to acetate. Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) are said to protect the liver against alcohol. We investigated the influence of ethanol and acetaldehyde on alcohol dehydrogenase (ADH)-containing human hepatoma cells (SK-Hep-1) and the protective effects of UDCA and TUDCA (0.01 and 0.1 mM). Cells were incubated with 100 and 200 mM ethanol, concentrations in a heavy drinker, or acetaldehyde. Treatment with acetaldehyde or ethanol resulted in a decrease of metabolic activity and viability of hepatocytes and an increase of cell membrane permeability. During simultaneous incubation with bile acids, the metabolic activity was better preserved by UDCA than by TUDCA. Due to its more polar character, acetaldehyde mostly damaged the superficial, more polar domain of the membrane. TUDCA reduced this effect, UDCA was less effective. Damage caused by ethanol was smaller and predominantly at the more apolar site of the cell membrane. In contrast, preincubation with TUDCA or UDCA strongly decreased metabolic activity and cell viability and led to an appreciable increase of membrane permeability. TUDCA and UDCA only in rather high concentrations reduce ethanol and acetaldehyde-induced toxicity in a different way, when incubated simultaneously with hepatocytes. In contrast, preincubation with bile acids intensified cell damage. Therefore, the protective effect of UDCA or TUDCA in alcohol- or acetaldehyde-treated SK-Hep-1 cells remains dubious.     

The effect of ethanol and acetaldehyde on [14C]pantothenate incorporation into CoA in cultured rat liver parenchymal cells

The effect of ethanol on [¹⁴C]pantothenate incorporation into CoA and on total CoA levels was measured in 3-day-old primary cultures of adult rat liver parenchymal cells. Ethanol decreased the incorporation of radioactivity into CoA a maximum of 67%, 5 mm ethanol was saturating for the inhibitory effect and 0.2 mm ethanol was sufficient for half-saturation. This inhibitory effect did not result from a loss of CoA precursors or from cell death. Ethanol concentrations up to 10 mm did not decrease the ATP content of cells or the total protein content of cells which adhered to the incubation flask. Ethanol (5 mm) had no effect on the cyteine + cystine content of the cells. Intracellular pantothenate concentrations were not affected by 5 mm ethanol, and increasing the pantothenate concentration did not affect ethanol inhibition. Ethanol inhibition of [¹⁴C]pantothenate conversion to CoA could be fully reversed by rinsing the cells free of ethanol. The ethanol inhibition could also be fully reversed by addition of 4-methylpyrazole, indicating that ethanol must be oxidized via alcohol dehydrogenase to exert its inhibitory effect. Acetaldehyde, the immediate product of alcohol dehydrogenase, was also an inhibitor of the incorporation of [¹⁴C]pantothenate into CoA; the maximum inhibition was 63%. Acetaldehyde concentrations maintained between 18 and 103 μm inhibited incorporation by 57%. The inhibition by acetaldehyde did not correlate well with changes in the NADH and NAD⁺ ratio of the cells (as determined by measuring changes in the lactate-to-pyruvate ratio). The ability of glucagon, dibutyryl cAMP + theophylline, or dexamethasone to stimulate [¹⁴C]pantothenate conversion to CoA was not decreased by the addition of ethanol or acetaldehyde, indicating that ethanol inhibition does not occur by reversal of the cAMP-mediated regulatory mechanism for CoA biosynthesis.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

Plasmodium falciparum activates endogenous Cl(-) channels of human erythrocytes by membrane oxidation.

Intraerythrocytic survival of the malaria parasite Plasmodium falciparum requires that host cells supply nutrients and dispose of waste products. This solute transport is accomplished by infection-induced new permeability pathways (NPP) in the erythrocyte membrane. Here, whole-cell patch-clamp and hemolysis experiments were performed to define properties of the NPP. Parasitized but not control erythrocytes constitutively expressed two types of anion conductances, differing in voltage dependence and sensitivity to inhibitors. In addition, infected but not control cells hemolyzed in isosmotic sorbitol solution. Both conductances and hemolysis of infected cells were inhibited by reducing agents. Conversely, oxidation induced identical conductances and hemolysis in non-infected erythrocytes. In conclusion, P.falciparum activates endogenous erythrocyte channels by applying oxidative stress to the host cell membrane.     

Influence of the Use of Statin on the Stability of Erythrocyte Membranes in Multiple Sclerosis

Multiple sclerosis (MS) probably occurs by oxidative, inflammatory and autoimmune mechanisms. This study investigated the influence of statin on the stability of erythrocyte membranes in MS patients. The population was composed of one group with simvastatin therapy (20 mg/day), another group without statin therapy and a healthy control group. The stability of erythrocytes was evaluated by the half-transition points, H₅₀ and D₅₀, obtained from the curves of hemolysis induced by hypotonic shock and ethanol action, respectively. Erythrocytes of MS patients were less stable against lysis by both chaotropes. This behavior may be merely a consequence of the lifestyle of MS patients or it may be intrinsically associated with the conjunct of factors responsible for the development of the disease. The use of statin by MS patients was associated with lower levels of LDL and total cholesterol, as expected, and with higher stability of erythrocytes against ethanol compared to the values of untreated MS patients.     

Inhibition of acetaminophen activation by ethanol and acetaldehyde in liver microsomes

Mechanisms of the inhibitory effect of ethanol on acetaminophen hepatotoxicity are controversial. We studied the effects of ethanol and acetaldehyde, an oxidative metabolite of ethanol, on NADPH-dependent acetaminophen-glutathione conjugate production in liver microsomes. Ethanol at concentrations as low as 2mM prevented the conjugate production noncompetitively. Acetaldehyde also inhibited acetaminophen-glutathione conjugate production at concentrations as low as 0.1mM that is comparable with those observed in vivo after social drinking. Acetaldehyde may be involved in ethanol-induced inhibition of acetaminophen hepatotoxicity.     

Ferriprotoporphyrin IX and cell lysis: a protective role for hydrogen peroxide

Two potentially lytic substances, ferriprotoporphyrin IX (FP) and hydrogen peroxide, may coexist and partially detoxify each other in sickle cells and in erythrocytes infected with malaria parasites. Since hydrogen peroxide can decompose FP, its effect on hemolysis induced by FP and by the complex of FP with chloroquine was investigated. Human erythrocytes suspended at a concentration of 0.5% in a 50 microM solution of FP underwent approximately 42% hemolysis during the course of 2 hours. Twenty-five micromolar chloroquine potentiated hemolysis to 99%, and preincubation of 50 microM FP with 25 microM hydrogen peroxide for 5 minutes reduced hemolysis to 4%. Mixing either FP or hydrogen peroxide first with chloroquine abolished the effect of hydrogen peroxide. Detoxification of FP by hydrogen peroxide may be an important protective mechanism in certain hemolytic anemias, and inhibition of detoxification could account for the effectiveness of chloroquine in malaria.     

Protective effect of green tea on erythrocyte membrane of different age rats intoxicated with ethanol

It is known that aging is characterized by changes in cell metabolism resulting in modification of the structure and function of cell membrane components which is mainly the consequence of reactive oxygen species action. These disturbances are also enhanced by different xenobiotics, e.g. ethanol. Therefore, the aim of this paper is to examine green tea influence on total antioxidant status (TAS) and on composition and electric charge of erythrocyte membrane phospholipids in ethanol intoxicated rats of various ages. Antioxidant abilities of erythrocytes were estimated by measuring TAS. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC, while the extent of erythrocytes lipid peroxidation was estimated by HPLC measurement of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels. Electrophoresis was used to determine the surface charge density of the rat erythrocyte membrane. It was shown that the process of aging was accompanied by a decrease in TAS and in the total amount of phospholipids as well as by enhancement of lipid peroxidation and increase in surface charge density of erythrocyte membrane. Ethanol administration caused, in term, decrease in TAS and increase in the level of all phospholipids and lipid peroxidation products. Ethanol as well significantly enhanced changes in surface charge density of erythrocyte membrane. The ingestion of green tea partially prevented decrease in erythrocyte antioxidant abilities observed during aging and ethanol intoxication. Moreover, long-term drinking of green tea protects the structure of the erythrocytes membrane disturbed during aging process and/or chronic ethanol intoxication.     

Effect of exogenous antioxidants on erythrocyte redox status and hepcidin content in disorders of iron metabolism regulation

In many diseases associated with impairments in iron metabolism, erythrocytes exhibit an increased sensitivity to oxidative stress induced in vitro. In this study we have examined the antioxidant status of erythrocytes from healthy donors and from 12 patients with disorders of iron homeostasis by measuring the extent of hemolysis induced in vitro by tert-butyl hydroperoxide (t-BHP). The extent of hemolysis observed with patient erythrocytes was significantly higher than that observed in experiments with erythrocytes from healthy donors. After therapeutic infusions of the antioxidants mexidol or emoxypin, oxidative hemolysis in patients was restored to normal values and blood hepcidin increased significantly as compared with its initial level. A significant correlation was observed between hepcidin concentration after treatment and t-BHP-induced hemolysis before treatment. These data suggest that antioxidants may exert a favorable effect on those at risk for iron overload disease.