Characterization of acatalasemic erythrocytes treated with low and high dose hydrogen peroxide. Hemolysis and aggregation

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.     

Glucose-6-phosphate dehydrogenase deficiency: a novel role for thyroxine in peroxide-induced hemolysis

G-6-PD deficient erythrocytes undergo hemolysis during exposure to hydrogen peroxide and thyroxine. Normal erythrocytes do not show this effect unless incubated in the absence of glucose. The inability of G-6-PD deficient cells to detoxify hydrogen peroxide apparently exposes them to the cytotoxic actions of thyroxine. It is suggested that the hemolysis and anemia associated with this genetic disorder is a consequence of the actions of both peroxide and thyroxine on erythrocyte membranes.     

Ferriprotoporphyrin IX binding substances and the mode of action of chloroquine against malaria

Bovine serum albumin and preparations of cell sap from malaria parasites and normal erythrocytes were tested for ability to protect cellular membranes against the toxicity of ferriprotoporphyrin IX (FP) and a chloroquine-FP complex. Suspensions of $\text{Plasmodium berghei}$ (approximately 7 × 10⁶ parasites per ml, isolated from saponin-lysed, infected erythrocytes) were used as a test system. Toxicity was monitored by measuring changes in turbidity of these suspensions at 700 nm. Parasite cell sap (0.56 mg protein per ml) and albumin (1 mg per ml) completely prevented the toxicity of 40 μM FP. Erythrocyte cell sap (8.6 mg of hemoglobin per ml) provided only partial protection from 40 μM FP. Neither the cell sap preparations nor albumin eliminated the toxicity of a chloroquine-FP complex formed from 20 μM chloroquine and 40 μM FP. These observations suggest that the cell sap preparations contain FP binding substances and that the mode of action of chloroquine may be to shunt FP away from a nontoxic complex with these substances and into a toxic chloroquine-FP complex.     

Heme polymerase: modulation by chloroquine treatment of a rodent malaria.

The biosynthesis of the beta-hematin of malarial pigment (hemozoin) is catalyzed by a newly discovered enzyme, heme polymerase, which is described for Plasmodium berghei in this report. This novel enzyme is present in the insoluble fraction of hemolysates of infected erythrocytes but is not present in normal erythrocytes. The substrate is ferriprotoporphyrin IX (FP) released from hemoglobin. At pH 5 and 37 degrees C the enzyme is saturated by 100 microM FP. The pH optimum is between 5 and 6 and the reaction is linear for 6 hours. All heme polymerase activity is destroyed by heating at 100 degrees C for 3 minutes. Chloroquine treatment of malarious mice reduces by 80 percent the activity of this enzyme, without inhibiting release of FP from hemoglobin, and thereby causes excess nonpolymerized, nonhemozoin FP to accumulate. Since the accumulated FP is accessible to bind chloroquine, we propose that it is the mediator of the antimalarial activity of chloroquine.     

Plasmodium falciparum malaria mimicking autoimmune hemolytic anemia during pregnancy.

A 30-year-old woman contracted Plasmodium falciparum malaria in the first trimester of her pregnancy while taking chloroquine for malaria prophylaxis. Her illness was characterized by hemolytic anemia with IgG1 coating of the surface of the erythrocytes and IgG3 in her serum. The hemolysis subsided following treatment of the malaria infection early in the third trimester. She delivered at term an infant who had hypoplasia of the right tibia and fibula and absence of the fifth ray of the right foot. The hemolytic process was attributed to the malaria infection, and the birth defect may have been related to the antimalarial therapy in the first trimester of pregnancy.     

Chloroquine accumulation by erythrocytes: a latent capability

Treatment of normal mouse erythrocytes with a nonspecific protease from streptomyces griseus activates a saturable process for chloroquine accumulation. The apparent dissociation constant for the interaction of chloroquine with the saturable component of this latent process is 50 nM. This value is similar to that of a high-affinity process of chloroquine accumulation which hitherto has been observed only in erythrocytes infected with malaria parasites. In addition, the capacity of the latent process, approximately 30 μmoles per Kg of erythrocytes, is sufficient to account for the accumulation of chloroquine by erythrocytes infected with malaria parasites. These findings demonstrate the plausibility of attributing the ability to accumulate chloroquine with high affinity to the erythrocyte host rather than to the parasite.     

A non-radiolabelled ferriprotoporphyrin IX biomineralisation inhibition test for the high throughput screening of antimalarial compounds

Intraerythrocytic malaria parasites produce large amounts of toxic ferriprotoporphyrin IX (FP) during their digestion of host cell haemoglobin. The inhibition of biomineralisation of FP to haemozoin (or beta-haematin) by antimalarial drugs underlies their mode of action. We have developed an in vitro microassay for testing the inhibition of biomineralisation by drugs. It is based on the detection by optical density measurement of solubilised beta-haematin remaining after contact with drugs. The assay uses a 192-microM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplates; it lasts 18-24h and requires a spectrophotometer. We determined by this assay the IC(50) of chloroquine phosphate (28microM) and quinine base (324microM) and showed that unlike previous methods it is insensitive to inorganic anions. We also determined the activity of synthetic dyes and plant extract to determinate the interference of coloured compounds on the accuracy of the test. We found that methylene blue, thionine (IC(50) 38 and 87microM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour.     

Antioxidant effect of dipyridamole (DIP) and its derivative RA 25 upon lipid peroxidation and hemolysis in red blood cells

The antioxidant effects of dipyridamol (DIP), a coronary vasodilator, and its derivative RA-25 were compared in intact red blood cells (RBC) and in isolated ghost membranes. Both compounds are quite effective antioxidants in cumene hydroperoxide-induced lipid peroxidation of RBC, showing a much smaller effect for hydrogen peroxide oxidation. The antioxidant effect of DIP was considerably higher than that of RA25. For isolated ghost membranes, the apparent IC50 (the drug concentration that produces 50% inhibition of lipid peroxidation) in cumene hydroperoxide-induced peroxidation was 25 microM, while the maximum protective effect of RA-25 was around 30% in the drug concentration range of 50-100 microM. The drugs can protect the oxidative hemolysis induced by cumene hydroperoxide with a lower effect when the hemolysis is induced by H2O2. The significant antioxidant effect against damages induced by cumene hydroperoxide suggests that DIP, due to its lipophilic character, can interact with RBC membranes, and the protective effect is associated with the binding of the drug to the membrane. On the other hand, RA-25 is more hydrophilic than DIP, binds to the membrane to a smaller extent, and, for this reason, has a lower antioxidant effect.     

In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8 +/- 1.2 microM) was significantly lower than its cytostatic activity; CC50 (261.3 +/- 14.5 microM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.     

In vitro studies on peroxidative changes leading to hemolysis of erythrocytes infested with malarial parasite Plasmodium vivax.

Blood erythrocytes of 25 confirrhed malarial patients infested with P. vivax were analyzed for peroxidation and hemolysis and results compared with 10 uninfected normal control samples. Results indicated significant increase in peroxide formation measured as malondialdehyde, both in presence and absence of H2O2, in parasite infested erythrocytes. These changes induced hemolysis of infected erythrocytes which was increased manifold in presence of H2O2 and could probably be the reason for extensive anemia reported in malaria.     

Variation in apoptosis mechanisms employed by malaria parasites: the roles of inducers, dose dependence and parasite stages

ABSTRACT: BACKGROUND: Plasmodium berghei ookinetes exhibit an apoptotic phenotype when developing within the mosquito midgut lumen or when cultured in vitro. Markers of apoptosis increase when they are exposed to nitric oxide or reactive oxygen species but high concentrations of hydrogen peroxide cause death without observable signs of apoptosis. Chloroquine and other drugs have been used to induce apoptosis in erythrocytic stages of Plasmodium falciparum and to formulate a putative pathway involving cysteine protease activation and mitochondrial membrane permeabilization; initiated, at least in the case of chloroquine, after its accumulation in the digestive vacuole causes leakage of the vacuole contents. The lack of a digestive vacuole in ookinetes prompted the investigation of the effect of chloroquine and staurosporine on this stage of the life cycle. Finally, the suggestion that apoptosis may have evolved as a strategy employed by ookinetes to increase the fitness of surviving parasites was explored by determining whether increasing the ecological triggers parasite density and nutrient depletion induced apoptosis. METHODS: Ookinetes were grown in culture then either exposed to hydrogen peroxide, chloroquine or staurosporine, or incubated at different densities and in different media. The proportion of ookinetes displaying positive markers for apoptosis in treated samples was compared with controls and results were analyzed using analysis of variance followed by a Turkey's test, or a Kruskal-Wallis test as appropriate. RESULTS: Hydrogen peroxide below 50 muM triggered apoptosis but cell membranes were rapidly compromised by higher concentrations, and the mode of death could not be defined. Both chloroquine and staurosporine cause a significant increase in ookinetes with condensed chromatin, caspase-like activity and, in the case of chloroquine, phosphatidylserine translocation and DNA fragmentation (not investigated for staurosporine). However, mitochondrial membrane potential remained intact. No relationship between ookinete density and apoptosis was detected but nutrient depletion significantly increased the proportion of ookinetes with chromatin condensation in four hours. CONCLUSIONS: It is proposed that both a mitochondrial and an amitochondrial apoptotic pathway may be involved, dependent upon the trigger that induces apoptosis, and that pathways may differ between erythrocytic stages and ookinetes, or between rodent and human malaria parasites.     

Effect of retinol on the hemolysis and lipid peroxidation in vitamin E deficiency

A study on the effect of retinolin vitro on the hemolysis of vitamin E deficient rat red blood cells showed that retinol enhanced the lysis of the E deficient cells as compared to the lysis of normal cells. The lipid peroxidation present during hydrogen peroxide induced lysis of E deficient cells was however markedly inhibited in the presence of retinol without affecting the rate of lysis. In an actively peroxidising system of non-enzymatic lipid peroxidation of rat liver or brain homogenates and of brain lysosomes incubated with human erythrocytes, no lysis was obtained; incorporation of retinol in such systems resulted in lysis but no peroxidation. Hydrogen peroxide generating substances almost completely inhibited the lysis of normal human erythrocytes by retinol, but linoleic acid hydroperoxide and auto-oxidised liver or brain homogenates and ox-brain liposomes increased the lysis. It is concluded that vitamin E deficient erythrocyte hemolysis may be augmented by retinol, an anti-oxidant, having a lytic function without the peroxidation of stromal lipids     

Antioxidative effect of melatonin on DNA and erythrocytes against free-radical-induced oxidation

The aim of this work is to investigate the antioxidative effect of melatonin (N-acetyl-5-methoxytryptamine) on the oxidation of DNA and human erythrocytes induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). First, the 50% inhibition concentration (IC50) of melatonin is measured by reacting with two radical species, i.e., 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS*+) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH). The IC50 of melatonin are 75microM and 300microM when melatonin reacts with ABTS*+ and DPPH, respectively. Especially, the reactions of melatonin with ABTS*+ and DPPH are the direct evidence for melatonin to trap radicals. Then, melatonin is applied to protect DNA and human erythrocytes against oxidative damage and hemolysis induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The presence of melatonin prolongs the occurrence of the oxidative damage of DNA and hemolysis of erythrocytes, generating an inhibition period (t(inh)). The proportional relationship between t(inh) and the concentration of melatonin ([MLT]) is treated by the chemical kinetic equation, t(inh)=(n/R(i))[MLT], in which n means the number of peroxyl radical trapped by an antioxidant, and R(i) stands for the initiation rate of the radical reaction. It is found that every molecule of melatonin can trap almost two radicals in protecting DNA and erythrocytes. Furthermore, quantum calculation proves that the indole-type radical derived from melatonin is much stable than amide-type radical. Finally, melatonin is able to accelerate hemolysis of erythrocytes induced by hemin, indicating that melatonin leads to the collapse of the erythrocyte membrane in the presence of hemin. This may provide detailed information for the usage of melatonin and helpful reference for the design of indole-related drugs.     

Factors influencing variable oxidative hemolysis of inbred mouse erythrocytes

The hemolysis of erythrocytes from certain inbred mouse strains (e.g., BALB/c) in response to hydrogen peroxide stress has been shown to be correlated with the type of hemoglobin beta chain (Kruckeberg, W.C., et al. (1987) Blood 70, 909-914). The characteristic hemolytic response of BALB/c red cells to oxidative stress resembles that of human red cells in that carbon monoxide and iron chelators inhibit hemolysis of both. Gross hemoglobin oxidation rates were similar in hemolytic (BALB/c) and nonhemolytic (C57BL/6) strains. The rate and degree of in vitro catalase inhibition by sodium azide was also the same for the two strains. Even in the presence of this catalase inhibitor the assayable hydrogen peroxide disappeared within seconds of its addition, yet hemolysis was not observed for about 15 min. The mechanism underlying this delay between hydrogen peroxide addition and disappearance and subsequent hemolysis is under investigation.     

Organic osmolyte permeabilities of the malaria-induced anion conductances in human erythrocytes

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl(-) conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (P(lactate)/P(Cl) = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC(50)s in the range of 0.1 and 1 microM, respectively. In contrast, the IC(50)s of NPPB and furosemide for the inward-rectifying current were >10 microM. Osmotic cell-shrinkage inhibited the inwardly but not the outwardly rectifying conductance. In conclusion, the parasite-induced outwardly-rectifying anion conductance allows permeation of lactate and neutral carbohydrates, whereas the inward rectifier seems largely impermeable to organic solutes. All together, these data should help to resolve ongoing controversy regarding the number of unique channels that exist in P. falciparum-infected erythrocytes.     

The state of ferriprotoporphyrin IX in malaria pigment

To evaluate the state of ferriprotoporphyrin IX (FP) in malaria pigment, mouse erythrocytes infected with Plasmodium berghei NYU-2 parasites were lysed by hypotonic shock, and hemoglobin and other soluble material were removed by extensive washing. The amount of FP recovered in the insoluble pellet was 2.1 mumol/ml of packed infected erythrocytes, of which approximately 1% was attributable to hemoglobin contamination. This crude preparation then was digested with a nonspecific protease from Streptomyces griseus and extracted with chloroform/methanol. The residue of insoluble dark brown material had the spectral and solubility properties characteristic of the FP of malaria pigment, and various different preparations contained from 82 to 99% of FP by weight. By elemental analysis, highly purified preparations contained no chlorine and had an oxygen content consistent with 1 mol of hydroxyl/mol of FP (oxygen content: calculated, 12.6%; found, 12.5%). In comparison to hematin purchased from Sigma, which had a measured oxygen content of 14.7%, the low oxygen form of hematin purified from malaria pigment was remarkably less soluble in ethanol, 3% sodium bicarbonate, and chloroform.     

Antimalarial properties of imipramine and amitriptyline

Dietary riboflavin deficiency is known to diminish malarial parasitemia. In this study, we determined whether imipramine and amitriptyline, drugs which inhibit riboflavin metabolism, have antimalarial efficacy. In addition, we evaluated whether these drugs, like other antimalarial agents, increase the hemolytic response to ferriprotoporphyrin IX (FP). The growth of Plasmodium falciparum (FCR3) in the absence and presence of these drugs (10 to 75 microM) was measured by determining (3H)hypoxanthine uptake by intra-erythrocytic parasites for 48 h in RPMI 1640 medium. The uptake of (3H)hypoxanthine was significantly reduced in a dose-dependent manner by both imipramine and amitriptyline. The IC50 values of imipramine and amitriptyline at 48 h were 56 and 45 microM, respectively. Both drugs enhanced hemolysis induced by FP (10 or 20 microM). No hemolysis by these drugs was detected in the absence of FP. It is concluded that the tricyclic antidepressants, imipramine and amitriptyline, possess substantial antimalarial properties.     

The effect of hypochlorite on human erythrocytes pretreated with X-radiation

Both hypochlorite and ionizing radiation induce oxidation processes of biomolecules. The effects are dependent to a large degree on the dose of the oxidizing agent. Previously we observed that split doses of gamma radiation caused lower hemolysis than the same but single doses. The critical factors influencing the occurrence of this effect were: the value of the first dose and the time between the doses. In this work we examined the effect of gamma radiation (40-400 Gy) on hemolysis of human erythrocytes induced by hypochlorite. Erythrocytes in PBS, hematocrit 2 %, were irradiated with doses of 40, 200 or 400 Gy. The dose-rate was 23.8 Gy/min. Cell suspensions were stirred during irradiation. After irradiation the erythrocytes were incubated for 1, 3 or 4 hours at room temperature and then hypochlorite was added to a 250 microM concentration. Control samples were erythrocytes treated only with NaOCl. The level of hemolysis was determined after NaOCl addition. Hemolysis of erythrocytes preirradiated with the dose of 400 Gy was lower than hemolysis of erythrocytes treated only with NaOCl. The effect was dependent on the time between the end of irradiation and the addition of NaOCl. In contrast, slightly higher hemolysis was observed for erythrocytes preirradiated with lower (40 or 200 Gy) doses of radiation. The observed effect is similar to that obtained for radiation-induced hemolysis. It suggests that ionizing radiation may induce structural and/or functional changes in erythrocytes, which make the cell more resistant to further oxidative damage.     

The Protective Effect of Hydrogen Peroxide on Erythrocyte Hemolysis Induced by Silver Nanoparticles

Biophysics - The mechanisms of regulation of adaptation of erythrocytes by hydrogen peroxide during hemolysis induced by silver nanoparticles have been studied using numerical modeling and...     

Relationship of chloroquine-induced redistribution of a neutral aminopeptidase to hemoglobin accumulation in malaria parasites

To study the relationship between neutral aminopeptidase activity and hemoglobin accumulation in malaria parasites, we treated mice infected with Plasmodium berghei NYU-2 with chloroquine intraperitoneally in doses ranging from 0.3 to 3 micromol per 25 g mouse. Preparations of infected erythrocytes (normalized to represent 1000 parasites per 1000 erythrocytes) hydrolyzed 1200 nmol of leucine-p-nitroanilide per minute per milliliter of packed erythrocytes, which was 10x more than that of uninfected preparations. The activity in infected preparations was distinguished by resistance to ferriprotoporphyrin IX and puromycin and susceptibility to inhibition by ethanol and Tris. Chloroquine treatment caused the activity in unwashed membrane ghosts of infected preparations to decrease by 50% despite an increase in total activity. Concomitantly, hemoglobin in washed membrane ghosts increased. Electron microscopy revealed that the hemoglobin was retained in endocytic vesicles. Chloroquine-induced redistribution of a neutral aminopeptidase may be the cause of hemoglobin accumulation in endocytic vesicles of malaria parasites.