Antioxidant effect of dipyridamole and its derivative RA-25 in mitochondria: correlation of activity and location in the membrane.

Dipyridamole (DIP), a coronary vasodilator, presents coactivator activity for a number of antitumor drugs as well as antioxidant activity in membrane systems. DIP and derivatives interact with membrane systems such as micelles, phospholipid monolayers and vesicles. The antioxidant effect of DIP and several derivatives upon iron-induced lipoperoxidation on mitochondria has been reported and a good correlation between the hydrophobicity and their protective effect was found (M.F. Nepomuceno et al., Free Radic. Biol. Med., 23 (1997) 1046-1054). In the present work an effort is made to better understand the role of DIP as inhibitor of Fe2+-induced lipid peroxidation in mitochondria. At low concentration, no significant effect on either state IV or state III respiration was found, discarding a possible direct interaction of DIP or RA-25 with the peripheral benzodiazepine receptor. The association constants for DIP and RA-25 in mitochondria were estimated, being 0.7 (mg/ml)-1 for DIP and 0.2 (mg/ml)-1 for RA-25. Oxygen consumption studies in the presence of FeSO4 showed that the antioxidant effect of DIP or RA-25 did not involved the initial step of Fe2+ oxidation. Our data strongly support the hypothesis that the antioxidant effect of both DIP and RA-25 is related to their partition in the lipid phase of the mitochondrial membrane and not to a specific interaction with membrane proteins. This protection may be due either to a direct inhibition of the propagation steps or a scavenger effect on the radicular species that would trigger the peroxidative process.     

Antioxidant effect of red wine polyphenols on red blood cells

The protective effect of red wine polyphenols against hydrogen peroxide (H(2)O(2))-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H(2)O(2), were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system.     

Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells

The present study has examined the effect of elevated glucose levels on membrane lipid peroxidation and osmotic fragility in human red blood cells (RBC). Defibrinated whole blood or RBC were incubated with varying concentrations of glucose at 37 degrees C for 24 h. RBC incubated with elevated levels of glucose showed a significantly increased membrane lipid peroxidation when compared with control RBC. A significant positive correlation was observed between the extent of glucose-induced membrane lipid peroxidation and the osmotic fragility of treated RBC. Glucose-induced membrane lipid peroxidation and osmotic fragility were blocked when RBC were pretreated with fluoride, an inhibitor of glucose metabolism; with vitamin E, an antioxidant; with para-chloromercurobenzoate and metyrapone, inhibitors of the cytochrome P-450 system; or with dimethylfurane, diphenylamine, and thiourea, scavengers of oxygen radicals. RBC treated with elevated glucose concentrations also showed an increase in NADPH levels. Exogenous addition of NADPH to normal RBC lysate induced membrane lipid peroxidation similar to that observed in the glucose-treated RBC. These data suggest that elevated glucose levels can cause the peroxidation of membrane lipids in human RBC.     

Electrical hemolysis of human and bovine red blood cells

Summary The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50% hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50% hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Antioxidant action of acteoside and its analogs on lipid peroxidation

Summary

We have investigated antioxidant actions of acteoside (ACT) and another natural phenylpropanoid glycoside, cistanoside F (CIS-F) on lipid peroxidation in rat liver mitochondria (RLM) and rat liver mitochondrial lipid (RLML) liposomes induced by Fe²⁺/ADP. A synthetic ACT analogue, TX-1847, was also examined. Oxygen consumption, the formation of thiobarbituric acid reactive substances (TBARs) and glutathione concentration were determined simultaneously during lipid peroxidation. The radical scavenging activity of the compounds was evaluated by using 1,1-diphenyl-2-picrylhydrazyl. ACT and its analogs produced dose-dependent inhibitions of mitochondrial and liposomal lipid peroxidation (ACT ≈ CIS-F > TX-1847). Their radical scavenging activities were ranked as follows: TX-1847 > ACT > CIS-F. ACT, CIS-F, and TX-1847 spared reduced glutathione (GSH) during mitochondrial lipid peroxidation. The radical scavenging activities of the compounds did not parallel their anti-peroxidative activities. The data are consistent with the idea that the inhibitory activities of phenylpropanoids were primarily due to a radical chain-breaking mechanism. The sugar moieties in ACT and CIS-F, and/or the conformational structure of the compounds, also seem to play an important role in their inhibitory effects on lipid peroxidation.     

Glutathione peroxidase and oxidative hemolysis in trout red blood cells

Red blood cells from the trout Salmo irideus contain several hemoglobin components that are prone to oxidation with production of oxygen radicals. The rate of hemolysis has been correlated to the extent of methemoglobin formation. A difference in the rate of hemolysis between red blood cells saturated with either CO or O2 was evident only when diminished glutathione peroxidase activity was observed. These results confirm the important role of this enzyme in providing protection against or repair of oxidative damage to the red cell membrane.     

Antioxidant enzyme activities and lipid peroxidation induced by eicosapentaenoic acid (EPA) in PC12 cells

Eicosapentaenoic acid (EPA) is one of the major dietary polyunsaturated fatty acids and induces apoptosis in several cancer cells. In this study, the EPA induced lipid peroxidation and response of antioxidative enzymes have been investigated in rat pheochromocytoma PC12 cells to elucidate the mechanisms of apoptosis induced by the polyunsaturated fatty acid EPA. We have analyzed superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and glutathione (GSH) contents in PC12 cells after exposure to different concentrations of EPA. Lipid peroxidation was shown to increase in the presence of EPA as an indication of the oxidative damage. Lipid peroxidation was enhanced by EPA in a dose-dependent manner, and the loss of cell viability was partially reversed by vitamin E. In the case of antioxidant enzyme activities, SOD and GPX activities and GSH contents increased significantly at 50 μmol/L EPA and were respectively 2.41-fold (p < 0.01), 3.49-fold (p < 0.05), and 1.43-fold (p < 0.05) higher than controls. The CAT activity at 10 μmol/L had the highest value and was increased by 25.83% (p < 0.05) compared to control. The results suggest that in PC12 cells the mechanism of apoptosis induced by EPA may be partly due to lipid peroxidation.     

Increased n-3 polyunsaturated fatty acid content of red blood cells from fish oil-fed rabbits increases in vitro lipid peroxidation, but decreases hemolysis.

In view of a possible relationship between fish oil, lipid peroxidation, and atherosclerosis, the in vitro lipid peroxidation susceptibility of red blood cells (RBCs) from rabbits on conventional (-FO) and fish oil-enriched diets (+FO) was investigated. The diet caused substantial increases in the RBC concentrations of n-3 polyunsaturated fatty acids (PUFAs), in combination with decreases in the concentration of oleic acid (18:1) and linoleic acid (18:2). Cumene hydroperoxide-induced oxidative stress led to increased overall fatty acid peroxidation in +FO RBCs compared with with -FO RBCs, as quantitated by GLC fatty acid analysis. However, the increased overall susceptibility to lipid peroxidation of +FO RBCs was not reflected in increased peroxidation of every individual fatty acid. This was observed for endogenous arachidonic acid (20:4) as well as, in separate experiments, for exogenously added parinaric acid (PnA). The increased cumene hydroperoxide-induced PUFA oxidation in +FO RBCs was accompanied by a lesser extent of hemolysis. To account for these observations, it is proposed that the increased n-3 PUFA content of +FO RBCs serves as an oxidizable buffer. The present data suggest that oxidation of fatty acids can occur until a critically low level of intact phospholipid in the RBC membrane is reached, after which the membrane destabilizes and hemolysis occurs. At the same time, the PUFA buffer in +FO RBCs could also prevent oxidative damage to specific membrane proteins, which could also help prevent cell lysis.     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Antioxidant effect of anthocyanin on enzymatic and non-enzymatic lipid peroxidation.

In vitro enzymatic and non-enzymatic polyunsaturated fatty acid peroxidation was significantly inhibited in a dose dependent manner by purified anthocyanin, a deep-red colour pigment from carrot cell culture. The kinetics showed that anthocyanin is a non-competitive inhibitor of lipid peroxidation. Anthocyanin has been found to be a potent antioxidant compared to classical antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxy toulene (BHT) and alpha tocopherol. This natural agent, in addition to imparting colour to the food, might prevent autooxidation of lipids as well as lipid peroxidation in biological systems.     

Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation

One of the recent approaches to enhance desiccation tolerance in red blood cells (RBCs) is by loading trehalose. This process has been shown to increase the recovery of lyophilized RBCs; conversely, it results in cellular damage including hemoglobin oxidation and loss of membrane integrity. The purpose of this study was to further investigate the extent of oxidative injury during the loading of trehalose into RBCs.RBCs were incubated in the absence (control) or presence of trehalose (0.8 mol/l) at 4 °C or 37 °C for different time scales. Oxidative damage was monitored by flow cytometry using dichlorofluorescin for reactive oxygen species formation, Annexin V-FITC for phosphatidylserine translocation and fluorescein-DHPE for lipid peroxidation. Percent methemoglobin, percent hemolysis and thiobarbituric acid reactive substances were measured by spectrophotometry. The extent of oxidative damage during trehalose loading is affected by the incubation temperature, incubation time and the presence of trehalose. Incubation at 4 °C was relatively innocuous; however, oxidative injury was evident at 37 °C in both RBC groups. The addition of trehalose is correlated with high osmotic pressure, which had minor effects during incubation at 4 °C, but seemed to have exacerbated the severity of cellular injury at 37 °C, as measured by higher levels of hemolysis, methemoglobin and lipid peroxidation.The process of trehalose-loading is problematic due to its requirement for prolonged incubations at 37 °C. These conditions are correlated with oxidative injury, even in the absence of trehalose. While trehalose is believed to be crucial for stabilizing biomembranes, the consequences of its introduction into the cells require further investigation.     

Detection of short-chain carbonyl products of lipid peroxidation from malaria-parasite (Plasmodium vinckei)-infected red blood cells exposed to oxidative stress.

Reversed-phase h.p.l.c. was used to detect 2,4-dinitrophenylhydrazine-reactive carbonyl products, which excludes malonaldehyde, in malaria-parasite (Plasmodium vinckei)-infected murine red blood cells (RBCs). A number of alkanals, 4-hydroxyalk-2-enals and alka-2,4-dienals were tentatively identified by comparison with authentic standards. The formation of 4-hydroxynon-2-enal, deca-2,4-dienal and hexanal was greater in P. vinckei-infected RBCs than in their uninfected counterparts and was increased by the presence of t-butyl hydroperoxide. Several of these aldehydes have previously been shown to be toxic to various types of cells, including P. falciparum, in vitro. The iron chelator desferrioxamine and the free-radical scavenger butylated hydroxyanisole inhibited the formation of these aldehydes. These experiments demonstrate that products of lipid peroxidation other than malonaldehyde are formed during the exposure of malaria-infected RBCs in vitro to drugs that generate reactive oxygen species and have anti-parasitic activity. The formation of products of this type during the natural course of malaria infection may have implications for the mechanisms underlying intra-RBC parasite death and the tissue damage associated with the disease.     

Stimulation of calcium-sodium exchange in dog red blood cells by hemolysis and resealing

Osmotic hemolysis and resealing greatly increase calcium influx in dog red blood cells. The resealed ghosts show a saturable calcium entry pathway with complex kinetics. As expected for a calcium-sodium exchanger, calcium uptake is stimulated by internal sodium and inhibited by external sodium. Compared to fresh, intact red cells the resealed ghost calcium-sodium exchanger is less responsive to quinidine and to alterations in medium tonicity. The differences in calcium uptake rate among cells from different donors are minimized in the ghost preparation. There are several ways to stimulate sodium-dependent calcium movements in these cells, of which hemolysis-resealing is the most potent. The results of these and previous studies suggest that dog red blood cells have a latent capacity for calcium-sodium exchange.     

Antioxidant status and lipid peroxidation in the blood of breast cancer patients of different ages

Oxidative stress is considered to be implicated in the pathophysiology of breast cancers. In this study we investigated the level of oxidative stress and antioxidant (AO) status in the blood of breast cancer patients of different ages. The level of lipid hydroperoxides (LP) was measured in blood plasma and the activities of copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) enzymes, as well as the level of total glutathione (GSH) and CuZnSOD protein were measured in blood cells of breast cancer patients and age-matched healthy subjects. Our results showed that breast carcinoma is related to increase of lipid peroxidation in plasma with concomitant decrease of AO defense capacity in blood cells, which becomes more pronounced during aging of the patients. Suppression of CuZnSOD activity related to breast cancer is most likely caused by decreased de novo synthesis of this enzyme. Similar patterns of suppression in CuZnSOD and CAT activities related to aging were recorded both in controls and patients. Age-related decrease in CuZnSOD activity seems not to be caused by altered protein levels of this enzyme. Suppression of AO enzymes associated with breast cancer and aging is most likely the cause of increased levels of reactive oxygen species (ROS). Our results indicate significant role of oxidative-induced injury in the breast carcinogenesis, particularly during the later stages of aging. Overall, our data support the importance of endogenous AOs in the etiology of breast cancer across all levels of predicted risk.     

Antioxidant effect of vitamin E and glutathione on lipid peroxidation in boar semen plasma

The protective effect of vitamin E and reduced glutathione (GSH) against lipid peroxidation in boar semen plasma was studied. The lipid peroxidation, measured by the test for thiobarbituric acid reactive substances (TBARS), doubled in the presence of the lipid peroxidation Fe²⁺-sodium ascorbate-inducing system. The ascorbate-induced TBARS were inhibited by about 62% through the water-soluble vitamin E analog (TROLOX) and about 57% by GSH. In the in vivo experiments, 7 wk of oraldl-α-tocopherol acetate (1000 IU/d/animal) administration caused a significant fall in the level of the semen plasma TBARS, from 2.2±0.09 to 1.2±0.13 nmol MDA/mL. The semen plasma superoxide dismutase (SOD) and GSSG tended to increase with the time of vitamin E administration, but the increment did not reach a significant level by the seventh week. The vitamin E supplementation significantly increased the number of spermatozoa per 1 cm³ of ejaculate. The protective role of vitamin E and GSH with respect to boar semen against fatty acid peroxidation and a positive influence of vitamin E supplementation on semen quality have been evidenced.     

Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on alpha-tocopherol

The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.     

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 effect of ionizing radiation on lipid peroxidation in rat blood

In experiments with 5-6 month male rats it was shown that whole-body gamma-irradiation (0.5 and 1.0 Gy) caused changes in the system of the antioxidant defence of the organism, the status of which influenced the intensity of lipid peroxidation in the blood.