Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation

The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.     

Detection of DNA damage in stressed trout nucleated erythrocytes using the comet assay: protection by nitroxide radicals

Because previous literature reports have demonstrated that nucleated trout erythrocytes in conditions of oxidative stress are subjected to both membrane damage and a decrease in the enzymatic defense systems (glutathione peroxidase), which in turn lead to hemolysis, the present study was undertaken to determine whether DNA may be affected too, prior to the hemolytic event. Impairment of DNA in stressed trout erythrocytes was assessed using the comet assay--a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. In addition, indolinic and quinolinic nitroxide radicals were included in the study to determine their efficacy as antioxidants against free-radical-induced DNA damage. The parameters, tail length, tail intensity, and tail moment, used as an index of DNA damage, have shown that trout erythrocytes exposed to oxidative stress experience DNA damage prior to hemolysis and that the nitroxides significantly prevent this damage. This result provides further information about the potential use of these compounds as antioxidants in biological systems.     

Delineation of supernumerary marker chromosomes in 38 patients

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.     

Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol

Previously we reported that eugenol (4-allyl-2-methoxyphenol) inhibits non-enzymatic peroxidation in liver mitochondria (E. Nagababu and N. Lakshmaiah, 1992, Biochemical Pharmacology. 43, 2393-2400). In the present study, we examined the effect of eugenol on microsomal mixed function oxidase mediated peroxidation using Fe+3-ADP-NADPH, carbon tetrachloride (CCL4)-NADPH and cumene hydroperoxide (CumOOH) systems. In the presence of eugenol the formation of thiobarbituric acid reactive substances (TBARS) was decreased in all the systems (IC50 values: 14 microM for Fe+3-ADP-NADPH, 4.0 microM for CCl4-NADPH and 15 microM for CumOOH). Oxygen uptake was also inhibited to a similar extent with Fe+3-ADP-NADPH and CumOOH systems. A comparative evaluation with other antioxidants showed that in Fe+3-ADP-NADPH and CumOOH systems, the antioxidant efficacy was in the order: butylated hydroxytoluene (BHT) > eugenol > alpha-tocopherol, while in CCl4-NADPH system the order was alpha-tocopherol > BHT > eugenol. Time course of inhibition by eugenol indicated interference in initiation as well as propagation of peroxidation. Eugenol did not inhibit cytochrome P-450 reductase activity but it inhibited P-450 - linked monooxygenase activities such as aminopyrine-N-demethylase, N-nitrosodimethylamine demethylase, benzo(a)pyrene hydroxylase and ethoxyresorufin-O-deethylase to different extents. However, CumOOH supported monooxygenases (aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase) required much higher concentrations of eugenol for inhibition. The concentration of eugenol required to inhibit monooxygenase activities was more than that required to inhibit peroxidation in all the systems. Eugenol elicited type 1 changes in the spectrum of microsomal cytochrome P-450. These results suggest that the inhibitory effect of eugenol on lipid peroxidation is predominantly due to its free radical quenching ability. Eugenol significantly protected against the degradation of cytochrome P-450 during lipid peroxidation with all the systems tested. These findings suggest that eugenol has the potential to be used as a therapeutic antioxidant. Further evaluation may throw more light on this aspect.     

Characterization of novel indenoindoles. Part I. Structure-activity relationships in different model systems of lipid peroxidation

Structure-activity relationships are presented for some representative compounds from a novel series of potent inhibitors of lipid peroxidation. The compounds are indenoindole derivatives with oxidation potentials in organic solvents of between 0.2 and 1.5 V. Two of these compounds, cis-5,5a,6,10b-tetrahydro-9-methoxy-7-methylindeno[2,1-b]indole (H 290/51) with an oxidation potential of 0.32 V and cis-4b,5,9b,10- tetrahydro-8-methoxy-6-methylindeno[1,2-b]indole (H 290/30) with an oxidation potential of 0.30 V, have been tested more extensively and compared with reference compounds in several pharmacological models of lipid peroxidation. The inhibitory potencies (pIC50) of the compounds in respect to Fe/Ascorbate-induced production of thiobarbituric acid-reactive substances (TBARS) in a suspension of purified soybean lecithin were calculated. These data are 8.2 for H 290/51; 8.0 for H 290/30; 5.6 for vitamin E; and 6.6 for butylated hydroxytoluene (BHT). In isolated rat renal tissue subjected to hypoxia and reoxygenation, the potency for inhibition of TBARS formation is 6.9 for H 290/51, 6.9 for H 290/30, and <5 for vitamin E. In oxidative modification of low-density lipoproteins (LDL) induced by mouse peritoneal macrophages, the corresponding pIC50 values for TBARS inhibition for each compound are: 8.7, 8.3, <5, and 6.9, respectively. It is concluded that the synthetic indenoindoles are potent antioxidants. The results suggest that indenoindoles such as H 290/51 and H 290/30 could be useful as therapeutic agents in pathophysiological situations where lipid peroxidation plays an important role.     

Studies of structure-activity relationship of nitroxide free radicals and their precursors as modifiers against oxidative damage

The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.     

Effects of dietary n-6 and n-3 lipids on antioxidant defense system in livers of exercised rats

OBJECTIVE: The present study was designed to investigate the effects of dietary n-6 and n-3 lipids and exercise on the activities of hepatic antioxidant enzymes and microsomal lipid composition and peroxidation in Fischer-344 male rats. METHODS: Weanling male Fischer-344 rats were fed ad libitum semipurified diets containing 10% corn oil (CO) or 10% fish oil (FO), with equal levels of antioxidants. After 2 months on the diets, weight-matched animals in each diet group were divided into sedentary (S) and exercised (Ex) groups, and the diets were continued. The animals in the exercise group were run on a treadmill 30 to 40 minutes to exhaustion 6 days/week for 2 months. At the end of 2 months, the rats were sacrificed and livers were collected; antioxidant enzymes were determined in the cytosol, fatty acid composition was analyzed in the microsomes, and vitamin E levels were analyzed in the sera. RESULTS: The rats in the FO-S group exhibited significantly higher liver cytosolic catalase activity, while their superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were significantly lower compared to the CO-S group. The GSH-Px activity was significantly higher in the FO-Ex group compared to FO-S group. The source of dietary lipids significantly influenced the fatty acid composition of the total lipids in the microsomes. Feeding the FO-based diet significantly increased 18:0 and n-3 fatty acids incorporation into the microsomes (18:3, 20:5, 22:5, and 22:6), whereas ingestion of CO resulted in a significant increase in 14:0, 14:1, 18:1, and n-6 fatty acids (18:2 and 20:4). The serum vitamin E levels were significantly higher in the CO groups, and exercise had no effect on vitamin E levels. Exercise significantly decreased the generation of thiobarbituric acid reactive substances (TBARS) by liver microsomes. Consumption of FO, which is highly susceptible to oxidation, did not show any significant changes in membrane lipid peroxidation. CONCLUSIONS: The present study suggests that feeding FO increases the activity of liver cytosolic catalase in FO-S rats and GSH-Px in FO-Ex rats. In addition, exercise significantly decreased the generation of TBARS by the liver microsomal lipids. Serum vitamin E levels were higher in the CO group and exercise did not alter vitamin E levels. This suggests that the amount of vitamin E included in the diets was possibly adequate to cope with the oxidative stress induced during exercise.     

Low doses of eicosapentaenoic acid, docosahexaenoic acid, and hypolipidemic eicosapentaenoic acid derivatives have no effect on lipid peroxidation in plasma

It was of interest to investigate the influence of both high doses of eicosapentaenoic acid (EPA) and low doses of 2- or 3-methylated EPA on the antioxidant status, as they all cause hypolipidemia, but the dose required is quite different. We fed low doses (250 mg/d/kg body wt) of different EPA derivatives or high doses (1500 mg/d/kg body wt) of EPA and DHA to rats for 5 and 7 d, respectively. The most potent hypolipidemic EPA derivative, 2,2-dimethyl-EPA, did not change the malondialdehyde content in liver or plasma. Plasma vitamin E decreased only after supplementation of those EPA derivatives that caused the greatest increase in the fatty acyl-CoA oxidase activity. Fatty acyl-CoA oxidase activity increased after administration of both EPA and DHA at high doses. High doses of EPA and DHA decreased plasma vitamin E content, whereas only DHA elevated lipid peroxidation. In liver, however, both EPA and DHA increased lipid peroxidation, but the hepatic level of vitamin E was unchanged. The glutathione-requiring enzymes and the glutathione level were unaffected, and no significant changes in the activities of xanthine oxidase and superoxide dismutase were observed in either low- or high-dose experiments. In conclusion, increased peroxisomal beta-oxidation in combination with high amounts of polyunsaturated fatty acids caused elevated lipid peroxidation. At low doses of polyunsaturated fatty acids, lipid peroxidation was unchanged, in spite of increased peroxisomal beta-oxidation, indicating that polyunsaturation is the most important factor for lipid peroxidation.     

Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro

This study aimed to explore the mechanisms and molecular parameters which control the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when oxidatively activated by horseradish peroxidase (HRP). Lipid peroxidation was measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by 3H labelling, stable products measured by HPLC, and cytotoxicity in hamster fibroblasts measured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membranes of hamster fibroblasts, and were cytotoxic after HRP activation to varying degrees. Cytotoxicity was not correlated with activation rate. The hydrophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loading fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox in the aqueous phase. However, two known oxidation products were nontoxic (the 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO* peroxyl radicals via the 3-CH*2 [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatment of hamster fibroblasts with 5-3H-IAA/HRP resulted in intracellular bound 3H. Together with earlier results, the new data point to unknown electrophilic oxidation products, reactive towards intracellular targets, being involved in cytotoxicity of the IAA/HRP combination, rather than direct attack of free radicals, excited states, or membrane lipid peroxidation.     

The in vitro antioxidant activity of trilinolein and other lipid-related natural substances as measured by enhanced chemiluminescence

There is abundant evidence for the premise that oxygen-derived free radicals (OFR) mediate ischemia/reperfusion injury to the myocardium. OFR scavengers such as superoxide dismutase can effectively reduce damage through lipid peroxidation during ischemia/reperfusion. Enhanced chemiluminescence, which has been used to measure OFR, was used to measure the antioxidant activity of fatty acids (palmitic and linoleic acid) and triglycerides (triolein, tristearin) and natural plant antioxidants (magnolol, catechin, trilinolein). Trilinolein, which has recently been isolated from natural products, as well as the well-known water soluble analogue of vitamin E-Trolox, were used as control. During pretreatment with chemicals, at concentrations of 10(-9) to 10(-7) M, enhanced chemiluminescence of linoleic acid (C 18:2) showed a dose-responsive reduction of OFR with a maximal mean reduction of -31.9% when compared to baseline. A saturated fatty acid such as palmitic acid (C 16:0) showed only relatively weak antioxidant activity at concentrations of 10(-7) to 10(-6) M with a maximum reduction of OFR of- 15.2% only. control chemicals such as trilinolein and Trolox showed significant antioxidant activity. At concentrations between 10(-10) and 10(-6) M and trilinolein has the most potent antioxidant activity with a maximal mean reduction of OFR of -48.0%, whereas Trolox showed only -39.2%. As for the natural plant antioxidants, only catechin showed potent antioxidant activity (-40%). Polyunsaturated triglycerides such as triolein (oleic acid, C 18:1) also possess significant OFR scavenging effect (-31.9%) whilst saturated triglycerides such as tristearin (stearic acid, C 18:0) had only relatively weak antioxidant activity (-15.2%). Generally, the antioxidant activity of unsaturated compounds is stronger than saturated compounds; double-bond existence may partially explain this phenomenon.     

The effect of certain vitamin deficiencies on hepatic drug metabolism

There is increasing evidence that the liver microsomal drug metabolizing system is affected by various vitamins such as ascorbic acid, riboflavin, and alpha-tocopherol. In regard to ascorbic acid deficiency there is a decrease in the quantity of hepatic microsomal electron transport components such as cytochrome P-450 and NADPH-cytochrome P-450 reductase, as well as decreases in a variety of drug enzyme reactions such as N-demethylation, O-demethylation, and steroid hydroxylation. In addition, young animals given high supplements of vitamin C have increased quantities of electron transport components and overall drug metabolism activities. Kinetic studies indicate no change in the apparent Km of N-demethylase, O-demethylase or hydroxylase for drug substrates in animals depleted or given high amounts of the vitamin. However, there are qualitative changes in both type I and II substrate-cytochrome P-450 binding. Ascorbic acid is not involved in microsomal lipid peroxidation or in any qualitative or quantitative change in phosphatidylcholine. Replenishing vitamin C-deficient animals with ascorbic acid required 3 to 7 days for the electron transport components and drug metabolism activities to return to normal levels. Induction with phenobarbital and 3-methylcholanthrene is not impaired in the deficient animal since drug metabolism activities are induced to the same extent as normal controls; however, the administration of delta-aminolevulinic acid, a precursor of heme synthesis, to deficient animals caused an increase in the quantity of cytochrome P-450. The effects of riboflavin deficiency on electron transport components and drug metabolism activities have been noted only in adult animals after prolonged periods of deficiency. Decreases in drug metabolism activities occur with both type I (aminopyrine and ethylmorphine) and type II (aniline) substrates. As was found with ascorbic acid deficiency, drug enzyme induction occurred to the same extent with phenobarbital in deficient and normal animals. In addition, it required from 10 to 15 days for the drug metabolism activities to return to normal levels when deficient animals were replenished with riboflavin. The effect of vitamin E on drug metabolism is specific in N-demethylase activities decrease while O-demethylase activities are not affected in the deficient state. This vitamin differs from ascorbic acid and riboflavin in that several laboratories have reported no quantitative decrease in cytochrome P-450, although there are some reports that it and delta-aminolevulinic acid dehydratase are lowered quantity of cytochrome in E-deficient animals. The effect of vitamin E, if any, on the P-450 is unresolved; an important question that requires further clarification. As with ascorbic acid there is no difference in the apparent Km of N-demethylase enzymes for varous substrates and the protective effect of vitamin E does not appear to be one of an antioxidant inhibiting microsomal lipid peroxidation.     

Involvement of vitamin E and protein thiols in the inhibition of microsomal lipid peroxidation by glutathione

Iron-ascorbate stimulated lipid peroxidation in rat liver microsomes can be inhibited by glutathione (GSH). The role of protein thiols and vitamin E in this process was studied in liver microsomes isolated from rats fed diets either sufficient or deficient in vitamin E and incubated at 37 degrees C under 100% O2. Lipid peroxidation was induced by adding 400 microM adenosine 5'-triphosphate, 2.5 to 20 microM FeCl3, and 450 microM ascorbic acid. One mL of the incubation mixture was removed at defined intervals for the measurement of thiobarbituric acid reactive substances (TBARS), protein thiols and vitamin E. In vitamin E sufficient microsomes, the addition of GSH enhanced the lag time prior to the onset of maximal TBARS accumulation and inhibited the loss of vitamin E. Treatment of these microsomes with the protein thiol oxidant diamide resulted in a 56% loss of protein thiols, but did not significantly change vitamin E levels. However, diamide treatment abolished the GSH-mediated protection against TBARS formation and loss of vitamin E during ascorbate-induced peroxidation. Liver microsomes isolated from rats fed a vitamin E deficient diet contained 40-fold less vitamin E and generated levels of TBARS similar to vitamin E sufficient microsomes at a 4-fold lower concentration of iron. GSH did not affect the lag time prior to the onset of maximal TBARS formation in vitamin E deficient microsomes although total TBARS accumulation was inhibited. Similar to what was previously found in vitamin E sufficient microsomes [Palamanda and Kehrer, (1992) Arch. Biochem. Biophys. 293, 103-109], GSH prevented the loss of protein thiols in vitamin E deficient microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebral metabolic monitoring experience in critical neurological patients

The effects of vitamin E on lipid peroxidation, intracellular free Ca2+ concentration ([Ca2+]i), and cell death were investigated in the postischemic immature cerebellum. Deprivation of oxygen and glucose for 10-min in a suspension of freshly dissociated granule cells from the cerebellum of 9-day-old male rat pups resulted in a recovery-induced consumption of cell nonenzymatic antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) and development of membrane lipid peroxidation as measured by the thiobarbituric acid method. The rate of lipid peroxidation of the postischemic cells was stimulated, not reduced, by treatment of the cells with vitamin E (5-30 microM alpha-tocopherol phosphate). In flow-cytometric studies a 10-min period of ischemia resulted in a small increase in intracellular calcium concentration, lipid peroxidation products and cell death, but in the presence of alpha-tocopherol the same treatment caused a dramatic increase in cell death, accompanied by a large increase in [Ca2+]i and lipid peroxidation products. Pretreatment of the cells with a mixture of three antioxidants (vitamin C/rutin/ubiquinol-10, 10/5/1) or nickel (Ni2+) reduced the alpha-tocopherol-induced increases in [Ca2+]i, and cell death. Hydrogen peroxide (1 mM) and the water-soluble analogue of vitamin E, trolox (50 microM), mimicked the effect of vitamin E on lipid peroxidation in the postischemic cells. Pretreatment of the cells with the intracellular Ca2+ chelator BAPTA-AM, reduced both the alpha-tocopherol-induced increase in [Ca2+]i and cell death. The effect of vitamin E on [Ca2+]i was age dependent and decreased abruptly during maturation of the cerebellum between the first and second weeks of life. Results of in vitro treatment of the immature cerebellar cells with the water-soluble form of vitamin E (alpha-tocopherol phosphate) suggest that, after consumption of cellular co-antioxidants, vitamin E may be converted to an alpha-tocopheroxyl radical, which act as a toxic prooxidant as cellular bioenergetics deteriorate.     

Synergistic effect of glycolic acid on the antioxidant activity of alpha-tocopherol and melatonin in lipid bilayers and in human skin homogenates

Considerable interest has been raised concerning the use of natural compounds in preventing skin aging and photoaging. In the idea that the combined action of agents increasing epidermal turnover with antioxidants could be advantageous in cosmetic and therapeutic treatments, we first investigated if alpha-glycolic acid affected or prevented the antioxidant activity of vitamin E and of melatonin, two compounds found beneficial as topical photoprotectant. Assays were carried out in vitro either in a biomimetic liposomal system, or in human skin homogenates. Lipid peroxidation was monitored spectrophotometrically by the time course of lipid hydroperoxide production in liposomes and by formation of TBA reactive substances (TBARS) in skin homogenates. Glycolic acid, at 25 microM to 1 mM, showed a mild, concentration-dependent antioxidant effect in liposomes, as evaluated by a slight decrease of the peroxidation rate, while, at 1 mM, reduced TBARS production in skin homogenates by 14%. Combinations of either vitamin E or melatonin with glycolic acid, in a 1:5 to 1:200 molar ratio, resulted in a clear synergistic protection of liposomes, more evident for the combination of glycolic acid with vitamin E. An amount of synergism up to 250% and up to 80% was evaluated with vitamin E and melatonin, respectively. Consumption rate of vitamin E during peroxidation of liposomes, in the absence or in the presence of glycolic acid, suggests that regeneration of vitamin E may in part explain the observed synergism. Synergistic antioxidant activity between vitamin E and glycolic acid was also observed in skin homogenates, whereas the effect of glycolic acid on the antioxidant activity of melatonin appeared additive. However, the combination of these three compounds inhibited TBARS production almost completely. Our data provide evidence that glycolic acid can strongly potentiate the antioxidant action of melatonin and vitamin E. This may suggest the advantage of combining alpha-glycolic acid with these antioxidants in skin designed preparations, both to improve penetration and availability of antioxidants to epidermal layers and to enhance their protective potential.     

A potent chain-breaking antioxidant activity of the cardiovascular drug dipyridamole

The antioxidant properties of the antithrombotic drug dipyridamole have been studied using lipid oxidation assays based on the generation of peroxy radicals by azo compounds. Dipyridamole was observed to prevent both peroxidation of arachidonic acid micelles in aqueous solution and peroxidation of methyl linoleate in organic solvents; in contrast to vitamin E, dipyridamole was found to scavenge both hydrophilic and hydrophobic radicals. The rate constant for the reaction of dipyridamole with methyl linoleate peroxyl radicals at 37 degrees C was calculated as 2 x 10(6) M-1s-1, in comparison to 1 x 10(6) M-1s-1 of vitamin E under the same conditions. The antioxidant efficiency of the drug was confirmed in experiments with radiolysis-induced oxidation and through measurements of malondialdehyde production and diene formation. As a result of radical scavenging, a relatively stable dipyridamole radical was formed that could be detected by electron spin resonance spectroscopy. The particular antioxidant properties of dipyridamole may explain the vasodilating and antiplatelet effects of this cardiovascular drug.     

Mechanism of brain protection by nitroxide radicals in experimental model of closed-head injury

Reactive oxygen-derived species were previously implicated in mediation of post-traumatic brain damage; however, the efficacy of traditional antioxidants in preventing/reversing the damage is sometimes limited. The present work focused on the mechanisms underlying the neuroprotective activity of cell permeable, nontoxic, antioxidants, namely stable nitroxide radicals in an experimental model of rat closed-head injury. Brain damage was induced by the weight-drop method and the clinical status was evaluated according to a neurological severity score at 1 h and 24 h, where the difference between these scores reflects the extent of recovery. The metal chelator deferoxamine as well as three nitroxide derivatives, differing in hydrophilicity and charge, and one hydroxylamine (a reduced nitroxide) facilitated the clinical recovery and decreased the brain edema. The nitroxides, but neither the hydroxylamine nor deferoxamine, protected the integrity of the blood-brain barrier. Superoxide dismutase also improved the clinical recovery but did not affect brain edema or the blood-brain barrier. The results suggest that by switching back and forth between themselves, the nitroxide and hydroxylamine act catalytically as self-replenishing antioxidants, and protect brain tissue by terminating radical-chain reactions, oxidizing deleterious metal ions, and by removal of intracellular superoxide.     

Antioxidant behaviors of vitamin E analogues in unilamellar vesicles

The antioxidant behaviors of vitamin E and its analogues, 2,2,5,7,8-pentamethyl-6-hydroxychroman and 1,2-diacyl-sn-glycero3-phospho-2'-(hydroxyethyl)-2'- 5',7',8'-tetramethyl-6'-hydroxychroman, were studied in unilamellar vesicles. The two analogues scavenged aqueous radicals generated from azo compounds more efficiently than vitamin E. On the other hand, vitamin E scavenged the lipid peroxyl radicals preferentially. It is concluded that the superior antioxidant activity of vitamin E is attributed to its location suitable for breaking the chain propagation reaction.     

Oxidative stress and cytotoxicity induced by ferric-nitrilotriacetate in HepG2 cells that express cytochrome P450 2E1

Iron can potentiate the toxicity of ethanol. Ethanol increases the content of cytochrome P450 2E1 (CYP2E1), which generates reactive oxygen species, and transition metals such as iron are powerful catalysts of hydroxyl radical formation and lipid peroxidation. Experiments were carried out to attempt to link CYP2E1, iron, and oxidative stress as a potential mechanism by which iron increases ethanol toxicity. The addition of ferric-nitrilotriacetate (Fe-NTA) to a HepG2 cell line expressing CYP2E1 decreased cell viability, whereas little effect was observed in control cells not expressing CYP2E1. Toxicity in the CYP2E1-expressing cells was markedly enhanced after the depletion of glutathione. Lipid peroxidation was increased by Fe-NTA, especially in cell extracts and medium from the CYP2E1-expressing cells. Toxicity was completely prevented by vitamin E or by 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, which also decreased the lipid peroxidation. Levels of ATP were lowered by Fe-NTA, and this was associated with a decreased rate of oxygen consumption by permeabilized cells with substrates donating electrons to complexes I, II, and IV of the respiratory chain. This mitochondrial damage was prevented by vitamin E. Toxicity was accompanied by DNA fragmentation, and this fragmentation was prevented by antioxidants. Overexpression of bcl-2 decreased the toxicity and DNA fragmentation produced by the combination of CYP2E1 plus Fe-NTA, as did a peptide inhibitor of caspase 3. These results suggest that elevated generation of reactive oxygen species in HepG2 cells expressing CYP2E1 leads to lipid peroxidation in the presence of iron, and the ensuing prooxidative state damages mitochondria, releasing factors that activate caspase 3, leading to a loss in cell viability and DNA fragmentation.     

Mitochondrial function is differentially affected upon oxidative stress

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in synaptosomes treated with ascorbate/iron. A loss of membrane integrity, evaluated by electron microscopy and by LDH leakage, was observed in peroxidized synaptosomes and it was prevented by pre-incubation with vitamin E (150 microM) and idebenone (50 microM). ATP levels decreased, in synaptosomes exposed to ascorbate/iron, as compared to controls. NADH-ubiquinone oxidoreductase (Cx I) and cytochrome c oxidase (Cx IV) activities were unchanged after ascorbate/iron treatment, whereas succinate-ubiquinone oxidoreductase (Cx II), ubiquinol cytochrome c reductase (Cx III) and ATP-synthase (Cx V) activities were reduced by 55%, 40%, and 55%, respectively. The decrease of complex II and ATP-synthase activities was prevented by reduced glutathione (GSH), whereas the other antioxidants tested (vitamin E and idebenone) were ineffective. However, vitamin E, idebenone and GSH prevented the reduction of complex III activity observed in synaptosomes treated with ascorbate/iron. GSH protective effect suggests that the oxidation of protein SH-groups is involved in the inhibition of complexes II, III and V activity, whereas vitamin E and idebenone protection suggests that membrane lipid peroxidation is also involved in the reduction of complex III activity. These results may indicate that the inhibition of the mitochondrial respiratory chain enzymatic complexes, that are differentially affected by oxidative stress, can be recovered by specific antioxidants.