Effect of the pyrrolopyrimidine lipid peroxidation inhibitor U-101033E on neuronal and astrocytic metabolism and infarct volume in rats with transient middle cerebral artery occlusion

The aim of the present study was to assess the effect of post ictal administration of the pyrrolopyrimidine lipid peroxidation inhibitor, U-101033E, on infarct volume and neuronal and astrocytic metabolism in rats with transient middle cerebral artery occlusion (MCAO).

Rats were subjected to 120 min of MCAO followed by 140 min of reperfusion and randomly assigned to control (n = 17) or U-101033E treatment (n = 16). Drug infusion started 5 min after MCAO and lasted 220 min with a 15 min interruption during the reperfusion procedure. Sixteen rats underwent diffusion weighted imaging 260 min after ictus, from which the apparent diffusion coefficient (ADC) was determined. Seventeen rats received an iv bolus injection of [1-¹³C]glucose and [1,2-¹³C]acetate 245 min after ictus. Tissue extracts from two brain regions representing penumbra and ischemic core were analyzed with ¹³C NMRS and HPLC.

U-101033E did not affect the volume of ischemic tissue estimated from the ADC maps. In the penumbra, U-101033E specifically decreased mitochondrial pyruvate metabolism via both pyruvate dehydrogenase and pyruvate carboxylase pathways. Thus, U-101033E impaired both neuronal and astrocytic mitochondrial pyruvate metabolism. At the same time anaerobic glucose usage was increased, leading to increased lactate labeling and content. Also alanine labeling was increased. The data do not support lactate as an important substrate for neuronal mitochondria in ischemia–reperfusion. A similar pattern of reduced mitochondrial pyruvate metabolism and increased cytosolic pyruvate metabolism was found in the irreversibly damaged ischemic core. The present study highlights the importance of other outcome measures than ischemic tissue volume for evaluation of drug efficacy in animal models, which in turn could increase the likelihood of success in clinical trials.     

6-Hydroxymelatonin converts Fe (III) to Fe (II) and reduces iron-induced lipid peroxidation

Disorders of iron accumulation are known to produce hepatotoxicity. Agents, which can reduce Fe(3+) to a more usable form Fe(2+) could potentially limit such damage. Since it has been previously demonstrated that the pineal secretory product, melatonin, is able to bind iron, we decided to investigate the potential protective properties of the principal melatonin metabolite and degradant, 6-hydroxymelatonin (6-OHM). Using adsorptive cathode stripping voltammetry (AdCSV) we showed that Fe(3+) in the presence of 6-OHM is converted to Fe(2+). We further demonstrated that 6-OHM reduces the Fe(2+)-induced rise in lipid peroxidation in rat liver homogenates. The results imply that 6-OHM facilitates the conversion of Fe(3+) to Fe(2+) which is a more biologically usable form of iron. While such a conversion could also potentially make more Fe(2+) available for driving the Fenton reaction and the consequent generation of the dangerous hydroxyl radical, 6-OHM is able to quench these radicals, thereby providing tissue protection.     

The action of a homologous series of ubiquinols on lipid peroxidation in brain mitochondrial and synaptosomal membranes

The effects of inhibition of ubiquinols and ubiquinones with various length of isoprenoid chain on the lipid peroxidation in membranes of brain mitochondria and synaptosomes were studied. The efficiency of inhibition effects of ubiquinols depends on the length of isoprenoid chain. Ubiquinols with shorter isoprenoid chains demonstrated more effective inhibition.     

Mitochondria and microsomal membranes have a free radical reductase activity that prevents chromanoxyl radical accumulation

Enzyme-dependent mechanisms which prevent accumulation of chromanoxyl radicals derived from the vitamin E analogue, 2,2,5,7,8-pentamethyl-6-hydroxycromane (PMC), were characterized in rat liver microsomal and mitochondrial membranes. The free radical oxidation product of PMC (chromanoxyl radical) was generated in membranes using either photochemical (uv light) or enzymatic (lipoxygenase and arachidonic acid) methods and detected by ESR. Substrates (NADH or NADPH) prevented accumulation of chromanoxyl radicals until the substrate was fully consumed. In microsomes, reduced glutathione increased the efficacy of NADPH in preventing the accumulation of the chromanoxyl radical, but was without effect in the absence of NADPH. Ascorbate also prevented accumulation of the chromanoxyl radical. It is concluded that rat liver microsomes and mitochondria have both enzymatic and non-enzymatic mechanisms for reducing chromanoxyl radicals.     

Protective properties of butanolic extract of the Calendula officinalis L. (marigold) against lipid peroxidation of rat liver microsomes and action as free radical scavenger

Calendula officinalis (marigold) has many pharmacological properties. It is used for the treatment of skin disorders, pain and also as a bactericide, antiseptic and anti-inflammatory. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to participate in the pathogenesis of various human diseases and may be involved in the conditions which C. officinalis is used to treat. The aim of this study was to investigate the relationship between the beneficial properties of this plant and its antioxidant action. The butanolic fraction (BF) was studied because it is non-cytotoxic and is rich in a variety of bioactive metabolites including flavonoids and terpenoids. Superoxide radicals (O(2)(*-)) and hydroxyl radicals (HO(*)) are observed in decreasing concentrations in the presence of increasing concentrations of BF with IC(50) values of 1.0 +/- 0.09 mg/ml and 0.5 +/- 0.02 mg/ml, respectively, suggesting a possible free radical scavenging effect. Lipid peroxidation in liver microsomes induced by Fe(2+)/ascorbate was 100% inhibited by 0.5 mg/ml of BF (IC(50) = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in microM Trolox equivalents) was 368.14 +/- 23.03 and its total antioxidant reactivity (TAR) was calculated to be 249.19 +/- 14.5 microM. The results obtained suggest that the butanolic fraction of C. officinalis possesses a significant free radical scavenging and antioxidant activity and that the proposed therapeutic efficacy of this plant could be due, in part, to these properties.     

Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage

Iron storage proteins, ferritin and haemosiderin, release iron to a range of chelators and reducing agents, including citrate, acetate and ascorbate. Released iron promotes both hydroxyl radical formation in the presence of hydrogen peroxide and lipid peroxidation in liposomes. Ferritin protein is modified in such reactions, both by free radical cleavage and addition reactions with aldehyde products of lipid peroxidation.     

Kinetics of lipid peroxidation in compartmentalized systems initiated by a water-soluble free radical source

Kinetic rate laws arising from theoretical expectations for the oxidation of lipids initiated by water-soluble free radicals in compartmentalized systems under different experimental conditions are deduced. In particular, the predictions for the kinetic reaction orders in: (a) intra-particle oxidizable compound concentration (at fixed number of particles and particle size), alpha; (b) number of particles or analytical lipid concentration (at fixed intra-particle concentration and particle size), beta and (c) initiator, gamma, are obtained. The reaction orders beta and gamma are determined by the fraction of initiator derived radicals captured by the particles (f) and the mean number of chain carrying radicals per particle () when the system reaches the steady state condition. Predicted orders in initiator range from 0 ( = 0.5) to 0.5 (f-->1; > > 1), while the order in number of particles ranges between 0.5 (f-->1; > > 1) and 1. These predictions are tested by measuring the kinetic law for the oxidation of SUV's egg yolk phosphatidylcholine vesicles initiated by the thermal decomposition of ABAP. The results indicate that, under the conditions employed, beta = 0.68 +/- 0.05 and gamma = 0.46 +/- 0.04. These values are close to those expected for a system in which > > 1 and the efficiency of capture is relatively high. This last condition is confirmed by estimating the efficiency of capture from a comparison of induction times elicited by similar concentrations of Trolox and alpha-tocopherol.     

C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro

C-Phycocyanin (from Spirulina platensis) effectively inhibited CCl(4)-induced lipid peroxidation in rat liver in vivo. Both native and reduced phycocyanin significantly inhibited peroxyl radical-induced lipid peroxidation in rat liver microsomes and the inhibition was concentration dependent with an IC(50) of 11.35 and 12.7 microM, respectively. The radical scavenging property of phycocyanin was established by studying its reactivity with peroxyl and hydroxyl radicals and also by competition kinetics of crocin bleaching. These studies have demonstrated that phycocyanin is a potent peroxyl radical scavenger with an IC(50) of 5.0 microM and the rate constant ratios obtained for phycocyanin and uric acid (a known peroxyl radical scavenger) were 1.54 and 3.5, respectively. These studies clearly suggest that the covalently linked chromophore, phycocyanobilin, is involved in the antioxidant and radical scavenging activity of phycocyanin.     

The lipid peroxidation product 4-hydroxynonenal is a potent inducer of the SOS response.

An important aspect of bacterial mutagenesis by several difunctional carbonyl compounds appears to be the induction of the SOS system. We tested the ability of a series of carbonyl compounds to induce expression of the SOS-regulated umu operon in Salmonella typhimurium TA1535/pSK1002. SOS-inducing potencies varied widely among the carbonyl compounds tested. 4-Hydroxynonenal, a product of lipid peroxidation, was the most potent SOS-inducer, with maximal induction observed at concentrations of 0.1-1 microM. Acrolein, crotonaldehyde and methacrolein induced little increase over background umu expression. Malondialdehyde, another product of lipid peroxidation, was a very weak SOS-inducer with a maximal response induced at a concentration of 28 mM. Substitution at the alpha-position of malondialdehyde, which abolishes frameshift mutagenicity, did not abolish SOS-inducing activity. Substitution of the hydroxyl group of malondialdehyde and alpha-methyl-malondialdehyde by a better leaving group (benzoyloxy) resulted in an approximately 250-fold higher SOS-inducing potency. Comparison of the present results to literature reports on bacterial mutagenicity indicates a poor correlation of the two properties between different classes of difunctional carbonyl compounds and even within the same class of difunctional carbonyl compounds.     

The importance of lipid solubility in antioxidants and free radical generating systems for determining lipoprotein peroxidation

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC₅₀ = 5.9 μM) and lazaroid (IC₅₀ = 5.0 μM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC₅₀ = 5.2 × 10³ μM) and trolox (IC₅ = 1.2 × 10³ μM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2′-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2′azobis (2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.     

Increase in free radical generation and lipid peroxidation following chemotherapy in patients with cancer

Several anti-cancer drugs are known to bring about their tumoricidal actions by a free radical dependent mechanism. Majority of the studies reported that adriamycin, mitomycin C, bleomycin, etc., augment free radical generation and lipid peroxidation process in vitro. Our results reported here suggest that following chemotherapy both stimulated and unstimulated human polymorphonuclear leukocytes generate increased amounts of superoxide anion and hydrogen peroxide. This was accompanied by increased formation of lipid peroxidation products as measured by thiobarbituric acid assay. These results confirm that many anti-cancer drugs augment free radical generation and lipid peroxidation even in an vivo situation.     

Protective properties of butanolic extract of the Calendula officinalis L.(marigold) against lipid peroxidation of rat liver microsomes and action as free radical scavenger

Abstract

Calendula officinalis (marigold) has many pharmacological properties. It is used for the treatment of skin disorders, pain and also as a bactericide, antiseptic and anti-inflammatory. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to participate in the pathogenesis of various human diseases and may be involved in the conditions which C. officinalis is used to treat. The aim of this study was to investigate the relationship between the beneficial properties of this plant and its antioxidant action. The butanolic fraction (BF) was studied because it is non-cytotoxic and is rich in a variety of bioactive metabolites including flavonoids and terpenoids. Superoxide radicals (O₂^?-) and hydroxyl radicals (HO^?) are observed in decreasing concentrations in the presence of increasing concentrations of BF with IC₅₀ values of 1.0 ± 0.09 mg/ml and 0.5 ± 0.02 mg/ml, respectively, suggesting a possible free radical scavenging effect. Lipid peroxidation in liver microsomes induced by Fe²⁺/ascorbate was 100% inhibited by 0.5 mg/ml of BF (IC₅₀ = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in μM Trolox equivalents) was 368.14 ± 23.03 and its total antioxidant reactivity (TAR) was calculated to be 249.19 ± 14.5 μM. The results obtained suggest that the butanolic fraction of C. officinalis possesses a significant free radical scavenging and antioxidant activity and that the proposed therapeutic efficacy of this plant could be due, in part, to these properties.     

Ferric(III) ions inhibits copper(II)/hydrogen peroxide-catalyzing lipid peroxidation in human erythrocyte membranes.

1. Effect of ferric ions (Fe3+) on the lipid peroxidation catalyzed by copper ions (Cu2+) and hydrogen peroxide (H2O2) was studied in human erythrocyte membranes. 2. The formation of thiobarbituric acid-reactive products elicited by CuCl2/H2O2 was inhibited by FeCl3 in a concentration-dependent manner; 0.25 mM FeCl3 were enough to cause 50% inhibition of the formation of peroxides. 3. The inhibitory effect of FeCl3 is not due to competition against Cu2+. 4. FeCl3 inhibited the initiation, but did not inhibit the propagation of Cu2+/H2O2-catalyzing lipid peroxidation. 5. In the heat- or trypsin-treated erythrocyte membranes, FeCl3 had no inhibitory effect on Cu2+/H2O2-catalyzing lipid peroxidation. 6. Sodium azide, an inhibitor of catalase, had no effect on the inhibitory effect of FeCl3. 7. These results suggest that a protein factor(s), which is not catalase, is involved in the inhibition of Cu2+/H2O2-catalyzing lipid peroxidation by Fe3+.     

The role of Fe2+-induced lipid peroxidation in the initiation of the mitochondrial permeability transition

Iron and iron complexes stimulate lipid peroxidation and formation of malondialdehyde (MDA). We have studied the effects of Fe2+ and ascorbate on mitochondrial permeability transition induced by phosphate and Ca2+. Iron is necessary for detectable MDA formation, but only Ca2+ and phosphate are necessary for the induction of membrane potential loss (Deltapsi) and Ca2+ release. Keeping the iron at a constant concentration and varying the Ca2+ level changed the mitochondrial Ca2+ retention times, but not the amount of MDA formation. The antioxidant butylated hydroxytoluene at low concentrations prevented MDA formation, but not mitochondrial Ca2+ release. Preincubation of mitochondria with Fe2+ decreased Ca2+ retention time in a concentration-dependent manner and facilitated Ca2+-stimulated MDA accumulation. Thus, Ca2+ phosphate-induced mitochondrial permeability transition (MPT) can be separated mechanistically from MDA accumulation. Lipid peroxidation products do not appear to participate in the initial phase of the permeability transition, but sensitize mitochondria toward MPT.     

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used ( 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.     

Nitric oxide inhibition of free radical-mediated lipid peroxidation in photodynamically treated membranes and cells

Of the numerous biological activities attributed to nitric oxide ((*)NO), relatively little is known about its ability to intercept lipid-derived free radicals and thus protect cells against the damaging effects of lipid peroxidation, particularly in photodynamic settings. To address this, we asked how the (*)NO donor spermine-NONOate (SPER/NO) would affect porphyrin (PpIX)-photosensitized, iron/ascorbate-amplified chain peroxidation in cholesterol (Ch)/phospholipid (0.8:1.0, mol/mol) liposomes. Several Ch oxidation products (ChOX) were monitored by high performance chromatographic techniques. When added immediately before irradiation, SPER/NO (0.4 mM) had no effect on accumulation of 5alpha-hydroperoxide, a primary singlet oxygen-derived ChOX, but strongly suppressed the secondary species arising from postphotooxidation chain reactions, including 7alpha/7beta-hydroperoxides, 7alpha/7beta-hydroxides, and 5,6-epoxides. Metabolism of exogenous 5-aminolevulinate to PpIX in COH-BR1 tumor cells sensitized them to ChOX photogeneration and necrotic photokilling. When present during irradiation, active (but not decomposed) SPER/NO strongly inhibited both effects. These findings support the hypothesis that suitably presented NO, by intercepting lipid-derived radicals, can antagonize the antitumor effects of photodynamic therapy and other oxidative therapies.     

Changes in lipid peroxidation and free radical scavengers in kidney of hypothyroid and hyperthyroid rats

Kidney weight was significantly decreased in hypothyroidism (induced by Na131I administration) and increased in hyperthyroidism (induced by thyroxine treatment) as compared to control in female Wistar rats. The tissue lipid peroxidation level remained unchanged in hyperthyroid rats but significantly increased in hypothyroid rats. Superoxide dismutase was decreased in both experimental groups but more so in hyperthyroid rats. Catalase was reduced significantly in hyperthyroid rats but remained unaffected in hypothyroid rats. Tissue glutathione peroxidase (GPx) activity was increased while reduced glutathione levels remained unaltered in both hypothyroid and hyperthyroid rats. Plasma GPx activity was significantly low in both the hypothyroid and hyperthyroid rats. The results suggest alterations in the oxidative stress in hypothyroid and hyperthyroid rat kidneys with concomitant changes of free radical scavengers.     

Channa punctata brain metallothionein is a potent scavenger of superoxide radicals and prevents hydroxyl radical-induced in vitro DNA damage

Mammalian brain metallothioneins (MTs) have been shown to scavenge free radicals. However, a similar role for fish brain MT has not been established yet. Previously, we have reported that MT from the liver of a freshwater fish, Channa punctata Bloch, had free-radical-scavenging activity in vitro. In this study, we report on the induction of MT in brain and other tissues of C. punctata treated with a low concentration of zinc chloride. We partially purified MT (Zn-MT)-rich fraction from the brain and studied its free-radical-scavenging and DNA damage attenuating effects. Zinc exposure showed significant MT induction in brain, gill, kidney, and liver. C. punctata brain MT efficiently scavenged superoxide radicals and also attenuated hydroxyl radical-mediated DNA damage. These findings suggest that fish brain MT has a free-radical-scavenging activity, and its expression may be regulated in response to stress and chemical exposure. C. punctata has been identified as a potent biomarker fish species. It is suggested that this fish species may be a good model for the study of MTs with regard to their regulatory and biomarker functions.     

The lipid peroxidation model for halogenated hydrocarbon toxicity. Kinetics of peroxyl radical processes involving fatty acids and Fe(III) porphyrins

The toxicity of halogenated alkanes originates from their metabolism by cytochrome P-450 which leads to the formation of reactive intermediates. In particular, peroxyl radicals derived from the halogenated compounds are believed to induce peroxidative chain degradation of lipids. To examine this hypothesis, radical reactions in a system involving FeIII-deuteroporphyrin as a model of cytochrome P-450, fatty acids or cholesterol, and carbon tetrachloride or the anesthetic agent halothane are studied by means of pulse radiolysis. It is shown that haloperoxyl radicals react with the fatty acids in competition with their reaction with the ferriporphyrin. Moreover, the secondary fatty acid peroxyl radicals also react efficiently with the porphyrin. A model for halogenated alkane toxicity is discussed in terms of these new findings. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 are emphasized.