Enhancement of Lipid Peroxidation by Indole-3-Acetic Acid and Derivatives: Substituent Effects

The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the Nl position. Rates of decarboxylation,pK_a values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.     

Lipid peroxidation induced by phenylbutazone radicals

Lipid peroxidation was investigated to evaluate the deleterious effect on tissues by phenylbutazone (PB). PB induced lipid peroxidation of microsomes in the presence of horseradish peroxidase and hydrogen peroxide (HRP-H2O2). The lipid peroxidation was completely inhibited by catalase but not by superoxide dismutase. Mannitol and dimethylsulfoxide had no effect. These results indicated no paticipation of superoxide and hydroxyl radical in the lipid peroxidation. Reduced glutathione (GSH) efficiently inhibited the lipid peroxidation. PB radicals emitted electron spin resonance (ESR) signals during the reaction of PB with HRP-H2O2. Microsomes and arachidonic acid strongly diminished the ESR signals, indicating that PB radicals directly react with unsaturated lipids of microsomes to cause thiobarbituric acid reactive substances. GSH sharply diminished the ESR signals of PB radicals, suggesting that GSH scavenges PB radicals to inhibit lipid peroxidation. Also, 2-methyl-2-nitrosopropan strongly inhibited lipid peroxidation. R-Phycoerythrin, a peroxyl radical detector substance, was decomposed by PB with HRP-H2O2. These results suggest that lipid peroxidation of microsomes is induced by PB radicals or peroxyl radicals, or both.     

Enhancement of peroxidase-induced lipid peroxidation by indole-3-acetic acid: effect of antioxidants

Summary

Indole-3-acetic acid (IAA) enhanced the peroxidase-induced lipid peroxidation in phosphatidylcholine liposomes, as measured by loss of fluorescence of cis-parinaric acid. α-Tocopherol or β-carotene in the lipid phase or ascorbate or Trolox in the aqueous phase inhibited the loss of fluorescence induced by the peroxidase + IAA system, but glutathione had only a small inhibitory effect. The peroxyl radical formed by one-electron oxidation of IAA, followed by decarboxylation and reaction with oxygen, is suggested to act as the initiator of lipid peroxidation. The protection by ascorbate or Trolox is explained by the reactivity of these compounds with the IAA indolyl radical, as shown by pulse radiolysis experiments, whereas the weak effect of glutathione agrees with its low reactivity towards the IAA-derived peroxyl radical and its precursors.     

Action of quinolinic and indolinonic aminoxyls as radical-scavenging antioxidants.

The kinetic studies on the actions of quinolinic and indolinonic aminoxyls in the oxidation of lipid peroxidation induced by free radicals were carried out to evaluate their antioxidant activity. These aminoxyls showed a similar reactivity toward peroxyl radical with alpha-tocopherol. The antioxidant efficacies of aminoxyls against oxidation of methyl linoleate in homogeneous solution were smaller than that of alpha-tocopherol. Hydroxylamine, a reduced form of aminoxyl, possessed a comparative antioxidant efficacy with alpha-tocopherol and was capable of suppressing the consumption of alpha-tocopherol. Aminoxyls showed more potent antioxidant activity than alpha-tocopherol against the oxidation of methyl linoleate micelles induced by peroxyl radical or by a combination of copper ion and hydrogen peroxide. These results suggest that quinolinic and indolinonic aminoxyls may act as potent antioxidants against lipid peroxidation, especially in the presence of a good reductant which reduces aminoxyl radicals to hydroxylamines.     

Reactivity of ebselen and related selenoorganic compounds with 1,2-dichloroethane radical cations and halogenated peroxyl radicals

The reactivity of ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, and structurally related analogues was studied by pulse radiolysis. The rate constant for the reaction of ebselen with trichloromethylperoxyl radicals was determined to be 2.9 X 10(8) M-1 s-1, while its sulfur analogue, 2-phenyl-1,2-benzisothiazol-3(2H)one, was oxidized at much lower rates, k less than or equal to 10(7) M-1 s-1. Among several derivatives studied, the only other compound that exhibited a high rate constant was 2-(methylseleno)-benzoic acid-N-phenylamide. Oxidation of ebselen by other halogenated peroxyl radicals was also carried out and revealed a direct relationship between rate constant and the degree of halogenation of the oxidant. The transient radicals generated during oxidation of ebselen and the analogues were characterized by optical absorption and conductivity measurements and were attributed to one-electron-oxidized radical cations. The oxidation potentials were determined by cyclic voltammetry. Comparative evaluation of the in vitro behavior during microsomal lipid peroxidation revealed ebselen to be the most potent antioxidant of the compounds investigated, 2-(Methylseleno)-benzoic acid-N-phenylamide, despite its high rate constant for oxidation by halogenated peroxyl radicals, was found to be a poor antioxidant. The rate constant of oxidation of ebselen by trichloromethylperoxyl radicals is comparable to that of alpha-tocopherol under similar conditions, underscoring the potential pharmacological interest of ebselen as an antioxidant.     

Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging

Oxidation of lipids has been implicated in the pathophysiology of atherosclerosis. It has been suggested that scavenging of lipid peroxyl radicals contribute to the antiatherosclerotic effects of naturally occurring compounds such as the isoflavones. This group of polyphenolics includes genistein and is present in relatively high concentrations in food products containing soy. Soy isoflavones are capable of inhibiting lipoprotein oxidation in vitro and suppressing formation of plasma lipid oxidation products in vivo. However, key aspects of the antioxidant mechanisms remain unknown. In this study the antioxidant effects of genistein and other soy isoflavones on lipid peroxidation initiated by mechanistically diverse oxidants was investigated. Although isoflavones inhibited lipid peroxidation stimulated by both metal-dependent and independent processes, the concentration required for these effects were relatively high compared to those found in vivo. Interestingly, however, isoflavones were not consumed and remained in the native state over the time during which inhibition of lipid peroxidation was observed. This was also the case under conditions where synergistic inhibition of LDL oxidation was observed with ascorbate. Furthermore, in an oxidation system driven solely by peroxyl radicals, isoflavones were found to be relatively poor peroxyl radical scavengers. Consistent with the apparent lack of reactivity with lipid-derived oxidants, isoflavones were also relatively resistant to oxidation mediated by the potent oxidant peroxynitrite. The potential antioxidant mechanisms of isoflavones are discussed in the context of possible reactivities of isoflavone-derived phenoxyl radicals.     

Lipid peroxidation initiated by superoxide-dependent hydroxyl radicals using complexed iron and hydrogen peroxide

Iron salts stimulate lipid peroxidation by decomposing lipid peroxides to produce alkoxyl and peroxyl radicals which initiate further oxidation. In aqueous solution ferrous salts produce OH. radicals, a reactive species able to abstract hydrogen atoms from unsaturated fatty acids, and so can initiate lipid peroxidation. When iron salts are added to lipids, containing variable amounts of lipid peroxide, the former reaction is favoured and OH. radicals contribute little to the observed rate of peroxidation. When iron is complexed with EDTA, however, lipid peroxide decomposition is prevented, but the complex reacts with hydrogen peroxide to form OH. radicals which are seen to initiate lipid peroxidation. Superoxide radicals appear to play an important part in reducing the iron complex.     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.     

Pulse radiolysis study of the interaction of retinoids with peroxyl radicals

Vitamin A (retinol) and its derivatives-retinal and retinoic acid-are known for their ability to inhibit lipid peroxidation. Antioxidant actions of retinoids have been attributed to chain-breaking by scavenging of peroxyl radicals. Based on chemical analysis of retinoic acid degradation products formed during microsomal lipid peroxidation, it was previously suggested that retinoids interact with peroxyl radicals forming free carbon-centered radical adducts. However, it can be argued that such a mode of antioxidant action of retinoids is not sufficient to fully explain their effectiveness at inhibiting lipid peroxidation, which in many systems is comparable to, or even exceeds, that of alpha-tocopherol. In order to elucidate the mechanism of interaction of retinoids with peroxyl radicals, (trichloromethyl)peroxyl radical was generated by pulse radiolysis, and its interactions with retinoids solubilized in Triton X-100 micelles were followed by kinetic absorption spectroscopy. All retinoids--retinol, retinal, and retinoic acid--interacted with the peroxyl radical, and at least two transient products were detected. One of these products, absorbing at 590 nm, was identified as retinoid cation radical. Therefore, we postulate that, apart from formation of radical adducts, retinoids may also scavenge peroxyl radicals by electron transfer.     

Anti-hemolytic and Peroxyl Radical Scavenging Activity of Organoselenium Compounds: An In Vitro Study

Selenium-containing amino acids, selenocystine (CysSeSeCys), methylselenocysteine (MeSeCys), and selenomethionine (SeMet) have been examined for anti-hemolytic and peroxyl radical scavenging ability. Effect of these compounds on membrane lipid peroxidation, release of hemoglobin, and loss of intracellular K⁺ ion as a consequence of peroxyl radicals-induced oxidation of human red blood cells were used to evaluate their anti-hemolytic ability. The peroxyl radicals were generated from thermal degradation of 2,2′-azobis(2-methylpropionamidine) dihydrochloride. Significant delay (t _eff) was observed in oxidative damage in the presence of the selenium compounds. From the IC₅₀ values for the inhibition of hemolysis, lipid peroxidation, and K⁺ ion leakage, the relative anti-hemolytic ability of the compounds were found to be in the order of CysSeSeCys > MeSeCys > SeMet. The anti-hemolytic abilities of the compounds, when compared with sodium selenite (Na₂SeO₃) under identical experimental conditions, were found to be better than Na₂SeO₃. Relative rate constants estimated for the reaction of MeSeCys and SeMet with peroxyl radicals by competition kinetics using ABTS²⁻ as a reference confirmed that all the compounds are efficient peroxyl radical scavengers. Comparison of the GPx-like activity of these compounds, by NADPH–GSH reductase coupled assay, indicated that CysSeSeCys exhibits the highest activity. Based on these results, it is concluded that among the compounds examined, CysSeSeCys, possessing the ability to reduce peroxyl radicals and hydroperoxides showed efficient anti-hemolytic activity.     

Singlet oxygen generation from the decomposition of alpha-linolenic acid hydroperoxide by cytochrome c and lactoperoxidase

Generation of singlet oxygen is first investigated in the decomposition of polyunsaturated lipid peroxide, alpha-linolenic acid hydroperoxide (LAOOH), by heme-proteins such as cytochrome c and lactoperoxidase. Chemiluminescence and electron spin resonance methods are used to confirm the singlet oxygen generation and quantify its yield. Decomposition products of LAOOH are characterized by HPLC-ESI-MS, which suggests that singlet oxygen is produced via the decomposition of a linear tetraoxide intermediate (Russell's mechanism). Free radicals formed in the decomposition are also identified by the electron spin resonance technique, and the results show that peroxyl, alkyl, and epoxyalkyl radicals are involved. The changes of cytochrome c and lactoperoxidase in the reaction are monitored by UV-visible spectroscopy, revealing the action of a monoelectronic and two-electronic oxidation for cytochrome c and lactoperoxidase, respectively. These results suggest that cytochrome c causes a homolytic reaction of LAOOH, generating alkoxyl radical and then peroxyl radical, which in turn releases singlet oxygen following the Russell mechanism, whereas lactoperoxidase leads to a heterolytic reaction of LAOOH, and the resulting ferryl porphyryl radical of lactoperoxidase abstracts the hydrogen atom from LAOOH to give peroxyl radical and then singlet oxygen. This observation would be important for a better understanding of the damage mechanism of cell membrane or lipoprotein by singlet oxygen and various radicals generated in the peroxidation and decomposition of lipids induced by heme-proteins.     

Effect of O-glycosilation on the antioxidant activity and free radical reactions of a plant flavonoid,chrysoeriol

Chrysoeriol and its glycoside (chrysoeriol-6-O-acetyl-4'-beta-D-glucoside) are two natural flavonoids extracted from the tropical plant Coronopus didymus. The aqueous solutions of both the flavonoids were tested for their ability to inhibit lipid peroxidation induced by gamma-radiation, Fe (III) and Fe (II). In all these assays chrysoeriol showed better protecting effect than the glycoside. The compounds were also found to inhibit enzymatically produced superoxide anion by xanthine/xanthine oxidase system; here the glycoside is more effective than the aglycone. The rate constants for the reaction of the compounds with superoxide anion determined by using stopped-flow spectrometer were found to be nearly same. Chrysoeriol glycoside reacts with DPPH radicals at millimolar concentration, but the aglycone showed no reaction. Using nanosecond pulse radiolysis technique, reactions of these compounds with hydroxyl, azide, haloperoxyl radicals and hydrated electron were studied. The bimolecular rate constants for these reactions and the transient spectra of the one-electron oxidized species indicated that the site of oxidation for the two compounds is different. Reaction of hydrated electron with the two compounds was carried out at pH 7, where similar reactivity was observed with both the compounds. Based on all these studies it is concluded that chrysoeriol exhibits potent antioxidant activity. O-glycosylation of chrysoeriol decreases its ability to inhibit lipid peroxidation and reaction with peroxyl radicals. However the glycoside is a more efficient scavenger of DPPH radicals and a better inhibitor of xanthine/xanthine oxidase than the aglycone.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

Medium effects on the antioxidant activity of dipyridamole

Very strong medium effects have been observed when testing the antioxidant activity of dipyridamole (DP) in different media such as benzene, tert-butanol, methanol solutions and egg yolk lecithin unilamellar and multilamellar vesicles. Actually, dipyridamole behaves as a very poor antioxidant in benzene while its ability to inhibit the lipid peroxidation reaction increases with increasing solvent polarity, being the highest in lipid vesicles. This behavior can not be rationalized on the basis of the classical chain breaking mechanism which operates in the case of phenolic and amine antioxidants and involving the transfer of a hydrogen atom to peroxyl radicals. An explanation is instead given in terms of an electron transfer reaction which leads to the oxidation of DP by the chain carrying peroxyl radical to give the dipyridamole cation radical, DP^+•, and the peroxyl anion LOO⁻, and whose rate constant is expected to increase in strongly polar media. EPR and electrochemical data supporting this interpretation have been collected.     

Carnosine and related dipeptides protect human ceruloplasmin against peroxyl radical-mediated modification

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. In a previous study, we showed that the aggregation of human ceruloplasmin was induced by peroxyl radicals. We investigated the effects of antioxidant dipeptides carnosine, homocarnosine and anserine on peroxyl radical-mediated ceruloplasmin modification. Carnosine, homocarnosine and anserine significantly inhibited the aggregation of CP induced by peroxyl radicals. When CP was incubated with peroxyl radicals in the presence of three compounds, ferroxidase activity, as measured by the activity staining method, was protected. All three compounds also inhibited the formation of dityrosine in peroxyl radicals-treated CP. The results suggest that carnosine and related compounds act as peroxyl radical scavenger to protect the protein modification. It is proposed that carnosine and related peptides might be explored as potential therapeutic agents for pathologies that involve CP modification mediated by peroxyl radicals generated in the lipid peroxidation.     

Sulfate anion free radical formation by the peroxidation of (Bi)sulfite and its reaction with hydroxyl radical scavengers

The one-electron oxidation of (bi)sulfite is catalyzed by peroxidases to yield the sulfur trioxide radical anion (SO3-), a predominantly sulfur-centered radical as shown by studies with 33S-labeled (bi)sulfite. This radical reacts with molecular oxygen to form a peroxyl radical. The subsequent reaction of this peroxyl radical with (bi)sulfite has been proposed to form the sulfate anion radical, which is nearly as strong an oxidant as the hydroxyl radical. We used the spin trapping electron spin resonance technique to provide for the first time direct evidence for sulfate anion radical formation during (bi)sulfite peroxidation. The sulfate anion radical is known to react with many compounds more commonly thought of as hydroxyl radical scavengers such as formate and ethanol. Free radicals derived from these scavengers are trapped in systems where (bi)sulfite peroxidation has been inhibited by these scavengers.     

Effect of functional groups on antioxidant properties of substituted selenoethers

Selenoethers attached to functional groups through propyl chain viz., bis(3-carboxypropyl)selenide (SeBA), bis(3-hydroxypropyl)selenide (SePOH) and bis(3-aminopropyl)selenide dihydrochloride (SePAm), have been examined for their ability to inhibit peroxyl radical mediated DNA damage, peroxyl radical scavenging ability and glutathione peroxidase (GPx) like activity. The DNA damage was monitored by gel electrophoresis, bimolecular rate constants for scavenging of model peroxyl radical were determined by pulse radiolysis and the GPx activity was followed by their ability to reduce hydrogen peroxide in the presence of glutathione utilizing NADPH decay and HPLC analysis. Among these compounds, SeBA showed maximum DNA protecting activity and it was also the most efficient in scavenging peroxyl radicals with the highest GPx mimicking activity. Quantum chemical calculations confirmed that SeBA with the highest energy level of HOMO (highest occupied molecular orbital) is the easiest to undergo oxidation and therefore exhibits better radical scavenging, GPx mimicking and DNA protecting activity than SePOH or SePAm.     

Antioxidant effect of manganese.

The antioxidant effects of manganese and other transition metals were studied as the inhibition of microsomal lipid peroxidation and crocin bleaching by peroxyl radicals. The peroxyl radical scavenging capacity was measured by competition kinetics analysis. While Zn(II), Ni(II), and Fe(II) were almost completely ineffective, Mn(II) and Co(II) showed a free radical scavenging capacity, exhibiting relative rate constant ratios respectively of 0.513 and 0.287. This indicates that Mn(II) is by far the most active. Therefore, the chain-breaking antioxidant capacity of Mn(II) seems to be related to the rapid quenching of peroxyl radicals according to the reaction R-OO. + Mn(II) + H(+)-->ROOH+Mn(III). The antioxidant mechanism is discussed considering the different reduction potentials of the examined cations.     

Reactivity toward oxygen radicals and antioxidant action of thiol compounds

Thiol compounds exert diverse functions in the defense network against oxidative stress in vivo. Above all, the role of glutathione in the enzymatic removal of hydrogen peroxide and lipid hydroperoxides has been well established. The scavenging of reactive free radicals is one of the many functions. In this study, the reactivities of several thiol compounds toward oxygen- and nitrogen-centered radicals were measured from their reaction with galvinoxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and also from their sparing effects on the decay of fluorescein, pyrogallol red, and BODIPY induced by peroxyl radicals. Furthermore, the antioxidant capacity against lipid peroxidation was assessed in the oxidation of methyl linoleate induced by free radicals in micelle systems. Cysteine, homocysteine, and glutathione exhibited considerable reactivity toward galvinoxyl, DPPH, and peroxyl radicals in this order but methionine did not. Bovine serum albumin (BSA) was less reactive toward these radicals than cysteine on molar base. Cysteine, homocysteine, and glutathione suppressed the oxidation of methyl linoleate in micelle systems, but methionine did not. The reactivity toward free radicals and antioxidant capacity of these thiol compounds were less than that of ascorbic acid, but higher than that of uric acid.     

Co-oxidation of xenobiotics: lipid peroxyl derivatives as mediators of metabolism

Systems which carry out peroxyl-dependent oxidations can serve as activation systems for carcinogenic compounds. Some function via classical peroxidase reactions in which an enzyme-derived oxidant performs the electron abstraction from or oxygen donation to the oxidizable substrate. This mechanism applies to the peroxidative activation of aromatic amines and of the phenolic compound diethylstilbestrol. These classical peroxidase reactions may be initiated by hydrogen peroxide or by organic peroxides, including lipid hydroperoxides. A different mechanism is involved in the oxygenation of polycyclic aromatic hydrocarbons and of aflatoxin B1. In these cases the oxidant is a peroxyl radical, and the reaction occurs by the direct, non-enzymatic interaction of the peroxyl radical and the oxidizable substrate. Most peroxyl radicals in biological systems are lipid-derived. The key reaction which distinguishes the peroxyl radical-dependent oxidations from the classical peroxidase reactions is the ability of the former to epoxidize activated carbon-carbon double bonds. The epoxidation of benzo[a]pyrene derivatives has been studied extensively in subcellular and whole cell and tissue systems, and is discussed as a model for this class of reaction. Determining the generality of this activation path and its role in vivo present the major questions to be answered in regard to the importance of these reactions in chemical carcinogenesis.