The requirement for iron (III) in the initiation of lipid peroxidation by iron (II) and hydrogen peroxide

The initiation of lipid peroxidation by Fe2+ and H2O2 (Fenton's reagent) is often proposed to be mediated by the highly reactive hydroxyl radical. Using Fe2+, H2O2, and phospholipid liposomes as a model system, we have found that lipid peroxidation, as assessed by malondialdehyde formation, is not initiated by the hydroxyl radical, but rather requires Fe3+ and Fe2+. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide and the bleaching of para-nitrosodimethylaniline confirmed the generation of the hydroxyl radical in this system. Accordingly, catalase and the hydroxyl radical scavengers mannitol and benzoate efficiently inhibited the generation and the detection of hydroxyl radical. However, catalase, mannitol, and benzoate could either stimulate or inhibit lipid peroxidation. These unusual effects were found to be consistent with their ability to modulate the extent of Fe2+ oxidation by H2O2 and demonstrated that lipid peroxidation depends on the Fe3+:Fe2+ ratio, maximal initial rates occurring at 1:1. These studies suggest that the initiation of liposomal peroxidation by Fe2+ and H2O2 is mediated by an oxidant which requires both Fe3+ and Fe2+ and that the rate of the reaction is determined by the absolute Fe3+:Fe2+ ratio.     

Inhibition of lipid peroxidation by a new family of iron chelators

Catechol derived siderophores are the most powerful currently known iron chelators. We have intended tripodal ligands built with o,o′ dihydroxy biaryl subunits (A, B, and C). We described antioxidant properties of this new family of iron chelators. Superoxidedependent hydroxyl radical system was used. Peroxidation of different lipid-containing systems (liposomes, erythrocyte membrane ghosts, tissue homogenates) were also investigated. The antioxidant properties of these new chelators have been related to that of desferrioxamine, as a reference compound. In general manner, the results depended mainly on the model used in the assay. However, C presents an antioxidant effect close to that of desferrioxamine.     

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

Abstract  

The purple acid phosphatases (PAPs) are the only binuclear metallohydrolases where the necessity for a heterovalent active site [Fe(III)–M(II) (M is Fe, Zn or Mn)] for catalysis has been established. The paradigm for the construction of PAP biomimetics, both structural and functional, is that the ligands possess characteristics which mimic those of the donor sites of the metalloenzyme and permit discrimination between trivalent and divalent metal ions. The donor atom set of the ligand 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino)acetic acid (H₃HPBA) mimics that of the active site of PAP although the iron(III) complex of this ligand has been characterized as the tetramer [Fe₄(HPBA)₂(μ-CH₃COO)₂(μ-O)(μ-OH)(OH₂)₂]ClO₄·5H₂O. The phosphoesterase-like activity of the complex in 1:1 acetonitrile/water has now been investigated using the substrate 2,4-bis(dinitrophenyl)phosphate. The pH dependence of the catalytic rate revealed a non-symmetric bell-shaped profile, with a finite but non-zero rate at high pH. Unlike the traditional approach usually employed to analyse these bell-shaped profiles, the approach used here involved incorporating additional species which contribute to the overall activity. Employing this approach, we show that the complex has a k _cat of 1.6 (±0.2) × 10⁻³ s⁻¹, three kinetically relevant pK _a values of 5.3, 6.2 and 8.4, with K _M of 7.4 ± 0.6 mM. The kinetic parameters are similar to those reported for heterovalent PAP biomimetics. Additionally, it is observed that, unlike the enzyme, the oxidation state is not the determining factor for catalytic activity.     

Inhibition of lipid peroxidation by prostaglandin oligomeric derivatives.

The inhibition of lipid peroxidation by oligomeric derivatives synthesized from prostaglandin E1 (PGE1) and PGB2 was studied using two rat models. In an in vitro model, the brain was exposed to decapitation-ischemia, the cortex was removed and homogenized, and the formation of thiobarbituric acid reactive substances (TBAR) was measured after exposing the homogenate to in vitro reoxygenation either in the presence or absence of oligomers. It was found that these oligomers could inhibit lipid peroxidation, and that their activities were higher than that of superoxide dismutase (SOD). In an in vivo administration model, either the oligomer or the vehicle was injected i.p. 30 min before decapitation. The brain was exposed to decapitation-ischemia, the cortex was homogenized and exposed to 'in vitro' reoxygenation, after which TBAR value was determined. Ester-type compounds had a greater activity than free-acid type compounds in inhibiting lipid peroxidation. A possible mechanism of the protective effect of these oligomers in ischemia/reperfusion injury may be to scavenge oxygen free radicals.     

Inhibition of nonenzymic lipid peroxidation by benzylisoquinoline alkaloids.

A group of benzylisoquinoline alkaloids, including five simple benzylisoquinolines, three phtalideisoquinolines, six aporphines, three protoberberines, and two benzophenanthridines, have been studied as inhibitors of lipid peroxidation stimulated by Fe2+/cysteine in rat liver microsomal fractions. Protopapaverine, apomorphine, laudanosoline, tetrahydroberberine, isoboldine, bulbocapnine, boldine, anonaine, glaucine, and stepholidine showed antiperoxidative effects, and structure-activity relationships were established. In simple benzylisoquinolines, the presence of phenolic hydroxyls or similar reactive groups is necessary for inhibition of peroxidation, while in aporphines and protoberberines nonhydroxylated compounds can exert antiperoxidative effects. The phtalideisoquinolines and benzophenanthridines tested were inactive.     

Inhibition of lipid peroxidation

Lipid peroxy radicals (ROO-) were detected by electron spin resonance (ESR) at low temperature after formation by addition of H₂O₂ into a suspension of mice lymphocites. If lymphocytes were treated with selenomethionine (Se-Met) prior to addition of H₂O₂, ROO-formation was inhibited in a fashion that was dependent on Se-Met concentration. Formation of ROO- in the spleen of mice was induced by⁶⁰Co irradiation. Animals that were supplemented with Na₂SeO₃ prior to irradiation exhibited a lower ROO-concentration than that of nontreated animals. Based on our experiments, we have concluded that Se has an oxygen-free radical scavenging effect. This should be a protective effect against lipid peroxy radical cellular attack.     

Inhibition of lipid peroxidation by S-nitrosoglutathione and copper

The antioxidant properties of S -nitrosoglutathione, a nitric oxide-derived product were studied in different experimental systems. By using the crocin bleaching test, S -nitrosoglutathione, in the presence of copper ions, shows an antioxidant capacity about six times higher than that of Trolox c and referable to the interception of peroxyl radicals by nitric oxide. Copper alone shows a modest inhibitory action, which is about seven times lower than that of Trolox c. S -nitrosoglutathione prevents lipid peroxidation induced by the well-known Fe 2+ /ascorbate system (IC 50 =450 μM) and the inhibitory effect is strongly reinforced by the presence of copper ions (IC 50 =6.5 μM). In addition, cumene hydroperoxide-induced lipid peroxidation is markedly decreased by S -nitrosoglutathione, provided that copper ions, maintained reduced by ascorbate, are present. Decomposition of S -nitrosoglutathione through metal catalysis and/or the presence of reducing agents and the consequent release of nitric oxide are of crucial importance for eliciting the antioxidant power. In this way, copper ions and/or reducing species with low antioxidant potency are able to promote the formation of an extremely strong antioxidant species such as nitric oxide.     

Inhibition of lipid peroxidation by alpha-tocopherolquinone and alpha-tocopherolhydroquinone

The antioxidant effect of alpha-tocopherolquinone and alpha-tocopherolhydroquinone was studied in liposomes and rat liver submitochondrial particles. Both alpha-tocopherolquinone and alpha-tocopherolhydroquinone inhibit lipid peroxidation induced by ascorbate/Fe2+ in liposomes and by cumene hydroperoxide in submitochondrial particles. Alpha-tocopherolhydroquinone is much more effective than alpha-tocopherolquinone in inhibiting lipid peroxidation. Submitochondrial particles, depleted of ubiquinones and reincorporated with alpha-tocopherolquinone, are protected from lipid peroxidation only in the presence of succinate. Alpha-tocopherolquinone cannot replace endogenous ubiquinones in the respiratory chain function, nevertheless it can be reduced by the mitochondrial respiratory chain substrates, presumably through the reduced ubiquinones.     

Effect of aldehydes on polyamine metabolism. III. Inhibition of S-adenosyl methionine decarboxylase (SAMD) by CCl4 and by aldehydes produced during lipid peroxidation

In isolated rat liver cells in which lipid peroxidation is stimulated by CCl4, a strong inhibition of S-adenosylmethionine decarboxylase (SAMD) activity occurs. Some purified aldehydes, which are produced during lipid peroxidation, are able to inhibit SAMD activity in Yoshida hepatoma cells. The most active aldehyde is hydroxypentenal (HPE). It inhibits by 50% SAMD activity at 0.5 mM concentration in entire hepatoma cells, or in hepatoma cell sap, and at 0.1 mM concentration in partially purified hepatoma cell sap fractions.     

Inhibition of superoxide and ferritin-dependent lipid peroxidation by ceruloplasmin

Ceruloplasmin (CP) was found to inhibit xanthine oxidase and ferritin-dependent peroxidation of phospholipid liposomes, as evidenced by decreased malondialdehyde formation. Ceruloplasmin was also shown to inhibit superoxide-mediated mobilization of iron from ferritin, in a concentration-dependent manner, as measured spectrophotometrically using the iron(II) chelator bathophenanthroline sulfonate. Ceruloplasmin failed to function as a peroxyl radical-scavenging antioxidant as evidenced by its inability to inhibit free radical-initiated peroxidation of linoleic acid, suggesting that CP inhibited lipid peroxidation by affecting the availability of ferritin-derived iron. In addition, CP scavenged xanthine oxidase-derived superoxide as measured spectrophotometrically via its effect on cytochrome c reduction. However, the extent of the superoxide scavenging of CP did not quantitatively account for its effects on iron release, suggesting that CP inhibits superoxide-dependent mobilization of ferritin iron independently of its ability to scavenge superoxide. The effects of CP and apoferritin on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. In the absence of apoferritin, CP exhibited a concentration-dependent prooxidant effect. However, CP-dependent, iron-catalyzed lipid peroxidation was inhibited by the addition of apoferritin. Apoferritin did not function as a peroxyl radical-scavenging antioxidant but was shown to incorporate iron in the presence of CP. These data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation largely via its ability to reincorporate reductively mobilized iron back into ferritin.     

Inhibition of iron/ascorbate-induced lipid peroxidation by an N-terminal peptide of bovine lactoferrin and its acylated derivatives.

Bovine lactoferrin (LF) and lactoferricin B (LFcin B), an antimicrobial peptide derived from bovine LF, inhibited thiobarbituric acid-reactive substance (TBARS) formation in a iron/ascorbate-induced liposomal phospholipid peroxidation system. The inhibition of TBARS formation occurred with N-acylated 9-mer peptides with a core sequence of LFcin B and, compared to LFcin B, their antioxidant effect was clearly observed at a concentration almost 100 times lower.     

Inhibition of S-(1,2-dichlorovinyl)-L-cysteine-induced lipid peroxidation by antioxidants in rabbit renal cortical slices: Dissociation of lipid peroxidation and toxicity

Precision-cut, rabbit renal slices were used to examine the effects of three novel antioxidants (U-74006, U-74500, and U-78517) on S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced lipid peroxidation and toxicity. Slices exposed to DCVC showed a dose- and time-dependent increase in lipid peroxidation (TBARS) and a decrease in cellular viability, as evidenced by the loss of intracellular potassium, during the course of a 3 hour incubation. Subsequent studies employed DCVC concentrations of 100 μM. Microemulsion formulations of U-78517, U-74500, and U-74006 (100 μM) inhibited DCVC-induced lipid peroxidation by 100±, 50±, and <5% (not significant), respectively. However, none of these antioxidants had a significant effect on DCVC-dependent cytotoxicity, as indicated by intracellular potassium release. The effects of U-78517, the most potent of the three antioxidants, were similar to those observed with two model antioxidants, diphenyl-p-phenylenedi-amine (DPPD) and the iron chelator, deferoxamine. Aminooxyacetic (AOAA), an inhibitor of renal cysteine conjugate β-lyase, had only a minimal effect on DCVC-induced lipid peroxidation, and no effect on toxicity. These data represent the first report of DCVC-induced lipid peroxidation in rabbit renal cortical slices, a system which has been widely used to investigate mechanisms of nephrotoxicity, including that induced by DCVC. Our results demonstrate that DCVC-induced lipid peroxidation in renal slices can be inhibited by a variety of antioxidant compounds operating by different mechanisms. Because inhibition of lipid peroxidation had minimal effect on DCVC-dependent cytotoxicity, the data suggest that DCVC-induced lipid peroxidation is not a major mechanism in the cytotoxicity induced by this compound.     

Inhibition of protein synthesis by products of lipid peroxidation

Effects of lipid peroxidation products on in vivo and in vitro protein synthesis have been studied. Malondialdehyde (MDA), a product, and a routinely used index of lipid peroxidation, inhibits in vivo protein synthesis in the two mosses, Tortula ruralis and Cratoneuron filicinum, and in pea (Pisum sativum) leaf discs. When wheat germ supernatant or poly(A)-rich mRNA of T. ruralis was incubated with MDA its subsequent activity in a cell-free protein-synthesizing system was reduced. When MDA was added directly to the in vitro protein-synthesizing mixture containing moss polyribosomes, the inhibition of amino acid incorporation was small. However, when simultaneous lipid peroxidation was allowed to occur along with in vitro protein synthesis there was a strong inhibition of amino acid incorporation and MDA accumulated in the reaction mixture indicating that products of lipid peroxidation other than, and apparently more toxic than, MDA were involved. It was concluded that lipid peroxidation inhibits protein synthesis probably by releasing toxic products which may react with and inactivate some components of the protein-synthesizing complex.     

Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins

The effects of all-zinc metallothionein (Zn-metallothionein) and predominantly cadmium metallothionein (Cd/Zn-metallothionein) on free radical lipid peroxidation have been investigated, using erythrocyte ghosts as the test system. When treated with xanthine and xanthine oxidase, Zn-metallothionein and Cd/Zn-metallothionein underwent thiolate group oxidation and metal ion release that was catalase-inhibitable, but superoxide dismutase-non-inhibitable. Similar treatment in the presence of ghosts and added Fe(III) resulted in metallothioneen oxidation that was significantly inhibited by superoxide dismutase. Ghosts incubated with xanthine/xanthine oxidase/Fe(III) underwent H₂O₂- and O₂⁻-dependent lipid peroxidation, as measured by thiobarbituric acid reactivity. Neither type of metallothionein had any effect on xanthine oxidase activity, but both strongly inhibited lipid peroxidation when added to the membranes concurrently with xanthine/xanthine oxidase/iron. This inhibition was far greater and more sustained than that caused by dithiothreitol at a concentration equivalent to that of metallothionein thiolate. Significant protection was also afforded when ghosts plus Cd/Zn-metallothionein or Zn/metallothionein were preincubated with H₂O₂ and Fe(III), and then subjected to vigorous peroxidation by the addition of xanthine and xanthine oxidase. These results could be mimicked by using Cd(II) or Zn(II) alone. Previous studies suggested that Zn(II) inhibits xanthine/xanthine oxidase/iron-driven lipid peroxidation in ghosts by interfering with iron binding and redox cycling. Therefore, the primary determinant of metallothionein proteciion appears to be metal release and subsequent uptake by the membranes. These results have important implications concerning the antioxidant role of metallothionein, a protein known to be induced by various prooxidant conditions.     

Inhibition of lipid peroxidation by the iron-binding protein lactoferrin

Lactoferrin containing physiological amounts of iron is an inhibitor of lipid peroxidation induced by iron(III) salts and ascorbic acid. It might therefore help to protect neutrophils, inflammatory foci and secretions from metal-ion-dependent oxidative damage.     

Inhibition of lipid peroxidation by spin labels. Relationships between structure and function

Inhibition of lipid peroxidation by nitroxide radicals and their corresponding hydroxylamines was investigated. The nitroxides were either oxazolidines or piperidines, differing in substitution of the backbone of the molecule (a five or six-membered ring structure, respectively). Concentration requirements for 50% inhibition of microsomal lipid peroxidation varied from 340 to 6 microM for the nitroxides, and from 120 to 3 microM for the hydroxylamines, correlating with lipophilicity and chemical structure. Intramembrane concentrations required for 50% inhibition was independent of lipophilicity when peroxidation was initiated with ADP-Fe2+ but increased with lipophilicity when peroxidation was initiated with t-butylhydroperoxide. During studies of the kinetics of the inhibition, two modes were seen: a delay or a decreased rate of the process. The former mode was seen with the more lipophilic inhibitors. The mechanism of inhibition was similar for all nitroxides and consisted of the following three major components: blocking of primary initiation, prevention of secondary (peroxide-dependent) initiation, and scavenging of various lipoid radicals in the membrane, the major mode of action of the hydroxylamines. Inhibitory efficiency was interpreted in terms of steric hindrance, diffusibility, regeneration of inhibitor, and ability to interact with hydrophilic sites in a hydrophobic environment.     

tert-butyl hydroperoxide-dependent microsomal release of iron and lipid peroxidation. II. Evidence for the involvement of nonheme, nonferritin iron in lipid peroxidation

In a previous study tert-butyl hydroperoxide (t-BOOH) was found to promote reductive release of nonheme, nonferritin iron from rat liver microsomes. The reaction was catalyzed by cytochrome P450 and was strictly contingent on the availability of ADP. In this study, t-BOOH was also found to promote microsomal lipid peroxidation, as evidenced by formation of malondialdehyde. t-BOOH-dependent lipid peroxidation was stimulated by ADP, and four lines of evidence suggested that such stimulation was mediated by reductive release and subsequent redox cycling of nonheme, nonferritin iron. First, lipid peroxidation was stimulated by the same concentration of ADP that promoted iron release. Second, depletion of nonheme, nonferritin iron by pretreatment of rats with phenobarbital decreased the stimulation of lipid peroxidation by ADP. Third, the effect of ADP was maximal when the concentration of t-BOOH was adjusted to values that yielded maximum iron release. Fourth, the effect of ADP was abolished by bathophenanthroline, which is known to chelate ferrous iron in a redox inactive form. These results suggest that the reductive release of nonheme, nonferritin iron exacerbates the deleterious effects of t-BOOH on microsomal lipids.     

NADPH-cytochrome-P-450 reductase promoted hydroxyl radical production by the iron(III)-ochratoxin A complex

The Fe3+ complex of ochratoxin A has been shown to produce hydroxyl radicals in the presence of NADPH and NADPH-cytochrome-P-450 reductase. ESR spin-trapping experiments carried out in the presence of the hydroxyl radical scavenger ethanol and the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide) produced ESR spectra characteristic of the hydroxyl radial-derived carbon-centered DMPO-alkoxyl radical adduct. Thus hydroxyl radicals produced by the Fe3(+)-ochratoxin A complex in the presence of an enzymatic reductase may be be partly responsible for ochratoxin A toxicity.     

The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

Inhibition of lipid peroxidation by heme-nonapeptide derived from cytochrome c

Heme-nonapeptide, derived from cytochrome c, inhibited both the NADPH- and NADH-dependent lipid peroxidation of brain microsomes but, in the case of liver microsomes, this inhibitory effect manifested itself in the presence of SKF-525A (a specific blocker of cytochrome P-450) only. Heme-nonapeptide prevented the transient accumulation of lipid peroxides in microsomes during lipid peroxidation. The oxygen consumption of microsomes in the presence of NADPH or NADH was stimulated by heme-nonapeptide. From these results we concluded that, in vitro, there are two independent mechanisms of lipid peroxidation in liver microsomes. It is suggested that, in vivo, the heme-peptide-sensitive mechanism, observed in brain microsomes, is more important.