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.     

Importance of Fe2+-ADP and the relative unimportance of OH in the mechanism of mitomycin C-induced lipid peroxidation

The mechanism of mitomycin C-induced lipid peroxidation has been studied at pH 7.5, using systems containing phospholipid membranes (liposomes) and an Fe3+-ADP complex with purified NADPH-cytochrome P-450 reductase. Both O2- and H2O2 are generated during the aerobic enzyme-catalyzed reaction in the presence of mitomycin C. Hydroxyl radical is formed in the reaction by the reduction of H2O2. This is catalyzed by the Fe2+-ADP complex in a phosphate buffer or to a lesser extent when in a Tris-HCl buffer. The reduction of Fe3+-ADP to Fe2+-ADP is mainly achieved by O2-. The resulting Fe2+-ADP in the presence of O2 forms a perferryl ion complex which is a powerful stimulator of lipid peroxidation. However, the formation of such an iron-oxygen complex is strongly inhibited by phosphate ions, which do not interfere with the generation of OH radicals. These findings suggest that, since lipid peroxidation occurs in a Tris-HCl buffer (but not in a phosphate buffer), the OH radical is unlikely to be involved in the observed lipid peroxidation process.     

The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation

Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions.     

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.     

NADPH- and adriamycin-dependent microsomal release of iron and lipid peroxidation

In a previous study (Minotti, G., 1989, Arch. Biochem. Biophys. 268, 398-403) NADPH-supplemented microsomes were found to reduce adriamycin (ADR) to semiquinone free radical (ADR-.), which in turn autoxidized at the expense of oxygen to regenerate ADR and form O2-. Redox cycling of ADR was paralleled by reductive release of membrane-bound nonheme iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. In the present study, iron release was found to increase with concentration of ADR in a superoxide dismutase- and catalase-insensitive manner. This suggested that membrane-bound iron was reduced by ADR-. with negligible contribution by O2-. or interference by its dismutation product H2O2. Following release from microsomes, Fe2+ was reconverted to Fe3+ via two distinct mechanisms: (i) catalase-inhibitable oxidation by H2O2 and (ii) catalase-insensitive autoxidation at the expense of oxygen, which occurred upon chelation by ADR and increased with the ADR:Fe2+ molar ratio. Malondialdehyde formation, indicative of membrane lipid peroxidation, was observed when approximately 50% of Fe2+ was converted to Fe3+. This occurred in presence of catalase and low concentrations of ADR, which prevented Fe2+ oxidation and favored only partial Fe2+ autoxidation, respectively. Lipid peroxidation was inhibited by superoxide dismutase via increased formation of H2O2 from O2-. and excessive Fe2+ oxidation. Lipid peroxidation was also inhibited by high concentrations of ADR, which favored maximum Fe2+ release but also caused excessive Fe2+ autoxidation via formation of very high ADR:Fe2+ molar ratios. These results highlighted multiple and diverging effects of ADR, O2-., and H2O2 on iron release, iron (auto-)oxidation and lipid peroxidation. Stimulation of malondialdehyde formation by catalase suggested that lipid peroxidation was not promoted by reaction of Fe2+ with H2O2 and formation of hydroxyl radical. The requirement for both Fe2+ and Fe3+ was indicative of initiation by some type of Fe2+/Fe3+ complex.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Induction of oxalate binding by lipid peroxidation in rat kidney mitochondria.

Enhanced oxalate binding (150-180% of control) was observed in kidney, liver, brain and heart, after subjecting them to lipid peroxidation in presence of iron. Kidney mitochondrial oxalate binding was stimulated by different promoters, and the order of stimulation was Fe2+ greater than t-BH greater than ascorbic acid greater than Fe3+ greater than H2O2. Oxalate binding was maximum when iron concentration was between 1-2 mM. The iron-induced oxalate binding was inhibited by reduced glutathione, beta-mercaptoethanol, alpha-tocopherol and hydroxyl ion scavengers, histidine and mannitol. Catalase inhibited both Fe(2+)-H2O2 induced oxalate binding and lipid peroxidation reactions, suggesting that the induced oxalate binding in mitochondria was mediated through the hydroxyl radical reaction mechanism.     

Cytochrome c-catalyzed membrane lipid peroxidation by hydrogen peroxide.

Cytochrome c(3+)-catalyzed peroxidation of phosphatidylcholine liposomes by hydrogen peroxide (H2O2) was indicated by the production of thiobarbituric acid reactive substances, oxygen consumption, and emission of spontaneous chemiluminescence. The iron chelator diethylenetriaminepentaacetic acid (DTPA) only partially inhibited peroxidation when H2O2 concentrations were 200 microM or greater. In contrast, iron compounds such as ferric chloride, potassium ferricyanide, and hemin induced H2O2-dependent lipid peroxidation which was totally inhibitable by DTPA. Cyanide and urate, which react at or near the cytochrome-heme, completely prevented lipid peroxidation, while hydroxyl radical scavengers and superoxide dismutase had very little or no inhibitory effect. Changes in liposome surface charge did not influence cytochrome c3+ plus H2O2-dependent peroxidation, but a net negative charge was critical in favoring cytochrome c(3+)-dependent, H2O2-independent lipid auto-oxidative processes. These results show that reaction of cytochrome c with H2O2 promotes membrane oxidation by more than one chemical mechanism, including formation of high oxidation states of iron at the cytochrome-heme and also by heme iron release at higher H2O2 concentrations. Cytochrome c3+ could react with mitochondrial H2O2 to yield "site-specific" mitochondrial membrane lipid peroxidation during tissue oxidant stress.     

Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity

Chronic ethanol consumption causes oxidative damage in the liver, and induction of cytochrome P450 2E1 (CYP2E1) is one pathway involved in oxidative stress produced by ethanol. The hepatic accumulation of iron and polyunsaturated fatty acids significantly contributes to ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment. The objective of this study was to analyze the effect of the green tea flavanol epigallocatechin-3-gallate (EGCG), which has been shown to prevent alcohol-induced liver damage, on CYP2E1-mediated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells). Treatment of E47 cells with arachidonic acid plus iron (AA + Fe) was previously reported to produce synergistic toxicity in E47 cells by a mechanism dependent on CYP2E1 activity and involving oxidative stress and lipid peroxidation. EGCG protected E47 cells against toxicity and loss of viability induced by AA+Fe; EGCG had no effect on CYP2E1 activity. Prevention of this toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species, a decrease in lipid peroxidation, and maintenance of intracellular glutathione in cells challenged by AA+Fe in the presence of EGCG. AA+Fe treatment caused a decline in the mitochondrial membrane potential, which was also blocked by EGCG. In conclusion, EGCG exerts a protective action on CYP2E1-dependent oxidative stress and toxicity that may contribute to preventing alcohol-induced liver injury, and may be useful in preventing toxicity by various hepatotoxins activated by CYP2E1 to reactive intermediates.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.     

Site-specific induction of lipid peroxidation by iron in charged micelles

Generation of hydroxyl radicals by the Fenton reaction resulted in lipid peroxidation of linoleic acid (LA) (H2O2-Fe2+-induced lipid peroxidation) in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles, but not in negatively charged sodium dodecyl sulfate (SDS) micelles. However, more OH radicals formed via the Fenton reaction were trapped by N-t-butyl-alpha-phenylnitrone (PBN) in SDS micelles than in TTAB micelles. When detergent-dispersed LA was contaminated with linoleic acid hydroperoxide (LOOH), lipid peroxidation was catalyzed by Fe2+ via reductive cleavage of LOOH (LOOH-Fe2+-induced lipid peroxidation), and Fe2+ was oxidized simultaneously in SDS micelles, even when H2O2 was not present. In contrast, LOOH-Fe2+-induced lipid peroxidation and simultaneous oxidation of Fe2+ were not observed in TTAB micelles. An ESR spectrum presumed to be due to an alkoxy radical trapped by PBN was also detected in SDS micelles, but not in TTAB micelles in the LOOH-Fe2+-induced lipid peroxidation system. The results are discussed in the light of the localization of iron, the unsaturated bonding moiety of LA, the OOH-group of LOOH, and the trapping site of PBN in different charged micelles.     

Microsomal reduction of low-molecular-weight Fe3+ chelates and ferritin: enhancement by adriamycin, paraquat, menadione, and anthraquinone 2-sulfonate and inhibition by oxygen

Reduction of iron is important in promoting xenobiotic-enhanced, microsomal lipid peroxidation, yet there is little evidence that Fe3+ chelates that promote lipid peroxidation can be reduced by the microsomal system. We have shown that rat liver microsomes catalyse NADPH-dependent reduction of Fe3+ without chelator, as well as Fe3+(ADP), Fe3+(ATP), Fe3+(citrate), Fe3+(EDTA), and ferrioxamine in N2. The NADPH oxidation that accompanied Fe3+ reduction was inhibited by CO for all chelates, except Fe3+ (EDTA). This implies that, except for Fe3+ (EDTA), cytochrome P450 was involved in reduction of the complexes. Adriamycin, paraquat, and anthraquinone 2-sulfonate (AQS) enhanced reduction of all the Fe3+ chelates, whereas menadione enhanced reduction only of Fe3+(ADP) and Fe3+(citrate). All the compounds enhanced oxidation of NADPH in the presence or absence of iron. This was not inhibited by CO, and the results are compatible with Fe3+ reduction occurring via the xenobiotic radicals produced by cytochrome P450 reductase. Microsomal reduction of the xenobiotics, except menadione, enabled the reduction and release of iron from ferritin. Fe3+ chelate reduction, both with and without xenobiotic, was inhibited by O2, although it still proceeded in air at 10-20% of the rate in N2. Iron-dependent lipid peroxidation was promoted by ADP and ATP, inhibited 50% by citrate, and completely inhibited by EDTA and desferrioxamine. Of the xenobiotics, only Adriamycin enhanced microsomal lipid peroxidation. These results indicate that the effects of chelators and xenobiotics on Fe3+ reduction do not correlate with lipid peroxidation and, although reduction is necessary, there must be other factors involved.     

渗透胁迫下稻苗中铁催化的膜脂过氧化作用

在－０．７ＭＰａ渗透胁迫下,水稻幼苗体内和Ｈ２Ｏ２大量产生,Ｆｅ２＋积累,膜脂过氧化作用加剧。水稻幼苗体内Ｆｅ２＋含量与膜脂过氧化产物ＭＤＡ含量呈极显著的正相关。外源Ｆｅ２＋、Ｆｅ３＋、Ｈ２Ｏ２、Ｆｅ２＋＋Ｈ２Ｏ２、ＤＤＴＣ均能刺激膜脂过氧化作用,而铁离子的螯合剂ＤＴＰＡ则有缓解作用。ＯＨ的清除剂苯甲酸钠和甘露醇能明显地抑制渗透胁迫下Ｆｅ２＋催化的膜脂过氧化作用。这都表明渗透胁迫下水稻幼苗体内铁诱导的膜脂过氧化作用主要是由于其催化Ｆｅｎｔｏｎ型Ｈａｂｅｒ－Ｗｅｉｓｓ反应形成ＯＨ所致。     

Peroxide-induced membrane damage in human erythrocytes

Erythrocytes exposed to H2O2 or t-butyl hydroperoxide (tBHP) exhibited lipid peroxidation and increased passive cation permeability. In the case of tBHP a virtually complete inhibition of both processes was caused by butylated hydroxytoluene (BHT), whereas pretreatment of the cells with CO increased both lipid peroxidation and K+ leakage. In the experiments with H2O2, on the other hand, both BHT and CO strongly inhibited lipid peroxidation, without affecting the increased passive cation permeability. These observations indicate different mechanisms of oxidative damage, induced by H2O2 and tBHP, respectively. The SH-reagent diamide strongly inhibited H2O2-induced K+ leakage, indicating the involvement of SH oxidation in this process. With tBHP, on the contrary, K+ leakage was not significantly influenced by diamide. Thiourea inhibited tBHP-induced K+ leakage, without affecting lipid peroxidation. Together with other experimental evidence this contradicts a rigorous interdependence of tBHP-induced lipid peroxidation and K+ leakage.     

Oxidation of ferrous iron during peroxidation of lipid substrates

Oxidation of Fe2+ in solution was dependent upon medium composition and the presence of lipid. The complete oxidation of Fe2+ in 0.9% saline was markedly accelerated in the presence of phosphate or EDTA and the ferrous oxidation product formed was readily recoverable as Fe2+ by ascorbate reduction. In contrast, in the presence of either brain synaptosomal membranes, phospholipid liposomes, fatty acid micelles or H2O2, less than 50% of the Fe2+ oxidized during an incubation could be recovered as Fe2+ via reduction with ascorbate. In the presence of unsaturated lipid, oxidation of Fe2+ was associated with peroxidation of lipid, as assessed by the uptake of O2 and formation of thiobarbituric acid-reactive products during incubations. Although relatively little Fe2+ oxidation or lipid peroxidation occurred in saline with synaptosomes or linoleic acid micelles during an incubation with Fe2+ alone, significant Fe2+ oxidation and lipid peroxidation occurred in incubations containing a 1:1 ratio of Fe2+ and Fe3+. Extensive Fe2+ oxidation and lipid peroxidation also occurred with Fe2+ alone in saline incubations with either linolenic or arachidonic acid acid micelles or liposomes prepared from dilinoleoylphosphatidylcholine. While a 1:1 ratio of Fe2+ and Fe3+ enhanced thiobarbituric acid-reactive product formation in incubations containing linolenic or arachidonic micelles, it reduced the rate of O2 consumption as compared with Fe2+ alone. The results demonstrate that oxidation of Fe2+ in incubations containing lipid substrates is linked to and accelerated by peroxidation of those substrates. Furthermore, the results suggest that oxidation of Fe2+ in the presence of lipid or H2O2 creates forms of iron which differ from those formed during simple Fe2+ autoxidation.     

Spin trapping agents (Tempol and POBN) protect HepG2 cells overexpressing CYP2E1 against arachidonic acid toxicity

Polyunsaturated fatty acids such as arachidonic acid were previously shown to be toxic to HepG2 cells expressing CYP2E1 by a mechanism involving oxidative stress and lipid peroxidation. This study investigated the effects of the spin trapping agents Tempol and POBN on the arachidonic acid toxicity. Arachidonic acid caused toxicity and induced lipid peroxidation and mitochondrial membrane damage in cells overexpressing CYP2E1 but had little or no effect in control cells not expressing CYP2E1. The toxicity appeared to be both apoptotic and necrotic in nature. 4-Hydroxy-[2,2,6,6-tetramethylpiperidine-1-oxyl] (Tempol) and alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) protected against the decrease in cell viability and the apoptosis and necrosis. These spin traps prevented the enhanced lipid peroxidation and the loss of mitochondrial membrane potential. Tempol and POBN had little or no effect on cellular viability or on CYP2E1 activity at concentrations which were protective. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently damages the mitochondrial membrane leading to a loss in cell viability when the cells are enriched with arachidonic acid. Tempol and POBN, which scavenge various radical intermediates, prevent in this way the enhanced lipid peroxidation, mitochondrial dysfunction, and the cell toxicity. Since oxidative stress appears to play a key role in ethanol hepatotoxicity, it may be of interest to evaluate whether such spin trapping agents are useful candidates for the prevention or improvement of ethanol-induced liver injury.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Phospholipid peroxidation induced by the catechol-Fe3+(Cu2+) complex: a possible mechanism of nigrostriatal cell damage

Ferric or cupric ions significantly promoted a peroxidative cleavage of unsaturated phospholipids in liposomes in vitro after coordinating with dopa and dopamine. Either alpha-tocopherol or desferrioxamine completely abolished the dopa-Fe3+ complex-induced phospholipid peroxidation, while superoxide dismutase, catalase, or sodium benzoate did not. A ferroxidase, ceruloplasmin, significantly inhibited the lipid peroxidation induced by the dopa-Fe3+ complex, indicating the importance of the reduction of the iron moiety in the complex for the lipid peroxidation. A possible mechanism of dopa-Fe3+ complex-induced phospholipid peroxidation is that oxene complexes, such as Fe(V) = O and Fe(IV) = O, produced abstract hydrogen atoms in unsaturated phospholipids to initiate lipid peroxidation.