Ferritin and superoxide-dependent lipid peroxidation

Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.     

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.     

Studies on the mechanism of the NADPH-catalyzed peroxidation of endogenous microsomal lipid.

The importance of metal chelation in the mechanism of microsomal lipid peroxidation has been studied using both phosphate- and sulfhydryl-containing compounds. The optimal concentration for maximum stimulation by each of these compounds has been determined, and the decrease in stimulation observe at concentrations above the maxima has been related to the ability of these compounds to form stable chelation complexes with non-heme iron. Of the compounds tested, only ADP and ATP facilitated the cooperative binding of NADPH to the membrane and thus suggested the possibility of three binding sites for NADPH. Neither of the other two phosphate-chelating agents (Pi or PPi) and neither of the two thiols (cysteine or dithiothreitol)facilitated cooperative binding of NADPH. These data suggested that the adenine ring of ADP or ATP is directly involved in the cooperativity of NADPH binding. They also emphasized that the binding of the chelation complex to the protein is an important parameter in the mechanism of the NADPH-catalyzed peroxidation of endogenous microsomal lipids. Furthermore, stimulation of the rat of lipid peroxidation by sulhydryl-containing compounds, by freezing thawing the microsomal protein, and by treatment of the protein with detergent may be due to a decrease in this cooperative binding effect. Since cysteine and deoxycholate as well as freezing and thawing alter membrane structure, the stimulation of lipid peroxidation seems to involve some alteration to the structure of the microsomal membrane prior to the onset of enzymatic lipid peroxidation.     

Iron binding to microsomes and liposomes in relation to lipid peroxidation

The effects of ADP, ATP, citrate and EDTA on iron-dependent microsomal and liposomal lipid peroxidation, and on 59FeCl3 binding to the lipid membranes were measured. The aim was to test if initiation of lipid peroxidation is a site-specific mechanism requiring bound iron. In the absence of chelator, iron was bound to both membranes. EDTA and citrate removed the iron and inhibited peroxidation. ATP and ADP stimulated peroxidation, but whereas ADP allowed only half of the iron to remain bound, all was removed by ATP. Chelators, therefore, cannot be simply influencing a site-specific mechanism. Their effects must relate to the reactivities of the different iron chelates as initiators of lipid peroxidation.     

Influence of histidine on lipid peroxidation in sarcoplasmic reticulum.

The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidine's effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo.     

The chelation of nonheme iron within sickle erythrocytes by the hydroxypyridinone chelator CP094.

Nonheme, nonferritin iron has been detected in membrane preparations from sickle erythrocytes and has been suggested to catalyze free radical reactions in these cells contributing to the development of membrane oxidation. In this study the hydroxypyridinone iron chelator, CP094, currently being evaluated as a potentially therapeutic chelator, and desferrioxamine have been studied for their abilities to chelate the nonheme iron within intact sickle erythrocytes under physiological conditions. The results suggest that CP094 can enter sickle erythrocytes, chelate nonheme iron and suppress membrane lipid peroxidation within a timescale in which desferrioxamine does not enter the cells. Suppression of lipid peroxidation showed no protective effect in an in vitro system inducing the formation of irreversibly sickled cells.     

Effect of ferritin-containing fractions with different iron loading on lipid peroxidation.

Ferritin-containing fractions with different degrees of iron loading were prepared. All ferritin fractions stimulated the peroxidation of bovine brain phospholipid liposomes, as measured by the formation of thiobarbituric acid-reactive material. This stimulation was increased in the presence of ascorbate. Iron salts of equivalent concentration to those of the ferritin fractions were more stimulatory to lipid peroxidation at the higher iron concentrations. None of the fractions inhibited ascorbate-dependent peroxidation in the presence of added iron salts.     

Effects of deferrioxamine on iron-catalyzed lipid peroxidation.

The kinetics of iron binding by deferrioxamine B mesylate and the ramifications of this process upon iron-catalyzed lipid peroxidation were assessed. The relative rates of Fe(III) binding by deferrioxamine varied for the chelators tested as follows: ADP greater than AMP greater than citrate greater than histidine greater than EDTA. The addition of a fivefold molar excess of deferrioxamine to that of Fe(III) did not result in complete binding (within 10 min) for any of the Fe(III) chelates tested except ADP:Fe(III). The rates of Fe(III) binding by deferrioxamine were greater at lower pH and when the competing chelator concentration was high in relationship to iron. The relatively slow binding of Fe(III) by deferrioxamine also affected lipid peroxidation, an iron-dependent process. The addition of deferrioxamine to an ascorbate- and ADP:Fe(III)-dependent lipid peroxidation system resulted in a time-dependent inhibition or stimulation of malondialdehyde formation (i.e., lipid peroxidation), depending on the ratio of deferrioxamine to iron. Converse to Fe(III), the rates of Fe(II) binding by deferrioxamine from the chelators tested above were rapid and complete (within 1 min), and resulted in the oxidation of Fe(II) to Fe(III). Lipid peroxidation dependent on Fe(II) autoxidation was stimulated by the addition of deferrioxamine. Malondialdehyde formation in this system was inhibited by the addition of catalase, and a similar extent of lipid peroxidation was achieved by substituting hydrogen peroxide for deferrioxamine. Collectively, these results suggest that the kinetics of Fe(III) binding by deferrioxamine is a slow, variable process, whereas Fe(II) binding is considerably faster. The binding of either valence of iron by deferrioxamine may result in variable effects on iron-catalyzed processes, such as lipid peroxidation, either via slow binding of Fe(III) or the rapid binding of Fe(II) with concomitant Fe(II) oxidation.     

NADPH-dependent drug redox cycling and lipid peroxidation in microsomes from human term placenta

1. NADPH-dependent iron and drug redox cycling, as well as lipid peroxidation process were investigated in microsomes isolated from human term placenta. 2. Paraquat and menadione were found to undergo redox cycling, catalyzed by NADPH:cytochrome P-450 reductase in placental microsomes. 3. The drug redox cycling was able to initiate microsomal lipid peroxidation in the presence of micromolar concentrations of iron and ethylenediaminetetraacetate (EDTA). 4. Superoxide was essential for the microsomal lipid peroxidation in the presence of iron and EDTA. 5. Drastic peroxidative conditions involving superoxide and prolonged incubation in the presence of iron were found to destroy flavin nucleotides, inhibit NADPH:cytochrome P-450 reductase and inhibit propagation step of lipid peroxidation. 6. Reactive oxo-complex formed between iron and superoxide is proposed as an ultimate species for the initiation of lipid peroxidation in microsomes from human term placenta as well as for the destruction of flavin nucleotides and inhibition of NADPH:cytochrome P-450 reductase as well as for impairment of promotion of lipid peroxidation under drastic peroxidative conditions.     

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.     

The role of an endogenous nonheme iron in microsomal redox reactions.

Microsomal membranes contain a nonheme iron which serves in vitro for the peroxidation of unsaturated lipids or the oxidation of several other chemicals. These redox reactions are reviewed in light of a recent identification of two or more iron-binding proteins in the microsomal milieu. Indirect evidence that the microsomal iron might serve in vivo for the synthesis of heme iron is also presented and discussed. Consistent with this, the newly identified iron proteins not only participate in redox reactions but also release their bound iron upon incubation with certain intermediates of heme synthesis.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.     

NADH-dependent reductive stress and ferritin-bound iron in allyl alcohol-induced lipid peroxidation in vivo: the protective effect of vitamin E.

The role of iron in allyl alcohol-induced lipid peroxidation and hepatic necrosis was investigated in male NMRI mice in vivo. Ferrous sulfate (0.36 mmol/kg) or a low dose of ally alcohol (0.6 mmol/kg) itself caused only minor lipid peroxidation and injury to the liver within 1 h. When FeSO4 was administered before allyl alcohol, lipid peroxidation and liver injury were potentiated 50-100-fold. Pretreatment with DL-tocopherol acetate 5 h before allyl alcohol protected dose-dependently against allyl alcohol-induced lipid peroxidation and liver injury in vivo. Products of allyl alcohol metabolism, i.e. NADH and acrolein, both mobilized trace amounts of iron from ferritin in vitro. Catalytic concentrations of FMN greatly facilitated the NADH-induced reductive release of ferritin-bound iron. NADH effectively reduced ferric iron in solution. Consequently, a mixture of NADH and Fe3+ or NADH and ferritin induced lipid peroxidation in mouse liver microsomes in vitro. Our results suggest that the reductive stress (excessive NADH formation) during allyl alcohol metabolism can release ferrous iron from ferritin and can reduce chelated ferric iron. These findings provide a rationale for the strict iron-dependency of allyl alcohol-induced lipid peroxidation and hepatotoxicity in mice in vivo and document iron mobilization and reduction as one of several essential steps in the pathogenesis.     

Microsomal lipid peroxidation: mechanisms of initiation. The role of iron and iron chelators

The role of iron and iron chelators in the initiation of microsomal lipid peroxidation has been investigated. It is shown that an Fe3+ chelate in order to be able to initiate enzymically induced lipid peroxidation in rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with O2; and (c) formation of a relatively stable perferryl radical. NADH can support lipid peroxidation in the presence of ADP-Fe3+ or oxalate-Fe3+ at rates comparable to those obtained with NADPH but requires 10 to 15 times higher concentrations of the Fe3+ chelates for maximal activity. The results are discussed in relation to earlier proposed mechanisms of microsomal lipid peroxidation.     

Effect of antioxidants on adriamycin-induced microsomal lipid peroxidation

Adriamycin (25 μM) stimulated NADPH-dependent microsomal lipid peroxidation about fourfold over control values. The tested antioxidants, zinc, superoxide dismutase, vitamin E, and desferrioxamine (Desferal) inhibited Adriamycin-enhanced lipid peroxidation to varying degrees. Others antioxidants, e.g., glutathione, catalase, and selenium, were found to have no effects. Our in vitro studies suggest that adriamycin effect is mediated by a complex oxyradical cascade involving superoxide, hydroxyl radical, and small amounts of iron.     

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions.     

Initiation of lipid peroxidation in submitochondrial particles: effect of respiratory inhibitors

Initiation of lipid peroxidation in the inner mitochondrial membrane was investigated using respiratory substrates and inhibitors and various iron chelates. An iron chelate was required for initiation of lipid peroxidation in the presence of either NADH or NADPH. The two nicotinamide nucleotides exhibited different activities in initiating lipid peroxidation with regard to concentration and to the effects of rotenone and rhein. Succinate and both nicotinamide nucleotides supported lipid peroxidation in the presence of thenoyl trifluoroacetone (TTFA), without a requirement for exogenously added iron. ADP stimulated lipid peroxidation in the case of NAD(P)H and TTFA, but inhibited it in the case of succinate and TTFA. Lipid peroxidation is thought to be enzymatically induced in both the NADH and the succinate dehydrogenase regions of the respiratory chain, and evidence is presented for a novel pathway of NADPH oxidation that may also be involved. Possible initiation mechanisms are discussed.     

Damage to DNA concurrent with lipid peroxidation in rat liver slices.

Lipid peroxidation and DNA damage were evaluated in liver slices incubated for 2 h at 37 degrees C with 1 mM-t-butyl hydroperoxide (t-BOOH), 1 mM-BrCCl3 or 50 microM-ferrous iron. t-BOOH induced the greatest amount of damage to DNA and increased the production of thiobarbituric acid-reactive substances (TBARS). Both phenomena depended on the incubation time. Ferrous iron induced both DNA damage and TBARS production, and BrCCl3 did not induce significant DNA damage and was the weakest TBARS inducer. Butylated hydroxytoluene at 1 mM inhibited both DNA damage and TBARS production. DNA damage and lipid peroxidation in liver slices were correlated, indicating that these events were concurrent.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.     

Reactions of Adriamycin with Microsomal Iron and Lipids

Iron plays a central role in oxidative injury, reportedly because it catalyzes superoxide- and hydrogen peroxide-dependent reactions yielding a powerful oxidant such as the hydroxyl radical. Iron is also thought to mediate the cardiotoxic and antitumour effects of adriamycin and related compounds. NADPH-supplemented microsomes reduce adriamycin to a semiquinone radical, which in turn re-oxidizes in the presence of oxygen to form superoxide and hence hydrogen peroxide. During this redox cycling membrane-bound nonheme iron undergoes superoxide dismutase- and catalase-insensitive reductive release. Membrane iron mobilization triggers lipid peroxidation, which is markedly enhanced by simultaneous addition of superoxide dismutase and catalase. The results indicate that : i) lipid peroxidation is mediated by the release of iron, yet the two reactions are governed by different mechanisms; and ii) oxygen radicals are not involved in or may actually inhibit adriamycin-induced lipid peroxidation. Microsomal iron delocalization and lipid peroxidation might represent oxyradical-independent mechanisms of adriamycin toxicity.