Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Ethanol-induced iron mobilization: role of acetaldehyde-aldehyde oxidase generated superoxide

Superoxide radicals, a species known to mobilize ferritin iron, and their interaction with catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury. The mechanism(s) by which ethanol metabolism generates free radicals and mobilizes catalytic iron, however, is not fully defined. In this investigation the role of hepatic aldehyde oxidase in the mobilization of catalytic iron from ferritin was studied in vitro. Iron mobilization due to the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was increased 100% by the addition of aldehyde oxidase. Iron release was favored by low pH and low oxygen concentration. Mobilization of iron due to acetaldehyde metabolism by aldehyde oxidase was completely inhibited by superoxide dismutase but not by catalase suggesting that superoxide radicals mediate mobilization. Acetaldehyde-aldehyde oxidase mediated reduction of ferritin iron was facilitated by incubation with menadione, an electron acceptor for aldehyde oxidase. Mobilization of ferritin iron due to the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism of alcohol-induced liver injury.     

On the limited ability of superoxide to release iron from ferritin

Reductive release of iron from ferritin may catalyze cytotoxic radical reactions like the Haber-Weiss reaction. The ability of .O2- to mobilize Fe(II) from ferritin was studied by using the xanthine/xanthine oxidase reaction, with and without superoxide dismutase, and with bathophenanthroline sulphonate as the chelator. Not more than one or two Fe(II)/ferritin molecules could be released by an .O2(-)-dependent mechanism, even after repeated exposures of ferritin to bursts of .O2-. The amount of releaseable iron depended on the size and the age of the iron core, but not on the iron content of the protein shell of ferritin which was manipulated by chelators and addition of FeCl3. The kinetic characteristics of the .O2(-)-mediated iron release indicated the presence of a small pool of readily available iron at the surface of the core. The very limited .O2(-)-dependent release of iron from ferritin is compatible with a protective role of ferritin against toxic iron-catalyzed reactions.     

Release of iron from ferritin by cardiotoxic anthracycline antibiotics

The use of the extremely effective anthracycline antitumor drugs adriamycin and daunomycin is limited by a severe, dose-dependent cardiomyopathy. Anthracycline-induced toxicity has been proposed to involve iron-dependent oxidative damage to biological macromolecules yet little is known regarding the availability of physiologic iron. We now report that, in the presence of NADPH-cytochrome P-450 reductase, these drugs undergo redox cycling to generate superoxide which mediates a slow, reductive release of iron from ferritin, the major intracellular iron storage protein. Anaerobically, the semiquinone free radical forms of adriamycin and daunomycin catalyze a very rapid, extensive release of iron from ferritin. In contrast, diaziquone, an aziridinyl quinone antitumorigenic agent which is less cardiotoxic, is unable to release iron from ferritin. Thus, the present studies suggest that the cardiomyopathy observed with the anthracyclines, and perhaps their antineoplastic activity as well, may be related to their ability to delocalize tissue iron, thereby contributing to the formation of strong oxidants capable of damaging critical cellular constituents.     

A Chelator is Required for Microsomal LIPID Peroxidation Following Reductive Ferritin-Iron Mobilisation

In the past, antioxidant and chelator studies have implicated a role for iron-dependent oxidative damage in tissues subjected to ischaemia followed by reperfusion. As ferritin is a major source of iron in non-muscular organs and therefore a potential source of the iron required for oxygen radical chemistry, we have determined conditions under which ferritin iron reduction leads to the formation of a pool of iron which is capable of catalysing lipid peroxidation. Under anaerobic conditions and in the presence of rat liver microsomes, flavin mononucleotide (FMN) catalysed the reduction of ferritin iron as shown by both continuous spectrophotometric measurements of tris ferrozine-Fe(II) complex formation and post-reaction Fe(II) determination. The presence of either ferrozine or citrate was not found to alter the time course or extent of ferritin reduction. In contrast, the addition of air to the reactants after a 20 min period of anaerobic reduction resulted in peroxidation of the microsome suspension (as determined with the 2-thiobarbituric acid test) only in the presence of a chelator such as citrate, ADP or nitrilotriacetic acid. These results support the concept that reduced ferritin iron can mediate oxidative damage during reperfusion of previously ischaemic tissues, provided that chelating agents such as citrate or ADP are present.     

Effect of a prolonged superoxide flux on transferrin and ferritin

The involvement of "free" iron in damage caused by oxidative stress is well recognized. Superoxide generated in a short burst and at a relatively high flux by the xanthine/xanthine oxidase couple is known to release iron from ferritin in the presence of phenanthroline derivatives as iron chelators. However, superoxide generation via xanthine oxidase is accompanied by the simultaneous direct generation of hydrogen peroxide and, in the presence of ferritin, there is also a superoxide-independent release of iron. In this study it was found that the iron chelator employed attenuates superoxide formation from the xanthine/xanthine oxidase couple. The reaction of ferritin and transferrin with a clean chemical source of superoxide, di(4-carboxybenzyl)hyponitrite (SOTS-1) was therefore investigated. The efficiency of superoxide-induced iron release from ferritin increases dramatically as the superoxide flux is decreased, reaching as high as 0.5 Fe per O2*-. Treatment of ferritin for 16 h with SOTS-1 yielded as many as 130 Fe atoms/ferritin molecule, which greatly exceeds the amount of possible "contaminating" iron absorbed on the protein shell.     

Reductive release of iron from ferritin by cation free radicals of paraquat and other bipyridyls

NADPH-cytochrome P-450 reductase-catalyzed reduction of paraquat promoted the release of iron from ferritin. Aerobically, iron release was inhibited approximately 60% by superoxide dismutase, whereas xanthine oxidase-dependent iron release was inhibited nearly 100%. This suggests that both superoxide and the paraquat cation radical can catalyze the release of iron from ferritin. Accordingly, under anaerobic conditions, the paraquat radical mediated a very rapid, complete release of iron from ferritin. Similarly, the cation free radicals of the closely related chemicals, diquat and benzyl viologen, also promoted iron release. ESR studies demonstrated that electron transfer from the paraquat cation radical to ferritin accounts for the reductive release of iron. The ferritin structure was not altered by exposure to the paraquat radical and also retained its ability to re-incorporate iron. These studies indicate that release of iron from ferritin may be a common feature contributing to free radical-mediated toxicities.     

Release of iron from ferritin by semiquinone, anthracycline, bipyridyl, and nitroaromatic radicals

The cytotoxicity of many xenobiotics is related to their ability to undergo redox reactions and iron dependent free radical reactions. We have measured the ability of a number of redox active compounds to release iron from the cellular iron storage protein, ferritin. Compounds were reduced to their corresponding radicals with xanthine oxidase/hypoxanthine under N₂ and the release of Fe²⁺ was monitored by complexation with ferrozine. Ferritin iron was released by a number of bipyridyl radicals including those derived from diquat and paraquat, the anthracycline radicals of adriamycin, daunorubicin and epirubicin, the semiquinones of anthraquinone-2-sulphonate, 1,5 and 2,6-dihydroxyanthraquinone, 1-hydroxyanthraquinone, purpurin, and plumbagin, and the nitroaromatic radicals of nitrofurantoin and metronidazole. In each case, iron release was more efficient than with an equivalent flux of superoxide. Introduction of air decreased the rate of iron release, presumably because the organic radicals reacted with O₂ to form superoxide. In air, iron release was inhibited by superoxide dismutase. Semiquinones of menadione, benzoquinone, duroquinone, anthraquinone 1,5 and 2,6-disulphonate, 1,4 naphthoquinone-2-sulphonate and naphthoquinone, when formed under N₂, were unable to release ferrin iron. In air, these systems gave low rates of superoxide dismutase-inhibitible iron release. Of the compounds investigated, those with a single electron reduction potential less than that of ferritin were able to release ferritin iron.     

Superoxide-dependent and -independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Implications for oxygen-free-radical-induced tissue destruction during ischaemia and inflammation.

Xanthine oxidase is able to mobilize iron from ferritin. This mobilization can be blocked by 70% by superoxide dismutase, indicating that part of its action is mediated by superoxide (O2-). Uric acid induced the release of ferritin iron at concentrations normally found in serum. The O2(-)-independent mobilization of ferritin iron by xanthine oxidase cannot be attributed to uric acid, because uricase did not influence the O2(-)-independent part and acetaldehyde, a substrate for xanthine oxidase, also revealed an O2(-)-independent part, although no uric acid was produced. Presumably the amount of uric acid produced by xanthine oxidase and xanthine is insufficient to release a measurable amount of iron from ferritin. The liberation of iron from ferritin by xanthine oxidase has important consequences in ischaemia and inflammation. In these circumstances xanthine oxidase, formed from xanthine dehydrogenase, will stimulate the formation of a non-protein-bound iron pool, and the O2(-)-produced by xanthine oxidase, or granulocytes, will be converted by 'free' iron into much more highly toxic oxygen species such as hydroxyl radicals (OH.), exacerbating the tissue damage.     

Mitochondrial superoxide mediates labile iron level: evidence from Mn-SOD-transgenic mice and heterozygous knockout mice and isolated rat liver mitochondria

Superoxide is the main reactive oxygen species (ROS) generated by aerobic cells primarily in mitochondria. It is also capable of producing other ROS and reactive nitrogen species (RNS). Moreover, superoxide has the potential to release iron from its protein complexes. Unbound or loosely bound cellular iron, known as labile iron, can catalyze the formation of the highly reactive hydroxyl radical. ROS/RNS can cause mitochondrial dysfunction and damage. Manganese superoxide dismutase (Mn-SOD) is the chief ROS-scavenging enzyme and thereby the primary antioxidant involved in protecting mitochondria from oxidative damage. To investigate whether mitochondrial superoxide mediates labile iron in vivo, the levels of labile iron were determined in the tissues of mice overexpressing Mn-SOD and heterozygous Mn-SOD-knockout mice. Furthermore, the effect of increased mitochondrial superoxide generation on labile iron levels was determined in isolated rat liver mitochondria exposed to various electron transport inhibitors. The results clearly showed that increased expression of Mn-SOD significantly lowered the levels of labile iron in heart, liver, kidney, and skeletal muscle, whereas decreased expression of Mn-SOD significantly increased the levels of labile iron in the same organs. In addition, the data showed that peroxidative damage to membrane lipids closely correlated with the levels of labile iron in various tissues and that altering the status of Mn-SOD did not alter the status of other antioxidant systems. Results also showed that increased ROS production in isolated liver mitochondria significantly increased the levels of mitochondrial labile iron. These findings constitute the first evidence suggesting that mitochondrial superoxide is capable of releasing iron from its protein complexes in vivo and that it could also release iron from protein complexes contained within the organelle.     

Protective effects of carnosine and homocarnosine on ferritin and hydrogen peroxide-mediated DNA damage

Previous studies have shown that one of the primary causes of increased iron content in the brain may be the release of excess iron from intracellular iron storage molecules such as ferritin. Free iron generates ROS that cause oxidative cell damage. Carnosine and related compounds such as endogenous histidine dipetides have antioxidant activities. We have investigated the protective effects of carnosine and homocarnosine against oxidative damage of DNA induced by reaction of ferritin with H(2)O(2). The results show that carnosine and homocarnosine prevented ferritin/H(2)O(2)-mediated DNA strand breakage. These compounds effectively inhibited ferritin/H(2)O(2)-mediated hydroxyl radical generation and decreased the mutagenicity of DNA induced by the ferritin÷H(2)O(2) reaction. Our results suggest that carnosine and related compounds might have antioxidant effects on DNA under pathophysiological conditions leading to degenerative damage such as neurodegenerative disorders.     

Nitric oxide does not promote iron release from ferritin

It has been previously reported that iron release from ferritin could be promoted by nitric oxide (NO) generated from sodium nitroprusside. It was thus proposed that some of the toxic effects of NO could be related to its ability to increase intracellular free iron concentrations and generate an oxidative stress. On the contrary, the iron exchange experiments reported here show that NO from S-nitrosothiols is unable to promote iron release from ferritin. The discrepancy may be explained by the disregarded ability of ferrozine, the ferrous trap used in the previous report, to mobilize iron both from ferritin and from sodium nitroprusside spontaneously.     

Ascorbate-mediated Iron Release from Ferritin in the Presence of Alloxan

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A·⁻) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A·⁻ is directly involved in iron reduction. Although release of iron from ferritin and generation of A·⁻ were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A·⁻ present, suggesting that other reductants in addition to A·⁻ (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A·⁻ is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A·⁻ contribute to GSH/alloxan-mediated iron release.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Iron-mediated oxidative stress plays an essential role in ferritin-induced cell death

Previously, we have demonstrated an apoptosis-inducing activity of an acidic, H-chain-rich isoferritin secreted from primary rat hepatocytes in vitro. Because this proapoptotic property may be responsible for the growth-inhibitory and immunosuppressive effects described for certain ferritin species, we aimed to address the mechanism by which ferritin can trigger cell death. Suggesting a pivotal role for iron, iron chelation by desferrioxamine significantly abrogates ferritin-mediated apoptosis and necrosis in primary rat hepatocytes and substantially lowers the extent of protein modification by 4-hydroxynonenal (HNE)—a major lipid peroxidation (LPO) product. Furthermore, supplementing the cultures with the radical-scavenging compound trolox also provided significant protection from ferritin-mediated apoptosis. Moreover, a significant increase in micronucleated cells upon exposure to ferritin indicates that ferritin also introduces damage to DNA. Based on these observations we therefore propose that endocytosis of extracellular ferritin increases the level of free ferrous iron in the lysosomal compartment, promoting Fenton chemistry-based oxidative stress involving LPO and increased lysosomal membrane permeability. Subsequently, the release of reactive lysosomal content leads to cellular damage, in particular modification of protein and DNA induced by HNE and other reactive aldehydic LPO products. Together, these effects will trigger apoptosis and necrosis based on the upregulation of p53, increased mitochondrial membrane permeability, and proapoptotic Fas signaling as described recently. In conclusion, based on their iron-storing ability, secreted acidic isoferritins may act as soluble mediators of oxidative stress under certain physiological and pathophysiological conditions.     

Nitric oxide mediates iron release from ferritin

Nitric oxide (NO) synthesis by cytotoxic activated macrophages has been postulated to result in a progressive loss of iron from tumor target cells as well as inhibition of mitochondrial respiration and DNA synthesis. In the present study, the addition of an NO-generating agent, sodium nitroprusside, to the iron storage protein ferritin resulted in the release of iron from ferritin and the released iron-catalyzed lipid peroxidation. Hemoglobin, which binds NO, and superoxide anion, which reacts with NO, inhibited nitroprusside-dependent iron release from ferritin, thereby providing evidence that NO can mobilize iron from ferritin. These results suggest that NO generation in vivo could lead to the mobilization of iron from ferritin disrupting intracellular iron homeostasis and increasing the level of reactive oxygen species.     

Oxidative modification of ferritin induced by hydrogen peroxide

Excess free iron generates oxidative stress that may contribute to the pathogenesis of various causes of neurodegenerative diseases. In this study, we assessed the modification of ferritin induced by H(2)O(2). When ferritin was incubated with H(2)O(2), the degradation of ferritin L-chain increased with the H(2)O(2) concentration whereas ferritin H-chain was remained. Free radical scavengers, azide, thiourea, and N-acetyl-(L)-cysteine suppressed the H(2)O(2)-mediated ferritin modification. The iron specific chelator, deferoxamine, effectively prevented H(2)O(2)-mediated ferritin degradation in modified ferritin. The release of iron ions from ferritin was increased in H(2)O(2) concentration-dependent manner. The present results suggest that free radicals may play a role in the modification and iron releasing of ferritin by H(2)O(2). It is assumed that oxidative damage of ferritin by H(2)O(2) may induce the increase of iron content in cells and subsequently lead to the deleterious condition.     

Dietary vitamin E reduces labile iron in rat tissues

Previous studies have shown that dietary vitamin E reduced generation and/or levels of superoxide. As superoxide has potential to release iron from its transport and storage proteins, and labile or available form of iron is capable of catalyzing the formation of reactive hydroxyl radicals, the effect of dietary vitamin E on labile iron pool was studied in rats. One-month-old Sprague-Dawley male and female rats were fed a basal vitamin E-deficient diet supplemented with 0, 20, 200, or 2,000 IU vitamin E/kg diet for 90 days. The levels of labile iron were measured in the liver, kidney, spleen, heart and skeletal muscle. Additionally, the levels of lipid peroxidation products were measured. The results showed that, except for labile iron in the heart of male rats, dietary vitamin E dose dependently reduced the levels of labile iron and lipid peroxidation products in all tissues of male and female rats. The findings suggest that dietary vitamin E may protect against oxidative tissue damage by reducing the generation and/or level of superoxide, which in turn attenuates the release of iron from its protein complexes.     

Superoxide-mediated release of iron from ferritin by some flavoenzymes

NADH-lipoamide dehydrogenase mobilized iron from ferritin under aerobic conditions. Superoxide dismutase strongly inhibited this mobilization, indicating that the superoxide radical is generated by the enzymatic reaction and release iron from ferritin. Addition of lipoamide as an electron acceptor to NADH-lipoamide dehydrogenase increased the release of iron from ferritin and this release was partially inhibited by superoxide dismutase. Similarly, addition of menadione (2-methyl-1, 4-naphthoquinone) as an electron acceptor to xanthine-xanthine oxidase promoted the release of iron from ferritin and this release was strongly inhibited by superoxide dismutase. These results suggest that dihydrolipoamide and semiquinone of menadione can react with oxygen to form the superoxide radical that mediates release of iron from ferritin.