In the presence of ferritin, visible light induces lipid peroxidation of the porcine photoreceptor outer segment

We studied the synergistic effect of visible light and ferritin on the lipid peroxidation on a fraction of porcine photoreceptor outer segment (POS). Reaction mixtures containing the POS fraction and horse spleen ferritin were irradiated under white fluorescent light mainly at 17,000 lx or incubated under dark conditions at 37°C. The lipid peroxidation was evaluated by both the thiobarbituric acid method and the ferrous oxidation/xylenol orange method. The irradiation-induced lipid peroxidation was affected by some experimental factors such as the irradiation dose and acidity of the material. When the irradiation was stopped, the lipid peroxidation was also stopped; thereafter, the re-irradiation induced lipid peroxidation. Moreover, this lipid peroxidation was inhibited by desferrioxamine, an iron chelator, or by dimethylthiourea, a hydroxyl radical scavenger, suggesting that the lipid peroxidation involves hydroxyl radicals generated via the Fenton reaction by iron ion released from ferritin. The lipid peroxidation did not take place under dark conditions or in the absence of ferritin. This study suggested the possibility that the visible light-induced lipid peroxidation of the POS fraction in the presence of ferritin may participate in the etiology of human retinal degenerative diseases as the human retina is exposed to light for life.     

In the presence of ferritin,visible light induces lipid peroxidation of the porcine photoreceptor outer segment

We studied the synergistic effect of visible light and ferritin on the lipid peroxidation on a fraction of porcine photoreceptor outer segment (POS). Reaction mixtures containing the POS fraction and horse spleen ferritin were irradiated under white fluorescent light mainly at 17,000 lx or incubated under dark conditions at 37°C. The lipid peroxidation was evaluated by both the thiobarbituric acid method and the ferrous oxidation/xylenol orange method. The irradiation-induced lipid peroxidation was affected by some experimental factors such as the irradiation dose and acidity of the material. When the irradiation was stopped, the lipid peroxidation was also stopped; thereafter, the re-irradiation induced lipid peroxidation. Moreover, this lipid peroxidation was inhibited by desferrioxamine, an iron chelator, or by dimethylthiourea, a hydroxyl radical scavenger, suggesting that the lipid peroxidation involves hydroxyl radicals generated via the Fenton reaction by iron ion released from ferritin. The lipid peroxidation did not take place under dark conditions or in the absence of ferritin. This study suggested the possibility that the visible light-induced lipid peroxidation of the POS fraction in the presence of ferritin may participate in the etiology of human retinal degenerative diseases as the human retina is exposed to light for life.     

Alloxan- and glutathione-dependent ferritin iron release and lipid peroxidation

The diabetogenic action of alloxan is believed to involve oxygen free radicals and iron. Incubation of glutathione (GSH) and alloxan with rat liver ferritin resulted in release of ferrous iron as assayed by spectrophotometric detection of ferrous-bathophenanthroline complex formation. Neither GSH nor alloxan alone mediated iron release from ferritin. Superoxide dismutase (SOD) and catalase did not affect initial rates of iron release whereas ceruloplasmin was an effective inhibitor of iron release. The reaction of GSH with alloxan resulted in the formation of the alloxan radical which was detected by ESR spectroscopy and by following the increase in absorbance at 310nm. In both instances, the addition of ferritin resulted in diminished alloxan radical detection. Incubation of GSH, alloxan, and ferritin with phospholipid liposomes also resulted in lipid peroxidation. Lipid peroxidation did not occur in the absence of ferritin. The rates of lipid peroxidation were not affected by the addition of SOD or catalase, but were inhibited by ceruloplasmin. These results suggest that the alloxan radical releases iron from ferritin and indicates that ferritin iron may be involved in alloxan-promoted lipid peroxidation.     

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.     

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.     

High-pressure enzyme kinetics. Lactate dehydrogenase in an optical cell that allows a reaction to be started under high pressure

Interactions of adriamycin with ferritin-bound iron have been investigated. It is demonstrated (i) that adriamycin stimulates an iron-dependent lipid peroxidation in submitochondrial particles in the presence of ferritin, and (ii) that incubation of adriamycin with ferritin results in a slow transfer of iron to adriamycin with formation of an adriamycin-iron complex. The results are discussed in relation to the possible role for intracellular iron in adriamycin toxicity.     

Ferritin, Lipid Peroxidation and Redox-Cycling Xenobiotics

A number of xenobiotics are toxic because they rcdox cycle and generate free radicals. Interaction with iron, either to produce reactive species such as the hydroxyl radical, or to promote lipid peroxidation, is an important factor in this toxicity. A potential biological source of iron is ferritin. The cytotoxic pyrimidines, dialuric acid, divicine and isouramil, readily release iron from ferritin and promote ferritin-dependent lipid peroxidation. Superoxide dismutase and GSH, which maintain the pyrimidines in their reduced form, enhance both iron release and lipid peroxidation. Microsomes plus NADPH can reduce a number of iron complexes, although not ferritin. Reduction of Adriamycin. paraquat or various quinones to their radicals by the microsomes enhances reduction of the iron complexes, and in some cases, enables iron release from ferritin. Adriamycin stimulates iron-dependent lipid peroxidation of the microsomes. Ferritin can provide the iron, and peroxidation is most pronounced at low PO₂. Compiexing agents that supress intraccllular iron reduction and lipid peroxidation may protect against the toxicity of Adriamycin.     

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.     

The role of superoxide and hydroxyl radicals in phospholipid peroxidation catalysed by iron salts

Iron(II) salts in aqueous solution, or iron(III) salts in the presence of an O√⁻₂ generating system, can activate dioxygen to produce hydroxyl radicals. These are detected indirectly by their ability to degrade deoxyribose with the formation of thiobarbituric acid-reactive (TBA) products. Iron salts also catalyse the peroxidation of phospholipids resulting in the formation of TBA-reactive products. Hydroxyl radicals were responsible for the degradation of deoxyribose but not for the observed peroxidation of phospholipid. The function of O√⁻₂ in both deoxyribose degradation and phospholipid peroxidation seems to be that of reducing iron(III) into iron(II).     

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.     

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.     

Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage

Iron storage proteins, ferritin and haemosiderin, release iron to a range of chelators and reducing agents, including citrate, acetate and ascorbate. Released iron promotes both hydroxyl radical formation in the presence of hydrogen peroxide and lipid peroxidation in liposomes. Ferritin protein is modified in such reactions, both by free radical cleavage and addition reactions with aldehyde products of lipid peroxidation.     

Iron release from ferritin and lipid peroxidation by radiolytically generated reducing radicals

Iron is involved in the formation of oxidants capable of damaging membranes, protein, and DNA. Using 137Cs gamma radiation, we investigated the release of iron from ferritin and concomitant lipid peroxidation by radiolytically generated reducing radicals, superoxide and the carbon dioxide anion radical. Both radicals released iron from ferritin with similar efficiencies and iron mobilization from ferritin required an iron chelator. Radiolytically generated superoxide anion resulted in peroxidation of phospholipid liposomes as measured by malondialdehyde formation only when ferritin was included as an iron source and the released iron was found to be chelated by the phospholipid liposomes.     

Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts

Aluminium salts do not themselves stimulate peroxidation of ox-brain phospholipid liposomes, but they greatly accelerate the peroxidation induced by iron(II) salts at acidic pH values. This effect of Al(III) is not seen at pH 7.4, perhaps because Al(III) salts form insoluble complexes at this pH in aqueous solution. Peroxidation of liposomes in the presence of Al(III) and Fe(II) salts is inhibited by the chelating agent desferrioxamine, and by EDTA and diethylenetriaminepentaacetic acid at concentrations greater than those of Fe(II) salt. Aluminium salts slightly stimulate the peroxidation of peroxide-depleted linolenic acid micelles, but they do not accelerate the peroxidation induced by addition of iron(II) salts to the micelles at acidic pH. Aluminium salts accelerate the peroxidation observed when human erythrocytes are treated with hydrogen peroxide at pH 7.4. Desferrioxamine decreases the peroxidation. We suggest that Al(III) ions produce an alteration in membrane structure that facilitates lipid peroxidation, and that the increased formation of fluorescent age pigments in the nervous system of patients exposed to toxic amounts of Al(III) may be related to this phenomenon. The ability of desferal to bind both iron (III) and aluminium(III) salts and to inhibit lipid peroxidation makes it an especially useful chelating agent in the treatment of 'aluminium overload'.     

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.     

In Vitro Studies of Ferritin Iron Release and Neurotoxicity

Abstract: The increase in brain iron associated with several neurodegenerative diseases may lead to an increased production of free radicals via the Fenton reaction. Intracellular iron is usually tightly regulated, being bound by ferritin in an insoluble ferrihydrite core. The neurotoxin 6-hydroxydopamine (6-OHDA) releases iron from the ferritin core by reducing it to the ferrous form. Iron release induced by 6-OHDA and structurally related compounds and two other dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium iodide (MPP⁺) and 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), were compared, to identify the structural characteristics important for such release. 1,2,4-Trihydroxybenzene (THB) was most effective in releasing ferritin-bound iron, followed by 6-OHDA, dopamine, catechol, and hydroquinone. Resorcinol, MPP⁺, and TaClo were ineffective. The ability to release iron was associated with a low oxidation potential. It is proposed that a low oxidation potential and an ortho -dihydroxyphenyl structure are important in the mechanism by which ferritin iron is mobilized. In the presence of ferritin, both 6-OHDA and THB strongly stimulated lipid peroxidation, an effect abolished by the addition of the iron chelator deferoxamine. These results suggest that ferritin iron release contributes to free radical-induced cell damage in vivo.     

Effects of Ceruloplasmin on Superoxide-Dependent Iron Release from Ferritin and Lipid Peroxidation

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin. suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum. and inhibited at higher concentrations. However. the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.     

Properties of human tissue isoferritins.

1. Human liver ferritin was separated by preparative isoelectric focusing into six fractions. 2. Except for the least acidic fraction the reactivity with antibody against spleen ferritin increased with rising pI, but with antibody against heart ferritin the reactivity decreased. 3. The highest iron content was found in the most acidic isoferritins and progressively decreased with rising pI. 4. Iron uptake was studied in apoferritin prepared from heart and liver ferritin fractions separated by ion-exchange chromatography. There was good correlation between the rate of iron uptake and pI. The most acidic fractions took up iron more rapidly than did the more basic ones. 5. Ferritin was prepared from heart, liver, spleen and kidney. There was little difference on isoelectric focusing between ferritin obtained from normal tissues and the corresponding iron-loaded tissues from patients who had received multiple blood transfusions. The iron-loaked heart ferritin invariably contained relatively more of the basic isoferritins. Normal and iron-overloaded heart ferritins were separated into isoferritin fractions by ion-exchange chromatography, and in each case there was a fall in iron content as the pI increased. The iron content of ferritin from the iron-overloaded heart was higher throughout than that from normal heart. 6. There is a relationship between the rate of iron uptake by apoferritin and pI, and this probably accounts for the variation in iron content of the isoferritins found in human liver and heart.     

Ferritin, a physiological iron donor for microsomal lipid peroxidation

In the process of lipid peroxidation of microsomes induced either by oxygen radicals generated by xanthine oxidase or by NADPH, ferritin is able to donate the necessary iron. The amount of ferritin necessary to catalyze the process of lipid peroxidation is in the physiological range. In contrast to the finding with phospholipid liposomes, catalase hardly stimulates the lipid peroxidation of microsomes.