Hispidulin: Antioxidant properties and effect on mitochondrial energy metabolism†

Hispidulin (6-methoxy-5,7,4′-trihydroxyflavone) and eupafolin (6-methoxy-5,7,3′,4′-tetrahydroxyflavone), are flavonoids found in the leaves of Eupatorium litoralle. They have recognized antioxidant and antineoplastic properties, although their action mechanisms have not been previously described. We now report the effects of hispidulin on the oxidative metabolism of isolated rat liver mitochondria (Mit) and have also investigated the prooxidant and antioxidant capacity of both flavonoids. Hispidulin (0.05–0.2 mM) decreased the respiratory rate in state III and stimulated it in state IV, when glutamate or succinate was used as oxidizable substrate. Hispidulin inhibited enzymatic activities between complexes I and III of the respiratory chain. In broken Mit hispidulin (0.2 mM) slightly inhibited ATPase activity (25%). However, when intact Mit were used, the flavonoid stimulated this activity by 100%. Substrate energized mitochondrial swelling was markedly inhibited by hispidulin. Both hispidulin and eupafolin were able to promote iron release from ferritin, this effect being more accentuated with eupafolin with the suggestion of a possible involvement of H₂O₂ in the process. Hispidulin was incapable of donating electrons to the stable free radical DPPH, while eupafolin reacted with it in a similar way to ascorbic acid. The results indicate that hispidulin as an uncoupler of oxidative phosphorylation, is able to release iron from ferritin, but has distinct prooxidant and antioxidant properties when compared to eupafolin.     

The interaction of ferritin and myoglobin as inductors of lipid peroxidation. The role of reactive oxygen and nitrogen species

The effect of iron dinitrosyl complexes, S-nitrosoglutathione, and glutathione on free radical oxidation of rat heart mitochondria induced by tert-butyl hydroperoxide and metmyoglobin or their combination with ferritin was studied. It was shown that iron dinitrosyl complexes or the combination of S-nitrosoglutathione and glutathione inhibited most effectively the peroxidation of mitochondrial membranes. It was found that ferritin stimulated the prooxidant action of metmyoglobin. Using EPR spectroscopy, it was established that, in conditions of O2*- generation, the destruction of iron dinitrosyl complexes took place. Iron dinitrosyl complexes also inhibited the formation of thiyl radicals, which appeared during O2*- generation in the system containing glutathione and S-nitrosoglutathione. It is essential that the formation of iron dinitrosyl complexes in this reaction system took place with the involvement of ferritin. It was proposed that the prooxidant action of ferritin and myoglobin could be inverted to the antioxidant one.     

Hispidulin protection against hepatotoxicity induced by bromobenzene in mice

The effects of the natural flavonoid hispidulin (6-methoxy-5, 7, 4′-trihydroxyflavone) on bromobenzene-induced hepatotoxicity in mice were investigated. We found a correlation between liver injury and hepatic lipid peroxidation besides a strong liver glutathione depletion due to the toxicant. Hispidulin at doses between 50 and 150 mg/kg i.p. compared favourably with the reference compound N-acetyl-L-cysteine for inhibition of liver injury and lipid peroxidation. The flavonoid at the highest dose tested was also able to counteract reduced glutathione depletion induced by bromobenzene in starved mice. These hepatoprotective effects can be related to the antioxidant properties of hispidulin.     

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.     

Oxidation of lecithin in the presence of dihydroquercetin and its complex with divalent iron ions

The ability of the flavonoid dihydroquercetin to prevent or accelerate the accumulation of reactive oxygen species and the metabolites of oxidative stress, carbonyl compounds has been studied. It has been shown on a model of oxidation of lecithin that dihydroquercetin exhibits a prooxidant effect in the alkaline region of pH, whereas at neutral and acidic pH values dihydroquercetin is an effective antioxidant. In the presence of ferrous iron ions, which catalyze the Fenton reaction, dihydroquercetin forms a complex with metal that shows the antioxidant activity in the region of high pH values. It has been found that the oxidation of lecithin in the presence of 20–200 μM ferrous iron is inhibited by dihydroquercetin to a concentration of 3.2 mM. At higher concentration of dihydroquercetin in the presence of ferrous iron, accumulation of malonic dialdehyde occurs, indicating the presence of the prooxidant activity of dihydroquercetin.     

Effects of reactive species of oxygen and nitrogen on iron ion release from ferritin and synthesis of dinitrosyl iron complexes

Using EPR spectroscopy it was established that Fe ions released from ferritin under the action of glutathione and superoxide took part in the formation of dinitrosyl complexes of iron with glutathione (DNIC). The reaction between O2-. and NO resulted in the formation of peroxynitrite, which oxidized glutathione to the thiyl radical. In these conditions, DNIC did not inhibit the formation of thiyl radicals but effectively slowed down the oxidative destruction of beta-carotene by peroxynitrite and free radicals of lipids. In the presence of glutathione, the inversion of the antioxidant properties of DNIC into prooxidant ones took place. S-nitrosoglutathione prevented this inversion and suppressed the free-radical oxidation of beta-carotene induced by ferritin. It was proposed that the equilibrium between S-nitrosoglutathione, DNIC, "free Fe" ions and ferritin may determine the balance between prooxidant and antioxidant processes in living organisms.     

Body iron is a contributor to oxidative damage of DNA

The transition metal iron is catalytically highly active in vitro, and not surprisingly, body iron has been suggested to promote oxidative stress in vivo. In the current analysis we studied the association of serum ferritin concentration and serum soluble transferrin receptor concentration with daily urinary 8-hydroxydeoxyguanosine excretion, a marker of oxidative stress, in 48 mildly dyslipidemic men in East Finland. In multivariate linear regression analyses allowing for age, smoking, body mass index and physical exercise, serum ferritin concentration predicted the excretion rate at B = 0.17 (95% CI 0.08-0.26, P = 0.001), and serum soluble transferrin receptor to ferritin concentration ratio (TfR/ferritin) predicted the excretion rate at B = - 0.13 (95% CI - 0.21 to - 0.05, P = 0.002). Our data suggest that body iron contributes to excess oxidative stress already at non-iron overload concentrations in these subjects.     

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.     

Prooxidant action of aluminum Ion – Stimulation of iron-mediated lipid peroxidation by aluminum

Prooxidant nature of aluminum ion was analyzed in relation to iron coordination. Aluminum ion effectively enhanced the formation of thiobarbituric acid-reactive substances as a marker of lipid peroxidation of microsomes from rat liver under the acidic conditions, and this metal further attenuated the antioxidant action of flavonoids such as quercetin and baicalein under neutral conditions. Autooxidation of ferrous ion was markedly inhibited by aluminum ion. Aluminum can act as a prooxidant by stabilizing reduced iron the initiating species for lipid peroxidation, and by inhibiting the antioxidant action of flavonoid.     

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.     

Ferritin-stimulated lipid peroxidation,lysosomal leak,and macroautophagy promote lysosomal “metastability” in primary hepatocytes determining in vitro cell survival

Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal “metastability” in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.     

The ferroxidase center is essential for ferritin iron loading in the presence of phosphate and minimizes side reactions that form Fe(III)-phosphate colloids

Ferritin iron loading was studied in the presence of physiological serum phosphate concentrations (1 mM), elevated serum concentrations (2–5 mM), and intracellular phosphate concentrations (10 mM). Experiments compared iron loading into homopolymers of H and L ferritin with horse spleen ferritin. Prior to studying the reactions with ferritin, a series of control reactions were performed to study the solution chemistry of Fe²⁺ and phosphate. In the absence of ferritin, phosphate catalyzed Fe²⁺ oxidation and formed soluble polymeric Fe(III)-phosphate complexes. The Fe(III)-phosphate complexes were characterized by electron microscopy and atomic force microscopy, which revealed spherical nanoparticles with diameters of 10–20 nm. The soluble Fe(III)-phosphate complexes also formed as competing reactions during iron loading into ferritin. Elemental analysis on ferritin samples separated from the Fe(III)-phosphate complexes showed that as the phosphate concentration increased, the iron loading into horse ferritin decreased. The composition of the mineral that does form inside horse ferritin has a higher iron/phosphate ratio (~1:1) than ferritin purified from tissue (~10:1). Phosphate significantly inhibited iron loading into L ferritin, due to the lack of the ferroxidase center in this homopolymer. Spectrophotometric assays of iron loading into H ferritin showed identical iron loading curves in the presence of phosphate, indicating that the ferroxidase center of H ferritin efficiently competes with phosphate for the binding and oxidation of Fe²⁺. Additional studies demonstrated that H ferritin ferroxidase activity could be used to oxidize Fe²⁺ and facilitate the transfer of the Fe³⁺ into apo transferrin in the presence of phosphate.     

Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation

We have previously observed that both Fe(II) and Fe(III) are required for lipid peroxidation to occur, with maximal rates of lipid peroxidation observed when the ratio of Fe(II) to Fe(III) is approximately one (J. R. Bucher et al. (1983) Biochem. Biophys. Res. Commun. 111, 777-784; G. Minotti and S. D. Aust (1987) J. Biol. Chem. 262, 1098-1104). Consistent with the requirement for both Fe(II) and Fe(III), ascorbate, by reducing Fe(III) to Fe(II), stimulated iron-catalyzed lipid peroxidation but when the ascorbate concentration was sufficient to reduce all of the Fe(III) to Fe(II), ascorbate inhibited lipid peroxidation. The rates of lipid peroxidation were unaffected by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers. Exogenously added H2O2 also either stimulated or inhibited ascorbate-dependent, iron-catalyzed lipid peroxidation apparently by altering the ratio of Fe(II) to Fe(III). Thus, it appears that the prooxidant effect of ascorbate is related to the ability of ascorbate to promote the formation of a proposed Fe(II):Fe(III) complex and not due to oxygen radical production. The antioxidant effect of ascorbate on iron-catalyzed lipid peroxidation may be due to complete reduction of iron.     

Eupafolin: Effect on mitochondrial energetic metabolism

This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3',4'-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25-200microM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated approximately 30% by eupafolin (100microM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA-Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.     

Fibrinogen is a co-antioxidant that supplements the vitamin E analog trolox in a model system

Objective: It appears that the atherosclerotic plaque is a prooxidant environment where some molecules that are normally antioxidants, including vitamins C and E, may act as prooxidants that contribute to atherosclerosis by oxidizing LDL. Some molecules can act as co-antioxidants to eliminate this prooxidant effect by recycling or other mechanisms of supplementation. Fibrinogen and other acute phase proteins found in the plaque are antioxidants. We hypothesized that fibrinogen can act as a co-antioxidant to supplement vitamin E thereby eliminating its oxidative effect under prooxidant conditions. We tested a model system for this hypothesis using the vitamin E analogue Trolox in a cell free system.

Methods: LDL was oxidized using 5 umol/l copper. Antioxidant conditions were achieved by adding the antioxidants immediately with LDL, while prooxidant conditions were created by adding antioxidants after a 40 min delay. Oxidation was monitored as the lag phase at 234 nm.

Results: Under antioxidant conditions, the protective effect of fibrinogen and Trolox combined together were about equal to the sum of the anitioxidant effects of each alone (additive), while under prooxidant conditions the combined protection was 54-200% greater (synergistic). These effects were different than those of vitamin C with Trolox in that under antioxidant conditions fibrinogen and Trolox were additive while vitamin C and Trolox showed strong synergistic effects, and in that unlike vitamin C and Trolox fibrinogen showed no prooxidant tendencies under prooxidant reaction conditions.

Conclusions: The data indicated that fibrinogen did act as a co-antioxidant to supplement Trolox and eliminate its prooxidant effect, most probably, by directly quenching the phenoxyl radical, because unlike vitamin C, fibrinogen did not appear to recycle vitamin E. But fibrinogen may act as a universal antioxidant, since unlike Trolox and vitamin C, it showed little tendency toward becoming a prooxidant.     

Safety of trivalent chromium complexes: no evidence for DNA damage in human HaCaT keratinocytes

Several studies have demonstrated beneficial effects of supplemental trivalent Cr in subjects with reduced insulin sensitivity with no documented signs of toxicity. However, recent studies have questioned the safety of supplemental trivalent Cr complexes. The objective of this study was to evaluate the cytotoxic and genotoxic potential of the Cr(III) complexes (histidinate, picolinate, and chloride) used as nutrient supplements compared with Cr(VI) dichromate. The cytotoxic and genotoxic effects of the Cr complexes were assessed in human HaCaT keratinocytes. The concentrations of Cr required to decrease cell viability were assessed by determining the ability of a keratinocyte cell line (HaCaT) to reduce tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. DNA damage using the Comet assay and the production of 8-hydroxy-2′-deoxyguanosine were also determined with and without hydrogen peroxide-induced stress. The LC50 for human cultured HaCaT keratinocytes was 50 μM for hexavalent sodium dichromate and more than 120-fold higher for Cr chloride (6 mM) and Cr histidinate (10 mM). For Cr picolinate at saturating concentration (120 μM) the LC50 was not attained. High Cr(III) concentrations, 250 μM Cr as Cr chloride and Cr histidinate and 120 μM Cr picolinate (highest amount soluble in the system), not only did not result in oxidative DNA damage but exhibited protective antioxidant effects when cells were exposed to hydrogen peroxide-induced oxidative stress. These data further support the low toxicity of trivalent Cr complexes used in nutrient supplements.     

Oxidative damage to ferritin by 5-aminolevulinic acid

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.     

Gene-specific oxidative lesions in aged rat brain detected by polymerase chain reaction inhibition assay

An exposure of isolated rat brain genomic DNA to oxidative stress in the form of iron salts (Fe²⁺) and ascorbate results in gene-specific DNA lesions detectable by a quantitative polymerase chain reaction (PCR) based assay in which PCR amplification efficiency of the affected genes (e.g. β-actin and p53) is grossly impaired. Such oxidative DNA lesions are prevented by hydroxyl radical scavengers like mannitol (20 mM) and sodium benzoate (20 mM) or by the antioxidant enzyme catalase (50 μg/ml) present in the incubation mixture during exposure to Fe²⁺ and ascorbate. When brain DNA isolated from young (4-6 months of age) and aged (20-24 months of age) rats are analyzed similarly by the PCR based method, the amplification levels of β-actin and p53 genes are noticeably decreased in the case of aged rat indicating an accumulation of gene-specific DNA lesions during brain aging.     

Role of phosphate in initial iron deposition in apoferritin

Ferritins from microorganisms to man are known to contain varying amounts of phosphate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe M?ssbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a significant influence on iron deposition. The presence of 1 mM phosphate during reconstitution of ferritin from apoferritin, Fe(II), and O2 accelerates the rate of oxidation of the iron 2-fold at pH 7.5. In the presence or absence of phosphate, the rate of oxidation at 0 degrees C follows simple first-order kinetics with respect to Fe(II) with half-lives of 1.5 +/- 0.3 or 2.8 +/- 0.2 min, respectively, consistent with a single pathway for iron oxidation when low levels of iron are added to the apoprotein. This pathway may involve a protein ferroxidase site where phosphate may bind iron(II), shifting its redox potential to a more negative value and thus facilitating its oxidation. Following oxidation, an intermediate mononuclear Fe(III)-protein complex is formed which exhibits a transient EPR signal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. In 0.5 mM Pi, the signal decays according to a single phase first-order process with a half-life near 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)