Multitissue analysis of exercise and metformin on doxorubicin-induced iron dysregulation.

Doxorubicin (DOX) is an effective chemotherapeutic treatment with lasting side effects in heart and skeletal muscle. DOX is known to bind with iron, contributing to oxidative damage resulting in cardiac and skeletal muscle toxicity. However, major cellular changes to iron regulation in response to DOX are poorly understood in liver, heart, and skeletal muscle. Additionally, two cotreatments, exercise (EX) and metformin (MET), were studied for their effectiveness in reducing DOX toxicity by ameliorating iron dysregulation and preventing oxidative stress. The purposes of this study were to 1) characterize the DOX-induced changes of the major iron regulation pathway in liver, heart, and skeletal muscle and 2) to determine whether EX and MET exert their benefits by minimizing DOX-induced iron dysregulation. Mice were assigned to receive saline or DOX (15 mg/kg) treatments, paired with either EX (5 days) or MET (500 mg/kg), and were euthanized 3 days after DOX treatment. Results suggest that the cellular response to DOX is protective against oxidative stress by reducing iron availability. DOX increased iron storage capacity through elevated ferritin levels in liver, heart, and skeletal muscle. DOX reduced iron transport capacity through reduced transferrin receptor levels in heart and skeletal muscle. EX and MET cotreatments had protective effects in the liver through reduced transferrin receptor levels. At 3 days after DOX, oxidative stress was mild, as shown by normal glutathione and lipid peroxidation levels. Together these results suggest that the cellular response to reduce iron availability in response to DOX treatment is sufficient to match oxidative stress.

Whole blood and urine bioactive Hepcidin-25 determination using liquid chromatography mass spectrometry.

Hepcidin is a small cysteine-rich signaling peptide that regulates blood serum iron concentrations [1-4]. Patients with chronic inflammation are known to have elevated levels of hepcidin in their blood and urine and often suffer from anemia as a result [5-10]. Measuring and quantifying the amount of active hepcidin in blood and urine can help to determine the cause and severity of the anemia thereby helping physicians determine the correct course of treatment [11-16]. We have developed a simple technique to isolate, chemically modify, and concentrate hepcidin from blood and urine coupled to high-pressure liquid chromatography mass spectrometry that can accurately and reproducibly measure and quantify the active hormone.

Tuning Ferritin's band gap through mixed metal oxide nanoparticle formation.

This study uses the formation of a mixed metal oxide inside ferritin to tune the band gap energy of the ferritin mineral. The mixed metal oxide is composed of both Co and Mn, and is formed by reacting aqueous Co2+ with [Formula: see text] in the presence of apoferritin. Altering the ratio between the two reactants allowed for controlled tuning of the band gap energies. All minerals formed were indirect band gap materials, with indirect band gap energies ranging from 0.52 to 1.30 eV. The direct transitions were also measured, with energy values ranging from 2.71 to 3.11 eV. Tuning the band gap energies of these samples changes the wavelengths absorbed by each mineral, increasing ferritin's potential in solar-energy harvesting. Additionally, the success of using [Formula: see text] in ferritin mineral formation opens the possibility for new mixed metal oxide cores inside ferritin.

Permanganate-based synthesis of manganese oxide nanoparticles in ferritin.

This paper investigates the comproportionation reaction of MnII with [Formula: see text] as a route for manganese oxide nanoparticle synthesis in the protein ferritin. We report that [Formula: see text] serves as the electron acceptor and reacts with MnII in the presence of apoferritin to form manganese oxide cores inside the protein shell. Manganese loading into ferritin was studied under acidic, neutral, and basic conditions and the ratios of MnII and permanganate were varied at each pH. The manganese-containing ferritin samples were characterized by transmission electron microscopy, UV/Vis absorption, and by measuring the band gap energies for each sample. Manganese cores were deposited inside ferritin under both the acidic and basic conditions. All resulting manganese ferritin samples were found to be indirect band gap materials with band gap energies ranging from 1.01 to 1.34 eV. An increased UV/Vis absorption around 370 nm was observed for samples formed under acidic conditions, suggestive of MnO2 formation inside ferritin.

A unified model for ferritin iron loading by the catalytic center: implications for controlling "free iron" during oxidative stress.

IRONING OUT THE DIFFERENCES: A unified model shows that the catalytic centers of human H ferritin and archaeal P. furiosus ferritin load iron according to the same mechanism. This model could help our understanding of the processes of controlling the various subcellular concentrations of iron during inflammation.

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 Fe(2+) and phosphate. In the absence of ferritin, phosphate catalyzed Fe(2+) 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 Fe(2+). Additional studies demonstrated that H ferritin ferroxidase activity could be used to oxidize Fe(2+) and facilitate the transfer of the Fe(3+) into apo transferrin in the presence of phosphate.

Oxido-reduction is not the only mechanism allowing ions to traverse the ferritin protein shell.

BACKGROUND: Most models for ferritin iron release are based on reduction and chelation of iron. However, newer models showing direct Fe(III) chelation from ferritin have been proposed. Fe(III) chelation reactions are facilitated by gated pores that regulate the opening and closing of the channels. SCOPE OF REVIEW: Results suggest that iron core reduction releases hydroxide and phosphate ions that exit the ferritin interior to compensate for the negative charge of the incoming electrons. Additionally, chloride ions are pumped into ferritin during the reduction process as part of a charge balance reaction. The mechanism of anion import or export is not known but is a natural process because phosphate is a native component of the iron mineral core and non-native anions have been incorporated into ferritin in vitro. Anion transfer across the ferritin protein shell conflicts with spin probe studies showing that anions are not easily incorporated into ferritin. To accommodate both of these observations, ferritin must possess a mechanism that selects specific anions for transport into or out of ferritin. Recently, a gated pore mechanism to open the 3-fold channels was proposed and might explain how anions and chelators can penetrate the protein shell for binding or for direct chelation of iron. CONCLUSIONS AND GENERAL SIGNIFICANCE: These proposed mechanisms are used to evaluate three in vivo iron release models based on (1) equilibrium between ferritin iron and cytosolic iron, (2) iron release by degradation of ferritin in the lysosome, and (3) metallo-chaperone mediated iron release from ferritin.

The many faces of the octahedral ferritin protein.

Iron is an essential trace nutrient required for the active sites of many enzymes, electron transfer and oxygen transport proteins. In contrast, to its important biological roles, iron is a catalyst for reactive oxygen species (ROS). Organisms must acquire iron but must protect against oxidative damage. Biology has evolved siderophores, hormones, membrane transporters, and iron transport and storage proteins to acquire sufficient iron but maintain iron levels at safe concentrations that prevent iron from catalyzing the formation of ROS. Ferritin is an important hub for iron metabolism because it sequesters iron during times of iron excess and releases iron during iron paucity. Ferritin is expressed in response to oxidative stress and is secreted into the extracellular matrix and into the serum. The iron sequestering ability of ferritin is believed to be the source of the anti-oxidant properties of ferritin. In fact, ferritin has been used as a biomarker for disease because it is synthesized in response to oxidative damage and inflammation. The function of serum ferritin is poorly understood, however serum ferritin concentrations seem to correlate with total iron stores. Under certain conditions, ferritin is also associated with pro-oxidant activity. The source of this switch from anti-oxidant to pro-oxidant has not been established but may be associated with unregulated iron release from ferritin. Recent reports demonstrate that ferritin is involved in other aspects of biology such as cell activation, development, immunity and angiogenesis. This review examines ferritin expression and secretion in correlation with anti-oxidant activity and with respect to these new functions. In addition, conditions that lead to pro-oxidant conditions are considered.

Spontaneous Formation of Melanin from Dopamine in the Presence of Iron.

Parkinson's disease is associated with degeneration of neuromelanin (NM)-containing substantia nigra dopamine (DA) neurons and subsequent decreases in striatal DA transmission. Dopamine spontaneously forms a melanin through a process called melanogenesis. The present study examines conditions that promote/prevent DA melanogenesis. The kinetics, intermediates, and products of DA conversion to melanin in vitro, and DA melanogenesis under varying levels of Fe3+, pro-oxidants, and antioxidants were examined. The rate of melanogenesis for DA was substantially greater than related catecholamines norepinephrine and epinephrine and their precursor amino acids tyrosine and l-Dopa as measured by UV-IR spectrophotometry. Dopamine melanogenesis was concentration dependent on the pro-oxidant species and Fe3+. Melanogenesis was enhanced by the pro-oxidant hydrogen peroxide (EC50 = 500 µM) and decreased by the antioxidants ascorbate (IC50 = 10 µM) and glutathione (GSH; IC50 = 5 µM). Spectrophotometric results were corroborated by tuning a fast-scan cyclic voltammetry system to monitor DA melanogenesis. Evoked DA release in striatal brain slices resulted in NM formation that was prevented by GSH. These findings suggest that DA melanogenesis occurs spontaneously under physiologically-relevant conditions of oxidative stress and that NM may act as a marker of past exposure to oxidative stress.

Hypoxia-inducible factor prolyl hydroxylase inhibitors: a paradigm shift for treatment of anemia in chronic kidney disease?

INTRODUCTION: The hypoxia-inducible factor prolyl hydroxylase (HIF-PH) pathway is responsible for regulating the biosynthesis of erythropoietin (EPO) and maintaining iron homeostasis. Investigational drugs that target the HIF-PH pathway are promising alternatives for treating anemia in Chronic Kidney Disease (CKD). AREAS COVERED: This review summarizes recent advances focused on the clinical development of HIF-PH inhibitors (HIF-PHIs) as potentially novel therapies in the treatment of anemia in CKD based on publications available on PubMed and restricted Google searches. We provide a comparison between HIF-PHIs regarding their pharmacokinetics, dosing regimens and safety concerns, structure-activity relationships, and alterations in key laboratory parameters observed in animal models and clinical trials. EXPERT OPINION: HIF-PHIs may be advantageous in some aspects compared to the conventional erythropoiesis-stimulating agents (ESAs). While ESAs could increase the risk of cardiovascular events due to rapid rises in ESA blood levels, HIF-PHIs have been reported to maintain EPO concentrations at levels that are closer to the normal physiological ranges. Although HIF-PHIs have been demonstrated to be relatively safe and effective in clinical trials, long-term safety data are needed in order to establish whether these therapeutic agents will lead to a major paradigm change in the treatment of anemia of CKD.

Nanocatalytic activity of clean-surfaced, faceted nanocrystalline gold enhances remyelination in animal models of multiple sclerosis.

Development of pharmacotherapies that promote remyelination is a high priority for multiple sclerosis (MS), due to their potential for neuroprotection and restoration of function through repair of demyelinated lesions. A novel preparation of clean-surfaced, faceted gold nanocrystals demonstrated robust remyelinating activity in response to demyelinating agents in both chronic cuprizone and acute lysolecithin rodent animal models. Furthermore, oral delivery of gold nanocrystals improved motor functions of cuprizone-treated mice in both open field and kinematic gait studies. Gold nanocrystal treatment of oligodendrocyte precursor cells in culture resulted in oligodendrocyte maturation and expression of myelin differentiation markers. Additional in vitro data demonstrated that these gold nanocrystals act via a novel energy metabolism pathway involving the enhancement of key indicators of aerobic glycolysis. In response to gold nanocrystals, co-cultured central nervous system cells exhibited elevated levels of the redox coenzyme nicotine adenine dinucleotide (NAD+), elevated total intracellular ATP levels, and elevated extracellular lactate levels, along with upregulation of myelin-synthesis related genes, collectively resulting in functional myelin generation. Based on these preclinical studies, clean-surfaced, faceted gold nanocrystals represent a novel remyelinating therapeutic for multiple sclerosis.

Citrate and albumin facilitate transferrin iron loading in the presence of phosphate.

Labile plasma iron (LPI) is redox active, exchangeable iron that catalyzes the formation of reactive oxygen species. Serum transferrin binds iron in a non-exchangeable form and delivers iron to cells. In several inflammatory diseases serum LPI increases but the reason LPI forms is unknown. This work evaluates possible pathways leading to LPI and examines potential mediators of apo transferrin iron loading to prevent LPI. Previously phosphate was shown to inhibit iron loading into apo transferrin by competitively binding free Fe3+. The reaction of Fe3+ with phosphate produced a soluble ferric phosphate complex. In this study we evaluate iron loading into transferrin under physiologically relevant phosphate conditions to evaluate the roles of citrate and albumin in mediating iron delivery into apo transferrin. We report that preformed Fe3+-citrate was loaded into apo transferrin and was not inhibited by phosphate. A competition study evaluated reactions when Fe3+ was added to a solution with citrate, phosphate and apo transferrin. The results showed citrate marginally improved the delivery of Fe3+ to apo transferrin. Studies adding Fe3+ to a solution with phosphate, albumin and apo transferrin showed that albumin improved Fe3+ loading into apo transferrin. The most efficient Fe3+ loading into apo transferrin in a phosphate solution occurred when both citrate and albumin were present at physiological concentrations. Citrate and albumin overcame phosphate inhibition and loaded apo transferrin equal to the control of Fe3+ added to apo transferrin. Our results suggest a physiologically important role for albumin and citrate for apo transferrin iron loading.

Rate of iron transfer through the horse spleen ferritin shell determined by the rate of formation of Prussian Blue and Fe-desferrioxamine within the ferritin cavity.

Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN)6]3-, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible. We report the rate of Fe2+ penetrating into the ferritin interior by adding external Fe2+ to [Fe(CN)6]3- encapsulated in the HoSF interior and measuring the rate of formation of the resulting encapsulated PB. The rate at which Fe2+ reacts with [Fe(CN)6]3- in the HoSF interior is much slower than the formation of free PB in solution and is proceeded by a lag period. We assume this lag period and the difference in rate represent the transfer of Fe2+ through the HoSF protein shell. The calculated diffusion coefficient, D approximately 5.8x10(-20) m2/s corresponds to the measured lag time of 10-20 s before PB forms within the HoSF interior. The activation energy for Fe2+ transfer from the outside solution through the protein shell was determined to be 52.9 kJ/mol by conducting the reactions at 10 approximately 40 degrees C. The reaction of Fe3+ with encapsulated [Fe(CN)6]4- also readily forms PB in the HoSF interior, but the rate is faster than the corresponding Fe2+ reaction. The rate for Fe3+ transfer through the ferritin shell was confirmed by measuring the rate of the formation of Fe-DES inside HoSF and an activation energy of 58.4 kJ/mol was determined. An attempt was made to determine the rate of iron (2+ and 3+) transit out from the ferritin interior by adding excess bipyridine or DES to PB trapped within the HoSF interior. However, the reactions are slow and occur at almost identical rates for free and HoSF-encapsulated PB, indicating that the transfer of iron from the interior through the protein shell is faster than the rate-limiting step of PB dissociation. The method described in this work presents a novel way of determining the rate of transfer of iron and possibly other small molecules through the ferritin shell.

Iron loading into ferritin can be stimulated or inhibited by the presence of cations and anions: a specific role for phosphate.

Phosphate and other oxo-anions have been shown to stimulate the rate of iron loading into ferritin (J. Polanams, A.D. Ray, R.K. Watt, Inorg. Chem. 44 (2005) 3203-3209). This study was undertaken to determine if accelerated iron loading was a specific effect for phosphate and closely associated oxo-anions or if it was a general anion effect. Controls were also performed with mono-valent cations to determine the effect of these cations on iron loading into ferritin. Cations were shown to slow the rate of iron loading into ferritin. Fluoride and iodide were shown to slow the iron loading process of ferritin. Sulfate was also shown to slow iron loading into ferritin to a more significant extent than the cations or halides tested. The trigonal planar oxo-anions, carbonate and nitrate, did not inhibit or stimulate iron loading. We conclude that the increased rate of iron loading into ferritin is specific to phosphate and other closely associated tetrahedral oxo-anion analogs, that the effect is driven by the insolubility of the iron and anion complex, and that in general, cations and anions slow the rate of iron loading into ferritin.

Synthesis of peptide-nanotube platinum-nanoparticle composites.

Nanotubes prepared by the self-assembly of D-Phe-D-Phe molecules are investigated by electron microscopy and Monte Carlo simulations; the nanotubes appear to be porous and are capable of forming novel peptide-nanotube platinum-nanoparticle composites.

The C-terminal regions have an important role in the activity of the ferroxidase center and the stability of Chlorobium tepidum ferritin.

The recombinant Chlorobium tepidum ferritin (rCtFtn) is able to oxidize iron using ferroxidase activity but its ferroxidase activity is intermediate between the H-chain human ferritin and the L-chain human ferritin. The rCtFtn has an unusual C-terminal region composed of 12 histidine residues, as well as aspartate and glutamate residues. These residues act as potential metal ion ligands, and the rCtFtn homology model predicts that this region projects inside the protein cage. The rCtFtn also lacks a conserved Tyr residue in position 19. In order to know if those differences are responsible for the altered ferroxidase properties of rCtFtn, we introduced by site-directed mutagenesis a stop codon at position 166 and a Tyr residue replaced Ala19 in the gene of rCtFtn (rCtFtn 166). The rCtFtn166 keeps the canonical sequence considered important for the activity of this family of proteins. Therefore, we expected that rCtFtn 166 would possess similar properties to those described for this protein family. The rCtFtn 166 is able to bind, oxidize and store iron; and its activity is inhibit by Zn(II) as was described for other ferritins. However, the rCtFtn 166 possesses a decrease ferroxidase activity and protein stability compared with the wild type rCtFtn. The analysis of the Ala19Tyr rCtFtn shows that this change does not affect the kinetic of iron oxidation. Therefore, these results indicate that the C-terminal regions have an important role in the activity of the ferroxidase center and the stability of rCtFtn.

The crystal structure of ferritin from Chlorobium tepidum reveals a new conformation of the 4-fold channel for this protein family.

Ferritins are ubiquitous iron-storage proteins found in all kingdoms of life. They share a common architecture made of 24 subunits of five α-helices. The recombinant Chlorobium tepidum ferritin (rCtFtn) is a structurally interesting protein since sequence alignments with other ferritins show that this protein has a significantly extended C-terminus, which possesses 12 histidine residues as well as several aspartate and glutamic acid residues that are potential metal ion binding residues. We show that the macromolecular assembly of rCtFtn exhibits a cage-like hollow shell consisting of 24 monomers that are related by 4-3-2 symmetry; similar to the assembly of other ferritins. In all ferritins of known structure the short fifth α-helix adopts an acute angle with respect to the four-helix bundle. However, the crystal structure of the rCtFtn presented here shows that this helix adopts a new conformation defining a new assembly of the 4-fold channel of rCtFtn. This conformation allows the arrangement of the C-terminal region into the inner cavity of the protein shell. Furthermore, two Fe(III) ions were found in each ferroxidase center of rCtFtn, with an average FeA-FeB distance of 3 Å; corresponding to a diferric µ-oxo/hydroxo species. This is the first ferritin crystal structure with an isolated di-iron center in an iron-storage ferritin. The crystal structure of rCtFtn and the biochemical results presented here, suggests that rCtFtn presents similar biochemical properties reported for other members of this protein family albeit with distinct structural plasticity.

Anion deposition into ferritin.

When the iron core of equine spleen ferritin is reduced, anions in solution cross the protein shell and enter the ferritin interior as part of a charge balancing reaction. Anion sequestration inside ferritin during iron core reduction was monitored using ion selective electrodes, inductively coupled plasma emission, and energy-dispersive X-ray spectroscopy. The requirement for anion translocation to the ferritin interior occurs because upon iron core reduction, two OH(-) ions per iron are released or neutralized inside ferritin leaving a net positive charge. Halides and oxoanions were tested as anionic substrates for this reaction. A general trend for the halides showed that the smaller halides accumulated inside ferritin in greater abundance than larger halides, presumably because the protein channels restrict the transfer of the larger anionic species. In contrast, oxoanion accumulation inside ferritin did not show selectivity based on size or charge. Vanadate and molybdate accumulated to the highest concentrations and nitrate, phosphate and tungstate showed poor accumulation inside ferritin. Fe(II) remains stably sequestered inside ferritin, as shown by electron microscopy and by column chromatography. Upon oxidation of the iron core, the anions are expelled from ferritin, and OH(-) ions coordinate to the Fe(III) to form the original Fe(O)OH mineral. Anion transport across the ferritin protein shell represents an important mechanism by which ferritin maintains proper charge balance inside the protein cavity.

Phosphate inhibits in vitro Fe3+ loading into transferrin by forming a soluble Fe(III)-phosphate complex: a potential non-transferrin bound iron species.

In chronic kidney diseases, NTBI can occur even when total iron levels in serum are low and transferrin is not saturated. We postulated that elevated serum phosphate concentrations, present in CKD patients, might disrupt Fe(3+) loading into apo-transferrin by forming Fe(III)-phosphate species. We report that phosphate competes with apo-transferrin for Fe(3+) by forming a soluble Fe(III)-phosphate complex. Once formed, the Fe(III)-phosphate complex is not a substrate for donating Fe(3+) to apo-transferrin. Phosphate (1-10mM) does not chelate Fe(III) from diferric transferrin under the conditions examined. Complexed forms of Fe(3+), such as iron nitrilotriacetic acid (Fe(3+)-NTA), and Fe(III)-citrate are not susceptible to this phosphate complexation reaction and efficiently deliver Fe(3+) to apo-transferrin in the presence of phosphate. This reaction suggests that citrate might play an important role in protecting against Fe(III), phosphate interactions in vivo. In contrast to the reactions of Fe(3+) and phosphate, the addition of Fe(2+) to a solution of apo-transferrin and phosphate lead to rapid oxidation and deposition of Fe(3+) into apo-transferrin. These in vitro data suggest that, in principle, elevated phosphate concentrations can influence the ability of apo-transferrin to bind iron, depending on the oxidation state of the iron.

A new approach to the ferritin iron core growth: influence of the H/L ratio on the core shape.

An electron microscopy study, in combination with modeling and image simulation, of four different reconstituted ferritin samples: recombinant human H and L homopolymers, and H and L heteropolymers of native L-subunit-rich horse spleen and H-subunit-rich human heart ferritins, points out the existence of a correlation between iron core shape and protein shell.