1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 Review Article The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: Insights into the potential of various iron therapies to induce oxidative and nitrosative stress Taija S. Koskenkorva-Frank a , Günter Weiss b , Willem H. Koppenol c , Susanna Burckhardt a,c,n Q1 a Chemical and Preclinical Research and Development, Vifor (International) Ltd., CH-9001 St. Gallen, Switzerland b Department of Internal Medicine VI, Infectious Diseases, Immunology, Rheumatology, Pneumology, Medical University of Innsbruck, Innsbruck, Austria c Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Zürich, Switzerland article info Article history: Received 17 July 2013 Received in revised form 5 September 2013 Accepted 5 September 2013 Keywords: Intravenous iron Nitrosative stress Oral iron Oxidative stress Reactive nitrogen species Reactive oxygen species Free radicals abstract Production of minute concentrations of superoxide (O 2 d) and nitrogen monoxide (nitric oxide, NO d ) plays important roles in several aspects of cellular signaling and metabolic regulation. However, in an inflammatory environment, the concentrations of these radicals can drastically increase and the antioxidant defenses may become overwhelmed. Thus, biological damage may occur owing to redox imbalance—a condition called oxidative and/or nitrosative stress. A complex interplay exists between iron metabolism, O 2 d, hydrogen peroxide (H 2 O 2 ), and NO d . Iron is involved in both the formation and the scavenging of these species. Iron deficiency (anemia) (ID(A)) is associated with oxidative stress, but its role in the induction of nitrosative stress is largely unclear. Moreover, oral as well as intravenous (iv) iron preparations used for the treatment of ID(A) may also induce oxidative and/or nitrosative stress. Oral administration of ferrous salts may lead to high transferrin saturation levels and, thus, formation of non- transferrin-bound iron, a potentially toxic form of iron with a propensity to induce oxidative stress. One of the factors that determine the likelihood of oxidative and nitrosative stress induced upon administration of an iv iron complex is the amount of labile (or weakly bound) iron present in the complex. Stable dextran-based iron complexes used for iv therapy, although they contain only negligible amounts of labile iron, can induce oxidative and/or nitrosative stress through so far unknown mechanisms. In this review, after summarizing the main features of iron metabolism and its complex interplay with O 2 d,H 2 O 2 , NO d , and other more reactive compounds derived from these species, the potential of various iron therapies to induce oxidative and nitrosative stress is discussed and possible underlying mechanisms are proposed. Understanding the mechanisms by which various iron formula- tions may induce oxidative and nitrosative stress will help us develop better tolerated and more efficient therapies for various dysfunctions of iron metabolism. & 2013 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.09.001 Abbreviations: ACD, anemia of chronic disease; AID, absolute iron deficiency; ARE, antioxidant-responsive elements; asc, ascorbic acid; DMT1, divalent metal transporter 1; eNOS, endothelial nitric oxide synthase; EPO, erythropoietin; FBXL5, F-box and leucine-rich repeat protein 5; FCM, ferric carboxymaltose; Fe-EDTA, sodium Fe(III) ethylenediaminetetraacetic acid; FG, ferric gluconate; FID, functional iron deficiency; FMX, ferumoxytol; FPN, ferroportin; GI, gastrointestinal; GPx, glutathione peroxidase; GSH, glutathione; Hb, hemoglobin; HIF, hypoxia-inducible factor; HO-1, heme oxygenase 1; ID(A), iron deficiency (anemia); IIM, iron isomaltoside 1000; IL, interleukin; iNOS, inducible nitric oxide synthase; IPC, iron polymaltose complex; IPCS, iron polymaltose complex similar; IRE, iron-regulatory element; IRP, iron-regulatory protein; IS, iron sucrose; ISS, iron sucrose similar; iv, intravenous; LIP, labile iron pool; LMWID, low-molecular-weight iron dextran; MDA, malondialdehyde, MPS, mononuclear phagocyte system, NF-κB, nuclear factor-κB; nNOS, neuronal nitric oxide synthase; Nramp1, natural resistance-associated macrophage protein 1; Nrf2, NF-E2-related factor 2; NTBI, non-transferrin-bound iron; PHD, prolyl hydroxylase; PSC, polyglucose sorbitol carboxymethyl ether; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; Steap3, six-transmembrane epithelial antigen of the prostate 3; TfR, transferrin receptor; TNF-α, tumor necrosis factor α; TRPML1, transient receptor potential cation channel, mucolipin subfamily, member 1; TSAT, transferrrin saturation; UTR, untranslated region; VHL, von Hippel–Lindau n Corresponding author: Fax: þ41 58 851 85 85. E-mail address: [email protected] (S. Burckhardt). i Please cite this article as: Koskenkorva-Frank, TS; et al. The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: Insights into the potential of various iron... stresshttp://dx.doi.org/10.1016/j.freeradbiomed.2013.09.001 Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎
The complex interplay of iron metabolism, reactive oxygen species, andreactive nitrogen species: Insights into the potential of various irontherapies to induce oxidative and nitrosative stress
Taija S. Koskenkorva-Frank a, Günter Weiss b, Willem H. Koppenol c,Susanna Burckhardt a,c,nQ1
a Chemical and Preclinical Research and Development, Vifor (International) Ltd., CH-9001 St. Gallen, Switzerlandb Department of Internal Medicine VI, Infectious Diseases, Immunology, Rheumatology, Pneumology, Medical University of Innsbruck, Innsbruck, Austriac Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Zürich, Switzerland
a r t i c l e i n f o
Article history:Received 17 July 2013Received in revised form5 September 2013Accepted 5 September 2013
Production of minute concentrations of superoxide (O2d�) and nitrogen monoxide (nitric oxide, NOd)
plays important roles in several aspects of cellular signaling and metabolic regulation. However, in aninflammatory environment, the concentrations of these radicals can drastically increase and theantioxidant defenses may become overwhelmed. Thus, biological damage may occur owing to redoximbalance—a condition called oxidative and/or nitrosative stress. A complex interplay exists betweeniron metabolism, O2
d� , hydrogen peroxide (H2O2), and NOd. Iron is involved in both the formation andthe scavenging of these species. Iron deficiency (anemia) (ID(A)) is associated with oxidative stress, butits role in the induction of nitrosative stress is largely unclear. Moreover, oral as well as intravenous (iv)iron preparations used for the treatment of ID(A) may also induce oxidative and/or nitrosative stress. Oraladministration of ferrous salts may lead to high transferrin saturation levels and, thus, formation of non-transferrin-bound iron, a potentially toxic form of iron with a propensity to induce oxidative stress.One of the factors that determine the likelihood of oxidative and nitrosative stress induced uponadministration of an iv iron complex is the amount of labile (or weakly bound) iron present in thecomplex. Stable dextran-based iron complexes used for iv therapy, although they contain only negligibleamounts of labile iron, can induce oxidative and/or nitrosative stress through so far unknownmechanisms. In this review, after summarizing the main features of iron metabolism and its complexinterplay with O2
d� , H2O2, NOd, and other more reactive compounds derived from these species, thepotential of various iron therapies to induce oxidative and nitrosative stress is discussed and possibleunderlying mechanisms are proposed. Understanding the mechanisms by which various iron formula-tions may induce oxidative and nitrosative stress will help us develop better tolerated and more efficienttherapies for various dysfunctions of iron metabolism.
0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.freeradbiomed.2013.09.001
Abbreviations: ACD, anemia of chronic disease; AID, absolute iron deficiency; ARE, antioxidant-responsive elements; asc, ascorbic acid; DMT1, divalent metal transporter 1;eNOS, endothelial nitric oxide synthase; EPO, erythropoietin; FBXL5, F-box and leucine-rich repeat protein 5; FCM, ferric carboxymaltose; Fe-EDTA, sodium Fe(III)ethylenediaminetetraacetic acid; FG, ferric gluconate; FID, functional iron deficiency; FMX, ferumoxytol; FPN, ferroportin; GI, gastrointestinal; GPx, glutathione peroxidase;GSH, glutathione; Hb, hemoglobin; HIF, hypoxia-inducible factor; HO-1, heme oxygenase 1; ID(A), iron deficiency (anemia); IIM, iron isomaltoside 1000; IL, interleukin;iNOS, inducible nitric oxide synthase; IPC, iron polymaltose complex; IPCS, iron polymaltose complex similar; IRE, iron-regulatory element; IRP, iron-regulatory protein;IS, iron sucrose; ISS, iron sucrose similar; iv, intravenous; LIP, labile iron pool; LMWID, low-molecular-weight iron dextran; MDA, malondialdehyde, MPS, mononuclearphagocyte system, NF-κB, nuclear factor-κB; nNOS, neuronal nitric oxide synthase; Nramp1, natural resistance-associated macrophage protein 1; Nrf2, NF-E2-related factor2; NTBI, non-transferrin-bound iron; PHD, prolyl hydroxylase; PSC, polyglucose sorbitol carboxymethyl ether; RNS, reactive nitrogen species; ROS, reactive oxygen species;SOD, superoxide dismutase; Steap3, six-transmembrane epithelial antigen of the prostate 3; TfR, transferrin receptor; TNF-α, tumor necrosis factor α; TRPML1, transientreceptor potential cation channel, mucolipin subfamily, member 1; TSAT, transferrrin saturation; UTR, untranslated region; VHL, von Hippel–Lindau
n Corresponding author: Fax: þ41 58 851 85 85.E-mail address: [email protected] (S. Burckhardt).
iPlease cite this article as: Koskenkorva-Frank, TS; et al. The complex interplay of iron metabolism, reactive oxygen species, and reactivenitrogen species: Insights into the potential of various iron... stresshttp://dx.doi.org/10.1016/j.freeradbiomed.2013.09.001
Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎
peroxide (H2O2), peroxynitrite (ONOO�), and oxidochlorate (1�)(OCl� , hypochlorite) [2]. The concentrations of these species arekept within a narrow range by balancing the rate of productionwith the rate of removal by enzymatic antioxidants, such assuperoxide dismutase (SOD), glutathione peroxidase (GPx), andcatalase, or nonenzymatic antioxidants, such as ascorbic acid (asc;vitamin C), α-tocopherol (vitamin E), glutathione (GSH), urate,carotenoids, cysteine, bilirubin, and flavonoids.
O2d� and NOd are produced by enzymes at low concentration
during normal cellular metabolism (Fig. 1) and, together withH2O2, which is produced by dismutation of O2
d� , these speciesplay an important role in cellular signaling and regulation [3].Interestingly, it has been proposed that O2
d� and H2O2 producedwithin cells and, in particular, within mitochondria can stimulatebeneficial responses to the cellular stresses induced by aging, suchas mitochondrial dysfunction and DNA damage [4]. Among others,O2
d� and H2O2 have been shown to induce autophagy as well asDNA base excision repair systems [5]. However, under inflamma-tory conditions an oxidative burst from activated neutrophils andmacrophages may lead to drastically elevated concentrations ofNOd and O2
d� [6,7] resulting in the formation of other morereactive oxidants such as ONOO� . Under these conditions, anti-oxidant defenses may become overwhelmed and biologicaldamage of lipids, proteins, and DNA may occur, a condition termedoxidative or nitrosative stress. Oxidative and/or nitrosative stresscan cause disruptions in normal cellular signaling mechanismsthereby severely compromising cell viability [8].
The production of O2d� and NOd can exceed the capacity of
detoxifying systems, resulting in oxidative stress [3] even undernormal conditions. In humans, oxidative and/or nitrosative stresshas been shown to be involved in many diseases and medicalconditions, such as cancer, diabetes, Parkinson disease, Alzheimerdisease, atherosclerosis, congestive heart failure, myocardialinfarction, or schizophrenia [3,8]. Interestingly, biological agingalso correlates with the accumulation of oxidized biomolecules inmany tissues [3,8].
Most of the iron in the body is bound in hemoglobin andmyoglobin, in which the metal is responsible for the reversiblebinding and release of oxygen. One of the most important proper-ties of iron, both for its essential role and for its toxicity in livingorganisms, is the ability to redox cycle under physiological condi-tions. Iron is also the essential component in a large number ofenzymes, which take advantage of its redox-cycling property tocarry out electron transfer, to activate dioxygen to hydroxylateand to oxidize substrates, and to catalyze a variety of reactions,such as DNA [9], NOd, and thyroid hormone (T3, T4) synthesis [10].Moreover, iron has been linked to the regulation of effectiveimmune responses against infection with various pathogens [11].However, because of its ability to redox cycle, iron may alsopromote formation of HOd or a reactive higher oxidation state ofiron (see Iron-induced generation of hydroxyl radicals), e.g., via theFenton reaction [2]. Therefore, to avoid metal-induced toxicity ironmust always be bound in a non-redox-active form, e.g., as found intransferrin and ferritin [9,12].
The interplay of iron with H2O2 and O2d� is complex, because
these species play important roles in regulating iron metabolism[13,14]. Similarly, a complex interaction exists between ironmetabolism, NOd, and, to a minor extent, ONOO� [15,16]. NOd
has a high affinity for Fe(II) and thus reacts with iron-containingproteins, affecting their activity [17]. NOd, ONOO� , and possiblyNO2
d, are also directly involved in the regulation of cellular ironhomeostasis by modulating the binding affinity of iron-regulatoryproteins (IRPs) to iron-responsive elements (IREs) [18–23]. Inaddition, cellular iron content influences macrophage activationand effector functions by inhibiting the activity of stimulatory
1 Ferrous sulfate and ferrous fumarate are used throughout this paper as theseare the common names of the two oral iron drugs. However, the terms “ferrous”and “ferric” to indicate Fe(II) and Fe(III), respectively, are outdated and do notconform to the current IUPAC recommendations [1].
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cytokine signals, such as interferon-γ (IFN-γ) [24–26]; by directlyinfluencing the expression of inducible nitric oxide synthase [27–29];and by promoting the expression of the anti-inflammatory cytokineinterleukin-10 (IL-10) [30] (see Oxidative and nitrosative stress iniron deficiency (anemia), Oral iron therapy and nitrosative stress,Intravenous iron therapy and nitrosative stress, and Alternativemechanisms for iv iron-induced oxidative and nitrosative stress).
It is clear from the above description that too much or too littleiron can be detrimental to the organism. Despite the tightlyregulated homeostatic mechanisms in the body to recycle ironefficiently and prevent its loss, iron deficiency (ID) is the mostcommon nutritional deficiency in the world [31]. When iron intakefails to meet physiological or increased demands for iron, e.g.,owing to bleeding or inadequate absorption, iron stores becomedepleted and ID develops. Subsequently, if this disequilibriumpersists, iron deficiency (anemia) (ID(A)) emerges because of aninsufficient supply of iron for hemoglobin synthesis during ery-thropoiesis [32].
Various types of ID(A) exist in clinical conditions. Commonclassification differentiates between absolute iron deficiency (AID),functional iron deficiency (FID), and iron sequestration [33]. AID is adeficit in total body iron. FID emerges when the mobilization of ironis not rapid enough to meet the increased demand as observedduring intense stimulation of erythropoiesis by endogenous erythro-poietin (EPO) or upon treatment with erythropoiesis-stimulatingagents [33]. Inflammation-driven iron sequestration and, subse-quently, anemia of chronic disease (ACD) are most often found inpatients with chronic inflammatory diseases, such as cancer, chronicheart failure, chronic infections, autoimmune disorders, or chronickidney disease (CKD), and result from iron retention in cells of themononuclear phagocyte system (MPS)2 [33,34]. All stages of ID(A) are associated with variable degrees of oxidative stress [35–37]and several studies have shown that nitrosative stress is also linkedto ID(A) [29,38–41].
The most appropriate modality for the management of ID and ID(A) depends on the underlying etiology. For example, oral irontherapy is often adequate to treat AID and mild to moderate casesof ID(A). In cases of severe ID(A), ACD, and FID, iv iron therapy isoften more effective [42]. A number of oral and iv iron preparationsare available. Oral iron preparations include iron(II) salts, polysac-charide iron(III) complexes, and combinations of iron, e.g., with folic
acid or vitamin C. These compounds (or combinations) differ in theirintestinal absorption mechanisms, efficacies, and side effect profiles.All iv iron preparations, on the other hand, are carbohydrate-stabilized polynuclear iron(III) (oxy)hydroxide complexes with var-ious sizes, stabilities, and types of carbohydrate. All these propertiesaffect the maximal single dose and rate of administration as well asthe safety profile [43]. In addition, depending on the type ofpreparation used, both oral [44–49] and intravenous [50–66] irontherapies can induce oxidative and/or nitrosative stress.
In this review we briefly summarize the current understandingof the mechanisms leading to oxidative and nitrosative stress aswell as the main features of iron metabolism and its complexinterplay with O2
d� , H2O2, and NOd. Moreover, we discuss thepotential of various iron therapies to induce oxidative and nitro-sative stress and describe possible underlying mechanisms. Under-standing the possible pathways by which different ironformulations may induce formation of these species is essentialfor developing safer and more efficient therapies for differentdysfunctions of iron metabolism.
Role of ROS and RNS in physiological and pathological cellularfunctions
An increasing amount of evidence describing the roles of ROSand RNS in various physiological and pathological conditions hasbeen published. In most cases, the abbreviation ROS refers to O2
d� ,H2O2, and HOd. RNS typically refers to NOd, ONOO� , and, lessfrequently, NO2
d. The use of these abbreviations is widespreadand, unfortunately, not always appropriate. The physicochemicalproperties and the reactivities of O2
d� , H2O2, and HOd, i.e., the so-called ROS, are very different. In brief, O2
d� reacts rapidly onlywith itself, with NOd, and with SOD. It does not react with aminoacids at a significant rate [67]. Most of the time, O2
d� acts as amild reductant, for instance, of Fe(III)–cytochrome c [68]. H2O2 isstable for many months, although it should thermodynamicallyproduce O2 and water. Only its reactions with metal centers andwith deprotonated cysteine residues are relevant. In contrast,HOd reacts at (near) diffusion-controlled rates with all biomole-cules [69]. Similar arguments can be made for the abbreviationRNS. Because of the distinct reactivity, the species responsible for agiven reaction can usually be identified and named explicitly,instead of talking generally about ROS and/or RNS. Following thisapproach, ROS and RNS are used only where appropriate in thisreview.
Fig. 1. Pathways leading to the formation of physiologically and pathologically relevant oxidants. Complex I, NADH dehydrogenase; Complex III, ubiquinol–cytochrome creductase; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase; NOX, nicotine adenine dinucleotide phosphate oxidase; SOD, superoxide dismutase; XO, xanthineoxidase.
2 he older term for MPS is reticuloendothelial system, which is used lesscommonly now since it has been recognized that, in contrast to former thinking,most endothelial cells are not macrophages.
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Various pathways lead to the formation of O2d� , H2O2, and NOd
in vivo (Fig. 1). These species are produced by enzymes, whereasmore reactive species are formed from these. Addition of oneelectron to O2 leads to the formation of O2
d� . The production ofO2
d� occurs mostly by Complexes I and III of the mitochondrialelectron transport chain. Under normal conditions, 1–3% of theelectrons in the electron transport chain leak out, generating O2
d�
[8]. In addition, O2d� is also produced by xanthine oxidase [70],
in activated neutrophils by nicotine adenine dinucleotide phos-phate (NADPH) oxidase [7], and by cytochrome P450 [71]. None-nzymatic formation of O2
d� is mediated by redox-reactivecompounds, such as the semiubiquinone of the mitochondrialelectron transport chain [8]. O2
d� is not very reactive, and the onlybiologically relevant reaction that may lead to damage of cellularcomponents is its diffusion-controlled reaction with NOd toproduce ONOO� [72] (see below).
H2O2 and O2 are formed by the SOD-catalyzed dismutation ofO2
d� (Fig. 1). In addition, large amounts of H2O2 are producedunder physiologic conditions in the peroxisomes [8]. Direct reac-tions of H2O2 with most organic compounds are slow unless theyare catalyzed by metal-containing complexes or proteins [2].In neutrophils, H2O2 and chloride (Cl�) are used by myeloperox-idase to produce hypochlorous acid (HOCl) [73] (Fig. 1). H2O2 isscavenged most efficiently by catalase, a ubiquitous enzyme thatcatalyzes its disproportionation to H2O and O2. In addition, GPxreduces H2O2 to water by using GSH as the electron donor.Oxidized glutathione is reduced back to GSH by glutathionereductase with NADPH as the electron donor. Finally, H2O2 isscavenged by peroxiredoxin in peroxisomes [74].
HOd is formed when Fe(II) reacts with H2O2 (Fenton reaction;Fig. 1) [2,75]. HOd is a very strong oxidizing agent reacting rapidlywith all molecules found in living cells [76]. Because of its fastreactions [67], it has a very short in vivo half-life and, thus, reactsvery close to its site of formation [8]. Among others, it can react withall components of DNA, damaging the purine and the pyrimidinebases as well as the deoxyribose backbone [77], thereby permanentlymodifying the genetic material, which is considered the first step inmutagenesis, carcinogenesis, and aging [8].
NOd is generated in tissues from L-arginine by three nitric oxidesynthase (NOS) isoforms, neuronal NOS (nNOS), endothelial NOS(eNOS), and inducible NOS (iNOS), which are cytochrome P450-like enzymes [78]. The reaction requires iron, O2, and reducingequivalents, such as flavins or tetrahydrobiopterin [79,80]. Underphysiological conditions, NOd is produced in nanomolar amountsby eNOS and nNOS for regulatory functions. These two isoformsare constitutively expressed in resting cells. Upon binding ofcalcium calmodulin they are activated to synthesize NOd, whichbinds to the heme of soluble guanylate cyclase to trigger theformation of cyclic guanosine monophosphate, a messenger mod-ulating an array of mediators, including various ion channels,phosphodiesterases, and protein kinases [81–83].
A large number of immune cells produce and respond to NOd.In monocytes and macrophages, iNOS expression is induced byinflammatory stimuli to produce micromolar amounts of NOd [84](Fig. 1). The regulation of iNOS expression occurs at the transcrip-tional and posttranscriptional levels by signaling pathways, inwhich, e.g., nuclear factor-κB (NF-κB) or mitogen-activated proteinkinases are involved [8].
NOd is a versatile molecule with a wide variety of roles inbiological processes [81,82,85]. Although NOd is often described asshort-lived and highly reactive, under most conditions it is not[72]. Moreover, the solubility of NOd in lipid media renders itreadily diffusible through membranes [82] with little consumptionor reaction, which makes it a good intracellular messenger[72]. Indeed, NOd is an important signaling molecule in diversephysiological processes, such as neurotransmission, regulation of
blood pressure, defense mechanisms, and smooth muscle relaxa-tion, as well as immune regulation [82]. It is also important fornonspecific host defense that helps to kill tumors and intracellularpathogens [84].
One of the characteristic features of NOd is its high-affinitybinding to heme and nonheme iron [82,86]. Moreover, oxidized orreduced forms of NOd can be produced under physiologicalconditions, which enable it to affect a variety of molecular targets[79,87]. The oxidized form of NOd, the nitrosonium ion (NOþ),is not stable in water. However, it may be generated in situ fromnitrite (NO2
�) under acidic conditions or bound to iron, and itreacts with sulfhydryl groups in proteins (S-nitrosation), leading toS-nitrosothiol formation [88]. The reduced form, nitroxyl (HNO),reacts with heme iron and thiols [88,89]. Depending on theirconcentration and the environment, both NOd and HNO can act aspro-oxidants as well as antioxidants.
Many of the toxic effects originally attributed to NOd are in factmediated by ONOO� , formed by the diffusion-controlled reactionof NOd with O2
d� [90,91] (Fig. 1). This important biological oxidantand nitrating agent is much more reactive than NOd and can causeDNA fragmentation, lipid peroxidation, and hydroxylation andnitration of tyrosine [2,92]. The rate constant of the reaction ofO2
d� with NOd is higher (k¼(1.670.3)�1010 M�1 s�1) [90] thanthat of its reaction with Cu,Zn-SOD (k¼(2�109 M�1 s�1) [93].However, under normal conditions, the NOd concentration is in thenanomolar range and NOd cannot compete with Cu,Zn-SOD for O2
d� .In contrast, under conditions that activate iNOS expression and thus,generation of NOd in micromolar concentrations, NOd can outcom-pete Cu,Zn-SOD and lead to the formation of ONOO� [72].
The reactions of ONOO� with biological targets are muchslower and more selective than those of HOd. At physiologicalpH, ONOO� (pKa¼6.8) is largely protonated and the main reactionof HOONO is its isomerization to nitrate (NO3
�) [94]. The debate ofwhether HOONO undergoes homolysis yielding NO2
d and HOd hasrecently been reviewed and it was concluded that homolysis maybe limited to an extent of ca. 5% [94]. In vivo, ONOO� reacts mainlywith carbon dioxide (CO2) to form mostly NO3
� and CO2 but alsoNO2
d and CO3d� [95] (Fig. 1), which are likely to contribute to
ONOO�-mediated oxidation of nucleic acids [96] and nitration oftyrosine [97] as well as tryptophan residues [98,99]. Based on anestimated concentration of ca. 5 mM and a rate constant of5.8�102 M�1 s�1, GSH is, after CO2, the second most importantsink for ONOO� in a cell [100].
Iron metabolism
Before reviewing the complex interplay between iron and oxida-tive/nitrosative stress we briefly summarize the major pathwaysinvolved in iron metabolism (see [9,101–105] for extensive review).Understanding these metabolic processes is vital for the appreciationof the role of iron in the generation of oxidative and nitrosative stressunder physiological conditions.
Because there is no active iron excretion process, iron home-ostasis is regulated by intestinal iron absorption [101] and bymacrophage iron recycling. The largest pathway of cellular ironefflux is iron recycling from senescent erythrocytes by macro-phages, which supplies 90–95% of all the iron needed in physio-logical processes and erythropoiesis [106,107]. In contrast,intestinal iron absorption compensates only for basal losses ofiron that result from, e.g., exfoliated mucosal cells, bile, andextravasated red blood cells [108].
In the gut, iron is taken up in a regulated way by duodenalenterocytes as Fe(II) via the divalent metal transporter 1 (DMT1)or as heme, possibly via the heme carrier protein 1 [101–105,109–111]. Depending on the body's requirements, Fe(II) is then
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exported from enterocytes by ferroportin (FPN) [112–114] andrapidly oxidized by hephaestin [115] or ceruloplasmin to Fe(III),which is tightly bound by transferrin. Transferrin has two high-affinity binding sites, each able to bind one Fe(III) ion. Underphysiological conditions, only about one-third of the iron-bindingcapacity of transferrin is saturated. Iron that is not exported by FPNis stored within the enterocyte in the form of ferritin [101–105]and subsequently lost when the enterocyte is exfoliated at thevillus tip [116].
Macrophages of the MPS in the liver, spleen, and bone marrowrecycle iron by phagocytosing senescent erythrocytes. In the pha-golysosomes, erythrocytes are degraded by hydrolytic enzymes andheme is obtained by proteolytic digestion of hemoglobin [108].Heme is then most probably transported to the cytosol via heme-responsive gene 1 and oxidized by heme oxygenase 1 (HO-1) toobtain Fe(II), which is transported from the macrophages to theplasma via FPN [106,117]. Alternatively, heme is transported directlyto the plasma via the feline leukemia virus subgroup C cellularreceptor [118,119] and bound by hemopexin [120]. As in theenterocytes, any iron that is not utilized for the cellular processesor exported via FPN can be retained within the macrophages asferritin [106].
Cellular uptake of iron proceeds via the transferrin receptor(TfR) pathway in endocytotic vesicles, where iron resides beforebeing released into the cytosol through DMT1 and/or transientreceptor potential cation channel, mucolipin subfamily, member 1(TRPML1) [110,111,121]. Before being exported to the cytosol Fe(III)has to be reduced to Fe(II) by the endosomal ferrireductase six-transmembrane epithelial antigen of the prostate 3 (Steap3) [122].
Within the cells iron is in a dynamic equilibrium mainly betweenfour compartments: vesicular iron, labile iron pool (LIP), functionaliron, and storage iron [123]. Lysosomal/endosomal iron representsthe largest fraction of vesicular and redox-active iron [124]. Withinthe lysosomal compartment, iron is constantly recycled from old ordamaged cell organelles and various macromolecules for the con-struction of new proteins during lysosomal autophagy. This results ina low-molecular-weight iron pool within the lysosomes, mostprobably redox-active Fe(II), which has the potential to generateHOd if exposed to hydrogen peroxide and consequently to lead tolysosomal membrane permeabilization [12].
The LIP, also known as the transit iron pool or cellularchelatable iron, plays an important role in the intracellular trans-port of iron between the vesicular, storage, and functional ironcompartments [123,125]. Iron in the LIP is redox-active Fe(II),which is loosely bound to proteins or low-molecular-weightligands, such as citrate [126]. In particular, Fe(II)–glutathione hasbeen recently suggested to be the dominant component of the LIP[126]. As discussed above, functional iron comprises various iron-containing proteins, enzymes, and prosthetic groups and, thus,participates in a variety of reactions in the cell [123].
Within the cell, iron is mainly stored within ferritin, anoligomeric protein forming a hollow shell, which can bind up to4500 Fe(III) ions in a nonredox, and thus nontoxic, soluble andbioavailable form [9]. Consequently, ferritin protects cells againstiron-mediated oxidative stress. In particular, ferritins rich in theH-subunit have been shown to act as important antioxidant agents[127]. Ferritin is continuously degraded via lysosomal autophagy,which makes iron constantly available. Some storage iron may alsobe found within lysosomes in the form of hemosiderin, whichresults from partial degradation of ferritin [128,129].
Interestingly, recent studies in budding yeast and mammaliancells have revealed a cytosolic iron-delivery system involving ironchaperones, such as Grx3-type monothiol glutaredoxins, poly(rC)-binding proteins, and the mitochondrial protein frataxin [130].These chaperones specifically bind iron and deliver it to targetapoproteins through direct protein–protein interactions. Although
more research is needed, it has been suggested that these chap-erones may represent the basic cellular machinery for intracellulariron delivery [130].
Regulation of iron metabolism
Hepcidin
Hepcidin is a 25-amino-acid peptide produced mainly by theliver. It is currently considered the key regulator of systemic ironhomeostasis as it orchestrates intestinal iron absorption and ironrecycling by the macrophages of the MPS [104]. Hepcidin acts bybinding to FPN, triggering its internalization and subsequentlysosomal degradation [131], a process that leads to accumulationof iron in the enterocytes, macrophages, and hepatocytes[104,132]. Moreover, hepcidin has also been shown to inhibitapical iron uptake in intestinal cells [133], a result supported byrecent in vitro and ex vivo studies, which demonstrated that anacute increase in hepcidin concentration reduces iron absorptionvia ubiquitin-dependent proteasome degradation of DMT1 [134].
Hepcidin production is regulated by iron availability, inflam-matory cytokines, hypoxia, and erythropoietic demand [103,135].Hepcidin expression is triggered via various mechanisms by highconcentrations of diferric transferrin, reflecting high iron levels inthe serum, as well as by high liver iron content [136,137] or byelevated cytokine levels, such as IL-6, under inflammatory condi-tions, which deprives invading microorganisms of iron [104]. Theupregulation of hepcidin in response to chronic inflammatorystimuli, such as cancer, infection, and autoimmunity, contributesto the development of ACD [34,138–141], whereas its down-regulation or a lack of response to hepcidin levels is associatedwith iron-overload disorders, such as hemochromatosis [103,142].Of interest, recent evidence suggests that minute amounts of H2O2
can induce hepcidin expression via activation of signal transducerand activator of transcription 3 signaling [143].
Hypoxia-inducible factors (HIFs)
HIFs are considered to have an important role in coupling ironsensing to regulation of iron homeostasis [144]. They have beenshown to regulate the expression of hepcidin, DMT1, duodenalcytochrome b, FPN, transferrin, TfR, ceruloplasmin, HO-1 [144],and δ-aminolevulinate synthase 2 [145]. HIFs themselves areregulated posttranslationally by prolyl hydroxylases (PHDs), a classof enzymes that require iron, O2, 2-oxoglutarate, and ascorbic acidto catalyze the hydroxylation of proline residues. This allows HIFsto bind to the von Hippel–Lindau (VHL)-E3 ubiquitin ligasecomplex and to subsequently induce the proteasomal degradationof the complex [146]. Thus, it is conceivable that ID(A) not onlydirectly leads to reduced PHD activity because of the lack ofavailable iron, but also causes decreased tissue oxygenation thatfurther diminishes PHD activity and thus VHL-mediated degradationof HIF-α factors. In addition, HIF-1 transcriptional activity is con-trolled by an additional Fe(II)- and 2-oxoglutarate-dependent dioxy-genase termed factor inhibiting HIF (FIH) [147]. During normoxia,FIH hydroxylates Asn803 within the C-terminal transactivationdomain (C-TAD) of human HIF-1α, which results in steric inhibitionof coactivator (p300/CREB binding protein) binding to the C-TAD,thus suppressing HIF-1 transcriptional activity [148].
In vivo studies have suggested that HIF-1α is able to bind toand negatively transactivate the hepcidin promoter, suggesting adirect repressor effect [149]. However, deletion of HIF-1α in theliver accounted for only a fractional hepcidin repression inresponse to an iron-deficient diet [149], and other in vivo andin vitro studies showed that HIF-1 does not directly activate
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hepcidin gene (HAMP) expression [150,151]. Interestingly, Choiet al. [151] showed that H2O2 suppressed hepcidin expression,whereas antioxidants prevented the hypoxia-mediated downre-gulation of hepcidin, suggesting that oxidative stress can mediatethe hypoxic repression of HAMP. A recent study supports thehypothesis that hepcidin is not a direct target of HIF-1α or HIF-2αand is also not indirectly regulated by HIF-1α or HIF-2α throughinduction of TfR [152]. Instead, HIF-2 has been shown to regulateEPO and key iron-related genes in the liver [153] and ironabsorption in the enterocytes [154]. Also, a recent study showedthat HIF-2 overexpression in double Vhlh/Hif1α hepatocyte-specific knockout mice does not downregulate hepcidin expres-sion directly, but rather indirectly, by increasing erythropoiesisand EPO production [154,155].
Iron-regulatory proteins
Iron homeostasis at the cellular level is maintained via a numberof regulatory pathways by IRP1 and IRP2. Both IRPs are ubiquitouslyexpressed cytosolic proteins that act by binding to IREs identified inthe untranslated regions (UTRs) of mRNAs of numerous proteinsinvolved in iron uptake, utilization, storage, and export, such asDMT1, TfR, ferritin, and FPN [156–158]. In general, both IRP1 andIRP2 bind to the respective target mRNA under low cytosolic ironconcentrations, an ability that is lost when cytosolic iron levels arehigh. The type of regulation depends on the location of the IRE inthe target mRNA. Binding of either IRP1 or IRP2 to the IRE in the5′ UTR prevents translation, whereas binding to the IRE in the3′ UTR increases mRNA stability. The binding activity of IRPs is alsocontrolled by several factors other than iron, for instance, tissueoxygen level, oxidative stress, and NOd [13–19,101,159–161].
Although both IRPs bind to the target mRNA when cytosoliciron levels are low, they respond to changing iron levels withdifferent mechanisms. IRP1 is a bifunctional protein. When highcytosolic iron levels are present, IRP1 is in its [4Fe–4S]-cluster-containing holo-form, which has cytosolic aconitase activity butcannot bind to IREs. Under low iron conditions, the [4Fe–4S]cluster is absent, and IRP1 loses its aconitase activity and acquiresIRE-binding capacity [101,157–161]. In contrast, IRP2 lacks the[4Fe–4S] cluster and is largely regulated by iron-mediated degra-dation. When iron levels are high, F-box and leucine-rich repeatprotein 5 (FBXL5) binds to its target motifs on IRP2 and induces itsproteasomal degradation. Under conditions of low iron levels, FBXL5itself is targeted for ubiquitination and degraded, which stabilizesIRP2 and allows its binding to IREs [162,163]. FBXL5 is regulated byboth iron and oxygen. At its N-terminus FBLX5 has a hemerythrin-like domain, which binds iron and oxygen, influencing its stability[162,163]. It has also been suggested that FBXL5 may degrade apo-IRP1, if [4Fe–4S]-cluster assembly is defective [162,163].
Iron-induced generation of hydroxyl radicals
Fe(II) reacts with H2O2 to form the reactive HOd radical (Fentonreaction; Fig. 1). As indicated by in vitro studies, instead of HOd,near neutral pH oxido-iron(IV) (FeO2þ) [164] or Fe(III)OOH may beformed [2]. The biological relevance of the Fenton reaction, and inparticular, the question of in which cellular compartment thisreaction is likely to take place, is a matter of discussion [2,12,165].
When iron is tightly bound by appropriate ligands in its Fe(III)form, such as in transferrin and ferritin, the Fenton reaction, whichis an inner-sphere reaction and requires direct binding of H2O2 toFe(II), cannot take place. Moreover, to prevent redox cycling thestability of the Fe(III) complex must be such that the reduction isthermodynamically not feasible. The electrode potentials of thecouples H2O2, Hþ/HOd, H2O and ascd�/Hasc� are þ0.39 [76] and
þ0.28 V [166], respectively. Thus, only a metal complex with anelectrode potential between these values can catalyze the formationof HOd. If physiologically relevant concentrations are taken intoaccount, this small window of redox opportunity widens consider-ably [167]. Even if the Fenton reaction is thermodynamically feasible,under normal physiological conditions (i.e., micromolar concentra-tions of iron and H2O2) it is kinetically unlikely, because of the fastreaction of H2O2 with catalase, GPx, and peroxiredoxin [2].
It has been suggested that the critical factor for Fenton-mediated HOd formation is the availability and abundance ofcytosolic LIP [123,168,169]. As summarized above (see Iron meta-bolism), the LIP concentration is influenced by cellular iron uptake,usage, storage, and export and is controlled via a coordinatedaction of IRPs, which, among others, regulate cellular ferritin andTfR concentrations [170]. Rupture of lysosomes and/or nonspecificuptake of plasma non-transferrin-bound iron (NTBI) can rapidlyincrease the LIP concentration [169].
In the acidic (pH 4–5) and reducing environment of thelysosomes, macromolecules and cellular organelles rich in ironare continuously degraded during autophagy. Thus, redox-activeiron may be present in high concentrations, which, based on thework by Brunk and co-workers, may be estimated in the range of15–50 mM [171–173]. Moreover, active catalase is absent in lyso-somes. Thus, when H2O2 diffuses into the lysosomes Fenton-mediated production of HOd or high-valent iron forms may takeplace and lead to peroxidation of membranes with subsequentrelease of redox-active iron into the cytosol [2,12]. The resultingincrease in LIP may lead to cell damage and, depending on theextent, may proceed to apoptosis or to necrosis [174]. Interestingly,autophagocytosed ferritin that is not fully saturated with iron hasbeen suggested to sequester redox-active iron inside the lyso-somes before being degraded, thus hindering Fenton-type chem-istry and increasing the lysosomal stability [175].
NTBI has been shown to exist in the plasma, at least in part, asFe(III) weakly bound to albumin or citrate with various iron/citratemolar ratios [176,177]. Kinetics data suggest that iron–citratecomplexes are not directly responsible for HOd generationin vivo [178]. Therefore, NTBI toxicity is more likely to derive fromnonselective uptake in highly vascular tissues, such as in the liver,the heart, the endocrine system, and the endothelial cells, where itincreases the LIP and thus may induce oxidative damage [123].
In vitro studies in rat hepatocytes and liver endothelial cellshave shown that redox-active iron pools are present in the nucleusand mitochondria [179]. Mitochondria are both the main source ofcellular O2
d� and H2O2 and the main assembly site of [Fe–S]clusters and heme- and iron-containing proteins, processes thatrequire a readily available mitochondrial iron pool [179]. Thus,mitochondria are also potential sources of Fenton-mediated oxi-dative stress [180]. Under normal physiological conditions, themitochondrial iron pool is kept at appropriate levels by theexpression of mitochondrial ferritin, which has been observed tohave a profound effect on cellular oxidative damage [181]. Mito-chondrial ferritin is expressed in various organs but in a limitednumber of cell types characterized by high metabolic activity andoxygen consumption, which suggests a role in protecting mito-chondria from iron-dependent oxidative damage [182]. Interest-ingly, it is not expressed in the spleen and the liver [182], whichindicates that it has no iron-storage function [182,183].
Regulation of iron metabolism by superoxide and hydrogenperoxide
O2d� and H2O2 have been shown to affect the regulation of iron
metabolism through IRPs. Reactions of IRP1 and IRP2 with these
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species provide an efficient way to control the LIP within the cellsand prevent further oxidative damage.
IRP1 function is influenced by O2d� and H2O2 via a number of
mechanisms that produce different responses. On one hand, IRP1mRNA-binding activity has been shown to be differentially modifiedin response to increased amounts of O2
d� and H2O2 [13,14,170].Whereas intracellular O2
d� generation resulted in IRP1 deactivationand associated changes in TfR mRNA and ferritin expression [13],addition of exogenous H2O2 to cells rapidly increased the mRNA-binding activity of IRP1 [184–186], possibly acting via membrane-transduced signaling rather than via oxidative stress or via directinteraction with IRP1 [184,187]. Oxidative modifications of theFBXL5-binding domain lead to IRP2 degradation by the proteasomeand, thus, affect the mRNA-binding activity of IRP2. Although anearlier study showed that IRP2 is not affected by exogenous H2O2
[170], a more recent study demonstrated that exogenous H2O2
increases the IRE-binding activity of IRP2, possibly via stabilizationof HIF-1α due to oxidation of Fe(II) to Fe(III), which would preventthe assembly of the Fe–O–Fe center of FBXL5 [188].
H2O2 has also been suggested to have effects on iron metabo-lism other than those related to its interaction with IRPs. Indeed,in vitro H2O2 exposure leads to IRP-dependent transient inhibitionof ferritin synthesis and upregulation of TfR, with consequentincreased uptake of transferrin, but also to IRP-independentincreased capacity to store iron in ferritin [184]. Nontoxic H2O2
concentrations (o5 μM), mimicking inflammatory conditions,stimulate TfR mRNA translation [189] without affecting IRP–IREbinding activity. Higher H2O2 doses, however, promote proteaso-mal degradation of ferritin [190].
Finally, in vitro experiments have shown that bolus addition ofH2O2 leads to a short-lived increase in HIF-1α levels as a con-sequence of rapid deactivation of PHD, possibly by oxidation of Fe(II) at the active site of the enzyme [191]. However, over longertime periods, H2O2-mediated activation of a ferrireductase, whichmodulates the availability of cellular Fe(II), may trigger theobserved substantial increase in PHD activity and thus the sub-sequent HIF-1α decline [191]. Considering the role of HIFs in theregulation of iron metabolism, these reactions have profoundimplications (see Hypoxia-inducible factors (HIFs)).
Iron metabolism and nitrogen monoxide/peroxynitrite
The interrelationships between iron metabolism and NOd arecomplex [15,16] and comprehensive reviews of this topic areavailable elsewhere [17,192,193]. Here, only the most relevantfeatures are highlighted.
Because of its high affinity for iron, in particular for Fe(II), NOd
reacts with several iron-containing proteins, thereby affectingtheir activity [17,194]. These include ribonucleotide reductase[195], ferritin [196], heme-containing proteins [81,197,198], ferro-chelatase [199], and other [Fe–S] proteins, such as mitochondrialaconitase and Complexes I and II of the electron transport chain[18,19]. Moreover, NOd may also inhibit the Fenton reaction [200].
NOd has an impact on cellular iron metabolism by targeting theIRP/IRE regulatory network, through which it can affect ironstorage, transport, and utilization [18,19,201,202]. In this context,perhaps the most important function of NOd is its ability toactivate the mRNA-binding activity of IRP1, most likely by bothdepleting intracellular iron and interacting with the [4Fe–4S]cluster of IRP1 [18,19,186,203–205]. NOd controls cellular ironexport and storage at the transcriptional as well as the posttran-scriptional level through an incoherent feed-forward loop inwhich an activator regulates both a gene and a repressor of thegene [206]. NOd upregulates ferritin and FPN transcription, possiblyvia the NF-E2-related factor 2 (Nrf2)/antioxidant-responsive
element (ARE) pathway, by stimulating binding of Nrf2 to the AREspresent in the promoter region of the ferritin and FPN genes[194,207,208]. This mechanism was recently shown to be partlyresponsible for the protective effect of NOd against infections withintracellular pathogens through increased cellular iron egress,which limits the availability of iron for intramacrophageal bacteria[209]. On the other hand, NOd-dependent IRP1 activation alsorepresses ferritin and FPN translation, which antagonizes thetranscriptional stimulation [18,19,205,206]. The production of NOd
has also been shown to induce the expression of HO-1 [210–212]via stimulation of the Nfr2/ARE complex [207], thus contributing tothe expansion of the LIP [210].
Whereas NOd activates the binding activity of IRP1 by [4Fe–4S]-cluster disruption, NOþ has been suggested to decrease its bindingactivity by S-nitrosation of critical thiol residues [194,203]. Simi-larly, IRP2 binding activity has been suggested to be decreased byNOþ-mediated S-nitrosation of cysteines in the FBXL5-bindingdomain. Such modification has been proposed to predispose IRP2to ubiquitination and consequent proteasomal degradation and toplay a role in controlling ferritin [194,213] and TfR synthesis [214].However, NOþ-mediated mechanisms are still a matter of con-troversy, resulting likewise from different experimental models.Indeed, convincing experimental evidence also suggests that theNOþ-mediated degradation of IRP2 occurs in the absence ofS-nitrosylation [215].
In addition to NOd, ONOO� is also capable of disrupting [Fe–S]clusters and may thereby inactivate enzymes containing this typeof cofactor [216–218]. In particular, ONOO� has been shown toinactivate aconitase via [4Fe–4S]-cluster disassembly [219–222],but concomitant activation of IRP1 has been a matter of discussion[220–222]. It has been suggested that, depending on its concen-tration and on the experimental conditions, ONOO� leads to theinactive, [3Fe–4S]-containing aconitase form or to the cluster-freemRNA-binding active form of IRP1 [220,222]. High concentrationsof ONOO� have been shown to lead to tyrosine nitration and/ordisulfide formation in IRP1 [219,221]. These modifications pre-clude mRNA-binding activity as well as the ability of the protein toswitch back to aconitase [219–222]. A dependence on the cell ironcontent has also been suggested: in iron-depleted cells, IRP1apoprotein lacking the [Fe–S] cluster has been shown to beinactivated most notably by ONOO� (but also by NOd) possiblyowing to damaging of a significant fraction of cysteine residuesthat mediate the mRNA-binding activity of IRP1 [220]. Finally, IRP2has also been reported to be inactivated by ONOO� , possiblybecause of oxidation of cysteine residues essential for mRNAbinding [219,220]. This process was shown not be influenced byintracellular iron levels [220].
After phagocytosis of foreign or infectious agents NOd productionby macrophages is induced upon activation of iNOS, which is a centralantimicrobial effector pathway [84]. A variety of systems have beenused to study iron metabolism in the MPS, but interpretation of theresults is difficult because of the specialized functions of various MPScells. Some of the conflicting results may be explained by macrophagepolarization, which leads to the differentiation of macrophages intotwo subsets, M1 and M2, which have distinct iron-handling mechan-isms [223,224] (see Alternative mechanisms for iv iron-inducedoxidative and nitrosative stress). Among others, macrophage polariza-tion influences the mRNA-binding activity of IRP1 and IRP2 [224] and,thus, may also explain the differences in the observed interactionsbetween NOd and iron metabolism. Moreover, commonly usedmacrophage cell lines, such as J774 and RAW264.7, do not expressfunctional natural resistance-associated macrophage protein 1(Nramp1) [225]. This transmembrane protein is expressed exclusivelyin monocytes and macrophages and transports Fe(II) bidirec-tionally between late endosomes/lysosomes and cytosol, thereby alsoregulating NOd formation [226–228].
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NOd production by macrophages has also been shown to causecontrolled cellular release of iron and GSH [229] as well asformation of intracellular dinitrosyl–iron complexes [230]. More-over, iron–nitrosyl complexes may be formed upon reaction ofNOd with [Fe–S] proteins, leading to the disruption of the [Fe–S]clusters and, thus, to inhibition of enzyme activity [86]. The NOd-mediated cellular release of iron is also observed upon interactionof activated macrophages with tumor cells [231–233].
Oxidative and nitrosative stress in iron deficiency (anemia)
ID(A) influences the activity of many essential iron-basedenzymes [234]. In vitro studies have suggested that a decrease inthe activity of Complex IV due to heme deficiency decreases theelectron flow to O2 and thus induces reduction of Complexes I–III[235]. Under these conditions, electrons leak from the electrontransport chain to form O2
d� [236,237]. In addition, oxidativestress induced by ID(A) may also be caused by inadequate tissueoxygen supply, which leads to increased concentrations of inflam-matory mediators that activate leukocytes and thus results inincreased levels of O2
d� , H2O2, HOCl, and NOd [35].In humans, the activity of the antioxidant enzymes erythrocyte
SOD and catalase, as well as GPx levels, have been found to besignificantly lower in patients with ID(A) relative to nonanemiccontrols, and they increased significantly as a result of ironreplacement therapy [36,37]. Similarly, iron therapy in patientswith ID(A) resulted in a decrease in oxidant activity and anincrease in total antioxidant capacity and catalase activity mea-sured in blood [238]. In anemic pregnant rats, an analysis of theantioxidant enzyme activities and malondialdehyde (MDA) levelsin the fetuses and placentas revealed that the activities of catalaseand GPx were decreased and that MDA levels were increasedcompared to nonanemic controls [239]. It was suggested that inthese anemic animals the lack of iron might have caused thereduced catalase activity and that the decrease in GPx expressioncould have resulted from GPx-specific pretranslational depressionof GPx mRNA levels during ID. In a similar study by the sameauthors, the unexpectedly high Cu,Zn-SOD values were suggestedto result from elevated NOd levels present in ID(A) and pregnancy[48], two conditions reported to upregulate Cu,Zn-SOD geneexpression [240].
A series of studies has shown upregulation of NOSs as well aselevated NOd production during ID(A) both in humans and in rats.In otherwise healthy humans, ID(A) has been shown to increaseNOd production [38]. In addition, the levels of renal and vasculareNOS and iNOS were elevated in anemic rats, coupled with amarked rise in the activity of these proteins [40]. Moreover,in vitro data have shown that low cellular iron levels lead toupregulation of iNOS mRNA and, thus, NOd formation [29], andiNOS gene expression has been found to be increased during ID inlimited DNA microarray data [41]. Finally, in two human brain celllines heme deficiency resulted in nNOS upregulation accompaniedby high levels of nitrate/nitrite [241]. In addition to the reducedactivity of antioxidant enzymes, enhanced NADPH oxidase, eNOS,and iNOS expression has been observed in the heart of anemic rats,suggesting that ONOO� may be formed in ID(A) and may inducenitrosative stress as reflected by elevated nitrotyrosine levels [39].
Although the mechanisms behind the observed NOS upregula-tion by low iron levels are not known, several possibilities havebeen suggested. For example, increased NOd production by nNOS,as a consequence of heme deficiency, might reflect the increase incytosolic Ca2þ under these conditions [241]. Alternatively, nNOSactivity may be increased in heme-deficient cells to remove excessO2 and to regenerate NADþ/NADPþ , as nNOS uses NADPH as acofactor [241].
Oxidative and nitrosative stress in iron-overload disorders
Because of the properties of iron, the observation that excessiveiron accumulation causes oxidative stress is easy to predict.A number of different iron overload disorders, grouped under thegeneral term of hereditary hemochromatosis, have been described.These diseases emerge from various genetic etiologies resultingfrom mutations in the HFE, hemojuvelin (HJV), hepcidin (HAMP),TFR2, or ferroportin (SLC40A1) genes, but all cause inappropriatelyhigh dietary iron absorption as a consequence of decreased hepci-din formation or increased FPN activity, which leads to accumula-tion of iron in tissues where it induces oxidative stress [142,242–246]. Alternatively, secondary iron overload may also be due totransfusional hemosiderosis in patients with thalassemia, sickle celldisease, and aplastic or sideroblastic anemia, but is also frequent inpatients with cancer or hematological malignancies that requirerepeated blood transfusions [245].
In the case of high dietary iron absorption in hereditaryhemochromatosis, iron is deposited mainly in hepatocytes and inthe parenchymal cells of other organs. Deposition in the MPSoccurs only when iron overload is far advanced [247], becausemacrophages and monocytes are iron depleted owing to reducedhepcidin formation [104,142]. In contrast, transfusional iron over-load is characterized by excessive accumulation of iron as hemo-siderin in the MPS, and iron storage in parenchymal cells of theliver and other organs may ensue only at a later stage [107,247].Both types of iron overload result in iron-mediated tissue damageand alterations in cellular function of parenchymal organs [248].
In many cases, patients with the above-mentioned iron over-load conditions have constantly high NTBI levels [123]. In addition,in both thalassemia and hemochromatosis, lysosomal accumula-tion of iron in the liver as hemosiderin has been demonstrated[249]. Although the mechanism is still largely unknown, highhemosiderin content correlates closely with impaired lysosomalintegrity [250] as well as tissue damage [251]. It has been shownthat experimental iron overload in rats induces noticeable mod-ifications in lysosomal morphology and an increase in intralyso-somal pH, alterations likely to be caused by iron-catalyzed lipidperoxidation [249].
Oral iron therapy
Ferrous (Fe(II)) salts, such as ferrous sulfate, ferrous fumarate,and ferrous gluconate, are commonly used oral iron preparations[43]. These compounds are frequently associated with significantgastrointestinal (GI) side effects, including nausea, vomiting,abdominal pain, and constipation [252], most probably causedby iron-induced oxidative stress in the gut. A number of casereports of GI mucosa injury upon long-term administration oftherapeutic ferrous sulfate doses have been published [253–258],highlighting the potential toxicity of this commonly used oral ironpreparation. Moreover, iron from ferrous sulfate is rapidlyabsorbed in the blood. As a consequence, high serum iron andtransferrin saturation (TSAT) levels are observed, and some of theiron is present in the form of NTBI (up to 6 μM), at least for 5 hafter administration of a 100-mg Fe dose [259–262].
Iron(III)-hydroxide polymaltose complex (IPC) is a soluble,stable, nonionic Fe(III) preparation with efficacy comparable tothat of ferrous compounds [263–265]. IPC consists of a polynuclearFe(III)-hydroxide core, which is stabilized by a number of sur-rounding noncovalently bound polymaltose molecules formingthe carbohydrate shell. This structure accounts for the solubilityof IPC over the entire pH range found in the GI tract and enables acontrolled uptake of iron [266], ensuring very low toxicity andgood tolerability [45,263,265]. Also with IPC, only part of the
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ingested iron dose is absorbed but, because of the redox stabilityof the complex, the fraction that remains within the gut seems lesslikely to induce oxidative stress [263]. Importantly, only very lowlevels of NTBI have been detected after oral administration oftherapeutic doses of IPC [259,261].
So-called IPC similars (IPCSs), i.e., copies of IPC, have beenintroduced in various markets [267]. Because of the complexity ofthe structure, which is largely defined by the manufacturingprocess [268], most IPCSs are not identical to the original IPC.Consequently, it has recently been shown that an IPCS withphysicochemical properties different from those of IPC, mostprobably because of structural dissimilarities caused by noniden-tical manufacturing processes, did not have the same biologicalproperties [269], as reflected by different intestinal iron absorptionkinetics in rats. Indeed, this IPCS was absorbed faster from the GItract, as indicated by higher serum iron and TSAT, and thus cancause iron deposition in different cellular compartments [269].
Direct comparisons of the potential of various oral iron preparationsto induce oxidative stress
Several studies have been conducted to assess the levels ofoxidative stress markers after treatment with various oral ironpreparations. Oxidative stress has been implicated both in the GItract and in organs such as the liver, kidneys, and heart. Because ofsignificant differences in mucosal iron uptake kinetics, iron distribu-tion in the tissues and the flux of iron from the intestinal lumen tothe blood vary greatly among classes of oral iron preparations, e.g.,Fe (II) salts and IPC (Table 1). As mentioned above (see Oral irontherapy), faster elevation of serum iron after oral iron administrationmay lead to full saturation of transferrin with iron, NTBI formation,and thus an increase in LIP, which may induce oxidative damage[123]. Serum iron, TSAT, and NTBI have been shown to increase inresponse to ferrous sulfate therapy [259,260,262], but not inresponse to IPC or sodium Fe(III)–ethylenediaminetetraacetic acid(Fe–EDTA) administration [259,261].
Several clinical studies have reported an increased level ofoxidative stress induced by iron therapy with ferrous sulfate orferrous fumarate. In a 70-day study in iron-deficient, nonanemicwomen receiving ferrous sulfate (98 mg Fe/day) for 8 weeks,
marked increases in plasma MDA level and breath ethane exhala-tion rate, i.e., biochemical indicators of lipid peroxidation, wereobserved at weeks 6 and 8 of oral iron therapy. At week 6 oftherapy, both parameters were more than 40% higher than atbaseline [270]. In another study in pregnant women, combinedsupplementation of ferrous fumarate (100 mg Fe/day) and vitaminC (500 mg/day) during the third trimester resulted in significantlyelevated thiobarbituric acid-reactive species and lower α-tocopherol plasma levels at delivery, suggesting a substantial levelof oxidative stress upon supplying a combination of therapeuticdoses of iron as ferrous fumarate and high doses of vitamin C[271]. In a 6-month, double-blind, placebo-controlled trial inhealthy men with low iron stores, the in vitro susceptibility of verylow and low density lipoproteins to oxidation was increased uponferrous sulfate treatment (180 mg Fe/day) by 8.8 and 12.8% comparedto placebo and IPC treatment (200 mg Fe/day), respectively [49].Moreover, treatment of patients with inflammatory bowel diseaseand iron deficiency without anemia for 14 days with ferrous sulfate(200 mg Fe/day) or IPC (200 mg Fe/day) has been reported to increasemarkers of oxidative tissue damage (MDA) only in the ferrous sulfategroup, whereas disease activity remained unaffected [45].
In rabbits, administration of an extremely high dose of ferroussulfate (230 mg Fe/kg body wt) has been shown to induce toxicity inthe GI tract, leading to gastric ulcers and erosions, whereas no effectswere observed after a comparable dose of IPC [43]. More recently,significant increases in oxidative stress markers in rats treated withferrous sulfate or iron amino chelate vs animals treated with IPC havebeen reported [47]. In anemic pregnant rats, IPC, ferrous fumarate,and ferrous sulfate treatments (2 mg Fe/kg body wt/day) demon-strated different results in terms of correcting oxidative stress inducedby ID(A) in dams, fetuses, and placentas. Most of the negative effectsassociated with ID(A) were resolved by IPC, whereas ferrous fumarateand, in particular, ferrous sulfate were found to elicit hepatic damageand oxidative stress in dams, fetuses, and placentas as well asinflammation and high levels of HIF-1α in the placenta [48].
Oral iron therapy and nitrosative stress
Little is known about the potential of oral iron therapy togenerate RNS and, thus, to induce nitrosative stress. However,
Table 1Summary of clinical and nonclinical studies that identified oxidative and/or nitrosative stress in association with oral iron therapy.
Study type Preparation Dose Duration Observations Ref.
Oxidative stressClinical (men with ID) FS 180 mg Fe/day 6
monthsIncrease in the susceptibility of plasma lipoproteinsto oxidation with FS
[48]
IPC 200 mg Fe/dayClinical (IBD patients) FS, IPC 200 mg Fe/day 14 days Increase in plasma MDA with FS [44]Nonclinical (rats) Carbonyl
ironDiet enriched with 3% (w/w)carbonyl iron ad libitum
4–12weeks
Manifest liver oxidative stress at 6 weeks [43]
Nonclinical (rats) FS 25 mg Fe/kg bw/day 28 days Increase in oxidative stress markers in the intestinal mucosawith FS and IAC
[46]
IAC 280 mg Fe/kg bw/dayIPC 280 mg Fe/kg bw/day
Nonclinical (pregnantrats with ID(A))
FS, FF, IPC 2 mg Fe/kg bw/day 21 days Increase in oxidative stress markers in the liver, heart,and kidneys (dams and fetuses) and in placentas with FF and FS
[47]
Nitrosative stressNonclinical (mousemodel of colitis)
Fe–EDTA 49 mg Fe/kg diet;245 mg Fe/kg diet
Intense nitrotyrosine staining of macrophages in the colonic mucosawith low dose; intense nitrotyrosine immunostaining of macrophagesand adjacent epithelial cells as well as intense iNOS staining of macrophagesin the colonic mucosa with high dose
[45]
Nonclinical (rats) Carbonyliron
Diet enriched with 3% (w/w)carbonyl iron ad libitum
(4–12weeks)
Upregulation of iNOS expression at 8–�12 weeks in the liver [43]
Abbreviations: bw, body weight; Fe–EDTA, Fe(III)–ethylenediaminetetraacetic acid; FF, ferrous fumarate; FS, ferrous sulfate; IAC, iron amino chelate; ID, iron deficiency; ID(A),iron deficiency (anemia); iNOS, inducible nitric oxide synthase; IPC, iron polymaltose complex; IBD, inflammatory bowel disease; MDA, malondialdehyde; w, weight.
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considering the close relationship between RNS and iron metabo-lism (see Iron metabolism and nitrogen monoxide/peroxynitrite),nitrosative stress upon oral administration of iron can be expectedparticularly in subjects suffering from diseases with underlyingchronic inflammation. Most of the evidence so far has beenobtained from hemochromatosis patients, nonclinical models ofiron overload (Table 1), and in vitro experiments.
Higher expression of liver iNOS has been described in heredi-tary hemochromatosis patients [272]. In addition, in a mousemodel of dextran sulfate sodium-induced colitis, oral iron therapy(Fe–EDTA) resulted in a marked increase in iron deposition on theepithelial surface of the colon and in the inflamed areas [46].Correspondingly, nitrotyrosine was detected in inflammatory cellsand in adjacent, damaged epithelial cells. Inflammatory cellsstained for iNOS were also frequently observed in areas witherosion and ulceration, and extensive mucosal infiltration bymacrophages was detected [46]. Interestingly, in a rat model ofexperimental chronic iron overload (diet enriched with 3% carbo-nyl iron) manifest liver oxidative stress at 6 weeks was followed byrecovery from oxidative stress at 8–12 weeks. This was accom-panied by upregulation of iNOS expression, suggesting a hepato-protective function for NOd during chronic iron overload [44],which may relate to functional effects of NOd on FPN-mediatediron export by Nrf2 activation [208,209].
Intravenous iron therapy
Most iv iron preparations are made of iron–carbohydratecomplexes consisting of a polynuclear Fe(III)-oxyhydroxide/oxidecore surrounded by a carbohydrate ligand, which stabilizes thecomplex and protects it against further polynuclearization[273,274]. Intravenous iron preparations currently on the marketin various countries include iron sucrose (IS), ferric carboxymal-tose (FCM), sodium ferric gluconate (FG), low-molecular-weightiron dextran (LMWID), ferumoxytol (FMX), and iron isomaltoside1000 (IIM). In addition, so-called iron sucrose similar (ISS) pre-parations, i.e., copies of IS, have been introduced in variousmarkets. However, because the physicochemical propertiesand thus the pharmacological activity of polynuclear iron com-plexes are strongly dependent on the extensive, multistep manu-facturing process, most ISSs are not identical to the originator IS[59,60,64,275–277].
The iron–carbohydrate complexes behave as prodrugs, becausethe iron has to be released from the polynuclear core to becomeavailable at its site of action. After iv administration, the variousiron complexes are metabolized via similar pathways. They aretaken up by resident macrophages of the MPS in the liver, spleen,and bone marrow [278,279], where they are degraded and thereleased iron is either stored or exported to plasma.
The metabolism of the various iron–carbohydrate complexeswithin macrophages and the storage and release kinetics of ironderived from intravenous iron preparations are not known indetail. Uptake of iron(–carbohydrate) complexes by macrophageshas been suggested to take place via endocytosis [124], and recentstudies have shown that dextran-coated iron-oxide nanoparticlescan also be taken up via a receptor-mediated pathway [280]. In thecase of weaker complexes, such as IS and FG, the carbohydrate islargely dissociated in the plasma, and essentially only the poly-nuclear iron core is taken up by the macrophages [281] (Fig. 2).The carbohydrate shell and the polynuclear iron core are likely tobe degraded within the endosome to release Fe(III), which is mostprobably reduced to Fe(II) by Steap3 [122]. Fe(II) is exported fromthe endosome to the cytosolic LIP possibly via the combinedactivities of DMT1/Nramp1/TRPML1 [11,282] and from the cytosolto the plasma via FPN [112] (Fig. 2). In the plasma, Fe(II) is oxidized
by ceruloplasmin and Fe(III) is bound by transferrin, whichdelivers iron to cells expressing TfR, especially to erythroidprecursors in the bone marrow for hemoglobin synthesis and tothe liver for storage [273,281,283]. Any excess iron that is notexported to the plasma transferrin will be stored in the macro-phages as intracellular ferritin (Fig. 2). A study investigating theeffects of iv IS3 injection into mice provided evidence for a stepwiseiron accumulation first in the spleen and subsequently in the liver,which was paralleled by a reduction in dietary iron absorption and anincreased hepatic hepcidin mRNA expression [284]. In contrast,another study showed that intraperitoneal administration of an irondextran complex led to acute increase in the liver iron contentwithout inducing hepcidin mRNA expression [285].
The physicochemical properties of the iron–carbohydrate com-plexes, such as molecular weight distribution and thermodynamicand kinetic stability, limit the maximal single therapeutic dose anddetermine the potential of the complexes to induce oxidativestress. The stability defines the amount of labile iron (ofteninappropriately called “free” iron) present in the iv iron formula-tion, i.e., the amount of iron that may be directly transferred totransferrin. If present in excess of the iron-binding capacity oftransferrin, labile iron may lead to the formation of NTBI (Fig. 2).Stable complexes such as the FCM, FMX, IIM, and LMWID have lowamounts of labile iron and thus can be given in high single doses[43,281,286,287]. More labile and weak complexes, such as IS andFG, contain a larger percentage of labile iron [287,288], and onlyadministration in small doses and/or slow rates ensures that theiron is taken up primarily by macrophages [43]. Last but not least,the type of carbohydrate ligand determines whether the liganddissociates before endocytosis (Fig. 2), a property that is likely toaffect the clearance rate of the complex from the plasma. More-over, the carbohydrate ligand also influences the potential toinduce immunogenic reactions [283,289].
Intravenous iron therapy and oxidative stress
The ability of various iv iron complexes to induce the formationof oxidative stress has been extensively studied in rats [59–64,290], in mice [65,291,292], in dogs [293], in avian embryos [294],ex vivo [294,295], and in a variety of cells [56,65,292,294–302]. Inaddition, several clinical studies on the induction of oxidativestress after intravenous iron administration have been publishedand reviewed [51,56,58,303–306] (Table 2).
Several in vitro studies have assessed the potential of ironcomplexes used for iv iron therapy to release labile, redox-activeiron [286,300,301], to induce formation of oxidants [297,299],and to cause various degrees of oxidative stress [296–298,300,302]and/or cytotoxicity toward various cells [65,292,294,295,302].Such reports, however, should be interpreted with caution. Cellsin culture are typically grown under ambient O2 tension thatresults from diffusion from a 21% O2 atmosphere (at sea level) andnot at the 2–4% O2 present in tissues. The rate of O2
d� productionby cellular enzymatic systems and by the electron transport chainis limited by the O2 concentration and will thus be higher incultured cells, imposing an oxidative stress to which cells adaptand which changes their properties (e.g., by activating antioxidantsystems) [307,308]. Thus, in vitro studies are not consideredinformative and are not discussed in this section.
The association between iv iron therapy and oxidative stressmay better be evaluated in vivo. However, it should be emphasizedthat most nonclinical in vivo studies have been conducted withiron doses that are significantly higher than doses used in clinicalpractice. As clearly seen in Table 2, all of the nonclinical studies
1001011021031041051061071081091101111121131141151161171181191201211221231241251261271281291301311323 Although Theurl et al. [284] talk about iron dextran, iron sucrose was used.
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were carried out with nonanemic animals and doses up to 10 timeshigher than those usually administered to anemic patients (depend-ing on the degree of iron deficiency and the iron preparation used).
Such a study design is usually necessary to compensate for theshorter blood half-life of the iv iron complex in rodents [307–309].Consequently, these studies [59–65,307,308] may be suitable only
Fig. 2. Model depicting the metabolism of various iv iron preparations and possible points of iron-induced oxidative/nitrosative stress. (A) Metabolism of weaker iron–carbohydrate complexes (e.g., iron sucrose and sodium ferric gluconate). Under the physiological conditions of the blood, the carbohydrate shell is largely dissociated fromthe iron core and subjected to renal elimination. Any labile iron is also dissociated and, depending on the dose, may result in saturation of transferrin and formation of NTBI.Nonselective uptake of NTBI by highly vascular tissue may lead to oxidative stress. The iron core is endocytosed by macrophages of the MPS and degraded within theendolysosomes. (B) Metabolism of ferric carboxymaltose. The carboxymaltose shell is partially degraded in the blood by α-amylase. After the endocytic uptake bymacrophages of the MPS, the iron–carbohydrate complex is degraded within the endolysosomes. (C) Metabolism of dextran-based iron–carbohydrate complexes. The iron–carbohydrate complex is endocytosed by macrophages of the MPS. Depending on the nature of the dextran (e.g., the chain length, branching, and substitution degree),O2
d� and/or NOd production within the endosomes/lysosomes may trigger depolymerization of carbohydrates via different mechanisms. Part of the iron–carbohydratecomplex may be retained within macrophages because of slow degradation of the carbohydrate shell, possibly leading to increased oxidative stress. Thermodynamically verystable iron cores may also require extended periods of time to be degraded within macrophages. (D) Metabolism of iron released from the iron–carbohydrate complexes.Liberated Fe(III) is reduced to Fe(II) by Steap3 and Fe(II) is transported via DMT1/Nramp1/TRPML1 to the cytosolic LIP. From the LIP, iron is delivered to mitochondria andtarget proteins, possibly by cytosolic iron chaperones, or exported from the cell by FPN. In the blood, Fe(II) is rapidly oxidized by hephaestin to Fe(III), which is tightly boundby transferrin. Any iron that is not utilized or exported can be retained within the macrophages as ferritin, which is continuously degraded via lysosomal autophagy.Saturation of ferritin may lead to reduced protection against iron-mediated oxidative stress and increased accumulation of redox-active iron within the lysosomes. WhenH2O2 diffuses into the lysosomes, Fenton-mediated production of HOd or high-valent iron forms may take place and lead to peroxidation of membranes and membranerupture. Rupture of lysosomes and/or nonspecific uptake of NTBI can also rapidly increase the LIP concentration, which may lead to cell damage and proceed to apoptosis orto necrosis. DMT1, divalent metal transporter 1; FPN, ferroportin; LIP, labile iron pool; Nramp1, natural resistance-associated macrophage protein 1; MPS, mononuclearphagocyte system; NTBI, non-transferrin-bound iron; TRPML1, transient receptor potential cation channel, mucolipin subfamily, member 1.
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Table 2Summary of clinical and nonclinical studies that identified oxidative and/or nitrosative stress in association with intravenous iron therapy.
Study type Preparation Dose Duration Observations Ref.
Oxidative stressClinical (HDpatients)
IS 100 mg Fe Single dose Decreased plasma SOD activity and increased plasma lipid peroxides [53]
Clinical (HDpatients)
IS 100 mg Fe Single dose Significant increase in two markers of lipid peroxidation: the ratio of plasmaMDA to cholesterol and the ratio of plasma total peroxides to cholesterol
[56]
Clinical (CKDpatients)
IS 100 mg Fe/week Two doses Transient increase in plasma concentration and urinary excretion rate of MDA [49]
Clinical (CKDpatients)
FG 125 mg Fe Single dose Increased postinfusion plasma MDA levels with all preparations;significant increase in plasma MDA levels with FG vs IS and LMWID
[52]
IS 100 mg FeLMWID 100 mg Fe
Clinical (HDpatients)
IS 100 mg Fe Single dose Significant increase in plasma ascorbic free radicaland protein carbonyls after IS, but not after LMWID
[57]
LMWID 100 mg FeClinical (HDpatients)
IS 100 mg Fe Single dose Increased H2O2 and O2d� production in isolated peripheral blood mononuclear
cells as well as increased lipid peroxidation in plasma[50]
Clinical (HDpatients)
FG 62.5 mg Fe Single dose Increased percentage of mononuclear cells producing O2d� with all
preparations[55]
IS 100 mg FeISS 100 mg FeLMWID 100 mg FeFCM 100 mg Fe
Nonclinical (rats) HMWID/LMWIDa
500 mg Fe/kg bw Single dose Significant increase in plasma and renal tissue MDA [65]
Nonclinical (rats) HMWID/LMWIDa
200 mg Fe/kg bw Single dose,ip
Significant increase in hepatic O2d� production and protein carbonyl levels [51]
Nonclinical (mice) IS 2 mg Fe Single dose Significantly increased MDA levels with all preparations in plasma, with FGand IS in renal cortex, and with IS in the heart
[64]
LMWIDFGIIMb
Nonclinical (rats) IS 40 mg Fe/kg bw/week Five doses Significant increase in catalase, MDA, SOD, and GPx activities and adecrease in GSH levels in the liver, heart, and kidneys
[58]
ISS test 1ISS test 2
Nonclinical (rats) IS 40 mg Fe/kg bw/week Five doses Significant increase in catalase, MDA, Cu,Zn-SOD, and GPx activityand a decrease in GSH:GSSG ratio in the liver, heart, and kidneys with ISS
[59]
ISSGNonclinical (rats) FG 40 mg Fe/kg bw/week Five doses Significant increase in catalase and MDA with FG; increased Cu,Zn-SOD
and GPx activities accompanied with a decrease in the GSH:GSSG ratio inthe liver, heart, and kidneys with FG, LMWID, and HMWID
[60]
ISLMWIDHMWIDFCM
Nonclinical (rats) IS 40 mg Fe/kg bw/week Five doses Significant increase in MDA levels and antioxidant enzyme activitiesaccompanied by significant reduction in GSH:GSSG ratio in the liver,heart, and kidneys with LMWID and FMX
[61]
LMWIDFCMFMX
Nonclinical (rats) IS 40 mg Fe/kg bw/week Five doses Significant increase in MDA levels and antioxidant activities as well assignificant reduction in GSH:GSSG ratio in the liver, heart, and kidneys with IIM
[62]
IIMNonclinical (rats) HMWID/
LMWIDa60 mg Fe/kg bw/every2 days
Five doses,ip
Clearly elevated protein oxidation in the liver; significant increase inα-enolase oxidation status in the liver accompanied by clearly decreasedα-enolase expression and activity
[54]
Nonclinical (rats) IS 40 mg Fe/kg bw/week Five doses Significant increase in MDA levels and antioxidant activities as well assignificant reduction in GSH:GSSG ratio in the liver, heart, and kidneys with allthe ISSs
[63]
ISSFERPISSFERIISSFEROISSENCIISSBACKISSLIB
Nonclinical (rats) IS 1 mg Fe/kg bw/week Five doses Significant increase in DNP levels in heart, lung, liver, and kidneytissues with all preparations
[280]
FCMFMXLMWID
Nitrosative stressNonclinical (rats) HMWID/
LMWIDa500 mg Fe/kg bw Single dose Significant increase in renal tissue nitrotyrosine and renal
eNOS and iNOS upregulation[65]
Nonclinical (rats) 200 mg Fe/kg bw Significant increase in hepatic NOd production [51]
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to compare potential safety issues related to different iron complexes,and the clinical relevance of the results is questionable although theyare important indicators for the clinical safety window.
The large majority of clinical studies that have analyzed theinduction of oxidative stress upon administration of an iv ironpreparation have been performed with IS and only a few havebeen performed with FG, LMWID [58], FCM, and ISSs [56]. To date,no clinical studies have analyzed the induction of oxidative stressafter FMX or IIM administration. Stefánsson et al. [58] summarizeda large number of human studies published between 1999 and2008 that report data on plasma or serum markers of oxidativestress upon iv injection of various iron preparations. The results ofthe various studies show some inconsistencies [58], an observa-tion that casts doubt on the validity of the study design andparameters selected to assess the level of oxidative stress [310].We refer to Stefánsson et al. [58] for these studies and will notinclude them in the following discussion.
Most of the indications of NTBI formation and generation ofoxidative stress upon administration of iv iron have been obtainedfrom studies with hemodialysis patients. High TSAT and/or NTBIlevels have been reported in hemodialysis patients receiving FG[311,312] or IS [311,313]. However, it has been shown thathemodialysis patients per se may present with higher than normalNTBI levels [311]. It is worth noting that these results have to becritically evaluated, because an international round robin for thequantification of serum NTBI showed that the values differ con-siderably between methods [314].
A few studies report increased levels of oxidative stress upon ivadministration of IS [50,54,57]. Yet in another study FG, but not ISor LMWID, was shown to raise postinfusion plasma MDA levels[53]. In another study, both IS and LMWID were reported to induceintracellular oxidant generation to similar extents and to increaseIL-6 levels in end-stage renal disease patients on hemodialysis,but significantly higher NTBI levels were observed only afteradministration of IS compared to LMWID [315]. Furthermore, itwas recently shown that standard doses of IS had only minor effectson oxidative stress levels and that no significant differences in lipidperoxidation were observed upon administration of a dose of 50 or100mg Fe [304]. Thus, it was suggested that the increase in lipidperoxidation products with iv iron during hemodialysis is a transientphenomenon that takes places early (1–2 h) after infusion and isindependent of the dose [304]. Indeed, NTBI has also been shownto increase transiently after iv IS administration during hemo-dialysis [316]. Taken together, such conflicting results in hemodialysispatients, especially in the case of IS, may be due to the selection of the
oxidative stress markers assessed. Moreover, hemodialysis alone hasbeen shown to cause a significant increase in oxidative stress,inflammation, and endothelial dysfunction [317,318], a feature thatmay contribute to the variability of the results.
In a rat model, administration of the currently marketeddextran-based iv iron complexes LMWID, IIM, and FMX (fiveweekly doses of 40 mg Fe/kg body wt) was found to result insignificantly increased levels of oxidative stress and proinflamma-tory cytokine markers in the liver, heart, and kidneys that werecomparable to those observed after FG administration [61] butsignificantly higher than those observed after administration ofFCM and IS [62,63]. LMWID and IIM also presented significantlyhigher Prussian blue-stainable Fe(III) deposits in the liver, heart,and kidneys than IS, FCM, and FMX, which, in contrast, presentedsignificantly higher ferritin levels in the same tissues [62,63].Whether these results have any significant physiological andpathological implications remains to be determined. In a similarrat model, weekly administration of LMWID, FMX, FCM, and IS in adose used in clinical practice (five weekly doses of 1 mg Fe/kgbody wt) was reported to increase oxidative stress in the heart,lung, kidney, and liver compared to control [290]. LMWID, FMX,and IIM are stable iron complexes that contain comparable,minimal amounts of labile iron [287]. Thus, labile iron is probablynot the primary cause for the observed toxicity profile of thesecomplexes [290]. Correspondingly, in humans, LMWID and IS(both at a single dose of 100 mg Fe) were shown to induce similarintracellular oxidant generation and IL-6 activation in end-stagerenal patients despite apparently different NTBI exposure profiles[315]. Therefore, it is reasonable to suggest that NTBI is not theonly factor that contributes to the iv iron-induced oxidative stress.
The rat model with five weekly doses of 40 mg Fe/kg body wtwas also used to assess the potential of ISSs to induce oxidativestress. Compared to IS, most of the ISSs showed significantlyhigher levels of oxidative stress and proinflammatory markers inthe liver, heart, and kidneys [59,60,64]. Moreover, iron distributionin these tissues was also different. Compared to IS, ISSs generallypresented with markedly reduced ferritin deposits, in particular inthe liver, and higher levels of iron (positive Prussian blue staining)in the Kupffer cells as well as in the surrounding tissue [59,60,64].
Intravenous iron therapy and nitrosative stress
In contrast to the large number of studies that assessed thepotential of iv iron therapy to induce oxidative stress, not much isknown about its potential to generate nitrosative stress. Only a few
Study type Preparation Dose Duration Observations Ref.
HMWID/LMWIDa
Single dose,ip
Nonclinical (rats) HMWID/LMWIDa
60 mg Fe/kg bw/every2 days
Five doses,ip
Elevated protein tyrosine nitration in the liver; significant increase in α-enolasenitration status in the liver accompanied by decreased α-enolase expression andactivity
[54]
Nonclinical (rats) IS 1 mg Fe/kg bw/week Five doses Significant increase in nitrotyrosine levels in heart, lung, liver,and kidney tissues with all preparations; LMWID induced over10-fold higher levels of nitrotyrosine than any other preparation
[280]
FCMFMXLMWID
Please see Stefansson et al. [57] for more studies. Abbreviations: bw, body weight; CKD, chronic kidney disease; DNP, dinitrophenyl; eNOS, endothelial nitric oxide synthase;FCM, ferric carboxymaltose; FG, ferric gluconate; FMX, ferumoxytol; GPx, glutathione oxidase; GSH, reduced glutathione; GSH:GSSG, reduced glutathione to oxidizedglutathione ratio; HD, hemodialysis; HMWID, high-molecular-weight iron dextran; IIM, iron isomaltoside 1000; iNOS, inducible nitric oxide synthase; ip, intraperitoneal;IS, iron sucrose; ISS, iron sucrose similar; LMWID, low-molecular-weight iron dextran; MDA, malondialdehyde; SOD, superoxide dismutase.
a Iron–dextran, molecular weight or preparation not specified.b No analysis of IIM effects in the renal cortex or heart.
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studies have analyzed nitrosative stress biomarkers in iron-overloadanimal models (Table 2).
In a rat model of chronic experimental hemosiderosis inducedby a high single dose of iv iron–dextran injection of 500 mg Fe/kgbody wt, analysis of renal tissue showed significantly increasednitrotyrosine levels and upregulation of eNOS and iNOS expression[66]. The same study also reported a mild proteinuria associatedwith significant glomerusclerosis, tubular atrophy, and interstitialfibrosis due to iron deposition in the glomeruli and proximaltubules. However, these pathologies may have been caused also byoxidative stress [66]. In another study, iron-overloaded rats (fiveintraperitoneal iron dextran injections of 60 mg Fe/kg body wt)showed a higher degree of nitration and oxidation of hepaticproteins and, in particular, a significant level of nitrated α-enolase,a key glycolytic enzyme [55].
In a recent study, five weekly doses of IS, FCM, FMX, and LMWID(1 mg Fe/kg body wt) were found to elicit nitrosative stress in healthyrats. Interestingly, nitrotyrosine levels were significantly higher in theheart, lung, kidney, and liver of LMWID-treated animals than inanimals treated with other iv iron preparations [290].
Alternative mechanisms for iv iron-induced oxidative andnitrosative stress
In most of the studies discussed above, the potential of iv ironpreparations to induce oxidative/nitrosative stress is assumed tobe correlated with the amount of labile iron present in thecomplexes and thus their likelihood of generating NTBI. However,as already mentioned, dextran-based iv iron complexes are stable,and thus the oxidative stress caused in nonclinical studies by thesepreparations [61–63] is likely to be induced by alternativemechanisms. Moreover, it has to be kept in mind that most ofthese rodent studies used very high dosages of iron that exceededmaximum dosages used in humans by several folds. In this section,we discuss a few other pathways that may explain the observedeffects.
As mentioned above (see Intravenous iron therapy), macro-phages of the MPS play a crucial role in the metabolism of iv iron–carbohydrate complexes. Even though the underlying mechanismis still poorly characterized, it is expected that the carbohydrateligand, complex stability, and particle size have important roles incomplex recognition, uptake, and degradation as well as ironrelease by macrophages [280]. This hypothesis is supported bypharmacokinetic studies that showed that the terminal half-livesof the more labile and weaker complexes, such as FG and IS, areshorter than those of the robust and strong complexes (FCM,LMWID, FMX, and IIM) [319]. However, complexes with dextran-based ligands (LMWID, FMX, and IIM), despite their variablesize (MW in the range of ca. 70–185 kDa), all have a longerterminal half-life than the non-dextran–iron complex FCM (MWca. 150 kDa), although all of these complexes are robust and strong[319].
Macrophages respond to microenvironmental signals and canadopt different functional profiles, from classical (or M1) activa-tion to the alternative (or M2) state [320], which have a profoundeffect on iron handling [224]. M1 macrophages have strongantimicrobial and tumoricidal capacity and produce high levelsof proinflammatory cytokines to increase their killing activity[320,321]. Moreover, they are characterized by increased ironretention [321]. M2 macrophages produce anti-inflammatorycytokines and have efficient phagocytic activity and high expres-sion of scavenger receptors, which contribute to inflammationresolution by clearing surrounding tissue of cell debris andoxidized lipoproteins [321]. Concerning iron metabolism, it hasbeen shown that the lower iron levels in M2 vs M1 macrophages
are primarily due to the upregulation of HO-1 and FPN in M2macrophages, resulting in greater iron export [320]. Interestingly,recent studies suggest that intracellular iron or heme can influencethe activation of macrophages [320,322–324]. Increased intrama-crophageal iron retention caused by hepcidin-mediated FPNdegradation has been suggested to facilitate the expression ofcytokines such as TNF-α [325]. In addition, increased iron levelshave been shown to enhance signaling through the NF-κB path-way [326]. However, iron retention by monocytes has also beensuggested to inhibit IFN-γ-mediated immune effector pathways,leading to diminished formation of TNF-α and NOd [25,27].
So far, only little is known about the effect of iv iron onmacrophage polarization. Administration of iv iron has beenshown to be linked to increased proinflammatory markers in bothhumans and animals. For example, upon administration of a singletherapeutic dose of IS in hemodialysis patients, a transientincrease in proinflammatory TNF-α and IL-6 via a short-termactivation of the NF-κB pathway and parallel transient storage ofiron in circulating monocytes have been observed [327]. In aprospective clinical study carried out in dialysis patients, admin-istration of IS (100 mg Fe/week) in addition to EPO resulted in asignificant decrease in circulating TNF-α levels after 90 days and ina significant increase in circulating anti-inflammatory IL-4 levelscompared to dialysis patients receiving EPO alone [328]. Thisresult suggests that IS downregulates the proinflammatoryimmune effector pathways in macrophages when given over alonger period of time [25–27]. Finally, a very recent study reportedthat administration of a single therapeutic dose of FCM (15 mg Fe/kg body wt, max 1 g Fe) in predialysis CKD patients with ID(A)did not have any acute (1 h) nor subacute (3 weeks and 3 months)effect on circulating concentrations of the studied inflammatorymarkers CRP and IL-6 [329]. Because of a lack of direct comparativestudies of the effects of various iv iron complexes in humans, it isdifficult to determine whether the observed results are indeed due toiron or if they represent a specific response to the administered iron–carbohydrate complex or its degradation products.
A few studies in healthy rats with relatively high dosage,however, have reported significant differences between proin-flammatory as well as oxidative stress markers after treatmentwith various iv iron preparations [59–64]. Interestingly, the stable,dextran-based iv iron complexes LMWID, FMX, and IIM led tosignificantly higher levels of proinflammatory as well as oxidativestress markers compared to the stable non-dextran-based FCM orthe more labile IS. Moreover, a recent study in a mouse chronicvenous leg ulcer model demonstrated that iron overloading byintraperitoneal administration of iron dextran (5 mg Fe/mouseevery 3 days for 21 days) induced an unrestrained proinflamma-tory M1 activation state and prevented the physiologic switch toanti-inflammatory M2 macrophages, thus leading to chronicinflammation [330]. Hence, it is conceivable that the type ofmacrophage activation after iv iron uptake depends on the ironpreparation and is related to the properties of the carbohydrateshell, polynuclear iron oxyhydroxide core, and/or the wholecomplex.
Early studies with radiolabeled iron dextran have shown that,in patients with ID, the rate of hemoglobin (Hb) synthesisincreased immediately after iv iron infusion and iron supplypermitted bone marrow production levels of Hb four to six timesthat of normal [331,332]. Interestingly, after approximately2 weeks, iron delivery to the bone marrow decreased substantially[331,332]. Similar results were observed when subjects with IDwere given large amounts of LMWID (1500–2550 mg Fe) as a total-dose iv infusion [333]. The tendency of iron to become progres-sively unavailable for Hb synthesis was postulated to be the resultof the disparate size of the iron dextran particles. Larger particleswere suggested to become partially or totally unavailable for iron
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iPlease cite this article as: Koskenkorva-Frank, TS; et al. The complex interplay of iron metabolism, reactive oxygen species, and reactivenitrogen species: Insights into the potential of various iron... stresshttp://dx.doi.org/10.1016/j.freeradbiomed.2013.09.001
release, leading to gradual iron deposits (Fig. 2). Moreover, thepresence of inflammation/infection as well as high loading of theMPS with iron dextran seemed to further impede iron utiliza-tion [331,333]. It is conceivable that formation of hemosiderinor iron dextran deposits may be linked to the oxidative stressobserved in the nonclinical studies discussed above [61–63].In iron-overloading syndromes hemosiderin is the predominantform of storage iron, which accumulates within the lysosomes,especially in the liver, but can also be found freely diffused inthe cytoplasm [334], and has been reported to lead to celldamage [129]. Furthermore, intracellular lipid peroxidationhas been shown to be induced or accelerated around hemosi-derin deposits [335] (Fig. 2). Last, rupture of lysosomes due toextensive iron loading and increased oxidative stress has beenshown to lead to apoptotic cell death or even necrosis [174](Fig. 2).
Accumulating evidence has shown that O2d� and NOd produc-
tion triggers depolymerization of carbohydrates within the endo-somes/lysosomes of antigen-presenting cells that can take placevia various mechanisms that depend on the fine structure of thepolysaccharide polymer [336,337]. Thus, it is likely that theseradical species may have a role in degrading the iron–carbohy-drate complex and that various carbohydrate shells may inducetheir production differentially. Dextran, which is the ligand inLMWID, has been shown to be depolymerized within the endo-some upon production of O2
d� and H2O2 [336]. Extensive in vivodegradation studies of the carbohydrate ligand are available onlyfor FMX. In this complex, the polynuclear iron core is coated withpolyglucose sorbitol carboxymethyl ether (PSC), a reduced andcarboxymethylated dextran. In rats receiving 14C-labeled FMX as asingle dose of 6 mg Fe/kg body wt, 90% of the labeled material wasdetected in the urine and in the feces after 14 days. The percentageof intact PSC was 81 and 24% in urine and in the feces, respectively[338]. Thus, part of the FMX complex might be retained withinmacrophages because of slow degradation of the carbohydrateshell (Fig. 2), resulting in the observed 10% retention of the 14Clabel. Other carboxydextran-coated iron oxide nanoparticleshave been shown to be retained by Kupffer cells for extendedperiods to degrade the carbohydrate shell, eliciting increasediron-mediated HOd generation and over 50% reduction of thecell number through apoptosis [339,340]. In the case of FCM, thecarboxymaltose ligand is partially degraded by α-amylase inblood [341] and IS, in turn, is dissociated to sucrose and the ironcore under the physiological conditions of the blood [342],before being taken up by the MPS (Fig. 2). Neither FCM nor IShas been shown to accumulate in significant amounts within themacrophages for extended periods of time, and iron utilizationhas been shown to be essentially quantitative [43,278,279,342],suggesting an efficient degradation of the complex withinthe MPS.
In addition to the carbohydrate shell the iron oxyhydroxide/oxide cores of the iron complexes also differ in size and structure.The properties of the core not only determine the amount of labileiron but may also influence its degradation rate within themacrophages. The half-life of in vitro hydrolysis under very acidicconditions (pH �1) of the various iron complexes was found torelate to their surface area and to decrease with decreasing coresize, i.e., FMX4FCM� IIM4LMWID4 IS4FG [287]. Thus, in addi-tion to a carbohydrate shell that may persist in cells of the MPS,it is possible that part of the FMX core, which is a thermodyna-mically stable polynuclear iron(III)-oxide, is retained in macro-phages for extended periods of time (Fig. 2). Indeed, a recent studyshowed that healthy adults without anemia or preexisting ironoverload receiving a single injection of FMX (5 mg Fe/kg body wt)presented with a remarkable variation in hepatic iron clearancerates [343]. Although the prescribing information of FMX warns of
potential interference with magnetic resonance imaging (MRI)tests for up to 3 months because FMX is superparamagnetic, 50%of the study cohort (i.e., three participants) were reported tohave extremely long complete elimination times of excess, MRI-detectable iron (411 months) [343]. Whether the iron(III)-oxy-hydroxide cores of IS, IIM, LMWID, and FCM are subjected tothe same phenomenon is currently not known and cannot beeasily assessed because the cores of these complexes are notsuperparamagnetic.
Conclusions
Under physiological conditions, there is a balance between theformation of ROS/RNS and their detoxification by enzymatic ornonenzymatic antioxidants. Iron plays a central role in the forma-tion and the scavenging of such species. Among these roles, theiron-catalyzed Fenton reaction may lead to formation of HOd,production of NOd is highly regulated by iron, and the ubiquitousantioxidant enzyme catalase is a heme-containing enzyme. Thus,it is understandable that dysfunction of normal iron homeostasis(i.e., not only iron overload but also ID(A)) causes oxidative/nitrosative stress by different pathways. Depending on the accom-panying medical condition, ID(A) is treated with oral or intrave-nous iron therapy, which may also cause oxidative stress.Moreover, in several situations that require iron administration,an elevated level of oxidative stress is already present, e.g., inhemodialysis, inflammatory bowel disease, cancer, and pregnancy[344,345].
The substantial differences in the physicochemical properties ofthe various oral and iv iron formulations result in variations inpharmacodynamics and pharmacokinetics between oral and iviron preparations. The propensity of an oral iron preparation toinduce oxidative stress depends on the mucosal iron uptakekinetics, the iron distribution in the tissues, and the flux of ironfrom the intestinal lumen to the blood. Ferrous salts, which arerapidly absorbed in the blood, lead to high serum iron and TSATlevels and thus elevated NTBI, which may induce oxidative stress.In contrast, iron from IPC is essentially taken up via a controlledmechanism leading to negligible levels of NTBI.
The likelihood of iv iron-induced oxidative/nitrosative stressdepends on various factors, including complex stability, carbohy-drate ligand and particle size, and the underlying medical condi-tion of the patient under treatment. More labile and weakercomplexes contain larger amounts of labile iron that may poten-tially lead to NTBI-induced oxidative stress. To avoid this, thesepreparations are usually administered in small single doses andover a longer period of time. In contrast, stable complexes have alow percentage of labile iron and thus, NTBI formation is less likelyeven when larger single doses are administered. However, recentstudies have also indicated that administration of stable androbust iron–carbohydrate complexes may lead to oxidative andnitrosative stress, although the underlying mechanisms remainlargely unknown. In conclusion, it is of pivotal importance tounderstand the possible mechanisms of how different iron com-plexes may induce formation of oxidants to enable development ofsafe and more efficacious therapies for dysfunctions of ironmetabolism.
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iPlease cite this article as: Koskenkorva-Frank, TS; et al. The complex interplay of iron metabolism, reactive oxygen species, and reactivenitrogen species: Insights into the potential of various iron... stresshttp://dx.doi.org/10.1016/j.freeradbiomed.2013.09.001
Acknowledgments
Illustrations were prepared with the help of SFL RegulatoryAffairs & Scientific Communication Ltd. T.S. Koskenkorva-Frankand S. Burckhardt are employees of Vifor (International) Ltd. G.Weiss has received speaker honoraria from Vifor Pharma Ltd. andPharmacosmos A/S. W.H. Koppenol has investigated the physico-chemical properties of iron–sucrose preparations in a studysponsored by Vifor (International) Ltd.
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