Molecular and cellular mechanisms of iron homeostasis and toxicity in mammalian cells

Journal of Inorganic Biochemistry 91 (2002) 9–18 www.elsevier.com / locate / jinorgbio

Focused Review

Molecular and cellular mechanisms of iron homeostasis and toxicity in mammalian cells ´ Robert R. Crichton*, Stephanie Wilmet, Rachida Legssyer, Roberta J. Ward Unite´ de Biochimie, Universite´ Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium Received 19 October 2001; received in revised form 22 April 2002; accepted 22 April 2002

Abstract Iron is an essential metal for almost all living organisms due to its involvement in a large number of iron-containing enzymes and proteins, yet it is also toxic. The mechanisms involved in iron absorption across the intestinal tract, its transport in serum and delivery to cells and iron storage within cells is briefly reviewed. Current views on cellular iron homeostasis involving the iron regulatory proteins IRP1 and IRP2 and their interactions with the iron regulatory elements, affecting either mRNA translation (ferritin and erythroid cell d-aminolaevulinate synthase) or mRNA stability (transferrin receptor) are discussed. The potential of Fe(II) to catalyse hydroxyl radical formation via the Fenton reaction means that iron is potentially toxic. The toxicity of iron in specific tissues and cell types (liver, macrophages and brain) is illustrated by studies with appropriate cellular and animal models. In liver, the high levels of cyoprotective enzymes and antioxidants, means that to observe toxic effects substantial levels of iron loading are required. In reticuloendothelial cells, such as macrophages, relatively small increases in cellular iron (2–3-fold) can affect cellular signalling, as measured by NO production and activation of the nuclear transcription factor NFkB, as well as cellular function, as measured by the capacity of the cells to produce reactive oxygen species when stimulated. The situation in brain, where anti-oxidative defences are relatively low, is highly regionally specific, where iron accumulation in specific brain regions is associated with a number of neurodegenerative diseases. In the brains of animals treated with either trimethylhexanoylferrocene or aluminium gluconate, iron and aluminium accumulate, respectively. With the latter compound, iron also increases, which may reflect an effect of aluminium on the IRP2 protein. Chelation therapy can reduce brain aluminium levels significantly, while iron can also be removed, but with greater difficulty. The prospects for chelation therapy in the treatment and possible prevention of neurodegenerative diseases is reviewed.  2002 Elsevier Science Inc. All rights reserved. Keywords: Iron; Toxicity; Chelation therapy; Neurodegenerative disease; Oxidative stress

1. Introduction The importance of well-defined amounts of iron for the survival, replication and differentiation of animals, plants and almost all micro organisms (one notable exception is the Lactobacillus family) is well established [1]. Iron deficiency is a general problem in biology, and in man is responsible for 400–500 million cases among the one-third of the world’s population who suffer from anaemia. However, iron in excess is toxic, particularly in man, where haemochromatosis is one of the most frequent genetic disorders, with an estimated carrier frequency of 1 in 200 in the Caucasian population of Northern European descent for C282Y homozygotes (greater than that of cystic fibrosis and phenylketonuria together). It is therefore *Corresponding author. Fax: 132-10-472-796. E-mail address: [email protected] (R.R. Crichton).

no surprise that iron homeostasis is a major preoccupation in human health and wellbeing. As we will see in the later part of this article, excess iron accumulation within tissues, cells and even organelles, can result in toxicity and is associated with pathological disorders. In addition to the classic iron loading found in genetic haemochromatosis and in secondary iron overload disorders, such as the thalassaemias, with their associated dysfunctions (reviewed in Ref. [1]), there are many other diseases associated with excess iron. For example, iron accumulation in the brain has been associated with Parkinson’s disease, Alzheimer’s disease, Huntington’s chorea and HIV encephalopathy (reviewed in Ref. [2]) and more recently in basal ganglia disease [3] and Hallervorden Spatz syndrome [4], while in Friedrich ataxia, excessive mitochondrial iron accumulation occurs particularly in brain and cardiac tissue [5]. In the rest of the introduction, we briefly review gastrointestinal iron absorption, iron transport and cellular uptake, and

0162-0134 / 02 / $ – see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00461-0

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iron storage. We then discuss current views on cellular iron homeostasis, before giving a brief account of the origins of iron toxicity.

1.1. Iron transport by intestinal mucosa cells Iron transport by intestinal mucosa cells is summarized in Fig. 1. From the time that the cells proliferate and ¨ differentiate in the crypts of Lieberkuhn, until their arrival in the absorptive part of the intestinal villus and their ultimate destruction, the total lifetime of a duodenal mucosal cell is not more than 2 days. During this time, as it moves from the crypts towards the tips of the villus, each mucosal cell sees only once or twice in its lifetime some iron passing by before it is released into the intestinal lumen. Normal subjects ingest approximately 12–18 mg of dietary Fe / day, mainly as Fe 31 , of which 1–2 mg is absorbed. Both of the major uptake systems for dietary iron, namely (i) haem and (ii) non-haem, require reduction of Fe 31 to Fe 21 (Fig. 1). This involves a putative haem receptor on the apical membrane of the enterocyte [6], and the transport of haem through the cytosol to the endoplasmic reticulum, where the microsomal haem oxygenase releases Fe 21 , porphobilinogen and CO [7]. In the second pathway, dietary Fe 31 can be reduced to Fe 21 by a recently identified duodenal ferric reductase, Dcytb [8] prior to its transport into the enterocyte via the divalent

cation transporter DMT1 (also known as DCT1 or Nramp2), which functions with concomitant transfer of H 1 [9]. Once Fe(II) has entered the mucosal cell it has only two choices: either to encounter a ferritin molecule, which incorporates iron as Fe(II), oxidising it to Fe(III), and traps the Fe(III) within the protein shell, or to be transported to the basolateral membrane. In the normal situation there are many ferritin molecules available, and much of the iron will be trapped in ferritin and retained in the mucosal cell, (ferritin acting as a buffer), resulting in a low mucosal transfer of iron. In iron deficiency, or in conditions of increased red blood cell production (erythropoiesis), mucosal cells hardly produce any ferritin and most of the iron entering the cell is available for transport across the cell to the basolateral membrane. Here the diffusion of Fe(II) across the basolateral membrane is facilitated by IREG1, a transmembrane iron transporter protein [10]. Hephaestin, a membrane-bound protein, promotes oxidation of Fe 21 to Fe 31 [11]. The Fe(III) so formed will be rapidly bound to plasma ligands, essentially to the major iron transport protein, apotransferrin, except in situations of saturation of the binding capacity of the plasma transporter, when it would also bind to other ligands, such as citrate, constituting the so-called non-transferrin bound iron (NTBI) pool. One of the important roles of hephaestin is to facilitate oxidation of Fe(II), which will allow rapid binding of iron to transferrin and its delivery to cells

Fig. 1. Schematic representation of iron absorption in normal subjects. The panel represents a mature intestinal mucosa cell. DMT15Divalent metal transporter 1; IREG15iron-regulated transporter 1; HFE5haemochromatosis gene product; b2m5b2-microglobulin; hephaestin5a putative multicopper ferroxidase; Cp5ceruloplasmin; NTBI5non transferrin bound iron; HemeOx5heme oxygenase I; Dcytb5duodenal cytochrome b, ferric reductase. In the enterocyte dietary iron is absorbed either directly as Fe(II) after reduction in the gastrointestinal tract by reductants like ascorbate, or after reduction of Fe(III) by the apical membrane ferrireductase Dcytb, via the divalent transporter DMT1. Alternatively, haem is taken up at the apical surface, perhaps via a receptor, and is degraded by haem oxygenase to release Fe(II). The enterocyte is programmed such that any excess iron retained within the enterocyte as ferritin, while iron needed is transferred to the circulation. This latter process is presumed to involve IREG1 and hephaestin at the basolateral membrane with incorporation of iron into apotransferrin. Alternatively ceruloplasmin in the serum can catalyse the oxidation of iron and its subsequent incorporation into transferrin. In the crypts of Lieberkuhn the transferrin receptor together with the haemochromatosis gene product may interact to ‘set’ the iron regulatory proteins (IRPs) which will then regulate the amount of iron absorbed by the enterocyte in its absorptive phase on the villus. HFE, TfR and b2-microglobulin localisation are suggested although their exact role in iron uptake awaits clarification. Adapted from Ref. [1].

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expressing IRP-regulated transferrin receptors, thus preventing endothelial damage and favouring the uptake of iron by other cells, particularly hepatocytes.

1.2. Iron transport by transferrin Iron in serum is present at concentration between 3 and 5 mg / ml in normal subjects and is predominantly bound to transferrin, with little associated with albumin or lowmolecular-weight species [1]. The bilobal transferrin molecule can bind tightly, but reversibly, two Fe 31 ions, with concomitant binding of two carbonate anions. The iron binding sites consist of four ligands contributed by the protein (two Tyr, one His and one Asp) with the final two ligands coming from the carbonate to give an almost ideal octahedral metal coordination with binding constants around 10 19 –10 20 M 21 . Recent high-resolution structures of the N-lobe of human serum transferrin supports the view that protonation of the carbonate anion is the first step in iron release [12]. Early pioneering studies by Katz using doubly labelled transferrin established that, whereas transferrin bound iron was rapidly cleared from the circulation (t 1 / 2 of the order of 1.7 h), the protein recycles many times (t 1 / 2 of the order of 7.6days) indicating that the transferrin molecule undergoes more than one hundred cycles of iron binding, transport and release before it is removed from the circulation [13]. It has become clear that iron uptake in almost all mammalian cells is mediated by transferrin receptors (TfRs), which are disulphide-linked homodimers consisting of two identical glycosylated subunits of Mr 90 kDa. The X-ray structure of the ectodomain of the human TfR has been determined [14].

1.3. Intracellular iron transport The transferrin-to-cell cycle by which iron is taken up from transferrin, and the iron-free apotransferrin released to the plasma for reutilisation, is now well established [1]. Diferric transferrin binds to its receptor at the cell surface and the transferrin–transferrin receptor complex is internalized in clathrin-coated vesicles. The vesicles lose their coat, and the resulting smooth vesicles fuse with endosomes. The interior of the endosomal compartment is maintained at a pH of approximately 5.5 by the action of an ATP-dependent proton pump in the endosomal membrane which pumps protons into the endosomal lumen from the cytosol. Iron is released from the transferrin– transferrin receptor as Fe 31 at this acidic pH within the endosomal compartment. The iron is transported out of the endosome by the divalent cation carrier DMT1 [15], the transmembrane iron transporter. We know little about the putative ferrireductase which is involved in the reduction of Fe 31 prior to its transport out of the endosome by DMT1. Apotransferrin, still bound to the transferrin receptor, then returns to the cell surface, where it is released

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into the circulation, completing a highly efficient cycle which takes iron into the cell essentially as diferric transferrin, and releases the iron-free protein for re-utilisation. Once iron has entered the cell, it enters a hypothetical low-molecular-weight pool or otherwise known as the chelatable iron pool. It can then become involved in a multitude of cellular processes, which include incorporation into a number of essential iron containing proteins, sequestration in ferritin and haemosiderin, as well as being available for chelation. Iron incorporation into the major iron storage protein, ferritin, involves oxidation of Fe 21 , and deposition of the iron within the hollow protein shell, essentially as ferrihydrite. Ferritin can store up to 4,500 iron atoms / molecule. In conditions of iron loading the ferritin is transferred to the lysosomes where it is transformed into the form known as haemosiderin [1,16].

1.4. Cellular iron homeostasis Iron homeostasis in mammalian cells is regulated by balancing iron uptake with intracellular storage and utilisation. This is achieved predominantly at the level of protein synthesis (translation of mRNA into protein) rather than at the level of transcription (mRNA synthesis), as in prokaryotes. Regulatory sequences (iron regulatory elements—IREs) are located in the non-coding or un-translated regions (UTRs) of the mRNA, at either the 59- and 39-extremities of the coding part of the mRNA sequence: IREs in the former are usually associated with the initiation of translation, in other words ribosome binding, whereas those at the 39-UTR are associated with mRNA stability and degradation, i.e., mRNA turnover. Two closely related cytosolic IRE-binding proteins (now known as iron regulatory proteins—IRPs), designated IRP1 and IRP-2, have been identified in many mammalian cell types, Fig. 2. They act as iron sensors, essentially by existing in two different conformations. When iron is in short supply, the apo-IRPs can bind to the IREs (Fig. 2), with high affinity. This prevents translation of the ferritin and erythroid cell d-aminolaevulinate synthase mRNAs, while protecting the mRNA for transferrin receptor against nuclease attack, and allowing the cells to take up iron. When iron supply to cells is increased, IRP-1 protein is inactivated, whereas IRP-2 is rapidly degraded (reviewed in Ref. [1]). The outcome is that ferritin and erythroid cell d-aminolaevulinate synthase are translated, whilst the transferrin receptor mRNA is degraded. The conversion of these two forms constitutes the iron sensor referred to above, which in iron replete cells allows ferritin and haem synthesis to take place while in iron deficient cells increases transferrin receptor synthesis.

1.5. Iron and oxidative stress Iron accumulation in tissues, (particularly if the labile

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Fig. 2. Production of transferrin receptor and ferritin is regulated at the level of mRNA by the iron regulatory proteins, IRP-1 and IRP-2. Iron responsive elements (IREs) in the mRNA of the transferrin receptor (TfR) are localized in the 39UTR, and in the 59UTR of ferritin mRNA. The system is designed to allow the cells to procure iron from the plasma, by expressing more transferrin receptors, if they need iron for production of proteins, and to protect cells against potentially toxic iron by expressing ferritin molecules, able to sequester Fe(III) within its core. The system is regulated by IRPs1 and 2. In iron deficiency the IRPs prevent RNase attack of the TfR mRNA, and inhibit production of ferritin by binding to the mRNAs encoding these proteins. If cellular iron levels are high, the IRPs no longer bind to mRNA resulting in the destruction the TfR mRNA and allowing translation of the ferritin mRNA. From Ref. [1].

iron pool is increased), is associated with tissue damage. Such low molecular weight iron can act as a catalyst in the Fenton reaction to potentiate oxygen toxicity by the generation of a wide range of free radical species, including hydroxyl radicals, OH ? Fe 21 1 H 2 O 2 → Fe 31 1 OH 2 1 OH ? Hydroxyl radicals are the most reactive free radical species known and have the ability to react with a wide range of cellular constituents, including amino acid residues and purine and pyrimidine bases of DNA, as well as attacking membrane lipids to initiate a free radical chain reaction known as lipid peroxidation. It is clear that reactive oxygen species, ROS, are generated within the cell as part of normal cellular mechanisms and that the cell is adequately provided with a range of cytoprotective enzymes and antioxidants to combat their toxicity. Such protective mechanisms do not act independently of each other but rather function co-operatively in the form of a cascade (Fig. 3). When marginally higher levels of ROS are generated in eukaryotic cells, certain response mechanisms are activated in order to combat their harmful effects. These effects may involve changes in gene expression in the cell, i.e., transcriptional changes. NFkB was the first transcriptional factor shown to respond directly to oxidative stress. In most cell types NFkB is present in the cytosol as an inactive heterodimer (p50 / p65 or p50 / c-rel) bound to a third subunit IkB. Removal of this inhibitory subunit is the

signal for the translocation of the transcription factor to the nucleus to bind to consensus DNA recognition sequences. NFkB plays a key role in the regulation of numerous genes involved in pathogen responses and cellular defence mechanisms (Fig. 4). These include many immunologically relevant genes, cytokines and cytokine receptors, growth factors and cell adhesion molecules, each of which contain functional NFkB binding sites in their promoter and enhancer regions. Increased accumulation of tissue iron has been associated with pathogenesis in a variety of diseases although the extent of any toxicity will, in part, be dictated by the localisation of the iron complex within the cell, i.e., cytosolic or lysosomal, its biochemical form, i.e., ferritin or haemosiderin [17], as well as the ability of the cell to prevent the generation and propagation of free radical species by the wide range of antioxidants and cytoprotective enzymes present in that cell [18].

1.6. Iron and reactive nitrogen species The reactive nitrogen species nitric oxide NO, formed from the nitric oxide synthase, iNOS, plays an important role in the cell as an antimicrobial agent. Its reaction with superoxide will form the peroxynitrite species, ONOO 2 which is extremely toxic to the cell. The influence of increasing cellular iron content has not be studied to any particular extent in vivo but may alter the ability of iNOS to adequately respond to cellular requirements. iNOS expression is controlled by free iron within the cell, in

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Fig. 3. Cascade showing co-operation between cytoprotective enzymes and antioxidants for scavenging reactive oxygen and reactive nitrogen species. From Ref. [1].

vitro iNOS transcription is lowered when iron loading is high with the opposite effect when iron content is low [19,20]. This is reputed to be due to the presence of an iron sensitive regulatory, NF-IL6 binding site, which is upstream on the iNOS gene [21].

1.7. Importance of cell type in susceptibility to oxidative stress Over the past years the importance of cell type in determining the extent of iron induced oxidative stress has become apparent. Cells, such as liver hepatocytes, which have high antioxidant protection will be less susceptible to iron, whereas those that have less, like many brain cells, will be more sensitive to iron catalysed oxidative stress (Table 1 and Table 2).

1.7.1. Hepatocytes Because of the high rate of metabolism in these cells,

high cytoprotection is encountered. Reduced glutathione, one of the major antioxidants is synthesised in hepatocytes. In various iron loading conditions, e.g., genetic haemochromatosis and thalassaemia, the hepatic iron content may increase 20–40-fold, with iron accumulating in the lysosomes of hepatocytes as haemosiderin, the degradation product of ferritin [16]. Such iron accumulation maybe associated with hepatic malfunction and fibrosis, although our previous studies showed no direct relationship between hepatic iron content and the extent of hepatic fibrosis in such patients [22]. This may be related in part to the high expression and translation of cytoprotective enzymes in hepatocytes, (Table 1) as well as to the extensive biosynthesis of glutathione (GSH), Table 2, such that ROS can be very efficiently scavenged.

1.7.2. Macrophages Macrophages are present in many tissues, including the liver (Kupffer cells), lungs (alveolar macrophages) spleen

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Fig. 4. Proposed mechanism for induction of NFkB proteins. From Ref. [1].

(free and fixed macrophages), nervous tissue (microglia), connective tissue (histiocytes), and the serous cavity (pleural and peritoneal macrophages). This class of cells has two important functions, firstly to orchestrate the host inflammatory response and secondly, in the regulation of iron homeostasis.

The role of the macrophages in iron homeostasis is critical as macrophages, predominantly in the spleen, are able to phagocytose senescent erythrocytes. Haem oxygenase is then involved in degrading haem, catabolising haem to Fe 21 , the bile pigment biliverdin, and the gaseous cellular messenger carbon monoxide. In the macrophage,

Table 1 Activities of cytoprotective enzymes in a variety of rat tissues Units / g tissue

Liver Spleen Brain Heart (aerobic muscle) Plantaris (anaerobic muscle)

SOD

Catalase

GPx

GRed

12,00063120 20226424 16796246 20606703

47.569.5 2.060.7 0.0460.002 0.9760.15

41.1612.3 11.261.7 0.6960.08 11.262.1

6.5962.6 1.760.54 2.6760.03 1.2860.52

0.0860.02

0.5460.39

0.4260.14

3986144

Results are mean6standard deviation of at least six determinations in different animals of body weight 201–300 g (from Ward et al. [18]). SOD: Superoxide dismutase; GPx: glutathione peroxdase; GRed: glutathione reductase.

R.R. Crichton et al. / Journal of Inorganic Biochemistry 91 (2002) 9 – 18 Table 2 Antioxidant content in a variety of rat tissues

Liver Spleen Brain Heart (aerobic muscle) Plantaris (anaerobic muscle)

a-Tocopherol (nmol / g tissue)

Glutathione (mmol / g tissue)

38068.5 62.4627.5 22.461.4 46.6620.9

5.662.1 3.0160.22 0.9060.09 2.6362.09

13.863.3

1.3360.23

Results are mean6standard deviation of at least six determinations in different animals of body weight 201–300 g (from Ward et al. [18]).

this free iron is incorporated into ferritin or released into the circulation to be bound by transferrin. It is crucial that the macrophage is able to retain iron during periods of adequate iron nutrition but be able to release it during times of iron paucity. Recent evidence of recurrent infection in patients with secondary iron loading syndromes indicates that the presence of excessive amounts of iron in the macrophage may adversely interfere with their role in the activation of NADPHoxidase and the generation of a wide range of reactive oxygen species. Inflammatory stimuli are associated with the activation of NADPH oxidase when a number of cytoplasmic proteins translocate to join the membrane bound component, a b-type cytochrome, resulting in the formation of a multi-component electron transfer system, which catalyses the reduction of molecular oxygen at the expense of NADPH. This is often referred to as the respiratory burst, whereby increased cellular oxygen uptake occurs, resulting in the generation of superoxide (O 2 2 ) as well as hydrogen peroxide and hydroxyl radicals. The production of NO is one of the major mechanisms in the macrophage’s defense against invading microorganisms. NO is synthesised from the enzyme iNOS (inducible nitric oxide synthase) within the macrophage. It is assumed that iNOS is cytoplasmically located, however, immuno-electron microscopic and biochemical analyses of primary mouse macrophages showed that one half of iNOS activity could be sedimented, in the form of vesicles (50–80 nm) that did not correspond to lysosomes or peroxisomes. It was suggested that these iNOS-positive vesicles may translocate to phagosomes [23]. The peroxynitrite anion (ONOO +), produced by the reaction between NO and superoxide anion, is important for killing bacteria and other microrganisms. Excessive production of peroxynitrite, however, can damage normal tissue by oxidation and nitration. In our recent studies we have investigated whether iron loading in vivo will alter the capacity of macrophages to respond to infection. Rats were iron loaded by repeated intraperitoneal injections of iron dextran (total iron5125 mg) in order to iron load macrophages [24] (Table 2). The iron content of the alveolar macrophages increased approx-

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imately twofold, from 16 to 29 ng / mg protein. However when primary cultures of these cells were stimulated with LPS / IFNg for 44 h, NO production was significantly reduced in the iron loaded macrophages [24]. Since, as already stated, there are NFkB binding sites on the iNOS gene, for the LPS and IFNg stimulants, the activation of this transcription factor was assayed in the nuclear pellet of iron loaded and control macrophages. Fig. 5 shows that iron loading alone induced NFkB while further activation with LPS / TNFa was unable to initiate any further increase [25]. In contrast, control macrophages showed a twofold increase in NFkB activation after stimulation. Such results indicate that iron accumulation may severely compromise macrophage function with respect to both NFkB activation and NO production. In addition, the excessive production of ROS observed by chemiluminescence (results not given), might indicate an imbalance in the ability of the cell to control production of ROS.

1.7.3. Brain As emphasized in Table 1 and Table 2, brain cells, including neurons, astrocytes and microglia, show a decreased ability to respond to oxidative stress, particularly with respect to their levels of glutathione and glutathione peroxidase, such that alteration in their iron status may predispose them to iron-induced oxidative stress. It is clear that with ageing there is a significant increase in iron stores in the brain [26] which, if localised in susceptible regions, could contribute to the pathogenesis of various neurodegenerative diseases. Astrocytes provide protection and trophic support to neurons, but like neurons are susceptible to oxidative stress. Decreased function of astrocytes resulting from oxidative stress could contribute to neurodegeneration. Iron-loading increases peroxide-induced oxidative stress in astrocytes, but induction of NO limits the effect of iron, suggesting an interaction between iron and NO [27].

Fig. 5. Activation of the nuclear transcription factor NFkB in control and iron-loaded alveolar macrophages with or without stimulation. From Ref. [37].

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1.8. Haem oxygenase—an important enzyme in oxidative stress Haem oxygenase 1 (HO-1) is an inducible enzyme that catalyses the rate limiting step in the degradation of haem to biliverdin, carbon monoxide and iron. Activation of HO-1 is an ubiquitous cellular response to oxidative stress [28]. Recent studies have suggested an important role for haem oxygenase 1 in the control of iNOS. Haem oxygenase was induced in bone-marrow derived macrophages by short term exposure to haemin, after which the cells were exposed LPS. NO production was decreased. It was therefore proposed that HO-1 inhibits iNOS in activated macrophages by decreasing haem availability for NOS synthesis [29]. HO-1 is considered to be protective against oxidative stress and its expression has been described in microglia, astrocytes and neurons in various pathological alterations in the brain. HO-1 is significantly increased in scrapie-infected animals, compared to an age matched control group, thereby suggesting that oxidative stress is closely associated with the pathogenesis of scrapie and might contribute to neurodegeneration in this disease [30]. Oxidative stress is also important in the pathogenesis of Parkinson’s disease. Altered iron content in the substantia nigra pars compacta is associated with impaired mitochondrial function, alteration in the antioxidant protective systems, namely SOD and GSH, as well as oxidative damage to lipids, proteins and DNA [31]. The increased iron is present in the form of H-ferritin and neuromelanin [26]. The exact role played by neuromelanin in the brain remains unclear, but it could be similar to haemosiderin, limiting the amount of iron available for free radical production [32]. Oxidative stress has also been proposed as a pathogenetic mechanism in Alzheimer’s disease. One mechanism of oxidative damage is the nitration of tyrosine residues in proteins, mediated by peroxynitrite breakdown [33]. Alterations in iron homeostasis have also been implicated in Alzheimer’s disease, possibly due to stabilisation of IRP-2 by aluminium (reviewed in Refs. [34,35]).

and cerebral cortex [37]. In the studies described here, male rats (Wistar strain, 100 g) were adapted to a powder diet containing THF (1 g / kg diet, with an iron content of 0.35 g Fe / kg) for 4 weeks. Subsequently they were administered an iron chelator (either i.p., 10 mg / kg, in the case of desferrioxamine B or by gavage, 30 mg / kg for CP24 (1-n-butyl-2-methyl-3-hydroxypyrid-4-one) and CP94 (1,2-diethyl-3-hydroxypyrid-4-one) every second day for 14 days. After iron loading with THF for 4 weeks, the iron contents of different brain regions was determined. Highest levels were found in the cerebellum (148 mg / g), cerebral cortex (119 mg / g) and striatum (119 mg / g) followed by substantia nigra (107 mg / g), hippocampus (78 mg / g) and brain stem (70 mg / g). After 2 weeks chelation with desferrioxamine, CP24 or CP94, some reduction of brain iron was seen in most brain regions (Fig. 6). The most effective result was obtained with desferrioxamine B in cerebellum, where an almost 60% decrease in iron content was observed. There was little difference between the two hydroxypyridones. Similar results were found with CP20 (Deferiprone  ). It should be noted that significant alterations in the levels of dopamine and its metabolites in the striatum were observed after CP94 treatment [37], which might reflect coordination of the labile active site iron of the key enzyme of dopamine metabolism, tyrosine hydroxylase. Similar effects were observed with acute administration of CP20 or desferrioxamine B at 100 mg / kg [37] but no such effects were observed with the orally active tridentate siderophore desferrithiocin or its desmethyl derivatives [38]. High concentrations of brain aluminium can be achieved by intraperitoneal injections of aluminium gluconate [39] for 8–12 weeks. This animal model was used to compare the efficacy of different chelators in mobilising brain aluminium and to establish whether perturbations of iron homeostasis occurred as brain aluminium concentration increased [40]. Chelator treatment was as above for

1.9. Importance of iron chelators to reduce oxidative stress There have been many attempts to find animal models which mimic iron overload in man in order to test new classes of iron chelators which are orally active, unlike the currently utilised desferrioxamine (Desferal  ). The iron containing compound 3,5,5-trimethylhexanoylferrocene (THF) is not absorbed in man, but in rodents goes directly to the liver where the iron is released and made available for haem synthesis and hepatic storage in ferritin and haemosiderin [36]. It also crosses the blood–brain barrier, resulting in significant iron loading in a number of specific locations in the brain, such as substantia nigra, cerebellum

Fig. 6. Iron mobilisation from ferrocene iron-loaded brain regions by the iron chelators desferrioxamine, CP24 and CP94. Results are expressed as a percentage of the untreated iron-loaded control group.

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References

Fig. 7. Aluminium mobilisation from aluminium gluconate-loaded brain regions by the chelators desferrioxamine, CP20 and CP94. Results are expressed as a percentage of the untreated aluminium-loaded control group. 15Frontal cortex; 25temporal cortex; 35parietal cortex; 45 hippocampus.

ferrocene iron-loaded animals, except that CP20 (1,2-dimethyl-3-hydroxypyrid-4-one) was used instead of CP24. In animals which had been loaded with aluminium gluconate, significant increases in brain iron content were also found, notably in various parts of the cerebral cortex (from four- to ninefold) and the hippocampus (fourfold) compared to untreated controls [40]. Aluminium contents varied from 14 to 25 mg / g wet weight in the cerebral cortex to 37 mg / g in the hippocampus. The results of aluminium chelation (Fig. 7) were significantly greater than for iron, particularly with the more hydrophobic hydroxypyridone CP94 which reduced aluminium levels in all four brain regions examined to 10% or less.

2. Conclusions and perspectives In conclusion, it seems that changes in iron homeostasis are a major contributary factor in a large number of diseases, both neurological and others. Long term oral chelation therapy at low doses could prevent the accumulation of both iron (and aluminium) in the brain as a function of ageing thus reducing the incidence of metal associated oxidative stress. This could be a promising approach to improving the quality of life of the ageing population and perhaps even more importantly could contribute to retarding the onset of the numerous neurological disorders associated with ageing, thus delaying the shipwreck of old age.

Acknowledgements This research has been funded by the EU grant QLK11999-00337.

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