Literature Review BI447

Academic Year 2014/15

THE IMPORTANT CONTRIBUTION OF IRON & OXIDATIVE STRESS TO CELL

DEATH

The important contribution of Iron & Oxidative Stress to Cell Death

Name: David Kirrane

ID Number: 11419558Academic Supervisor: Prof. Afshin SamaliCourse: Undenominated Science.Word Count: 4834

ABSTRACT

Iron is an important transition metal required for diverse biological functions. High levels of iron can create pro-oxidant conditions of oxidative stress characterised by the formation of excessive reactive oxygen species (ROS). It has been observed that elevated levels of iron and ROS play roles in the induction of diverse cell death pathways including a new cell death phenotype that requires the presence of iron known as ferroptosis. Although progress has been made to elucidate their cytotoxic effects, a further comprehensive understanding of how iron and oxidative stress contribute to cell death is required as these factors are often implicated in the pathogenesis of diverse diseases.

INTRODUCTION

Iron is an essential element in life processes being the most abundant transition metal in the body [1]. Iron has roles in the transport of oxygen and is a component of many enzymes including the class of oxidase enzymes [5]. While this micronutrient is vital for life, excessive intracellular accumulation of the free or labile redox-active transition metal is toxic to cells and tissues as it is observed to augment the production of reactive oxygen species (ROS) [3]. This increased accumulation of ROS can overwhelm the antioxidant defences in the cell, culminating in pro-oxidant conditions known as oxidative stress [1]. ROS is observed to cause oxidative damage to several biomolecules in the cell namely DNA, proteins and phospholipids [2]. The organelle involved in cellular respiration, the mitochondria, has been identified as the major source of the endogenous toxic partially reduced oxygen species [2]. As iron and ROS accumulate in aberrant, toxic levels within cells, they have been implicated in various disease states such as iron overload disorders [3] as well as severe neurodegenerative disorders [4]. It is thus imperative that the levels of intracellular iron are carefully regulated by iron homeostasis at a systemic and cellular level so that there is a sufficient provision of this transition metal to all cells while preventing deleterious superfluous iron accumulation [3]. It is the disruption of the balance between safe iron and oxidant levels in the body that is the prelude to many of the aforementioned chronic pathological conditions and degenerative states [3].

Of particular interest to this review is the particular role that iron and oxidative stress induced by ROS play in causing cell death. Various lines of scientific research have provided considerable evidentiary support that iron and ROS act in the induction and mediation of cell death [5]. The body of scientific literature in the accompanying bibliography of this literature review will attest to this observation. Increased cellular iron and ROS levels have been observed to contribute to the induction of apoptosis, necrosis and autophagic cell death which will be discussed later in this review. A new form of cell death that is iron-dependent and is a form of oxidative death, that is distinct from the established apoptotic, necrotic and autophagic death mechanisms [6]. This form of cell death is known as ferroptosis [6]. Improved understanding of how iron and oxidative stress contribute to cell death is of upmost importance in treating diverse pathological states, injuries, traumas and neurodegenerative disorders that are linked to these putative causative factors of cytotoxicity [5]. The role of iron chelation therapy in treating iron overload disorders and other degenerative disorders

will be discussed [7].There will also be a discussion of the new therapeutic approaches to treat these diseases such as specialised antioxidants that are targeted to cellular locations that reduce the accumulation of oxidants [5]. Another new therapeutic intervention that is showing potential is small molecules that enhance ROS intracellular accumulation that have been demonstrated to be cytotoxic to different cancers in vitro [5]. However there needs to be a greater elucidation of how iron induces cell death and the molecular targets of ROS that are involved in these cell death pathways [5].

Iron Importance in Life Processes

Iron in Biology

Iron is one of the most abundant transition metals in the body [3]. It acts as an important micronutrient and cofactor a variety of physiological processes that are necessary to sustain life. This transition metal is found in many protein complexes such as in iron sulphur complexes (i.e. the 4Fe-4S complex) and catalyses electron transport that results in the production of energy. Iron also can participate in oxidation and reduction reactions as it can switch between different oxidation states [9]. It also is an essential cofactor for the synthesis of DNA as it is an essential component of the enzyme ribonucleotide reductase [9]. Iron is also stated to have a role in regulating gene expression and is an integral component of the active sites of oxidases & oxygenases such as NADH oxidase (NOX), xanthine oxidase, lipoxygenase, cytochrome P450 enzymes [5].

The best known function of iron in biological systems is its transport of oxygen as it is incorporated in the heme prosthetic group of haemoglobin [9].

Iron catalyses conditions of oxidative stress

Reactive Oxygen Species (ROS), cellular antioxidant defences and Oxidative Stress

Reactive Oxygen Species (ROS) are chemically reactive intermediates of oxygen that represent partially reduced oxygen radical species [11]. These include superoxide anion (O2

-), Hydrogen peroxide (H2O2), organic hydroperoxides, alkoxy and peroxyl radicals as well as the extremely reactive hydroxyl radical (HO●) [5, 13]. A free radical is defined as a chemical entity that possesses a reactive and unstable lone pair of electrons in its valence orbital [11]. An important endogenous source of ROS is the mitochondrion [2, 3]. The majority of cellular ROS are produced indirectly as a consequence of aerobic respiration as the components of the electron transport chain can leak electrons to O2 prematurely ahead of its tetravalent reduction to water by cytochrome oxidase [2]. Complex I and Complex III of the electron transport chain produce small amounts of O2

- as around 2% of molecular oxygen is reduced to this intermediate. The production of O2

- is a single electron or univalent reduction of molecular oxygen and it represents the initial molecule in ROS formation [2]. NOX enzymes can also catalyse the production of O2

- [2]. O2- is relatively stable oxygen radical intermediate with

limited reactivity that is rapidly converted or dismutated into H2O2 by the detoxifying enzyme Superoxide Dismutase (SOD) [3]. H2O2 has a limited reactivity but can diffuse

through biological membranes [3]. The reaction of O2- with H2O2 via the Haber-Weiss

reaction or the reaction of H2O2 with free divalent transition metals results in the production of the cytotoxic HO● [3]. This toxic radical is reactive and causes oxidative damage to various biomolecules such as nucleic acids, proteins, lipids [2]. Reactive Nitrogen Species (RNS) such as Nitric Oxide radical and peroxynitrite (ONOO-) are also formed in the cell [3,]. Other enzymatic sources of ROS include xanthine oxidase , monoamine oxidase, lipoxygenase, cytochrome P450 [5 , 13].

Various enzymatic and non-enzymatic antioxidant defences exist that scavenge ROS and maintain low concentrations of these oxidants [13]. Detoxifying enzymes include the H2O2 inactivators such as catalase, peroxiredoxin, glutathione linked enzymes such as glutathione peroxidases (Gpx 1 & Gpx4) that reduce H2O2 and lipid peroxides, glutathione reductase, thioredoxin & SOD [2, 3, 13]. Non-enzymatic antioxidants that neutralise and remove excess ROS include water soluble antioxidants such as glutathione, ascorbic acid that react with ROS in the cytoplasm. Also there exist lipophilic antioxidants such as Vitamin E and α-tocopherol that scavenge lipid peroxides produced at cellular membranes as a result of oxidant induced membrane damage [13]. An imbalance that results in excessive ROS formation that exceeds the cellular antioxidant, detoxifying defences results in pro-oxidant conditions called oxidative stress [1, 3]. As we will see intracellular iron levels play a role in mediating cellular oxidative stress.

Figure 1. a & b. The formation and detoxification of Reactive Oxygen Species (ROS) from enzymatic sources. c. Iron chelators used in clinical studies as a therapeutic intervention and in experimental studies on the role of iron in cell death. Taken from [5].

Excessive levels of free iron can catalyse Oxidative Stress

Iron exists in cellular conditions predominantly in two oxidation states the oxidised Ferric ion (Fe3+) and the reduced Ferrous iron (Fe2+) and readily converts between these two oxidation states in aqueous solutions and is thus redox active [7]. In normal physiological circumstances a labile iron pool (LIP) is found in the cytosol with redox-active poorly coordinated iron chelated to low molecular weight compounds such as ATP, citrate, pyrophosphates. The levels of LIP are a reflection of the cellular iron status [3]. Excessive amounts of free redox-active iron ions can participate in oxidation and reduction reactions and therefore are involved in dangerous Fenton chemistry reactions in which ferrous ions react with hydrogen peroxide to produce toxic hydroxyl free radicals [1, 3].

Fe2+ + H2O2 Fe3+ + ●OH + OH- [3].

As a result of this potentially hazardous property of iron, iron content must be carefully regulated at a cellular and systemic level. Specialised proteins exist that are involved in iron uptake, storage and release [1, 3 ,10]. Iron is absorbed from the intestine by enterocytes at the apical membrane and are first reduced by the ferrireductase duodenal Cytochrome B from ferric to ferrous iron that is then transported into the enterocytes by Divalent Metal Transporter 1 (DMT1) [1, 3, 10]. Iron efflux is facilitated by the iron exporter protein ferroportin as iron is released from the basolateral membrane into plasma. As Fe2+ is released into plasma it is oxidised by the ferroxidases hephaestin and ceruloplasmin to Fe3+. Two molecules of Fe3+ then bind with high affinity to the plasma iron carrier protein transferrin (Tf). Diferric Transferrin delivers iron into cells by binding to the cell surface transferrin receptor (TfR) and the Fe-Tf-TfR complex enters the cell by receptor mediated endocytosis and releases reduced Fe2+ from the acidic endosome into the cytoplasm. Large amounts of free iron are sequestered by the cellular iron storage protein Ferritin [10]. This endogenous iron chelator stores up to 4,500 atoms of Fe and consists of 24 subunits of Heavy chain (H-chain) and Light Chain (L-chain) [1, 3]. The H-chain has intrinsic ferroxidase activity that maintains iron in its nontoxic state while the L-chain has a nucleating centre that stores the iron atoms [1, 3]. The peptide hormone hepcidin regulates iron release by binding to ferroportin and causing ferroportin to endocyse into the cell and targets it for lysosomal degradation [10]. As there currently is no knowledge of the existence of an effective cellular iron excretory pathway, free iron can accumulate and cause deleterious effects to the cell by mediating oxidative stress [3]. Iron overloading is associated with many diseases typified by deregulated iron homeostasis, excessive iron absorption by the intestine , cirrhosis of the liver, hepatotoxicity, fibrosis, cardiomyopathy and endocrine disorders [1, 3, 5].Hereditary hemochromatosis, juvenile hemochromatosis are examples of these iron overload diseases [1, 3, 5].

Iron and Oxidative Stress in Cell Death

Of particular interest to this review is the apparent role that iron and ROS have in mediating cell death. The observation that iron and oxidative stress play a role in inducing cell death was determined in many experimental studies in which the cell death phenotype was prevented or attenuated by iron chelators and antioxidants. This field of study is still under active investigation but has made progress in recent times by key new discoveries particularly a new death phenotype that is iron dependent known as ferroptosis [6].

Cell Death

Cell death can be classified by morphological and biochemical features [14, 15]. Apoptosis is a form of programmed cell death (PCD) with morphological features such as reduction in cell size, membrane blebbing, chromatin condensation, DNA fragmentation, formation of apoptotic bodies with phosphatidylserine translocation to the outer leaflet of the plasma membrane [14, 15]. There are two pathways regulating apoptosis the intrinsic pathway( mitochondrial) and the extrinsic or death receptor pathway. The intrinsic pathway of apoptosis involves proapoptotic members of the Bcl-2 protein family such as Bak and Bax translocating from the cytosol to the mitochondria and inducing mitochondrial outer membrane permeabilisation (MOMP). This causes a release of cytochrome c from the mitochondria which complexes with Apaf-1 in the apoptosome which activates the initiator caspase 9 which subsequently activates downstream effector caspases that execute the death program [15]. The extrinsic pathway involves ligands such as Fas and TNFα binding to the TNF superfamily of death receptors with recruitment of death domain containing adaptor proteins TRADD, FADD and pro-caspase 8 to form the Death Inducing Signaling Complex (DISC). This signalling eventually causes the activation of downstream caspases which cleave molecular targets [15]. Autophagic cell death is characterised by grand engulfment of cytoplasmic constituents into a large double membrane enclosed vesicle called the autophagosome without chromatin condensation. The autophagosome subsequently fuses with the lysosome forming the autolysosome which degrades organelles and macromolecules by lysosomal hydrolytic enzymes [15].

Necrosis is an accidental form of cell death caused by injury. It is characterised by a gain in cellular volume, swelling of organelles, rupture or lysis of the plasma membrane and release of cytoplasmic constituents into the surrounding extracellular fluid eliciting an inflammatory response [ 14, 15].

The Cytotoxicity of Oxidative Stress

Conditions of oxidative stress can be detrimental to cellular viability as excessive ROS or Reactive Nitrogen Species (RNS) can cause damage to various tissues by oxidatively modifying proteins, nucleic acids and lipid peroxidation of biological membranes. The noxious effects of these reactive species can activate various cell death signalling by regulated physiological means or as a result of trauma (apoptosis and necrosis respectively) [16]. Basal levels of ROS are important for physiological signal transduction pathways but aberrant levels of these oxidants have been implicated in diverse pathologies and injuries such as cardiovascular disease, neurodegenerative diseases and inflammatory diseases [16]. Cell death induced by ROS or RNS can come about by causing the loss of mitochondrial function and membrane integrity [16]. Ischemia/reperfusion injury in which there is an initial restriction of blood flow resulting in hypoxic conditions followed by reoxygenation. Reperfusion causes production of excess ROS which can kill cells by necrotic and apoptotic death pathways [16].

As mitochondria are the principal sources of intracellular ROS they are also sensitive targets of oxidative damage as the mitochondrial DNA is close to the electron transport chain where ROS are produced and can become mutated [2]. This can cause loss of mitochondrial function. ROS have been demonstrated to facilitate the opening of the mitochondrial permeability transition (MTP), a pore in the inner mitochondrial membrane that causes ATP depletion, changes in mitochondrial morphology, loss of mitochondrial membrane potential and necrosis [2]. Mitochondrial ROS have been implicated in cell death as increased superoxide production occurs by a mutation in the ribosomal 12S rRNA, the A1555G mutation. This causes the ribosome to be hypermethylated which interferes with normal mitochondrial translation. The superoxide anions activate a signalling pathway from the mitochondria to the nucleus involving the activation of AMPK which upregulates the transcription factor E2F1 that increases the expression of proapoptotic genes [5]. This cell death was prevented experimentally by the overexpression of the detoxifying mitochondrial SOD enzyme establishing ROS as the causative factor of cell death [5]. Loss or depletion of mitochondrial antioxidant enzymes such as MnSOD, Gpx4, peroxiredoxin, Trx2 reduces cellular viability and can cause embryonic lethality [2]. Cytochrome c is released from the inner mitochondrial membrane into the cytosol when its bound partner the negatively charged lipid cardiolipin is oxidised by the peroxidase activity of cytochrome c [2]. ROS such as H2O2

enhances or facilitates the cytochrome detachment by oxidising cardiolipin as large amounts of ROS produced by the cleavage of the p75 subunit of mitochondrial electron transport complex I by caspases coordinate with cytochrome c release [5]. ROS accumulation causes TNFα mediated cell death by oxidising the catalytic cysteines of the MAP kinase phosphatases to sulfenic acid, inhibiting their function and prolonging the activation of c-Jun N-terminal kinases (JNK) [17]. This event causes apoptotic and necrotic cell death that is prevented by antioxidants such as butylated hydroxyanisole (BHA) [17].

A mechanism of how oxidative stress induces cell death has been proposed in mice that are depleted of the lipid peroxide scavenging enzyme Glutathione peroxidase 4 (Gpx4) [18]. The loss of this enzyme causes extensive lipid peroxidation mediated by 12/15 lipoxygenase which causes a caspase independent cell death with apoptosis -inducing factor (AIF)

translocation from the mitochondria to the nucleus causing DNA fragmentation and lethality [18]. Glutathione (GSH) is a major water soluble antioxidant produced in the body of diverse organism that acts to eliminate intracellular oxidants and prevent oxidative stress [12, 15]. Glutathione is a tripeptide molecule composed of the amino acids cysteine, glycine and glutamate (γ- glutamyl-L-cysteinylglycine) [12, 15]. The rate at which GSH is synthesised is dependent on the availability of cysteine as the thiol group acts as an electron donor for GSH linked enzymes [12, 15, 18]. The function of GSH is to maintain a reducing environment in the cell to retain normal cellular redox homeostasis by engaging in thiol disulphide exchange reactions, reducing disulphide bonds in oxidised proteins [15, 18]. Depletion of glutathione by impaired synthesis (cysteine depletion, treatment with GSH depleting agents, absence of GSH synthesising and reducing enzymes) or by its oxidation to GSSG by oxidants can lead to oxidative stress and cell death [12, 15]. GSH depletion is observed in many diseases associated with oxidative stress such as Parkinson’s disease [12, 15].

The Cytotoxicity of Iron

Labile redox-active iron has been established as a critical inducer of cytotoxicity as the lysosomal compartment is the principal source of this harmful pool of iron in the cell due to its autophagic catabolism of iron containing cellular material such as ferritin and metalloproteins [5, 19]. Labile ferrous iron reacts with H2O2 that diffuses into the lysosomal compartment to initiate apoptotic & necrotic cell death by OH● induced lysosomal membrane peroxidation, destabilization & rupture, releasing acidic hydrolases, Fe2+, ROS into the cytosol to initiate apoptosis or necrosis [19]. Treatment with the anticancer agent doxorubicin (DOX) causes an intracellular accumulation of ROS, killing cancer cells as well as endothelial cells by apoptosis [20]. It has been shown that apoptosis is induced by increased expression of Transferrin Receptor and increased uptake of cellular iron as treatment with cell permeable iron chelators such as dexaroxane and cell permeable antioxidants such as ebselen result in inhibition of cellular iron uptake, oxidant formation & apoptotic cell death and reduced TfR expression [20]. Also treatment with the anti-TfR monoclonal antibody IgA prevented iron uptake, oxidative stress and cell death.

Acidic H-chain isoferritin has been demonstrated to induce apoptotic cell death in hepatocytes [21]. Iron and oxidative stress were exhibited to play important roles in this ferritin-induced death as treatment with the iron chelator desferrioxamine (DFO) and the antioxidant Trolox significantly protected against apoptotic and necrotic death. These agents reduced the formation of 4-hydroxynonenal (a lipid peroxide) - modified proteins and micronucleated cells that are characteristic of oxidative DNA damage [21]. Endocytosis of ferritin and its degradation by the lysosomes increasing the labile redox active iron pool and causing lysosomal membrane permeabilisation has been reasoned to be the mechanism behind this ferritin-induced cell death pathway [21].

Figure 2. The proposed mechanism of how ferritin induces cell death by its endocytosis and lysosomal degradation increasing the labile redox-active iron pool and inducing apoptotic and necrotic death by catalyzing oxidative stress. Taken from [22].

Necrotic cell death was induced in rat hepatocytes after they were cold incubated and then rewarmed after an extended period [21] as evidenced by lactate dehydrogenase (LDH) leakage into the cell culture, substantial cell membrane damage, ATP depletion and lack of morphological and biochemical features of apoptosis. Iron and oxidative stress were shown to be pivotal in inducing necrosis as DFO and a host of antioxidants incubated before cold storage – N-acetyl cysteine, reduced glutathione and Trolox abrogated this cell death [22]. Also the sensitivity of skin cells to UVA-induced necrotic death was determined by their intracellular levels of labile redox active iron as keratinocytes possess significantly lower levels of basal labile iron levels and lower levels of labile iron released after UVA irradiation than fibroblasts. Keratinocytes showed stronger resistance to necrosis than fibroblasts and the link was established between their respective labile iron levels and extent of susceptibility to necrotic cell death [23]. Iron release from the lysosome and its translocation to the mitochondria facilitated the opening of the MTP that causes cyclophilin-dependent necrotic death and accounts for the ability of iron chelators to mitigate ischemia –reperfusion necrotic death [5]. The entry of iron into primary mouse cortical neurons via Divalent Metal transporter 1 (DMT1) was shown to induce excitotoxic cell death upon treatment of N-methyl-D-aspartate. The oxidative stress and resulting cell death may be enhanced by generation of ROS by mitochondrial NOX enzymes [5].

Infusion of ferrous chloride into the rat brain has been shown to induce brain injury by autophagic cell death [24]. Ultrastructural features of autophagic cell death such as autophagosomal vesicles as well as increased conversion of the mammalian protein homolog of the yeast autophagy gene ATG8 LC3-II which is associated with autophagosomal membrane from LC3-I (cytosolic) were detected by transmission electron microscopy and Western blotting respectively after ferrous iron treatment in the contralateral cortex [24]. Other markers of autophagy were upregulated such as ATG-5 and Beclin-1 -which is required for autophagosome generation and elongation after ferrous iron injection to the brain. Autophagic cell death has been proposed as a mechanism of iron-induced brain injury in situations such as intracerebral hemorrhage (ICH) as iron accumulates in toxic amounts in the brain after this trauma and in iron overload disorders affecting the central nervous system [24].

Ferroptosis- a new iron-dependent form of cell death

A new form of cell death that is dependent on the presence of iron has recently been proposed [6]. This cell death phenotype can be distinguished on the basis of morphology, biochemistry and genetics from apoptotic, autophagic and some forms of necrotic death [5, 6]. This cell death is characterized as an iron dependent oxidative death as there is an accumulation of cytosolic and lipid ROS that overwhelms the antioxidant defenses of the cell. This cell death pathway is called ferroptosis [6]. It has a unique genetic program, unique morphology characterized by a shrunken mitochondria. Treatment with iron chelators (DFO, Ciclopirox olamine) and ROS scavenging antioxidants (Trolox) were shown to prevent this cell death [6]. This lethal pathway was discovered as a result of small synthetic compounds such as Erastin, sulfasalazine inducing selective killing of mutagenic RAS cell lines [6]. The precise mechanism of how Erastin, sulfasalazine induce ferroptotic cell death is the inhibition of the system xc

- cystine/glutamate antiporter located on the cell surface. This prevents in the uptake of cystine, the oxidized form of the amino acid cysteine into the cell. Interruption of cystine cellular entry deprives glutathione of its reactive thiol, reducing its synthesis [5, 6]. Loss of GSH results in the inability of the cell to effectively defend the cell against excessive oxidant formation sensitizing the cell to oxidative stress and cell death [5, 6]. The precise role that iron plays in this cell death pathway has yet to be further elucidated but inhibition of iron-dependent NOX enzymes partially prevented ferroptosis in some cancer cell lines [6]. It is possible that an iron-dependent enzyme might be involved in this pathway [6]. Ferroptosis shares some features in common with glutamate induced neuronal death in inhibition of system xc

- and subsequent cystine and GSH depletion and oxidative stress. However the latter cell death is activated by high concentrations of glutamate and involves Ca2+ cellular influx, lipoxygenase activity, mitochondrial fragmentation and release of AIF from the mitochondria [5].

Figure 3. The mechanism of ferroptotic cell death mediated by Erastin and Sulfasalazine and excitotoxic death in cultured neurons induced by Glutamate. Taken from [6].

Iron-dependent nonoxidative cell death

In the yeast S. cerevisiae that possess mutations in the vacuolar iron transporter Ccc1, iron overload induces cell death that cannot be rescued by overexpressing SOD or catalase or growing the cells in anaerobic conditions [5]. This phenomenon indicates that oxidative stress by accumulating excessive ROS may not be a prerequisite for cell death pathways that are iron-dependent [5]. This nonoxidative cell death may occur by iron incorrectly binding to an essential enzyme (enzyme mismetallation), inactivating it and triggering cell death. Another explanation is that high levels of iron activates a serine-palmitoyl transferase complex (SPT) resulting in the production of sphingolipid long chain bases and long-chain phosphates. The sphingolipid accumulation results in the activation of a signaling kinase cascade and the eventual outcome- cell death with myriocin and Orm2 overexpression inhibiting this process [5]. However the details behind this cell death pathway remain unclear.

Figure 4. Iron overload induces cell death in S. cerevisiae without an accompanying ROS accumulation and oxidative stress. Excessive levels of iron could cause enzyme mismetallation and inactivate the enzyme, triggering cell death. High levels of iron can also activate a sphingolipid long-chain base and long-chain phosphates synthetic pathway that activates a signaling cascade that induces cell death. Taken from [5].

Therapeutic interventions

Iron Chelation Therapy

Multiple acute diseases and traumas are associated with dyshomeostasis of iron with cellular iron accumulating to high toxic pro-oxidant levels that cannot be excreted from the body [3, 5, 7]. An attractive therapeutic approach is to remove or deplete excess free intracellular iron from the body by coordinating this metal to chelating agents that are excreted from the body in this complex [25]. This is the basis of Iron Chelation Therapy. Iron chelators have been discussed above in their use in experimental studies to elucidate the role of iron in mediating cell death.

However iron chelators have shown to have beneficial uses in clinical studies in ameliorating diverse iron-induced injuries and pathologies [5, 7, 25]. Iron chelators are used in the treatment of secondary iron overload in patients with thalassemia anemia that receive repeated red blood cell transfusions [7, 25]. Deferoxamine (DFO) was the first clinically relevant iron chelator that is a hexadendate ligand that binds Fe (III) ions. It is a hydrophilic ligand that enters the cell by endocytosis and primarily localizes to the lysosomal compartment [3]. This chelator is administered intravenously and has a harsh regimen which is the reason why orally bioavailable iron chelators are favoured over DFO [7, 25]. Oral iron chelators such as the tridendate ligand Deferasirox and the bidendate ligand Deferiprone have been subsequently developed and have been used widely as therapeutic interventions in iron overload diseases & neurodegenerative diseases [7, 25]. Iron mismanagement or deregulated iron homeostasis is implicated in many neurodegenerative diseases such as Parkinson’s disease, Friedrich’s ataxia, Alzheimer’s disease [4, 25, 27]. Numerous iron chelators have been shown to have neuroprotective roles in these pathologies such as DFO and VK-28 preventing substantia nigral dopaminergic neuronal degeneration induced by 6-OHDA neurotoxin treatment as well as VK-28 and M30 prevention of neuronal loss in mouse models treated with UPS [27].

Clioquinol treatment and overexpression of the natural iron chelator H-ferritin has also been demonstrated to protect against neuronal loss or degeneration in Parkinson’s disease [5]. Deferoxamine and deferiprone treatment has been shown to potentially prevent neuronal cell loss in Parkinson’s disease by mitophagy of damaged mitochondria in dopaminergic neurons that produce ROS [5]. Treatment of DFO in ischemia-reperfusion affected hepatocytes resulted in less oxidative damage and prevention of hepatocytic necrosis [28].

For iron chelators to be optimal they must be cell permeable, bind with high affinity and selectivity to iron, cross the blood brain barrier, do not bind to iron containing enzymes and inhibit their function and not bind to other divalent metals disturbing their homeostatic distribution [7, 26]. Iron prochelators such as BSIH are converted to the active cell permeable iron chelator SIH only upon exposure to H2O2, distinguishing the difference between healthy iron and deleterious iron [26].

Mitochondrial targeted antioxidants and ROS producing small molecules

The treatment of various pathologies associated with oxidative stress has relied on manipulating the heightened levels of oxidants to safe physiological levels with the use of ROS scavenging antioxidants [29]. Traditionally these antioxidants employed included vitamin E and Coenzyme Q, however treatment with these antioxidants has only reaped limited effectiveness in ameliorating diseases and oxidative injury. A novel therapeutic strategy exploits the fact that the mitochondria is the major source of ROS production in the cell by designing synthetic antioxidants that preferentially concentrate at the mitochondria. These synthetic antioxidants include an antioxidant moiety conjugated to a positively charged lipophilic triphenylphosphonium ion including mitoVit and MitoQ (contains ubiquinone as the antioxidant) that concentrates the antioxidant to the negatively charged membrane of the mitochondrial matrix [5, 29]. Treatment of MitoQ has shown clinically beneficial effects in mouse model of Alzheimer’s disease [5].

Small molecules such as parthenolide, piperlongumine and phenyl etylisothiocyanate have been demonstrated to kill cancer cells by the formation of excessive ROS, increasing oxidative stress and reducing the synthesis of glutathione by impairment of the glutathione antioxidant defenses in the cell [5]. This is beneficial in targeting various cancers that have highly reduced cellular environments due to high levels of NADPH and high expressions of enzymes that synthesize glutathione [5].

Future Perspectives

Although much progress has been made in elucidating the cytotoxic roles of iron and oxidative stress in diverse cell death pathways, the precise mechanism of how iron is inducing cell death either via excessive ROS generation or by non-oxidative means remains yet to be discovered. Although the role of oxidative stress in cell death in disease states and trauma is firmly appreciated, the source of ROS and its targets in cell death pathways are unclear [5]. The precise mechanism of how iron is mediating cell death either by free radical generation and oxidative damage, by an iron-dependent enzyme or by alternative non-oxidative means remains unclear [5]. The use of investigative probes will enrich and facilitate the study of the roles of iron and oxidative stress in cell death with particular interest in their contribution to iron overload diseases, neurodegenerative diseases, diverse traumas and injuries [5].

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