1. Literature Review BI447
Academic Year 2014/15
THE IMPORTANT CONTRIBUTION OF IRON &
OXIDATIVE STRESS TO CELL DEATH
Name: David Kirrane
ID Number: 11419558
Academic Supervisor: Prof. Afshin Samali
Course: UndenominatedScience.
Word Count: 4834

2. The important contribution of Iron & Oxidative Stress to Cell Death
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

3. 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

4. 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].

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].

6. 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].

7. 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

8. 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].

9. 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].

10. 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].

11. 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.

12. 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].

13. 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].

14. 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].

15. BIBLIOGRAPHY
1. Puntarulo S: Iron, oxidative stress and human health. Mol Aspects Med 2005, 26:299-
312.
2. Orrenius S, Gogvadze V, Zhivotovsky B: Mitochondrial oxidative stress: implications
for cell death. Annu Rev Pharmacol Toxicol 2007, 47:143-183.
3. Galaris D, Pantopoulos K: Oxidative stress and iron homeostasis: mechanistic and
health aspects. Crit Rev Clin Lab Sci 2008, 45:1-23.
4. Salvador GA, Uranga RM, Giusto NM: Iron and mechanisms of neurotoxicity. Int J
Alzheimers Dis. 2010, 2011: 1-10.
5. Dixon SJ, Stockwell BR: The role of iron and reactive oxygen species in cell death. Nat
Chem Biol 2014, 10:9-17.
6. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN,
Bauer AJ, Cantley AM, Yang WS, et al.: Ferroptosis: an iron-dependent form of
nonapoptotic cell death. Cell 2012, 149:1060-1072.
7. Nick H: Iron chelation, quo vadis? Curr Opin Chem Biol 2007, 11:419-423.
8. Lieu PT, Heiskala M, Peterson PA, Yang Y: The roles of iron in health and disease. Mol
Aspects Med 2001, 22:1-87.
9. Frey PA, Reed GH: The ubiquity of iron. ACS Chem Biol 2012, 7:1477-1481.
10. Andrews NC, Schmidt PJ: Iron homeostasis. Annu Rev Physiol 2007, 69:69-85.
11. Fridovich I: Fundamental aspects of reactive oxygen species, or what's the matter
with oxygen? Ann N Y Acad Sci 1999, 893:13-18.
12. Mytilineou C, Kramer BC, Yabut JA: Glutathione depletion and oxidative stress.
Parkinsonism Relat Disord 2002, 8:385-387.
13. Noori S: An Overview of Oxidative Stress and Antioxidant Defensive System.
Scientific Reports 2012, 1 (8).
14. Kroemer G et al: Classification of cell death: recommendations of the Nomenclature
Committee on Cell Death. Cell Death Differ. 2005, 12:1463-1467.
15. Franco R, Cidlowski JA: Apoptosis and glutathione: beyond an antioxidant. Cell
Death Differ. 2009, 16:1303-1314.
16. Ryter SW, Kim HP, Hoetzel A, Park JW, Nakahira K, Wang X, Choi AM: Mechanisms
of cell death in oxidative stress. Antioxid Redox Signal. 2007, 9(1):49-89.
17. Kamata H, Honda SI, Maeda S, Chang L, Hirata H, Karin M: Reactive Oxygen Species
Promote TNFα-Induced Death and Sustained JNK Activation by Inhibiting MAP
Kinase Phosphatases. Cell 2005, 120:649-661.
18. Seiler A et al: Glutathione Peroxidase 4 Senses and Translates Oxidative Stress into
12/15 Lipoxygenase Dependent- and AIF-Mediated Cell Death. Cell Metabolism 2008,
8:237-248.